Search results for: nucleotide sequencing

Authors: A. Díaz-Roa, P. I. Silva Junior, F. J. Bello

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Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Dipterous maggots release diverse proteins and peptides contained in larval excretion and secretion (ES) products playing a key role in digestion. The most important mechanism for combating infection using larval therapy depends on larval ES. These larvae are protected against infection by a diverse spectrum of antimicrobial peptides (AMPs), one already known like lucifensin. Special interest in these peptides has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue and kill different bacteria during larval therapy. The action of larvae on wounds occurs through 3 mechanisms of action: removal of necrotic tissue, stimulation of granulation tissue, and antibacterial action of larval ES. Some components of the ES include calcium, urea, allantoin ammonium bicarbonate and reducing the viability of Gram positive and Gram negative bacteria. The Lucilia sericata fly larvae have been the most used, however, we need to evaluate new species that could potentially be similar or more effective than fly above. This study was thus aimed at identifying and characterizing S. magellanica AMPs contained in ES products for the first time and compared them with the common fly used L. sericata. These products were obtained from third-instar larvae taken from a previously established colony. For the first analysis, ES fractions were separate by Sep-Pak C18 disposable columns (first step). The material obtained was fractionated by RP-HPLC by using Júpiter C18 semi-preparative column. The products were then lyophilized and their antimicrobial activity was characterized by incubation with different bacterial strains. The first chromatographic analysis of ES from L. sericata gives 6 fractions with antimicrobial activity against Gram-positive bacteria Micrococus luteus, and 3 fractions with activity against Gram-negative bacteria Pseudomonae aeruginosa while the one from S. magellanica gaves 1 fraction against M. luteus and 4 against P. aeruginosa. Maybe one of these fractions could correspond to the peptide already known from L. sericata. These results show the first work for supporting further experiments aimed at validating S. magellanica use in larval therapy. We still need to search if we find some new molecules, by making mass spectrometry and ‘de novo sequencing’. Further studies are necessary to identify and characterize them to better understand their functioning.

Keywords: antimicrobial peptides, larval therapy, Lucilia sericata, Sarconesiopsis magellanica

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Authors: Miaomiao Zhang, Sabrina Beckmann, Haluk Ertan, Rocky Chau, Mike Manefield

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Phenazines are a large class of nitrogen-containing aromatic heterocyclic compounds, which are almost exclusively produced by bacteria from diverse genera including Pseudomonas and Streptomyces. Phenazine-1-carboxylic acid (PCA) as one of 'core' phenazines are converted from chorismic acid before modified to other phenazine derivatives in different cells. Phenazines have attracted enormous interests because of their multiple roles on biocontrol, bacterial interaction, biofilm formation and fitness of their producers. However, in spite of ecological importance, degradation as a part of phenazines’ fate only have extremely limited attention now. Here, to isolate PCA-degrading bacteria, 200 mg L-1 PCA was supplied as sole carbon, nitrogen and energy source in minimal mineral medium. Quantitative PCR and Reverse-transcript PCR were employed to study abundance and activity of functional gene MFORT 16269 in PCA degradation, respectively. Intermediates and products of PCA degradation were identified with LC-MS/MS. After enrichment and isolation, a PCA-degrading strain was selected from soil and was designated as Rhodanobacter sp. PCA2 based on full 16S rRNA sequencing. As determined by HPLC, strain PCA2 consumed 200 mg L-1 (836 µM) PCA at a rate of 17.4 µM h-1, accompanying with significant cells yield from 1.92 × 105 to 3.11 × 106 cells per mL. Strain PCA2 was capable of degrading other phenazines as well, including phenazine (4.27 µM h-1), pyocyanin (2.72 µM h-1), neutral red (1.30 µM h-1) and 1-hydroxyphenazine (0.55 µM h-1). Moreover, during the incubation, transcript copies of MFORT 16269 gene increased significantly from 2.13 × 106 to 8.82 × 107 copies mL-1, which was 2.77 times faster than that of the corresponding gene copy number (2.20 × 106 to 3.32 × 107 copies mL-1), indicating that MFORT 16269 gene was activated and played roles on PCA degradation. As analyzed by LC-MS/MS, decarboxylation from the ring structure was determined as the first step of PCA degradation, followed by cleavage of nitrogen-containing ring by dioxygenase which catalyzed phenazine to nitrosobenzene. Subsequently, phenylhydroxylamine was detected after incubation for two days and was then transferred to aniline and catechol. Additionally, genomic and proteomic analyses were also carried out for strain PCA2. Overall, the findings presented here showed that a newly isolated strain Rhodanobacter sp. PCA2 was capable of degrading phenazines through decarboxylation and cleavage of nitrogen-containing ring, during which MFORT 16269 gene was activated and played important roles.

