Search results for: mitochondrial 16s rRNA gene

Authors: Y. Pilehvar-Soltanahmadi, F. Badrzadeh, N. Zarghami, S. Jalilzadeh-Tabrizi, R. Zamani

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Herbal compounds such as curcumin which decrease telomerase and gene expression have been considered as beneficial tools for lung cancer treatment. In this article, we compared the effects of pure curcumin and curcumin-loaded NIPAAm-MAA nanoparticles on telomerase and PinX1 gene expression in a lung cancer cell line. A tetrazolium-based assay was used for determination of cytotoxic effects of curcumin on the Calu-6 lung cancer cell line and telomerase and pinX1 gene expression was measured with real-time PCR. MTT assay showed that Curcumin-loaded NIPAAm-MAA inhibited the growth of the Calu-6 lung cancer cell line in a time and dose-dependent manner. Our q-PCR results showed that the expression of telomerase gene was effectively reduced as the concentration of curcumin-loaded NIPAAm-MAA increased while expression of the PinX1 gene became elevated. The results showed that curcumin loaded NIPAAm-MAA exerted cytotoxic effects on the Calu-6 cell line through down-regulation of telomerase and stimulation of pinX1 gene expression. NIPPAm-MAA could be the good carrier for such kinds of hydrophobic agent.

Keywords: curcumin, NIPAAm-MAA, PinX1, telomerase, lung cancer cells

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Authors: S. S. Singh, A. Bhattacharya, S. Bhattacharya

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The eukaryotic exosome complex plays a pivotal role in RNA biogenesis, maturation, surveillance and differential expression of various RNAs in response to varying environmental signals. The exosome is composed of evolutionary conserved nine core subunits and the associated exonucleases Rrp6 and Rrp44. Rrp6p is crucial for the processing of rRNAs, other non-coding RNAs, regulation of polyA tail length and termination of transcription. Rrp6p, a 3’-5’ exonuclease is required for degradation of 5’-external transcribed spacer (ETS) released from the rRNA precursors during the early steps of pre-rRNA processing. In the parasitic protist Entamoeba histolytica in response to growth stress, there occurs the accumulation of unprocessed pre-rRNA and 5’ ETS sub fragment. To understand the processes leading to this accumulation, we looked for Rrp6 and the exosome subunits in E. histolytica, by in silico approaches. Of the nine core exosomal subunits, seven had high percentage of sequence similarity with the yeast and human. The EhRrp6 homolog contained exoribonuclease and HRDC domains like yeast but its N- terminus lacked the PMC2NT domain. EhRrp6 complemented the temperature sensitive phenotype of yeast rrp6Δ cells suggesting conservation of biological activity. We showed 3’-5’ exoribonuclease activity of EhRrp6p with in vitro-synthesized appropriate RNAs substrates. Like the yeast enzyme, EhRrp6p degraded unstructured RNA, but could degrade the stem-loops slowly. Furthermore, immunolocalization revealed that EhRrp6 was nuclear-localized in normal cells but was diminished from nucleus during serum starvation, which could explain the accumulation of 5’ETS during stress. Our study shows functional conservation of EhRrp6p in E.histolytica, an early-branching eukaryote, and will help to understand the evolution of exosomal components and their regulatory function.

Keywords: entamoeba histolytica, exosome complex, rRNA processing, Rrp6

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Authors: Fitria D. Ayuningtyas, Anggraini Barlian

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Green turtle (Chelonia mydas) is one of TSD (Temperature-dependent Sex Determination, TSD) animals which sex is determined by the egg’s incubation temperature. GSD (Genotypic Sex Determination) homologous genes such as Wilms’ Tumor (Wt1) and Forkhead Box L2 (FoxL2) play a role in TSD animal sex determination process. Wt1 plays a role in both male pathway, as a transcription factor for Sf1 gene and in female pathway, as a transcription factor for Dax1. FoxL2 plays a role specifically in female sex determination, and known as transcriptional factor for Aromatase gene. Until now, research on the pattern of Wt1 and FoxL2 genes expression in C.mydas has not been conducted yet. The aim of this research is to know the pattern of Wt1 and FoxL2 genes expression in Mesonephros-Gonad (MG) complexes of Chelonia mydas embryos incubated in masculinizing temperature (MT) and feminizing temperature (FT). Eggs of C.mydas incubated in 3 different stage of TSP (Thermosensitive Period) at masculinizing temperature (26±10C, MT) and feminizing temperature (31±10C FT). Mesonefros-gonad complexes were isolated at Pre-TSP stage (FT at days 14th, MT at days 24th), TSP stage (FT at days 24th, MT at days 36th) and differentiated stage (FT at days 40th, MT at days 58th). RNA from mesonephros-gonad (MG) complexes were converted into cDNA by RT-PCR process, and the pattern of Wt1 and FoxL2 genes expression is analyzed by quantitative Real Time PCR (qPCR) method, β-actin gene is used as an internal control. The pattern of Wt1 gene expression in Pre-TSP stage was almost the same between MG complexes incubated at MT or FT, while TSP and differentiation stage, the pattern of Wt1 gene expression in MG complexes incubated at MT or FT was increased. Wt1 gene expression of MG complexes that incubated at FT was higher than at MT. There was a difference pattern between Wt1 gene expression in this research compared to the previous research in protein level. It could be assumed that the difference caused by post-transcriptional regulation mechanisms before mRNA of Wt1 gene translated into protein structure. The pattern of FoxL2 gene expression in Pre-TSP stage was almost the same between MG complexes that incubated at MT and FT, and increased in both TSP and differentiated stage. The FoxL2 gene expression in MG complexes that incubated in FT is higher than MT on TSP and differentiated stage. Based on the results of this research, it can be assumed that Wt1 and FoxL2 gene were expressed in MG complexes that incubated both at MT and FT since Pre-TSP stage. The pattern of Wt1 gene expression was increased in every stage of gonadal development, and so do the pattern of FoxL2 gene expression. Wt1 and FoxL2 gene expressions were higher in MG complexes incubated at FT than MT.

