Molecular Biology International The latest articles from Hindawi Publishing Corporation © 2016 , Hindawi Publishing Corporation . All rights reserved. SCAR Marker for Identification and Discrimination of Commiphora wightii and C. myrrha Wed, 16 Mar 2016 13:12:42 +0000 Commercially important Commiphora species are drought-tolerant plants and they are leafless for most of the year. Therefore, it is necessary to develop some molecular marker for the identification. Intended for that, in the present study, species-specific, sequence-characterized amplified regions (SCAR) markers were developed for proficient and precise identification of closely related species Commiphora wightii and C. myrrha, which may ensure the quality, safety, and efficacy of medicines made from these plants through adulterous mixing of these plants. Two species-specific RAPD amplicons were selected, gel-purified, cloned, and sequenced after screening of 20 RAPD primers. The sequence of 979 and 590 nucleotides (Genebank accession numbers K90051 and K90052) was used for development of 4 SCAR markers, namely, Sc1P, Sc1Pm, Sc2P, and Sc2Pm. Out of them, the Sc1Pm was specific for C. wightii, while Sc2P discriminated both the Commiphora species. These markers are first reported and will be useful for rapid identification of closely related Commiphora wightii and C. myrrha species. Pramod Kumar Sairkar, Anjana Sharma, and N. P. Shukla Copyright © 2016 Pramod Kumar Sairkar et al. All rights reserved. Molecular Characterization of Human Rotavirus from Children with Diarrhoeal Disease in Sokoto State, Nigeria Wed, 09 Mar 2016 13:35:58 +0000 This study was conducted to detect and characterize prevalent human group A rotavirus strains from 200 diarrheic children in Sokoto, Nigeria, by ELISA, monoclonal antibody (Mab) serotyping and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) techniques. Rotavirus was detected in 25.5% of the children. The G-serotypes observed in circulation were G4: 16 (59.3%), G1: 4 (14.8%), G2: 3 (11.1%), G3: 3 (11.1%), and G12: 1 (3.7%). The monoclonal antibody (Mab) serotyping detected G1 and G3 but did not detect G4 and G2 serotypes. The Mab typing of the G1 and G3 serotypes was consistent with the result of the RT-PCR. The VP4 genotypes detected were P 3 (13%), P 11 (47.8%), and the rare human P genotype (P), found in 9 patients (39.1%). Nine strains identified with the common G and P combinations were G4 P 5 (56%), G4 P 1 (11%), G1 P 2 (22%), and G3 P 1 (11%), while seven strains with unusual combinations or rare G or P genotypes identified were G12 P 1 (14%), G2 P 2 (29%), and G4 P 4 (57%). To our knowledge this is the first molecular study of human rotavirus and report of rare human G and P serotypes in Sokoto State. B. R. Alkali, A. I. Daneji, A. A. Magaji, L. S. Bilbis, and F. Bande Copyright © 2016 B. R. Alkali et al. All rights reserved. Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli Sun, 07 Feb 2016 13:12:43 +0000 Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L−1) compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein’s functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform. Nagendra Suryanarayana, Vanlalhmuaka, Bharti Mankere, Monika Verma, Kulanthaivel Thavachelvam, and Urmil Tuteja Copyright © 2016 Nagendra Suryanarayana et al. All rights reserved. FOXO3a Gene Polymorphism Associated with Asthma in Indian Population Wed, 09 Dec 2015 13:12:09 +0000 Asthma is a chronic inflammatory disorder delineated by a heightened immunological response due to environmental or genetic factors. Single nucleotide polymorphism studies have shown that FOXO3a plays a pivotal role in maintaining immunoregulation. Polymorphism in FOXO3a has been linked to inflammatory diseases such as chronic obstructive pulmonary disease (COPD), Rheumatoid Arthritis, and Crohn’s disease suggesting that FOXO3a may be associated with asthma. Airway inflammation in asthma is characterized by activation of T helper type 2 (Th2) T cells and Foxo family members are reported to play critical roles in the suppression of T cell activation. Thus this study was undertaken to investigate an association between single nucleotide polymorphism of the FOXO3a (rs13217795, C>T transition) gene and asthma in Indian population. To our knowledge we are the first ones reporting an association between FOXO3a and asthma. Shravani Barkund, Tejas Shah, Nikhil Ambatkar, Maithili Gadgil, and Kalpana Joshi Copyright © 2015 Shravani Barkund et al. All rights reserved. Redox Changes Induced by General Anesthesia in Critically Ill