BioMed Research International: Molecular Biology The latest articles from Hindawi © 2017 , Hindawi Limited . All rights reserved. Enhancement of Angiogenesis by Ultrasound-Targeted Microbubble Destruction Combined with Nuclear Localization Signaling Peptides in Canine Myocardial Infarction Sun, 12 Nov 2017 00:00:00 +0000 Objective. This study aimed to develop a gene delivery system using ultrasound-targeted microbubbles destruction (UTMD) combined with nuclear localization signal (NLS) and investigate its efficacy and safety for therapeutic angiogenesis in canine myocardial infarction (MI) model. Methods. Fifty MI dogs were randomly divided into 5 groups and transfected with Ang-1 gene plasmid: (i) group A: only injection of microbubbles and Ang-1 plasmid; (ii) group B: only UTMD mediated gene transfection; (iii) group C: UTMD combined with classical NLS mediated gene transfection; (iv) group D: UTMD combined with mutational NLS mediated transfection; and (v) group E: UTMD combined with classical NLS in the presence of a nucleus transport blocker. The mRNA and protein expression of Ang-1 gene, microvessel density (MVD) cardiac troponin I (cTnI), and cardiac function were determined after transfection. Results. The expression of mRNA and protein of Ang-1 gene in group C was significantly higher than that of the other groups (all P < 0.01). The MVD of group C was 10.2-fold of group A and 8.1-fold of group E (P < 0.01). The cardiac function in group C was significant improvement without cTnI rising. Conclusions. The gene delivery system composed of UTMD and NLS is efficient and safe. Jingjing Cui, Qing Deng, Qing Zhou, Sheng Cao, Nan Jiang, Yijia Wang, Jinling Chen, Bo Hu, and Tuantuan Tan Copyright © 2017 Jingjing Cui et al. All rights reserved. Inhibitors of Histone Deacetylases Are Weak Activators of the FMR1 Gene in Fragile X Syndrome Cell Lines Wed, 25 Oct 2017 00:00:00 +0000 Fragile X syndrome is the most common cause of inherited intellectual disability in humans. It is a result of CGG repeat expansion in the 5′ untranslated region (5′ UTR) of the FMR1 gene. This gene encodes the FMRP protein that is involved in neuronal development. Repeat expansion leads to heterochromatinization of the promoter, gene silencing, and the subsequent absence of FMRP. To date, there is no specific therapy for the syndrome. All treatments in clinic practice provide symptomatic therapy. The development of drug therapy for Fragile X syndrome treatment is connected with the search for inhibitors of enzymes that are responsible for heterochromatinization. Here, we report a weak transcriptional activity of the FMR1 gene and the absence of FMRP protein after Fragile X syndrome cell lines treatment with two FDA approved inhibitors of histone deacetylases, romidepsin and vorinostat. We demonstrate that romidepsin, an inhibitor of class I histone deacetylases, does not activate FMR1 expression in patient cell cultures, whereas vorinostat, an inhibitor of classes I and II histone deacetylases, activates a low level of FMR1 expression in some patient cell lines. Alexander A. Dolskiy, Vladimir O. Pustylnyak, Andrey A. Yarushkin, Natalya A. Lemskaya, and Dmitry V. Yudkin Copyright © 2017 Alexander A. Dolskiy et al. All rights reserved. Effects of LncRNA BC168687 siRNA on Diabetic Neuropathic Pain Mediated by P2X7 Receptor on SGCs in DRG of Rats Tue, 24 Oct 2017 09:32:34 +0000 Diabetic neuropathic pain (DNP), one of the early symptoms of diabetic neuropathy, relates to metabolic disorders induced by high blood glucose, neurotrophic vascular ischemia and hypoxia, and autoimmune factors. This study was aimed at exploring the effects of long noncoding RNA (lncRNA) BC168687 siRNA on DNP mediated by P2X7 receptor on SGCs in DRG of rats. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of rats, the expression levels of P2X7 mRNA and protein in the DRG, and nitric oxide (NO) in the serum were, respectively, detected in our study. Our experimental results showed that the level of BC168687 mRNA in DNP group was markedly higher than that of control group; the MWT and TWL of DNP + BC168687 si group were significantly increased, and the expression levels of P2X7 in DRG and the concentrations of NO in serum of DNP + BC168687 si group were decreased compared to those of the DNP group. In conclusion, lncRNA BC168687 may participate in the pathogenesis of DNP mediated by P2X7 receptor, which will provide a novel way for the study of the pathogenesis of diabetes mellitus complicated with neuropathic pain and its prevention and treatment. Chenglong Liu, Jia Tao, Hui Wu, Yixin Yang, Qiang Chen, Zeyu Deng, Jiandi Liu, and Changshui Xu Copyright © 2017 Chenglong Liu et al. All rights reserved. Combined Transcriptomic Analysis Revealed AKR1B10 Played an Important Role in Psoriasis through the Dysregulated Lipid Pathway and Overproliferation of Keratinocyte Tue, 24 Oct 2017 06:58:07 +0000 RNA-seq has enabled in-depth analysis of the pathogenesis of psoriasis on the transcriptomic level, and many biomarkers have been discovered to be related to the immune response, lipid metabolism, and keratinocyte proliferation. However, few studies have combined analysis from various datasets. In this study, we integrated different psoriasis RNA-seq datasets to reveal the pathogenesis of psoriasis through the analysis of differentially expressed genes (DEGs), pathway analysis, and functional annotation. The revealed biomarkers were further validated through proliferation phenotypes. The results showed that DEGs were functionally related to lipid metabolism and keratinocyte differentiation dysregulation. The results also showed new biomarkers, such as AKR1B10 and PLA2G gene families, as well as pathways that include the PPAR signaling pathway, cytokine-cytokine receptor interaction, alpha-linoleic acid metabolism, and glycosphingolipid biosynthesis. Using siRNA knockdown assays, we further validated the role that the AKR1B10 gene plays in proliferation. Our study demonstrated not only the dysfunction of the AKR1B10 gene in lipid metabolizing but also its important role in the overproliferation and migration of keratinocyte, which provided evidence for further therapeutic uses for psoriasis. Yunlu Gao, Xuemei Yi, and Yangfeng Ding Copyright © 2017 Yunlu Gao et al. All rights reserved. miR-195-5p Suppresses the Proliferation, Migration, and Invasion of Oral Squamous Cell Carcinoma by Targeting TRIM14 Tue, 24 Oct 2017 00:00:00 +0000 MicroRNAs (miRNAs) play an essential role in tumor biological processes through interacting with specific gene targets. The involvement of miR-195-5p in cell proliferation, invasion, and migration has been demonstrated in several cancer cell lines, while its function in oral squamous cell carcinoma (OSCC) remains unclear. Here we find that miR-195-5p expression is lower in OSCC than in nontumor tissues, while its overexpression in cell lines can lead to the promotion of apoptosis and the reduction of cell growth, migration, and invasion. Moreover, we identify the tripartite motif-containing protein (TRIM14) as a target of miR-195-5p. Therefore, we reason that the tumor suppressor role of miR-195-5p in OSCC is dependent on the interaction with TRIM14. Tong Wang, Yipeng Ren, Ruixun Liu, Juntao Ma, Yueyi Shi, Lei Zhang, and Rongfa Bu Copyright © 2017 Tong Wang et al. All rights reserved. Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico Thu, 19 Oct 2017 00:00:00 +0000 We had previously reported presence of histone deacetylase 6 (HDAC6) in sperm and demonstrated its tubulin deacetylase activity and role in sperm motility in rat. In the present study we report its abundant expression in testis, epididymis, accessory sex organs, brain, and adrenal. In the testis, HDAC6 transcript and protein were observed throughout development. We therefore cloned the gene from rat testis using primers for hdac6 (accession number XM_228753.8) in order to determine the role of acetylation/deacetylation in spermatogenesis. The cloned rat hdac6 gene is ~3.5 kb with 28 exons and 1152 amino acids. We noted 4 single nucleotide polymorphisms (SNPs) on exons 2 (G/A), 5 (A/G), 7 (T/C), and 26 (G/T), respectively, in this sequence when compared to XM_228753.8. These were further validated at both cDNA and gene level. These SNPs resulted in 2 amino acids changes, namely, glycine → arginine and valine → phenylalanine at protein level. Cloned hdac6 overexpressed in HEK293T cells demonstrated significant overexpression by IIF. Alpha-tubulin acetylation analysis of the overexpressed cell lysate demonstrated that the protein was bioactive. This is the first study showing the ontogenic expression in the testis and reporting experimentally validated sequence of rat HDAC6 and its structural and functional annotation in silico. This sequence has been submitted to GenBank (Accession number Rattus KY009929.1). Pratibha Verma, Omshree Shetty, Sweta Parab, Karen Menezes, and Priyanka Parte Copyright © 2017 Pratibha Verma et al. All rights reserved. Biochemical and Genetical Responses of Phoenix dactylifera L. to Cadmium Stress Thu, 19 Oct 2017 00:00:00 +0000 The cadmium (Cd), a heavy metal, causes toxicity, which leads to hampering the growth and development of the plant. The molecular and biochemical approaches were used for the investigation of antioxidant system response and genotoxicity in date palm (Phoenix dactylifera L.) cv. Sagai in pot experiment having Cd. The root length was more affected than the shoot length as more accumulation of Cd occurs in roots. Fresh weights of root and shoot were reduced significantly in treated plants as compared to the control. The proline content was increased at low concentration of Cd (300 µM-CdCl2) than the medium and high concentrations (600 and 900 µM-CdCl2), respectively. The thiobarbituric acid reactive substances (TBARS) content was increased at 600 and 900 µM-CdCl2 compared to the plants treated at 300 µM-CdCl2 and controls. Antioxidant enzymatic assay was performed under Cd stress and compared with control plants. The catalase (CAT) and superoxide dismutase (SOD) activities were found to be high in plants treated with CdCl2 at 300 µM compared to at 600 and 900 µM-CdCl2, respectively. The genotoxicity of Cd was assessed using the inter-simple sequence repeat (ISSR) marker where all treated and control plants were clustered into three main groups based on genetic similarity. P. dactylifera plants were found to be more divergent at high Cd stress as compared to control and plants treated at low concentration of Cd. Fahad Al-Qurainy, Salim Khan, Mohamed Tarroum, Mohammad Nadeem, Saleh Alansi, and Aref Alshameri Copyright © 2017 Fahad Al-Qurainy et al. All rights reserved. Barcoding the Dendrobium (Orchidaceae) Species and Analysis of the Intragenomic Variation Based on the Internal Transcribed Spacer 2 Tue, 17 Oct 2017 08:58:17 +0000 Many species belonging to the genus Dendrobium are of great commercial value. However, their difficult growth conditions and high demand have caused many of these species to become endangered. Indeed, counterfeit Dendrobium products are common, especially in medicinal markets. This study aims to assess the suitability of the internal transcribed spacer 2 (ITS2) region as a marker for identifying Dendrobium and to evaluate its intragenomic variation in Dendrobium species. In total, 29,624 ITS2 copies from 18 species were obtained using 454 pyrosequencing to evaluate intragenomic variation. In addition, 513 ITS2 sequences from 26 Dendrobium species were used to assess its identification suitability. The highest intragenomic genetic distance was observed in Dendrobium chrysotoxum (0.081). The average intraspecific genetic distances of each species ranged from 0 to 0.032. Phylogenetic trees based on ITS2 sequences showed that most Dendrobium species are monophyletic. The intragenomic and intraspecies divergence analysis showed that greater intragenomic divergence is mostly correlated with larger intraspecific variation. As a major ITS2 variant becomes more common in genome, there are fewer intraspecific variable sites in ITS2 sequences at the species level. The results demonstrated that the intragenomic multiple copies of ITS2 did not affect species identification. Xiaoyue Wang, Xiaochen Chen, Pei Yang, Lili Wang, and Jianping Han Copyright © 2017 Xiaoyue Wang et al. All rights reserved. Fibroblast Growth Factor 21 Promotes C2C12 Cells Myogenic Differentiation by Enhancing Cell Cycle Exit Wed, 04 Oct 2017 08:36:42 +0000 Fibroblast growth factor 21 (FGF21), a secretion protein, functions as a pivotal regulator of energy metabolism and is being considered as a therapeutic candidate in metabolic syndromes. However, the roles of FGF21 in myogenic differentiation and cell cycle remain obscure. In this study, we investigated the function of FGF21 in myogenesis and cell cycle exit using C2C12 cell line. Our data showed that the expression of myogenic genes as well as cell cycle exit genes was increased after FGF21 overexpression, and FGF21 overexpression induces cell cycle arrest. Moreover, cell cycle genes were decreased in FGF21 overexpression cells while they were increased in FGF21 knockdown cells. Further, FGF21/P53/p21/Cyclin-CDK has been suggested as the key pathway for cell cycle exit mediated by FGF21 in C2C12 cells. Also, we deduce that FGF21 promotes the initiation of myogenic differentiation mainly through enhancing cell cycle exit of C2C12 cells. Taken together, our results demonstrated that FGF21 promotes cell cycle exit and enhances myogenic differentiation of C2C12 cells. This study provided new evidence that FGF21 promotes myogenic differentiation, which could be useful for better understanding the roles of FGF21 in myogenesis. Xinyi Liu, Yongliang Wang, Shuhong Zhao, and Xinyun Li Copyright © 2017 Xinyi Liu et al. All rights reserved. Cell Signaling Pathway in 12-O-Tetradecanoylphorbol-13-acetate-Induced LCN2 Gene Transcription in Esophageal Squamous Cell Carcinoma Mon, 02 Oct 2017 00:00:00 +0000 LCN2 is involved in various cellular functions, including transport of small hydrophobic molecules, protection of MMP9 from proteolytic degradation, and regulating innate immunity. LCN2 is elevated in multiple human cancers, frequently being associated with tumor size, stage, and invasiveness. Our previous studies have shown that LCN2 expression could be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in esophageal squamous cell carcinoma (ESCC) by the binding of five nucleoproteins (MISP, KLF10, KLF15, PPP1R18, and RXRβ) at a novel TPA-responsive element (TRE), at −152~−60 bp of the 5′ flanking region of the LCN2 promoter. However, much is unknown about whether these proteins can respond to TPA stimulation to regulate LCN2 transactivation and which cell signaling pathways mediate this process. In this study, expression plasmids encoding these five nucleoproteins were stably transfected into EC109 cells. Then, stable transfectant was characterized by a Dual-Luciferase Reporter Assay System. RT-PCR, real-time PCR, western blotting, specific kinase inhibitor treatment, and bioinformatics analyses were applied in this study. We found that MISP, KLF10, KLF15, PPP1R18, and RXRβ proteins could strongly respond to TPA stimulation and activate LCN2 transcriptional expression. MEK, ERK, JNK, and P38 kinases were involved in the LCN2 transactivation. Furthermore, the MEK-ERK signal pathway plays a major role in this biological process but does not involve PKCα signaling. Lingying Meng, Muting Wang, Zepeng Du, Zhongmin Fang, Bingli Wu, Jianyi Wu, Wenming Xie, Jian Shen, Tianxiang Zhu, XieE Xu, Liandi Liao, Liyan Xu, Enmin Li, and Bin Lan Copyright © 2017 Lingying Meng et al. All rights reserved. Angelica sinensis Suppresses Body Weight Gain and Alters Expression of the FTO Gene in High-Fat-Diet Induced Obese Mice Wed, 20 Sep 2017 00:00:00 +0000 The root of Angelica sinensis (RAS) is a traditional Chinese medicine used for preventing and treating various diseases. In this study, we assessed RAS supplementation effects on body weight and the FTO gene expression and methylation status in a high-fat-diet (HFD) induced obese mouse model. Female obese mice were divided into groups according to RAS dosage in diet as follows: normal diet, HFD diet (HC), HFD with low-dosage RAS (DL), HFD with medium-dosage RAS (DM), and HFD with high-dosage RAS (DH). After RAS supplementation for 4 weeks, body weight suppression and FTO expression in DH mice were significantly higher than in HC mice, whereas no significant change in FTO expression was detected between DM and DL mice or in their offspring. Bisulfite sequencing PCR (BSP) revealed that the CpG island in the FTO promoter was hypermethylated up to 95.44% in the HC group, 91.67% in the DH group, and 90.00% in the normal diet group. Histological examination showed that adipocytes in the DH group were smaller than those in the HC group, indicating a potential role of RAS in obesity. This study indicated that RAS could ameliorate obesity induced by HFD and that the molecular mechanism might be associated with the expression of the FTO gene. Tao Zhong, Xiao-Yue Duan, Hao Zhang, Li Li, Hong-Ping Zhang, and Lili Niu Copyright © 2017 Tao Zhong et al. All rights reserved. The miRNA Expression Profile in Acute Myocardial Infarct Using Sheep Model with Left Ventricular Assist Device Unloading Mon, 11 Sep 2017 00:00:00 +0000 This study attempted to establish miRNA expression profiles in acute myocardial infarct (AMI) sheep model with left ventricular assist device (LVAD) unloading. AMI was established in sheep model and FW-II type axial flow pump was implanted to maintain continuous unloading for 3 days. The cardiomyocyte survival, inflammatory cell infiltration, and myocardial fibrosis were detected by tissue staining, and cardiomyocyte apoptosis was detected by TUNEL assay. High throughput sequencing technique was used to detect miRNA expression in cardiomyocytes and to establish miRNA expression profile. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were established. miRNA sequencing results identified 152 known mature miRNAs and 1582 new mature miRNAs. The unloading and control groups differentially expressed genes, of which RT-PCR verified oar-miR-19b and oar-miR-26a. The GO and KEGG pathway annotation and enrichment established that the regulating functions and signaling pathways of these miRNAs were closely related to cardiovascular diseases (CVD). In this study, LVAD effectively reduced the cell death degree of cardiomyocyte in MI. The established miRNA expression profiles of AMI and LVAD intervention in this study suggest that the expression profile could be used to explore the unknown miRNA and the regulatory mechanisms involved in AMI. Xiaoqian Yan, Yu Gan, Haibo Chen, Guangmao Liu, Shengshou Hu, and Jianye Zhou Copyright © 2017 Xiaoqian Yan et al. All rights reserved. Evaluation of the Bacterial Diversity in the Human Tongue Coating Based on Genus-Specific Primers for 16S rRNA Sequencing Sun, 20 Aug 2017 00:00:00 +0000 The characteristics of tongue coating are very important symbols for disease diagnosis in traditional Chinese medicine (TCM) theory. As a habitat of oral microbiota, bacteria on the tongue dorsum have been proved to be the cause of many oral diseases. The high-throughput next-generation sequencing (NGS) platforms have been widely applied in the analysis of bacterial 16S rRNA gene. We developed a methodology based on genus-specific multiprimer amplification and ligation-based sequencing for microbiota analysis. In order to validate the efficiency of the approach, we thoroughly analyzed six tongue coating samples from lung cancer patients with different TCM types, and more than 600 genera of bacteria were detected by this platform. The results showed that ligation-based parallel sequencing combined with enzyme digestion and multiamplification could expand the effective length of sequencing reads and could be applied in the microbiota analysis. Beili Sun, Dongrui Zhou, Jing Tu, and Zuhong Lu Copyright © 2017 Beili Sun et al. All rights reserved. The uS8, uS4, eS31, and uL14 Ribosomal Protein Genes Are Dysregulated in Nasopharyngeal Carcinoma Cell Lines Sun, 16 Jul 2017 00:00:00 +0000 The association of ribosomal proteins with carcinogenesis of nasopharyngeal carcinoma (NPC) has been established in a limited subset of ribosomal protein genes. To date, three ribosomal protein genes, eL27 (L27), eL41 (L41), and eL43 (L37a), have been found to be differentially expressed in cell lines derived from NPC tumors. This raises the possibility of more ribosomal protein genes that could be associated with NPC. In this study, we investigated the expression profiles of eight ribosomal protein genes, uS8 (S8), uS4 (S9), eS31 (S27a), eL6 (L6), eL18 (L18), uL14 (L23), eL24 (L24), and eL30 (L30), in six NPC-derived cell lines (HONE-1, SUNE1, HK1, TW01, TW04, and C666-1). Their expression levels were compared with that of a nonmalignant nasopharyngeal epithelial cell line (NP69) using quantitative real-time PCR (RT-qPCR) assay. Of the eight genes studied, the expressions of four ribosomal protein genes uS8 (S8), uS4 (S9), eS31 (S27a), and uL14 (L23) were found to be significantly downregulated in NPC cell lines relative to NP69. Our findings provide novel empirical evidence of these four ribosomal protein genes as NPC-associated genetic factors and reinforce the relevance of ribosomal proteins in the carcinogenesis of nasopharyngeal cancer. Edmund Ui-Hang Sim, Kher-Lee Ng, Choon-Weng Lee, and Kumaran Narayanan Copyright © 2017 Edmund Ui-Hang Sim et al. All rights reserved. Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics Wed, 12 Jul 2017 00:00:00 +0000 Nucleic acid extraction (NAE) plays a vital role in molecular biology as the primary step for many downstream applications. Many modifications have been introduced to the original 1869 method. Modern processes are categorized into chemical or mechanical, each with peculiarities that influence their use, especially in point-of-care diagnostics (POC-Dx). POC-Dx is a new approach aiming to replace sophisticated analytical machinery with microanalytical systems, able to be used near the patient, at the point of care or point of need. Although notable efforts have been made, a simple and effective extraction method is still a major challenge for widespread use of POC-Dx. In this review, we dissected the working principle of each of the most common NAE methods, overviewing their advantages and disadvantages, as well their potential for integration in POC-Dx systems. At present, it seems difficult, if not impossible, to establish a procedure which can be universally applied to POC-Dx. We also discuss the effects of the NAE chemicals upon the main plastic polymers used to mass produce POC-Dx systems. We end our review discussing the limitations and challenges that should guide the quest for an efficient extraction method that can be integrated in a POC-Dx system. Nasir Ali, Rita de Cássia Pontello Rampazzo, Alexandre Dias Tavares Costa, and Marco Aurelio Krieger Copyright © 2017 Nasir Ali et al. All rights reserved. Experimental Verification of CAPN1 and CAST Gene Polymorphisms in Different Generations of Da-Heng Broilers Wed, 21 Jun 2017 00:00:00 +0000 The micromolar calcium activated neutral protease (CAPN1) and calpastatin (CAST) have been widely regarded as genes related to muscle growth and meat tenderness. The objective of this study was to verify the association of SNPs of CAPN1 and CAST genes with carcass and tenderness traits and search the possible change patterns of SNPs in CAPN1 and CAST genes in six generations of broiler breeding process for growth rate, efficiency, and reproduction, during the third generation and the ninth generation, respectively. We found that, for CAPN1, genetic effects between SNPs (G3535A, C7198A) and meat tenderness were similar in different generations, while SNP3 (G7324A) was a novel polymorphism and had significant association with carcass and tenderness traits () in this study. Furthermore, there was significant association between SNP4 (G9950A) and carcass indexes instead of tenderness traits () which was consistent in the two generations. Moreover, although SNP6 (G37868A) of CAST had no relevance to carcass traits or tenderness traits in the third generation, it showed significant association with LW and CW in the ninth generation (). Yu-Guang Zhou, Yong Xiong, Chao-Wu Yang, Xiao-Song Jiang, Jin-Shan Ran, Jie Jin, Ye Wang, Dan Lan, Peng Ren, Yao-Dong Hu, and Yi-Ping Liu Copyright © 2017 Yu-Guang Zhou et al. All rights reserved. Genetic Variation and Its Reflection on Posttranslational Modifications in Frequency Clock and Mating Type a-1 Proteins in Sordaria fimicola Wed, 21 Jun 2017 00:00:00 +0000 Posttranslational modifications (PTMs) occur in all essential proteins taking command of their functions. There are many domains inside proteins where modifications take place on side-chains of amino acids through various enzymes to generate different species of proteins. In this manuscript we have, for the first time, predicted posttranslational modifications of frequency clock and mating type a-1 proteins in Sordaria fimicola collected from different sites to see the effect of environment on proteins or various amino acids pickings and their ultimate impact on consensus sequences present in mating type proteins using bioinformatics tools. Furthermore, we have also measured and walked through genomic DNA of various Sordaria strains to determine genetic diversity by genotyping the short sequence repeats (SSRs) of wild strains of S. fimicola collected from contrasting environments of two opposing slopes (harsh and xeric south facing slope and mild north facing slope) of Evolution Canyon (EC), Israel. Based on the whole genome sequence of S. macrospora, we targeted 20 genomic regions in S. fimicola which contain short sequence repeats (SSRs). Our data revealed genetic variations in strains from south facing slope and these findings assist in the hypothesis that genetic variations caused by stressful environments lead to evolution. Rabia Arif, Faiza Akram, Tazeen Jamil, Hamid Mukhtar, Siu Fai Lee, and Muhammad Saleem Copyright © 2017 Rabia Arif et al. All rights reserved. The Effect of Osteopontin on Microglia Sun, 18 Jun 2017 08:06:50 +0000 Osteopontin (OPN) is a proinflammatory cytokine that can be secreted from many cells, including activated macrophages and T-lymphocytes, and is widely distributed in many tissues and cells. OPN, a key factor in tissue repairing and extracellular matrix remodeling after injury, is a constituent of the extracellular matrix of the central nervous system (CNS). Recently, the role of OPN in neurodegenerative diseases has gradually caused widespread concern. Microglia are resident macrophage-like immune cells in CNS and play a vital role in both physiological and pathological conditions, including restoring the integrity of the CNS and promoting the progression of neurodegenerative disorders. Microglia’s major function is to maintain homeostasis and the normal function of the CNS, both during development and in response to CNS injury. Although the functional mechanism of OPN in CNS neurodegenerative diseases has yet to be fully elucidated, most studies suggest that OPN play a role in pathogenesis of neurodegenerative diseases or in neuroprotection by regulating the activation and function of microglia. Here, we summarize the functions of OPN on microglia in response to various stimulations in vitro and in vivo. Huan Yu, Xiaohong Liu, and Yisheng Zhong Copyright © 2017 Huan Yu et al. All rights reserved. No Significant Difference between Plasma miRNAs and Plasma-Derived Exosomal miRNAs from Healthy People Thu, 01 Jun 2017 07:27:19 +0000 Background. MicroRNAs (miRNAs) are small noncoding RNAs that are involved in many biological regulation processes. Studies have reported that miRNAs are enriched in human plasma and plasma-derived exosome as novel diagnostic biomarkers. The aim of this study was to determine whether the miRNA expression levels are different between plasma and plasma-derived exosome. Methods. We sequenced and quantified the miRNAs in plasma and exosome from healthy blood samples and validated three miRNAs in the two groups of lung cancer samples by qRT-PCR. Results. The sequencing results showed that only several of miRNAs were differential, while the qRT-PCR further validated that most of them did not have the consistent differences. However, the levels of two upregulated miRNAs (miR-181b-5p and miR-21-5p) in lung cancer were significantly higher in exosomes than plasma. Conclusions. To the best of our knowledge, this study is the first to compare the expression levels of miRNAs between plasma and exosome in healthy blood samples. Our data suggested that the miRNA levels were similar in the two parts of the healthy people, whereas the two onco-miRNAs were significantly enriched in the exosome of lung cancer patients. Fei Tian, Yanting Shen, Zhenzhu Chen, Rui Li, and Qinyu Ge Copyright © 2017 Fei Tian et al. All rights reserved. Isolation and Abiotic Stress Resistance Analyses of a Catalase Gene from Ipomoea batatas (L.) Lam Tue, 30 May 2017 10:06:00 +0000 As an indicator of the antioxidant capability of plants, catalase can detoxify reactive oxygen species (ROS) generated by environmental stresses. Sweet potato is one of the top six most important crops in the world. However, its catalases remain largely unknown. In this study, a catalase encoding gene, IbCAT2 (accession number: KY615708), was identified and cloned from sweet potato cv. Xushu 18. It contained a 1479 nucleotides’ open reading frame (ORF). S-R-L, Q-K-L, and a putative calmodulin binding domain were located at the C-terminus of IbCAT2, which suggests that IbCAT2 could be a peroxisomal catalase. Next-generation sequencing (NGS) based quantitative analyses showed that IbCAT2 was mainly expressed in young leaves and expanding tuberous roots under normal conditions. When exposed to 10% PEG6000 or 200 mmol/L NaCl solutions, IbCAT2 was upregulated rapidly in the first 11 days and then downregulated, although different tissues showed different degree of change. Overexpression of IbCAT2 conferred salt and drought tolerance in Escherichia coli and Saccharomyces cerevisiae. The positive response of IbCAT2 to abiotic stresses suggested that IbCAT2 might play an important role in stress responses. Bin Yong, Xiaoyan Wang, Pan Xu, Haiyan Zheng, Xueting Fei, Zixi Hong, Qinqin Ma, Yuzhi Miao, Xianghua Yuan, Yusong Jiang, and Huanhuan Shao Copyright © 2017 Bin Yong et al. All rights reserved. Effect of Gamma Rays on Sophora davidii and Detection of DNA Polymorphism through ISSR Marker Mon, 22 May 2017 09:01:48 +0000 Sophora davidii (Franch.) Kom. ex Pavol is an important medicinal plant and a feeding scrub with ecological value. The effects of different gamma irradiation doses (20–140 Kr) on seed germination and seedling morphology were investigated in S. davidii, and intersimple sequence repeat (ISSR) markers were used to identify the DNA polymorphism among mutants. Significant variations were observed for seed germination, stem diameter, and number of branches per plant. The improved agronomic traits, such as stem diameter and number of branches per plant, were recorded at 80 Kr dose and 20 Kr dose for seed germination. ISSR analysis generated in total 183 scorable fragments, of which 94 (51.37%) were polymorphic. The percentage of polymorphism ranged from 14.29 to 93.33 with an average of 45.69%. Jaccard’s coefficients of dissimilarity varied from 0.6885 to 1.000, indicative of the level of genetic variation among the mutants. The constructed dendrogram grouped the entities into five clusters. Consequently, it was concluded that gamma rays irradiation of seeds generates a sufficient number of induced mutations and that ISSR analysis offered a useful molecular marker for the identification of mutants. Puchang Wang, Yu Zhang, Lili Zhao, Bentian Mo, and Tianqiong Luo Copyright © 2017 Puchang Wang et al. All rights reserved. Piper betle L. Modulates Senescence-Associated Genes Expression in Replicative Senescent Human Diploid Fibroblasts Wed, 17 May 2017 08:12:15 +0000 Piper betle (PB) is a traditional medicine that is widely used to treat different diseases around Asian region. The leaf extracts contain various bioactive compounds, which were reported to have antidiabetic, antibacterial, anti-inflammatory, antioxidant, and anticancer effects. In this study, the effect of PB aqueous extracts on replicative senescent human diploid fibroblasts (HDFs) was investigated by determining the expressions of senescence-associated genes using quantitative PCR. Our results showed that PB extracts at 0.4 mg/ml can improve cell proliferation of young (143%), presenescent (127.3%), and senescent (157.3%) HDFs. Increased expressions of PRDX6, TP53, CDKN2A, PAK2, and MAPK14 were observed in senescent HDFs compared to young and/or presenescent HDFs. Treatment with PB extracts modulates the transcriptional profile changes in senescent HDFs. By contrast, expressions of SOD1 increased, whereas GPX1, PRDX6, TP53, CDKN2A, PAK2, and MAPK14 were decreased in PB-treated senescent HDFs compared to untreated senescent HDFs. In conclusion, this study indicates the modulation of PB extracts on senescence-associated genes expression of replicative senescent HDFs. Further studies warrant determining the mechanism of PB in modulating replicative senescence of HDFs through these signaling pathways. Lina Wati Durani, Shy Cian Khor, Jen Kit Tan, Kien Hui Chua, Yasmin Anum Mohd Yusof, and Suzana Makpol Copyright © 2017 Lina Wati Durani et al. All rights reserved. Exogenous Expressions of FTO Wild-Type and R316Q Mutant Proteins Caused an Increase in HNRPK Levels in 3T3-L1 Cells as Demonstrated by DIGE Analysis Sun, 07 May 2017 00:00:00 +0000 Fat mass and obesity-associated protein is an enzyme that oxidatively demethylates DNA. Although there are numerous studies regarding the catalytic function of FTO, the overall existence or absence of FTO on cellular proteome has not been investigated. This study investigated the changes in the soluble proteome of 3T3-L1 cells upon expression of the WT and the mutant (R316Q) FTO proteins. Protein extracts prepared from 3T3-L1 cells expressing either the WT or the mutant FTO proteins were used in DIGE experiments. Analysis of the data revealed the number of spots matched to every member and there were 350 ± 20 spots with 30.5% overall mean coefficient of variation. Eleven regulated protein spots were excised from the gels and identified by MALDI-TOF/TOF. One of the identified proteins was heterogeneous nuclear ribonucleoprotein K, which displayed more than 2.6- and 3.7-fold increases in its abundance in the WT and the mutant FTO expressing cells, respectively. Western blot analysis validated these observations. This is the first study revealing the presence of a parallel increase in expressions of FTO and HNRNPK proteins. This increase may codictate the metabolic changes occurring in the cell and may attribute a significance to HNRNPK in FTO-associated transformations. Nil Guzel, Murat Kasap, Aylin Kanli, Gurler Akpinar, M. Dogan Gulkac, and Kubra Karaosmanoglu Copyright © 2017 Nil Guzel et al. All rights reserved. Extracellular Signal-Regulated Kinase 5 is Required for Low-Concentration H2O2-Induced Angiogenesis of Human Umbilical Vein Endothelial Cells Sun, 30 Apr 2017 13:25:20 +0000 Background. The aim of this study was to assess the effects of low concentrations of H2O2 on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro and explore the underlying mechanisms. Methods. HUVECs were cultured and stimulated with different concentrations of H2O2. Flow cytometric analysis was used to select an optimal concentration of H2O2 for the following experiments. Cell proliferation, migration, and tubule formation were evaluated by Cell Counting Kit-8 (CCK-8) assays, scratch wound assays, and Matrigel tubule formation assays, respectively. For gain and loss of function studies, constitutively active MEK5 (CA-MEK5) and ERK5 shRNA lentiviruses were used to activate or knock down extracellular signal-regulated kinase 5 (ERK5). Results. We found that low concentrations of H2O2 promoted HUVECs proliferation, migration, and tubule formation. ERK5 in HUVECs was significantly activated by H2O2. Enhanced ERK5 activity significantly amplified the proangiogenic effects of H2O2; in contrast, ERK5 knock-down abrogated the effects of H2O2. Conclusions. Our results confirmed that low concentrations of H2O2 promoted HUVECs angiogenesis in vitro, and ERK5 is an essential mediator of this process. Therefore, ERK5 may be a potential therapeutic target for promoting angiogenesis and improving graft survival. Shan Jiang, Dongxin Zhang, Hong Huang, Yonghong Lei, Yan Han, and Weidong Han Copyright © 2017 Shan Jiang et al. All rights reserved. The Critical Role of Long Noncoding RNA in Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Thu, 27 Apr 2017 10:53:55 +0000 Objective. Long noncoding RNAs (lncRNAs) have been demonstrated to regulate many biological processes including differentiation. However, their role in osteogenic differentiation was poorly known. Materials and Methods. In this study, we first globally profiled the differentially expressed lncRNAs and mRNAs during osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSCs). Bioinformatics analysis was performed to further analyze these significantly changed molecules. Then the role of lncRNA ENST00000502125.2 in the osteogenic differentiation was determined. Results. A number of lncRNAs and mRNAs were significantly differentially expressed during hBMMSC osteogenic differentiation. Among them, 433 lncRNAs and 956 mRNAs were continuously upregulated, while 232 lncRNAs and 229 mRNAs were continuously downregulated. Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that carbohydrate derivative binding and complement and coagulation cascades were most correlated molecular function and pathway, respectively. Downregulation of lncRNA ENST00000502125.2 promoted the osteogenic differentiation of hBMMSCs, and opposite results were found when lncRNA ENST00000502125.2 was upregulated. Conclusions. lncRNAs play a critical role in the osteogenic differentiation of hBMMSCs and targeting lncRNA ENST00000502125.2 might be a promising strategy to promote osteogenic differentiation. Xiaoling Qiu, Bo Jia, Xiang Sun, Weitao Hu, Hongxing Chu, Shuaimei Xu, and JianJiang Zhao Copyright © 2017 Xiaoling Qiu et al. All rights reserved. MicroRNA Expression Varies according to Glucose Tolerance, Measurement Platform, and Biological Source Wed, 26 Apr 2017 06:28:04 +0000 Dysregulated microRNA (miRNA) expression is observed during type 2 diabetes (T2D), although the consistency of miRNA expression across measurement platform and biological source is uncertain. Here we report miRNA profiling in the whole blood and serum of South African women with different levels of glucose tolerance, using next generation sequencing (NGS) and quantitative real time PCR (qRT-PCR). Whole blood-derived miRNAs from women with newly diagnosed T2D (), impaired glucose tolerance (IGT) (), and normal glucose tolerance (NGT) () were subjected to NGS, whereafter transcript levels of selected miRNAs were quantified in the whole blood and serum of these women using qRT-PCR. Of the five significantly differentially expressed miRNAs identified by NGS, only the directional increase of miR-27b in women with IGT compared to NGT was confirmed in whole blood and serum, using qRT-PCR. Functional enrichment of miR-27b gene targets identified biological pathways associated with glucose transport and insulin regulation. In conclusion, this study showed poor correlation in miRNA expression profiled using NGS and qRT-PCR and in whole blood and serum. The consistent increased expression of miR-27b in women with IGT compared to NGT across measurement platform and biological source holds potential as a biomarker for risk stratification in our population. S. Dias, S. Hemmings, C. Muller, J. Louw, and C. Pheiffer Copyright © 2017 S. Dias et al. All rights reserved. Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells Sun, 19 Mar 2017 09:13:53 +0000 Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome. Hiroshi Sato, Hiroki Kato, Haruyoshi Yamaza, Keiji Masuda, Huong Thi Nguyen Nguyen, Thanh Thi Mai Pham, Xu Han, Yuta Hirofuji, and Kazuaki Nonaka Copyright © 2017 Hiroshi Sato et al. All rights reserved. Overexpression of CaAPX Induces Orchestrated Reactive Oxygen Scavenging and Enhances Cold and Heat Tolerances in Tobacco Mon, 13 Mar 2017 07:05:43 +0000 Ascorbate peroxidase (APX) acts indispensably in synthesizing L-ascorbate (AsA) which is pivotal to plant stress tolerance by detoxifying reactive oxygen species (ROS). Enhanced activity of APX has been shown to be a key step for genetic engineering of improving plant tolerance. However it needs a deeper understanding on the maintenance of cellular ROS homeostasis in response to stress. In this study, we identified and characterized an APX (CaAPX) gene from Camellia azalea. Quantitative real-time PCR (qRT-PCR) analysis showed that CaAPX was expressed in all tissues and peaked in immature green fruits; the expression levels were significantly upregulated upon cold and hot stresses. Transgenic plants displayed marked enhancements of tolerance under both cold and heat treatments, and plant growth was correlated with CaAPX expression levels. Furthermore, we monitored the activities of several ROS-scavenging enzymes including Cu/Zn-SOD, CAT, DHAR, and MDHAR, and we showed that stress tolerance was synchronized with elevated activities of ROS-scavenging. Moreover, gene expression analysis of ROS-scavenging enzymes revealed a role of CaAPX to orchestrate ROS signaling in response to temperature stresses. Overall, this study presents a comprehensive characterization of cellular response related to CaAPX expression and provides insights to breed crops with high temperature tolerances. Jiangying Wang, Bin Wu, Hengfu Yin, Zhengqi Fan, Xinlei Li, Sui Ni, Libo He, and Jiyuan Li Copyright © 2017 Jiangying Wang et al. All rights reserved. Characterization and Expression Profiling Analysis of Calmodulin Genes in Response to Salt and Osmotic Stresses in Pear (Pyrus bretschneideri Rehd.) and in Comparison with Arabidopsis Wed, 08 Mar 2017 08:07:44 +0000 A genome-wide identification and cloning of CaM genes in pear was conducted and in compared with Arabidopsis that indicated a conserved expansion of CaM genes in pear, and PbCaMs and AtCaMs had a similar distribution of cis-elements and expressions in response to salt and osmotic stress. In particular, PbCaM1 and PbCaM3 were both significantly upregulated in response to salt and osmotic stress in pear. Jun Tang, Jing Lin, Xiaogang Li, Qingsong Yang, Qunkang Cheng, Zong-Ming (Max) Cheng, and Youhong Chang Copyright © 2017 Jun Tang et al. All rights reserved. Genotypic Diversity of Mycobacterium tuberculosis Clinical Isolates in the Multiethnic Area of the Xinjiang Uygur Autonomous Region in China Tue, 28 Feb 2017 13:51:21 +0000 Objectives. We studied the genetic diversity of clinical isolates from patients with tuberculosis in the multiethnic area of Xinjiang autonomous region in China. A total of 311 clinical M. tuberculosis isolates were collected in 2006 and 2011 and genotyped by two genotyping methods. All isolates were grouped into 68 distinct spoligotypes using the spoligotyping method. The Beijing family was dominant, followed by T1 and CAS. MIRU-VNTR results showed that a total of 195 different VNTR types were identified. Ten of the 15 loci were highly or moderately discriminant according to their HGDI scores, and 13 loci had good discriminatory power in non-Beijing family strains, whereas only two loci had good discriminatory power in Beijing family strains. Chi-square tests demonstrated that there were no correlations between four characteristics (sex, age, type of case, and treatment history) and the Beijing family. In summary, Beijing family strains were predominant in Xinjiang, and the locus-set was suitable for genotyping all Xinjiang strains, but not for the Beijing family strains. Thus, these data suggested that different genotype distributions may exist in different regions; MLVA locus-sets should be adjusted accordingly, with newly added loci to increase resolution if necessary. Jie Liu, Junlian Li, Jiao Liu, Xiuqin Zhao, Lulu Lian, Haican Liu, Bing Lu, Qin Yu, Jingrui Zhang, Yingcheng Qi, and Kanglin Wan Copyright © 2017 Jie Liu et al. All rights reserved. PTEN Reduced UVB-Mediated Apoptosis in Retinal Pigment Epithelium Cells Wed, 22 Feb 2017 07:24:21 +0000 Age-related macular degeneration (AMD) is a leading cause of blindness and progressive loss of central vision in the elderly population. The important factor of AMD pathogenesis is the degeneration of retinal pigment epithelial (RPE) cells by oxidative stress. Inactivation of PTEN can disrupt intercellular adhesion in the RPE cells, but the mechanism of oxidative stress is less known. Here we presented evidence that UVB-mediated oxidative stress induced apoptosis in ARPE-19 cells. Downregulation of the expression of PTEN in UVB-irradiative RPE cells triggered DNA damage and increased the level of UVB-induced apoptosis by activating p53-dependent pathway. However, overexpression of PTEN increased cell survival by suppressing p-H2A in response to DNA damage and apoptosis. When using Pifithrin-α (one of p53 inhibitors), the level of p53-dependent apoptosis was significantly lower than untreated, which suggested that p53 was possibly involved in PTEN-dependent apoptosis. Thus, it elucidated the molecular mechanisms of UVB-induced damage in RPE cells and may offer an alternative therapeutic target in dry AMD. Jia He, Chongde Long, Zixin Huang, Xin Zhou, Xielan Kuang, Lanying Liu, Huijun Liu, Yan Tang, Yuting Fan, Jie Ning, Xinqi Ma, Qingjiong Zhang, and Huangxuan Shen Copyright © 2017 Jia He et al. All rights reserved. Proteomic Analysis of Liver Proteins in a Rat Model of Chronic Restraint Stress-Induced Depression Wed, 15 Feb 2017 00:00:00 +0000 Depression is a global mental disorder disease and greatly threatened human health and stress is considered to be one of the important factors that lead to depression. In this study, we used newly developed iTRAQ labeling and high performance liquid chromatography (HPLC) and mass spectrum united analysis technology obtained the 2176 accurate proteins. Successively, we used the GO analysis and IPA software to analyze the 98 differentially expressed proteins of liver in depression rats due to chronic restraint stress, showing a map of proteomics analysis of liver proteins from the aspects of related functions, disease and function analysis, canonical pathway analysis, and associated network. This study provide important information for comprehensively understanding the mechanisms of dysfunction or injury in the liver in depression. Cong Li, Zhengguang Guo, Ronghua Zhao, Wei Sun, and Ming Xie Copyright © 2017 Cong Li et al. All rights reserved. Identification of Three Novel Splicing Variants and Expression Analysis of Chicken GPR1 Gene Sun, 22 Jan 2017 00:00:00 +0000 GPR1 is a G protein-coupled receptor that plays critical roles in eukaryotic cells: typically, response to glucose stimulation, lipid accumulation, and transmitting nutrition signals to cAMP pathway. However, the alternative splicing of the GPR1 gene and its expression pattern in chicken tissues and ovarian follicles were unknown. In our current study, we used RACE-PCR to identify three GPR1 variants, including the full-length variant (GPR1-va1) and two alternatively spliced variants (GPR1-va2, GPR1-vb). Quantitative real-time PCR examined the expression pattern of GPR1 mRNA in chicken tissues and ovarian follicles. The result reveals that the coding sequence of the three variants cDNA is 1053, 1053, and 627 bp in length, encoding 350, 350, and 208 amino acids, respectively. The three variants of GPR1 show similar tissue distributions; GPR1 expression was abundant in the abdominal fat, lung, and heart. With the follicular development, the expression of GPR1 gene gradually increased, and GPR1-va1 and GPR1-va2 spliced variants expression in F2 were significantly higher than in F5, F4, and prehierarchical follicles (). Taken together, we found three novel variants of GPR1, and the results of GPR1 expression profiling in adipose tissues and ovarian follicles suggest that GPR1 may play a significant role in the lipid accumulation and progression of follicular development. Xueyou Zhang, Qihai Xiao, Kai Tian, Yan Wang, Xiaoling Zhao, Huadong Yin, Diyan Li, and Qing Zhu Copyright © 2017 Xueyou Zhang et al. All rights reserved. Effect of R119G Mutation on Human P5CR1 Dynamic Property and Enzymatic Activity Wed, 18 Jan 2017 07:46:18 +0000 Pyrroline-5-carboxylate reductase (P5CR1) is a universal housekeeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)+. The enzymatic cycle between P5C and proline is important for function in amino acid metabolism, apoptosis, and intracellular redox potential balance in mitochondria. Autosomal recessive cutis laxa (ARCL) results from a mutation in P5CR1 encoded by PYCR1. Specifically, the R119G mutation is reported to be linked to ARCL although it has not yet been characterized. We synthesized R119G P5CR1 and compared it to WT P5CR1. Foldx prediction of WT and R119G mutant P5CR1 protein stability suggests that the R119G mutation could significantly reduce protein stability. We also performed enzymatic activity assays to determine how the mutation impacts P5CR1 enzymatic function. The results of these experiments show that mutagenesis of R119 to G decreases P5CR1 catalytic efficiency for 3,4-dehydro-L-proline relative to WT. Mutagenesis and kinetic studies reveal that the activity of the mutant decreases as temperature increases from 5°C to 37°C, with almost no activity at 37°C, indicating that this mutation impairs P5CR1 function in vivo. Conversely, WT P5CR1 retains its activity after incubation at 37°C and has essentially no remaining activity at 75°C. Taken together, our experimental results indicate the R119G mutation could be an involving pathomechanism for ARCL. Linhua Li, Yujia Ye, Peng Sang, Yirui Yin, Wei Hu, Jing Wang, Chao Zhang, Deyun Li, Wen Wan, Rui Li, Longjun Li, Linling Ma, Yuehui Xie, and Zhaohui Meng Copyright © 2017 Linhua Li et al. All rights reserved. Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions Tue, 03 Jan 2017 00:00:00 +0000 Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000 and 160,000, and vesicle-depleted plasma supernatant of healthy donors and non-small cell lung cancer (NSCLC) patients. It was determined that 16,000 blood plasma vesicles were enriched with cell-free mitochondria and with a set of mitochondrial RNAs. The variable RNA set of blood plasma 160,000 pellets reflected the prominent contribution of U1, U5, and U6 small nuclear RNAs’ fragments and at the same time was characterized by a remarkable depletion of small nucleolar RNAs. Besides microRNAs, the variety of fragments of mRNAs and snoRNAs dominated in the set of circulating RNAs differentially expressed in blood fractions of NSCLC patients. Taken together, our data emphasize that not only extracellular microRNAs but also circulating fragments of messenger and small nuclear/nucleolar RNAs represent prominent classes of circulating regulatory ncRNAs as well as promising circulating biomarkers for the development of disease diagnostic approaches. Anna V. Savelyeva, Elena V. Kuligina, Dmitry N. Bariakin, Vadim V. Kozlov, Elena I. Ryabchikova, Vladimir A. Richter, and Dmitry V. Semenov Copyright © 2017 Anna V. Savelyeva et al. All rights reserved. Molecular Phylogenetics 2016 Thu, 29 Dec 2016 09:33:10 +0000 Vassily Lyubetsky, William H. Piel, and Peter F. Stadler Copyright © 2016 Vassily Lyubetsky et al. All rights reserved. Genome-Wide Identification of Long Noncoding RNAs in Human Intervertebral Disc Degeneration by RNA Sequencing Tue, 20 Dec 2016 09:51:54 +0000 Long noncoding RNAs (lncRNAs) are emerging as crucial players in a myriad of biological processes. However, the precise mechanism and functions of most lncRNAs are poorly characterized. In this study, we presented genome-wide identification of lncRNAs in the patients with intervertebral disc degeneration (IDD) and spinal cord injury (control) using RNA sequencing (RNA-seq). A total of 124.6 million raw reads were yielded using Hiseq 2500 platform and approximately 88% clean reads could be aligned to human reference genome in both IDD and control groups. RNA-seq profiling indicated that 1,854 lncRNAs were differentially expressed (log2 fold change ≥ 1 or , ), in which 1,530 could potentially target 6,386 genes via cis-regulatory effects. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for these target genes suggested that lncRNAs were involved in diverse pathways, such as lysosome, focal adhesion, and MAPK signaling. In addition, a competing endogenous RNA (ceRNA) network was constructed for analyzing the function of lncRNAs. Further, quantitative real time PCR (qRT-PCR) was used to confirm the differentially expressed lncRNAs and ceRNA network. In conclusion, our results present the first global identification of lncRNAs in IDD and may provide candidate diagnostic biomarkers for IDD treatment. Bo Zhao, Minjuan Lu, Dong Wang, Haopeng Li, and Xijing He Copyright © 2016 Bo Zhao et al. All rights reserved. Burkholderia contaminans Biofilm Regulating Operon and Its Distribution in Bacterial Genomes Wed, 14 Dec 2016 16:57:11 +0000 Biofilm formation by Burkholderia spp. is a principal cause of lung chronic infections in cystic fibrosis patients. A “lacking biofilm production” (LBP) strain B. contaminans GIMC4587:Bct370-19 has been obtained by insertion modification of clinical strain with plasposon mutagenesis. It has an interrupted transcriptional response regulator (RR) gene. The focus of our investigation was a two-component signal transduction system determination, including this RR. B. contaminans clinical and LBP strains were analyzed by whole genome sequencing and bioinformatics resources. A four-component operon (BiofilmReg) has a key role in biofilm formation. The relative location (i.e., by being separated by another gene) of RR and histidine kinase genes is unique in BiofilmReg. Orthologs were found in other members of the Burkholderiales order. Phylogenetic analysis of strains containing BiofilmReg operons demonstrated evidence for earlier inheritance of a three-component operon. During further evolution one lineage acquired a fourth gene, whereas others lost the third component of the operon. Mutations in sensor domains have created biodiversity which is advantageous for adaptation to various ecological niches. Different species Burkholderia and Achromobacter strains all demonstrated similar BiofilmReg operon structure. Therefore, there may be an opportunity to develop a common drug which is effective for treating all these causative agents. Olga L. Voronina, Marina S. Kunda, Natalia N. Ryzhova, Ekaterina I. Aksenova, Andrey N. Semenov, Yulia M. Romanova, and Alexandr L. Gintsburg Copyright © 2016 Olga L. Voronina et al. All rights reserved. A Novel In Vitro System for Comparative Analyses of Bone Cells and Bacteria under Electrical Stimulation Sun, 04 Dec 2016 09:52:03 +0000 Electrical stimulation is a promising approach to enhance bone regeneration while having potential to inhibit bacterial growth. To investigate effects of alternating electric field stimulation on both human osteoblasts and bacteria, a novel in vitro system was designed. Electric field distribution was simulated numerically and proved by experimental validation. Cells were stimulated on Ti6Al4V electrodes and in short distance to electrodes. Bacterial growth was enumerated in supernatant and on the electrode surface and biofilm formation was quantified. Electrical stimulation modulated gene expression of osteoblastic differentiation markers in a voltage-dependent manner, resulting in significantly enhanced osteocalcin mRNA synthesis rate on electrodes after stimulation with 1.4. While collagen type I synthesis increased when stimulated with 0.2, it decreased after stimulation with 1.4. Only slight and infrequent influence on bacterial growth was observed following stimulations with 0.2 and 1.4 after 48 and 72 h, respectively. In summary this novel test system is applicable for extended in vitro studies concerning definition of appropriate stimulation parameters for bone cell growth and differentiation, bacterial growth suppression, and investigation of general effects of electrical stimulation. Thomas Josef Dauben, Josefin Ziebart, Thomas Bender, Sarah Zaatreh, Bernd Kreikemeyer, and Rainer Bader Copyright © 2016 Thomas Josef Dauben et al. All rights reserved. Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis Tue, 22 Nov 2016 13:29:57 +0000 The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40–45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment. Hasnain Hussain and Nikson Fatt-Ming Chong Copyright © 2016 Hasnain Hussain and Nikson Fatt-Ming Chong. All rights reserved. TRIM25 Identification in the Chinese Goose: Gene Structure, Tissue Expression Profiles, and Antiviral Immune Responses In Vivo and In Vitro Tue, 22 Nov 2016 07:02:13 +0000 The retinoic acid-inducible gene I (RIG-I) and the RIG-I-like receptor (RLR) protein play a critical role in the interferon (IFN) response during RNA virus infection. The tripartite motif containing 25 proteins (TRIM25) was reported to modify caspase activation and RIG-I recruitment domains (CARDs) via ubiquitin. These modifications allow TRIM25 to interact with mitochondrial antiviral signaling molecules (MAVs) and form CARD-CARD tetramers. Goose TRIM25 was cloned from gosling lungs, which possess a 1662 bp open reading flame (ORF). This ORF encodes a predicted 554 amino acid protein consisting of a B-box domain, a coiled-coil domain, and a PRY/SPRY domain. The protein sequence has 89.25% sequence identity with Anas platyrhynchos TRIM25, 78.57% with Gallus gallus TRIM25, and 46.92% with Homo sapiens TRIM25. TRIM25 is expressed in all gosling and adult goose tissues examined. QRT-PCR revealed that goose TRIM25 transcription could be induced by goose IFN-α, goose IFN-γ, and goose IFN-λ, as well as a35 s polyinosinic-polycytidylic acid (poly(I:C)), oligodeoxynucleotides 2006 (ODN 2006), and resiquimod (R848) in vitro; however, it is inhibited in H9N2 infected goslings for unknown reasons. These data suggest that goose TRIM25 might play a positive role in the regulation of the antiviral immune response. Yunan Wei, Hao Zhou, Anqi Wang, Lipei Sun, Mingshu Wang, Renyong Jia, Dekang Zhu, Mafeng Liu, Qiao Yang, Ying Wu, Kunfeng Sun, Xiaoyue Chen, Anchun Cheng, and Shun Chen Copyright © 2016 Yunan Wei et al. All rights reserved. Polymorphism of 41 kD Flagellin Gene and Its Human B-Cell Epitope in Borrelia burgdorferi Strains of China Tue, 15 Nov 2016 07:56:38 +0000 The 41 kD flagellin of Borrelia burgdorferi (B. burgdorferi) is a major component of periplasmic flagellar filament core and a good candidate for serodiagnosis in early stage of Lyme disease. Here, we chose 89 B. burgdorferi strains in China, amplified the gene encoding the 41 kD flagellin, and compared the sequences. The results showed that genetic diversity presented in the 41 kD flagellin genes of all 89 strains among the four genotypes of B. burgdorferi, especially in the genotype of B. garinii. Some specific mutation sites for each genotype of the 41 kD flagellin genes were found, which could be used for genotyping B. burgdorferi strains in China. Human B-cell epitope analysis showed that thirteen of 15 nonsynonymous mutations occurred in the epitope region of 41 kD flagellin and thirty of 42 B-cell epitopes were altered due to all 13 nonsynonymous mutations in the epitope region, which may affect the function of the antigen. Nonsynonymous mutations and changed human B-cell epitopes exist in 41 kD flagellin of B. burgdorferi sensu lato strains; these changes should be considered in serodiagnosis of Lyme disease. Huixin Liu, Wei Liu, Xuexia Hou, Lin Zhang, Qin Hao, and Kanglin Wan Copyright © 2016 Huixin Liu et al. All rights reserved. The Relationship between MC1R Mutation and Plumage Color Variation in Pigeons Sun, 13 Nov 2016 14:26:59 +0000 The polymorphisms of MC1R gene play a crucial role in coat color variation in mammals; however, the relationship is still unclear in pigeons. In this study, we sequenced 741 bp fragment of the MC1R for 39 individuals with five plumage color patterns (gray plumage, ; black plumage, ; white plumage, ; spotted plumage, ; red plumage, ). A total of three single nucleotide polymorphisms (SNPs) were detected, including G199A, G225A, and A466G, which subsequently determined four haplotypes (H1–H4). Among them, H1 is the predominant haplotype. Association analysis revealed that H1 and H3 were significantly associated with the black plumage trait (), while the H4 was significantly associated with gray plumage trait (). Furthermore, only diplotype H1H1 was significantly associated with black and gray traits of pigeons. Collectively, our study suggested an association between genetic variation of MC1R and plumage color in pigeon. Jin-Shan Ran, Xiao-Yan You, Jie Jin, Yu-Guang Zhou, Ye Wang, Dan Lan, Peng Ren, and Yi-Ping Liu Copyright © 2016 Jin-Shan Ran et al. All rights reserved. Resistance of Permafrost and Modern Acinetobacter lwoffii Strains to Heavy Metals and Arsenic Revealed by Genome Analysis Tue, 04 Oct 2016 06:52:22 +0000 We performed whole-genome sequencing of five permafrost strains of Acinetobacter lwoffii (frozen for 15–3000 thousand years) and analyzed their resistance genes found in plasmids and chromosomes. Four strains contained multiple plasmids (8–12), which varied significantly in size (from 4,135 to 287,630 bp) and genetic structure; the fifth strain contained only two plasmids. All large plasmids and some medium-size and small plasmids contained genes encoding resistance to various heavy metals, including mercury, cobalt, zinc, cadmium, copper, chromium, and arsenic compounds. Most resistance genes found in the ancient strains of A. lwoffii had their closely related counterparts in modern clinical A. lwoffii strains that were also located on plasmids. The vast majority of the chromosomal resistance determinants did not possess complete sets of the resistance genes or contained truncated genes. Comparative analysis of various A. lwoffii and of A. baumannii strains discovered a number of differences between them: (i) chromosome sizes in A. baumannii exceeded those in A. lwoffii by about 20%; (ii) on the contrary, the number of plasmids in A. lwoffii and their total size were much higher than those in A. baumannii; (iii) heavy metal resistance genes in the environmental A. lwoffii strains surpassed those in A. baumannii strains in the number and diversity and were predominantly located on plasmids. Possible reasons for these differences are discussed. Sofia Mindlin, Anatolii Petrenko, Anton Kurakov, Alexey Beletsky, Andrey Mardanov, and Mayya Petrova Copyright © 2016 Sofia Mindlin et al. All rights reserved. Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas Thu, 15 Sep 2016 08:48:43 +0000 The shell of the pearl oyster (Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization. Xing-Xia Li, Wen-Chao Yu, Zhong-Qiang Cai, Cheng He, Na Wei, Xiao-Tong Wang, and Xi-Qing Yue Copyright © 2016 Xing-Xia Li et al. All rights reserved. RNA-Seq Analysis Reveals Candidate Targets for Curcumin against Tetranychus cinnabarinus Thu, 08 Sep 2016 13:42:18 +0000 Tetranychus cinnabarinus is an important agricultural pest with a broad host range. We previously identified curcumin as a promising acaricidal compound against T. cinnabarinus. However, the acaricidal mechanism of curcumin remains unknown. In this study, RNA-seq was employed to analyze the transcriptome changes in T. cinnabarinus treated with curcumin or the solvent. A total of 105,706,297 clean sequence reads were generated by sequencing, with more than 90% of the reads successfully mapped to the reference sequence. The RNA-seq identified 111 and 96 differentially expressed genes between curcumin- and solvent-treated mites at 24 and 48 h after treatment, respectively. GO enrichment analysis of differentially expressed genes showed that the cellular process was the dominant group at both time points. Finally, we screened 23 differentially expressed genes that were functionally identical or similar to the targets of common insecticide/acaricides or genes that were associated with mite detoxification and metabolism. Calmodulin, phospholipase A2, and phospholipase C were activated upon curcumin treatment suggesting that the calcium channel related genes might play important roles in mite’s response to curcumin. Overall our results revealed the global transcriptional changes in T. cinnabarinus after curcumin treatment to enable further identification of the targets of curcumin in mites. Xuejiao Liu, Dousheng Wu, Yongqiang Zhang, Hong Zhou, Ting Lai, and Wei Ding Copyright © 2016 Xuejiao Liu et al. All rights reserved. 18S Ribosomal RNA Evaluation as Preanalytical Quality Control for Animal DNA Thu, 08 Sep 2016 11:08:15 +0000 The 18S ribosomal RNA (rRNA) gene is present in all eukaryotic cells. In this study, we evaluated the use of this gene to verify the presence of PCR-amplifiable host (animal) DNA as an indicator of sufficient sample quality for quantitative real-time PCR (qPCR) analysis. We compared (i) samples from various animal species, tissues, and sample types, including swabs; (ii) multiple DNA extraction methods; and (iii) both fresh and formalin-fixed paraffin-embedded (FFPE) samples. Results showed that 18S ribosomal RNA gene amplification was possible from all tissue samples evaluated, including avian, reptile, and FFPE samples and most swab samples. A single swine rectal swab, which showed sufficient DNA quantity and the demonstrated lack of PCR inhibitors, nonetheless was negative by 18S qPCR. Such a sample specifically illustrates the improvement of determination of sample integrity afforded by inclusion of 18S rRNA gene qPCR analysis in addition to spectrophotometric analysis and the use of internal controls for PCR inhibition. Other possible applications for the described 18S rRNA qPCR are preselection of optimal tissue specimens for studies or preliminary screening of archived samples prior to acceptance for biobanking projects. Cory Ann Leonard, Marina L. Meli, Marilisa Novacco, and Nicole Borel Copyright © 2016 Cory Ann Leonard et al. All rights reserved. Phenolic Compounds and In Vitro Antibacterial and Antioxidant Activities of Three Tropic Fruits: Persimmon, Guava, and Sweetsop Thu, 25 Aug 2016 16:15:38 +0000 In our previous study, we have found that persimmon, guava, and sweetsop owned considerably high antioxidant activity and contained high total phenolic contents as well. In order to further supply information on the antibacterial and antioxidant activity of these three tropic fruits, they were extracted by 80% methanol. We then examined the extractions about their phenolic compounds and also studied the extractions and phenolic contents about their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against twelve targeted pathogens including 8 standard strains (Staphylococcus aureus, Bacillus cereus, Staphylococcus epidermidis, Monilia albican, Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Pseudomonas aeruginosa) and 4 multidrug-resistant strains (methicillin-resistant Staphylococcus aureus, ESBLs-producing Escherichia coli, carbapenems-resistant Pseudomonas aeruginosa, and multidrug-resistant Acinetobacter baumannii), which are common and comprehensive in clinic. We also employed two ways, that is, FRAP and TEAC, to evaluate their antioxidant activities, using ultraviolet and visible spectrophotometer. Our study indicated that the three tropical fruits possessed obvious antioxidant and antibacterial activity, which supported the possibility of developing the fruits into new natural resource food and functional food as well as new natural antimicrobial agent and food preservatives. Moreover, phenolic compounds detected in the fruits could be used as a potential natural antibacterial agent and antioxidant. Li Fu, WenQing Lu, and XiaoMin Zhou Copyright © 2016 Li Fu et al. All rights reserved. Altered Clock and Lipid Metabolism-Related Genes in Atherosclerotic Mice Kept with Abnormal Lighting Condition Thu, 18 Aug 2016 13:54:40 +0000 Background. The risk of atherosclerosis is elevated in abnormal lipid metabolism and circadian rhythm disorder. We investigated whether abnormal lighting condition would have influenced the circadian expression of clock genes and clock-controlled lipid metabolism-related genes in ApoE-KO mice. Methods. A mouse model of atherosclerosis with circadian clock genes expression disorder was established using ApoE-KO mice (ApoE-KO LD/DL mice) by altering exposure to light. C57 BL/6J mice (C57 mice) and ApoE-KO mice (ApoE-KO mice) exposed to normal day and night and normal diet served as control mice. According to zeitgeber time samples were acquired, to test atheromatous plaque formation, serum lipids levels and rhythmicity, clock genes, and lipid metabolism-related genes along with Sirtuin 1 (Sirt1) levels and rhythmicity. Results. Atherosclerosis plaques were formed in the aortic arch of ApoE-KO LD/DL mice. The serum lipids levels and oscillations in ApoE-KO LD/DL mice were altered, along with the levels and diurnal oscillations of circadian genes, lipid metabolism-associated genes, and Sirt1 compared with the control mice. Conclusions. Abnormal exposure to light aggravated plaque formation and exacerbated disorders of serum lipids and clock genes, lipid metabolism genes and Sirt1 levels, and circadian oscillation. Zhu Zhu, Bingxuan Hua, Zhanxian Shang, Gongsheng Yuan, Lirong Xu, Ermin Li, Xiaobo Li, Ning Sun, Zuoqin Yan, Ruizhe Qian, and Chao Lu Copyright © 2016 Zhu Zhu et al. All rights reserved. Is Hydrogen Peroxide a Suitable Apoptosis Inducer for All Cell Types? Tue, 09 Aug 2016 13:00:21 +0000 Hydrogen peroxide is currently the most widely used apoptosis inducer due to its broad cytotoxic efficacy against nearly all cell types. However, equivalent cytotoxicity is achieved over a wide range of doses, although the reasons for this differential sensitivity are not always clear. In this study, three kinds of cells, the 293T cell line, primary fibroblasts, and terminally differentiated myocardial cells, were treated with a wide range of H2O2 doses. Times to apoptosis initiation and end were measured cytochemically and the changes in expression of caspase-9, P53, NF-B, and RIP were determined by RT-PCR. The 293T cell line was the most sensitive to H2O2, undergoing necroptosis and/or apoptosis at all concentrations from 0.1 to 1.6 mM. At > 0.4 mM, H2O2 also caused necroptosis in primary cells. At < 0.4 mM, however, primary cells exhibited classic signs of apoptosis, although they tended to survive for 36 hours in < 0.2 mM H2O2. Thus, H2O2 is a broadly effective apoptosis inducer, but the dose range differs by cell type. For cell lines, a low dose is required and the exposure time must be reduced compared to primary cells to avoid cell death primarily by necroptosis or necrosis. Jinmei Xiang, Chunyun Wan, Rui Guo, and Dingzong Guo Copyright © 2016 Jinmei Xiang et al. All rights reserved. Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System Thu, 04 Aug 2016 09:07:37 +0000 Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system. Mohammed N. Baeshen, Thamer A. F. Bouback, Mubarak A. Alzubaidi, Roop S. Bora, Mohammed A. T. Alotaibi, Omar T. O. Alabbas, Sultan M. Alshahrani, Ahmed A. M. Aljohani, Rayan A. A. Munshi, Ahmed Al-Hejin, Mohamed M. M. Ahmed, Elrashdy M. Redwan, Hassan A. I. Ramadan, Kulvinder S. Saini, and Nabih A. Baeshen Copyright © 2016 Mohammed N. Baeshen et al. All rights reserved. Stress Signaling Responses in Plants Mon, 01 Aug 2016 07:41:50 +0000 Marta Wilton Pereira Leite de Vasconcelos, Paloma Koprovski Menguer, Yanbo Hu, Luis Fernando Revers, and Raul Antonio Sperotto Copyright © 2016 Marta Wilton Pereira Leite de Vasconcelos et al. All rights reserved. Cloning and Expression Analysis of One Gamma-Glutamylcysteine Synthetase Gene (Hbγ-ECS1) in Latex Production in Hevea brasiliensis Thu, 23 Jun 2016 10:41:09 +0000 Rubber tree is a major commercial source of natural rubber. Latex coagulation is delayed by thiols, which belong to the important type of antioxidants in laticifer submembrane, and is composed of glutathione (GSH), cysteine, and methionine. The rate-limiting enzyme, γ-ECS, plays an important role in regulating the biosynthesis of glutathione under any environment conditions. To understand the relation between γ-ECS and thiols and to correlate latex flow with one-time tapping and continuous tapping, we cloned and derived the full length of one γ-ECS from rubber tree latex (Hbγ-ECS1). According to qPCR analysis, the expression levels of Hbγ-ECS1 were induced by tapping and Ethrel stimulation, and the expression was related to thiols content in the latex. Continuous tapping induced injury, and the expression of HbγECS1 increased with routine tapping and Ethrel-stimulation tapping (more intensive tapping). According to expression in long-term flowing latex, the gene was related to the duration of latex flow. HbγECS1 was expressed in E. coli Rosetta using pET-sumo as an expression vector and the recombinant enzyme was purified; then we achieved 0.827 U/mg specific activity and about 66 kDa molecular weight. The present study can help us understand the complex role of Hbγ-ECS in thiols biosynthesis, which is influenced by tapping. Wei Fang, Luo Shi Qiao, Wu Ming, Qiu Jian, Yang Wen Feng, Gao Hong Hua, and Xiao Xian Zhou Copyright © 2016 Wei Fang et al. All rights reserved. High Glucose-Induced PC12 Cell Death by Increasing Glutamate Production and Decreasing Methyl Group Metabolism Sun, 19 Jun 2016 08:59:02 +0000 Objective. High glucose- (HG-) induced neuronal cell death is responsible for the development of diabetic neuropathy. However, the effect of HG on metabolism in neuronal cells is still unclear. Materials and Methods. The neural-crest derived PC12 cells were cultured for 72 h in the HG (75 mM) or control (25 mM) groups. We used NMR-based metabolomics to examine both intracellular and extracellular metabolic changes in HG-treated PC12 cells. Results. We found that the reduction in intracellular lactate may be due to excreting more lactate into the extracellular medium under HG condition. HG also induced the changes of other energy-related metabolites, such as an increased succinate and creatine phosphate. Our results also reveal that the synthesis of glutamate from the branched-chain amino acids (isoleucine and valine) may be enhanced under HG. Increased levels of intracellular alanine, phenylalanine, myoinositol, and choline were observed in HG-treated PC12 cells. In addition, HG-induced decreases in intracellular dimethylamine, dimethylglycine, and 3-methylhistidine may indicate a downregulation of methyl group metabolism. Conclusions. Our metabolomic results suggest that HG-induced neuronal cell death may be attributed to a series of metabolic changes, involving energy metabolism, amino acids metabolism, osmoregulation and membrane metabolism, and methyl group metabolism. Minjiang Chen, Hong Zheng, Tingting Wei, Dan Wang, Huanhuan Xia, Liangcai Zhao, Jiansong Ji, and Hongchang Gao Copyright © 2016 Minjiang Chen et al. All rights reserved. Involvement of Hormone- and ROS-Signaling Pathways in the Beneficial Action of Humic Substances on Plants Growing under Normal and Stressing Conditions Sun, 05 Jun 2016 06:43:12 +0000 The importance of soil humus in soil fertility has been well established many years ago. However, the knowledge about the whole mechanisms by which humic molecules in the rhizosphere improve plant growth remains partial and rather fragmentary. In this review we discuss the relationships between two main signaling pathway families that are affected by humic substances within the plant: one directly related to hormonal action and the other related to reactive oxygen species (ROS). In this sense, our aims are to try the integration of all these events in a more comprehensive model and underline some points in the model that remain unclear and deserve further research. Andrés Calderín García, Maite Olaetxea, Leandro Azevedo Santos, Verónica Mora, Roberto Baigorri, Marta Fuentes, Angel Maria Zamarreño, Ricardo Luis Louro Berbara, and José María Garcia-Mina Copyright © 2016 Andrés Calderín García et al. All rights reserved. Ethylene-Induced Vinblastine Accumulation Is Related to Activated Expression of Downstream TIA Pathway Genes in Catharanthus roseus Sun, 29 May 2016 07:47:46 +0000 We selected different concentrations of ethephon, to stress C. roseus. We used qRT-PCR and HPLC followed by PCA to obtain comprehensive profiling of the vinblastine biosynthesis in response to ethephon. Based on our findings, the results showed that the high concentration of ethephon had a positive effect at both transcriptional and metabolite level. Meanwhile, there was a remarkable decrease of hydrogen peroxide content and a promoted peroxidase activity in leaves. The loading plot combination with correlation analysis suggested that CrPrx1 could be regarded as a positive regulator and interacts with ethylene response factor (ERF) to play a key role in vinblastine content and peroxidase (POD) activity. This study provides the foundation for a better understanding of the regulation and accumulation of vinblastine in response to ethephon. Xi Wang, Ya-Jie Pan, Bo-Wen Chang, Yan-Bo Hu, Xiao-Rui Guo, and Zhong-Hua Tang Copyright © 2016 Xi Wang et al. All rights reserved. Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples Thu, 26 May 2016 13:56:39 +0000 Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings. Dilip Chandu, Sudakshina Paul, Mathew Parker, Yelena Dudin, Jennifer King-Sitzes, Tim Perez, Don W. Mittanck, Manali Shah, Kevin C. Glenn, and Olaf Piepenburg Copyright © 2016 Dilip Chandu et al. All rights reserved. Phylogeographic Structure of a Tethyan Relict Capparis spinosa (Capparaceae) Traces Pleistocene Geologic and Climatic Changes in the Western Himalayas, Tianshan Mountains, and Adjacent Desert Regions Tue, 24 May 2016 13:58:08 +0000 Complex geological movements more or less affected or changed floristic structures, while the alternation of glacials and interglacials is presumed to have further shaped the present discontinuous genetic pattern of temperate plants. Here we consider Capparis spinosa, a xeromorphic Tethyan relict, to discuss its divergence pattern and explore how it responded in a stepwise fashion to Pleistocene geologic and climatic changes. 267 individuals from 31 populations were sampled and 24 haplotypes were identified, based on three cpDNA fragments (trnL-trnF, rps12-rpl20, and ndhF). SAMOVA clustered the 31 populations into 5 major clades. AMOVA suggests that gene flow between them might be restricted by vicariance. Molecular clock dating indicates that intraspecific divergence began in early Pleistocene, consistent with a time of intense uplift of the Himalaya and Tianshan Mountains, and intensified in mid-Pleistocene. Species distribution modeling suggests range reduction in the high mountains during the Last Glacial Maximum (LGM) as a result of cold climates when glacier advanced, while gorges at midelevations in Tianshan appear to have served as refugia. Populations of low-altitude desert regions, on the other hand, probably experienced only marginal impacts from glaciation, according to the high levels of genetic diversity. Qian Wang, Ming-Li Zhang, and Lin-Ke Yin Copyright © 2016 Qian Wang et al. All rights reserved. Association between Polymorphisms in Interleukins 4 and 13 Genes and Chronic Periodontitis in a Han Chinese Population Mon, 18 Apr 2016 07:26:01 +0000 Chronic periodontitis (CP) is one of the most common chronic inflammatory diseases and cytokines play a pivotal role in the regulation of immune response. Interleukin-4 (IL-4) and interleukin-13 (IL-13) are anti-inflammatory cytokines and several polymorphisms of them have been proved involved in periodontal disease. This study aimed to evaluate whether three single nucleotide polymorphisms (SNPs), rs2070874 and rs2243248 from IL4 and rs1800925 from IL13, are associated with CP in a Han Chinese population consisting of 440 moderate or severe CP patients and 324 healthy controls. Genomic DNA extracted from buccal epithelial cells of the included participants were genotyped using a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry method. No significant association between rs2070874 or rs1800925 and CP was found, while the frequencies of rs2243248 and two haplotypes C-G-T and C-T-T showed significant differences between the two groups. The results suggest that the polymorphism rs2243248 and haplotypes C-G-T and C-T-T may be associated with CP susceptibility in the present Han Chinese population. Dong Chen, Tian-liang Zhang, and Xia Wang Copyright © 2016 Dong Chen et al. All rights reserved. Reconstructing the Phylogeny of Capsosiphon fulvescens (Ulotrichales, Chlorophyta) from Korea Based on rbcL and 18S rDNA Sequences Sun, 17 Apr 2016 13:48:29 +0000 Capsosiphon fulvescens is a filamentous green algae in the class Ulvophyceae. It has been consumed as food with unique flavor and soft texture to treat stomach disorders and hangovers, and its economic value justifies studying its nutritional and potential therapeutic effects. In contrast to these applications, only a few taxonomic studies have been conducted on C. fulvescens. In particular, classification and phylogenetic relationships of the C. fulvescens below the order level are controversial. To determine its phylogenetic position in the class, we used rbcL and 18S rDNA sequences as molecular markers to construct phylogenetic trees. The amplified rbcL and 18S rDNA sequences from 4 C. fulvescens isolates (Jindo, Jangheung, Wando, and Koheung, Korea) were used for phylogenetic analysis by employing three different phylogenetic methods: neighbor joining (NJ), maximum parsimony (MP), and maximum likelihood (ML). The rbcL phylogenetic tree showed that all taxa in the order Ulvales were clustered as a monophyletic group and resolved the phylogenetic position of C. fulvescens in the order Ulotrichales. The significance of our study is that the 18S rDNA phylogenetic tree shows the detailed taxonomic position of C. fulvescens. In our result, C. fulvescens is inferred as a member of Ulotrichaceae, along with Urospora and Acrosiphonia. Sang-Mi Sun, Seung Hwan Yang, Kirill S. Golokhvast, Bao Le, and Gyuhwa Chung Copyright © 2016 Sang-Mi Sun et al. All rights reserved. Decipher the Molecular Response of Plant Single Cell Types to Environmental Stresses Sun, 20 Mar 2016 12:36:55 +0000 The analysis of the molecular response of entire plants or organs to environmental stresses suffers from the cellular complexity of the samples used. Specifically, this cellular complexity masks cell-specific responses to environmental stresses and logically leads to the dilution of the molecular changes occurring in each cell type composing the tissue/organ/plant in response to the stress. Therefore, to generate a more accurate picture of these responses, scientists are focusing on plant single cell type approaches. Several cell types are now considered as models such as the pollen, the trichomes, the cotton fiber, various root cell types including the root hair cell, and the guard cell of stomata. Among them, several have been used to characterize plant response to abiotic and biotic stresses. In this review, we are describing the various -omic studies performed on these different plant single cell type models to better understand plant cell response to biotic and abiotic stresses. Mehrnoush Nourbakhsh-Rey and Marc Libault Copyright © 2016 Mehrnoush Nourbakhsh-Rey and Marc Libault. All rights reserved. Leaf Proteome Analysis Reveals Prospective Drought and Heat Stress Response Mechanisms in Soybean Sun, 13 Mar 2016 14:33:37 +0000 Drought and heat are among the major abiotic stresses that affect soybean crops worldwide. During the current investigation, the effect of drought, heat, and drought plus heat stresses was compared in the leaves of two soybean varieties, Surge and Davison, combining 2D-DIGE proteomic data with physiology and biochemical analyses. We demonstrated how 25 differentially expressed photosynthesis-related proteins affect RuBisCO regulation, electron transport, Calvin cycle, and carbon fixation during drought and heat stress. We also observed higher abundance of heat stress-induced EF-Tu protein in Surge. It is possible that EF-Tu might have activated heat tolerance mechanisms in the soybean. Higher level expressions of heat shock-related protein seem to be regulating the heat tolerance mechanisms. This study identifies the differential expression of various abiotic stress-responsive proteins that regulate various molecular processes and signaling cascades. One inevitable outcome from the biochemical and proteomics assays of this study is that increase of ROS levels during drought stress does not show significant changes at the phenotypic level in Davison and this seems to be due to a higher amount of carbonic anhydrase accumulation in the cell which aids the cell to become more resistant to cytotoxic concentrations of H2O2. Aayudh Das, Moustafa Eldakak, Bimal Paudel, Dea-Wook Kim, Homa Hemmati, Chhandak Basu, and Jai S. Rohila Copyright © 2016 Aayudh Das et al. All rights reserved. Drought Tolerance Is Correlated with the Activity of Antioxidant Enzymes in Cerasus humilis Seedlings Mon, 07 Mar 2016 07:47:23 +0000 Cerasus humilis, grown in the northern areas of China, may experience water deficit during their life cycle, which induces oxidative stress. Our present study was conducted to evaluate the role of oxidative stress management in the leaves of two C. humilis genotypes, HR (drought resistant) and ND4 (drought susceptible), when subjected to a long-term soil drought (WS). The HR plants maintained lower membrane injury due to low ROS and MDA accumulation compared to ND4 plants during a long-term WS. This is likely attributed to global increase in the activities of superoxide dismutase (SOD) isoenzymes and enzymes of the ascorbate-glutathione (AsA-GSH) cycle and maintenance of ascorbate (AsA) levels. Consistent closely with enzymes activities, the expression of cytosolic ascorbate peroxidase (cAPX) and dehydroascorbate reductase (DHAR) followed a significant upregulation, indicating that they were regulated at the transcriptional level for HR plants exposed to WS. In contrast, ND4 plants exhibited high ROS levels and poor antioxidant enzyme response, leading to enhanced membrane damage during WS conditions. The present study shows that genotypic differences in drought tolerance could be likely attributed to the ability of C. humilis plants to induce antioxidant defense under drought conditions. Jing Ren, Li Na Sun, Qiu Yan Zhang, and Xing Shun Song Copyright © 2016 Jing Ren et al. All rights reserved. Transcriptome Analysis of Ramie (Boehmeria nivea L. Gaud.) in Response to Ramie Moth (Cocytodes coerulea Guenée) Infestation Mon, 29 Feb 2016 13:32:40 +0000 The ramie moth Cocytodes coerulea Guenée (RM) is an economically important pest that seriously impairs the yield of ramie, an important natural fiber crop. The molecular mechanisms that underlie the ramie-pest interactions are unclear up to date. Therefore, a transcriptome profiling analysis would aid in understanding the ramie defense mechanisms against RM. In this study, we first constructed two cDNA libraries derived from RM-challenged (CH) and unchallenged (CK) ramie leaves. The subsequent sequencing of the CH and CK libraries yielded 40.2 and 62.8 million reads, respectively. Furthermore, de novo assembling of these reads generated 26,759 and 29,988 unigenes, respectively. An integrated assembly of data from these two libraries resulted in 46,533 unigenes, with an average length of 845 bp per unigene. Among these genes, 24,327 (52.28%) were functionally annotated by predicted protein function. A comparative analysis of the CK and CH transcriptome profiles revealed 1,980 differentially expressed genes (DEGs), of which 750 were upregulated and 1,230 were downregulated. A quantitative real-time PCR (qRT-PCR) analysis of 13 random selected genes confirmed the gene expression patterns that were determined by Illumina sequencing. Among the DEGs, the expression patterns of transcription factors, protease inhibitors, and antioxidant enzymes were studied. Overall, these results provide useful insights into the defense mechanism of ramie against RM. Liangbin Zeng, Airong Shen, Jia Chen, Zhun Yan, Touming Liu, Zhaodong Xue, and Yongting Yu Copyright © 2016 Liangbin Zeng et al. All rights reserved. Genome-Wide Transcriptome Analysis of Cadmium Stress in Rice Mon, 29 Feb 2016 12:02:33 +0000 Rice growth is severely affected by toxic concentrations of the nonessential heavy metal cadmium (Cd). To elucidate the molecular basis of the response to Cd stress, we performed mRNA sequencing of rice following our previous study on exposure to high concentrations of Cd (Oono et al., 2014). In this study, rice plants were hydroponically treated with low concentrations of Cd and approximately 211 million sequence reads were mapped onto the IRGSP-1.0 reference rice genome sequence. Many genes, including some identified under high Cd concentration exposure in our previous study, were found to be responsive to low Cd exposure, with an average of about 11,000 transcripts from each condition. However, genes expressed constitutively across the developmental course responded only slightly to low Cd concentrations, in contrast to their clear response to high Cd concentration, which causes fatal damage to rice seedlings according to phenotypic changes. The expression of metal ion transporter genes tended to correlate with Cd concentration, suggesting the potential of the RNA-Seq strategy to reveal novel Cd-responsive transporters by analyzing gene expression under different Cd concentrations. This study could help to develop novel strategies for improving tolerance to Cd exposure in rice and other cereal crops. Youko Oono, Takayuki Yazawa, Hiroyuki Kanamori, Harumi Sasaki, Satomi Mori, Hirokazu Handa, and Takashi Matsumoto Copyright © 2016 Youko Oono et al. All rights reserved. Plant Responses to High Frequency Electromagnetic Fields Sun, 14 Feb 2016 12:17:11 +0000 High frequency nonionizing electromagnetic fields (HF-EMF) that are increasingly present in the environment constitute a genuine environmental stimulus able to evoke specific responses in plants that share many similarities with those observed after a stressful treatment. Plants constitute an outstanding model to study such interactions since their architecture (high surface area to volume ratio) optimizes their interaction with the environment. In the present review, after identifying the main exposure devices (transverse and gigahertz electromagnetic cells, wave guide, and mode stirred reverberating chamber) and general physics laws that govern EMF interactions with plants, we illustrate some of the observed responses after exposure to HF-EMF at the cellular, molecular, and whole plant scale. Indeed, numerous metabolic activities (reactive oxygen species metabolism, α- and β-amylase, Krebs cycle, pentose phosphate pathway, chlorophyll content, terpene emission, etc.) are modified, gene expression altered (calmodulin, calcium-dependent protein kinase, and proteinase inhibitor), and growth reduced (stem elongation and dry weight) after low power (i.e., nonthermal) HF-EMF exposure. These changes occur not only in the tissues directly exposed but also systemically in distant tissues. While the long-term impact of these metabolic changes remains largely unknown, we propose to consider nonionizing HF-EMF radiation as a noninjurious, genuine environmental factor that readily evokes changes in plant metabolism. Alain Vian, Eric Davies, Michel Gendraud, and Pierre Bonnet Copyright © 2016 Alain Vian et al. All rights reserved. Molecular Typing Characteristic and Drug Susceptibility Analysis of Mycobacterium tuberculosis Isolates from Zigong, China Thu, 11 Feb 2016 12:27:42 +0000 China is one of the 22 countries with high TB burden worldwide, and Sichuan contained the second-largest number of TB cases among all of the Chinese provinces. But the characteristics of Mycobacterium tuberculosis circulated in Zigong, Sichuan, were still unknown. To investigate the character and drug resistance profile, 265 clinical isolates were cultured from tuberculosis patient’s sputum samples in the year of 2010, of which the genetic profile was determined by using Spoligotyping and MIRU-VNTR typing methods, and the drug sensibility testing to the four first-line and four second-line antituberculosis (anti-TB) drugs was performed by using proportion method on Lowenstein-Jensen (L-J) media. The major Spoligotype was Beijing family (143/265, 53.96%), followed by T (80/265, 30.19%) and H (9/265, 3.40%) genotypes; the total Hunter-Gaston discrimination index (HGDI) of the 24 loci MIRU-VNTR was 0.9995. About 27.17% (72/265) of the isolates were resistant to at least one of the eight tested anti-TB drugs, and for Beijing and non-Beijing family isolates the proportion of drug resistance was 28.47% (41/144) and 25.62% (31/121), respectively. That is, the most prevalent genotype here was Beijing family, and the 24 loci VNTR analysis could supply a high resolution for genotyping, and Beijing and non-Beijing isolates had no difference () for drug resistance. Hai-Can Liu, Jian-Ping Deng, Hai-Yan Dong, Ti-Quan Xiao, Xiu-Qin Zhao, Zheng-Dong Zhang, Yi Jiang, Zhi-Guang Liu, Qun Li, and Kang-Lin Wan Copyright © 2016 Hai-Can Liu et al. All rights reserved. MyD88 Polymorphisms and Association with Susceptibility to Salmonella Pullorum Thu, 31 Dec 2015 08:59:19 +0000 Myeloid differentiation primary response gene 88 (MYD88), a universal adapter protein, plays an important role in activating the nuclear factor-κB (NF-κB) and regulating the expression of proinflammatory genes like tumor necrosis factor (TNF) and interleukin-1 (IL-1), which were highly involved in Salmonella Pullorum infection. To detect the relationship between polymorphisms of the MyD88 gene and Salmonella Pullorum disease, we screened the coding region (CDS) of the MYD88 gene by DNA pool construction and sequencing based on case-control study. Eight single nucleotide polymorphisms (SNPs) in the sequenced fragment (5 exons), 7 known loci and one novel mutation named G4810372T (SNP8), were found in the fifth exon. In addition, we found 7 nonsynonymous substitutions. The allele frequency of only one SNP, g.4810191C > T (SNP1), was significantly different () between case and control groups. The genotype frequencies of SNP1 (g.4810191C > T) and SNP3 (g.4810257G > T) were of significant difference between the case and the control groups (). Collectively, SNPs of the MyD88 gene were significantly associated with susceptibility to Salmonella Pullorum infection, which can be used as a disease-resistant marker in chicken. These results provided a theoretical basis for future research on chicken breeding by marker-assisted selection. Xian-Qing Liu, Fei Wang, Jie Jin, Yu-Guang Zhou, Jin-Shan Ran, Ze-Qing Feng, Yan Wang, and Yi-Ping Liu Copyright © 2015 Xian-Qing Liu et al. All rights reserved. Role of TLR4-Mediated PI3K/AKT/GSK-3β Signaling Pathway in Apoptosis of Rat Hepatocytes Mon, 07 Dec 2015 10:59:42 +0000 We investigated the mechanism of the Toll-like receptor 4- (TLR4-) mediated PI3K/AKT/GSK-3β signaling pathway in rat hepatocytes apoptosis induced by LPS. The cultured rat hepatocytes were treated with LPS alone or first pretreated with TLR4 inhibitor, AKT inhibitor, and GSK-3β inhibitor, respectively, and then stimulated with the same dose of LPS. Cell viability, cell apoptotic rate, and apoptosis morphology were assessed; the level of P-, P-GSK-, and active Caspase-3 and the ratio of Bax/Bcl-2 were evaluated. The results indicated that cell viability decreased, while cell apoptotic rate increased with time after LPS stimulation. The expression of P- and P-GSK- in the LPS group decreased compared with the control, while the level of active Caspase-3 and the ratio of Bax/Bcl-2 were significantly increased. These effects were attenuated by pretreatment with CLI-095. In addition, the apoptotic ratio decreased after pretreatment with LiCl but increased following pretreatment with LY294002. The expression of P- further decreased following pretreatment with LY294002 and the expression of P-GSK- increased following pretreatment with LiCl. Moreover, pretreatment with CLI-095 weakened LPS-induced nuclear translocation of GSK-3β. Our findings suggest that the TLR4-mediated PI3K/AKT/GSK-3β signaling pathway is present in rat hepatocytes and participates in apoptosis of BRL-3A cells. Xian Zhang, Daorong Jiang, Wei Jiang, Min Zhao, and Jianhe Gan Copyright © 2015 Xian Zhang et al. All rights reserved. Antibody-Based Assays for Phenotyping of Extracellular Vesicles Thu, 03 Dec 2015 09:07:12 +0000 Extracellular vesicles (EVs) are a heterogeneous population of membrane-enclosed vesicles. EVs are recognized as important players in cell-to-cell communication and are described to be involved in numerous biological and pathological processes. The fact that EVs are involved in the development and progression of several diseases has formed the basis for the use of EV analysis in a clinical setting. As the interest in EVs has increased immensely, multiple techniques have been developed aiming at characterizing these vesicles. These techniques characterize different features of EVs, like the size distribution, enumeration, protein composition, and the intravesicular cargo (e.g., RNA). This review focuses on techniques that exploit the specificity and sensitivity associated with antibody-based assays to characterize the protein phenotype of EVs. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. Thus, protein profiling of EVs holds great diagnostic and prognostic potential. Lotte Hatting Pugholm, Anne Louise Schacht Revenfeld, Evo Kristina Lindersson Søndergaard, and Malene Møller Jørgensen Copyright © 2015 Lotte Hatting Pugholm et al. All rights reserved. Evaluation of MicroRNAs Regulating Anoikis Pathways and Its Therapeutic Potential Mon, 26 Oct 2015 06:19:02 +0000 Dysregulation of microRNAs (miRNAs) has been implicated in almost every known survival mechanisms utilized by cancer cells. One of such mechanisms, anoikis resistance, plays a pivotal role in enabling metastasis by allowing cancer cells to circumvent cell death induced by lack of attachment. Understanding how miRNAs regulate the various anoikis pathways has become the research question of increasing number of studies published in the past years. Through these studies, a growing list of miRNAs has been identified to be important players in promoting either anoikis or resistance to anoikis. In this review, we will be focusing on these miRNAs and how the findings from those studies can contribute to novel therapeutic strategies against cancer progression. We will be examining miRNAs that have been found to promote anoikis sensitivity in numerous cancer types followed by miRNAs that inhibit anoikis. In addition, we will also be taking a look at major signaling pathways involved in the action of the each of these miRNAs to gain a better understanding on how miRNAs regulate anoikis. Sharan Malagobadan and Noor Hasima Nagoor Copyright © 2015 Sharan Malagobadan and Noor Hasima Nagoor. All rights reserved. Reference Gene Selection for qPCR Normalization of Kosteletzkya virginica under Salt Stress Sun, 25 Oct 2015 11:28:07 +0000 Kosteletzkya virginica (L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene in K. virginica which showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA), β-actin (ACT), α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and 18SrRNA was assessed to be the most stable reference gene in this study. However, TUA was identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies in K. virginica. Xiaoli Tang, Hongyan Wang, Chuyang Shao, and Hongbo Shao Copyright © 2015 Xiaoli Tang et al. All rights reserved. Ameliorative Effect of Zinc Oxide Nanoparticles on Antioxidants and Sperm Characteristics in Streptozotocin-Induced Diabetic Rat Testes Sun, 25 Oct 2015 10:39:06 +0000 The present study investigated the impact of zinc oxide nanoparticles (ZnONPs) on the oxidative status and sperm characteristics in diabetic rat testicular tissue. Forty male albino rats were used in this study; 10 of them served as a control and 30 rats were injected with a single dose (100 mg/kg) of streptozotocin intraperitoneally. They were subdivided into diabetic, diabetic + ZnONPs (10 mg/kg B.W.), and diabetic and cotreated with ZnONPs + insulin groups. The sperm count and motility were assessed. The activity and mRNA expression of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GRD), and Glutathion-S-Transferase (GST) were determined in the testicular tissue. Malondialdehyde (MDA) and reduced glutathione (GSH) levels were estimated in the testicular tissue. Sperm count and motility increased in ZnONPs treated diabetic rats. A significant increase in the activity and mRNA expression of SOD, CAT, GPx, GRD, and GST was shown in ZnONPs treated diabetic rats. MDA significantly decreased, while GSH increased in testicular tissue of ZnONPs treated diabetic rats. It was concluded that ZnONPs either alone or in combination with insulin have the ability to increase the sperm count and motility and protect the testicular tissue against the oxidative stress induced by diabetes in rats. Mohamed Afifi, Omar A. Almaghrabi, and Naif Mohammed Kadasa Copyright © 2015 Mohamed Afifi et al. All rights reserved. Establishment of the MethyLight Assay for Assessing Aging, Cigarette Smoking, and Alcohol Consumption Thu, 22 Oct 2015 14:06:54 +0000 The environmental factors such as aging, smoking, and alcohol consumption have been reported to influence DNA methylation (DNAm). However, the versatility of DNAm measurement by DNAm array systems is low in clinical use. Thus, we developed the MethyLight assay as a simple method to measure DNAm. In the present study, we isolated peripheral blood DNA from 33 healthy volunteers and selected cg25809905, cg02228185, and cg17861230 as aging, cg23576855 as smoking, and cg02583484 as alcohol consumption biomarkers. The predicted age by methylation rates of cg25809905 and cg17861230 significantly correlated with chronological age. In immortalized B-cells, DNAm rates of two sites showed a younger status than the chronological age of donor. On the other hand, the predicted age of the patients with myocardial infarction (MI) was not accelerated. The methylation rate of cg23576855 was able to discriminate the groups based on the smoking status. The DNAm rate of cg02583484 was reduced in subjects with habitual alcohol consumption compared to that of subjects without habitual alcohol consumption. In conclusion, our MethyLight assay system reconfirms that aging, smoking, and alcohol consumption influenced DNAm in peripheral blood in the Japanese. This MethyLight system will facilitate DNAm measurement in epidemiological and clinical studies. Kosuke Endo, Jiawei Li, Michio Nakanishi, Takashi Asada, Masahiro Ikesue, Yoichi Goto, Yasue Fukushima, and Naoharu Iwai Copyright © 2015 Kosuke Endo et al. All rights reserved. Analysis of the Oxidative Stress Status in Nonspecific Vaginitis and Its Role in Vaginal Epithelial Cells Apoptosis Mon, 19 Oct 2015 13:14:40 +0000 Nonspecific vaginitis (NSV), also named bacterial vaginosis, is one of the most common genital system diseases in women during their reproductive years. The specific pathogenic mechanism of NSV is not clear yet. Upon the balance alteration, large amount of reactive oxidant species (ROS) is generated and accumulated in the genital tract, and thus resulting in oxidative stress, which has been reported to be an important trigger of mitochondrial pathway cell apoptosis. In this study, the antioxidant secretion level and antioxidant enzyme activity in the vaginal discharge were evaluated to analyze the oxidative status in the vaginal tract of NSV patients. The effect of oxidative stress on the vaginal mucosa epithelial cell apoptosis was then studied. The role of oxidative stress on NSV development was uncovered; thus open new direction for the prevention and treatment of NSV by providing antiradical agents was revealed. Zhaojie Chen, Zhen Zhang, Haiyan Zhang, and Beibei Xie Copyright © 2015 Zhaojie Chen et al. All rights reserved. Decreased Splenic T-Lymphocytes in Apolipoprotein M Gene Deficient Mice Mon, 12 Oct 2015 08:48:50 +0000 Spleen T-lymphocytes, especially CD4+ T-cells, have been demonstrated to be involved in broad immunomodulation and host-defense activity in vivo. Apolipoprotein M gene (apoM) may have an important role in the regulation of immunoprocess and inflammation, which could be hypothesized to the apoM containing sphingosine-1-phosphate (S1P). In the present study we demonstrate that the splenic CD4+ T-lymphocytes were obviously decreased in the apoM gene deficient (apoM−/−) mice compared to the wild type (apoM+/+). Moreover, these mice were treated with lipopolysaccharide (LPS) and it was found that even more pronounced decreasing CD4+ T-lymphocytes occurred in the spleen compared to the apoM+/+ mice. The similar phenomena were found in the ratio of CD4+/CD8+ T-lymphocytes. After administration of LPS, the hepatic mRNA levels of tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) were markedly increased; however, there were no statistical differences observed between apoM+/+ mice and apoM−/− mice. The present study demonstrated that apoM might facilitate the maintenance of CD4+ T-lymphocytes or could modify the T-lymphocytes subgroups in murine spleen, which may further explore the importance of apoM in the regulation of the host immunomodulation, although the detailed mechanism needs continuing investigation. Zhigang Wang, Guanghua Luo, Yuehua Feng, Lu Zheng, Hongyao Liu, Yun Liang, Zhonghua Liu, Peng Shao, Maria Berggren-Söderlund, Xiaoying Zhang, and Ning Xu Copyright © 2015 Zhigang Wang et al. All rights reserved. Testosterone Depletion Induces Demethylation of Murine Reelin Promoter CpG Dinucleotides: A Preliminary Study Wed, 07 Oct 2015 09:27:13 +0000 Schizophrenia (SZ) is a debilitating mental disorder characterized by psychotic events, abnormal social behavior, false beliefs, and auditory hallucinations. Hypermethylation of the promoter region of reelin (RELN), a gene involved in regulation of neuronal positioning during telencephalic development, is strongly associated with low protein expression in several cortical structures and promoter hypermethylation in brain from postmortem SZ subjects. Recent experimental data suggests that testosterone is able to promote RELN demethylation, although no direct evidence of hormonal influence on reelin promoter methylation was obtained. We investigated if reduced levels of plasma testosterone in adult male mice lead to Reln promoter demethylation. Animals were administered with flutamide, an antiandrogenic compound, and reelin promoter methylation was assessed using methylationspecific PCR using bisulfite DNA from cerebellum. We found that flutamide was able to significantly lower plasma testosterone when compared to control mice, and treatment did not influence animal survival and body weight. We also show that low plasma testosterone was associated with demethylation of a cytosine residue located at −860 in reelin promoter region. These preliminary data suggest that androgenic hormones can influence cerebral reelin demethylation. To our knowledge, this is the first experimental approach directly linking testosterone depletion and RELN promoter methylation. Victor Augusto Moraes da Silva, Marília de Souza Dantas, Leonardo Agostinho de Castro Silva, Juliana Garcia Carneiro, and Bruno Luiz Fonseca Schamber-Reis Copyright © 2015 Victor Augusto Moraes da Silva et al. All rights reserved. Genetic Diversity of Mycobacterium tuberculosis Complex Isolated from Tuberculosis Patients in Bahir Dar City and Its Surroundings, Northwest Ethiopia Mon, 28 Sep 2015 13:52:39 +0000 The knowledge of the diversity of strains of Mycobacterium tuberculosis complex (MTBC) species in a specific geographical region can contribute to the control of tuberculosis (TB). This study was conducted to identify the MTBC isolates to the species and spoligotype international type (SIT) level by spoligotyping. A total of 168 MTBC isolates were recovered from TB patients, spoligotyped, and their patterns were compared with those of the strains registered in the SITVIT2 database. Of 168 isolates spoligotyped, 89 patterns were identified. Ninety-eight isolates were clustered into 19 strain groups with clustering percentage of 58.3%. Forty-four strains matched the preexisting SITs in the SITVIT2 database. The dominant strains were SIT289, SIT134, and SIT3411, comprising 16.7% (28/168), 7.14% (12/168), and 4.76% (8/168) of the isolates, respectively. Euro-American (51.2%), East-African-Indian (34.5%), and M. africanum (9.52%) were the major lineages identified. Two strains of M. bovis were isolated from TB lymphadenitis cases. The high percentage of clustered strains of M. tuberculosis could suggest that a small number of lineages of M. tuberculosis are causing the disease in the area while isolation of M. bovis could suggest its zoonotic potential. Additionally, identification of M. africanum requires further confirmation by tools with a better discriminatory power. Anwar Nuru, Gezahegne Mamo, Adane Worku, Aschalew Admasu, Girmay Medhin, Rembert Pieper, and Gobena Ameni Copyright © 2015 Anwar Nuru et al. All rights reserved. Efficient Mitochondrial Genome Editing by CRISPR/Cas9 Thu, 10 Sep 2015 05:48:07 +0000 The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing. Areum Jo, Sangwoo Ham, Gum Hwa Lee, Yun-Il Lee, SangSeong Kim, Yun-Song Lee, Joo-Ho Shin, and Yunjong Lee Copyright © 2015 Areum Jo et al. All rights reserved. Identification of Type II Interferon Receptors in Geese: Gene Structure, Phylogenetic Analysis, and Expression Patterns Thu, 06 Aug 2015 13:23:52 +0000 Interferon γ receptor 1 (IFNGR1) and IFNGR2 are two cell membrane molecules belonging to class II cytokines, which play important roles in the IFN-mediated antiviral signaling pathway. Here, goose IFNGR1 and IFNGR2 were cloned and identified for the first time. Tissue distribution analysis revealed that relatively high levels of goose IFNγ mRNA transcripts were detected in immune tissues, including the harderian gland, cecal tonsil, cecum, and thymus. Relatively high expression levels of both IFNGR1 and IFNGR2 were detected in the cecal tonsil, which implicated an important role of IFNγ in the secondary immune system of geese. No specific correlation between IFNγ, IFNGR1, and IFNGR2 expression levels was observed in the same tissues of healthy geese. IFNγ and its cognate receptors showed different expression profiles, although they appeared to maintain a relatively balanced state. Furthermore, the agonist R848 led to the upregulation of goose IFNγ but did not affect the expression of goose IFNGR1 or IFNGR2. In summary, trends in expression of goose IFNγ and its cognate receptors showed tissue specificity, as well as an age-related dependency. These findings may help us to better understand the age-related susceptibility to pathogens in birds. Hao Zhou, Shun Chen, Yulin Qi, Qin Zhou, Mingshu Wang, Renyong Jia, Dekang Zhu, Mafeng Liu, Fei Liu, Xiaoyue Chen, and Anchun Cheng Copyright © 2015 Hao Zhou et al. All rights reserved. Mmu-miR-27a-5p-Dependent Upregulation of MCPIP1 Inhibits the Inflammatory Response in LPS-Induced RAW264.7 Macrophage Cells Thu, 30 Jul 2015 12:10:03 +0000 Lipopolysaccharide (LPS) stimulates macrophages to release proinflammatory cytokines. MicroRNAs (miRNAs) are short noncoding RNAs that are involved in inflammatory reaction. Our previously study identified the downregulated expression of mmu-miR-27a-5p in RAW267.4 cells treated with LPS. To dissect the mechanism that mmu-miR-27a-5p regulates target genes and affects proinflammatory cytokine secretion more clearly, based on previous bioinformatics prediction data, one of the potential target genes, MCPIP1 was observed to be upregulated with qRT-PCR and western blot. Luciferase reporter assays were performed to further confirm in silico prediction and determine that MCPIP1 is the target of mmu-miR-27-5p. The results suggested that mmu-miR-27a-5p directly targeted the 3′-UTR of MCPIP1 and the interaction between mmu-miR-27-5p and the 3′-UTR of MCPIP1 is sequence-specific. MCPIP1 overexpression decreased the secretion of IL-6, IL-1β, and IL-10 in macrophage cells stimulated with LPS. Our findings may provide the important information for the precise roles of mmu-miR-27a-5p in the macrophage inflammatory response to LPS stimulation in the future. Ying Cheng, Li Du, Hanwei Jiao, Huapei Zhu, Kailian Xu, Shiyu Guo, Qiaoyun Shi, Tianjing Zhao, Feng Pang, Xiaoxiao Jia, and Fengyang Wang Copyright © 2015 Ying Cheng et al. All rights reserved. Posttranscriptional Regulation and RNA Binding Proteins in Cancer Biology Mon, 27 Jul 2015 09:54:16 +0000 Claudia Ghigna, Luca Cartegni, Peter Jordan, and Maria Paola Paronetto Copyright © 2015 Claudia Ghigna et al. All rights reserved. An Association Rule Mining Approach to Discover lncRNAs Expression Patterns in Cancer Datasets Mon, 27 Jul 2015 07:26:45 +0000 In the past few years, the role of long noncoding RNAs (lncRNAs) in tumor development and progression has been disclosed although their mechanisms of action remain to be elucidated. An important contribution to the comprehension of lncRNAs biology in cancer could be obtained through the integrated analysis of multiple expression datasets. However, the growing availability of public datasets requires new data mining techniques to integrate and describe relationship among data. In this perspective, we explored the powerness of the Association Rule Mining (ARM) approach in gene expression data analysis. By the ARM method, we performed a meta-analysis of cancer-related microarray data which allowed us to identify and characterize a set of ten lncRNAs simultaneously altered in different brain tumor datasets. The expression profiles of the ten lncRNAs appeared to be sufficient to distinguish between cancer and normal tissues. A further characterization of this lncRNAs signature through a comodulation expression analysis suggested that biological processes specific of the nervous system could be compromised. Paolo Cremaschi, Roberta Carriero, Stefania Astrologo, Caterina Colì, Antonella Lisa, Silvia Parolo, and Silvia Bione Copyright © 2015 Paolo Cremaschi et al. All rights reserved. Posttranscriptional Regulation of Splicing Factor SRSF1 and Its Role in Cancer Cell Biology Sun, 26 Jul 2015 10:00:54 +0000 Over the past decade, alternative splicing has been progressively recognized as a major mechanism regulating gene expression patterns in different tissues and disease states through the generation of multiple mRNAs from the same gene transcript. This process requires the joining of selected exons or usage of different pairs of splice sites and is regulated by gene-specific combinations of RNA-binding proteins. One archetypical splicing regulator is SRSF1, for which we review the molecular mechanisms and posttranscriptional modifications involved in its life cycle. These include alternative splicing of SRSF1 itself, regulatory protein phosphorylation events, and the role of nuclear versus cytoplasmic SRSF1 localization. In addition, we resume current knowledge on deregulated SRSF1 expression in tumors and describe SRSF1-regulated alternative transcripts with functional consequences for cancer cell biology at different stages of tumor development. Vânia Gonçalves and Peter Jordan Copyright © 2015 Vânia Gonçalves and Peter Jordan. All rights reserved. Splicing Regulators and Their Roles in Cancer Biology and Therapy Sun, 26 Jul 2015 08:18:18 +0000 Alternative splicing allows cells to expand the encoding potential of their genomes. In this elegant mechanism, a single gene can yield protein isoforms with even antagonistic functions depending on the cellular physiological context. Alterations in splicing regulatory factors activity in cancer cells, however, can generate an abnormal protein expression pattern that promotes growth, survival, and other processes, which are relevant to tumor biology. In this review, we discuss dysregulated alternative splicing events and regulatory factors that impact pathways related to cancer. The SR proteins and their regulatory kinases SRPKs and CLKs have been frequently found altered in tumors and are examined in more detail. Finally, perspectives that support splicing machinery as target for the development of novel anticancer therapies are discussed. Maria Roméria da Silva, Gabriela Alves Moreira, Ronni Anderson Gonçalves da Silva, Éverton de Almeida Alves Barbosa, Raoni Pais Siqueira, Róbson Ricardo Teixera, Márcia Rogéria Almeida, Abelardo Silva Júnior, Juliana Lopes Rangel Fietto, and Gustavo Costa Bressan Copyright © 2015 Maria Roméria da Silva et al. All rights reserved. Splicing Regulation: A Molecular Device to Enhance Cancer Cell Adaptation Sun, 26 Jul 2015 08:07:28 +0000 Alternative splicing (AS) represents a major resource for eukaryotic cells to expand the coding potential of their genomes and to finely regulate gene expression in response to both intra- and extracellular cues. Cancer cells exploit the flexible nature of the mechanisms controlling AS in order to increase the functional diversity of their proteome. By altering the balance of splice isoforms encoded by human genes or by promoting the expression of aberrant oncogenic splice variants, cancer cells enhance their ability to adapt to the adverse growth conditions of the tumoral microenvironment. Herein, we will review the most relevant cancer-related splicing events and the underlying regulatory mechanisms allowing tumour cells to rapidly adapt to the harsh conditions they may face during the occurrence and development of cancer. Vittoria Pagliarini, Chiara Naro, and Claudio Sette Copyright © 2015 Vittoria Pagliarini et al. All rights reserved. Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage Sun, 26 Jul 2015 08:04:58 +0000 Synthesis of the p53 tumor suppressor increases following DNA damage. This increase and subsequent activation of p53 are essential for the protection of normal cells against tumorigenesis. We previously discovered an internal ribosome entry site (IRES) that is located at the 5′-untranslated region (UTR) of p53 mRNA and found that the IRES activity increases following DNA damage. However, the mechanism underlying IRES-mediated p53 translation in response to DNA damage is still poorly understood. In this study, we discovered that translational control protein 80 (TCP80) has increased binding to the p53 mRNA in vivo following DNA damage. Overexpression of TCP80 also leads to increased p53 IRES activity in response to DNA damage. TCP80 has increased association with RNA helicase A (RHA) following DNA damage and overexpression of TCP80, along with RHA, leads to enhanced expression of p53. Moreover, we found that MCF-7 breast cancer cells with decreased expression of TCP80 and RHA exhibit defective p53 induction following DNA damage and diminished expression of its downstream target PUMA, a proapoptotic protein. Taken together, our discovery of the function of TCP80 and RHA in regulating p53 IRES and p53 induction following DNA damage provides a better understanding of the mechanisms that regulate IRES-mediated p53 translation in response to genotoxic stress. Marie-Jo Halaby, Yan Li, Benjamin R. Harris, Shuxia Jiang, W. Keith Miskimins, Margot P. Cleary, and Da-Qing Yang Copyright © 2015 Marie-Jo Halaby et al. All rights reserved. Emerging Roles of MicroRNAs in EGFR-Targeted Therapies for Lung Cancer Sun, 26 Jul 2015 07:58:10 +0000 Lung cancer is a leading cause of cancer mortality worldwide. Several molecular pathways underlying mechanisms of this disease have been partly elucidated, among which the epidermal growth factor receptor (EGFR) pathway is one of the well-known signaling cascades that plays a critical role in tumorigenesis. Dysregulation of the EGFR signaling is frequently found in lung cancer. The strategies to effectively inhibit EGFR signaling pathway have been mounted for developing anticancer therapeutic agents. However, most anti-EGFR-targeted agents fail to repress cancer progression because of developing drug-resistance. Therefore, studies of the mechanisms underpinning the resistance toward anti-EGFR agents may provide important findings for lung cancer treatment using anti-EGFR therapies. Recently, increasing numbers of miRNAs are correlated with the drug resistance of lung cancer cells to anti-EGFR agents, indicating that miRNAs may serve as novel targets and/or promising predictive biomarkers for anti-EGFR therapy. In this paper, we summarize the emerging role of miRNAs as regulators to modulate the EGFR signaling and the resistance of lung cancer cells to anti-EGFR therapy. We also highlight the evidence supporting the use of miRNAs as biomarkers for response to anti-EGFR agents and as novel therapeutic targets to circumvent the resistance of lung cancer cells to EGFR inhibitors. Fei Han, Jinxi He, Feng Li, Jiali Yang, Jun Wei, William C. Cho, and Xiaoming Liu Copyright © 2015 Fei Han et al. All rights reserved. Unravelling the RNA-Binding Properties of SAFB Proteins in Breast Cancer Cells Sun, 26 Jul 2015 07:50:19 +0000 Scaffold attachment factor B1 (SAFB1) and SAFB2 proteins are oestrogen (ER) corepressors that bind to and modulate ER activity through chromatin remodelling or interaction with the basal transcription machinery. SAFB proteins also have an internal RNA-recognition motif but little is known about the RNA-binding properties of SAFB1 or SAFB2. We utilised crosslinking and immunoprecipitation (iCLIP) coupled with high-throughput sequencing to enable a transcriptome-wide mapping of SAFB1 protein-RNA interactions in breast cancer MCF-7 cells. Analysis of crosslinking frequency mapped to transcript regions revealed that SAFB1 binds to coding and noncoding RNAs (ncRNAs). The highest proportion of SAFB1 crosslink sites mapped to ncRNAs, followed by intergenic regions, open reading frames (ORFs), introns, and 3′ or 5′ untranslated regions (UTR). Furthermore, we reveal that SAFB1 binds directly to RNA and its binding is particularly enriched at purine-rich sequences not dissimilar to the RNA-binding motifs for SR proteins. Using RNAi, we also show, for the first time, that single depletion of either SAFB1 or SAFB2 leads to an increase in expression of the other SAFB protein in both MCF-7 and MDA-MD231 breast cancer cells. Elaine Hong, Andrew Best, Hannah Gautrey, Jas Chin, Anshuli Razdan, Tomaz Curk, David J. Elliott, and Alison J. Tyson-Capper Copyright © 2015 Elaine Hong et al. All rights reserved. SAM68: Signal Transduction and RNA Metabolism in Human Cancer Sun, 26 Jul 2015 07:32:41 +0000 Alterations in expression and/or activity of splicing factors as well as mutations in cis-acting splicing regulatory sequences contribute to cancer phenotypes. Genome-wide studies have revealed more than 15,000 tumor-associated splice variants derived from genes involved in almost every aspect of cancer cell biology, including proliferation, differentiation, cell cycle control, metabolism, apoptosis, motility, invasion, and angiogenesis. In the past decades, several RNA binding proteins (RBPs) have been implicated in tumorigenesis. SAM68 (SRC associated in mitosis of 68 kDa) belongs to the STAR (signal transduction and activation of RNA metabolism) family of RBPs. SAM68 is involved in several steps of mRNA metabolism, from transcription to alternative splicing and then to nuclear export. Moreover, SAM68 participates in signaling pathways associated with cell response to stimuli, cell cycle transitions, and viral infections. Recent evidence has linked this RBP to the onset and progression of different tumors, highlighting misregulation of SAM68-regulated splicing events as a key step in neoplastic transformation and tumor progression. Here we review recent studies on the role of SAM68 in splicing regulation and we discuss its contribution to aberrant pre-mRNA processing in cancer. Paola Frisone, Davide Pradella, Anna Di Matteo, Elisa Belloni, Claudia Ghigna, and Maria Paola Paronetto Copyright © 2015 Paola Frisone et al. All rights reserved. Evaluation of Genetic Variations in Maize Seedlings Exposed to Electric Field Based on Protein and DNA Markers Wed, 17 Jun 2015 11:23:57 +0000 The current study analyzed proteins and nuclear DNA of electric fields (ELF) exposed and nonexposed maize seedlings for different exposure periods using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isozymes, random amplified polymorphic DNA (RAPD), and comet assay, respectively. SDS-PAGE analysis revealed total of 46 polypeptides bands with different molecular weights ranging from 186.20 to 36.00 KDa. It generated distinctive polymorphism value of 84.62%. Leucine-aminopeptidase, peroxidase, and catalase isozymes showed the highest values of polymorphism (100%) based on zymograms number, relative front (), and optical intensity while esterase isozyme generated polymorphism value of 83.33%. Amino acids were analyzed using high-performance liquid chromatography, which revealed the presence of 17 amino acids of variable contents ranging from 22.65% to 28.09%. RAPD revealed that 78 amplified DNA products had highly polymorphism value (95.08%) based on band numbers, with variable sizes ranging from 120 to 992 base pairs and band intensity. Comet assay recorded the highest extent of nuclear DNA damage as percentage of tailed DNA (2.38%) and tail moment unit (5.36) at ELF exposure of maize nuclei for 5 days. The current study concluded that the longer ELF exposing periods had genotoxic stress on macromolecules of maize cells and biomarkers used should be augmented for reliable estimates of genotoxicity after exposure of economic plants to ELF stressors. Asma A. AL-Huqail and Ekram Abdelhaliem Copyright © 2015 Asma A. AL-Huqail and Ekram Abdelhaliem. All rights reserved. Molecular Phylogenetics 2014 Tue, 02 Jun 2015 07:12:34 +0000 Vassily Lyubetsky, William H. Piel, and Peter F. Stadler Copyright © 2015 Vassily Lyubetsky et al. All rights reserved. Comparative Analysis of Apicoplast-Targeted Protein Extension Lengths in Apicomplexan Parasites Sun, 31 May 2015 16:47:04 +0000 In general, the mechanism of protein translocation through the apicoplast membrane requires a specific extension of a functionally important region of the apicoplast-targeted proteins. The corresponding signal peptides were detected in many apicomplexans but not in the majority of apicoplast-targeted proteins in Toxoplasma gondii. In T. gondii signal peptides are either much diverged or their extension region is processed, which in either case makes the situation different from other studied apicomplexans. We propose a statistic method to compare extensions of the functionally important regions of apicoplast-targeted proteins. More specifically, we provide a comparison of extension lengths of orthologous apicoplast-targeted proteins in apicomplexan parasites. We focus on results obtained for the model species T. gondii, Neospora caninum, and Plasmodium falciparum. With our method, cross species comparisons demonstrate that, in average, apicoplast-targeted protein extensions in T. gondii are 1.5-fold longer than in N. caninum and 2-fold longer than in P. falciparum. Extensions in P. falciparum less than 87 residues in size are longer than the corresponding extensions in N. caninum and, reversely, are shorter if they exceed 88 residues. Alexandr V. Seliverstov, Oleg A. Zverkov, Svetlana N. Istomina, Sergey A. Pirogov, and Philip S. Kitsis Copyright © 2015 Alexandr V. Seliverstov et al. All rights reserved. Lengths of Orthologous Prokaryotic Proteins Are Affected by Evolutionary Factors Sun, 31 May 2015 12:55:48 +0000 Proteins of the same functional family (for example, kinases) may have significantly different lengths. It is an open question whether such variation in length is random or it appears as a response to some unknown evolutionary driving factors. The main purpose of this paper is to demonstrate existence of factors affecting prokaryotic gene lengths. We believe that the ranking of genomes according to lengths of their genes, followed by the calculation of coefficients of association between genome rank and genome property, is a reasonable approach in revealing such evolutionary driving factors. As we demonstrated earlier, our chosen approach, Bubble-sort, combines stability, accuracy, and computational efficiency as compared to other ranking methods. Application of Bubble Sort to the set of 1390 prokaryotic genomes confirmed that genes of Archaeal species are generally shorter than Bacterial ones. We observed that gene lengths are affected by various factors: within each domain, different phyla have preferences for short or long genes; thermophiles tend to have shorter genes than the soil-dwellers; halophiles tend to have longer genes. We also found that species with overrepresentation of cytosines and guanines in the third position of the codon (GC3 content) tend to have longer genes than species with low GC3 content. Tatiana Tatarinova, Bilal Salih, Jennifer Dien Bard, Irit Cohen, and Alexander Bolshoy Copyright © 2015 Tatiana Tatarinova et al. All rights reserved. miR-1322 Binding Sites in Paralogous and Orthologous Genes Sun, 31 May 2015 09:52:45 +0000 We searched for 2,563 microRNA (miRNA) binding sites in 17,494 mRNA sequences of human genes. miR-1322 has more than 2,000 binding sites in 1,058 genes with ratio of 85% and more. miR-1322 has 1,889 binding sites in CDSs, 215 binding sites in 5′ UTRs, and 160 binding sites in 3′ UTRs. From two to 28 binding sites have arranged localization with the start position through three nucleotides of each following binding site. The nucleotide sequences of these sites in CDSs encode oligopeptides with the same and/or different amino acid sequences. We found that 33% of the target genes encoded transcription factors. miR-1322 has arranged binding sites in the CDSs of orthologous MAMLD1, MAML2, and MAML3 genes. These sites encode a polyglutamine oligopeptide ranging from six to 47 amino acids in length. The properties of miR-1322 binding sites in orthologous and paralogous target genes are discussed. Raigul Niyazova, Olga Berillo, Shara Atambayeva, Anna Pyrkova, Aigul Alybayeva, and Anatoly Ivashchenko Copyright © 2015 Raigul Niyazova et al. All rights reserved. Phyloproteomic Analysis of 11780 Six-Residue-Long Motifs Occurrences Sun, 31 May 2015 09:43:41 +0000 How is it possible to find good traits for phylogenetic reconstructions? Here, we present a new phyloproteomic criterion that is an occurrence of simple motifs which can be imprints of evolution history. We studied the occurrences of 11780 six-residue-long motifs consisting of two randomly located amino acids in 97 eukaryotic and 25 bacterial proteomes. For all eukaryotic proteomes, with the exception of the Amoebozoa, Stramenopiles, and Diplomonadida kingdoms, the number of proteins containing the motifs from the first group (one of the two amino acids occurs once at the terminal position) made about 20%; in the case of motifs from the second (one of two amino acids occurs one time within the pattern) and third (the two amino acids occur randomly) groups, 30% and 50%, respectively. For bacterial proteomes, this relationship was 10%, 27%, and 63%, respectively. The matrices of correlation coefficients between numbers of proteins where a motif from the set of 11780 motifs appears at least once in 9 kingdoms and 5 phyla of bacteria were calculated. Among the correlation coefficients for eukaryotic proteomes, the correlation between the animal and fungi kingdoms (0.62) is higher than between fungi and plants (0.54). Our study provides support that animals and fungi are sibling kingdoms. Comparison of the frequencies of six-residue-long motifs in different proteomes allows obtaining phylogenetic relationships based on similarities between these frequencies: the Diplomonadida kingdoms are more close to Bacteria than to Eukaryota; Stramenopiles and Amoebozoa are more close to each other than to other kingdoms of Eukaryota. O. V. Galzitskaya and M. Yu. Lobanov Copyright © 2015 O. V. Galzitskaya and M. Yu. Lobanov. All rights reserved. A Database of Plastid Protein Families from Red Algae and Apicomplexa and Expression Regulation of the moeB Gene Sun, 31 May 2015 09:34:01 +0000 We report the database of plastid protein families from red algae, secondary and tertiary rhodophyte-derived plastids, and Apicomplexa constructed with the novel method to infer orthology. The families contain proteins with maximal sequence similarity and minimal paralogous content. The database contains 6509 protein entries, 513 families and 278 nonsingletons (from which 230 are paralog-free, and among the remaining 48, 46 contain at maximum two proteins per species, and 2 contain at maximum three proteins per species). The method is compared with other approaches. Expression regulation of the moeB gene is studied using this database and the model of RNA polymerase competition. An analogous database obtained for green algae and their symbiotic descendants, and applications based on it are published earlier. Oleg A. Zverkov, Alexandr V. Seliverstov, and Vassily A. Lyubetsky Copyright © 2015 Oleg A. Zverkov et al. All rights reserved. Advanced Glycation End Products Enhance Macrophages Polarization into M1 Phenotype through Activating RAGE/NF-κB Pathway Sun, 31 May 2015 08:12:08 +0000 Atherosclerotic lesions are accelerated in patients with diabetes. M1 (classically activated in contrast to M2 alternatively activated) macrophages play key roles in the progression of atherosclerosis. Since advanced glycation end products (AGEs) are major pathogenic factors and active inflammation inducers in diabetes mellitus, this study assessed the effects of AGEs on macrophage polarization. The present study showed that AGEs significantly promoted macrophages to express IL-6 and TNF-α. M1 macrophage markers such as iNOS and surface markers including CD11c and CD86 were significantly upregulated while M2 macrophage markers such as Arg1 and CD206 remained unchanged after AGEs stimulation. AGEs significantly increased RAGE expression in macrophages and activated NF-κB pathway, and the aforementioned effects were partly abolished by administration of anti-RAGE antibody or NF-κB inhibitor PDTC. In conclusion, our results suggest that AGEs enhance macrophage differentiation into proinflammatory M1 phenotype at least partly via RAGE/NF-κB pathway activation. Xian Jin, Tongqing Yao, Zhong’e Zhou, Jian Zhu, Song Zhang, Wei Hu, and Chengxing Shen Copyright © 2015 Xian Jin et al. All rights reserved. Molecular Biogeography of Tribe Thermopsideae (Leguminosae): A Madrean-Tethyan Disjunction Pattern with an African Origin of Core Genistoides Sun, 31 May 2015 07:23:32 +0000 Thermopsideae has 45 species and exhibits a series of interesting biogeographical distribution patterns, such as Madrean-Tethyan disjunction and East Asia-North America disjunction, with a center of endemism in the Qinghai-Xizang Plateau (QTP) and Central Asia. Phylogenetic analysis in this paper employed maximum likelihood using ITS, rps16, psbA-trnH, and trnL-F sequence data; biogeographical approaches included BEAST molecular dating and Bayesian dispersal and vicariance analysis (S-DIVA). The results indicate that the core genistoides most likely originated in Africa during the Eocene to Oligocene, ca. 55-30 Ma, and dispersed eastward to Central Asia at ca. 33.47 Ma. The origin of Thermopsideae is inferred as Central Asian and dated to ca. 28.81 Ma. Ammopiptanthus is revealed to be a relic. Birth of the ancestor of Thermopsideae coincided with shrinkage of the Paratethys Sea at ca. 30 Ma in the Oligocene. The Himalayan motion of QTP uplift of ca. 20 Ma most likely drove the diversification between Central Asia and North America. Divergences in East Asia, Central Asia, the Mediterranean, and so forth, within Eurasia, except for Ammopiptanthus, are shown to be dispersals from the QTP. The onset of adaptive radiation at the center of the tribe, with diversification of most species in Thermopsis and Piptanthus at ca. 4-0.85 Ma in Tibet and adjacent regions, seems to have resulted from intense northern QTP uplift during the latter Miocene to Pleistocene. Ming-Li Zhang, Jian-Feng Huang, Stewart C. Sanderson, Ping Yan, Yu-Hu Wu, and Bo-Rong Pan Copyright © 2015 Ming-Li Zhang et al. All rights reserved. Phylogeography of Pteronotropis signipinnis, P. euryzonus, and the P. hypselopterus Complex (Teleostei: Cypriniformes), with Comments on Diversity and History of the Gulf and Atlantic Coastal Streams Sun, 31 May 2015 07:15:55 +0000 The cyprinid genus Pteronotropis is endemic to southeastern Gulf of Mexico and Atlantic Ocean of North America. Never before has the genus been demonstrated to be monophyletic. We investigate both the phylogenetic relationships and the phylogeography of some species in the genus using mitochondrial ND2 sequences. In no analysis is the genus resolved as monophyletic if Notropis harperi is not included in the genus. Biogeographic and phylogeographic evaluations are conducted with Pteronotropis, including P. signipinnis, P. euryzonus, and the P. hypselopterus complex. Patterns of relationships and population genetic analyses support divergences within multiple clades both at the species level and within species that are tied to abiotic changes in the region. Replicated patterns across clades are observed, as well as patterns previously found in other taxa. Pteronotropis hypselopterus is likely not a natural grouping as populations from some drainages form clades more closely related to other species of the genus. The general patterns of relationships indicate likely cryptic species not currently recognized. Finally, the patterns of species relationships and clades and population structuring within species serve as another example of replicated divergences in the biodiversity east and west of the Mobile Bay. Richard L. Mayden and Jason Allen Copyright © 2015 Richard L. Mayden and Jason Allen. All rights reserved. Structural and Population Polymorphism of RT-Like Sequences in Avian Schistosomes Trichobilharzia szidati (Platyhelminthes: Digenea: Schistosomatidae) Sun, 31 May 2015 07:10:23 +0000 Recently we developed the genus-specific markers of the avian schistosomes of the genus Trichobilharzia, the causative agents of human cercarial dermatitis. The 7 novel genome sequences of T. franki, T. regenti, and T. szidati revealed similarity with genome repeat region of African schistosome Schistosoma mansoni. In the present work we analyzed the 37 new T. szidati sequences to study intragenome variability and host specificity for the parasite from three localities of East Europe. DNAs were isolated from cercariae or single sporocysts obtained from 6 lymnaeid snails Lymnaea stagnalis and L. palustris from Belarus and Russia. All sequences formed three diverged groups, one of which consists of the sequences with multiple deletions; other groups involved two paralogous copies with stop codons and frameshift mutations. Strong association between geographical distribution and snail host specificity cannot be established. All studied sequences have homology with the reverse transcriptase domain (RT) of Penelope-like elements (PLE) of S. mansoni and S. japonicum and new members of RT family were identified. We proposed that three diverged groups RT sequences of T. szidati are results of duplication or transposition of PLE during parasite evolution. Implications of the retroelement dynamics in the life history of avian schistosomes are discussed. S. K. Semyenova, G. G. Chrisanfova, A. S. Guliaev, A. P. Yesakova, and A. P. Ryskov Copyright © 2015 S. K. Semyenova et al. All rights reserved. The Variability of the Order Burkholderiales Representatives in the Healthcare Units Sun, 31 May 2015 06:57:45 +0000 Background and Aim. The order Burkholderiales became more abundant in the healthcare units since the late 1970s; it is especially dangerous for intensive care unit patients and patients with chronic lung diseases. The goal of this investigation was to reveal the real variability of the order Burkholderiales representatives and to estimate their phylogenetic relationships. Methods. 