Keywords: chelonia mydas, FoxL2, gene expression, TSD, Wt1

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Authors: Debasmita Dubey, Rajesh Kumar Meher, Smruti Pragya Samal, Pradeep Kumar Naik

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Background: To determine antioxidant properties and anticancer activity by ROS and mitochondrial transmembrane potential mediated apoptosis against MCF7, MDA-MB-231, cell line. Methods: Leaf sample was extracted using methanol by microwave digestion technique. The antioxidant properties of the methanolic extract were determined by a DPPH scavenging assay. In vitro anticancer activity, mitochondrial transmembrane potential, apoptosis activity and DNA fragmentation study, as well as intracellular ROS activity of most potential leaf extract, were also determined by using the MDA-MB-231cell line. In vivo animal toxicity study was carried out using mice model. Results: Methanolic leaf extract has shown the highest antioxidant, as well as anticancer activity, is based on the assay conducted. For the identification of active phytochemicals from methanolic extract, High-resolution mass spectroscopy-LCMS was used. In vitro cytotoxicity study against MCF-7 and MDA-MB-231 cell line and IC 50 value was found to be 37.5µg/ml. From histopathological studies, no toxicity in liver and kidney tissue was identified. Conclusion: This plant tuber can be used as a regular diet to reduce the chance of breast cancer. Further, more studies should be conducted to isolate and identify the responsible compound.

Keywords: human breast adenocarcinoma, ROS, mitochondrial transmembrane, apoptosis

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Authors: Stephy Saavedra, Annsy C. Arredondo, Gisele Monteiro, Adalberto Pessoa Jr, Carol N. Flores-Fernandez, Amparo I. Zavaleta

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L-asparaginase from bacterial sources is used in leukemic treatment and food industry. This enzyme is classified based on its affinity towards L-asparagine and L-glutamine. Likewise, ansZ genes express L-asparaginase with higher affinity to L-asparagine. The aim of this work was to clone and express of ansZ gene from Bacillus sp. CH11 isolated from Chilca salterns in Peru. The gene encoding L-asparaginase was cloned into pET15b vector and transformed in Escherichia coli BL21 (DE3) pLysS. The expression was carried out in a batch culture using LB broth and 0.5 mM IPTG. The recombinant L-asparaginase showed a molecular weight of ~ 39 kDa by SDS PAGE and a specific activity of 3.19 IU/mg of protein. The cloning and expression of ansZ gene from this halotolerant Bacillus sp. CH11 allowed having a biological input to improve a future scaling-up.

Keywords: ansZ gene, Bacillus sp, Chilca salterns, recombinant L-asparaginase

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Authors: Sirous Eydivandi

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Diacylglycerol-O-acyltransferase (DGAT1) gene encodes diacylglycerol transferase enzyme that plays an important role in glycerol lipid metabolism. DGAT1 is considered to be the key enzyme in controlling the synthesis of triglycerides in adipocytes. This enzyme catalyzes the final step of triglyceride synthesis (transform triacylglycerol (DAG) into triacylglycerol (TAG). A total of 20 DGAT1 gene sequences and corresponding amino acids belonging to 4 species include cattle, goats, sheep and yaks were analyzed, and the differentiation within and among the species was also studied. The length of the DGAT1 gene varies greatly, from 1527 to 1785 bp, due to deletion, insertion, and stop codon mutation resulting in elongation. Observed genetic diversity was higher among species than within species, and Goat had more polymorphisms than any other species. Novel amino acid variation sites were detected within several species which might be used to illustrate the functional variation. Differentiation of the DGAT1 gene was obvious among species, and the clustering result was consistent with the taxonomy in the National Center for Biotechnology Information.

Keywords: DGAT1gene, bioinformatic, ruminnants, biotechnology information

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Authors: K. Petrosyan, R. Piwowarczyk, K. Ruraż, S. Thijs, J. Vangronsveld, W. Kaca

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Holoparasitic plants are flowering heterotrophic angiosperms which with the help of an absorbing organ - haustorium, attach to another plant, the so-called the host. Due to the different hosts, unusual lifestyle, lack of roots, chlorophylls and photosynthesis, these plants are interesting and unique study objects for global biodiversity. The seeds germination of the parasitic plants also is unique: they germinate only in response to germination stimulants, namely strigolactones produced by the root of an appropriate host. Resistance of the seeds on different environmental conditions allow them to stay viable in the soil for more than 20 years. Among the wide range of plant protection mechanisms the endophytic communities have a specific role. In this way, they have the potential to mitigate the impacts of adverse conditions such as soil salinization. The major objective of our study was to compare the bacterial endo-microbiomes from seeds of two holoparasitic plants from Orobanchaceae family, Cistanche – C. armena (Armenia) and C. phelypaea (Portugal) – from saline habitats different in soil water status. The research aimed to perform how environmental conditions influence on the diversity of the bacterial communities of C. armena and C. phelypaea seeds. This was achieved by comparison of the endophytic microbiomes of two species and isolation of culturable bacteria. A combination of culture-dependent and molecular techniques was employed for the identification of the seed endomicrobiome (culturable and unculturable). Using the V3-V4 hypervariable region of the 16S rRNA gene, four main taxa were identified: Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, but the relative proportion of the taxa was different in each type of seed. Generally, sixteen phyla, 323 genera and 710 bacterial species were identified, mainly Gram negative, halotolerant bacteria with an environmental origin. However, also some unclassified and unexplored taxonomic groups were found in the seeds of both plants. 16S rRNA gene sequencing analysis from both species identified the gram positive, endospore forming, halotolerant and alkaliphile Bacillus spp. which suggests that the endophytic bacteria of examined seeds possess traits that are correlated with the natural habitat of their hosts. The cultivable seed endophytes from C. armena and C. phelypaea were rather similar, notwithstanding the big distances between their growth habitats - Armenia and Portugal. Although the seed endophytic microbiomes of C. armena and C. phelypaea contain a high number of common bacterial taxa, also remarkable differences exist. We demonstrated that the environmental conditions or abiotic stresses influence on diversity of the bacterial communities of holoparasiotic seeds. To the best of our knowledge the research is the first report of endophytes from seeds of holoparasitic Cistanche armena and C. phelypaea plants.