Patients with Multiple Traumas Thu, 26 Nov 2015 09:34:48 +0000 The critically ill polytrauma patient is a constant challenge for the trauma team due to the complexity of the complications presented. Intense inflammatory response and infections, as well as multiple organ dysfunctions, significantly increase the rate of morbidity and mortality in these patients. Moreover, due to the physiological and biochemical imbalances present in this type of patients, the bioproduction of free radicals is significantly accelerated, thus installing the oxidative stress. In the therapeutic management of such patients, multiple surgical interventions are required and therefore they are being subjected to repeated general anesthesia. In this paper, we want to present the pathophysiological implications of oxidative stress in critically ill patients with multiple traumas and the implications of general anesthesia on the redox mechanisms of the cell. We also want to summarize the antioxidant treatments able to reduce the intensity of oxidative stress by modulating the biochemical activity of some cellular mechanisms. Marius Papurica, Alexandru Florin Rogobete, Dorel Sandesc, Raluca Dumache, Radu Nartita, Mirela Sarandan, Alina Carmen Cradigati, Loredana Luca, Corina Vernic, and Ovidiu Horea Bedreag Copyright © 2015 Marius Papurica et al. All rights reserved. Evaluation of Loop-Mediated Isothermal Amplification Assay for the Detection of Pneumocystis jirovecii in Immunocompromised Patients Thu, 19 Nov 2015 08:38:26 +0000 Pneumocystis pneumonia (PCP) is one of the common opportunistic infection among HIV and non-HIV immunocompromised patients. The lack of a rapid and specific diagnostic test necessitates a more reliable laboratory diagnostic test for PCP. In the present study, the loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Pneumocystis jirovecii. 185 clinical respiratory samples, including both BALF and IS, were subjected to GMS staining, nested PCR, and LAMP assay. Of 185 respiratory samples, 12/185 (6.5%), 41/185 (22.2%), and 49/185 (26.5%) samples were positive by GMS staining, nested PCR, and LAMP assay, respectively. As compared to nested PCR, additional 8 samples were positive by LAMP assay and found to be statistically significant () with the detection limit of 1 pg. Thus, the LAMP assay may serve as a better diagnostic tool for the detection of P. jirovecii with high sensitivity and specificity, less turn-around time, operational simplicity, single-step amplification, and immediate visual detection. Preeti Singh, Sundeep Singh, Bijay Ranjan Mirdha, Randeep Guleria, Sanjay Kumar Agarwal, and Anant Mohan Copyright © 2015 Preeti Singh et al. All rights reserved. Assessment of Immunotoxicity of Dextran Coated Ferrite Nanoparticles in Albino Mice Tue, 20 Oct 2015 13:56:23 +0000 In this study, dextran coated ferrite nanoparticles (DFNPs) of size <25 nm were synthesized, characterized, and evaluated for cytotoxicity, immunotoxicity, and oxidative stress by in vitro and in vivo methods. Cytotoxicity was performed in vitro using splenocytes with different concentrations of DFNPs. Gene expression of selected cytokines (IL-1, IL-10, and TNF β) secretion by splenocytes was evaluated. Also, 100 mg of DFNPs was injected intraperitoneally to 18 albino mice for immunological stimulations. Six animals each were sacrificed at the end of 7, 14, and 21 days. Spleen was subjected to immunotoxic response and liver was analyzed for antioxidant parameters (lipid peroxidation, reduced glutathione, glutathione peroxidase, superoxide dismutase, and glutathione reductase). The results indicated that DFNPs failed to induce any immunological reactions and no significant alternation in antioxidant defense mechanism. Also, mRNA expression of the cytokines revealed an increase in IL-10 expression and subsequent decreased expression of IL-1 and TNF β. Eventually, DNA sequencing of liver actin gene revealed base alteration in nonconserved regions (10–20 bases) of all the treated groups when compared to control samples. Hence, it can be concluded that the DFNPs were nontoxic at the cellular level and nonimmunotoxic when exposed intraperitoneally to mice. Santhakumar Syama, Viswanathan Gayathri, and Parayanthala Valappil Mohanan Copyright © 2015 Santhakumar Syama et al. All rights reserved. Electrostatic Interactions between Complement Regulator CD46(SCR1-2) and Adenovirus Ad11/Ad21 Fiber Protein Knob Wed, 19 Aug 2015 14:00:15 +0000 Adenoviruses bind to a variety of human cells to cause infection. Both the B2 adenovirus 11 and B1 