Keywords: decarboxylation, MFORT16269 gene, phenazine-1-carboxylic acid degradation, Rhodanobacter sp. PCA2

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Authors: Louise H. Gracioso, Marcela P.G. Baltazar, Ingrid R. Avanzi, Bruno Karolski, Luciana J. Gimenes, Claudio O. Nascimento, Elen A. Perpetuo

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Introduction: Higher copper concentrations promote a selection pressure on organisms such as plants, fungi and bacteria, which allows surviving only the resistant organisms to the contaminated site. This selective pressure keeps only the organisms most resistant to a specific condition and subsequently increases their bioremediation potential. Despite the bacteria importance for biosphere maintenance, it is estimated that only a small fraction living microbial species has been described and characterized. Due to the molecular biology development, tools based on analysis 16S ribosomal RNA or another specific gene are making a new scenario for the characterization studies and identification of microorganisms in the environment. News identification of microorganisms methods have also emerged like Biotyper (MALDI / TOF), this method mass spectrometry is subject to the recognition of spectroscopic patterns of conserved and features proteins for different microbial species. In view of this, this study aimed to isolate bacteria resistant to copper present in a Copper Processing Area (Sossego Mine, Canaan, PA) and identifies them in two different methods: Recent (spectrometry mass) and conventional. This work aimed to use them for a future bioremediation of this Mining. Material and Methods: Samples were collected at fifteen different sites of five periods of times. Microorganisms were isolated from mining wastes by culture enrichment technique; this procedure was repeated 4 times. The isolates were inoculated into MJS medium containing different concentrations of chloride copper (1mM, 2.5mM, 5mM, 7.5mM and 10 mM) and incubated in plates for 72 h at 28 ºC. These isolates were subjected to mass spectrometry identification methods (Biotyper – MALDI/TOF) and 16S gene sequencing. Results: A total of 105 strains were isolated in this area, bacterial identification by mass spectrometry method (MALDI/TOF) achieved 74% agreement with the conventional identification method (16S), 31% have been unsuccessful in MALDI-TOF and 2% did not obtain identification sequence the 16S. These results show that Biotyper can be a very useful tool in the identification of bacteria isolated from environmental samples, since it has a better value for money (cheap and simple sample preparation and MALDI plates are reusable). Furthermore, this technique is more rentable because it saves time and has a high performance (the mass spectra are compared to the database and it takes less than 2 minutes per sample).

Keywords: copper mining area, bioremediation, microorganisms, identification, MALDI/TOF, RNA 16S

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Authors: Maryam Beiranvand

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Multi-drug resistance in various microorganisms has increased globally in many healthcare facilities. Less effective antimicrobial activity of drug therapies for infection control becomes trouble. Since 1980, no new type of antimicrobial drug has been identified, even though combinations of antibiotic drugs have been discovered almost every decade. Between 1981 and 2006, over 70% of novel pharmaceuticals and chemical agents came from natural sources. Microorganisms have yielded almost 22,000 natural compounds. The identification of antimicrobial components from endophytes bacteria could help overcome the threat posed by multi-drug resistant strains. The project aims to analyze and identify antimicrobial peptides isolated from entophytic bacteria and their activity against multidrug-resistant Gram-negative bacteria in South Korea. Endophytic Paenibacillus polymyxa. 4G3 isolated from the plant, Gynura procumbery exhibited considerable antimicrobial activity against Methicillin-resistant Staphylococcus aureus, and Escherichia coli. The Rapid Annotations using Subsystems Technology showed that the total size of the draft genome was 5,739,603bp, containing 5178 genes with 45.8% G+C content. Genome annotation using antiSMASH version 6.0.0 was performed, which predicted the most common types of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS). In this study, diethyl aminoethyl cellulose (DEAEC) resin was used as the first step in purifying for unknown peptides, and then the target protein was identified using hydrophilic and hydrophobic solutions, optimal pH, and step-by-step tests for antimicrobial activity. This crude was subjected to C18 chromatography and elution with 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol, respectively. Only the fraction eluted with 20% -60% methanol demonstrated good antimicrobial activity against MDR E. coli. The concentration of the active fragment was measured by the Brad-ford test, and Protein A280 - Thermo Fisher Scientific at the end by examining the SDS PAGE Resolving Gel, 10% Acrylamide and purity were confirmed. Our study showed that, based on the combined results of the analysis and purification. P polymyxa. 4G3 has a high potential exists for producing novel functions of polymyxin E and bacitracin against bacterial pathogens.

