Search results for: genomic

Authors: András Farkas, István Molnár, Jan Vrána, Veronika Burešová, Petr Cápal, András Cseh, Márta Molnár-Láng, Jaroslav Doležel

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Species of Aegilops played a central role in the evolution of wheat and are sources of traits related to yield quality and tolerance against biotic and abiotic stresses. These wild genes and alleles are desirable to use in crop improvement programs via introgressive hybridization. However, the success of chromosome mediated gene transfer to wheat are hampered by the pour knowledge on the genome structure of Aegilops relative to wheat and by the low number of cost-effective molecular markers specific for Aegilops chromosomes. The COS markers specific for genes conserved throughout evolution in both sequence and copy number between Triticeae/Aegilops taxa and define orthologous regions, thus enabling the comparison of regions on the chromosomes of related species. The present study compared individual chromosomes of Aegilops umbellulata (UU), Ae. comosa (MM), Ae. speltoides (SS) and Ae. caudata (CC) purified by flourescent labelling with oligonucleotid SSR repeats and biparametric flow cytometry with wheat by identifying orthologous chromosomal regions by COS markers. The linear order of bin-mapped COS markers along the wheat D chromosomes was identified by the use of chromosome-specific sequence data and virtual gene order. Syntenic regions of wheat identifying genome rearrangements differentiating the U, M, S or C genomes from the D genome of wheat were detected. The conserved orthologous set markers assigned to Aegilops chromosomes promise to accelerate gene introgression by facilitating the identification of alien chromatin. The syntenic relationships between the Aegilops species and wheat will facilitate the targeted development of new markers specific for U, M, S and C genomic regions and will contribute to the understanding of molecular processes related to the evolution of Aegilops.

Keywords: Aegilops, cos-markers, flow-sorting, wheat

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Authors: Saranya Ganapathy, Megha N. Parajulee, Michael San Francisco, Hong Zhang

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Insect pests are a serious threat to agricultural productivity. Use of chemical pesticides, the predominant control method thus far, has resulted in environmental damage, pest resurgence, and negative effects on non-target species. Genetically modified (GM) crops offer a promising alternative, and Bacillus thuringiensis endotoxin genes have played a major role in this respect. However, to overcome insect tolerance issues and to broaden the target range, it is critical to identify alternative-insecticidal toxins working through novel mechanisms. Our research group has identified a kinase from Chilo iridescent virus (CIV; Family Iridoviridae) that has insecticidal activity and designated it as ISTK (Iridovirus Serine/Threonine Kinase). A 35 kDa truncated form of ISTK, designated iridoptin, was obtained during expression and purification of ISTK in the yeast system. This yeast-expressed CIV toxin induced 50% mortality in cotton aphids and 100% mortality in green peach aphids (GPA). Optimized viral genes (o-ISTK and o-IRI) were stably transformed into the model plant, Arabidopsis. PCR analysis of genomic DNA confirmed the presence of the gene insert (oISTK/oIRI) in selected transgenic lines. The further screening was performed to identify the PCR positive lines that showed expression of respective toxins at the polypeptide level using Western blot analysis. The stable lines expressing either of these two toxins induced moderate to very high mortality in GPAs and significantly affected GPA development and fecundity. The aphicidal potential of these transgenic Arabidopsis lines will be presented.

Keywords: Chilo iridescent virus, insecticidal toxin, iridoviruses, plant-incorporated protectants, serine/threonine kinase

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Authors: Alisa Kazarina, Guntis Gerhards, Elina Petersone-Gordina, Ilva Pole, Viktorija Igumnova, Janis Kimsis, Valentina Capligina, Renate Ranka

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Human body is inhabited by a vast number of microorganisms, collectively known as the human microbiome, and there is a tremendous interest in evolutionary changes in human microbial ecology, diversity and function. The field of paleomicrobiology, study of ancient human microbiome, is powered by modern techniques of Next Generation Sequencing (NGS), which allows extracting microbial genomic data directly from archaeological sample of interest. One of the major techniques is 16S rRNA gene sequencing, by which certain 16S rRNA gene hypervariable regions are being amplified and sequenced. However, some limitations of this method exist including the taxonomic precision and efficacy of different regions used. The aim of this study was to evaluate the phylogenetic sensitivity of different 16S rRNA gene hypervariable regions for microbiome studies in the archaeological samples. Towards this aim, archaeological bone samples and corresponding soil samples from each burial environment were collected in Medieval cemeteries in Latvia. The Ion 16S™ Metagenomics Kit targeting different 16S rRNA gene hypervariable regions was used for library construction (Ion Torrent technologies). Sequenced data were analysed by using appropriate bioinformatic techniques; alignment and taxonomic representation was done using Mothur program. Sequences of most abundant genus were further aligned to E. coli 16S rRNA gene reference sequence using MEGA7 in order to identify the hypervariable region of the segment of interest. Our results showed that different hypervariable regions had different discriminatory power depending on the groups of microbes, as well as the nature of samples. On the basis of our results, we suggest that wider range of primers used can provide more accurate recapitulation of microbial communities in archaeological samples. Acknowledgements. This work was supported by the ERAF grant Nr. 1.1.1.1/16/A/101.

Keywords: 16S rRNA gene, ancient human microbiome, archaeology, bioinformatics, genomics, microbiome, molecular biology, next-generation sequencing

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Authors: Amr A. El Hanafy, Muhammad I. Qureshi, Jamal Sabir, Mohamed Mutawakil, Mohamed M. Ahmed, Hassan El Ashmaoui, Hassan Ramadan, Mohamed Abou-Alsoud, Mahmoud Abdel Sadek

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β-Lactoglobulin (β-LG) is the dominant non-casein whey protein found in bovine milk and of most ruminants. The amino acid sequence of β-LG along with its 3-dimensional structure illustrates linkage with the lipocalin superfamily. Preliminary studies in goats indicated that milk yield can be influenced by polymorphism in genes coding for whey proteins. The aim of this study is to identify and evaluate the incidence of functional polymorphisms in the exonic and intronic portions of β-LG gene in native Saudi goat breeds (Ardi, Habsi, and Harri). Blood samples were collected from 300 animals (100 for each breed) and genomic DNA was extracted using QIAamp DNA extraction Kit. A fragment of the β-LG gene from exon 7 to 3’ flanking region was amplified with pairs of specific primers. Subsequent digestion with Sac II restriction endonuclease revealed two alleles (A and B) and three different banding patterns or genotypes i.e. AA, AB and BB. The statistical analysis showed that β-LG AA genotype had higher milk yield than β-LG AB and β-LG BB genotypes. Nucleotide sequencing of the selected β-LG fragments was done and submitted to GenBank NCBI (Accession No. KJ544248, KJ588275, KJ588276, KJ783455, KJ783456 and KJ874959). Two already established SNPs in exon 7 (+4601 and +4603) and one fresh SNP in the 3’ UTR region were detected in the β-LG fragments with designated AA genotype. The polymorphisms in exon 7 did not produce any amino acid change. Phylogenetic analysis on the basis of nucleotide sequences of native Saudi goats indicated evolutional similarity with the GenBank reference sequences of goat, Bubalus bubalis and Bos taurus.