adenovirus 21 use protein knobs to bind to complement regulator CD46(SCR1-2) in order to gain entry into host cells. In each complex, the two proteins are highly negatively charged but bind to each other at an interface with oppositely charged surface patches. We computationally generated single-alanine mutants of charged residues in the complexes CD46(SCR1-2)-Ad11k and CD46(SCR1-2)-Ad21k. We used electrostatic clustering and Poisson-Boltzmann free energy calculations to propose a hypothesis on the role of electrostatics in association. Our results delineate specific interfacial electrostatic interactions that are critical for association in both CD46(SCR1-2)-Ad11k and CD46(SCR1-2)-Ad21k. These results will serve as a predictive tool in the selection of mutants with desired binding affinity in experimental mutagenesis studies. This study will also serve as a foundation for the design of inhibitors to treat adenovirus infections. Carl Z. Chen, Ronald D. Gorham Jr., Zied Gaieb, and Dimitrios Morikis Copyright © 2015 Carl Z. Chen et al. All rights reserved. Systems Medicine: The Application of Systems Biology Approaches for Modern Medical Research and Drug Development Tue, 18 Aug 2015 09:51:21 +0000 The exponential development of highly advanced scientific and medical research technologies throughout the past 30 years has arrived to the point where the high number of characterized molecular agents related to pathogenesis cannot be readily integrated or processed by conventional analytical approaches. Indeed, the realization that several moieties are signatures of disease has partly led to the increment of complex diseases being characterized. Scientists and clinicians can now investigate and analyse any individual dysregulations occurring within the genomic, transcriptomic, miRnomic, proteomic, and metabolomic levels thanks to currently available advanced technologies. However, there are drawbacks within this scientific brave new age in that only isolated molecular levels are individually investigated for their influence in affecting any particular health condition. Since their conception in 1992, systems biology/medicine focuses mainly on the perturbations of overall pathway kinetics for the consequent onset and/or deterioration of the investigated condition/s. Systems medicine approaches can therefore be employed for shedding light in multiple research scenarios, ultimately leading to the practical result of uncovering novel dynamic interaction networks that are critical for influencing the course of medical conditions. Consequently, systems medicine also serves to identify clinically important molecular targets for diagnostic and therapeutic measures against such a condition. Duncan Ayers and Philip J. Day Copyright © 2015 Duncan Ayers and Philip J. Day. All rights reserved. Identification of Putative Molecular Markers Associated with Root Traits in Coffea canephora Pierre ex Froehner Tue, 03 Mar 2015 09:38:22 +0000 Coffea canephora exhibit poor root system and are very sensitive to drought stress that affects growth and production. Deeper root system has been largely empirical as better avoidance to soil water limitation in drought condition. The present study aimed to identify molecular markers linked to high root types in Coffea canephora using molecular markers. Contrasting parents, L1 valley with low root and S.3334 with high root type, were crossed, and 134 F1 individuals were phenotyped for root and associated physiological traits (29 traits) and genotyped with 41 of the 320 RAPD and 9 of the 55 SSR polymorphic primers. Single marker analysis was deployed for detecting the association of markers linked to root associated traits by SAS software. There were 13 putative RAPD markers associated with root traits such as root length, secondary roots, root dry weight, and root to shoot ratio, in which root length associated marker OPS1850 showed high phenotypic variance of 6.86%. Two microsatellite markers linked to root length (CPCM13400) and root to shoot ratio (CM211300). Besides, 25 markers were associated with more than one trait and few of the markers were associated with positively related physiological traits and can be used in marker assisted trait selection. Devaraja Achar, Mallikarjuana G. Awati, M. Udayakumar, and T. G. Prasad Copyright © 2015 Devaraja Achar et al. All rights reserved. Interleukin-6 c.