Keywords: endophytic bacteria, antimicrobial activity, antimicrobial peptide, whole genome sequencing analysis, multi -drug resistance gram negative bacteria

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Authors: Samantha A. Cintron, Janet Pierce, Mihaela E. Sardiu, Diane Mahoney, Jill Peltzer, Bhanu Gupta, Qiuhua Shen

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High output heart failure (HOHF) is characterized a high output state resulting from an underlying disease process and is commonly caused by obesity. As obesity levels increase, more individuals will be at risk for obesity-related HOHF. However, the underlying pathophysiologic mechanisms of obesity-related HOHF are not well understood and need further research. The aim of the study was to describe the differences in leukocyte transcriptomes of morbidly obese patients with HOHF and those with non-HOHF. In this cross-sectional study, the study team collected blood samples, demographics, and clinical data of six patients with morbid obesity and HOHF and six patients with morbid obesity and non-HOHF. The study team isolated the peripheral blood leukocyte RNA and applied stranded total RNA sequencing. Differential gene expression was calculated, and Ingenuity Pathway Analysis software was used to interpret the canonical pathways, functional changes, upstream regulators, and mechanistic and causal networks that were associated with the significantly different leukocyte transcriptomes. The study team identified 116 differentially expressed genes; 114 were upregulated, and 2 were downregulated in the HOHF group (Benjamini-Hochberg adjusted p-value ≤ 0.05 and log2(fold-change) of ±1). The differentially expressed genes were involved with cell proliferation, mitochondrial function, erythropoiesis, erythrocyte stability, and apoptosis. The top upregulated canonical pathways associated with differentially expressed genes were autophagy, adenosine monophosphate-activated protein kinase signaling, and senescence pathways. Upstream regulator GATA Binding Protein 1 (GATA1) and a network associated with nuclear factor kappa-light chain-enhancer of activated B cells (NF-kB) were also identified based on the different leukocyte transcriptomes of morbidly obese patients with HOHF and non-HOHF. To the author’s best knowledge, this is the first study that reported the differential gene expression in patients with obesity-related HOHF and demonstrated the unique pathophysiologic mechanisms underlying the disease. Further research is needed to determine the role of cellular function and maintenance, inflammation, and iron homeostasis in obesity-related HOHF.

Keywords: cardiac output, heart failure, obesity, transcriptomics

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Authors: Christine Y. Yeh, Brian D. Piening, Sarah M. Totten, Kimberly Kukurba, Wenyu Zhou, Kevin P. F. Contrepois, Gucci J. Gu, Sharon Pitteri, Michael Snyder

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Three million deaths worldwide are attributed to obesity. However, the biomolecular mechanisms that describe the link between adiposity and subsequent disease states are poorly understood. Insulin resistance characterizes approximately half of obese individuals and is a major cause of obesity-mediated diseases such as Type II diabetes, hypertension and other cardiovascular diseases. This study makes use of longitudinal quantitative and high-throughput multi-omics (genomics, epigenomics, transcriptomics, glycoproteomics etc.) methodologies on blood samples to develop multigenic and multi-analyte signatures associated with weight gain and insulin resistance. Participants of this study underwent a 30-day period of weight gain via excessive caloric intake followed by a 60-day period of restricted dieting and return to baseline weight. Blood samples were taken at three different time points per patient: baseline, peak-weight and post weight loss. Patients were characterized as either insulin resistant (IR) or insulin sensitive (IS) before having their samples processed via longitudinal multi-omic technologies. This comparative study revealed a wealth of biomolecular changes associated with weight gain after using methods in machine learning, clustering, network analysis etc. Pathways of interest included those involved in lipid remodeling, acute inflammatory response and glucose metabolism. Some of these biomolecules returned to baseline levels as the patient returned to normal weight whilst some remained elevated. IR patients exhibited key differences in inflammatory response regulation in comparison to IS patients at all time points. These signatures suggest differential metabolism and inflammatory pathways between IR and IS patients. Biomolecular differences associated with weight gain and insulin resistance were identified on various levels: in gene expression, epigenetic change, transcriptional regulation and glycosylation. This study was not only able to contribute to new biology that could be of use in preventing or predicting obesity-mediated diseases, but also matured novel biomedical informatics technologies to produce and process data on many comprehensive omics levels.