16S rDNA and genes of the Burkholderia cenocepacia complex (Bcc) Multi Locus Sequence Typing (MLST) scheme were used for the bacteria detection. Results. A huge diversity of genome size and organization was revealed in the order Burkholderiales that may prove the adaptability of this taxon’s representatives. The following variability of the Burkholderiales in Russian healthcare units has been revealed: Burkholderiaceae (Burkholderia, Pandoraea, and Lautropia), Alcaligenaceae (Achromobacter), and Comamonadaceae (Variovorax). The Burkholderia genus was the most diverse and was represented by 5 species and 16 sequence types (ST). ST709 and 728 were transmissible and often encountered in cystic fibrosis patients and in hospitals. A. xylosoxidans was estimated by 15 genotypes. The strains of first and second ones were the most numerous. Conclusions. Phylogenetic position of the genus Lautropia with smaller genome is ambiguous. The Bcc MLST scheme is applicable for all Burkholderiales representatives for resolving the epidemiological problems. Olga L. Voronina, Marina S. Kunda, Natalia N. Ryzhova, Ekaterina I. Aksenova, Andrey N. Semenov, Anna V. Lasareva, Elena L. Amelina, Alexandr G. Chuchalin, Vladimir G. Lunin, and Alexandr L. Gintsburg Copyright © 2015 Olga L. Voronina et al. All rights reserved. Biochemical and Molecular Phylogenetic Study of Agriculturally Useful Association of a Nitrogen-Fixing Cyanobacterium and Nodule Sinorhizobium with Medicago sativa L. Sun, 31 May 2015 06:56:15 +0000 Seed inoculation with bacterial consortium was found to increase legume yield, providing a higher growth than the standard nitrogen treatment methods. Alfalfa plants were inoculated by mono- and binary compositions of nitrogen-fixing microorganisms. Their physiological and biochemical properties were estimated. Inoculation by microbial consortium of Sinorhizobium meliloti T17 together with a new cyanobacterial isolate Nostoc PTV was more efficient than the single-rhizobium strain inoculation. This treatment provides an intensification of the processes of biological nitrogen fixation by rhizobia bacteria in the root nodules and an intensification of plant photosynthesis. Inoculation by bacterial consortium stimulates growth of plant mass and rhizogenesis and leads to increased productivity of alfalfa and to improving the amino acid composition of plant leaves. The full nucleotide sequence of the rRNA gene cluster and partial sequence of the dinitrogenase reductase (nifH) gene of Nostoc PTV were deposited to GenBank (JQ259185.1, JQ259186.1). Comparison of these gene sequences of Nostoc PTV with all sequences present at the GenBank shows that this cyanobacterial strain does not have 100% identity with any organisms investigated previously. Phylogenetic analysis showed that this cyanobacterium clustered with high credibility values with Nostoc muscorum. E. V. Karaushu, I. V. Lazebnaya, T. R. Kravzova, N. A. Vorobey, O. E. Lazebny, D. A. Kiriziy, O. P. Olkhovich, N. Yu. Taran, S. Ya. Kots, A. A. Popova, E. Omarova, and O. A. Koksharova Copyright © 2015 E. V. Karaushu et al. All rights reserved. Signs of Selection in Synonymous Sites of the Mitochondrial Cytochrome b Gene of Baikal Oilfish (Comephoridae) by mRNA Secondary Structure Alterations Sun, 31 May 2015 06:51:24 +0000 Studies over the past decade have shown a significant role of synonymous mutations in posttranscriptional regulation of gene expression, which is particularly associated with messenger RNA (mRNA) secondary structure alterations. Most studies focused on prokaryote genomes and the nuclear genomes of eukaryotes while little is known about the regulation of mitochondrial DNA (mtDNA) gene expression. This paper reveals signs of selection in synonymous sites of the mitochondrial cytochrome b gene (Cytb) of Baikal oilfish or golomyankas (Comephoridae) directed towards altering the secondary structure of the mRNA and probably altering the character of mtDNA gene expression. Our findings are based on comparisons of intraspecific genetic variation patterns of small golomyanka (Comephorus dybowski) and two genetic groups of big golomyanka (Comephorus dybowskii). Two approaches were used: (i) analysis of the distribution of synonymous mutations between weak-AT (W) and strong-GC (S) nucleotides within species and groups in accordance with mutation directions from central to peripheral haplotypes and (ii) approaches based on the predicted mRNA secondary structure. Veronika I. Teterina, Anatoliy M. Mamontov, Lyubov V. Sukhanova, and Sergei V. Kirilchik Copyright © 2015 Veronika I. Teterina et al. All rights reserved. Molecular Systematics of the Phoxinin Genus Pteronotropis (Otophysi: Cypriniformes) Sun, 31 May 2015 06:25:42 +0000 The genus Pteronotropis is widely distributed along the gulf slope of eastern North America from Louisiana to Florida and rivers in South Carolina along the Atlantic slope. Pteronotropis have very distinctive, flamboyant coloration. The habitats most frequently associated with these species include heavily vegetated backwater bayous to small sluggish or flowing tannin-stained streams. Although Pteronotropis is recognized as a valid genus, no phylogenetic analysis of all the species has corroborated its monophyly. In recent years, four additional species have been either described or elevated from synonymy: P. merlini, P. grandipinnis, P. stonei, and P. metallicus, with the wide-ranging P. hypselopterus complex. To examine relationships within this genus and test its monophyly, phylogenetic analyses were conducted using two nuclear genes, recombination activating gene 1, RAG1, and the first intron of S7 ribosomal protein gene in both maximum parsimony and Bayesian analyses. In no analysis was Pteronotropis, as currently recognized, recovered as monophyletic without the inclusion of the currently recognized Notropis harperi, herein referred to as Pteronotropis. Two major clades are supported: one inclusive of P. hubbsi, P. welaka, and P. harperi and the second inclusive of P. signipinnis, P. grandipinnis, P. hypselopterus plus P. merlini sister to P. euryzonus, and P. metallicus plus P. stonei. Richard L. Mayden and Jason S. Allen Copyright © 2015 Richard L. Mayden and Jason S. Allen. All rights reserved. Mcl-1 Is a Novel Target of miR-26b That Is Associated with the Apoptosis Induced by TRAIL in HCC Cells Thu, 21 May 2015 08:50:14 +0000 Aim. To investigate the role of miR-26b and Mcl-1 in TRAIL-inducing cell death in hepatocellular carcinoma. Methods. The expression of miR-26b and Mcl-1 in HCC was detected by RT-qPCR and western blot. The regulation of Mcl-1 by miR-26b was determined by luciferase reporter assay. MTT and flow cytometry were employed to detect the cell viability and apoptosis. Results. miR-26b is commonly downregulated in HCC cell lines compared with the LO2 cell line. In contrast, the Mcl-1 expression is upregulated in HCC cell lines. Bioinformatic analysis identified a putative target site in the Mcl-1 mRNA for miR-26b and luciferase reporter assay showed that miR-26b directly targeted the 3′-UTR (3′-Untranslated Regions) of Mcl-1 mRNA. Transfection of miR-26b mimics suppressed Mcl-1 expression in HCC cells and sensitized the cancer cells to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) cytotoxicity. In addition, transfection of HCC cells with Mcl-1 expression plasmid abolished the sensitization effect of miR-26b to TRAIL-inducing apoptosis. Conclusions. Our study showed that miR-26b was a negative regulator of Mcl-1 gene and sensitized TRAIL-inducing apoptosis in HCC cells, suggesting that the miR-26b-Mcl-1 pathway might be a novel target for the treatment of HCC. Chunlin Jiang, Jianting Long, Baoxian Liu, Xiaoyan Xie, and Ming Kuang Copyright © 2015 Chunlin Jiang et al. All rights reserved. Identification, Characterization, and Developmental Expression Pattern of Type III Interferon Receptor Gene in the Chinese Goose Sun, 10 May 2015 10:59:38 +0000 Interferons, as the first line of defense against the viral infection, play an important role in innate immune responses. Type III interferon (IFN-λ) was a newly identified member of IFN family, which plays IFN-like antiviral activity. Towards a better understanding of the type III interferon system in birds, type III interferon lambda receptor (IFNLR1) was first identified in the Chinese goose. In this paper, we had cloned 1952 bp for goose IFNLR1 (goIFNLR1), including an ORF of 1539 bp, encoding a 512-amino acid protein with a 20 aa predict signal peptide at its N terminal and a 23 aa transmembrane region. The predicted amino acid sequence of goIFNLR1 has 90%, 73%, and 34% identity with duck IFNLR1 (predicted sequence), chicken IFNLR1, and human IFNLR1, respectively. And the age-related tissue distribution of goIFNLR1 was identified by Real Time quantitative PCR (RT-qPCR), we found that the goIFNLR1 has a mainly expression in epithelium-rich tissues similar to other species’, such as small intestinal, lung, liver, and stomach. Moreover, a relatively high expression of goIFNLR1 was also observed in the secondary immune tissues (harderian gland and cecal tonsil). The identification and tissue distribution of goIFNLR1 will facilitate further study of the role of IFN-λ in goose antiviral defense. Qin Zhou, Shun Chen, Yulin Qi, Hao Zhou, Mingshu Wang, Renyong Jia, Dekang Zhu, Mafeng Liu, Fei Liu, Xiaoyue Chen, Xue Zhou, and Anchun Cheng Copyright © 2015 Qin Zhou et al. All rights reserved. miRNA Profiles of Tubular Cells: Diagnosis of Kidney Injury Thu, 30 Apr 2015 08:34:50 +0000 MicroRNAs (miRNAs) are small noncoding RNAs of 18–23 nucleotides that regulate gene expression. Recently, plasma miRNAs have been investigated as biomarkers for various physiological and pathological conditions. The present study details the conserved miRNA expression profiles of tubular tissues, and discusses whether they could be used to distinguish between proximal tubule injury, diagnose acute kidney injury (AKI), and the early-stage renal tubular dysfunction. miRNA expression was assessed with miRNA array and real-time reverse transcription polymerase chain reaction using the TaqMan system. The expression profiles of miR-200a/b/c, miR-145, miR-192, miR-194, miR-216a/b, miR-217, and miR-449a in human and rat tubular tissues such as the kidneys, lung, small intestine, and various exocrine glands were adequate for discriminating tubular tissues. In the kidney, miR-192 and miR-194 were highly expressed, whereas miR-145 and miR-449a were absent. miR-145 and miR-449a were relatively specifically expressed in small intestine and lung, respectively. Therefore, the combined levels of miR-200a/b/c, miR-192, and miR-194 in plasma were very useful in diagnosing AKI induced by contact freezing in mice. Moreover, urinary miR-200a levels were useful for the diagnosis of renal tubular dysfunction in Dahl salt-sensitive rat with high salt administration. Our results indicate that miRNA expression profiles are useful as biomarkers for identification of various kidney injuries. Naoko Kito, Kosuke Endo, Masahiro Ikesue, Huachun Weng, and Naoharu Iwai Copyright © 2015 Naoko Kito et al. All rights reserved. DNA Methylation and Histone Modifications Are the Molecular Lock in Lentivirally Transduced Hematopoietic Progenitor Cells Sun, 19 Apr 2015 09:48:13 +0000 Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV. Siew Ching Ngai, Rozita Rosli, Akram Al Abbar, and Syahril Abdullah Copyright © 2015 Siew Ching Ngai et al. All rights reserved. IFN-CSP Inhibiting Hepatitis B Virus in HepG2.2.15 Cells Involves JAK-STAT Signal Pathway Mon, 16 Mar 2015 09:00:58 +0000 Frequent and high-dose administration of interferon to patients with viral hepatitis results in various side effects. In our previous study, a novel liver-targeting interferon (IFN-CSP) combining Plasmodium region I peptide with IFNα2b was successfully designed and expressed in the Escherichia coli expression systems. This targeting would target the IFNα2b specifically to the liver, thus reducing the adverse events. In the present study, we further investigated the anti-HBV effects and molecular mechanisms of recombinant IFN-CSP in HepG2.2.15 cell line. Hepatitis B surface antigen (HBsAg) and HBe antigen (HBeAg) in the culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA). HBV-DNA was measured by real-time quantitative PCR. HBV core protein was assayed by immunofluorescent and western blot analysis. The expressions of signal transducers and transactivator 1 (STAT1), STAT2, IFN regulatory factor 9 (IRF-9), and 2′-5′-oligoadenylate synthetase 1 (OAS1) were investigated by the reverse transcription PCR and western blot analysis. Results indicate IFN-CSP efficiently inhibited HBsAg and HBeAg secretion, HBV-DNA replication, and HBV core protein expression in HepG2.2.15 cells. The anti-HBV mechanisms involve activation of JAK-STAT signaling and increase of the anti-HBV protein OAS expression. IFN-CSP could be a good substitute for IFNα2b for anti-HBV treatment. Xuemei Lu, Jie Wang, Xiaobao Jin, Yanting Huang, Wenting Zeng, and Jiayong Zhu Copyright © 2015 Xuemei Lu et al. All rights reserved. MicroRNA and mRNA Features of Malignant Pleural Mesothelioma and Benign Asbestos-Related Pleural Effusion Sun, 01 Feb 2015 13:13:18 +0000 Introduction. We investigated the expression of microRNAs and mRNAs in pleural tissues from patients with either malignant pleural mesothelioma or benign asbestos-related pleural effusion. Methods. Fresh frozen tissues from a total of 18 malignant pleural mesothelioma and 6 benign asbestos-related pleural effusion patients were studied. Expression profiling of mRNA and microRNA was performed using standard protocols. Results. We discovered significant upregulation of multiple microRNAs in malignant pleural mesothelioma compared to benign asbestos-related pleural effusion. Hsa-miR-484, hsa-miR-320, hsa-let-7a, and hsa-miR-125a-5p were able to discriminate malignant from benign disease. Dynamically regulated mRNAs were also identified. MET was the most highly overexpressed gene in malignant pleural mesothelioma compared to benign asbestos-related pleural effusion. Integrated analyses examining microRNA-mRNA interactions suggested multiple altered targets within the Notch signaling pathway. Conclusions. Specific microRNAs and mRNAs may have diagnostic utility in differentiating patients with malignant pleural mesothelioma from benign asbestos-related pleural effusion. These studies may be particularly helpful in patients who reside in a region with a high incidence of mesothelioma. Guntulu Ak, Sandra C. Tomaszek, Farhad Kosari, Muzaffer Metintas, James R. Jett, Selma Metintas, Huseyin Yildirim, Emine Dundar, Jie Dong, Marie Christine Aubry, Dennis A. Wigle, and Charles F. Thomas Jr. Copyright © 2015 Guntulu Ak et al. All rights reserved. MicroRNAs and Cardiovascular Diseases Sun, 01 Feb 2015 12:32:02 +0000 Coronary artery diseases (CAD) and heart failure have high mortality rate in the world, although much progress has been made in this field in last two decades. There is still a clinical need for a novel diagnostic approach and a therapeutic strategy to decrease the incidence of CAD. MicroRNAs (miRNAs) are highly conserved noncoding small RNA molecules that regulate a large fraction of the genome by binding to complementary messenger RNA sequences, resulting in posttranscriptional gene silencing. Recent studies have shown that specific miRNAs are involved in whole stage of atherosclerosis, from endothelium dysfunction to plaque rupture. These findings suggest that miRNAs are potential biomarkers in early diagnosis and therapeutic targets in CAD. In the present review, we highlight the role of miRNAs in every stage of atherosclerosis, and discuss the prospects of miRNAs in the near future. Tsuyoshi Nishiguchi, Toshio Imanishi, and Takashi Akasaka Copyright © 2015 Tsuyoshi Nishiguchi et al. All rights reserved. Diagnosis of Neisseria gonorrhoeae Using Molecular Beacon Sat, 31 Jan 2015 12:30:25 +0000 Neisseria gonorrhoeae is an important sexually transmitted diseases (STD) causing pathogen worldwide. Due to absence of an affordable diagnostic assay, routine screening of gonococcal infection becomes impossible in developing countries where infection rates are maximum. Treatment is given on the basis of symptoms alone which leads to spread of infection. Thus, development of a rapid, sensitive, specific, and PCR based visual diagnostic assay suitable for developing countries, required for better disease management, is aimed at in present study. Endocervical swabs were collected from patients visiting gynecology department of various hospitals in Delhi. In-house PCR based assay was developed and modified to visual assay using molecular beacon for end-point detection. It was evaluated against Roche AMPLICOR NG kit and rmp gene. Specificity of beacon was confirmed by competition experiments. Diagnostic test was 98.21% specific and 99.59% sensitive whereas negative and positive predicted value were 99.40% and 98.78%, respectively. We also observed that twice the concentration (2X) of premix was stable at 4°C for 4 months and dry swab samples gave concordant results with that of wet swabs. These features make the test best suitable for routine diagnosis of genital infections in developing countries. Divya Sachdev, Achchhe Lal Patel, Subash Chandra Sonkar, Indu Kumari, and Daman Saluja Copyright © 2015 Divya Sachdev et al. All rights reserved. Correlation between Serum Levels of High Mobility Group Box-1 Protein and Pancreatitis: A Meta-Analysis Wed, 28 Jan 2015 08:05:39 +0000 Background. Aberrant expression of high mobility group box-1 protein (HMGB1) contributes to the progression of various inflammatory diseases. This meta-analysis focused on the clinical significance of serum HMGB1 levels in pancreatitis patients, with the goal of building a novel diagnostic score model. Method. We conducted a meta-analysis by searching in the PubMed, Embase, Web of Science, Cochrane Library, CISCOM, CINAHL, Google Scholar, China BioMedicine (CBM), and China National Knowledge Infrastructure (CNKI) databases without any language restrictions. Studies were pooled and standard mean difference (SMD) and its corresponding 95% confidence intervals (95% CIs) were calculated. Version 12.0 STATA software was used for statistical analysis. Results. We performed a final analysis of 841 subjects from 12 clinical case-control studies. The meta-analysis results showed a positive association between serum HMGB1 levels and the progression of pancreatitis. In the subgroup analysis by country, high serum level of HMGB1 may be related to pancreatitis progression in China, Korea, Hungary, and Japan populations (all ). Conclusion. The present meta-analysis indicated that serum HMGB1 level was statistically elevated in patients with pancreatitis, and thus serum levels of HMGB1 could be determined to be a useful biomarker for pancreatitis patients. Yan Lin, Lian-Jie Lin, Yu Jin, Yong Cao, Ying Zhang, Chang-Qing Zheng, Jia-Li Liu, and Sheng-Li Yang Copyright © 2015 Yan Lin et al. All rights reserved. Recent Novel High-Tech Researches in Molecular Biology Wed, 28 Jan 2015 07:01:17 +0000 Calvin Yu-Chian Chen, Kuo-Chen Chou, James David Adams, Tai-Ping Fan, and Gerhard Litscher Copyright © 2015 Calvin Yu-Chian Chen et al. All rights reserved. Transgenic Sugarcane Resistant to Sorghum mosaic virus Based on Coat Protein Gene Silencing by RNA Interference Thu, 22 Jan 2015 14:21:12 +0000 As one of the critical diseases of sugarcane, sugarcane mosaic disease can lead to serious decline in stalk yield and sucrose content. It is mainly caused by Potyvirus sugarcane mosaic virus (SCMV) and/or Sorghum mosaic virus (SrMV), with additional differences in viral strains. RNA interference (RNAi) is a novel strategy for producing viral resistant plants. In this study, based on multiple sequence alignment conducted on genomic sequences of different strains and isolates of SrMV, the conserved region of coat protein (CP) genes was selected as the target gene and the interference sequence with size of 423 bp in length was obtained through PCR amplification. The RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence and cp4-epsps herbicide-tolerant gene was transferred to sugarcane cultivar ROC22 via Agrobacterium-mediated transformation. After herbicide screening, PCR molecular identification, and artificial inoculation challenge, anti-SrMV positive transgenic lines were successfully obtained. SrMV resistance rate of the transgenic lines with the interference sequence was 87.5% based on SrMV challenge by artificial inoculation. The genetically modified SrMV-resistant lines of cultivar ROC22 provide resistant germplasm for breeding lines and can also serve as resistant lines having the same genetic background for study of resistance mechanisms. Jinlong Guo, Shiwu Gao, Qinliang Lin, Hengbo Wang, Youxiong Que, and Liping Xu Copyright © 2015 Jinlong Guo et al. All rights reserved. Positive Association of Vitamin D Receptor Gene Variations with Multiple Sclerosis in South East Iranian Population Tue, 20 Jan 2015 12:42:30 +0000 Among the factors postulated to play a role in MS susceptibility, the role of vitamin D is outstanding. Since the function of vitamin D receptor (VDR) represents the effect of vitamin D on the body and genetic variations in VDR gene may affect its function, we aim to highlight the association of two VDR gene polymorphisms with MS susceptibility. In current study, we recruited 113 MS patients and 122 healthy controls. TaqI (rs731236) and ApaI (rs7975232) genetic variations in these two groups were evaluated using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. All genotype and allele frequencies in both variations showed association with the disease status. However, to find the definite connection between genetic variations in VDR gene and MS disease in a population of South East of Iran, more researches on gene structure and its function with regard to patients’ conditions are required. Mehrnaz Narooie-Nejad, Maryam Moossavi, Adam Torkamanzehi, and Ali Moghtaderi Copyright © 2015 Mehrnaz Narooie-Nejad et al. All rights reserved. Next-Generation Technologies for Multiomics Approaches Including Interactome Sequencing Mon, 12 Jan 2015 06:23:55 +0000 The development of high-speed analytical techniques such as next-generation sequencing and microarrays allows high-throughput analysis of biological information at a low cost. These techniques contribute to medical and bioscience advancements and provide new avenues for scientific research. Here, we outline a variety of new innovative techniques and discuss their use in omics research (e.g., genomics, transcriptomics, metabolomics, proteomics, and interactomics). We also discuss the possible applications of these methods, including an interactome sequencing technology that we developed, in future medical and life science research. Hiroyuki Ohashi, Mai Hasegawa, Kentaro Wakimoto, and Etsuko Miyamoto-Sato Copyright © 2015 Hiroyuki Ohashi et al. All rights reserved. Potential Inhibitors for Isocitrate Lyase of Mycobacterium tuberculosis and Non-M. tuberculosis: A Summary Thu, 08 Jan 2015 09:08:04 +0000 Isocitrate lyase (ICL) is the first enzyme involved in glyoxylate cycle. Many plants and microorganisms are relying on glyoxylate cycle enzymes to survive upon downregulation of tricarboxylic acid cycle (TCA cycle), especially Mycobacterium tuberculosis (MTB). In fact, ICL is a potential drug target for MTB in dormancy. With the urge for new antitubercular drug to overcome tuberculosis treat such as multidrug resistant strain and HIV-coinfection, the pace of drug discovery has to be increased. There are many approaches to discovering potential inhibitor for MTB ICL and we hereby review the updated list of them. The potential inhibitors can be either a natural compound or synthetic compound. Moreover, these compounds are not necessary to be discovered only from MTB ICL, as it can also be discovered by a non-MTB ICL. Our review is categorized into four sections, namely, (a) MTB ICL with natural compounds; (b) MTB ICL with synthetic compounds; (c) non-MTB ICL with natural compounds; and (d) non-MTB ICL with synthetic compounds. Each of the approaches is capable of overcoming different challenges of inhibitor discovery. We hope that this paper will benefit the discovery of better inhibitor for ICL. Yie-Vern Lee, Habibah A. Wahab, and Yee Siew Choong Copyright © 2015 Yie-Vern Lee et al. All rights reserved. Targeted Next Generation Sequencing Identifies Novel Mutations in RP1 as a Relatively Common Cause of Autosomal Recessive Rod-Cone Dystrophy Tue, 06 Jan 2015 12:59:36 +0000 We report ophthalmic and genetic findings in families with autosomal recessive rod-cone dystrophy (arRCD) and RP1 mutations. Detailed ophthalmic examination was performed in 242 sporadic and arRCD subjects. Genomic DNA was investigated using our customized next generation sequencing panel targeting up to 123 genes implicated in inherited retinal disorders. Stringent filtering coupled with Sanger sequencing and followed by cosegregation analysis was performed to confirm biallelism and the implication of the most likely disease causing variants. Sequencing identified 9 RP1 mutations in 7 index cases. Eight of the mutations were novel, and all cosegregated with severe arRCD phenotype, found associated with additional macular changes. Among the identified mutations, 4 belong to a region, previously associated with arRCD, and 5 others in a region previously associated with adRCD. Our prevalence studies showed that RP1 mutations account for up to 2.5% of arRCD. These results point out for the necessity of sequencing RP1 when genetically investigating sporadic and arRCD. It further highlights the interest of unbiased sequencing technique, which allows investigating the implication of the same gene in different modes of inheritance. Finally, it reports that different regions of RP1 can also lead to arRCD. Said El Shamieh, Elise Boulanger-Scemama, Marie-Elise Lancelot, Aline Antonio, Vanessa Démontant, Christel Condroyer, Mélanie Letexier, Jean-Paul Saraiva, Saddek Mohand-Saïd, José-Alain Sahel, Isabelle Audo, and Christina Zeitz Copyright © 2015 Said El Shamieh et al. All rights reserved. Plants Coping Abiotic and Biotic Stresses: A Tale of Diligent Management Mon, 05 Jan 2015 09:51:47 +0000 Hatem Rouached, Sikander Pal, Shimon Rachmilevitch, Marc Libault, and Lam-Son Phan Tran Copyright © 2015 Hatem Rouached et al. All rights reserved. DT2008: A Promising New Genetic Resource for Improved Drought Tolerance in Soybean When Solely Dependent on Symbiotic N2 Fixation Mon, 05 Jan 2015 06:16:23 +0000 Water deficit is one of the major constraints for soybean production in Vietnam. The soybean breeding research efforts conducted at the Agriculture Genetics Institute (AGI) of Vietnam resulted in the development of promising soybean genotypes, suitable for the drought-stressed areas in Vietnam and other countries. Such a variety, namely, DT2008, was recommended by AGI and widely used throughout the country. The aim of this work was to assess the growth of shoots, roots, and nodules of DT2008 versus Williams 82 (W82) in response to drought and subsequent rehydration in symbiotic association as a means to provide genetic resources for genomic research. Better shoot, root, and nodule growth and development were observed in the cultivar DT2008 under sufficient, water deficit, and recovery conditions. Our results represent a good foundation for further comparison of DT2008 and W82 at molecular levels using high throughput omic technologies, which will provide huge amounts of data, enabling us to understand the genetic network involved in regulation of soybean responses to water deficit and increasing the chances of developing drought-tolerant cultivars. Saad Sulieman, Chien Van Ha, Maryam Nasr Esfahani, Yasuko Watanabe, Rie Nishiyama, Chung Thi Bao Pham, Dong Van Nguyen, and Lam-Son Phan Tran Copyright © 2015 Saad Sulieman et al. All rights reserved. Interaction Analysis through Proteomic Phage Display Thu, 11 Sep 2014 09:10:37 +0000 Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. Gustav N. Sundell and Ylva Ivarsson Copyright © 2014 Gustav N. Sundell and Ylva Ivarsson. All rights reserved. Applications of Flow Cytometry to Characterize Bacterial Physiological Responses Tue, 09 Sep 2014 13:09:09 +0000 Although reports of flow cytometry (FCM) applied to bacterial analysis are increasing, studies of FCM related to human cells still vastly outnumber other reports. However, current advances in FCM combined with a new generation of cellular reporter probes have made this technique suitable for analyzing physiological responses in bacteria. We review how FCM has been applied to characterize distinct physiological conditions in bacteria including responses to antibiotics and other cytotoxic chemicals and physical factors, pathogen-host interactions, cell differentiation during biofilm formation, and the mechanisms governing development pathways such as sporulation. Since FCM is suitable for performing studies at the single-cell level, we describe how this powerful technique has yielded invaluable information about the heterogeneous distribution of differently and even specialized responding cells and how it may help to provide insights about how cell interaction takes place in complex structures, such as those that prevail in bacterial biofilms. Verónica Ambriz-Aviña, Jorge A. Contreras-Garduño, and Mario Pedraza-Reyes Copyright © 2014 Verónica Ambriz-Aviña et al. All rights reserved. Female Aging Alters Expression of Human Cumulus Cells Genes that Are Essential for Oocyte Quality Wed, 03 Sep 2014 08:41:20 +0000 Impact of female aging is an important issue in human reproduction. There was a need for an extensive analysis of age impact on transcriptome profile of cumulus cells (CCs) to link oocyte quality and developmental potential with patient’s age. CCs from patients of three age groups were analyzed individually using microarrays. RT-qPCR validation was performed on independent CC cohorts. We focused here on pathways affected by aging in CCs that may explain the decline of oocyte quality with age. In CCs collected from patients >37 years, angiogenic genes including ANGPTL4, LEPR, TGFBR3, and FGF2 were significantly overexpressed compared to patients of the two younger groups. In contrast genes implicated in TGF-β signaling pathway such as AMH, TGFB1, inhibin, and activin receptor were underexpressed. CCs from patients whose ages are between 31 and 36 years showed an overexpression of genes related to insulin signaling pathway such as IGFBP3, PIK3R1, and IGFBP5. A bioinformatic analysis was performed to identify the microRNAs that are potential regulators of the differentially expressed genes of the study. It revealed that the pathways impacted by age were potential targets of specific miRNAs previously identified in our CCs small RNAs sequencing. Tamadir Al-Edani, Said Assou, Alice Ferrières, Sophie Bringer Deutsch, Anna Gala, Charles-Henri Lecellier, Ounissa Aït-Ahmed, and Samir Hamamah Copyright © 2014 Tamadir Al-Edani et al. All rights reserved. The Characteristics of Ubiquitous and Unique Leptospira Strains from the Collection of Russian Centre for Leptospirosis Tue, 02 Sep 2014 12:40:51 +0000 Background and Aim. Leptospira, the causal agent of leptospirosis, has been isolated from the environment, patients, and wide spectrum of animals in Russia. However, the genetic diversity of Leptospira in natural and anthropurgic foci was not clearly defined. Methods. The recent MLST scheme was used for the analysis of seven pathogenic species. 