Keywords: β-Lactoglobulin, Saudi goats, PCR-RFLP, functional polymorphism, nucleotide sequencing, phylogenetic analysis

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

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Dilated cardiomyopathy (DCM) is a primary myocardial disease, it is characterized by progressive systolic dysfunction due to cardiac chamber dilatation and inefficient myocardial contractility with estimated prevalence of 37 in 100 000 people. It is the most frequent cause of heart failure and cardiac transplantation in young adults. About one-third of all patients have a suspected familial disease indicating a genetic basis of DCM. Many candidate gene studies in humans have tested the association of single nucleotide polymorphisms (SNPs) in various genes coding for proteins with a known cardiovascular function. In our study we present the results of ZBTB17 gene rs10927875 polymorphism genotyping in relation to dilated cardiomyopathy in Slovak population. The study included 78 individuals, 39 patients with DCM and 39 healthy control persons. The mean age of patients with DCM was 50.7±11.5 years; the mean age of individuals in control group was 51.3±9.8 years. Risk factors detected at baseline in each group included age, sex, body mass index, smoking status, diabetes and blood pressure. Genomic DNA was extracted from leukocytes by a standard methodology and screened for rs10927875 polymorphism in intron of ZBTB17 gene using Real-time PCR method (Step One Applied Biosystems). The distribution of investigated genotypes for rs10927875 polymorphism in the group of patients with DCM was as follows: CC (89.74%), CT (10.26%), TT (0%), and the distribution in the control group: CC (92.31%), CT (5.13%), and TT (2.56%). Using the chi-square (χ2) test we compared genotype and allele frequencies between patients and controls. There was no difference in genotype or allele frequencies in ZBTB17 gene rs10927875 polymorphism between patients and control group (χ2=3.028, p=0.220; χ2=0.264, p=0.608). Our results represent an initial study, it can be considered as preliminary and first of its kind in Slovak population. Further studies of ZBTB17 gene polymorphisms of more numerous files and additional functional investigations are needed to fully understand the role of genetic associations.

Keywords: dilated cardiomyopathy, SNP polymorphism, ZBTB17 gene, bioscience

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Authors: Ramin Ghasemi Shayan, Morteza Janebifam

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Tumors are regularly inhomogeneous. Provincial varieties in death, metabolic action, multiplication and body part are watched. There’s expanding proof that strong tumors may contain subpopulations of cells with various genotypes and phenotypes. These unmistakable populaces of malignancy cells can connect during a serious way and may contrast in affectability to medications. Most tumors show organic heterogeneity1–3 remembering heterogeneity for genomic subtypes, varieties inside the statement of development variables and genius, and hostile to angiogenic factors4–9 and varieties inside the tumoural microenvironment. These can present as contrasts between tumors in a few people. for instance, O6-methylguanine-DNA methyltransferase, a DNA fix compound, is hushed by methylation of the quality advertiser in half of glioblastoma (GBM), adding to chemosensitivity, and improved endurance. From the outset, there includes been specific enthusiasm inside the usage of dissemination weighted imaging (DWI) and dynamic complexity upgraded MRI (DCE-MRI). DWI sharpens MRI to water dispersion inside the extravascular extracellular space (EES) and is wiped out with the size and setup of the cell populace. Additionally, DCE-MRI utilizes dynamic obtaining of pictures during and after the infusion of intravenous complexity operator. Signal changes are additionally changed to outright grouping of differentiation permitting examination utilizing pharmacokinetic models. PET scan modality gives one of a kind natural particularity, permitting dynamic or static imaging of organic atoms marked with positron emanating isotopes (for example, 15O, 18F, 11C). The strategy is explained to a colossal radiation portion, which points of confinement rehashed estimations, particularly when utilized together with PC tomography (CT). At long last, it's of incredible enthusiasm to quantify territorial hemoglobin state, which could be joined with DCE-CT vascular physiology estimation to create significant experiences for understanding tumor hypoxia.

Keywords: heterogeneity, computerized tomography scan, magnetic resonance imaging, PET

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Authors: Othman E. Othman, Hassan R. Darwish, Amira M. Nowier

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Lactoferrin (LF) is a multifunctional protein involved in economically production traits like milk protein composition and skeletal structure in small ruminants including sheep and goat. So, LF gene - with its genetic polymorphisms associated with production traits - is considered a candidate genetic marker used in marker-assisted selection in goats. This study aimed to identify the different alleles and genotypes of this gene in three Egyptian goat breeds using PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing. Genomic DNA was extracted from 120 animals belonging to Barki, Zaraibi, and Damascus goat breeds. Using specific primers, PCR amplified 247-bp fragments from exon 2 of LF goat gene. The PCR products were subjected to Single-Strand Conformation Polymorphism (SSCP) technique. The results showed the presence of two genotypes GG and AG in the tested animals. The frequencies of both genotypes varied among the three tested breeds with the highest frequencies of GG genotype in all tested goat breeds. The sequence analysis of PCR products representing these two detected genotypes declared the presence of an SNP (single nucleotide polymorphisms) substitution (G/A) among G and A alleles of this gene. The association between different LF genotypes and milk composition as well as body measurement was estimated. The comparison showed that the animals possess AG genotypes are superior over those with GG genotypes for different parameters of milk protein compositions and skeletal structures. This finding declared that allele A of LF gene is considered the promising marker for the productive traits in goat. In conclusion, the Egyptian goat breeds will be needed to enhance their milk protein composition and growth trait parameters through the increasing of allele A frequency in their herds depending on the superior production traits of this allele in goats.

Keywords: lLactoferrin gene, PCR-SSCP, SNPs, Egyptian goat

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Authors: M. Borgie, Z. Dagher, F. Ledoux, A. Verdin, F. Cazier, H. Greige, P. Shirali, D. Courcot

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In October 2013, the International Agency for Research on Cancer (IARC) classified outdoor air pollution and fine particulate matter (PM2.5) as carcinogenic to humans. Despite the clearly relationship established by epidemiological studies between PM exposure and the onset of respiratory and cardiovascular diseases, uncertainties remain about the physiopathological mechanisms responsible for these diseases. The aim of this work was to evaluate the toxicological effects of two samples of atmospheric PM2.5 collected at urban and rural sites on human bronchial epithelial cells, BEAS-2B, especially to investigate the metabolic activation of organic compounds, the alteration of epigenetic mechanisms (i.e. microRNAs genes expression), the phosphorylation of H2AX and the telomerase activity. Our results showed a significant increase in CYP1A1, CYP1B1, and AhRR genes expression, miR-21 gene expression, H2AX phosphorylation and telomerase activity in BEAS-2B cells after their exposure to PM2.5, both in a dose and site-dependent manner. These results showed that PM2.5, especially urban PM, are able to induce the expression of metabolizing enzymes which can provide metabolic biotransformation of organic compounds into more toxic and carcinogenic metabolites, and to induce the expression of the oncomiR miR-21 which promotes cell growth and enhances tumor invasion and metastasis in lung cancer. In addition, our results have highlighted the role of PM2.5 in the activation of telomerase, which can maintain the telomeres length and subsequently preventing cell death, and have also demonstrated the ability of PM2.5 to induce DNA breaks and thus to increase the risk of mutations or chromosomal translocations that lead to genomic instability. All these factors may contribute to cell abnormalities, and thus the development of cancer.