Keywords: microbiome, parasitic plant, salinity, seeds

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Authors: Mohammadreza Hajialigol, Nargues Falahi Charkhabi, Fatemeh Shahryari, Saadat Sarikhani

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BACKGROUND AND OBJECTIVES Walnut canker is characterized by brown to blackish roundish blotches on the trunks and main branches, necrosis of inner bark and bleeding with dark brown to black-colored exudates. The present study aimed to identify the causative agents of walnut decline by their phenotypic features, approval of pathogenicity, the partial sequencing of the housekeeping genes in Razavi Khorasan. MATERIAL AND METHODS Ten Symptomatic samples were collected from walnut orchards of Razavi Khorasan in 2019. Pathogenicity of all isolated strains was carried out on walnut immature fruits cv. ‘Hartley’ and young green twigs of cv. ‘Chandler’. All pathogenic strains were subjected to physiological, morphological and biochemical tests. 16S rRNA and housekeeping genes (fusA, leuS, and pyrG) were partially amplified and sequenced. RESULTS Eight strains were able to cause necrosis and a dark-colored region in the mesocarp of immature walnut fruits, and three representative strains caused necrosis on young inoculated twigs. Strains utilized starch, however, did not utilized esculin, Tween 20, Tween 80, and gelatin. The partial 16S rRNA gene sequence of strain KH7 indicated 99.63 % similarity to that of Citrobacter braakii ATCC5113T. The phylogenetic analyses based on the partial sequencing of three housekeeping genes, fusA (633 bp), pyrG (305), and leuS (640 bp), demonstrated that strains KH1, KH3, and KH7 belong to C. braakii species in a monophyletic clade with high bootstrap support. CONCLUSION To the best of our knowledge, this is the first report of C. braakii as a new plant pathogen which cause walnut decline. Identification of bacteria associated with walnut decline will eventually improve our understanding of the etiology of the disease and may result in improved management techniques for control.

Keywords: emerging pathogens, Iran, juglans regia, MLSA

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Authors: M. Ottenbrite, G. Yilmaz, J. Devenish, M. Kang, H. Dan, M. Lin, C. Lau, C. Carrillo, K. Bessonov, J. Nash, E. Topp, J. Guan

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Consumption of food contaminated by antibiotic-resistant (AR) bacteria may lead to the transmission of AR genes in the gut microbiota and cause AR bacterial infection, a significant public health concern. However, information is limited on if and how background microbes from the food matrix (food microbes) may influence resistance transmission. Thus, we assessed the colonization of a β-lactam resistant Salmonella Heidelberg strain (donor) and a β-lactam susceptible S. Typhimurium strain (recipient) and the transfer of the resistance genes in the mouse gut in the presence or absence of food microbes that were derived from washing freshly-harvested carrots. Mice were pre-treated with streptomycin and then inoculated with both donor and recipient bacteria or recipient only. Fecal shedding of the donor, recipient, and transconjugant bacteria was enumerated using selective culture techniques. Transfer of AR genes was confirmed by whole genome sequencing. Gut microbial composition was determined by 16s rRNA amplicon sequencing. Significantly lower numbers of donors and recipients were shed from mice that were inoculated with food microbes compared to those without food microbe inoculation. S. Typhimurium transconjugants were only recovered from mice without inoculation of food microbes. A significantly higher survival rate was in mice with vs. without inoculation of food microbes. The results suggest that the food microbes may compete with both the donor and recipient Salmonella, limit their growth and reduce transmission of the β-lactam resistance gene in the mouse gut.

Keywords: antibiotic resistance, gene transfer, gut microbiota, Salmonella infection

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Authors: Anna Höfer, Johannes Herrmann, Patrick Meybohm, Christopher Lotz

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Mitochondria are pivotal for energy supply and regulation of cellular functions. Deficiencies of mitochondrial metabolism have been implicated in diverse stressful conditions including infections. Platelets are key mediators for thrombo-inflammation during development and resolution of acute respiratory distress syndrome (ARDS). Previous data point to an exhausted platelet phenotype in critically-ill patients with coronavirus 19 disease (COVID-19) impacting the course of disease. The objective of this work was to characterize platelet mitochondrial metabolism in patients suffering from COVID-19 ARDSA longitudinal analysis of platelet mitochondrial metabolism in 24 patients with COVID-19 induced ARDS compared to 35 healthy controls (ctrl) was performed. Blood samples were analyzed at two time points (t1=day 1; t2=day 5-7 after study inclusion). The activity of mitochondrial citrate synthase was photometrically measured. The impact of oxidative stress on mitochondrial permeability was assessed by a photometric calcium-induced swelling assay and the activity of superoxide dismutase (SOD) by a SOD assay kit. The amount of protein carbonylation and the activity of mitochondria complexes I-IV were photometrically determined. Levels of interleukins (IL)-1α, IL-1β and tumor necrosis factor (TNF-) α were measured by a Multiplex assay kit. Median age was 54 years, 63 % were male and BMI was 29.8 kg/m2. SOFA (12; IQR: 10-15) and APACHE II (27; IQR: 24-30) indicated critical illness. Median Murray Score was 3.4 (IQR: 2.8-3.4), 21/24 (88%) required mechanical ventilation and V-V ECMO support in 14/24 (58%). Platelet counts in ARDS did not change during ICU stay (t1: 212 vs. t2: 209 x109/L). However, mean platelet volume (MPV) significantly increased (t1: 10.6 vs. t2: 11.9 fL; p<0.0001). Citrate synthase activity showed no significant differences between ctrl and ARDS patients. Calcium induced swelling was more pronounced in patients at t1 compared to t2 and to ctrl (50µM; t1: 0.006 vs. ctrl: 0.016 ΔOD; p=0.001). The amount of protein carbonylation as marker for irreversible proteomic modification constantly increased during ICU stay and compared to ctrl., without reaching significance. In parallel, superoxid dismutase activity gradually declined during ICU treatment vs. ctrl (t2: - 29 vs. ctrl.: - 17 %; p=0.0464). Complex I analysis revealed significantly stronger activity in ARDS vs. ctrl. (t1: 0.633 vs. ctrl.: 0.415 ΔOD; p=0.0086). There were no significant differences in complex II, III or IV activity in platelets from ARDS patients compared to ctrl. IL-18 constantly increased during the observation period without reaching significance. IL-1α and TNF-α did not differ from ctrl. However, IL-1β levels were significantly elevated in ARDS (t1: 16.8; t2: 16.6 vs. ctrl.: 12.4 pg/mL; p1=0.0335, p2=0.0032). This study reveals new insights in platelet mitochondrial metabolism during COVID-19 caused ARDS. it data point towards enhanced platelet activity with a pronounced turnover rate. We found increased activity of mitochondria complex I and evidence for enhanced oxidative stress. In parallel, protective mechanisms against oxidative stress were narrowed with elevated levels of IL-1β likely causing a pro-apoptotic environment. These mechanisms may contribute to platelet exhaustion in ARDS.