Keywords: insulin resistance, multi-omics, next generation sequencing, proteogenomics, type ii diabetes

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Authors: Nuha Mansour Alhazmi, Magda Mohamed Aly, Fardus M. Bokhari, Ahmed Bahieldin, Sherif Edris

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Out of 30 fungal isolates, 2 new isolates were proven to degrade poly-β-hydroxybutyrate (PHB). Enzyme assay for these isolates indicated the optimal environmental conditions required for depolymerase enzyme to induce the highest level of biopolymer degradation. The two isolates were basically characterized at the morphological level as Trichoderma asperellum (isolate S1), and Aspergillus fumigates (isolate S2) using standard approaches. The aim of the present study was to characterize these two isolates at the molecular level based on the highly diverged rRNA gene(s). Within this gene, two domains of the ribosome large subunit (LSU) namely internal transcribed spacer (ITS) and 26S were utilized in the analysis. The first domain comprises the ITS1/5.8S/ITS2 regions ( > 500 bp), while the second domain comprises the D1/D2/D3 regions ( > 1200 bp). Sanger sequencing was conducted at Macrogen (Inc.) for the two isolates using primers ITS1/ITS4 for the first domain, while primers LROR/LR7 for the second domain. Sizes of the first domain ranged between 594-602 bp for S1 isolate and 581-594 bp for S2 isolate, while those of the second domain ranged between 1228-1238 bp for S1 isolate and 1156-1291 for S2 isolate. BLAST analysis indicated 99% identities of the first domain of S1 isolate with T. asperellum isolates XP22 (ID: KX664456.1), CTCCSJ-G-HB40564 (ID: KY750349.1), CTCCSJ-F-ZY40590 (ID: KY750362.1) and TV (ID: KU341015.1). BLAST of the first domain of S2 isolate indicated 100% identities with A. fumigatus isolate YNCA0338 (ID: KP068684.1) and strain MEF-Cr-6 (ID: KU597198.1), while 99% identities with A. fumigatus isolate CCA101 (ID: KT877346.1) and strain CD1621 (ID: JX092088.1). Large numbers of other T. asperellum and A. fumigatus isolates and strains showed high level of identities with S1 and S2 isolates, respectively, based on the diversity of the first domain. BLAST of the second domain of S1 isolate indicated 99 and 100% identities with only two strains of T. asperellum namely TR 3 (ID: HM466685.1) and G (ID: KF723005.1), respectively. However, other T. species (ex., atroviride, hamatum, deliquescens, harzianum, etc.) also showed high level of identities. BLAST of the second domain of S2 isolate indicated 100% identities with A. fumigatus isolate YNCA0338 (ID: KP068684.1) and strain MEF-Cr-6 (ID: KU597198.1), while 99% identities with A. fumigatus isolate CCA101 (ID: KT877346.1) and strain CD1621 (ID: JX092088.1). Large numbers of other A. fumigatus isolates and strains showed high level of identities with S2 isolate. Overall, the results of molecular characterization based on rRNA diversity for the two isolates of T. asperellum and A. fumigatus matched those obtained by morphological characterization. In addition, ITS domain proved to be more sensitive than 26S domain in diversity profiling of fungi at the species level.