Keywords: BEAS-2B cells, carcinogenesis, epigenetic alterations and genotoxicity, PM2.5

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Authors: Shringika Soni, Utkarsh Jain, Nidhi Chauhan

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Aptamers are recently discovered ~80-100 bp long artificial oligonucleotides that not only demonstrated their applications in therapeutics; it is tremendously used in diagnostic and sensing application to detect different biomarkers and drugs. Synthesizing aptamers for proteins or genomic template is comparatively feasible in laboratory, but drugs or other chemical target based aptamers require major specification and proper optimization and validation. One has to optimize all selection, amplification, and characterization steps of the end product, which is extremely time-consuming. Therefore, we performed asymmetric PCR (polymerase chain reaction) for random oligonucleotides pool synthesis, and further use them in Systematic evolution of ligands by exponential enrichment (SELEX) for anti-psychotic drugs based aptamers synthesis. Anti-psychotic drugs are major tranquilizers to control psychosis for proper cognitive functions. Though their low medical use, their misuse may lead to severe medical condition as addiction and can promote crime in social and economical impact. In this work, we have approached the in-vitro SELEX method for ssDNA synthesis for anti-psychotic drugs (in this case ‘target’) based aptamer synthesis. The study was performed in three stages, where first stage included synthesis of random oligonucleotides pool via asymmetric PCR where end product was analyzed with electrophoresis and purified for further stages. The purified oligonucleotide pool was incubated in SELEX buffer, and further partition was performed in the next stage to obtain target specific aptamers. The isolated oligonucleotides are characterized and quantified after each round of partition, and significant results were obtained. After the repetitive partition and amplification steps of target-specific oligonucleotides, final stage included sequencing of end product. We can confirm the specific sequence for anti-psychoactive drugs, which will be further used in diagnostic application in clinical and forensic set-up.

Keywords: anti-psychotic drugs, aptamer, biosensor, ssDNA, SELEX

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Authors: Dina Athariah, Desriani Desriani, Bugi Ratno Budiarto, Abinawanto Abinawanto, Dwi Wulandari

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Breast cancer is a regulated by many genes. Defect in PIK3CA gene especially at position of exon 9 (E542K and E545K), called hot spot mutation induce early transformation of breast cells. The early detection of breast cancer based on mutation profile of this hot spot region would be hampered by the existence of pseudogene, marked by its substitution mutation at base 1658 (E545A) and deletion at 1659 that have been previously proven in several cancers. To the best of the authors’ knowledge, until recently no studies have been reported about pseudogene phenomenon in breast cancer. Here, we reported PCR optimization to to obtain true exon 9 of PIK3CA gene from its pseudogene hence increasing the validity of data. Material and methods: two genomic DNA with Dev and En code were used in this experiment. Two pairs of primer were design for Standard PCR method. The size of PCR products for each primer is 200bp and 400bp. While other primer was designed for Nested-PCR followed with DNA sequencing method. For Nested-PCR, we optimized the annealing temperature in first and second run of PCR, and the PCR cycle for first run PCR (15x versus 25x). Result: standard PCR using both primer pairs designed is failed to detect the true PIK3CA gene, appearing a substitution mutation at 1658 and deletion at 1659 of PCR product in sequence chromatogram indicated pseudogene. Meanwhile, Nested-PCR with optimum condition (annealing temperature for the first round at 55oC, annealing temperatung for the second round at 60,7oC with 15x PCR cycles) and could detect the true PIK3CA gene. Dev sample were identified as WT while En sample contain one substitution mutation at position 545 of exon 9, indicating amino acid changing from E to K. For the conclusion, pseudogene also exists in breast cancer and the apllication of optimazed Nested-PCR in this study could detect the true exon 9 of PIK3CA gene.

Keywords: breast cancer, exon 9, hotspot mutation, PIK3CA, pseudogene

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Authors: Saad Mahmud Khan, Sarah El Hajj Chehadeh, Mehera Abdulrahman, Wael Osman, Habiba Al Safar

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Obesity is a non-communicable disease that is widely prevalent with approximately 600 million people classified as obese worldwide. Its etiology is multifactorial and involves a complex interplay between genes and the environment. Over the past few decades, obesity rates among the Emirati population have been increasing. The aim of this study was to investigate the association of candidate gene single nucleotide polymorphisms (SNPs), namely the fat mass and obesity associated (FTO) gene SNP rs9939609 and Vitamin D Receptor (VDR) gene SNP rs1544410, with obesity in the UAE population. Methods: This is a case-control study in which 414 individuals were enrolled during their routine visit to endocrinology clinics in Abu Dhabi, United Arab Emirates between the period of June 2012 and December 2013. Several biochemical tests and clinical assessments along with a lifestyle questionnaire for each participant were completed at the clinic. Genomic DNA was extracted from saliva samples of 201 obese, 114 overweight and 99 normal subjects. Genotyping for the variants was performed using TaqMan assay. Results: The mean Body Mass Index (BMI) ± SD for the obese, overweight, and normal subjects was 35.76 ± 4.54, 27.53 ± 1.45 and 22.69 ± 1.84 kg/m2, respectively. Increasing BMI values were associated with an increase in values for systolic blood pressure, diastolic blood pressure, HbA1c, and triglycerides. The SNP rs9939609 in the FTO gene was found to be significantly associated with the BMI (p=0.028), with the minor allele A having a clear additive effect on BMI values. No significant association was detected between BMI and rs1544410 of the VDR gene. Conclusions: Our study findings indicate that the minor allele A of the rs9939609 has a significant association with increasing BMI values. In addition, our findings support the fact that increasing BMI is associated with increasing risks of other comorbidities such as higher blood pressure, poorer glycemic control and higher triglycerides.

Keywords: body mass index, FTO gene, obesity, rs9939609, United Arab Emirates

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Authors: Neelma Ashraf

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Actinomycetes, particularly genus Streptomyces is of great importance due to their role in the discovery of new natural products, particularly antimicrobial secondary metabolites in the medicinal science and biotechnology industry. Different Streptomyces strains were isolated from Helianthus annuus plants and tested for antibacterial and antifungal activities. The most promising five strains were chosen for further investigation, and growth conditions for antibiotic synthesis were optimised. The supernatants were extracted in different solvents, and the extracted products were analyzed using liquid chromatography-mass spectrometry (LC-MS) and biological testing. From one of the potent strains Streptomyces globusus sp. BR123, a compound lavendamycin was identified using these analytical techniques. In addition, this potent strain also produces a strong antifungal polyene compound with a quasimolecular ion of 2072. Streptomyces sp. BR123 was genome sequenced because of its promising antimicrobial potential in order to identify the gene cluster responsible for analyzed compound “lavendamycin”. The genome analysis yielded candidate genes responsible for the production of this potent compound. The genome sequence of 8.15 Mb of Streptomyces sp. isolate BR123 with a GC content of 72.63% and 8103 protein coding genes was attained. Many antimicrobial, antiparasitic, and anticancerous compounds were detected through multiple biosynthetic gene clusters predicted by in-Silico analysis. Though, the novelty of metabolites was determined through the insignificant resemblance with known biosynthetic gene clusters. The current study gives insight into the bioactive potential of Streptomyces sp. isolate BR123 with respect to the synthesis of bioactive secondary metabolites through genomic and spectrometric analysis. Moreover, the comparative genome study revealed the connection of isolate BR123 with other Streptomyces strains, which could expand the knowledge of this genus and the mechanism involved in the discovery of new antimicrobial metabolites.