Keywords: acute respiratory distress syndrome (ARDS), coronavirus 19 disease (COVID-19), oxidative stress, platelet mitochondrial metabolism

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Authors: Ankita Shukla, Tiratha Raj Singh

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Colorectal cancer (CRC) imposes serious mortality burden worldwide and it has been increasing for past consecutive years. Continuous efforts have been made so far to diagnose the disease condition and to identify the root cause for it. In this study, we performed the pathway level as well as the differential gene expression studies for CRC. We analyzed the gene expression profile GSE24514 from Gene Expression Omnibus (GEO) along with the gene pathways involved in the CRC. This analysis helps us to understand the behavior of the genes that have shown differential expression through their targeted pathways. Pathway analysis for the targeted genes covers the wider area which therefore decreases the possibility to miss the significant ones. This will prove to be beneficial to expose the ones that have not been given attention so far. Through this analysis, we attempt to understand the various neighboring genes that have close relationship to the targeted one and thus proved to be significantly controlling the CRC. It is anticipated that the identified hub and neighboring genes will provide new directions to look at the pathway level differently and will be crucial for the regulatory processes of the disease.

Keywords: mismatch repair, microsatellite instability, carcinogenesis, morbidity

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Authors: Dona Pamoda W. Jayatunga, Veer Bala Gupta, Eugene Hone, Ralph N. Martins

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Being a neurodegenerative disorder, Alzheimer’s disease (AD) affects a majority of the elderly demented worldwide. The key risk factors for AD are age, metabolic syndrome, allele status of APOE gene, head injuries and lifestyle. The progressive nature of AD is characterized by symptoms of multiple cognitive deficits exacerbated over time, leading to death within a decade from clinical diagnosis. However, it is revealed that AD originates via a prodromal phase that spans from one to few decades before symptoms first manifest. The key pathological hallmarks of AD brains are deposition of amyloid beta (Aβ) plaques and neurofibrillary tangles (NFT). However, the yet unknown etiology of the disease fails to distinguish mitochondrial dysfunction between a cause or an outcome. The absence of early diagnosis tools and definite therapies for AD have permitted recruits of nutraceutical-based approaches aimed at reducing the risk of AD by modulating lifestyle or be used as preventive tools during AD prodromal state before widespread neurodegeneration begins. The objective of the present study was to investigate beneficial effects of luteolin, a plant-based flavone compound, against AD. The neuroprotective effects of luteolin on amyloid beta (Aβ) (1-42)-induced neurotoxicity was measured using cultured human neuroblastoma BE(2)-M17 cells. After exposure to 20μM Aβ (1-42) for 48 h, the neuroblastoma cells exhibited marked apoptotic death. Co-treatment of 20μM Aβ (1-42) with luteolin (0.5-5μM) significantly protected the cells against Aβ (1-42)-induced toxicity, as assessed by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4sulfophenyl)-2H-tetrazolium, inner salt; MTS] reduction assay and the lactate dehydrogenase (LDH) cell death assay. The results suggest that luteolin prevents Aβ (1-42)-induced apoptotic neuronal death. However, further studies are underway to determine its protective mechanisms in AD including the activity against tau hyperphosphorylation and mitochondrial dysfunction.

Keywords: Aβ (1-42)-induced toxicity, Alzheimer’s disease, luteolin, neuroblastoma cells

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Authors: I. Boroňová, J. Bernasovská, J. Kmec, E. Petrejčíková

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Dilated cardiomyopathy (DCM) is a severe cardiovascular disorder characterized by progressive systolic dysfunction due to cardiac chamber dilatation and inefficient myocardial contractility often leading to chronic heart failure. Recently, a genome-wide association studies (GWASs) on DCM indicate that the ZBTB17 gene rs10927875 single nucleotide polymorphism is associated with DCM. The aim of the study was to identify the distribution of ZBTB17 gene rs10927875 polymorphism in 50 Slovak patients with DCM and 80 healthy control subjects using the Custom Taqman®SNP Genotyping assays. Risk factors detected at baseline in each group included age, sex, body mass index, smoking status, diabetes and blood pressure. The mean age of patients with DCM was 52.9±6.3 years; the mean age of individuals in control group was 50.3±8.9 years. The distribution of investigated genotypes of rs10927875 polymorphism within ZBTB17 gene in the cohort of Slovak patients with DCM was as follows: CC (38.8%), CT (55.1%), TT (6.1%), in controls: CC (43.8%), CT (51.2%), TT (5.0%). The risk allele T was more common among the patients with dilated cardiomyopathy than in normal controls (33.7% versus 30.6%). The differences in genotype or allele frequencies of ZBTB17 gene rs10927875 polymorphism were not statistically significant (p=0.6908; p=0.6098). The results of this study suggest that ZBTB17 gene rs10927875 polymorphism may be a risk factor for susceptibility to DCM in Slovak patients with DCM. Studies of numerous files and additional functional investigations are needed to fully understand the roles of genetic associations.

Keywords: ZBTB17 gene, rs10927875 polymorphism, dilated cardiomyopathy, cardiovascular disorder

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Authors: Gül Ebru Orhun

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A half diallel crossing design was carried out during 2011 and 2012 growing seasons under Çanakkale-Turkey ecological conditions. In this research, 20 F1 maize hybrids obtained by 6x6 half diallel crossing were used. Gene action for protein content and grain yield traits were explored in half set involving six elite inbred lines. According to the results diallel analysis dominance and additive gene variances were determined for protein content. Variance/Co-variance graphs revealed for grain yield and protein content traits. In this study, inheritance of grain yield and protein content demonstrated over-dominance type of gene action.