Keywords: Aspergillus fumigates, Trichoderma asperellum, PHB, degradation, BLAST, ITS, 26S, rRNA

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Authors: Selisha A. Sooklal, Phelelani Mpangase, Shaun Aron, Karl Rumbold

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Organically bound fluorine (C-F bond) is extremely rare in nature. Despite this, the first fluorinated secondary metabolite, fluoroacetate, was isolated from the plant Dichapetalum cymosum (commonly known as Gifblaar). However, the enzyme responsible for fluorination (fluorinase) in Gifblaar was never isolated and very little progress has been achieved in understanding this process in higher plants. Fluorinated compounds have vast applications in the pharmaceutical, agrochemical and fine chemicals industries. Consequently, an enzyme capable of catalysing a C-F bond has great potential as a biocatalyst in the industry considering that the field of fluorination is virtually synthetic. As with any biocatalyst, a range of these enzymes are required. Therefore, it is imperative to expand the exploration for novel fluorinases. This study aimed to gain molecular insights into secondary metabolite biosynthesis in Gifblaar using a high-throughput sequencing-based approach. Mechanical wounding studies were performed using Gifblaar leaf tissue in order to induce expression of the fluorinase. The transcriptome of the wounded and unwounded plant was then sequenced on the Illumina HiSeq platform. A total of 26.4 million short sequence reads were assembled into 77 845 transcripts using Trinity. Overall, 68.6 % of transcripts were annotated with gene identities using public databases (SwissProt, TrEMBL, GO, COG, Pfam, EC) with an E-value threshold of 1E-05. Sequences exhibited the greatest homology to the model plant, Arabidopsis thaliana (27 %). A total of 244 annotated transcripts were found to be differentially expressed between the wounded and unwounded plant. In addition, secondary metabolic pathways present in Gifblaar were successfully reconstructed using Pathway tools. Due to lack of genetic information for plant fluorinases, a transcript failed to be annotated as a fluorinating enzyme. Thus, a local database containing the 5 existing bacterial fluorinases was created. Fifteen transcripts having homology to partial regions of existing fluorinases were found. In efforts to obtain the full coding sequence of the Gifblaar fluorinase, primers were designed targeting the regions of homology and genome walking will be performed to amplify the unknown regions. This is the first genetic data available for Gifblaar. It has provided novel insights into the mechanisms of metabolite biosynthesis and will allow for the discovery of the first eukaryotic fluorinase.

Keywords: biocatalyst, fluorinase, gifblaar, transcriptome

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Authors: Mike Wei, Nebu Koshy, Koen van Besien, Giorgio Inghirami, Steven M. Horwitz

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T-cell prolymphocytic leukemia (T-PLL) is a rare hematologic disease characterized by a T-cell phenotype, rapid progression, and poor prognosis with median survival of less than a year. Alemtuzumab-based chemotherapy has increased the rate of complete remissions but these are often short-lived, and allogeneic transplant is considered the only curative therapy. In recent studies, JAK3 activating mutations have been identified in T-cell cancers, with T-PLL having the highest rate of JAK3 mutations (30 – 42%). As such, T-PLL is a model disease for evaluating the utility of JAK3 inhibitors. We present a case of a 64-year-old man with relapsed-refractory T-PLL. He was initially treated with alemtuzumab and obtained complete response and was consolidated with matched unrelated donor stem cell transplant. His disease stayed in remission for approximately 1.5 years before relapse, which was then treated with a clinical trial of romidepsin-lenalidomide (partial responses then progression at 6 months) and later alemtuzumab. Due to complications of myelosuppression and CMV reactivation, his treatment was interrupted leading to disease progression. The doubling time of lymphocyte count was approximately 20 days and over a span of 60 days the lymphocyte count rose from 8 x 109/L to 68 x 109/L. Exon sequencing showed a JAK3 mutation. The patient consented to and was treated with FDA-approved tofacitinib (initially 5 mg BID, increased to 10 mg BID after 15 days of treatment). An initial decrease in lymphocyte count was followed by progression. In vitro treatment of the patient’s cells showed modest effects of tofacitinib and ruxolitinib as single agents, in the range of doxorubicin, but synergy between the agents. After 40 days of treatment with tofacitinib and with a lymphocyte count of 150 x 109/L, ruxolitinib (5mg BID) was added. Over the 60 days since dual inhibition was started, the lymphocyte count has stabilized. The patient has remained completely asymptomatic during treatment with tofacitinib and ruxolitinib. Neutrophil count has remained normal. Platelet count and hemoglobin have however declined from ~50 x109/L to ~30 x109/L and from 11 g/dL to 8.1 g/dL respectively, since the introduction of ruxolitinib. The stabilization in lymphocyte count confirms the clinical activity of JAK inhibitors in T-PLL as suggested by the presence of JAK3 mutations and by in-vitro assays. It also suggests clinical synergy between ruxolitinib and tofacitinib in this setting. Prospective studies of JAK inhibitors in PLL patients with formal dose-finding studies are needed.