Keywords: streptomyces, secondary metabolites, genome, biosynthetic gene clusters, high performance liquid chromatography, mass spectrometry

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Authors: Abhaydeep Pandey, Shweta Shukla, Saptamita Goswami, Bhaswati Bandyopadhyay, Vishnampettai Ramachandran, Sudhanshu Vrati, Arup Banerjee

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Dengue virus infection is now considered as one of the most important mosquito-borne infection in human. The virus is known to promote vascular permeability, cerebral edema leading to Dengue hemorrhagic fever (DHF) or Dengue shock syndrome (DSS). Dengue infection has known to be endemic in India for over two centuries as a benign and self-limited disease. In the last couple of years, the disease symptoms have changed, manifesting severe secondary complication. So far, Delhi has experienced 12 outbreaks of dengue virus infection since 1997 with the last reported in 2014-15. Without specific antivirals, the case management of high-risk dengue patients entirely relies on supportive care, involving constant monitoring and timely fluid support to prevent hypovolemic shock. Nonetheless, the diverse clinical spectrum of dengue disease, as well as its initial similarity to other viral febrile illnesses, presents a challenge in the early identification of this high-risk group. WHO recommends the use of warning signs to identify high-risk patients, but warning signs generally appear during, or just one day before the development of severe illness, thus, providing only a narrow window for clinical intervention. The ability to predict which patient may develop DHF and DSS may improve the triage and treatment. With the recent discovery of high throughput RNA sequencing allows us to understand the disease progression at the genomic level. Here, we will collate the results of RNA-Sequencing data obtained recently from PBMC of different categories of dengue patients from India and will discuss the possible role of deregulated genes and long non-coding RNAs NEAT1 for development of disease progression.

Keywords: long non-coding RNA (lncRNA), dengue, peripheral blood mononuclear cell (PBMC), nuclear enriched abundant transcript 1 (NEAT1), dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS)

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Authors: Neera Kapoor

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Background: Knockdown Resistance (KDR) is one of the mechanisms of insecticide resistance in insects caused by the reduced target site sensitivity i.e. voltage gated sodium channel (VGSC) rendering it less sensitive to the toxic effects of DDT and pyrethroids. In this study, we evaluated insecticide susceptibility and its underlying KDR mechanism in eight Ae. aegypti and five Ae. albopictus field populations. Methodology: Field population was collected from four different geographical regions of India covering 18 districts of ten states. For genotyping of twelve KDR alleles in Ae. aegypti field populations, three PCR based assays were used; with DNA sequencing; ASPCR; PCR-RFLP. Genomic DNA was isolated, and three partial domains (II, III, and IV) of VGSC were amplified and sequenced. Results: Molecular screening for common KDR mutations, revealed the presence of five mutations viz. S989P, V1016G, T1520I, F1534C/L. Two novel mutations were observed, first at T1520 (ACC) residue where a C > T substitution at the second position of codon results in amino acid change to Isoleucine (ATC). Second mutation was an alternative point mutation at F1534 (TTC) residue where a substitution of T > C at the first position of codon results in an amino acid change to Leucine (CTC). ASPCRs were not accurate, so three PCR-RFLP assays were developed for genotyping of five KDR alleles in Ae. aegypti; viz. T1520I, F1534C/L. Representative samples of all genotypes (n=200) were sequenced to validate the newly developed PCR based assays for Ae. aegypti. Genotyping results showed that 989P is linked to 1016G and novel mutation 1520I was always found with 1534C allele. Conclusion: Present study confirmed the presence of DDT and pyrethroid resistance among Ae. aegypti populations in India and for the first time reported KDR mutations in this species from India including two novel mutations. Results of present study lead us to infer that, at least five KDR mutations (S989P, V1016G, T1530I, F1534C, and F1534L) can be seen as a potential marker for DDT/pyrethroid resistance.

Keywords: F1534C, F1534L, S989P, T1530I, V1016G

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Authors: Alok Kumar Yadav, Saurabh Saraswat, Preeti Sirohi, Manjoo Rani, Sameer Srivastava, Manish Pratap Singh, Nand K. Singh

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The development of antibiotic resistance in bacteria is increasing at an alarming rate, and this is considered as one of the most serious threats in the history of medicine, and an alternative solution should be derived so as to tackle this problem. In many countries, people use the medicinal plants for the treatment of various diseases as these are cheaper, easily available and least toxic. Syzygium cumini is used for the treatment of various kinds of diseases but their mechanism of action is not reported. The antimicrobial activity of Syzygium cumini was tested by the well diffusion assay and zone of inhibition was reported to be 20.06 mm as compared to control with MIC of 0.3 mg/ml. Genomic DNA fragmentation of Bacillus subtilis revealed apoptosis and FE-SEM indicate cell wall cracking on several intervals of time. Propidium iodide staining results showed that few bacterial cells were stained in the control and population of stained cells increase after exposing them for various period of time. Flow cytometric kinetic data analysis on the membrane permeabilization in bacterial cell showed the significant contribution of antimicrobial potential of the seed extract on antimicrobial-induced permeabilization. Two components of Syzygium cumini methanolic seed extract was found to be quite active against four enzymes like PDB ID- 1W5D, 4OX3, 3MFD and 5E2F which have a very crucial role in membrane synthesis in Bacillus subtilis by in silico analysis. Through in silico analysis, lupeol showed highest binding energy for macromolecule 1W5D and 4OX3 whereas stigmasterol showed the highest binding energy for macromolecule 3MFD and 5E2F respectively. It showed that methanolic seed extract of Syzygium cumini can be used for the inhibition of foodborne infections caused by Bacillus subtilis and also as an alternative of prevalent antibiotics.

Keywords: antibiotics, Bacillus subtilis, inhibition, Syzygium cumini

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Authors: Debabrata Biswas

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The current trend in the development of antibiotic resistance by multiple bacterial pathogens has resulted in a troubling loss of effective antibiotic options for human. The emergence of multi-drug-resistant pathogens has necessitated higher dosages and combinations of multiple antibiotics, further exacerbating the problem of antibiotic resistance. Zoonotic bacterial pathogens, such as Salmonella, Campylobacter, Shiga toxin-producing Escherichia coli (such as enterohaemorrhagic E. coli or EHEC), and Listeria are the most common and predominant foodborne enteric infectious agents. It was observed that these pathogens gained/developed their ability to survive in the presence of antibiotics either in farm animal gut or farm environment and researchers believe that therapeutic and sub-therapeutic antibiotic use in farm animal production might play an important role in it. The mechanism of action of antimicrobial components used in farm animal production in genomic interplay in the gut and farm environment, has not been fully characterized. Even the risk of promoting the exchange of mobile genetic elements between microbes specifically pathogens needs to be evaluated in depth, to ensure sustainable farm animal production, safety of our food and to mitigate/limit the enteric infection with multiple antibiotic resistant bacterial pathogens. Due to the consumer’s demand and considering the current emerging situation, many countries are in process to withdraw antibiotic use in farm animal production. Before withdrawing use of the sub-therapeutic antibiotic or restricting the use of therapeutic antibiotics in farm animal production, it is essential to find alternative natural antimicrobials for promoting the growth of farm animal and/or treating animal diseases. Further, it is also necessary to consider whether that compound(s) has the potential to trigger the acquisition or loss of genetic materials in zoonotic and any other bacterial pathogens. Development of alternative therapeutic and sub-therapeutic antimicrobials for farm animal production and food processing and preservation and their effective implementation for sustainable strategies for farm animal production as well as the possible risk for horizontal gene transfer in major enteric pathogens will be focus in the study.