454 pyrosequencing technology was the base of the whole genome sequencing (WGS). Results. The most wide spread and prevalent Leptospira species in Russia were L. interrogans, L. kirschneri, and L. borgpetersenii. Five STs, common for Russian strains: 37, 17, 199, 110, and 146, were identified as having a longtime and ubiquitous distribution in various geographic areas. Unexpected properties were revealed for the environmental Leptospira strain Bairam-Ali. WGS of this strain genome suggested that it combined the features of the pathogenic and nonpathogenic strains and may be a reservoir of the natural resistance genes. Results of the comparative analysis of rrs and rpoB genes and MLST loci for different Leptospira species strains and phenotypic and serological properties of the strain Bairam-Ali suggested that it represented separate Leptospira species. Conclusions. Thus, the natural and anthropurgic foci supported ubiquitous Leptospira species and the pool of genes important for bacterial adaptivity to various conditions. Olga L. Voronina, Marina S. Kunda, Ekaterina I. Aksenova, Natalia N. Ryzhova, Andrey N. Semenov, Evgeny M. Petrov, Lubov V. Didenko, Vladimir G. Lunin, Yuliya V. Ananyina, and Alexandr L. Gintsburg Copyright © 2014 Olga L. Voronina et al. All rights reserved. Curcumin, a Natural Antioxidant, Acts as a Noncompetitive Inhibitor of Human RNase L in Presence of Its Cofactor 2-5A In Vitro Tue, 02 Sep 2014 00:00:00 +0000 Ribonuclease L (RNase L) is an antiviral endoribonuclease of the innate immune system, which is induced and activated by viral infections, interferons, and double stranded RNA (dsRNA) in mammalian cells. Although, RNase L is generally protective against viral infections, abnormal RNase L expression and activity have been associated with a number of diseases. Here, we show that curcumin, a natural plant-derived anti-inflammatory active principle, inhibits RNase L activity; hence, it may be exploited for therapeutic interventions in case of pathological situations associated with excess activation of RNase L. Ankush Gupta and Pramod C. Rath Copyright © 2014 Ankush Gupta and Pramod C. Rath. All rights reserved. Multifunctional Role of ATM/Tel1 Kinase in Genome Stability: From the DNA Damage Response to Telomere Maintenance Thu, 28 Aug 2014 15:27:02 +0000 The mammalian protein kinase ataxia telangiectasia mutated (ATM) is a key regulator of the DNA double-strand-break response and belongs to the evolutionary conserved phosphatidylinositol-3-kinase-related protein kinases. ATM deficiency causes ataxia telangiectasia (AT), a genetic disorder that is characterized by premature aging, cerebellar neuropathy, immunodeficiency, and predisposition to cancer. AT cells show defects in the DNA damage-response pathway, cell-cycle control, and telomere maintenance and length regulation. Likewise, in Saccharomyces cerevisiae, haploid strains defective in the TEL1 gene, the ATM ortholog, show chromosomal aberrations and short telomeres. In this review, we outline the complex role of ATM/Tel1 in maintaining genomic stability through its control of numerous aspects of cellular survival. In particular, we describe how ATM/Tel1 participates in the signal transduction pathways elicited by DNA damage and in telomere homeostasis and its importance as a barrier to cancer development. Enea Gino Di Domenico, Elena Romano, Paola Del Porto, and Fiorentina Ascenzioni Copyright © 2014 Enea Gino Di Domenico et al. All rights reserved. Molecular Characterization and Screening for Sheath Blight Resistance Using Malaysian Isolates of Rhizoctonia solani Thu, 28 Aug 2014 09:45:27 +0000 Two field isolates of Rhizoctonia solani were isolated from infected paddy plants in Malaysia. These isolates were verified via ITS-rDNA analysis that yielded ~720 bp products of the ITS1-5.8S-ITS4 region, respectively. The sequenced products showed insertion and substitution incidences which may result in strain diversity and possible variation in disease severity. These strains showed some regional and host-specific relatedness via Maximum Likelihood and further phylogenetic analysis via Maximum Parsimony showed that these strains were closely related to R. solani AG1-1A (with 99-100% identity). Subsequent to strain verification and analysis, these isolates were used in the screening of twenty rice varieties for tolerance or resistance to sheath blight via mycelial plug method where both isolates (1801 and 1802) showed resistance or moderate resistance to Teqing, TETEP, and Jasmine 85. Isolate 1802 was more virulent based on the disease severity index values. This study also showed that the mycelial plug techniques were efficient in providing uniform inoculum and humidity for screening. In addition this study shows that the disease severity index is a better mode of scoring for resistance compared to lesion length. These findings will provide a solid basis for our future breeding and screening activities at the institution. Kalaivani Nadarajah, Nurfarahana Syuhada Omar, Marhamah Md. Rosli, and Ong Shin Tze Copyright © 2014 Kalaivani Nadarajah et al. All rights reserved. Retrotransposon-Based Molecular Markers for Analysis of Genetic Diversity within the Genus Linum Wed, 27 Aug 2014 11:27:15 +0000 SSAP method was used to study the genetic diversity of 22 Linum species from sections Linum, Adenolinum, Dasylinum, Stellerolinum, and 46 flax cultivars. All the studied flax varieties were distinguished using SSAP for retrotransposons FL9 and FL11. Thus, the validity of SSAP method was demonstrated for flax marking, identification of accessions in genebank collections, and control during propagation of flax varieties. Polymorphism of Fl1a, Fl1b, and Cassandra insertions were very low in flax varieties, but these retrotransposons were successfully used for the investigation of Linum species. Species clusterization based on SSAP markers was in concordance with their taxonomic division into sections Dasylinum, Stellerolinum, Adenolinum, and Linum. All species of sect. Adenolinum clustered apart from species of sect. Linum. The data confirmed the accuracy of the separation in these sections. Members of section Linum are not as closely related as members of other sections, so taxonomic revision of this section is desirable. L. usitatissimum accessions genetically distant from modern flax cultivars were revealed in our work. These accessions are of utmost interest for flax breeding and introduction of new useful traits into flax cultivars. The chromosome localization of Cassandra retrotransposon in Linum species was determined. Nataliya V. Melnikova, Anna V. Kudryavtseva, Alexander V. Zelenin, Valentina A. Lakunina, Olga Yu. Yurkevich, Anna S. Speranskaya, Alexey A. Dmitriev, Anastasia A. Krinitsina, Maxim S. Belenikin, Leonid A. Uroshlev, Anastasiya V. Snezhkina, Asiya F. Sadritdinova, Nadezda V. Koroban, Alexandra V. Amosova, Tatiana E. Samatadze, Elena V. Guzenko, Valentina A. Lemesh, Anastasya M. Savilova, Olga A. Rachinskaia, Natalya V. Kishlyan, Tatiana A. Rozhmina, Nadezhda L. Bolsheva, and Olga V. Muravenko Copyright © 2014 Nataliya V. Melnikova et al. All rights reserved. Phytoliths in Taxonomy of Phylogenetic Domains of Plants Wed, 27 Aug 2014 10:32:18 +0000 We discuss, from the aspect of phylogeny, the interrelationships of the phytolith types in plants from the main taxonomical groups (algae, lichens, horsetails, gymnosperms, and floral plants) with homologues of known proteins of biomineralization. Phytolith morphotypes in various phylogenetic plant domains have different shapes. We found that, in ancient types of plants (algae, horsetails, and gymnosperms), there are fewer different phytolith morphotypes compared to more modern plants (floral plants). The phytolith morphotypes in primitive plants are generally larger than the morphotypes in more highly organized plants. We found that the irregular ruminate and irregular smooth morphotypes are the two most frequently encountered phytolith morphotypes in the tested plants (from algae to floral plants). These two morphotypes probably have a universal role. Silacidins, silicon transporters, silicateins, silaffins, and silicase homologues are often found in the major taxonomic groups of plants. Red algae had the smallest number of homologues of the biomineralization proteins (70–80), Monocotyledonous: 142, Coniferous: 166, Mosses: 227, and Dicotyledones: 336. Kirill S. Golokhvast, Ivan V. Seryodkin, Vladimir V. Chaika, Alexander M. Zakharenko, and Igor E. Pamirsky Copyright © 2014 Kirill S. Golokhvast et al. All rights reserved. Quantitative Analysis of Pork and Chicken Products by Droplet Digital PCR Wed, 27 Aug 2014 07:58:26 +0000 In this project, a highly precise quantitative method based on the digital polymerase chain reaction (dPCR) technique was developed to determine the weight of pork and chicken in meat products. Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of species-specific DNAs in meat products. However, it is limited in amplification efficiency and relies on standard curves based Ct values, detecting and quantifying low copy number target DNA, as in some complex mixture meat products. By using the dPCR method, we find the relationships between the raw meat weight and DNA weight and between the DNA weight and DNA copy number were both close to linear. This enabled us to establish formulae to calculate the raw meat weight based on the DNA copy number. The accuracy and applicability of this method were tested and verified using samples of pork and chicken powder mixed in known proportions. Quantitative analysis indicated that dPCR is highly precise in quantifying pork and chicken in meat products and therefore has the potential to be used in routine analysis by government regulators and quality control departments of commercial food and feed enterprises. Yicun Cai, Xiang Li, Rong Lv, Jielin Yang, Jian Li, Yuping He, and Liangwen Pan Copyright © 2014 Yicun Cai et al. All rights reserved. Binding Sites of miR-1273 Family on the mRNA of Target Genes Tue, 26 Aug 2014 13:27:17 +0000 This study examined binding sites of 2,578 miRNAs in the mRNAs of 12,175 human genes using the MirTarget program. It found that the miRNAs of miR-1273 family have between 33 and 1,074 mRNA target genes, with a free hybridization energy of 90% or more of its maximum value. The miR-1273 family consists of miR-1273a, miR-1273c, miR-1273d, miR-1273e, miR-1273f, miR-1273g-3p, miR-1273g-5p, miR-1273h-3p, and miR-1273h-5p. Unique miRNAs (miR-1273e, miR-1273f, and miR-1273g-3p) have more than 400 target genes. We established 99 mRNA nucleotide sequences that contain arranged binding sites for the miR-1273 family. High conservation of each miRNA binding site in the mRNA of the target genes was found. The arranged binding sites of the miR-1273 family are located in the 5′UTR, CDS, or 3′UTR of many mRNAs. Five repeating sites containing some of the miR-1273 family’s binding sites were found in the 3′UTR of several target genes. The oligonucleotide sequences of miR-1273 binding sites located in CDSs code for homologous amino acid sequences in the proteins of target genes. The biological role of unique miRNAs was also discussed. Anatoly Ivashchenko, Olga Berillo, Anna Pyrkova, and Raigul Niyazova Copyright © 2014 Anatoly Ivashchenko et al. All rights reserved. tasiRNA-ARF Pathway Moderates Floral Architecture in Arabidopsis Plants Subjected to Drought Stress Tue, 26 Aug 2014 13:24:16 +0000 In plants, miRNAs and siRNAs, such as transacting siRNAs (ta-siRNAs), affect their targets through distinct regulatory mechanisms. In this study, the expression profiles of small RNAs (smRNAs) in Arabidopsis plants subjected to drought, cold, and high-salinity stress were analyzed using 454 DNA sequencing technology. Expression of three groups of ta-siRNAs (TAS1, TAS2, and TAS3) and their precursors was downregulated in Arabidopsis plants subjected to drought and high-salinity stress. Analysis of ta-siRNA synthesis mutants and mutated ARF3-overexpressing plants that escape the tasiRNA-ARF target indicated that self-pollination was hampered by short stamens in plants under drought and high-salinity stress. Microarray analysis of flower buds of rdr6 and wild-type plants under drought stress and nonstressed conditions revealed that expression of floral development- and auxin response-related genes was affected by drought stress and by the RDR6 mutation. The overall results of the present study indicated that tasiRNA-ARF is involved in maintaining the normal morphogenesis of flowers in plants under stress conditions through fine-tuning expression changes of floral development-related and auxin response-related genes. Akihiro Matsui, Kayoko Mizunashi, Maho Tanaka, Eli Kaminuma, Anh Hai Nguyen, Maiko Nakajima, Jong-Myong Kim, Dong Van Nguyen, Tetsuro Toyoda, and Motoaki Seki Copyright © 2014 Akihiro Matsui et al. All rights reserved. Phylogenetic Information Content of Copepoda Ribosomal DNA Repeat Units: ITS1 and ITS2 Impact Mon, 18 Aug 2014 08:19:35 +0000 The utility of various regions of the ribosomal repeat unit for phylogenetic analysis was examined in 16 species representing four families, nine genera, and two orders of the subclass Copepoda (Crustacea). Fragments approximately 2000 bp in length containing the ribosomal DNA (rDNA) 18S and 28S gene fragments, the 5.8S gene, and the internal transcribed spacer regions I and II (ITS1 and ITS2) were amplified and analyzed. The DAMBE (Data Analysis in Molecular Biology and Evolution) software was used to analyze the saturation of nucleotide substitutions; this test revealed the suitability of both the 28S gene fragment and the ITS1/ITS2 rDNA regions for the reconstruction of phylogenetic trees. Distance (minimum evolution) and probabilistic (maximum likelihood, Bayesian) analyses of the data revealed that the 28S rDNA and the ITS1 and ITS2 regions are informative markers for inferring phylogenetic relationships among families of copepods and within the Cyclopidae family and associated genera. Split-graph analysis of concatenated ITS1/ITS2 rDNA regions of cyclopoid copepods suggested that the Mesocyclops, Thermocyclops, and Macrocyclops genera share complex evolutionary relationships. This study revealed that the ITS1 and ITS2 regions potentially represent different phylogenetic signals. Maxim V. Zagoskin, Valentina I. Lazareva, Andrey K. Grishanin, and Dmitry V. Mukha Copyright © 2014 Maxim V. Zagoskin et al. All rights reserved. Methylation-Associated Gene Silencing of RARB in Areca Carcinogens Induced Mouse Oral Squamous Cell Carcinoma Sun, 17 Aug 2014 11:01:55 +0000 Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptor β (RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis. Zi-Lun Lai, Yung-An Tsou, Shin-Ru Fan, Ming-Hsui Tsai, Hsiao-Ling Chen, Nai-Wen Chang, Ju-Chien Cheng, and Chuan-Mu Chen Copyright © 2014 Zi-Lun Lai et al. All rights reserved. The Properties of Binding Sites of miR-619-5p, miR-5095, miR-5096, and miR-5585-3p in the mRNAs of Human Genes Mon, 04 Aug 2014 11:08:23 +0000 The binding of 2,578 human miRNAs with the mRNAs of 12,175 human genes was studied. It was established that miR-619-5p, miR-5095, miR-5096, and miR-5585-3p bind with high affinity to the mRNAs of the 1215, 832, 725, and 655 genes, respectively. These unique miRNAs have binding sites in the coding sequences and untranslated regions of mRNAs. The mRNAs of many genes have multiple miR-619-5p, miR-5095, miR-5096, and miR-5585-3p binding sites. Groups of mRNAs in which the ordering of the miR-619-5p, miR-5095, miR-5096, and miR-5585-3p binding sites differ were established. The possible functional and evolutional properties of unique miRNAs are discussed. Anatoly Ivashchenko, Olga Berillo, Anna Pyrkova, Raigul Niyazova, and Shara Atambayeva Copyright © 2014 Anatoly Ivashchenko et al. All rights reserved. The Antileukemia Activity of Natural Product HQ17(3) Is Possibly Associated with Downregulation of miR-17-92 Cluster Tue, 22 Jul 2014 09:07:15 +0000 The compound 10′(Z),13′(E),15′(E)-heptadecatrienylhydroquinone [HQ17(3)] was purified from the sap of the lacquer tree Rhus succedanea. HQ17(3) has cytotoxic effect on cancer cells and can inhibit topoisomerase (topo) IIα activity. We treated various cancer cells with different doses of HQ17(3) and found that leukemia cells were most sensitive to HQ17(3). After analysis of microRNA (miRNA) profiling, we found that treatment with HQ17(3) caused downregulation of miR-17-92 cluster in some leukemia cells. These changes partially restored the normal levels from leukemia-specific miRNA expression signature. Messenger RNAs of tumor suppressor proteins, such as pRB, PTEN, and Dicer, are targets of miR-17-92 cluster. Their protein levels were increased after the treatment. c-Myc is a regulatory protein for miR-17-92 gene. Similar to topo IIα, we found that c-Myc decreased its activity after the HQ17(3) treatment, which may explain the downregulation of miR-17-92 cluster. Combined with 5-fluorouracil, NaAsO2, or ABT-737, HQ17(3) elicited additive inhibitory effects on leukemia cells. In conclusion, the high sensitivity of leukemia cells to HQ17(3) may be associated with the reduction of topo IIα and c-Myc activities, as well as with the downregulation of the miR-17-92 cluster expression. Ya-Chun Liao, Tzu-Heng Lin, Chih-Ying Chen, Shwu-Bin Lin, and Lo-Chun Au Copyright © 2014 Ya-Chun Liao et al. All rights reserved. Effects of Crude Extracts from Medicinal Herbs Rhazya stricta and Zingiber officinale on Growth and Proliferation of Human Brain Cancer Cell Line In Vitro Tue, 22 Jul 2014 07:37:49 +0000 Hitherto, limited clinical impact has been achieved in the treatment of glioblastoma (GBMs). Although phytochemicals found in medicinal herbs can provide mankind with new therapeutic remedies, single agent intervention has failed to bring the expected outcome in clinical trials. Therefore, combinations of several agents at once are gaining increasing attractiveness. In the present study, we investigated the effects of crude alkaloid (CAERS) and flavonoid (CFEZO) extracts prepared from medicinal herbs, Rhazya stricta and Zingiber officinale, respectively, on the growth of human GBM cell line, U251. R. stricta and Z. officinale are traditionally used in folkloric medicine and have antioxidant, anticarcinogenic, and free radical scavenging properties. Combination of CAERS and CFEZO treatments synergistically suppressed proliferation and colony formation and effectively induced morphological and biochemical features of apoptosis in U251 cells. Apoptosis induction was mediated by release of mitochondrial cytochrome c, increased Bax : Bcl-2 ratio, enhanced activities of caspase-3 and -9, and PARP-1 cleavage. CAERS and CFEZO treatments decreased expression levels of nuclear NF-κBp65, survivin, XIAP, and cyclin D1 and increased expression level of p53, p21, and Noxa. These results suggest that combination of CAERS and CFEZO provides a useful foundation for studying and developing novel chemotherapeutic agents for the treatment of GBM. Ayman I. Elkady, Rania Abd El Hamid Hussein, and Osama A. Abu-Zinadah Copyright © 2014 Ayman I. Elkady et al. All rights reserved. Protein Kinase D3 Is Essential for Prostratin-Activated Transcription of Integrated HIV-1 Provirus Promoter via NF-κB Signaling Pathway Mon, 21 Jul 2014 00:00:00 +0000 Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation. The molecular mechanism of this activation, however, is far from clear. Here, we show that the protein kinase D3 (PKD3) is essential for prostratin-induced transcription activation of latent HIV-1 provirus. First, silencing PKD3, but not the other members of PKD family, blocked prostratin-induced transcription of HIV-1. Second, overexpressing the constitutively active form of PKD3, but not the wild-type or kinase-dead form of PKD3, augmented the expression of HIV-1. Consistent with this observation, we found that prostratin could trigger PKD3 activation by inducing the phosphorylation of its activation loop. In addition, we identified PKC of the novel PKC subfamily as the upstream kinase for this phosphorylation. Finally, the activation effect of PKD3 on HIV-1 transcription was shown to depend on the presence of κB element and the prostratin-induced activation of NF-κB, as indicated by the fact that silencing PKD3 blocked prostratin-induced NF-κB activation and NF-κB-dependent HIV-1 transcription. Therefore, for the first time, PKD3 is implicated in the transcription activation of latent HIV-1 provirus, and our results revealed a molecular mechanism of prostratin-induced HIV-1 transcription via PKC/PKD3/NF-κB signaling pathway. Huiping Wang, Xinxing Zhu, Ying Zhu, Jiangfang Liu, Xiangming Hu, Yu Wang, Sijia Peng, Yanheng Chen, Ruichuan Chen, Feng Ding, and Runzhong Liu Copyright © 2014 Huiping Wang et al. All rights reserved. Recent Advances in Polyamine Metabolism and Abiotic Stress Tolerance Sun, 20 Jul 2014 00:00:00 +0000 Global warming is an alarming problem in agriculture and its effect on yield loss has been estimated to be five per cent for every degree centigrade rise in temperature. Plants exhibit multiple mechanisms like optimizing signaling pathway, involvement of secondary messengers, production of biomolecules specifically in response to stress, modulation of various metabolic networks in accordance with stress, and so forth, in order to overcome abiotic stress factors. Many structural genes and networks of pathway were identified and reported in plant systems for abiotic stress tolerance. One such crucial metabolic pathway that is involved in normal physiological function and also gets modulated during stress to impart tolerance is polyamine metabolic pathway. Besides the role of structural genes, it is also important to know the mechanism by which these structural genes are regulated during stress. Present review highlights polyamine biosynthesis, catabolism, and its role in abiotic stress tolerance with special reference to plant systems. Additionally, a system based approach is discussed as a potential strategy to dissect the existing variation in crop species in unraveling the interacting regulatory components/genetic determinants related to PAs mediated abiotic stress tolerance. Parimalan Rangan, Rajkumar Subramani, Rajesh Kumar, Amit Kumar Singh, and Rakesh Singh Copyright © 2014 Parimalan Rangan et al. All rights reserved. Molecular Cloning and Characterization of Novel Phytocystatin Gene from Turmeric, Curcuma longa Mon, 14 Jul 2014 08:11:51 +0000 Phytocystatin, a type of protease inhibitor (PI), plays major roles in plant defense mechanisms and has been reported to show antipathogenic properties and plant stress tolerance. Recombinant plant PIs are gaining popularity as potential candidates in engineering of crop protection and in synthesizing medicine. It is therefore crucial to identify PI from novel sources like Curcuma longa as it is more effective in combating against pathogens due to its novelty. In this study, a novel cDNA fragment encoding phytocystatin was isolated using degenerate PCR primers, designed from consensus regions of phytocystatin from other plant species. A full-length cDNA of the phytocystatin gene, designated CypCl, was acquired using 5′/3′ rapid amplification of cDNA ends method and it has been deposited in NCBI database (accession number KF545954.1). It has a 687 bp long open reading frame (ORF) which encodes 228 amino acids. BLAST result indicated that CypCl is similar to cystatin protease inhibitor from Cucumis sativus with 74% max identity. Sequence analysis showed that CypCl contains most of the motifs found in a cystatin, including a G residue, LARFAV-, QxVxG sequence, PW dipeptide, and SNSL sequence at C-terminal extension. Phylogenetic studies also showed that CypCl is related to phytocystatin from Elaeis guineensis. Seow-Neng Chan, Norliza Abu Bakar, Maziah Mahmood, Chai-Ling Ho, and Noor Azmi Shaharuddin Copyright © 2014 Seow-Neng Chan et al. All rights reserved. Analysis of Differential miRNA Expression in Primary Tumor and Stroma of Colorectal Cancer Patients Thu, 10 Jul 2014 00:00:00 +0000 Microarray technology was used to profile miRNA expression in primary tumor and stromal tissue from paraffin embedded material of 51 patients with colorectal cancer. 26 miRNAs resulted differentially expressed with at least 2-fold change in tumor tissue with respect to stroma (16 more expressed in the tumor and 10 more expressed in the stroma). 10/26 were confirmed as differentially expressed at qRTPCR: miR-200c-3p, miR-141-3p, miR-200b-3p, miR-200a-3p, miR-1246, miR-92a-3p, miR-194-5p, miR-192-5p, miR-3651-5p, and miR-574-3p. No significant association was found between miRNA expressions and stage at diagnosis, site of primary tumor, first site of metastasis, progression-free, or overall survival. Giuseppina Della Vittoria Scarpati, Enrica Calura, Mariacristina Di Marino, Chiara Romualdi, Luca Beltrame, Umberto Malapelle, Giancarlo Troncone, Alfonso De Stefano, Stefano Pepe, Sabino De Placido, Maurizio D'Incalci, Sergio Marchini, and Chiara Carlomagno Copyright © 2014 Giuseppina Della Vittoria Scarpati et al. All rights reserved. Cloning, Expression, Purification, and Characterization of Glutaredoxin from Antarctic Sea-Ice Bacterium Pseudoalteromonas sp. AN178 Mon, 07 Jul 2014 11:06:23 +0000 Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide. In this study, a full-length Grx gene (PsGrx) with 270 nucleotides was isolated from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178. It encoded deduced 89 amino acid residues with the molecular weight 9.8 kDa. Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC. Recombinant PsGrx (rPsGrx) stably expressed in E. coli BL21 was purified to apparent homogeneity by Ni-affinity chromatography. rPsGrx exhibited optimal activity at 30°C and pH 8.0 and showed 25.5% of the activity at 0°C. It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl. These results indicated that rPsGrx was a typical cold active protein with low thermostability. Quanfu Wang, Yanhua Hou, Yonglei Shi, Xiao Han, Qian Chen, Zhiguo Hu, Yuanping Liu, and YuJin Li Copyright © 2014 Quanfu Wang et al. All rights reserved. Comparison of Two Methods of RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissue Specimens Thu, 03 Jul 2014 11:50:46 +0000 The present study aimed to compare two different methods of extracting RNA from formalin-fixed paraffin-embedded (FFPE) specimens of diffuse large B-cell lymphoma (DLBCL). We further aimed to identify possible influences of variables—such as tissue size, duration of paraffin block storage, fixative type, primers used for cDNA synthesis, and endogenous genes tested—on the success of amplification from the samples. Both tested protocols used the same commercial kit for RNA extraction (the RecoverAll Total Nucleic Acid Isolation Optimized for FFPE Samples from Ambion). However, the second protocol included an additional step of washing with saline buffer just after sample rehydration. Following each protocol, we compared the RNA amount and purity and the amplification success as evaluated by standard PCR and real-time PCR. The results revealed that the extra washing step added to the RNA extraction process resulted in significantly improved RNA quantity and quality and improved success of amplification from paraffin-embedded specimens. Gisele Rodrigues Gouveia, Suzete Cleusa Ferreira, Jerenice Esdras Ferreira, Sheila Aparecida Coelho Siqueira, and Juliana Pereira Copyright © 2014 Gisele Rodrigues Gouveia et al. All rights reserved. Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine Genome Wed, 02 Jul 2014 07:43:10 +0000 Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming, especially in some important organ development. Osteoponin (OPN) is an extracellular-matrix protein involved in heart and bone development and diseases. In this study, we investigated the correlation between OPN mRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay. Aberrant methylation of porcine OPN was frequently found in different tissues of somatic nuclear transferred cloning pigs, and bisulfite sequence data suggested that the OPN promoter region −2615 to −2239 nucleotides (nt) may be a crucial regulation DNA element. In pig ear fibroblast cell culture study, the demethylation of OPN promoter was found in dose-dependent response of 5-aza-dc treatment and followed the OPN mRNA reexpression. In cloned pig study, discrepant expression pattern was identified in several cloned pig tissues, especially in brain, heart, and ear. Promoter assay data revealed that four methylated CpG sites presenting in the −2615 to −2239 nt region cause significant downregulation of OPN promoter activity. These data suggested that methylation in the OPN promoter plays a crucial role in the regulation of OPN expression that we found in cloned pigs genome. Chih-Jie Shen, Yung-An Tsou, Hsiao-Ling Chen, Hung-Jin Huang, Shinn-Chih Wu, Winston T. K. Cheng, Calvin Yu-Chian Chen, and Chuan-Mu Chen Copyright © 2014 Chih-Jie Shen et al. All rights reserved. Potential Smoothened Inhibitor from Traditional Chinese Medicine against the Disease of Diabetes, Obesity, and Cancer Tue, 01 Jul 2014 07:03:00 +0000 Nowadays, obesity becomes a serious global problem, which can induce a series of diseases such as type 2 diabetes mellitus, cancer, cardiovascular disease, metabolic syndrome, and stoke. For the mechanisms of diseases, the hedgehog signaling pathway plays an important role in body patterning during embryogenesis. For this reason, smoothened homologue (Smo) protein had been indicated as the drug target. In addition, the small-molecule Smo inhibitor had also been used in oncology clinical trials. To improve drug development of TCM compounds, we aim to investigate the potent lead compounds as Smo inhibitor from the TCM compounds in TCM Database@Taiwan. The top three TCM compounds, precatorine, labiatic acid, and 2,2′-[benzene-1,4-diylbis(methanediyloxybenzene-4,1-diyl)]bis(oxoacetic acid), have displayed higher potent binding affinities than the positive control, LY2940680, in the docking simulation. After MD simulations, which can optimize the result of docking simulation and validate the stability of H-bonds between each ligand and Smo protein under dynamic conditions, top three TCM compounds maintain most of interactions with Smo protein, which keep the ligand binding stable in the binding domain. Hence, we propose precatorine, labiatic acid, and 2,2′-[benzene-1,4-diylbis(methanediyloxybenzene-4,1-diyl)]bis(oxoacetic acid) as potential lead compounds for further study in drug development process with the Smo protein. Kuan-Chung Chen, Mao-Feng Sun, Hsin-Yi Chen, Cheng-Chun Lee, and Calvin Yu-Chian Chen Copyright © 2014 Kuan-Chung Chen et al. All rights reserved. Investigation of Estrogen Receptor (ESR1) for Breast Cancer from Traditional Chinese Medicine Thu, 26 Jun 2014 10:22:36 +0000 Recently, an important topic of breast cancer had been published in 2013. In this report, estrogen receptor (ESR1) had defined the relation of hormone-cause breast cancer. The screening of traditional Chinese medicine (TCM) database has found the molecular compounds by simulating molecular docking and molecular dynamics to regulate ESR1. S-Allylmercaptocysteine and 5-hydroxy-L-tryptophan are selected according to the highest docking score than that of other TCM compounds and Raloxifene (control). The simulation from molecular dynamics is helpful in analyzing and detecting the protein-ligand interactions. After a comparing the control and the Apo form, then based on the docking poses, hydrophobic interactions, hydrogen bond and structure variations, this research postulates that S-allylmercaptocysteine may be more appropriate than other compounds for protein-ligand interaction. Tzu-Chieh Hung, Wen-Yuan Lee, Kuen-Bao Chen, Yueh-Chiu Chan, and Calvin Yu-Chian Chen Copyright © 2014 Tzu-Chieh Hung et al. All rights reserved. Muscle-Type Specific Autophosphorylation of CaMKII Isoforms after Paced Contractions Thu, 26 Jun 2014 08:42:27 +0000 We explored to what extent isoforms of the regulator of excitation-contraction and excitation-transcription coupling, calcium/calmodulin protein kinase II (CaMKII) contribute to the specificity of myocellular calcium sensing between muscle types and whether concentration transients in its autophosphorylation can be simulated. CaMKII autophosphorylation at Thr287 was assessed in three muscle compartments of the rat after slow or fast motor unit-type stimulation and was compared against a computational model (CaMuZclE) coupling myocellular calcium dynamics with CaMKII Thr287 phosphorylation. Qualitative differences existed between fast- (gastrocnemius medialis) and slow-type muscle (soleus) for the expression pattern of CaMKII isoforms. Phospho-Thr287 content of δA CaMKII, associated with nuclear functions, demonstrated a transient and compartment-specific increase after excitation, which contrasted to the delayed autophosphorylation of the sarcoplasmic reticulum-associated βM CaMKII. In soleus muscle, excitation-induced δA CaMKII autophosphorylation demonstrated frequency dependence (P = 0.02). In the glycolytic compartment of gastrocnemius medialis, CaMKII autophosphorylation after excitation