Keywords: protein, maize, inheritance, gene action

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Authors: Ryan D. Martinus

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Inflammation of the endothelium is an important process leading to diabetic atherosclerosis. However, the molecular mechanisms by which diabetes contributes to endothelial inflammation remain to be established. Using In-vitro cultured Human cells and Hsp60 specific ELISA assays, we show that Hsp60 is not only induced in Human monocyte cells under hyperglycaemic conditions but that the Hsp60 is also secreted from these cells. Furthermore, we also demonstrate that the Hsp60 secreted from these monocyte cells is also able to activate Toll-like receptor-4 (TLR4) from Human endothelial cells. This suggests that a potential link may exist between the hyperglycaemia-induced expression of Hsp60 in monocyte cells and vascular inflammation. Circulating levels of Hsp60 due to mitochondrial stress in diabetes patients could, therefore, be an important modulator of inflammation in endothelial cells and thus contribute to the increased incidences of atherosclerosis in diabetes mellitus.

Keywords: mitochondria, Hsp60, inflammation, diabetes mellitus

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Authors: Ashraf Ali, Kriti Upadhyay, Puja Sohal, Anant Mohan, Randeep Guleria

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Background: Gene silencing by aberrant promoter hypermethylation is common in lung cancer and is an initiating event in its development. Aim: To evaluate the gene promoter hypermethylation frequency in serum and tissue of lung cancer patients. Method: 95 newly diagnosed untreated advance stage lung cancer patients and 50 cancer free matched controls were studied. Bisulfite modification of tissue and serum DNA was done; modified DNA was used as a template for methylation-specific PCR analysis. Survival was assessed for one year. Results: Of 95 patients, 82% were non-small cell lung cancer (34% squamous cell carcinoma, 34% non-small cell lung cancer and 14% adenocarcinoma) and 18% were small cell lung cancer. Biopsy revealed that tissue of 89% and 75% of lung cancer patients and 85% and 52% of controls had promoter hypermethylated for MGMT (p=0.35) and p16(p<0.001) gene, respectively. In serum, 33% and 49% of lung cancer patients and 28% and 43% controls were positive for MGMT and p16 gene. No significant correlation was found between survival and clinico-pathological parameters. Conclusion: High gene promoter methylation frequency of p16 gene in tissue biopsy may be linked with early stages of carcinogenesis. Appropriate follow-up is required for confirmation of this finding.

Keywords: lung cancer, MS- PCR, methylation, molecular biology

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Authors: Maria Luisa Barcena De Arellano, Sofya Pozdniakova, Pavelas Karkacas, Anja Kuhl, Istvan Baczko, Yury Ladilov, Vera Regitz-Zagrosek

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Introduction: Aging is associated with deterioration of the physiological function, leading to systemic inflammation and mitochondrial dysfunction that promote the development of cardiovascular diseases. Sex differences in aging-related cardiovascular diseases have been postulated. However, their precise mechanisms remain unclear. In the current study, we aimed to investigate the sex difference in the age-related alteration in Sirt1-AMPK signaling and its relation to the mitochondrial biogenesis and inflammation. Methods: Male and female human non-disease lateral left ventricular wall tissue (young (17–40 years; n= 7 male and 7 female) and old (50–68 years; n= 9 male and 8 female)) were used. qRT-PCR, western blot and immunohistochemistry assays were performed for expression analyses of Sirt1, AMPK, pAMPK, ac-Ku70, TFAM, PGC-1α, Sirt3, SOD2 and catalase. CD68 was used as a marker for macrophages and the ratio of IL-12:IL10 (pro-inflammatory phenotype (high IL-12/low IL-10) and anti-inflammatory phenotype (low IL-12/high IL-10) was used to examine the inflammatory stage in the heart. Results: Sirt1 expression was significantly higher in young females compared to young males, whereas in aged hearts Sirt1 expression was significantly downregulated in females, but not in males. In line with the Sirt1 downregulation in aged females, acetylation of nuclear Ku70, a direct target of Sirt1, in aged female hearts was significantly elevated. The activity of AMPK was significantly decreased in aged individuals, however no sex differences in the AMPK expression or activity were found in young or old individuals. The expression of mitochondrial proteins TOM40, SOD2 and Sirt3 was significantly higher in young females compared to young males, while in aged female hearts SOD2 and TOM40 were downregulated. In addition, the expression of catalase, a key cytosolic and mitochondrial anti-oxidative enzyme was significantly higher in young females and this female sex benefit was lost in aged hearts. In addition, the number of cardiac macrophages was significantly increased in old female, but not in male hearts. Consistently, the pro-inflammatory shift in old females was further confirmed by differences in the IL12/IL10 ratio in young female cardiac tissue in a favour of the anti-inflammatory mediator IL-10 (ratio 1:4) compared to young males (ratio 1:1). The anti-inflammatory environment in the heart was lost in aged females (ratio 1:1). Conclusion: Aging leads to the significant downregulation of Sirt1 expression and elevated acetylation of Ku70 in female, but not in male hearts. Furthermore, a beneficial upregulation of mitochondrial and anti-oxidative proteins in young females is lost with aging. Moreover, the malfunctions in the expression of Sirt1 and mitochondrial proteins in aged female hearts is accompanied by a significant pro-inflammatory shift. The study provides a molecular basis for the increased incidence of cardiovascular diseases in old women.