Keywords: food safety, natural antimicrobial, sustainable farming, antibiotic resistance

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Authors: Tanakrit Doltanakarn

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Introduction: These days, cannabis is being accepted in many countries due to the fact that cannabis could be use in medical. The medical cannabis is used to treat and reduce the pain many diseases. For example, neuropathic pain, Parkinson, autism disorders, cancer pain reduce the adverse effect of chemotherapy, diabetes, and migraine. Active ingredients in cannabis that modulate patients' perceptions of their conditions include Δ9‐tetrahydrocannabinol (THC), cannabidiol (CBD), flavonoids, and terpenes. However, there is an adverse effect of cannabis, cardiovascular effects, psychosis, schizophrenia, mood disorder, and cognitive alternation. These effects are from the THC and CBD ingredients in the cannabis. The metabolize processes of delta-9 THC to 11-OH-delta 9 -THC (inactive form), THC were cause of adverse effects. Interestingly, the distributions of CYP2C9 gene (CYP2C9*2 and CYP2C9*3, poor metabolizer) that might affect incidences of adverse effects in patients who treated with medical cannabis. Objective: The aim of this study we want to investigate the association between genetic polymorphism of CYP2C9 frequency and Thai patients who treated with medical cannabis. Materials and Methods:We recruited sixty-five unrelated Thai patients from the College of Pharmacy, Rangsit University. DNA were extracted using Genomic DNA Mini Kit. Genotyping of CYP2C9*2 (430C>T, rs1799853) and CYP2C9*3 (1075A>C, rs1057910) were genotyped by the TaqMan Real-time PCR assay. Results: Among these 31 medicals cannabis-induced ADRs patients, they were diagnosed with 22 (33.85%) tachycardia and 3 (4.62%) arrhythmia. There were 34 (52.31%) medical cannabis-tolerant controls who were included in this study.40 (61.53%) Thai patients were female, and 25 (38.46%) were male, with median age of 57 (range 27 – 87) years. In this study, we found none of the medical cannabis-induced ADRs carried CYP2C9*2 variant along with medical cannabis-tolerant control group. CYP2C9*3 variant (intermediate metabolizer, IM) was found just only one of thirty-one (3.23%) in the medical cannabis-induced ADRs and two of thirty-fourth (5.88%) in the tolerant controls. Conclusions: Thus, the distribution of CYP2C9 alleles offer a comprehensive view of pharmacogenomics marker in Thai population that could be used as a reference for worldwide to investigate the pharmacogenomics application.

Keywords: medical cannabis, adverse effect, CYP2C9, thai patients

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Authors: Clara Franch de la Cal, Christopher J Morris, Michael McArthur

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Cystic fibrosis (CF) is an autosomal recessive genetic disorder characterized by abnormal transport of chloride and sodium across the lung epithelium, leading to thick and viscous secretions. Within which CF patients suffer from repeated bacterial pulmonary infections, with Pseudomonas aeru-ginosa (PA) eliciting the greatest inflammatory response, causing an irreversible loss of lung func-tion that determines morbidity and mortality. The cell wall of PA is a permeability barrier to many antibacterials and the rise of Mutli-Drug Resistant strains (MDR) is eroding the efficacy of the few remaining clinical options. In addition when PA infection becomes established it forms an antibi-otic-resistant biofilm, embedded in which are slow growing cells that are refractive to drug treat-ment. Making the development of new antibacterials a major challenge. This work describes the development of new type of nanoparticulate oligonucleotide antibacterial capable of tackling PA infections, including MDR strains. It is being developed to both block growth and prevent biofilm formation. These oligonucleotide therapeutics, Transcription Factor Decoys (TFD), act on novel genomic targets by capturing key regulatory proteins to block essential bacterial genes and defeat infection. They have been successfully transfected into a wide range of pathogenic bacteria, both in vitro and in vivo, using a proprietary delivery technology. The surfactant used self-assembles with TFD to form a nanoparticle stable in biological fluids, which protects the TFD from degradation and preferentially transfects prokaryotic membranes. Key challenges are to adapt the nanoparticle so it is active against PA in the context of biofilms and to formulate it for administration by inhalation. This would allow the drug to be delivered to the respiratory tract, thereby achieving drug concentrations sufficient to eradicate the pathogenic organisms at the site of infection.

Keywords: antibacterials, transcriptional factor decoys (TFDs), pseudomonas aeruginosa

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Authors: R. Hasterok, A. Betekhtin, N. Borowska, A. Braszewska-Zalewska, E. Breda, K. Chwialkowska, R. Gorkiewicz, D. Idziak, J. Kwasniewska, M. Kwasniewski, D. Siwinska, A. Wiszynska, E. Wolny

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In contrast to animals, the organisation of plant genomes at the cytomolecular level is still relatively poorly studied and understood. However, the Brachypodium genus in general and B. distachyon in particular represent exceptionally good model systems for such study. This is due not only to their highly desirable ‘model’ biological features, such as small nuclear genome, low chromosome number and complex phylogenetic relations, but also to the rapidly and continuously growing repertoire of experimental tools, such as large collections of accessions, WGS information, large insert (BAC) libraries of genomic DNA, etc. Advanced cytomolecular techniques, such as fluorescence in situ hybridisation (FISH) with evermore sophisticated probes, empowered by cutting-edge microscope and digital image acquisition and processing systems, offer unprecedented insight into chromatin organisation at various phases of the cell cycle. A good example is chromosome painting which uses pools of chromosome-specific BAC clones, and enables the tracking of individual chromosomes not only during cell division but also during interphase. This presentation outlines the present status of molecular cytogenetic analyses of plant genome structure, dynamics and evolution using B. distachyon and some of its relatives. The current projects focus on important scientific questions, such as: What mechanisms shape the karyotypes? Is the distribution of individual chromosomes within an interphase nucleus determined? Are there hot spots of structural rearrangement in Brachypodium chromosomes? Which epigenetic processes play a crucial role in B. distachyon embryo development and selective silencing of rRNA genes in Brachypodium allopolyploids? The authors acknowledge financial support from the Polish National Science Centre (grants no. 2012/04/A/NZ3/00572 and 2011/01/B/NZ3/00177)

Keywords: Brachypodium, B. distachyon, chromosome, FISH, molecular cytogenetics, nucleus, plant genome organisation

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Authors: Aoxue Yang, Weili Tian, Yonghe Wu, Haikun Liu