-174G>C Polymorphism and Periodontitis in a Brazilian Population Thu, 04 Dec 2014 07:17:47 +0000 Aim. Periodontitis is an inflammatory disease that affects the teeth supporting structures, triggered by periodontal pathogens, and is influenced by environmental and genetic factors. Genes encoding molecules related to the immune response, such as cytokine, are the main candidates for polymorphisms analysis and may be possibly associated with this pathology. A G/C promoter polymorphism on the IL6 gene has been shown to affect basal IL-6 levels. The aim of this study was to investigate the association between the IL6 c.-174G>C polymorphism and periodontitis in individuals from Vitória da Conquista, Bahia, Brazil. Material and Methods. Three hundred and thirty individuals (134 cases, 196 controls) were genotyped for the IL6 c.-174G>C by MS-PCR technique. Concentrations of salivary IL-6 were determined by ELISA method. Results. The IL6 c.-174G>C polymorphism was associated with periodontitis when comparing the distribution of genotypes between patients with periodontitis and control subjects. The GC genotype appeared as a protective factor for periodontitis. Results showed increased levels of salivary IL-6 in periodontitis patients. Nevertheless, there was no relationship between the concentrations of IL-6 and genotypes when comparing the case and control groups. Conclusions. Our data indicate an association between IL6 c.-174G>C polymorphism and periodontitis and showed that IL-6 may be considered an important marker for periodontitis. Fernanda Gabriela Teixeira, Samir Andrade Mendonça, Kamilla Menezes Oliveira, Djanilson Barbosa dos Santos, Lucas Miranda Marques, Maise Mendonça Amorim, and Raquel de Souza Gestinari Copyright © 2014 Fernanda Gabriela Teixeira et al. All rights reserved. mTOR Signaling in Protein Translation Regulation: Implications in Cancer Genesis and Therapeutic Interventions Thu, 20 Nov 2014 12:51:19 +0000 mTOR is a central nutrient sensor that signals a cell to grow and proliferate. Through distinct protein complexes it regulates different levels of available cellular energy substrates required for cell growth. One of the important functions of the complex is to maintain available amino acid pool by regulating protein translation. Dysregulation of mTOR pathway leads to aberrant protein translation which manifests into various pathological states. Our review focuses on the role mTOR signaling plays in protein translation and its physiological role. It also throws some light on available data that show translation dysregulation as a cause of pathological complexities like cancer and the available drugs that target the pathway for cancer treatment. Mehvish Showkat, Mushtaq A. Beigh, and Khurshid I. Andrabi Copyright © 2014 Mehvish Showkat et al. All rights reserved. MCM Paradox: Abundance of Eukaryotic Replicative Helicases and Genomic Integrity Sun, 19 Oct 2014 08:43:02 +0000 As a crucial component of DNA replication licensing system, minichromosome maintenance (MCM) 2–7 complex acts as the eukaryotic DNA replicative helicase. The six related MCM proteins form a heterohexamer and bind with ORC, CDC6, and Cdt1 to form the prereplication complex. Although the MCMs are well known as replicative helicases, their overabundance and distribution patterns on chromatin present a paradox called the “MCM paradox.” Several approaches had been taken to solve the MCM paradox and describe the purpose of excess MCMs distributed beyond the replication origins. Alternative functions of these MCMs rather than a helicase had also been proposed. This review focuses on several models and concepts generated to solve the MCM paradox coinciding with their helicase function and provides insight into the concept that excess MCMs are meant for licensing dormant origins as a backup during replication stress. Finally, we extend our view towards the effect of alteration of MCM level. Though an excess MCM constituent is needed for normal cells to withstand stress, there must be a delineation of the threshold level in normal and malignant cells. This review also outlooks the future prospects to better understand the MCM biology. Mitali Das, Sunita Singh, Satyajit Pradhan, and Gopeshwar Narayan Copyright © 2014 Mitali Das et al. All rights reserved. An Improved Method for Soil DNA Extraction to Study the Microbial Assortment within Rhizospheric Region Mon, 15 Sep 2014 05:55:46 +0000 The need for identification of soil microbial community mainly depends on direct extraction of DNA from soil, a multifaceted environment that is a major pool for microbial genetic diversity. The soil DNA extraction procedures usually suffer from two major problems, namely, inappropriate rupturing of cells