Keywords: inflammation, mitochondrial dysfunction, aging, Sirt1-AMPK axis

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Authors: M. D. Asemoloye, S. G. Jonathan, A. Rafiq, O. J. Olawuyi, D. O. Adejoye

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Calmodulin is one of the intracellular calcium proteins that regulates large spectrum of enzymes and cellular functions including metabolism of cyclic nucleotides and glycogen. The potentials of calmodulin gene in fungi necessitates their genetic response and their strong cassette of enzyme secretions for pesticide degradation. Therefore, this study was carried out to investigate the ‘Transcriptomic’ response of calmodulin encoding genes in Talaromyces fungi in response to 2, 2-dichlorovinyl dimethyl phosphate (DDVP or Dichlorvos) an organophosphate pesticide and γ-Hexachlorocyclohexane (Lindane) an organochlorine pesticide. Fungi strains isolated from rhizosphere from grasses rhizosphere in pesticide polluted sites were subjected to percentage incidence test. Two most frequent fungi were further characterized using ITS gene amplification (ITS1 and ITS4 combinations), they were thereafter subjected to In-vitro DDVP and lindane tolerance tests at different concentrations. They were also screened for presence and expression of calmodulin gene (caM) using RT-PCR technique. The two Talaromyces strains had the highest incidence of 50-72% in pesticide polluted site, they were both identified as Talaromyces astroroseus asemoG and Talaromyces purpurogenum asemoN submitted in NCBI gene-bank with accession numbers KY488464 and KY488468 respectively. T. astroroseus KY488464 tolerated DDVP (1.23±0.023 cm) and lindane (1.11±0.018 cm) at 25 % concentration while T. purpurogenum KY488468 tolerated DDVP (1.33±0.061 cm) and lindane (1.54±0.077 cm) at this concentration. Calmodulin gene was detected in both strains, but RT-PCR expression of caM gene revealed at 900-1000 bp showed an under-expression of caM in T. astrorosues KY488464 but overexpressed in T. purpurogenum KY488464. Thus, the calmodulin gene response of these fungal strains to both pesticides could be considered in monitoring the potentials of fungal strains to pesticide tolerance and bioremediation of pesticide in polluted soil.

Keywords: Calmodulin gene, pesticide, RT-PCR, talaromyces, tolerance

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Authors: Diana C. El Assal, Fatima Monteiro, Caroline May, Peter Barbuti, Silvia Bolognin, Averina Nicolae, Hulda Haraldsdottir, Lemmer R. P. El Assal, Swagatika Sahoo, Longfei Mao, Jens Schwamborn, Rejko Kruger, Ines Thiele, Kathrin Marcus, Ronan M. T. Fleming

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Degeneration of substantia nigra pars compacta dopaminergic neurons is one of the hallmarks of Parkinson's disease. These neurons have a highly complex axonal arborisation and a high energy demand, so any reduction in ATP synthesis could lead to an imbalance between supply and demand, thereby impeding normal neuronal bioenergetic requirements. Synaptic mitochondria exhibit increased vulnerability to dysfunction in Parkinson's disease. After biogenesis in and transport from the cell body, synaptic mitochondria become highly dependent upon oxidative phosphorylation. We applied a systems biochemistry approach to identify the metabolic pathways used by neuronal mitochondria for energy generation. The mitochondrial component of an existing manual reconstruction of human metabolism was extended with manual curation of the biochemical literature and specialised using omics data from Parkinson's disease patients and controls, to generate reconstructions of synaptic and somal mitochondrial metabolism. These reconstructions were converted into stoichiometrically- and fluxconsistent constraint-based computational models. These models predict that Parkinson's disease is accompanied by an increase in the rate of glycolysis and a decrease in the rate of oxidative phosphorylation within synaptic mitochondria. This is consistent with independent experimental reports of a compensatory switching of bioenergetic pathways in the putamen of post-mortem Parkinson's disease patients. Ongoing work, in the context of the SysMedPD project is aimed at computational prediction of mitochondrial drug targets to slow the progression of neurodegeneration in the subset of Parkinson's disease patients with overt mitochondrial dysfunction.

Keywords: bioenergetics, mitochondria, Parkinson's disease, systems biochemistry

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Authors: Sarah Devi Silvian, Hanna Shobrina Iqomatul Haq, Fara Cholidatun Nabila, Agustin Krisna Wardani

Abstract:

Bacteria ability to survive in antibiotic is controlled by the presence of gene that encodes the antibiotic resistance protein. The instability of the antibiotic resistance gene can be observed by exposing the bacteria under the lethal dose of antibiotic. Low concentration of antibiotic can induce mutation, which may take a role in bacterial adaptation through the antibiotic concentration. Lactobacillus casei FNCC 0090 is one of the probiotic bacteria that has an ability to survive in tetracycline by expressing the tetM gene. The aims of this study are to observe the possibilities of mutation happened in L.casei FNCC 0090 by exposing in sub-lethal dose of tetracycline and also observing the instability of the tetM gene by comparing the sequence between the wild type and mutant. L.casei FNCC 0090 has a lethal dose in 60 µg/ml, low concentration is applied to induce the mutation, the range from 10 µg/ml, 15 µg/ml, 30 µg/ml, 45 µg/ml, and 50 µg/ml. L.casei FNCC 0090 is exposed to the low concentration from lowest to the highest concentration to induce the adaptation. Plasmid is isolated from the highest concentration culture which is 50 µg/ml by using modified alkali lysis method with the addition of lysozyme. The tetM gene is isolated by using PCR (Polymerase Chain Reaction) method, then PCR amplicon is purified and sequenced. Sequencing is done on both samples, wild type and mutant. Both sequences are compared and the mutations can be traced in the presence of nucleotides changes. The changing of the nucleotides means that the tetM gene is instable.

Keywords: L. casei FNCC 0090, probiotic, tetM, tetracycline

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Authors: Yusra Tariq

Abstract:

The Grass 37 gene is presently known in tomatoes, which are the source of healthy substances such as ascorbic acid, polyphenols, carotenoids and nutrients. It has a significant impact on the growth and development of humans. The GRASS 37 gene is a plant Transcription factor group assuming significant parts in various reactions of different Abiotic stresses such as (drought, salinity, thermal stresses, temperature, and bright waves) which could highly affect the growth. Tomatoes are very sensitive to temperature, and their growth or production occurs optimally in a temperature range from 21 C to 29.5 C during the daytime and from 18.5 C to 21 C during the night. This protein acts as a positive regulator of salt stress response and abscisic acid signaling. This study summarizes the structure characterized by molecular formula and protein-binding domains by different bioinformatics tools such as Expasy translate tool, Expasy Portparam, Swiss Prot and Inter Pro Scan, Clustal W tool regulatory procedure of GRASS gene components, also their reactions to both biotic and Abiotic stresses.