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Brain tumor stem cells (BTSCs) are resistant to therapy and give rise to recurrent tumors. These rare and elusive cells are likely to disseminate during cancer progression, and some may enter dormancy, remaining viable but not increasing. The identification of dormant BTSCs is thus necessary to design effective therapies for glioblastoma (GBM) patients. Little progress has been made in therapeutic treatment of glioblastoma in the last decade despite rapid progress in molecular understanding of brain tumors1. Here we show that the stress hormone glucocorticoid is essential for the maintenance of brain tumor stem cells (BTSCs), which are resistant to conventional therapy. The glucocorticoid receptor (GR) regulates metabolic plasticity and chemoresistance of the dormant BTSC via controlling expression of GPD1 (glycerol-3-phosphate dehydrogenase 1), which is an essential regulator of lipid metabolism in BTSCs. Genomic, lipidomic and cellular analysis confirm that GR/GPD1 regulation is essential for BTSCs metabolic plasticity and survival. We further demonstrate that the GR agonist dexamethasone (DEXA), which is commonly used to control edema in glioblastoma, abolishes the effect of chemotherapy drug temozolomide (TMZ) by upregulating GPD1 and thus promoting tumor cell dormancy in vivo, this provides a mechanistic explanation and thus settle the long-standing debate of usage of steroid in brain tumor patient edema control. Pharmacological inhibition of GR/GPD1 pathway disrupts metabolic plasticity of BTSCs and prolong animal survival, which is superior to standard chemotherapy. Patient case study shows that GR antagonist mifepristone blocks tumor progression and leads to symptomatic improvement. This study identifies an important mechanism regulating cancer stem cell dormancy and provides a new opportunity for glioblastoma treatment.

Keywords: cancer stem cell, dormancy, glioblastoma, glycerol-3-phosphate dehydrogenase 1, glucocorticoid receptor, dexamethasone, RNA-sequencing, phosphoglycerides.

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Authors: Zuliani Mahmood, Thirumulu Ponnuraj Kannan, Yean Yean Chan, Salahddin A. Al-Hudhairy

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Caries is the most common childhood disease which develops due to disturbances in the physiological equilibrium in the dental plaque resulting in demineralization of tooth structures. Plaque and dentine samples were collected from three different tooth surfaces representing caries progression (intact, over carious lesion and dentine) in children with early childhood caries (ECC, n=36). In caries free (CF) children, plaque samples were collected from sound tooth surfaces at baseline and after one year (n=12). The genomic DNA was extracted from all samples and subjected to 16S rRNA PCR amplification. The end products were cloned into pCR®2.1-TOPO® Vector. Five randomly selected positive clones collected from each surface were sent for sequencing. Identification of the bacterial clones was performed using BLAST against GenBank database. In the ECC group, the frequency of Lactobacillus sp. detected was significantly higher in the dentine surface (p = 0.031) than over the cavitated lesion. The highest frequency of bacteria detected in the intact surfaces was Fusobacterium nucleatum subsp. polymorphum (33.3%) while Streptococcus mutans was detected over the carious lesions and dentine surfaces at a frequency of 33.3% and 52.7% respectively. Fusobacterium nucleatum subsp. polymorphum was also found to be highest in the CF group (41.6%). Follow up at the end of one year showed that the frequency of Corynebacterium matruchotii detected was highest in those who remained caries free (16.6%), while Porphyromonas catoniae was highest in those who developed caries (25%). In conclusion, Streptococcus mutans and Porphyromonas catoniae are strongly associated with caries progression, while Lactobacillus sp. is restricted to deep carious lesions. Fusobacterium nucleatum subsp. polymorphum and Corynebacterium matruchotii may play a role in sustaining the healthy equilibrium in the dental plaque. These identified bacteria show promise as potential biomarkers in diagnosis which could help in the management of dental caries in children.

Keywords: early childhood caries, genotypic identification, oral bacteria, 16S rRNA

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Authors: Jasbir Singh, Devender Kumar, Davender Redhu, Surender Kumar, Vandana Bhardwaj

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Single Nucleotide polymorphism (SNP) of proteosomal gene complex is involved in the pathogenesis of hepatitis B Virus (HBV) infection. Some of such proteosomal gene complex are large multifunctional proteins (LMP) and antigen associated transporters that help in antigen presentation. Both are involved in intracellular processing and presentation of viral antigens in association with Major Histocompatability Complex (MHC) Class I molecules. A total of hundred each of hepatitis B virus infected and control samples from northern India were studied. Genomic DNA was extracted from all studied samples and PCR-RFLP method was used for genotyping at different positions of LMP genes. Genotypes at a given position were inferred from the pattern of bands and genotype frequencies and haplotype frequencies were also calculated. Homozygous SNP {A>C} was observed at codon 145 of LMP7 gene and having a protective role against HBV as there was statistically significant high distribution of this SNP among controls than cases. Heterozygous SNP {A>C} was observed at codon 145 of LMP7 gene and made individuals more susceptible to HBV infection as there was statistically significant high distribution of this SNP among cases than control. SNP {T>C} was observed at codon 60 of LMP2 gene but statistically significant differences were not observed among controls and cases. For codon 145 of LMP7 and codon 60 of LMP2 genes, four haplotypes were constructed. Haplotype I (LMP2 ‘C’ and LMP7 ‘A’) made individuals carrying it more susceptible to HBV infection as there was statistically significant high distribution of this haplotype among cases than control. Haplotype II (LMP2 ‘C’ and LMP7 ‘C’) made individuals carrying it more immune to HBV infection as there was statistically significant high distribution of this haplotype among control than cases. Thus it can be concluded that homozygous SNP {A>C} at codon 145 of LMP7 and Haplotype II (LMP2 ‘C’ and LMP7 ‘C’) has a protective role against HBV infection whereas heterozygous SNP {A>C} at codon 145 of LMP7 and Haplotype I (LMP2 ‘C’ and LMP7 ‘A’) made individuals more susceptible to HBV infection.

Keywords: Hepatitis B Virus, single nucleotide polymorphism, low molecular weight proteins, transporters associated with antigen presentation

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Authors: Hung-Chieh Chen, Kuo-Hui Wang, Tsu-Lin Yeh

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Proteomics represent the performance of genomic complement proteins and the protein level on functional genomics. This study adopted proteomic strategies for comparing serum proteins among three groups: elite sprinter (sprint runner group, SR), long-distance runners (long-distance runner group, LDR), and the untrained control group (control group, CON). Purposes: This study aims to identify elite sprinters and long-distance runners’ serum protein and to provide a comparison of their serum proteome’ composition. Methods: Serum protein fractionations that separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by a quantitative nano-LC-MS/MS-based proteomic profiling. The one-way analysis of variance (ANOVA) and Scheffe post hoc comparison (α= 0.05) was used to determine whether there is any significant difference in each protein level among the three groups. Results: (1) After analyzing the 307 identified proteins, there were 26 unique proteins in the SR group, and 18 unique proteins in the LDR group. (2) For the LDR group, 7 coagulation function-associated proteins’ expression levels were investigated: vitronectin, serum paraoxonase/arylesterase 1, fibulin-1, complement C3, vitamin K-dependent protein, inter-alpha-trypsin inhibitor heavy chain H3 and von Willebrand factor, and the findings show the seven coagulation function-associated proteins were significantly lower than the group of SR. (3) Comparing to the group of SR, this study found that the LDR group’s expression levels of the 2 antioxidant proteins (afamin and glutathione peroxidase 3) were also significantly lower. (4) The LDR group’s expression levels of seven immune function-related proteins (Ig gamma-3 chain C region, Ig lambda-like polypeptide 5, clusterin, complement C1s subcomponent, complement factor B, complement C4-A, complement C1q subcomponent subunit A) were also significantly lower than the group of SR. Conclusion: This study identified the potential serum protein markers for elite sprinters and long-distance runners. The changes in the regulation of coagulation, antioxidant, or immune function-specific proteins may also provide further clinical applications for these two different track athletes.