and contamination with humic substances. In the present study, five protocols for single type of rhizospheric soil were investigated and their comparison indicated that the inclusion of 120 mM phosphate buffered saline (PBS) for washing and mannitol in the lysis buffer allowed the processing of soil sample in minimal time with no specific equipment requirement. Furthermore, DNA purity and yield were also improved, which allowed the exploitation of genetic potential of soil microbes within soil sample thereby facilitating the amplification of metagenomic DNA. The effectiveness of methods was analyzed using random amplification of polymorphic DNA. The banding patterns revealed that both the abundance and the composition of indigenous microbial community depend on the DNA recovery method. Faria Fatima, Neelam Pathak, and Smita Rastogi Verma Copyright © 2014 Faria Fatima et al. All rights reserved. Human Epidermal Growth Factor Receptor 2 (HER2) in Cancers: Overexpression and Therapeutic Implications Sun, 07 Sep 2014 08:11:44 +0000 Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family having tyrosine kinase activity. Dimerization of the receptor results in the autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptors and initiates a variety of signaling pathways leading to cell proliferation and tumorigenesis. Amplification or overexpression of HER2 occurs in approximately 15–30% of breast cancers and 10–30% of gastric/gastroesophageal cancers and serves as a prognostic and predictive biomarker. HER2 overexpression has also been seen in other cancers like ovary, endometrium, bladder, lung, colon, and head and neck. The introduction of HER2 directed therapies has dramatically influenced the outcome of patients with HER2 positive breast and gastric/gastroesophageal cancers; however, the results have been proved disappointing in other HER2 overexpressing cancers. This review discusses the role of HER2 in various cancers and therapeutic modalities available targeting HER2. Nida Iqbal and Naveed Iqbal Copyright © 2014 Nida Iqbal and Naveed Iqbal. All rights reserved. Regulatory Variants and Disease: The E-Cadherin −160C/A SNP as an Example Tue, 02 Sep 2014 09:55:38 +0000 Single nucleotide polymorphisms (SNPs) occurring in noncoding sequences have largely been ignored in genome-wide association studies (GWAS). Yet, amounting evidence suggests that many noncoding SNPs especially those that are in the vicinity of protein coding genes play important roles in shaping chromatin structure and regulate gene expression and, as such, are implicated in a wide variety of diseases. One of such regulatory SNPs (rSNPs) is the E-cadherin (CDH1) promoter −160C/A SNP (rs16260) which is known to affect E-cadherin promoter transcription by displacing transcription factor binding and has been extensively scrutinized for its association with several diseases especially malignancies. Findings from studying this SNP highlight important clinical relevance of rSNPs and justify their inclusion in future GWAS to identify novel disease causing SNPs. Gongcheng Li, Tiejun Pan, Dan Guo, and Long-Cheng Li Copyright © 2014 Gongcheng Li et al. All rights reserved. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase Sun, 17 Aug 2014 12:13:49 +0000 As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition. Peter McInerney, Paul Adams, and Masood Z. Hadi Copyright © 2014 Peter McInerney et al. All rights reserved. The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons Thu, 10 Jul 2014 00:00:00 +0000 In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis. Ingo C. Starke, Wilfried Vahjen, Robert Pieper, and Jürgen Zentek Copyright © 2014 Ingo C. Starke et al. All rights reserved. Expression of TPM1, a Novel Sarcomeric Isoform of the TPM1 Gene, in Mouse Heart and Skeletal Muscle Thu, 24 Apr 2014 13:56:25 +0000 We have investigated the expression of TPM1α and TPM1κ in mouse striated muscles. TPM1α and TMP1κ were amplified from the cDNA of mouse heart by using conventional RT-PCR. We have cloned the PCR amplified DNA and determined the nucleotide sequences. Deduced amino acid sequences show that there are three amino acid changes in mouse exon 2a when compared with the human TPM1κ. However, the deduced amino acid sequences of human TPM1α and mouse TPM1α are identical. Conventional RT-PCR data as well as qRT-PCR data, calculating both absolute copy number and relative expression, revealed that the expression of TPM1κ is significantly lower compared to TPM1α in both mouse heart and skeletal muscle. It was also found that