Keywords: GRAS37, gene, bioinformatics, tool

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Authors: Vanessa Funk, Steven Blum, Stephanie Cole, Jorge Maciel, Matthew Lux

Abstract:

Paper-based synthetic gene circuits offer a new paradigm for programmable, fieldable biodetection. We demonstrate that by freeze-drying gene circuits with in vitro expression machinery, we can use complimentary RNA sequences to trigger colorimetric changes upon rehydration. We have successfully utilized both green fluorescent protein and luciferase-based reporters for easy visualization purposes in solution. Through several efforts, we are aiming to use this new platform technology to address a variety of needs in portable detection by demonstrating several more expression and reporter systems for detection functions on paper. In addition to RNA-based biodetection, we are exploring the use of various mechanisms that cells use to respond to environmental conditions to move towards all-hazards detection. Examples include explosives, heavy metals for water quality, and toxic chemicals.

Keywords: cell-free lysates, detection, gene circuits, in vitro

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Authors: Paul Shize Li, Frank Alber

Abstract:

A fundamental task of clinical genomics is to unravel the functions of genes and their associations with disorders. Although experimental biology has made efforts to discover and elucidate the molecular mechanisms of individual genes in the past decades, still about 40% of human genes have unknown functions, not to mention the diseases they may be related to. For those biologists who are interested in a particular gene with unknown functions, a powerful computational method tailored for inferring the functions and disease relevance of uncharacterized genes is strongly needed. Studies have shown that genes strongly linked to each other in multiple biological networks are more likely to have similar functions. This indicates that the densely connected subgraphs in multiple biological networks are useful in the functional and phenotypic annotation of uncharacterized genes. Therefore, in this work, we have developed an integrative network approach to identify the frequent local clusters, which are defined as those densely connected subgraphs that frequently occur in multiple biological networks and consist of the query gene that has few or no disease or function annotations. This is a local clustering algorithm that models multiple biological networks sharing the same gene set as a three-dimensional matrix, the so-called tensor, and employs the tensor-based optimization method to efficiently find the frequent local clusters. Specifically, massive public gene expression data sets that comprehensively cover dynamic, physiological, and environmental conditions are used to generate hundreds of gene co-expression networks. By integrating these gene co-expression networks, for a given uncharacterized gene that is of biologist’s interest, the proposed method can be applied to identify the frequent local clusters that consist of this uncharacterized gene. Finally, those frequent local clusters are used for function and disease annotation of this uncharacterized gene. This local tensor clustering algorithm outperformed the competing tensor-based algorithm in both module discovery and running time. We also demonstrated the use of the proposed method on real data of hundreds of gene co-expression data and showed that it can comprehensively characterize the query gene. Therefore, this study provides a new tool for annotating the uncharacterized genes and has great potential to assist clinical genomic diagnostics.

Keywords: local tensor clustering, query gene, gene co-expression network, gene annotation

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Authors: Alieh Farshbaf

Abstract:

Introduction: Current anti-retroviral drugs have some restrictions for treatment of HIV-1 infection. The efficacy of retroviral drugs is not same in different infected patients and the virus rebound from latent reservoirs after stopping them. Recently, the engineering of stem cells and gene therapy provide new approaches to eliminate some drug problems by induction of resistance to HIV-1. Literature review: Up to now, AIDS-restriction genes (ARGs) were suitable candidate for gene and cell therapies, such as cc-chemokine receptor-5 (CCR5). In this manner, CCR5 provide effective cure in Berlin and Boston patients by inducing of HIV-1 resistance with allogeneic stem cell transplantation. It is showed that Zinc Finger Nuclease (ZFN) could induce HIV-1 resistance in stem cells of infected patients by homologous recombination or non-end joining mechanism and eliminate virus loading after returning the modified cells. Then, gene modification by HIV restriction factors, as TRIM5, introduced another gene candidate for HIV by interfering in infection process. These gene modifications/editing provided by stem cell futures that improve treatment in refractory disease such as HIV-1. Conclusion: Although stem cell transplantation has some complications, but in compare to retro-viral drugs demonstrated effective cure by elimination of virus loading. On the other hand, gene therapy is cost-effective for an infected patient than retroviral drugs payment in a person life-long. The results of umbilical cord blood stem cell transplantation showed that gene and cell therapy will be applied easier than previous treatment of AIDS with high efficacy.

Keywords: stem cell, AIDS, gene modification, cell engineering

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Authors: Habib Onsori, Somayeh Akrami, Mohammad Rahmati

Abstract:

Deafness is the most common sensory disorder with the frequency of 1/1000 in many populations. Mutations in the GJB2 (CX26) gene at the DFNB1 locus on chromosome 13q12 are associated with congenital hearing loss. Approximately 80% of congenital hearing loss cases are recessively inherited and 15% dominantly inherited. Mutations of the GJB2 gene, encoding gap junction protein Connexin 26 (Cx26), are the most common cause of hereditary congenital hearing loss in many countries. This report presents two cases of different mutations from Iranian patients with bilateral hearing loss. DNA studies were performed for the GJB2 gene by PCR and sequencing methods. In one of them, direct sequencing of the gene showed a heterozygous T→C transition at nucleotide 604 resulting in a cysteine to arginine amino acid substitution at codon 202 (C202R) in the fourth extracellular domain (TM4) of the protein. The analyses indicate that the C202R mutation appeared de novo in the proband with a possible dominant effect (GenBank: KF 638275). In the other one, DNA sequencing revealed a compound heterozygous mutation (35delG, 363delC) in the Cx26 gene that is strongly associated with congenital non-syndromic hearing loss (NSHL). So screening the mutations for hearing loss individuals referring to genetics counseling centers before marriage and or pregnancy is recommended.

Keywords: CX26, deafness, GJB2, mutation

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Authors: Ekaterina M. Myasnikova, Andrey A. Makashov, Alexander V. Spirov

Abstract:

First manifestation of a segmentation pattern in the early Drosophila development is the formation of expression domains (along with the main embryo axis) of genes belonging to the trunk gene class. Highly variable expression of genes from gap family in early Drosophila embryo is strongly reduced by the start of gastrulation due to the gene cross-regulation. The dynamics of gene expression is described by a gene circuit model for a system of four gap genes. It is shown that for the formation of a steep and stationary border by the model it is necessary that there existed a nucleus (modeling point) in which the gene expression level is constant in time and hence is described by a stationary equation. All the rest genes expressed in this nucleus are in a dynamic equilibrium. The mechanism of border formation associated with the existence of a stationary nucleus is also confirmed by the experiment. An important advantage of this approach is that properties of the system in a stationary nucleus are described by algebraic equations and can be easily handled analytically. Thus we explicitly characterize the cross-regulation properties necessary for the robustness and formulate the conditions providing this effect through the properties of the initial input data. It is shown that our formally derived conditions are satisfied for the previously published model solutions.