Keywords: biomarkers, coagulation, immune response, oxidative stress

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Authors: Reema K. AlEssa, Sahar Alshomer, Abdullah Alfaleh, Sultan ALkhenaizan, Mohammed Albalwi

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Ichthyosis is a disorder of abnormal keratinization, characterized by excessive scaling, and consists of more than twenty subtypes varied in severity, mode of inheritance, and the genes involved. There is insufficient data in the literature about the epidemiology and characteristics of ichthyosis locally. Our aim is to identify the histopathological features and genetic profile of ichthyosis. Method: It is an observational retrospective case series study conducted in March 2020, included all patients who were diagnosed with Ichthyosis and confirmed by histological and molecular findings over the last 20 years in King Abdulaziz Medical City (KAMC), Riyadh, Saudi Arabia. Molecular analysis was performed by testing genomic DNA and checking genetic variations using the AmpliSeq panel. All disease-causing variants were checked against HGMD, ClinVar, Genome Aggregation Database (gnomAD), and Exome Aggregation Consortium (ExAC) databases. Result: A total of 60 cases of Ichthyosis were identified with a mean age of 13 ± 9.2. There is an almost equal distribution between female patients 29 (48%) and males 31 (52%). The majority of them were Saudis, 94%. More than half of patients presented with general scaling 33 (55%), followed by dryness and coarse skin 19 (31.6%) and hyperlinearity 5 (8.33%). Family history and history of consanguinity were seen in 26 (43.3% ), 13 (22%), respectively. History of colloidal babies was found in 6 (10%) cases of ichthyosis. The most frequent genes were ALOX12B, ALOXE3, CERS3, CYP4F22, DOLK, FLG2, GJB2, PNPLA1, SLC27A4, SPINK5, STS, SUMF1, TGM1, TGM5, VPS33B. Most frequent variations were detected in CYP4F22 in 16 cases (26.6%) followed by ALOXE3 6 (10%) and STS 6 (10%) then TGM1 5 (8.3) and ALOX12B 5 (8.3). The analysis of molecular genetic identified 23 different genetic variations in the genes of ichthyosis, of which 13 were novel mutations. Homozygous mutations were detected in the majority of ichthyosis cases, 54 (90%), and only 1 case was heterozygous. Few cases, 4 (6.6%) had an unknown type of ichthyosis with a negative genetic result. Conclusion: 13 novel mutations were discovered. Also, about half of ichthyosis patients had a positive history of consanguinity.

Keywords: ichthyosis, genetic profile, molecular characterization, congenital ichthyosis

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Authors: Islam Nowisser, Noha Farag, Mohamed El Azizi

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Helicobacter pylori is a highly important human pathogen which inhabits about 50% of the population worldwide. Infection with this bacteria is very hard to treat, with high probability of recurrence. H. pylori causes severe gastric diseases, including peptic ulcer, gastritis, and gastric cancer, which has been linked to chronic inflammation. The infection has been reported to be associated with high levels of pro-inflammatory cytokines, especially IL-1β and TNF-α. The aim of the current study is to investigate the molecular mechanisms by which H. pylori activates NLRP3 inflammasome and its contribution to Il-1 β production in an innate cellular model. H. pylori PMSS1 and G27 standard strains, as well as the PMSS1 isogenic mutant strain PMSS1ΔVacA and G27ΔVacA, G27ΔCagA in addition to clinical isolates obtained from biopsy samples from the antrum and corpus mucosa of chronic gastritis patients, were used to establish infection in RAW-264.7 macrophages. The production levels of TNF-α and IL-1β was assessed using ELISA. Since expression of these cytokines is often regulated by the transcription factor complex, nuclear factor-kB (NF-kB), the activation of NF-κB in H. pylori infected cells was also evaluated by luciferase assay. Genomic DNA was extracted from bacterial cultures of H. pylori clinical isolates as well as the standard strains and their corresponding mutants, where they were evaluated for the cagA pathogenicity island and vacA expression. The correlation between these findings and expression of the cagA Pathogenicity Island and vacA in the bacteria was also investigated. The results showed IL-1β, and TNF-α production significantly increased in raw macrophages following H. pylori infection. The cagA+ and vacA+ H. pylori strains induced significant production of IL-1β compared to cagA- and vacA- strains. The activation pattern of NF-κB was correlated in the isolates to their cagA and vacA expression profiles. A similar finding could not be confirmed for TNF-α production. Our study shows the ability of H. pylori to activate NF-kB and induce significant IL-1β production as a possible mechanism for the augmented inflammatory response seen in subjects infected with cagA+ and vacA+ H. pylori strains that would lead to the progression to more severe form of the disease.

Keywords: Helicobacter pylori, IL-1β, inflammatory cytokines, nuclear factor KB, TNF-α

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Authors: Mohamed Shafi Bin Mahboob Ali

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Introduction: Synchronous tumor is defined as the presence of more than one primary malignant lesion in the same patient at the indexed diagnosis. It is a rare occurrence, especially in the spectrum of colorectal cancer, which accounts for less than 4%. The underlying pathology of a synchronous tumor is thought to be due to a genomic factor, which is microsatellite instability (MIS) with the involvement of BRAF, KRAS, and the GSRM1 gene. There are no specific sites of occurrence for the synchronous colorectal tumor, but many studies have shown that a synchronous tumor has about 43% predominance in the ascending colon with rarity in the sigmoid colon. Case Report: We reported a case of a young lady in the middle of her 30's with no family history of colorectal cancer that was diagnosed with a synchronous adenocarcinoma at the descending colon and rectosigmoid region. The lady's presentation was quite perplexing as she presented to the district hospital initially with simple, uncomplicated hemorrhoids and constipation. She was then referred to our center for further management as she developed a 'football' sized right gluteal swelling with a complete intestinal obstruction and bilateral lower-limb paralysis. We performed a CT scan and biopsy of the lesion, which found that the tumor engulfed the sacrococcygeal region with more than one primary lesion in the colon as well as secondaries in the liver. The patient was operated on after a multidisciplinary meeting was held. Pelvic exenteration with tumor debulking and anterior resection were performed. Postoperatively, she was referred to the oncology team for chemotherapy. She had a tremendous recovery in eight months' time with a partial regain of her lower limb power. The patient is still under our follow-up with an improved quality of life post-intervention. Discussion: Synchronous colon cancer is rare, with an incidence of 2.4% to 12.4%. It has male predominance and is pathologically more advanced compared to a single colon lesion. Down staging the disease by means of chemoradiotherapy has shown to be effective in managing this tumor. It is seen commonly on the right colon, but in our case, we found it on the left colon and the rectosigmoid. Conclusion: Managing a synchronous colon tumor could be challenging to surgeons, especially in deciding the extent of resection and postoperative functional outcomes of the bowel; thus, individual treatment strategies are needed to tackle this pathology.