was blunted. In silico assessment emphasized the importance of mitochondrial calcium buffer capacity for excitation-induced CaMKII autophosphorylation but did not predict its isoform specificity. The findings expose that CaMKII autophosphorylation with paced contractions is regulated in an isoform and muscle type-specific fashion and highlight properties emerging for phenotype-specific regulation of CaMKII. Wouter Eilers, Wouter Gevers, Daniëlle van Overbeek, Arnold de Haan, Richard T. Jaspers, Peter A. Hilbers, Natal van Riel, and Martin Flück Copyright © 2014 Wouter Eilers et al. All rights reserved. CELLCOUNTER: Novel Open-Source Software for Counting Cell Migration and Invasion In Vitro Thu, 26 Jun 2014 07:33:01 +0000 Transwell Boyden chamber based migration/invasion assay is a simple and extensively used approach for the characterization of cell motility in vitro. Cell motility is quantified by counting the number of cells that pass through the filter membrane. The counting is usually performed manually, which is laborious and error prone. We have therefore developed CELLCOUNTER, an application that is capable of recognizing and counting the total number of cells through an intuitive graphical user interface. The counting can be performed in batch, and the counting results can be visualized and further curated manually. CELLCOUNTER will be helpful in streamlining the experimental process and improving the reliability of the data acquisition. Xiaoni Li, Hongshun Yang, Hailiang Huang, and Tao Zhu Copyright © 2014 Xiaoni Li et al. All rights reserved. In Silico Investigation of Potential TRAF6 Inhibitor from Traditional Chinese Medicine against Cancers Wed, 25 Jun 2014 13:23:11 +0000 It has been indicated that tumor necrosis factor receptor-associated factor-6 (TRAF6) will upregulate the expression of hypoxia-inducible factor-1α (HIF-1α) and promote tumor angiogenesis. TRAF6 proteins can be treated as drug target proteins for a differentiation therapy against cancers. As structural disordered disposition in the protein may induce the side-effect and reduce the occupancy for ligand to bind with target protein, PONDR-Fit protocol was performed to predict the disordered disposition in TRAF6 protein before virtual screening. TCM compounds from the TCM Database@Taiwan were employed for virtual screening to identify potent compounds as lead compounds of TRAF6 inhibitor. After virtual screening, the MD simulation was performed to validate the stability of interactions between TRAF6 proteins and each ligand. The top TCM compounds, tryptophan, diiodotyrosine, and saussureamine C, extracted from Saussurea lappa Clarke, Bos taurus domesticus Gmelin, and Lycium chinense Mill., have higher binding affinities with target protein in docking simulation. However, the docking pose of TRAF6 protein with tryptophan is not stable under dynamic condition. For the other two TCM candidates, diiodotyrosine and saussureamine C maintain the similar docking poses under dynamic conditions. Hence, we propose the TCM compounds, diiodotyrosine and saussureamine C, as potential candidates as lead compounds for further study in drug development process with the TRAF6 protein against cancer. Kuan-Chung Chen, Wen-Yuan Lee, Hsin-Yi Chen, and Calvin Yu-Chian Chen Copyright © 2014 Kuan-Chung Chen et al. All rights reserved. In Silico Investigation of Potential Pyruvate Kinase M2 Regulators from Traditional Chinese Medicine against Cancers Wed, 25 Jun 2014 09:37:22 +0000 A recent research in cancer research demonstrates that tumor-specific pyruvate kinase M2 (PKM2) plays an important role in chromosome segregation and mitosis progression of tumor cells. To improve the drug development of TCM compounds, we aim to identify potent TCM compounds as lead compounds of PKM2 regulators. PONDR-Fit protocol was utilized to predict the disordered disposition in the binding domain of PKM2 protein before virtual screening as the disordered structure in the protein may cause the side effect and downregulation of the possibility of ligand to bind with target protein. MD simulation was performed to validate the stability of interactions between PKM2 proteins and each ligand after virtual screening. The top TCM compounds, saussureamine C and precatorine, extracted from Lycium chinense Mill. and Abrus precatorius L., respectively, have higher binding affinities with target protein in docking simulation than control. They have stable H-bonds with residues A:Lys311 and some other residues in both chains of PKM2 protein. Hence, we propose the TCM compounds, saussureamine C and precatorine, as potential candidates as lead compounds for further study in drug development process with the PKM2 protein against cancer. Kuan-Chung Chen, Kuen-Bao Chen, Hsin-Yi Chen, and Calvin Yu-Chian Chen Copyright © 2014 Kuan-Chung Chen et al. All rights reserved. In Silico Investigation of Traditional Chinese Medicine Compounds to Inhibit Human Histone Deacetylase 2 for Patients with Alzheimer’s Disease Mon, 23 Jun 2014 12:14:33 +0000 Human histone deacetylase 2 (HDAC2) has been identified as being associated with Alzheimer’s disease (AD), a neuropathic degenerative disease. In this study, we screen the world’s largest Traditional Chinese Medicine (TCM) database for natural compounds that may be useful as lead compounds in the search for inhibitors of HDAC2 function. The technique of molecular docking was employed to select the ten top TCM candidates. We used three prediction models, multiple linear regression (MLR), support vector machine (SVM), and the Bayes network toolbox (BNT), to predict the bioactivity of the TCM candidates. Molecular dynamics simulation provides the protein-ligand interactions of compounds. The bioactivity predictions of pIC50 values suggest that the TCM candidatesm, (−)-Bontl ferulate, monomethylcurcumin, and ningposides C, have a greater effect on HDAC2 inhibition. The structure variation caused by the hydrogen bonds and hydrophobic interactions between protein-ligand interactions indicates that these compounds have an inhibitory effect on the protein. Tzu-Chieh Hung, Wen-Yuan Lee, Kuen-Bao Chen, Yueh-Chiu Chan, Cheng-Chun Lee, and Calvin Yu-Chian Chen Copyright © 2014 Tzu-Chieh Hung et al. All rights reserved. In Silico Investigation of Potential mTOR Inhibitors from Traditional Chinese Medicine for Treatment of Leigh Syndrome Mon, 23 Jun 2014 11:28:49 +0000 A recent research demonstrates that the inhibition of mammalian target of rapamycin (mTOR) improves survival and health for patients with Leigh syndrome. mTOR proteins can be treated as drug target proteins against Leigh syndrome and other mitochondrial disorders. In this study, we aim to identify potent TCM compounds from the TCM Database@Taiwan as lead compounds of mTOR inhibitors. PONDR-Fit protocol was employed to predict the disordered disposition in mTOR protein before virtual screening. After virtual screening, the MD simulation was employed to validate the stability of interactions between each ligand and mTOR protein in the docking poses from docking simulation. The top TCM compounds, picrasidine M and acerosin, have higher binding affinities with target protein in docking simulation than control. There have H-bonds with residues Val2240 and π interactions with common residue Trp2239. After MD simulation, the top TCM compounds maintain similar docking poses under dynamic conditions. The top two TCM compounds, picrasidine M and acerosin, were extracted from Picrasma quassioides (D. Don) Benn. and Vitex negundo L. Hence, we propose the TCM compounds, picrasidine M and acerosin, as potential candidates as lead compounds for further study in drug development process with the mTOR protein against Leigh syndrome and other mitochondrial disorders. Kuan-Chung Chen, Wen-Yuan Lee, Hsin-Yi Chen, and Calvin Yu-Chian Chen Copyright © 2014 Kuan-Chung Chen et al. All rights reserved. Extended Genetic Diversity of Bovine Viral Diarrhea Virus and Frequency of Genotypes and Subtypes in Cattle in Italy between 1995 and 2013 Sun, 22 Jun 2014 13:26:24 +0000 Genetic typing of bovine viral diarrhea virus (BVDV) has distinguished BVDV-1 and BVDV-2 species and an emerging putative third species (HoBi-like virus), recently detected in southern Italy, signaling the occurrence of natural infection in Europe. Recognizing the need to update the data on BVDV genetic variability in Italy for mounting local and European alerts, a wide collection of UTR sequences (n = 371) was selected to identify the frequency of genotypes and subtypes at the herd level. BVDV-1 had the highest frequency, followed by sporadic BVDV-2. No novel HoBi-like viruses were identified. Four distribution patterns of BVDV-1 subtypes were observed: highly prevalent subtypes with a wide temporal-spatial distribution (1b and 1e), low prevalent subtypes with a widespread geographic distribution (1a, 1d, 1g, 1h, and 1k) or a restricted geographic distribution (1f), and sporadic subtypes detected only in single herds (1c, 1j, and 1l). BVDV-1c, k, and l are reported for the first time in Italy. A unique genetic variant was detected in the majority of herds, but cocirculation of genetic variants was also observed. Northern Italy ranked first for BVDV introduction, prevalence, and dispersion. Nevertheless, the presence of sporadic variants in other restricted areas suggests the risk of different routes of BVDV introduction. Camilla Luzzago, Stefania Lauzi, Erika Ebranati, Monica Giammarioli, Ana Moreno, Vincenza Cannella, Loretta Masoero, Elena Canelli, Annalisa Guercio, Claudio Caruso, Massimo Ciccozzi, Gian Mario De Mia, Pier Luigi Acutis, Gianguglielmo Zehender, and Simone Peletto Copyright © 2014 Camilla Luzzago et al. All rights reserved. Treatment of Cardiovascular Disease by Traditional Chinese Medicine against Pregnane X Receptor Sun, 22 Jun 2014 06:00:21 +0000 Recently, cardiovascular disease, also known as loop circulatory system diseases or disorders, is one of the serious diseases including heart disease, stroke, atherosclerosis, myocardial infarction, hypertension, hypotension, and thrombosis. Human pregnane X receptor, PXR, plays a crucial role in exogenous and endobiotic metabolism for rabbit, rat, mouse, and human. The PXR activation can protect the blood vessels from damage of hazardous substances. In this study we aim to investigate the potent lead compounds as PXR receptor agonist against cardiovascular disease. To improve drug development of TCM compounds, we aim to investigate the potent lead compounds as PXR agonists from the TCM compounds in TCM Database@Taiwan. The top three TCM compounds, bis(4-hydroxybenzyl) ether mono-β-D-glucopyranoside (BEMG), ixerisoside, and tangshenoside II, have displayed higher potent binding affinities than the positive control, PNU-142721, in the docking simulation. After MD simulations, which can optimize the result of docking simulation and validate the stability of H-bonds between each ligand and PXR protein under dynamic conditions, top TCM compounds, BEMG and tangshenoside II, maintain most of interactions with PXR protein, which keep the ligand binding stable in the binding domain. Hence, we propose BEMG and tangshenoside II as potential lead compounds for further study in drug development process with the PXR protein. Kuen-Bao Chen, Hsin-Yi Chen, Kuan-Chung Chen, and Calvin Yu-Chian Chen Copyright © 2014 Kuen-Bao Chen et al. All rights reserved. Drug Design of Cyclin-Dependent Kinase 2 Inhibitor for Melanoma from Traditional Chinese Medicine Thu, 19 Jun 2014 15:43:40 +0000 One has found an important cell cycle controller. This guard can decide the cell cycle toward proliferation or quiescence. Cyclin-dependent kinase 2 (CDK2) is a unique target among the CDK family in melanoma therapy. We attempted to find out TCM compounds from TCM Database@Taiwan that have the ability to inhibit the activity of CDK2 by systems biology. We selected Tetrahydropalmatine, Reserpiline, and (+)-Corydaline as the candidates by docking and screening results for further survey. We utilized support vector machine (SVM), multiple linear regression (MLR) models and Bayesian network for validation of predicted activity. By overall analysis of docking results, predicted activity, and molecular dynamics (MD) simulation, we could conclude that Tetrahydropalmatine, Reserpiline, and (+)-Corydaline had better binding affinity than the control. All of them had the ability to inhibit the activity of CDK2 and might have the opportunity to be applied in melanoma therapy. Hsin-Chieh Tang and Calvin Yu-Chian Chen Copyright © 2014 Hsin-Chieh Tang and Calvin Yu-Chian Chen. All rights reserved. An Investigation of Small GTPases in relation to Liver Tumorigenesis Using Traditional Chinese Medicine Thu, 19 Jun 2014 15:42:00 +0000 Recently, an important topic of liver tumorigenesis had been published in 2013. In this report, Ras and Rho had defined the relation of liver tumorigenesis. The traditional Chinese medicine (TCM) database has been screened for molecular compounds by simulating molecular docking and molecular dynamics to regulate Ras and liver tumorigenesis. Saussureamine C, S-allylmercaptocysteine, and Tryptophan are selected based on the highest docking score than other TCM compounds. The molecular dynamics are helpful in the analysis and detection of protein-ligand interactions. Based on the docking poses, hydrophobic interactions, and hydrogen bond variations, this research surmises are the main regions of important amino acids in Ras. In addition to the detection of TCM compound efficacy, we suggest Saussureamine C is better than the others for protein-ligand interaction. Tzu-Chieh Hung, Wen-Yuan Lee, Kuen-Bao Chen, Yueh-Chiu Chan, and Calvin Yu-Chian Chen Copyright © 2014 Tzu-Chieh Hung et al. All rights reserved. The Inhibition of Folylpolyglutamate Synthetase (folC) in the Prevention of Drug Resistance in Mycobacterium tuberculosis by Traditional Chinese Medicine Thu, 19 Jun 2014 07:35:48 +0000 Tuberculosis (TB) is an infectious disease caused by many strains of mycobacteria, but commonly Mycobacterium tuberculosis. As a possible method of reducing the drug resistance of M. tuberculosis, this research investigates the inhibition of Folylpolyglutamate synthetase, a protein transcript from the resistance association gene folC. After molecular docking to screen the traditional Chinese medicine (TCM) database, the candidate TCM compounds, with Folylpolyglutamate synthetase, were selected by molecular dynamics. The 10,000 ps simulation in association with RMSD analysis and total energy and structural variation defined the protein-ligand interaction. The selected TCM compounds Saussureamine C, methyl 3-O-feruloylquinate, and Labiatic acid have been found to inhibit the activity of bacteria and viruses and to regulate immunity. We also suggest the possible pathway in protein for each ligand. Compared with the control, similar interactions and structural variations indicate that these compounds might have an effect on Folylpolyglutamate synthetase. Finally, we suggest Saussureamine C is the best candidate compound as the complex has a high score, maintains its structural composition, and has a larger variation value than the control, thus inhibiting the drug resistance ability of Mycobacterium tuberculosis. Tzu-Chieh Hung, Kuen-Bao Chen, Wen-Yuan Lee, and Calvin Yu-Chian Chen Copyright © 2014 Tzu-Chieh Hung et al. All rights reserved. Possible Inhibitor from Traditional Chinese Medicine for the β Form of Calcium-Dependent Protein Kinase Type II in the Treatment of Major Depressive Disorder Wed, 18 Jun 2014 09:30:30 +0000 Recently, an important topic of major depressive disorder (MDD) had been published in 2013. MDD is one of the most prevalent and disabling mental disorders. Consequently, much research is being undertaken into the causes and treatment. It has been found that inhibition of the β form of calcium/calmodulin-dependent protein kinase type II (β-CaMKII) can ameliorate the disorder. Upon screening the traditional Chinese medicine (TCM) database by molecular docking, sengesterone, labiatic acid, and methyl 3-O-feruloylquinate were selected for molecular dynamics. After 20 ns simulation, the RMSD, total energy, and structure variation could define the protein-ligand interaction. Furthermore, sengesterone, the principle candidate compound, has been found to have an effect on the regulation of emotions and memory development. In structure variation, we find the sample functional group of important amino acids make the protein stable and have limited variation. Due to similarity of structure variations, we suggest that these compounds may have an effect on β-CaMKII and that sengesterone may have a similar efficacy as the control. However labiatic acid may be a stronger inhibitor of β-CaMKII based on the larger RMSD and variation. Tzu-Chieh Hung, Wen-Yuan Lee, Kuen-Bao Chen, Hung-Jin Huang, Yueh-Chiu Chan, Cheng-Chun Lee, and Calvin Yu-Chian Chen Copyright © 2014 Tzu-Chieh Hung et al. All rights reserved. Insight into HIV of IFN-Induced Myxovirus Resistance 2 (MX2) Expressed by Traditional Chinese Medicine Wed, 18 Jun 2014 08:21:54 +0000 Recently, an important topic of the acquired immunodeficiency syndrome (AIDS) had been published in 2013. In this report, the expression of the IFN-induced myxovirus resistance 2 (MX2) had been defined the function to kill the human immunodeficiency virus (HIV). The screening from the Traditional Chinese Medicine (TCM) database by simulating molecular docking and molecular dynamics could select candidate compounds, which may express MX2 against HIV. Saussureamine C, Crotalaburnine, and Precatorine are selected based on the highest docking score and other TCM compounds. The data from molecular dynamics are helpful in the analysis and detection of protein-ligand interactions. According to the docking poses, hydrophobic interactions, and hydrogen bond with structure variations, this research could assess the interaction between protein and ligand interaction. In addition to the detection of TCM compound efficacy, we suggest that Saussureamine C is better than the others in protein-ligand interaction and the structural variation to express MX2. Tzu-Chieh Hung, Wen-Yuan Lee, Kuen-Bao Chen, Yueh-Chiu Chan, and Calvin Yu-Chian Chen Copyright © 2014 Tzu-Chieh Hung et al. All rights reserved. Sequence-Based Appraisal of the Genes Encoding Neck and Carbohydrate Recognition Domain of Conglutinin in Blackbuck (Antilope cervicapra) and Goat (Capra hircus) Mon, 16 Jun 2014 09:24:53 +0000 Conglutinin, a collagenous C-type lectin, acts as soluble pattern recognition receptor (PRR) in recognition of pathogens. In the present study, genes encoding neck and carbohydrate recognition domain (NCRD) of conglutinin in goat and blackbuck were amplified, cloned, and sequenced. The obtained 488 bp ORFs encoding NCRD were submitted to NCBI with accession numbers KC505182 and KC505183. Both nucleotide and predicted amino acid sequences were analysed with sequences of other ruminants retrieved from NCBI GenBank using DNAstar and Megalign5.2 software. Sequence analysis revealed maximum similarity of blackbuck sequence with wild ruminants like nilgai and buffalo, whereas goat sequence displayed maximum similarity with sheep sequence at both nucleotide and amino acid level. Phylogenetic analysis further indicated clear divergence of wild ruminants from the domestic ruminants in separate clusters. The predicted secondary structures of NCRD protein in goat and blackbuck using SWISSMODEL ProtParam online software were found to possess 6 beta-sheets and 3 alpha-helices which are identical to the result obtained in case of sheep, cattle, buffalo, and nilgai. However, quaternary structure in goat, sheep, and cattle was found to differ from that of buffalo, nilgai, and blackbuck, suggesting a probable variation in the efficiency of antimicrobial activity among wild and domestic ruminants. Sasmita Barik, Chandra Mohan Sidappa, Mohini Saini, Ramesh Doreswamy, Asit Das, Anil K. Sharma, and Praveen K. Gupta Copyright © 2014 Sasmita Barik et al. All rights reserved. Phosphate/Zinc Interaction Analysis in Two Lettuce Varieties Reveals Contrasting Effects on Biomass, Photosynthesis, and Dynamics of Pi Transport Sun, 15 Jun 2014 12:09:05 +0000 Inorganic phosphate (Pi) and Zinc (Zn) are essential nutrients for normal plant growth. Interaction between these elements has been observed in many crop plants. Despite its agronomic importance, the biological significance and genetic basis of this interaction remain largely unknown. Here we examined the Pi/Zn interaction in two lettuce (Lactuca sativa) varieties, namely, “Paris Island Cos” and “Kordaat.” The effects of variation in Pi and Zn supply were assessed on biomass and photosynthesis for each variety. Paris Island Cos displayed better growth and photosynthesis compared to Kordaat under all the conditions tested. Correlation analysis was performed to determine the interconnectivity between Pi and Zn intracellular contents in both varieties. Paris Island Cos showed a strong negative correlation between the accumulation levels of Pi and Zn in shoots and roots. However, no relation was observed for Kordaat. The increase of Zn concentration in the medium causes a decrease in dynamics of Pi transport in Paris Island Cos, but not in Kordaat plants. Taken together, results revealed a contrasting behavior between the two lettuce varieties in terms of the coregulation of Pi and Zn homeostasis and provided evidence in favor of a genetic basis for the interconnection of these two elements. Nadia Bouain, Mushtak Kisko, Aida Rouached, Myriam Dauzat, Benoit Lacombe, Nibras Belgaroui, Tahar Ghnaya, Jean-Claude Davidian, Pierre Berthomieu, Chedly Abdelly, and Hatem Rouached Copyright © 2014 Nadia Bouain et al. All rights reserved. Evaluation of Correlation of Cell Cycle Proteins and Ki-67 Interaction in Paranasal Sinus Inverted Papilloma Prognosis and Squamous Cell Carcinoma Transformation Thu, 12 Jun 2014 12:53:58 +0000 The recurrent sinonasal inverted papilloma (IP) could be transformed to sinonasal squamous cell carcinoma. We use protein expression patterns by immunohistochemical method to see whether the expression of p53, p16, p21, and p27 belongs to cell-cycle-regulators and PCNA (proliferating cell nuclear antigen) and Ki-67 the proliferation markers in sixty patients with sinonasal inverted papilloma, and 10 of them with squamous cell carcinoma transformation. Significantly elevated levels of Ki-67, p27, and PCNA in IP with squamous cell carcinoma transformation of sinonasal tract compared with inverted papilloma were revealed. No variation of p16, p21, PLUNC (palate, lung, and nasal epithelium clone protein) and p53 expression was correlated to sinonasal IP malignant transformation by multivariate survey. However, we found elevated PLUNC expression in IPs with multiple recurrences. Finally, we found that PCNA, p27 may interact with CDK1 which promote IP cell proliferation and correlate to sinonasal squamous cell carcinoma. Ki-67 could work throughout the cell cycles to cause malignant transformation. In conclusion, this is a first study showing the correlation of Ki-67, PCNA interacted with CDK1 might lead to malignant transformation. Elevated PLUNC expression in the sinonasal IPs was related to multiple recurrences in human. Yung-An Tsou, Hung-Jin Huang, Tang-Chuan Wang, Chih-Jaan Tai, Chuan-Mu Chen, and Calvin Yu-Chian Chen Copyright © 2014 Yung-An Tsou et al. All rights reserved. Investigation of Potent Lead for Acquired Immunodeficiency Syndrome from Traditional Chinese Medicine Thu, 12 Jun 2014 11:47:14 +0000 Acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV), has become, because of the rapid spread of the disease, a serious global problem and cannot be treated. Recent studies indicate that VIF is a protein of HIV to prevent all of human immunity to attack HIV. Molecular compounds of traditional Chinese medicine (TCM) database filtered through molecular docking and molecular dynamics simulations to inhibit VIF can protect against HIV. Glutamic acid, plantagoguanidinic acid, and Aurantiamide acetate based docking score higher with other TCM compounds selected. Molecular dynamics are useful for analysis and detection ligand interactions. According to the docking position, hydrophobic interactions, hydrogen bonding changes, and structure variation, the study try to select the efficacy of traditional Chinese medicine compound Aurantiamide acetate is better than the other for protein-ligand interactions to maintain the protein composition, based on changes in the structure. Tzu-Chieh Hung, Wen-Yuan Lee, Kuen-Bao Chen, Yueh-Chiu Chan, and Calvin Yu-Chian Chen Copyright © 2014 Tzu-Chieh Hung et al. All rights reserved. Apoptosis and Molecular Targeting Therapy in Cancer Thu, 12 Jun 2014 08:22:29 +0000 Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction. Mohamed Hassan, Hidemichi Watari, Ali AbuAlmaaty, Yusuke Ohba, and Noriaki Sakuragi Copyright © 2014 Mohamed Hassan et al. All rights reserved. Lead Screening for HIV-1 Integrase (IN) Inhibited by Traditional Chinese Medicine Wed, 11 Jun 2014 09:13:44 +0000 Human immunodeficiency virus causes the acquired immunodeficiency syndrome (AIDS) and becomes a serious world-wide problem because of this disease's rapid propagation and incurability. Integrase strand transfer inhibitors (INSTIs) supports HIV have rapid drug resistance for antitreatment. Screening the traditional Chinese medicine (TCM) database by simulating molecular docking and molecular dynamics may select molecular compounds to inhibit INSTIs against HIV drug resistance. (S)-cathinone and (1S,2S)-norpseudoephedrine are selected based on structure and ligand-based drugs are designed and then get higher bioactivity predicted score from SVM than Raltegravir and other TCM compounds. The molecular dynamics are helpful in the analysis and detection of protein-ligand interactions. According to the docking poses, hydrophobic interactions and hydrogen bond variations define the main regions of important amino acids in integrase. In addition to the detection of TCM compound efficacy, we suggest (1S,2S)-norpseudoephedrine is better than the others based on the analysis of interaction and the effect on the structural variation. Tzu-Chieh Hung, Wen-Yuan Lee, Kuen-Bao Chen, Yueh-Chiu Chan, and Calvin Yu-Chian Chen Copyright © 2014 Tzu-Chieh Hung et al. All rights reserved. Lead Screening for CXCR4 of the Human HIV Infection Receptor Inhibited by Traditional Chinese Medicine Thu, 05 Jun 2014 15:58:05 +0000 The acquired immunodeficiency syndrome (AIDS) is a serious worldwide disease caused by the human immunodeficiency virus (HIV) infection. Recent research has pointed out that the G protein-coupled chemokine receptor CXCR4 and the coreceptor C-C chemokine receptor type 5 (CCR5) are important targets for HIV infection. The traditional Chinese medicine (TCM) database has been screened for candidate compounds by simulating molecular docking and molecular dynamics against HIV. Saussureamine C, 5-hydroxy-L-tryptophan, and diiodotyrosine are selected based on the highest docking score. The molecular dynamics is helpful in the analysis and detection of protein-ligand interactions. According to the analysis of docking poses, hydrophobic interactions, hydrogen bond variations, and the comparison of the effect on CXCR4 and CCR5, these results indicate Saussureamine C may have better effect on these two receptors. But for some considerations, diiodotyrosine could make the largest variation and may have some efficacy contrary to expectations. Tzu-Chieh Hung, Wen-Yuan Lee, Kuen-Bao Chen, and Calvin Yu-Chian Chen Copyright © 2014 Tzu-Chieh Hung et al. All rights reserved. Potential Mitochondrial Isocitrate Dehydrogenase R140Q Mutant Inhibitor from Traditional Chinese Medicine against Cancers Thu, 05 Jun 2014 12:11:34 +0000 A recent research of cancer has indicated that the mutant of isocitrate dehydrogenase 1 and 2 (IDH1 and 2) genes will induce various cancers, including chondrosarcoma, cholangiocarcinomas, and acute myelogenous leukemia due to the effect of point mutations in the active-site arginine residues of isocitrate dehydrogenase (IDH), such as IDH1/R132, IDH2/R140, and IDH2/R172. As the inhibition for those tumor-associated mutant IDH proteins may induce differentiation of those cancer cells, these tumor-associated mutant IDH proteins can be treated as a drug target proteins for a differentiation therapy against cancers. In this study, we aim to identify the potent TCM compounds from the TCM Database@Taiwan as lead compounds of IDH2 R140Q mutant inhibitor. Comparing to the IDH2 R140Q mutant protein inhibitor, AGI-6780, the top two TCM compounds, precatorine and abrine, have higher binding affinities with target protein in docking simulation. After MD simulation, the top two TCM compounds remain as the same docking poses under dynamic conditions. In addition, precatorine is extracted from Abrus precatorius L., which represents the cytotoxic and proapoptotic effects for breast cancer and several tumor lines. Hence, we propose the TCM compounds, precatorine and abrine, as potential candidates as lead compounds for further study in drug development process with the IDH2 R140Q mutant protein against cancer. Wen-Yuan Lee, Kuan-Chung Chen, Hsin-Yi Chen, and Calvin Yu-Chian Chen Copyright © 2014 Wen-Yuan Lee et al. All rights reserved. Treatment of Rheumatoid Arthritis with Traditional Chinese Medicine Wed, 04 Jun 2014 10:01:59 +0000 Rheumatoid arthritis (RA) is a chronic inflammatory disease that will affect quality of life and, working efficiency, and produce negative thoughts for patients. Current therapy of RA is treated with disease-modifying antirheumatic drugs (DMARDs). Although most of these treatment methods are effective, most patients still have a pleasant experience either due to poor efficacy or side effects or both. Interleukin-6 receptor (IL6R) is important in the pathogenesis of RA. In this study, we would like to detect the potential candidates which inhibit IL6R against RA from traditional Chinese medicine (TCM). We use TCM compounds from the TCM Database@Taiwan for virtually screening the potential IL6R inhibitors. The TCM candidate compound, calycosin, has potent binding affinity with IL6R protein. The molecular dynamics simulation was employed to validate the stability of interaction in the protein complex with calycosin. The analysis indicates that protein complex with calycosin is more stable. In addition, calycosin is known to be one of the components of Angelica sinensis, which has been indicated to have an important role in the treatment of rheumatoid arthritis. Therefore, calycosin is a potential candidate as lead compounds for further study in drug development process with IL6R protein against rheumatoid arthritis. Wen-Yuan Lee, Hsin-Yi Chen, Kuan-Chung Chen, and Calvin Yu-Chian Chen Copyright © 2014 Wen-Yuan Lee et al. All rights reserved. An Overview of the Spindle Assembly Checkpoint Status in Oral Cancer Tue, 03 Jun 2014 11:11:38 +0000 Abnormal chromosome number, or aneuploidy, is a common feature of human solid tumors, including oral cancer. Deregulated spindle assembly checkpoint (SAC) is thought as one of the mechanisms that drive aneuploidy. In normal cells, SAC prevents anaphase onset until all chromosomes are correctly aligned at the metaphase plate thereby ensuring genomic stability. Significantly, the activity of this checkpoint is compromised in many cancers. While mutations are rather rare, many tumors show altered expression levels of SAC components. Genomic alterations such as aneuploidy indicate a high risk of oral cancer and cancer-related mortality, and the molecular basis of these alterations is largely unknown. Yet, our knowledge on the status of SAC components in oral cancer remains sparse. In this review, we address the state of our knowledge regarding the SAC defects and the underlying molecular mechanisms in oral cancer, and discuss their therapeutic relevance, focusing our analysis on the core components of SAC and its target Cdc20. José Henrique Teixeira, Patrícia Manuela Silva, Rita Margarida Reis, Inês Moranguinho Moura, Sandra Marques, Joana Fonseca, Luís Silva Monteiro, and Hassan Bousbaa Copyright © 2014 José Henrique Teixeira et al. All rights reserved. p120 Modulates LPS-Induced NF-κB Activation Partially through RhoA in Bronchial Epithelial Cells Tue, 03 Jun 2014 08:14:43 +0000 p120-Catenin (p120) is an adherens junction protein recognized to regulate cell-cell adhesion. Emerging evidence indicates that p120 may also play an important role in inflammatory responses, and the regulatory mechanisms are still unknown. In the present study, we showed that p120 was associated with airway inflammation. p120 downregulation induced nuclear factor-κB (NF-κB) activation, accompanied with IκBα degradation, p65 nuclear translocation, and increased expression of interleukin-8 (IL-8) in lipopolysaccharide (LPS)- treated C57BL mice and human bronchial epithelial cells (BECs). Moreover, we first found that p120 directly coprecipitated with RhoA in BECs. After LPS stimulation, although total RhoA and p120-bound RhoA were unchanged, RhoA activity was increased. Y27632, a ROCK inhibitor, could partially inhibit nuclear translocation of p65. Overexpression of p120 inactivated RhoA and NF-κB in BECs, whereas p120 loss significantly increased RhoA activity, p65 nuclear translocation, and IL-8 expression. Taken together, our study supports the regulatory role of p120 in airway inflammation and reveals that p120 may modulate NF-κB signaling partially through RhoA. Lingzhi Qin, Shenghui Qin, Yanli Zhang, Chao Zhang, Heng Ma, Naping Li, Liwei Liu, Xi Wang, and Renliang Wu Copyright © 2014 Lingzhi Qin et al. All rights reserved. Lead Discovery for Alzheimer’s Disease Related Target Protein RbAp48 from Traditional Chinese Medicine Mon, 02 Jun 2014 07:18:05 +0000 Deficiency or loss of function of Retinoblastoma-associated proteins (RbAp48) is related with Alzheimer’s disease (AD), and AD disease is associated with age-related memory loss. During normal function, RbAp48 forms a complex with the peptide FOG-1 (friend of GATA-1) and has a role in gene transcription, but an unstable complex may affect the function of RbAp48. This study utilizes the world’s largest traditional Chinese