Keywords: tofacitinib, ruxolitinib, T-cell prolymphocytic leukemia, JAK3

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Authors: Philippa Saunders, Claire Fletcher

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INTRODUCTION: Prostate cancer is the most prevalent malignancy affecting Western males. It is initially an androgen-dependent disease: androgens bind to the androgen receptor and drive the expression of genes that promote proliferation and evasion of apoptosis. Despite reduced androgen dependence in advanced prostate cancer, androgen receptor signaling remains a key driver of growth. Androgen deprivation therapy (ADT) is, therefore, a first-line treatment approach and works well initially, but resistance inevitably develops. Abiraterone and Enzalutamide are drugs widely used in ADT and are androgen synthesis and androgen receptor signaling inhibitors, respectively. The shortage of other treatment options means acquired resistance to these drugs is a major clinical problem. MicroRNAs (miRs) are important mediators of post-transcriptional gene regulation and show altered expression in cancer. Several have been linked to the development of resistance to ADT. Manipulation of such miRs may be a pathway to breakthrough treatments for advanced prostate cancer. This study aimed to validate ADT resistance-implicated miRs and their clinically relevant targets. MATERIAL AND METHOD: Small RNA-sequencing of Abiraterone- and Enzalutamide-resistant C42 prostate cancer cells identified subsets of miRs dysregulated as compared to parental cells. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) was used to validate altered expression of candidate ADT resistance-implicated miRs 195-5p, 497-5p and 29a-5p in ADT-resistant and -responsive prostate cancer cell lines, patient-derived xenografts (PDXs) and primary prostate cancer explants. RESULTS AND DISCUSSION: This study suggests a possible role for miR-497-5p in the development of ADT resistance in prostate cancer. MiR-497-5p expression was increased in ADT-resistant versus ADT-responsive prostate cancer cells. Importantly, miR-497-5p expression was also increased in Enzalutamide-treated, castrated (ADT-mimicking) PDXs versus intact PDXs. MiR-195-5p was also elevated in ADT-resistant versus -responsive prostate cancer cells, while there was a drop in miR-29a-5p expression. Candidate clinically relevant targets of miR-497-5p in prostate cancer were identified by mining AGO-PAR-CLIP-seq data sets and may include AVL9 and FZD6. CONCLUSION: In summary, this study identified microRNAs that are implicated in prostate cancer resistance to androgen deprivation therapy and could represent novel therapeutic targets for advanced disease.

Keywords: microRNA, androgen deprivation therapy, Enzalutamide, abiraterone, patient-derived xenograft

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Authors: Gianmaria Califano, Júlio Augusto Lucena Maciel, Olfa Zarrouk, Miguel Damasio, Jose Silvestre, Ana Margarida Fortes

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Global warming, with rapid and sudden changes in meteorological conditions, is one of the major constraints to ensuring agricultural and crop resilience in the Mediterranean regions. Several strategies are being adopted to reduce the pressure of drought stress on grapevines at regional and local scales: improvements in the irrigation systems, adoption of interline cover crops, and adaptation of pruning techniques. However, still, more can be achieved if also microbial compartments associated with plants are considered in crop management. It is known that the microbial community change according to several factors such as latitude, plant variety, age, rootstock, soil composition and agricultural management system. Considering the increasing pressure of the biotic and abiotic stresses, it is of utmost necessity to also evaluate the effects of drought on the microbiome associated with the grapevine, which is a commercially important crop worldwide. In this study, we characterize the diversity and the structure of the microbial community under three long-term irrigation levels (100% ETc, 50% ETc and rain-fed) in a drought-tolerant grapevine cultivar present worldwide, Syrah. To avoid the limitations of culture-dependent methods, amplicon sequencing with target primers for bacteria and fungi was applied to the same soil samples. The use of the DNeasy PowerSoil (Qiagen) extraction kit required further optimization with the use of lytic enzymes and heating steps to improve DNA yield and quality systematically across biological treatments. Target regions (16S rRNA and ITS genes) of our samples are being sequenced with Illumina technology. With bioinformatic pipelines, it will be possible to obtain a characterization of the bacterial and fungal diversity, structure and composition. Further, the microbial communities will be assessed for their functional activity, which remains an important metric considering the strong inter-kingdom interactions existing between plants and their associated microbiome. The results of this study will lay the basis for biotechnological applications: in combination with the establishment of a bacterial library, it will be possible to explore the possibility of testing synthetic microbial communities to support plant resistance to water scarcity.