the expression level of TPM1κ transcripts in mouse heart is higher than it is in skeletal muscle. To the best of our knowledge, this is the first report of the expression of TPM1κ in mammalian skeletal muscle. Syamalima Dube, Lauren Panebianco, Amr A. Matoq, Henry N. Chionuma, Christopher R. Denz, Bernard J. Poiesz, and Dipak K. Dube Copyright © 2014 Syamalima Dube et al. All rights reserved. Primer Based Approach for PCR Amplification of High GC Content Gene: Mycobacterium Gene as a Model Mon, 24 Mar 2014 16:58:57 +0000 The genome of Mycobacterium is rich in GC content and poses problem in amplification of some genes, especially those rich in the GC content in terminal regions, by standard/routine PCR procedures. Attempts have been made to amplify three GC rich genes of Mycobacterium sp. (Rv0519c and Rv0774c from M. tuberculosis and ML0314c from M. leprae). Out of these three genes, Rv0774c gene was amplified with normal primers under standard PCR conditions, while no amplification was observed in case of Rv0519c and ML0314c genes. In the present investigation a modified primer based approach was successfully used for amplification of GC rich sequence of Rv0519c through codon optimization without changing the native amino acid sequence. The strategy was successfully confirmed by redesigning the standard primers with similar modifications followed by amplification of ML0314c gene. Arbind Kumar and Jagdeep Kaur Copyright © 2014 Arbind Kumar and Jagdeep Kaur. All rights reserved. The Role of Suppressors of Cytokine Signalling in Human Neoplasms Sun, 16 Mar 2014 13:46:47 +0000 Suppressors of cytokine signalling 1–7 (SOCS1–7) and cytokine-inducible SH2-containing protein (CIS) are a group of intracellular proteins that are well known as JAK-STAT and several other signalling pathways negative feedback regulators. More recently several members have been identified as tumour suppressors and dysregulation of their biological roles in controlling cytokine and growth factor signalling may contribute to the development of many solid organ and haematological malignancies. This review explores their biological functions and their possible tumour suppressing role in human neoplasms. Walid Sasi, Anup K. Sharma, and Kefah Mokbel Copyright © 2014 Walid Sasi et al. All rights reserved. A Synthetic Interaction between CDC20 and RAD4 in Saccharomyces cerevisiae upon UV Irradiation Sun, 23 Feb 2014 09:50:56 +0000 Regulation of DNA repair can be achieved through ubiquitin-mediated degradation of transiently induced proteins. In Saccharomyces cerevisiae, Rad4 is involved in damage recognition during nucleotide excision repair (NER) and, in conjunction with Rad23, recruits other proteins to the site of damage. We identified a synthetic interaction upon UV exposure between Rad4 and Cdc20, a protein that modulates the activity of the anaphase promoting complex (APC/C), a multisubunit E3 ubiquitin ligase complex. The moderately UV sensitive Δrad4 strain became highly sensitive when cdc20-1 was present, and was rescued by overexpression of CDC20. The double mutant is also deficient in elicting RNR3-lacZ transcription upon exposure to UV irradiation or 4-NQO compared with the Δrad4 single mutant. We demonstrate that the Δrad4/cdc20-1 double mutant is defective in double strand break repair by way of a plasmid end-joining assay, indicating that Rad4 acts to ensure that damaged DNA is repaired via a Cdc20-mediated mechanism. This study is the first to present evidence that Cdc20 may play a role in the degradation of proteins involved in nucleotide excision repair. Bernadette Connors, Lauren Rochelle, Asela Roberts, and Graham Howard Copyright © 2014 Bernadette Connors et al. All rights reserved. Cancer Stem Cells Accountability in Progression of Head and Neck Squamous Cell Carcinoma: The Most Recent Trends! Wed, 19 Feb 2014 12:07:22 +0000 Cancer stem cells (CSCs) play a major role in local recurrence and metastatic spread in head and neck squamous cell carcinomas (HNSCC). Evidence suggests that cancer stem cells are resistant to conventional therapy. So the emerging concepts of the role of cancer stem cells in the pathobiology of HNSCC should be understood carefully to be able to create new paradigms in treatment plans. Samapika Routray and Neeta Mohanty Copyright © 2014 Samapika Routray and Neeta Mohanty. All rights reserved. Sequence Characterization of Mitochondrial 12S rRNA Gene in Mouse Deer (Moschiola indica) for PCR-RFLP Based Species Identification Mon, 23 Dec 2013 10:04:21 +0000 Mitochondrial 12S rRNA has proven