medicine (TCM) database and virtual screening to provide potential compounds for RbAp48 binding. A molecular dynamics (MD) simulation was employed to understand the variations after protein-ligand interaction. FOG1 was found to exhibit low stability after RbAp48 binding; the peptide displayed significant movement from the initial docking position, a phenomenon which matched the docking results. The protein structure of the other TCM candidates was not variable during MD simulation and had a greater stable affinity for RbAp48 binding than FOG1. Our results reveal that the protein structure does not affect ligand binding, and the top three TCM candidates Bittersweet alkaloid II, Eicosandioic acid, and Perivine might resolve the instability of the RbAp48-FOG1 complex and thus be used in AD therapy. Hung-Jin Huang, Cheng-Chun Lee, and Calvin Yu-Chian Chen Copyright © 2014 Hung-Jin Huang et al. All rights reserved. Primary Genetic Investigation of a Hyperlipidemia Model: Molecular Characteristics and Variants of the Apolipoprotein E Gene in Mongolian Gerbil Sun, 01 Jun 2014 00:00:00 +0000 The objective of this work was to establish a novel Mongolian gerbil (Meriones unguiculatus) hyperlipidemia model and to investigate its susceptibility genetic basis. Two rodent (gerbil and rat) hyperlipidemia models were induced by feeding a high fat/high-cholesterol (HF/HC) diet. There were significant increases of serum total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in gerbils within a 4-week modeling period. About 10–30% of >8-month-old individuals developed hyperlipidemia spontaneously. The apolipoprotein E (ApoE) gene was cloned by merging a sequence of rapid amplification of cDNA ends (RACE) and nested polymerase chain reaction products. The results revealed an open reading frame of 948 bp, encoding a protein of 298 amino acids. The gene without a 5′-UTR region in the first intron was highly homologous to human Apo-A-I and rat Apo-A-IV. The distribution of expression of the ApoE gene in liver, brain, heart, lung, kidney, and adrenal gland was detected by an ABC immunohistochemical procedure. Three single nucleotide polymorphisms (SNPs; C97T, G781T, and A1774T) were first found using PCR-single-strand conformation polymorphism (PCR-SSCP) in a closed population containing 444 animals. Correlation analysis confirmed that new SNPs , age, and gender were associated significantly () with hyperlipidemia. Yuehuan Liu, Jiusheng Wu, Qiaojuan Shi, Honggang Guo, Huazhong Ying, and Ningying Xu Copyright © 2014 Yuehuan Liu et al. All rights reserved. The Carbon-Nitrogen Balance of the Nodule and Its Regulation under Elevated Carbon Dioxide Concentration Wed, 28 May 2014 13:09:02 +0000 Legumes have developed a unique way to interact with bacteria: in addition to preventing infection from pathogenic bacteria like any other plant, legumes also developed a mutualistic symbiotic relationship with one gender of soil bacteria: rhizobium. This interaction leads to the development of a new root organ, the nodule, where the differentiated bacteria fix for the plant the atmospheric dinitrogen (atmN2). In exchange, the symbiont will benefit from a permanent source of carbon compounds, products of the photosynthesis. The substantial amounts of fixed carbon dioxide dedicated to the symbiont imposed to the plant a tight regulation of the nodulation process to balance carbon and nitrogen incomes and outcomes. Climate change including the increase of the concentration of the atmospheric carbon dioxide is going to modify the rates of plant photosynthesis, the balance between nitrogen and carbon, and, as a consequence, the regulatory mechanisms of the nodulation process. This review focuses on the regulatory mechanisms controlling carbon/nitrogen balances in the context of legume nodulation and discusses how the change in atmospheric carbon dioxide concentration could affect nodulation efficiency. Marc Libault Copyright © 2014 Marc Libault. All rights reserved. XRCC1 Arg399Gln and Arg194Trp Polymorphisms and Risk of Systemic Lupus Erythematosus in an Iranian Population: A Pilot Study Mon, 26 May 2014 06:39:41 +0000 Background. Evidences are suggesting that DNA damage is implicated in development of systemic lupus erythematosus (SLE). Therefore we focused on two common XRCC1 polymorphisms (Arg399Gln and Arg194Trp) in SLE susceptibility in South East of Iran. Methods. Peripheral blood DNA was extracted from 163 SLE patients and 180 healthy controls. PCR-restriction fragment length polymorphism method was used for genotyping of XRCC1 Arg399Gln and Arg194Trp polymorphisms. Results. The frequency of Arg/Gln genotype of the XRCC1 Arg399Gln polymorphism was significantly lower in SLE patients than controls. Moreover, lower frequency of Arg/Gln genotype was found in SLE patients with malar rash compared to patients without this manifestation. No association was observed between XRCC1 Arg194Trp polymorphism and increased risk of SLE in studied population. Haplotype analysis revealed no correlation between four haplotypes of XRCC1 Arg399Gln and Arg194Trp polymorphisms and SLE risk. Conclusion. These findings suggest that XRCC1 399 Arg/Gln heterozygous genotype plays a protective role in SLE susceptibility. Saeedeh Salimi, Milad Mohammadoo-khorasani, Ehsan Tabatabai, Mahnaz Sandoughi, Zahra Zakeri, and Anoosh Naghavi Copyright © 2014 Saeedeh Salimi et al. All rights reserved. EMMPRIN Expression in Oral Squamous Cell Carcinomas: Correlation with Tumor Proliferation and Patient Survival Wed, 21 May 2014 09:39:21 +0000 The aim of our study was to explore the clinicopathological and prognostic significance of extracellular matrix metalloproteinase inducer (EMMPRIN) expression in oral squamous cell carcinomas (OSCC), and its relation with the proliferative tumor status of OSCC. We examined EMMPRIN and Ki-67 proteins expression by immunohistochemistry in 74 cases with OSCC. Statistical analysis was conducted to examine their clinicopathological and prognostic significance in OSCC. EMMPRIN membrane expression was observed in all cases, with both membrane and cytoplasmic tumor expression in 61 cases (82.4%). EMMPRIN overexpression was observed in 56 cases (75.7%). Moderately or poorly differentiated tumors showed EMMPRIN overexpression more frequently than well-differentiated tumors . Overexpression of EMMPRIN was correlated with high Ki-67 expression . In the multivariate analysis, EMMPRIN overexpression reveals an adverse independent prognostic value for cancer-specific survival (CSS) . Our results reveal that EMMPRIN protein is overexpressed in more than two-thirds of OSCC cases, especially in high proliferative and less differentiated tumors. The independent value of EMMPRIN overexpression in CSS suggests that this protein could be used as an important biological prognostic marker for patients with OSCC. Moreover, the high expression of EMMPRIN makes it a possible therapeutic target in OSCC patients. Luís Silva Monteiro, Maria Leonor Delgado, Sara Ricardo, Fernanda Garcez, Barbas do Amaral, José Júlio Pacheco, Carlos Lopes, and Hassan Bousbaa Copyright © 2014 Luís Silva Monteiro et al. All rights reserved. Computational Design of Apolipoprotein E4 Inhibitors for Alzheimer’s Disease Therapy from Traditional Chinese Medicine Wed, 21 May 2014 06:26:28 +0000 Apolipoprotein E4 (Apo E4) is the major genetic risk factor in the causation of Alzheimer’s disease (AD). In this study we utilize virtual screening of the world’s largest traditional Chinese medicine (TCM) database and investigate potential compounds for the inhibition of ApoE4. We present the top three TCM candidates: Solapalmitine, Isodesacetyluvaricin, and Budmunchiamine L5 for further investigation. Dynamics analysis and molecular dynamics (MD) simulation were used to simulate protein-ligand complexes for observing the interactions and protein variations. Budmunchiamine L5 did not have the highest score from virtual screening; however, the dynamics pose is similar to the initial docking pose after MD simulation. Trajectory analysis reveals that Budmunchiamine L5 was stable over all simulation times. The migration distance of Budmunchiamine L5 illustrates that docked ligands are not variable from the initial docked site. Interestingly, Arg158 was observed to form H-bonds with Budmunchiamine L5 in the docking pose and MD snapshot, which indicates that the TCM compounds could stably bind to ApoE4. Our results show that Budmunchiamine L5 has good absorption, blood brain barrier (BBB) penetration, and less toxicity according to absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction and could, therefore, be safely used for developing novel ApoE4 inhibitors. Hung-Jin Huang, Hsin-Yi Chen, Cheng-Chun Lee, and Calvin Yu-Chian Chen Copyright © 2014 Hung-Jin Huang et al. All rights reserved. Chromium (VI) Uptake and Tolerance Potential in Cotton Cultivars: Effect on Their Root Physiology, Ultramorphology, and Oxidative Metabolism Wed, 14 May 2014 09:46:04 +0000 Chromium (Cr) is present in our environment as a toxic pollutant, which needs to be removed using phytoremediation technology. In present study, two transgenic cotton cultivars (J208, Z905) and their hybrid line (ZD14) were used to explore their Cr uptake and tolerance potential using multiple biomarkers approach. Four different levels of Cr (CK, 10, 50, and 100 μM) were applied. Cr caused a significant reduction in root/shoot length, number of secondary roots, and root fresh and dry biomasses at 100 μM. Cr accumulated more in roots and was found higher in hybrid line (ZD14) as compared with its parent lines (J208, Z905) at all Cr stress levels (10, 50, and 100 μM). Cr translocation was less than 1 in all cultivars. Ultrastructural studies at 100 μM Cr showed an increase in number of nuclei and vacuoles and presence of Cr dense granules in dead parts of the cell (vacuoles/cell wall). Malondialdehyde (MDA), hydrogen peroxide (H2O2), total soluble proteins, superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) as a whole were upregulated with elevated levels of Cr. Higher Cr uptake by roots, accelerated metabolism, and Cr sequestration in dead parts of the cell indicate that these cotton cultivars can be useful for Cr accumulation and tolerance. M. K. Daud, Lei Mei, M. T. Variath, Shafaqat Ali, Cheng Li, M. T. Rafiq, and S. J. Zhu Copyright © 2014 M. K. Daud et al. All rights reserved. Solanine Induces Mitochondria-Mediated Apoptosis in Human Pancreatic Cancer Cells Sun, 11 May 2014 09:03:51 +0000 Steroid alkaloids have been suggested as potential anticancer compounds. However, the underlying mechanisms of how steroid alkaloids inhibit the tumor growth are largely unknown. Here, we reported that solanine, a substance of steroid alkaloids, has a positive effect on the inhibition of pancreatic cancer cell growth in vitro and in vivo. In pancreatic cancer cells and nu/nu nude mice model, we found that solanine inhibited cancer cells growth through caspase-3 dependent mitochondrial apoptosis. Mechanically, solanine promotes the opening of mitochondrial membrane permeability transition pore (MPTP) by downregulating the Bcl-2/Bax ratio; thereafter, Cytochrome c and Smac are released from mitochondria into cytosol to process the caspase-3 zymogen into an activated form. Moreover, we found that the expression of tumor metastasis related proteins, MMP-2 and MMP-9, was also decreased in the cells treated with solanine. Therefore, our results suggested that solanine was an effective compound for the treatment of pancreatic cancer. Hongwei Sun, Chongqing Lv, Longlong Yang, Yingxiu Wang, Qingshun Zhang, Suhui Yu, Hongru Kong, Meng Wang, Jianming Xie, Chunwu Zhang, and Mengtao Zhou Copyright © 2014 Hongwei Sun et al. All rights reserved. In Silico Design of BACE1 Inhibitor for Alzheimer’s Disease by Traditional Chinese Medicine Thu, 08 May 2014 07:10:11 +0000 The -site APP cleaving enzyme 1 (BACE1) is an important target for causing Alzheimer’s disease (AD), due to the brain deposition peptide amyloid beta (A) require cleavages of amyloid precursor protein (APP) by BACE1 and -secretase, but treatments of AD still have side effect in recent therapy. This study utilizes the world largest traditional Chinese medicine (TCM) database and database screening to provide potential BACE1 inhibited compound. Molecular dynamics (MD) simulation was carried out to observe the dynamics structure after ligand binding. We found that Triptofordin B1 has less toxicity than pyrimidine analogue, which has more potent binding affinity with BACE1. For trajectory analysis, all conformations are tending to be stable during 5000 ps simulation time. In dynamic protein validation, the residues of binding region are still stable after MD simulation. For snapshot comparison, we found that Triptofordin B1 could reduce the binding cavity; the results reveal that Triptofordin B1 could bind to BACE1 and better than control, which could be used as potential lead drug to design novel BACE1 inhibitor for AD therapy. Hung-Jin Huang, Cheng-Chun Lee, and Calvin Yu-Chian Chen Copyright © 2014 Hung-Jin Huang et al. All rights reserved. Possible Association of IL-4 VNTR Polymorphism with Susceptibility to Preeclampsia Mon, 28 Apr 2014 12:48:52 +0000 Preeclampsia (PE) is a pregnancy-specific disorder that results in maternal mortality and morbidity. Growing evidence indicated that cytokines are involved in the pathogenesis of PE and interleukin-4 VNTR polymorphism could be implicated in altering the PE risk. The aim of this study was to evaluate the possible association between IL-4 VNTR polymorphism and susceptibility to PE in Iranian population for the first time. Genetic polymorphism was evaluated in 192 PE and 186 healthy control women by polymerase chain reaction method. We found that the VNTR polymorphism of IL-4 gene has significantly increased the risk of preeclampsia (RP2/RP1 versus RP1/RP1, OR, 2.8 [95% CI, 1.7 to 8.8]; and RP2/RP2 versus RP1/RP1; ). The results showed that carriage of IL-4 VNTR RP2 allele has positive association with preeclampsia susceptibility. Saeedeh Salimi, Milad Mohammadoo-Khorasani, Minoo Yaghmaei, Mojgan Mokhtari, and Maryam Moossavi Copyright © 2014 Saeedeh Salimi et al. All rights reserved. Current Advances in Molecular Phylogenetics Mon, 07 Apr 2014 09:37:53 +0000 Vassily Lyubetsky, William H. Piel, and Dietmar Quandt Copyright © 2014 Vassily Lyubetsky et al. All rights reserved. Phylogenetic Analysis of Entomoparasitic Nematodes, Potential Control Agents of Flea Populations in Natural Foci of Plague Thu, 03 Apr 2014 10:58:16 +0000 Entomoparasitic nematodes are natural control agents for many insect pests, including fleas that transmit Yersinia pestis, a causative agent of plague, in the natural foci of this extremely dangerous zoonosis. We examined the flea samples from the Volga-Ural natural focus of plague for their infestation with nematodes. Among the six flea species feeding on different rodent hosts (Citellus pygmaeus, Microtus socialis, and Allactaga major), the rate of infestation varied from 0 to 21%. The propagation rate of parasitic nematodes in the haemocoel of infected fleas was very high; in some cases, we observed up to 1,000 juveniles per flea specimen. Our study of morphology, life cycle, and rDNA sequences of these parasites revealed that they belong to three distinct species differing in the host specificity. On SSU and LSU rRNA phylogenies, these species representing three genera (Rubzovinema, Psyllotylenchus, and Spilotylenchus), constitute a monophyletic group close to Allantonema and Parasitylenchus, the type genera of the families Allantonematidae and Parasitylenchidae (Nematoda: Tylenchida). We discuss the SSU-ITS1-5.8S-LSU rDNA phylogeny of the Tylenchida with a special emphasis on the suborder Hexatylina. E. I. Koshel, V. V. Aleshin, G. A. Eroshenko, and V. V. Kutyrev Copyright © 2014 E. I. Koshel et al. All rights reserved. A New Nested Allele-Specific Multiplex Polymerase Chain Reaction Method for Haplotyping of VKORC1 Gene to Predict Warfarin Sensitivity Sun, 30 Mar 2014 12:16:06 +0000 The vitamin K epoxide reductase complex 1 gene (VKORC1) is commonly assessed to predict warfarin sensitivity. In this study, a new nested allele-specific multiplex polymerase chain reaction (PCR) method that can simultaneously identify single nucleotide polymorphisms (SNPs) at VKORC1 381, 861, 5808, and 9041 for haplotype analysis was developed and validated. Extracted DNA was amplified in the first PCR DNA, which was optimized by investigating the effects of varying the primer concentrations, annealing temperature, magnesium chloride concentration, enzyme concentration, and the amount of DNA template. The amplification products produced from the first round of PCR were used as templates for a second PCR amplification in which both mutant and wild-type primers were added in separate PCR tubes, followed by optimization in a similar manner. The final PCR products were resolved by agarose gel electrophoresis and further analysed by using a VKORC1 genealogic tree to infer patient haplotypes. Fifty patients were identified to have H1H1, one had H1H2, one had H1H7, 31 had either H1H7 or H1H9, one had H1H9, eight had H7H7, and one had H8H9 haplotypes. This is the first method that is able to infer VKORC1 haplotypes using only conventional PCR methods. Yung An Chua, Wan Zaidah Abdullah, Zukurnai Yusof, and Siew Hua Gan Copyright © 2014 Yung An Chua et al. All rights reserved. Reconciliation of Gene and Species Trees Thu, 27 Mar 2014 06:44:11 +0000 The first part of the paper briefly overviews the problem of gene and species trees reconciliation with the focus on defining and algorithmic construction of the evolutionary scenario. Basic ideas are discussed for the aspects of mapping definitions, costs of the mapping and evolutionary scenario, imposing time scales on a scenario, incorporating horizontal gene transfers, binarization and reconciliation of polytomous trees, and construction of species trees and scenarios. The review does not intend to cover the vast diversity of literature published on these subjects. Instead, the authors strived to overview the problem of the evolutionary scenario as a central concept in many areas of evolutionary research. The second part provides detailed mathematical proofs for the solutions of two problems: (i) inferring a gene evolution along a species tree accounting for various types of evolutionary events and (ii) trees reconciliation into a single species tree when only gene duplications and losses are allowed. All proposed algorithms have a cubic time complexity and are mathematically proved to find exact solutions. Solving algorithms for problem (ii) can be naturally extended to incorporate horizontal transfers, other evolutionary events, and time scales on the species tree. L. Y. Rusin, E. V. Lyubetskaya, K. Y. Gorbunov, and V. A. Lyubetsky Copyright © 2014 L. Y. Rusin et al. All rights reserved. A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms Wed, 05 Mar 2014 00:00:00 +0000 A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process. Zhiyuan Wu, Yunqing Zhang, Xinju Zhang, Xiao Xu, Zhihua Kang, Shibao Li, Chen Zhang, Bing Su, and Ming Guan Copyright © 2014 Zhiyuan Wu et al. All rights reserved. Noncoding RNAs: Emerging Players in Muscular Dystrophies Tue, 04 Mar 2014 09:46:56 +0000 The fascinating world of noncoding RNAs has recently come to light, thanks to the development of powerful sequencing technologies, revealing a variety of RNA molecules playing important regulatory functions in most, if not all, cellular processes. Many noncoding RNAs have been implicated in regulatory networks that are determinant for skeletal muscle differentiation and disease. In this review, we outline the noncoding RNAs involved in physiological mechanisms of myogenesis and those that appear dysregulated in muscle dystrophies, also discussing their potential use as disease biomarkers and therapeutic targets. Germana Falcone, Alessandra Perfetti, Beatrice Cardinali, and Fabio Martelli Copyright © 2014 Germana Falcone et al. All rights reserved. Sex Differences in Constitutive Autophagy Thu, 27 Feb 2014 15:58:50 +0000 Sex bias has been described nowadays in biomedical research on animal models, although sexual dimorphism has been confirmed widely under pathological and physiological conditions. The main objective of our work was to study the sex differences in constitutive autophagy in spinal cord and skeletal muscle tissue from wild type mice. To examine the influence of sex on autophagy, mRNA and proteins were extracted from male and female mice tissues. The expressions of microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (p62), markers to monitor autophagy, were analyzed at 40, 60, 90, and 120 days of age. We found significant sex differences in the expression of LC3 and p62 in both tissues at these ages. The results indicated that sex and tissue specific differences exist in constitutive autophagy. These data underlined the need to include both sexes in the experimental groups to minimize any sex bias. Sara Oliván, Ana Cristina Calvo, Raquel Manzano, Pilar Zaragoza, and Rosario Osta Copyright © 2014 Sara Oliván et al. All rights reserved. Role of Noncoding RNAs in the Regulation of P-TEFb Availability and Enzymatic Activity Wed, 19 Feb 2014 10:09:00 +0000 P-TEFb is a transcriptional factor that specifically regulates the elongation step of RNA polymerase II-dependent transcription and its activity strictly required for Human Immunodeficiency Virus (HIV) infection and during cardiac differentiation. P-TEFb role has emerged as a crucial regulator of transcription elongation and its activity found finely tuned in vivo at transcriptional level as well as posttranscriptionally by dynamic association with different multisubunit molecular particles. Both physiological and pathological cellular signals rapidly converge on P-TEFb regulation by modifying expression and activity of the complex to allow cells to properly respond to different stimuli. In this review we will give a panoramic view on P-TEFb regulation by noncoding RNAs in both physiological and pathological conditions. Giuliana Napolitano, Luigi Lania, and Barbara Majello Copyright © 2014 Giuliana Napolitano et al. All rights reserved. Insulin Receptor Substrate-1 (IRS-1) Gly927Arg: Correlation with Gestational Diabetes Mellitus in Saudi Women Mon, 17 Feb 2014 12:24:40 +0000 Pregnant women with gestational diabetes mellitus (GDM) and type 2 diabetes mellitus (T2DM) share a common pathophysiology associated with similar risk factors. Genetic variants used to determine the risk of developing T2DM might also be associated with the prevalence of GDM. The aim of the present study was to scrutinize the relationship between the G972R polymorphism of the insulin receptor substrate-1 (IRS-1) gene with GDM in the Saudi female population. This is a case-control study that monitored 500 Saudi women. Subjects with GDM () were compared with non-GDM () controls. We opted to evaluate rs1801278 polymorphism in the IRS1 gene, which plays a critical role in the insulin-signaling pathway. Genotyping was performed with the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. The frequency of the rs1801278 polymorphism was significantly higher in women with GDM than in women with non-GDM (for TT + CT versus CC: ). Additionally, there was a significant increase in the frequency of the Arg-encoding mutant allele from GDM to non-GDM (for T versus C: ). Our results suggest that the rs1801278 polymorphism in the IRS-1 gene is involved in the occurrence of GDM in the Saudi population. Khalid Khalaf Alharbi, Imran Ali Khan, Zeinab Abotalib, and Malak Mohammed Al-Hakeem Copyright © 2014 Khalid Khalaf Alharbi et al. All rights reserved. The Role of PinX1 in Growth Control of Breast Cancer Cells and Its Potential Molecular Mechanism by mRNA and lncRNA Expression Profiles Screening Mon, 03 Feb 2014 11:13:11 +0000 As a major tumor suppressor gene, the role of PinX1 in breast cancer and its molecular mechanism remain unclear. In this study, overexpression of PinX1 was generated in 3 breast cancer cell lines, and knockdown of PinX1 was performed in a nontumorigenic breast cell line. The regulation of PinX1 on cell proliferation and cell cycle was observed. A microarray-based lncRNA and mRNA expression profile screening was also performed. We found a lower growth rate, G0/G1 phase arrest, and S phase inhibition in the PinX1 overexpressed breast cancer cells, while a higher growth rate, decreased G0/G1 phase, and increased S phase rate in the PinX1 knocked-down nontumorigenic breast cell. A total of 977 mRNAs and 631 lncRNAs were identified as differentially expressed transcripts between PinX1 overexpressed and control MCF-7 cells. Further analysis identified the involvement of these mRNAs in 52 cancer related pathways and various other biological processes. 11 enhancer-like lncRNAs and 25 lincRNAs with their adjacent mRNA pairs were identified as coregulated transcripts. Our results confirmed the role of PinX1 as a major tumor suppressor gene in breast cancer cell lines and provided information for further research on the molecular mechanisms of PinX1 in tumorigenesis. Rong Shi, Jue-Yu Zhou, Hui Zhou, Zhen Zhao, Sang-Hua Liang, Wen-Ling Zheng, and Wen-Li Ma Copyright © 2014 Rong Shi et al. All rights reserved. Differences in Brain Transcriptomes of Closely Related Baikal Coregonid Species Wed, 29 Jan 2014 00:00:00 +0000 The aim of this work was to get deeper insight into genetic factors involved in the adaptive divergence of closely related species, specifically two representatives of Baikal coregonids—Baikal whitefish (Coregonus baicalensis Dybowski) and Baikal omul (Coregonus migratorius Georgi)—that diverged from a common ancestor as recently as 10–20 thousand years ago. Using the Serial Analysis of Gene Expression method, we obtained libraries of short representative cDNA sequences (tags) from the brains of Baikal whitefish and omul. A comparative analysis of the libraries revealed quantitative differences among ~4% tags of the fishes under study. Based on the similarity of these tags with cDNA of known organisms, we identified candidate genes taking part in adaptive divergence. The most important candidate genes related to the adaptation of Baikal whitefish and Baikal omul, identified in this work, belong to the genes of cell metabolism, nervous and immune systems, protein synthesis, and regulatory genes as well as to DTSsa4 Tc1-like transposons which are widespread among fishes. Oksana S. Bychenko, Lyubov V. Sukhanova, Tatyana L. Azhikina, Timofey A. Skvortsov, Tuyana V. Belomestnykh, and Eugene D. Sverdlov Copyright © 2014 Oksana S. Bychenko et al. All rights reserved. Brd4 and HEXIM1: Multiple Roles in P-TEFb Regulation and Cancer Wed, 29 Jan 2014 00:00:00 +0000 Bromodomain-containing protein 4 (Brd4) and hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) are two opposing regulators of the positive transcription elongation factor b (P-TEFb), which is the master modulator of RNA polymerase II during transcriptional elongation. While Brd4 recruits P-TEFb to promoter-proximal chromatins to activate transcription, HEXIM1 sequesters P-TEFb into an inactive complex containing the 7SK small nuclear RNA. Besides regulating P-TEFb’s transcriptional activity, recent evidence demonstrates that both Brd4 and HEXIM1 also play novel roles in cell cycle progression and tumorigenesis. Here we will discuss the current knowledge on Brd4 and HEXIM1 and their implication as novel therapeutic options against cancer. Ruichuan Chen, Jasper H. N. Yik, Qiao Jing Lew, and Sheng-Hao Chao Copyright © 2014 Ruichuan Chen et al. All rights reserved. Molecular Characterization of a Recombinant Manganese Superoxide Dismutase from Lactococcus lactis M4 Mon, 27 Jan 2014 12:38:58 +0000 A superoxide dismutase (SOD) gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression of sodA under T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172). Boon Hooi Tan, Thean Chor Leow, Hooi Ling Foo, and Raha Abdul Rahim Copyright © 2014 Boon Hooi Tan et al. All rights reserved. DNA Methylation Pattern in Overweight Women under an Energy-Restricted Diet Supplemented with Fish Oil Wed, 22 Jan 2014 12:50:44 +0000 Dietary factors modulate gene expression and are able to alter epigenetic signatures in peripheral blood mononuclear cells (PBMC). However, there are limited studies about the effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) on the epigenetic mechanisms that regulate gene expression. This research investigates the effects of n-3-rich fish oil supplementation on DNA methylation profile of several genes whose expression has been reported to be downregulated by n-3 PUFA in PBMC: CD36, FFAR3, CD14, PDK4, and FADS1. Young overweight women were supplemented with fish oil or control in a randomized 8-week intervention trial following a balanced diet with 30% energy restriction. Fatty acid receptor CD36 decreased DNA methylation at CpG +477 due to energy restriction. Hypocaloric diet-induced weight loss also reduced the methylation percentages of CpG sites located in CD14, PDK4, and FADS1. The methylation patterns of these genes were only slightly affected by the fish oil supplementation, being the most relevant to the attenuation of the weight loss-induced decrease in CD36 methylation after adjusting by baseline body weight. These results suggest that the n-3 PUFA-induced changes in the expression of these genes in PBMC are not mediated by DNA methylation, although other epigenetic mechanisms cannot be discarded. Cátia Lira do Amaral, Fermín I. Milagro, Rui Curi, and J. Alfredo Martínez Copyright © 2014 Cátia Lira do Amaral et al. All rights reserved. Erratum to “Use of Metarhizium anisopliae Chitinase Genes for Genotyping and Virulence Characterization” Mon, 20 Jan 2014 00:00:00 +0000 Saliou Niassy, Sevgan Subramanian, Sunday Ekesi, Joel L. Bargul, Jandouwe Villinger, and Nguya K. Maniania Copyright © 2014 Saliou Niassy et al. All rights reserved. Changes in Bacterial Population of Gastrointestinal Tract of Weaned Pigs Fed with Different Additives Sun, 19 Jan 2014 08:12:32 +0000 This study aimed to provide novel insights into the gastrointestinal microbial diversity from different gastrointestinal locations in weaning piglets using PCR-restriction fragment length polymorphism (PCR-RFLP). Additionally, the effect of different feed additives was analyzed. Thirty-two piglets were fed with four different diets: a control group and three enriched diets, with avilamycin, sodium butyrate, and a plant extract mixture. Digesta samples were collected from eight different gastrointestinal segments of each animal and the bacterial population was analysed by a PCR-RFLP technique that uses 16S rDNA gene sequences. Bacterial diversity was assessed by calculating the number of bands and the Shannon-Weaver index. Dendrograms were constructed to estimate the similarity of bacterial populations. A higher bacterial diversity was detected in large intestine compared to small intestine. Among diets, the most relevant microbial diversity differences were found between sodium butyrate and plant extract mixture. Proximal jejunum, ileum, and proximal colon were identified as those segments that could be representative of microbial diversity in pig gut. Results indicate that PCR-RFLP technique allowed detecting modifications on the gastrointestinal microbial ecology in pigs fed with different additives, such as increased biodiversity by sodium butyrate in feed. Mercè Roca, Miquel Nofrarías, Natàlia Majó, Ana María Pérez de Rozas, Joaquim Segalés, Marisol Castillo, Susana María Martín-Orúe, Anna Espinal, Joan Pujols, and Ignacio Badiola Copyright © 2014 Mercè Roca et al. All rights reserved. Regulation of CDK9 Activity by Phosphorylation and Dephosphorylation Sun, 12 Jan 2014 14:00:54 +0000 HIV-1 transcription is regulated by CDK9/cyclin T1, which, unlike a typical cell cycle-dependent kinase, is regulated by associating with 7SK small nuclear ribonuclear protein complex (snRNP). While the protein components of this complex are well studied, the mechanism of the complex formation is still not fully understood. The association of CDK9/cyclin T1 with 7SK snRNP is, in part, regulated by a reversible CDK9 phosphorylation. Here, we present a comprehensive review of the kinases and phosphatases involved in CDK9 phosphorylation and discuss their role in regulation of HIV-1 replication and potential for being targeted for drug development. We propose a novel pathway of HIV-1 transcription regulation via CDK9 Ser-90 phosphorylation by CDK2 and CDK9 Ser-175 dephosphorylation by protein phosphatase-1. Sergei Nekhai, Michael Petukhov, and Denitra Breuer Copyright © 2014 Sergei Nekhai et al. All rights reserved. Isolation and Expression Analysis of Novel Silicon Absorption Gene from Roots of Mangrove (Rhizophora apiculata) via Suppression Subtractive Hybridization Wed, 01 Jan 2014 21:41:53 +0000 Silicon (Si) is the second most abundant element in soil after oxygen. It is not an essential element for plant growth and formation but plays an important role in increasing plant tolerance towards different kinds of abiotic and biotic stresses. The molecular mechanism of Si absorption and accumulation may differ between plants, such as monocotyledons and dicotyledons. Silicon absorption and accumulation in mangrove plants are affected indirectly by some proteins rich in serine and proline amino acids. The expression level of the genes responsible for Si absorption varies in different parts of plants. In this study, Si is mainly observed in the epidermal roots’ cell walls of mangrove plants compared to other parts. The present work was carried out to discover further information on Si stress responsive genes in Rhizophora apiculata, using the suppression subtractive hybridization technique. To construct the cDNA library, two-month-old seedlings were exposed to 0.5, 1, and 1.5 mM SiO2 for 15 hrs and for 1 to 6 days resulting in a total of 360 high quality ESTs gained. Further examination by RT-PCR and real-time qRT-PCR showed the expression of a candidate gene of serine-rich protein. Mahbod Sahebi, Mohamed M. Hanafi, Siti Nor Akmar Abdullah, Mohd Y. Rafii, Parisa Azizi, Naghmeh Nejat, and Abu Seman Idris Copyright © 2014 Mahbod Sahebi et al. All rights reserved.