Keywords: microbiome, metabarcoding, soil, vinegrape, syrah, global warming, crop sustainability

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Authors: Thilina Ranaweera, Enes Makalic, John L. Hopper, Adrian Bickerstaffe

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Data are the primary asset of biomedical researchers, and the engine for both discovery and research translation. As the volume and complexity of research datasets increase, especially with new technologies such as large single nucleotide polymorphism (SNP) chips, so too does the requirement for software to manage, process and analyze the data. Researchers often need to execute complicated queries and conduct complex analyzes of large-scale datasets. Existing tools to analyze such data, and other types of high-dimensional data, unfortunately suffer from one or more major problems. They typically require a high level of computing expertise, are too simplistic (i.e., do not fit realistic models that allow for complex interactions), are limited by computing power, do not exploit the computing power of large-scale parallel architectures (e.g. supercomputers, GPU clusters etc.), or are limited in the types of analysis available, compounded by the fact that integrating new analysis methods is not straightforward. Solutions to these problems, such as those developed and implemented on parallel architectures, are currently available to only a relatively small portion of medical researchers with access and know-how. The past decade has seen a rapid expansion of data management systems for the medical domain. Much attention has been given to systems that manage phenotype datasets generated by medical studies. The introduction of heterogeneous genomic data for research subjects that reside in these systems has highlighted the need for substantial improvements in software architecture. To address this problem, we have developed SPARK, an enabling and translational system for medical research, leveraging existing high performance computing resources, and analysis techniques currently available or being developed. It builds these into The Ark, an open-source web-based system designed to manage medical data. SPARK provides a next-generation biomedical data management solution that is based upon a novel Micro-Service architecture and Big Data technologies. The system serves to demonstrate the applicability of Micro-Service architectures for the development of high performance computing applications. When applied to high-dimensional medical datasets such as genomic data, relational data management approaches with normalized data structures suffer from unfeasibly high execution times for basic operations such as insert (i.e. importing a GWAS dataset) and the queries that are typical of the genomics research domain. SPARK resolves these problems by incorporating non-relational NoSQL databases that have been driven by the emergence of Big Data. SPARK provides researchers across the world with user-friendly access to state-of-the-art data management and analysis tools while eliminating the need for high-level informatics and programming skills. The system will benefit health and medical research by eliminating the burden of large-scale data management, querying, cleaning, and analysis. SPARK represents a major advancement in genome research technologies, vastly reducing the burden of working with genomic datasets, and enabling cutting edge analysis approaches that have previously been out of reach for many medical researchers.

Keywords: biomedical research, genomics, information systems, software

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Authors: Glenn Auld Knierim

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Automated robotic winding of high-temperature superconductors (HTS) addresses precision, efficiency, and reliability critical to the commercialization of products. Today’s HTS materials are mature and commercially promising but require manufacturing attention. In particular to the exaggerated rectangular cross-section (very thin by very wide), winding precision is critical to address the stress that can crack the fragile ceramic superconductor (SC) layer and destroy the SC properties. Damage potential is highest during peak operations, where winding stress magnifies operational stress. Another challenge is operational parameters such as magnetic field alignment affecting design performance. Winding process performance, including precision, capability for geometric complexity, and efficient repeatability, are required for commercial production of current HTS. Due to winding limitations, current HTS magnets focus on simple pancake configurations. HTS motors, generators, MRI/NMR, fusion, and other projects are awaiting robotic wound solenoid, planar, and spherical magnet configurations. As with conventional power cables, full transposition winding is required for long length alternating current (AC) and pulsed power cables. Robotic production is required for transposition, periodic swapping of cable conductors, and placing into precise positions, which allows power utility required minimized reactance. A full transposition SC cable, in theory, has no transmission length limits for AC and variable transient operation due to no resistance (a problem with conventional cables), negligible reactance (a problem for helical wound HTS cables), and no long length manufacturing issues (a problem with both stamped and twisted stacked HTS cables). The Infinity Physics team is solving manufacturing problems by developing automated manufacturing to produce the first-ever reliable and utility-grade commercial SC cables and magnets. Robotic winding machines combine mechanical and process design, specialized sense and observer, and state-of-the-art optimization and control sequencing to carefully manipulate individual fragile SCs, especially HTS, to shape previously unattainable, complex geometries with electrical geometry equivalent to commercially available conventional conductor devices.