Keywords: drosophila, gap genes, reaction-diffusion model, robustness

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Authors: Ogbonnaya O. Iroanya, Samson T. Fakorede, Osamudiamen J. Edosa, Hadiat A. Azeez

Abstract:

The human mitochondrial DNA (mtDNA) is about 17 kbp circular DNA fragments found within the mitochondria together with smaller fragments of 1200 bp known as the control region. Knowledge of variation within populations has been employed in forensic and molecular anthropology studies. The study was aimed at investigating the polymorphic nature of the two hypervariable segments (HVS) of the mtDNA, i.e., HVS1 and HVS2, and to determine the haplogroup distribution among individuals resident in Lagos, Southwestern Nigeria. Peripheral blood samples were obtained from sixty individuals who are not related maternally, followed by DNA extraction and amplification of the extracted DNA using primers specific for the regions under investigation. DNA amplicons were sequenced, and sequenced data were aligned and compared to the revised Cambridge Reference Sequence (rCRS) GenBank Accession number: NC_012920.1) using BioEdit software. Results obtained showed 61 and 52 polymorphic nucleotide positions for HVS1 and HVS2, respectively. While a total of three indels mutation were recorded for HVS1, there were seven for HVS2. Also, transition mutations predominate nucleotide change observed in the study. Genetic diversity (GD) values for HVS1 and HVS2 were estimated to be 84.21 and 90.4%, respectively, while random match probability was 0.17% for HVS1 and 0.89% for HVS2. The study also revealed mixed haplogroups specific to the African (L1-L3) and the Eurasians (U and H) lineages. New polymorphic sites obtained from the study are promising for human identification purposes.

Keywords: hypervariable region, indels, mitochondrial DNA, polymorphism, random match probability

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Authors: Arfan Ali, Idrees Ahmad Nasir

Abstract:

Potato (Solanum tuberosum) is an important cash crop and popular vegetable in Pakistan and throughout the world. Cold storage of potatoes accelerates the conversion of starch into reduced sugars (glucose and fructose). This process causes dry mass and bitter taste in the potatoes that are not acceptable to end consumers. In the current study, the phosphofructokinase B gene was cloned into the pET-30 vector for protein expression and the pCambia-1301 vector for plant expression. Amplification of a 930bp product from an E. coli strain determined the successful isolation of the phosphofructokinase B gene. Restriction digestion using NcoI and BglII along with the amplification of the 930bp product using gene specific primers confirmed the successful cloning of the PfkB gene in both vectors. The protein was expressed as a His-PfkB fusion protein. Western blot analysis confirmed the presence of the 35 Kda PfkB protein when hybridized with anti-His antibodies. The construct Fani-01 was evaluated transiently using a histochemical gus assay. The appearance of blue color in the agroinfiltrated area of potato leaves confirmed the successful expression of construct Fani-01. Further, the area displaying gus expression was evaluated for PfkB expression using ELISA. Moreover, PfkB gene expression evaluated through transient expression determined successful gene expression and highlighted its potential utilization for stable expression in potato to reduce sweetening due to long-term storage.

Keywords: potato, Solanum tuberosum, transformation, PfkB, anti-sweetening

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Authors: Syed Sameer Aga, Saniya Nissar

Abstract:

The Xeroderma pigmentosum group D gene (XPD) plays a key role in nucleotide excision repair (NER) pathway of the damaged DNA. Genetic polymorphisms in the coding region of the XPD gene may alter DNA repair capacity of the protein and hence can modulate the risk of colorectal cancer (CRC) risk. The aim of the study was to determine the genetic association of XPD Lys751Gln polymorphism with the risk of colorectal cancer (CRC) development. 120 CRC patients and 160 normal controls were assessed for genotype frequencies of XPD Lys751Gln polymorphism using PCR-RFLP technique. We observed a significant association (p < 0.05) between the XPD Lys751Gln polymorphism and the risk of developing CRC (p < 0.05). Additionally, Gln/Gln genotype of the XPD gene doubled the risk for the development of CRC [p < 0.05; OR=2.25 95% CI (1.07-4.7)]. Our results suggest that there is a significant association between the XPD Lys751Gln polymorphism and the risk of CRC.

Keywords: colorectal cancer, polymorphism, RFLP, DNA Repair, NER, XPD

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Authors: Rafael Estrada, Cristian Ruiz Rueda

Abstract:

Carbapenems are last-resort antibiotics used in clinical settings to treat antibiotic-resistant bacterial infections. Thus, the emergence and spread of resistance to carbapenems is a major public health concern. Here, we have studied a carbapenem-resistant Aeromonas veronii strain previously isolated from a water sample from Sam Simeon Creek (Hearst San Simeon State Park, CA). Analysis of this isolate using disk-diffusion, CarbaNP, eCIM and mCIM assays revealed that it was resistant to amoxicillin-clavulanic acid and all carbapenems tested and that this isolate produced a potentially novel carbapenemase of the Metallo-β-lactamase family. Whole genome sequencing analysis revealed that this A. veronii isolate carries a novel variant of the blacₚₕₐ class β-carbapenemase gene that was closely related to the blacₚₕₐ₇ gene of Aeromonas jandaei. This isolate also carried a novel variant of the blaₒₓₐ class D carbapenemase gene that was most closely related to the blaₒₓₐ-₉₁₂ gene found in other Aeromonas veronii isolates. Finally, we also identified a novel class C β-lactamase gene moderately related to the blaFₒₓ-₁₇ gene of Providencia stuartii and other blaFₒₓ variants identified in Klebsiella pneumoniae, Escherichia coli and other Enterobacteriaceae. Overall, our findings reveal that environmental isolates are an important reservoir of multiple carbapenemases and other β-lactamases of clinical significance.

Keywords: β-lactamases, carbapenem, antibiotic-resistant, aeromonas veronii

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