Keywords: synchronous, colon, tumor, adenocarcinoma

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Authors: Janneth Gonzalez, Andres Pinzon Velasco, Maria Angarita, Nicolas Mendoza

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Astrocytes play an important role in various processes in the brain, including pathological conditions such as neurodegenerative diseases. Recent studies have shown that the increase in saturated fatty acids such as palmitic acid (PA) triggers pro-inflammatory pathways in the brain. The use of synthetic neurosteroids such as tibolone has demonstrated neuro-protective mechanisms. However, there are few studies on the neuro-protective mechanisms of tibolone, especially at the systemic (omic) level. In this study, we performed the integration of multi-omic data (transcriptome and proteome) into a human astrocyte genomic scale metabolic model to study the astrocytic response during palmitate treatment. We evaluated metabolic fluxes in three scenarios (healthy, induced inflammation by PA, and tibolone treatment under PA inflammation). We also use control theory to identify those reactions that control the astrocytic system. Our results suggest that PA generates a modulation of central and secondary metabolism, showing a change in energy source use through inhibition of folate cycle and fatty acid β-oxidation and upregulation of ketone bodies formation.We found 25 metabolic switches under PA-mediated cellular regulation, 9 of which were critical only in the inflammatory scenario but not in the protective tibolone one. Within these reactions, inhibitory, total, and directional coupling profiles were key findings, playing a fundamental role in the (de)regulation in metabolic pathways that increase neurotoxicity and represent potential treatment targets. Finally, this study framework facilitates the understanding of metabolic regulation strategies, andit can be used for in silico exploring the mechanisms of astrocytic cell regulation, directing a more complex future experimental work in neurodegenerative diseases.

Keywords: astrocytes, data integration, palmitic acid, computational model, multi-omics, control theory

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Authors: Saima Saleem, Zubair Abbasi, Abdul Hameed, Mansoor Ahmed Khan, Navid Rashid Qureshi, Abid Azhar

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Oral Squamous Cell Carcinoma (OSCC) is the leading cause of death in the developing countries like Pakistan. This problem aggravates because of the excessive use of available chewing products. In spite of widespread information on their use and purported legislations against their use the Pakistani markets are classical examples of selling chewable carcinogenic mutagens. Reported studies indicated that these products are rich in reactive oxygen species (ROS) and polyphenols. TP53 gene is involved in the suppression of tumor. It has been reported that somatic mutations caused by TP53 gene are the foundation of the cancer. This study aims to find the loss of TP53 functions due to mutation/polymorphism caused by genomic alteration and interaction with tobacco and its related ingredients. Total 260 tissues and blood specimens were collected from OSCC patients and compared with age and sex matched controls. Mutations in exons 2-11 of TP53 were examined by PCR-SSCP. Samples showing mobility shift were directly sequenced. Two mutations were found in exon 4 at nucleotide position 108 and 215 and one in exon 7 at nucleotide position 719 of the coding sequences in patient’s tumor samples. These results show that substitution of proline with arginine at codon 72 and serine with threonine at codon 240 of p53 protein. These polymorphic changes, found in tumor samples of OSCC, could be involved in loss of heterozygocity and apoptotic activity in the binding domain of TP53. The model of the mutated TP53 gene elaborated a nonfunctional unfolded p53 protein, suggesting an important role of these mutations in p53 protein inactivation and malfunction. This nonfunctional 3D model also indicates that exogenous tobacco related carcinogens may act as DNA-damaging agents affecting the structure of DNA. The interpretations could be helpful in establishing the pathways responsible for tumor formation in OSCC patients.

Keywords: TP53 mutation/polymorphism, OSCC, PCR-SSCP, direct DNA sequencing, 3D structure

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Authors: Benjamin A. Ha, Marco Morselli, Xinhui Paige Zhang, Elizabeth A. C. Heath-Heckman, Jonathan B. Puritz, David K. Jacobs

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Next-generation sequencing has enhanced the ability to acquire massive amounts of sequence data to address classic population genetic questions for non-model organisms. Targeted approaches allow for cost effective or more precise analyses of relevant sequences; although, many such techniques require a known genome and it can be costly to purchase probes from a company. This is challenging for non-model organisms with no published genome and can be expensive for large population genetic studies. Expressed exome capture sequencing (EecSeq) synthesizes probes in the lab from expressed mRNA, which is used to capture and sequence the coding regions of genomic DNA from a pooled suite of samples. A normalization step produces probes to recover transcripts from a wide range of expression levels. This approach offers low cost recovery of a broad range of genes in the genome. This research project expands on EecSeq to investigate if mRNA from one taxon may be used to capture relevant sequences from a series of increasingly less closely related taxa. For this purpose, we propose to use the endangered Northern Tidewater goby, Eucyclogobius newberryi, a non-model organism that inhabits California coastal lagoons. mRNA will be extracted from E. newberryi to create probes and capture exomes from eight other taxa, including the more at-risk Southern Tidewater goby, E. kristinae, and more divergent species. Captured exomes will be sequenced, analyzed bioinformatically and phylogenetically, then compared to previously generated phylogenies across this group of gobies. This will provide an assessment of the utility of the technique in cross-species studies and for analyzing low genetic variation within species as is the case for E. kristinae. This method has potential applications to provide economical ways to expand population genetic and evolutionary biology studies for non-model organisms.

Keywords: coastal lagoons, endangered species, non-model organism, target capture method

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Authors: Farahnaz Sadat Golestan Hashemi, Mohd Razi Ismail, Mohd Y. Rafii

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A huge revolution has emerged in genome engineering by the discovery of CRISPR (clustered regularly interspaced palindromic repeats) and CRISPR-associated system genes (Cas) in bacteria. The function of type II Streptococcus pyogenes (Sp) CRISPR/Cas9 system has been confirmed in various species. Other S. thermophilus (St) CRISPR-Cas systems, CRISPR1-Cas and CRISPR3-Cas, have been also reported for preventing phage infection. The CRISPR1-Cas system interferes by cleaving foreign dsDNA entering the cell in a length-specific and orientation-dependant manner. The S. thermophilus CRISPR3-Cas system also acts by cleaving phage dsDNA genomes at the same specific position inside the targeted protospacer as observed in the CRISPR1-Cas system. It is worth mentioning, for the effective DNA cleavage activity, RNA-guided Cas9 orthologs require their own specific PAM (protospacer adjacent motif) sequences. Activity levels are based on the sequence of the protospacer and specific combinations of favorable PAM bases. Therefore, based on the specific length and sequence of PAM followed by a constant length of target site for the three orthogonals of Cas9 protein, a well-organized procedure will be required for high-throughput and accurate mining of possible target sites in a large genomic dataset. Consequently, we created a reliable procedure to explore potential gRNA sequences for type I (Streptococcus thermophiles), II (Streptococcus pyogenes), and III (Streptococcus thermophiles) CRISPR/Cas systems. To mine CRISPR target sites, four different searching modes of sgRNA binding to target DNA strand were applied. These searching modes are as follows: i) coding strand searching, ii) anti-coding strand searching, iii) both strand searching, and iv) paired-gRNA searching. The output of such procedure highlights the power of comparative genome mining for different CRISPR/Cas systems. This could yield a repertoire of Cas9 variants with expanded capabilities of gRNA design, and will pave the way for further advance genome and epigenome engineering.

Keywords: CRISPR/Cas systems, gRNA mining, Streptococcus pyogenes, Streptococcus thermophiles

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