to be a useful molecular marker for better conservation and management of the endangered species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the mitochondrial 12S rRNA gene has proven to be a reliable and efficient tool for the identification of different Indian deer species of family cervidae. In the present study, mitochondrial 12S rRNA gene sequence of mouse deer (Moschiola indica) belonging to the family Tragulidae was characterized and analysed in silico for its use in species identification. Genomic DNA was isolated from the hair follicles and mitochondrial 12S rRNA gene was amplified using universal primers. PCR product was cloned and sequenced for the first time. The sequence of mouse deer showed 90.04, 90.08, 90.04, 91.2, 90.04, and 90.08% identities with sika deer, sambar, hog deer, musk deer, chital, and barking deer, respectively. Restriction mapping in Lasergene (DNAstar Inc., Madison, WI, USA) revealed that mouse deer mitochondrial 12S rRNA gene sequence can be differentiated from the other deer species in PCR-RFLP using RsaI, DdeI, BsrI, and BstSFI. With the help of predicted pattern, mouse deer can be identified using genomic DNA from a variety of biomaterials, thereby providing molecular aid in wildlife forensics and conservation of the species. Chandra Mohan Siddappa, Mohini Saini, Asit Das, Ramesh Doreswamy, Anil K. Sharma, and Praveen K. Gupta Copyright © 2013 Chandra Mohan Siddappa et al. All rights reserved. Investigation of the Association between Genetic Polymorphism of Microsomal Epoxide Hydrolase and Primary Brain Tumor Incidence Mon, 16 Dec 2013 09:18:07 +0000 mEH is a critical biotransformation enzyme that catalyzes the conversion of xenobiotic epoxide substrates into more polar diol metabolites: it is also capable of inactivating a large number of structurally different molecules. Two polymorphisms affecting enzyme activity have been described in the exon 3 and 4 of the mEH gene. The hypothesis of this study is that inherent genetic susceptibility to a primary brain tumor is associated with mEH gene polymorphisms. The polymorphisms of the mEH gene were determined with PCR-RFLP techniques and 255 Turkish individuals. Our results indicate that the frequency of the mEH exon 4 polymorphism (in controls) is significantly higher than that of primary brain tumor patients (OR = 1.8, 95% CI = 1.0–3.4). This report, however, failed to demonstrate a significant association between mEH exon 3 polymorphism and primary brain tumor susceptibility in this population. Analysis of patients by both histological types of primary brain tumor and gene variants showed no association, although analysis of family history of cancer between cases and controls showed a statistically significant association (, ). Our results marginally support the hypothesis that genetic susceptibility to brain tumors may be associated with mEPHX gene polymorphisms. Ali Aydin, Hatice Pinarbasi, and Mustafa Gurelik Copyright © 2013 Ali Aydin et al. All rights reserved. The RASSF1 Gene and the Opposing Effects of the RASSF1A and RASSF1C Isoforms on Cell Proliferation and Apoptosis Tue, 12 Nov 2013 15:11:27 +0000 RASSF1A has been demonstrated to be a tumor suppressor, while RASSF1C is now emerging as a growth promoting protein in breast and lung cancer cells. To further highlight the dual functionality of the RASSF1 gene, we have compared the effects of RASSF1A and RASSF1C on cell proliferation and apoptosis in the presence of TNF-α. Overexpression of RASSF1C in breast and lung cancer cells reduced the effects of TNF-α on cell proliferation, apoptosis, and MST1/2 phosphorylation, while overexpression of RASSF1A had the opposite effect. We also assessed the expression of RASSF1A and RASSF1C in breast and lung tumor and matched normal tissues. We found that RASSF1A mRNA levels are significantly higher than RASSF1C mRNA levels in all normal breast and lung tissues examined. In addition, RASSF1A expression is significantly downregulated in 92% of breast tumors and in 53% of lung tumors. Conversely, RASSF1C was upregulated in 62% of breast tumors and in 47% of lung tumors. Together, these findings suggest that RASSF1C, unlike RASSF1A, is not a tumor suppressor but instead may play a role in stimulating survival in breast and lung cancer cells. Mark E. Reeves, Matthew Firek, Shin-Tai Chen, and Yousef Amaar Copyright © 2013 Mark E. Reeves et al. All rights reserved. Communication and the Emergence of Collective Behavior in Living Organisms: A Quantum Approach Wed, 30 Oct 2013 13:47:07 +0000 Intermolecular interactions within living organisms have been found to occur not as individual independent events but as a part of a collective array of interconnected events. The problem of the emergence of this collective dynamics and of the correlated biocommunication therefore arises. In the present paper we review the proposals given within the paradigm of modern molecular biology and those given by some holistic approaches to biology. In recent times, the collective behavior of ensembles of microscopic units (atoms/molecules) has been addressed in the conceptual framework of Quantum Field Theory. The possibility of producing physical states where all the components of the ensemble move in unison has been recognized. In such cases, electromagnetic fields trapped within the ensemble appear. In the present paper we present a scheme based on Quantum Field Theory where molecules are able to move in phase-correlated unison among them and with a self-produced electromagnetic field. Experimental corroboration of this scheme is presented. Some consequences for future biological developments are discussed. Marco Bischof and Emilio Del Giudice Copyright © 2013 Marco Bischof and Emilio Del Giudice. All rights reserved. The Transcriptomics of Secondary Growth and Wood Formation in Conifers Tue, 29 Oct 2013 13:27:55 +0000 In the last years, forestry scientists have adapted genomics and next-generation sequencing (NGS) technologies to the search for candidate genes related to the transcriptomics of secondary growth and wood formation in several tree species. Gymnosperms, in particular, the conifers, are ecologically and economically important, namely, for the production of wood and other forestry end products. Until very recently, no whole genome sequencing of a conifer genome was available. Due to the gradual improvement of the NGS technologies and inherent bioinformatics tools, two draft assemblies of the whole genomes sequence of Picea abies and Picea glauca arose in the current year. These draft genome assemblies will bring new insights about the structure, content, and evolution of the conifer genomes. Furthermore, new directions in the forestry, breeding and research of conifers will be discussed in the following. The identification of genes associated with the xylem transcriptome and the knowledge of their regulatory mechanisms will provide less time-consuming breeding cycles and a high accuracy for the selection of traits related to wood production and quality. Ana Carvalho, Jorge Paiva, José Louzada, and José Lima-Brito Copyright © 2013 Ana Carvalho et al. All rights reserved. GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents Sun, 28 Apr 2013 17:44:01 +0000 Quantitative real-time PCR (qPCR) is broadly used to detect and quantify nucleic acid targets. In order to determine cell copy number and genome equivalents, a suitable reference gene that is present in a defined number in the genome is needed, preferably as a single copy gene. For most organisms, a variable number of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes have been reported. However, it has been suggested that a single-copy of the GAPDH pseudogene is present in the feline genome and that a GAPDH assay can therefore be used to quantify feline genomic DNA (gDNA). The aim of this study was to determine whether one or more GAPDH pseudogenes are present in the feline genome and to provide a suitable alternative qPCR system for the quantification of feline cell copy number and genome equivalents. Bioinformatics and sequencing results revealed that not just one but several closely related GAPDH-like sequences were present in the cat genome. We thus identified, developed, optimized, and validated an alternative reference gene assay using feline albumin (fALB). Our data emphasize the need for an alternative reference gene, apart from the GAPDH pseudogene, for the normalization of gDNA levels. We recommend using the fALB qPCR assay for future studies. A. Katrin Helfer-Hungerbuehler, Stefan Widmer, and Regina Hofmann-Lehmann Copyright © 2013 A. Katrin Helfer-Hungerbuehler et al. All rights reserved. Prevention of Lysosomal Storage Diseases and Derivation of Mutant Stem Cell Lines by Preimplantation Genetic Diagnosis Wed, 26 Dec 2012 07:53:51 +0000 Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research. Gheona Altarescu, Rachel Beeri, Rachel Eiges, Silvina Epsztejn-Litman, Talia Eldar-Geva, Deborah Elstein, Ari Zimran, Ehud J. Margalioth, Ephrat Levy-Lahad, and Paul Renbaum Copyright © 2012 Gheona Altarescu et al. All rights reserved.