Keywords: automated winding manufacturing, high temperature superconductor, magnet, power cable

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Authors: Swati Gautam, Salman Akhtar, S. P. Jaiswar, Amita Jain

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Background: FGTB is now a major global health problem mostly in developing countries including India. In humans, Mycobacterium Tuberculosis (M.tb) is a causating agent of infection. High index of suspicion is required for early diagnosis due to asymptomatic presentation of FGTB disease. In macrophages Toll Like Receptor-2 (TLR-2) is one which mediated host’s immune response to M.tb. The expression of TLR-2 on macrophages is important to determine the fate of innate immune responses to M.tb. TLR-2 have two work. First its high expression on macrophages worsen the outer of infection and another side, it maintains M.tb to its dormant stage avoids activation of M.tb from latent phase. Single Nucleotide Polymorphism (SNP) of TLR-2 gene plays an important role in susceptibility to TB among different populations and subsequently, in the development of infertility. Methodology: This Case-Control study was done in the Department of Obs and Gynae and Department of Microbiology at King George’s Medical University, U.P, Lucknow, India. Total 300 subjects (150 Cases and 150 Controls) were enrolled in the study. All subjects were enrolled only after fulfilling the given inclusion and exclusion criteria. Inclusion criteria: Age 20-35 years, menstrual-irregularities, positive on Acid-Fast Bacilli (AFB), TB-PCR, (LJ/MGIT) culture in Endometrial Aspiration (EA). Exclusion criteria: Koch’s active, on ATT, PCOS, and Endometriosis fibroid women, positive on Gonococal and Chlamydia. Blood samples were collected in EDTA tubes from cases and healthy control women (HCW) and genomic DNA extraction was carried out by salting-out method. Genotyping of TLR2 genetic variants (Arg753Gln and Arg677Trp) were performed by using single amplification refractory mutation system (ARMS) PCR technique. PCR products were analyzed by electrophoresis on 1.2% agarose gel and visualized by gel-doc. Statistical analysis of the data was performed using the SPSS 16.3 software and computing odds ratio (OR) with 95% CI. Linkage Disequiliribium (LD) analysis was done by SNP stats online software. Results: In TLR-2 (Arg753Gln) polymorphism significant risk of FGTB observed with GG homozygous mutant genotype (OR=13, CI=0.71-237.7, p=0.05), AG heterozygous mutant genotype (OR=13.7, CI=0.76-248.06, p=0.03) however, G allele (OR=1.09, CI=0.78-1.52, p=0.67) individually was not associated with FGTB. In TLR-2 (Arg677Trp) polymorphism a significant risk of FGTB observed with TT homozygous mutant genotype (OR= 0.020, CI=0.001-0.341, p < 0.001), CT heterozygous mutant genotype (OR=0.53, CI=0.33-0.86, p=0.014) and T allele (OR=0.463, CI=0.32-0.66, p < 0.001). TT mutant genotype was only found in FGTB cases and frequency of CT heterozygous more in control group as compared to FGTB group. So, CT genotype worked as protective mutation for FGTB susceptibility group. In haplotype analysis of TLR-2 genetic variants, four possible combinations, i.e. (G-T, A-C, G-C, and A-T) were obtained. The frequency of haplotype A-C was significantly higher in FGTB cases (0.32). Control group did not show A-C haplotype and only found in FGTB cases. Conclusion: In conclusion, study showed a significant association with both genetic variants of TLR-2 of FGTB disease. Moreover, the presence of specific associated genotype/alleles suggest the possibility of disease severity and clinical approach aimed to prevent extensive damage by disease and also helpful for early detection of disease.

Keywords: ARMS, EDTA, FGTB, TLR

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Authors: Francesco Pantano, Marta Fogolari, Michele Iuliani, Sonia Simonetti, Silvia Cavaliere, Marco Russano, Fabrizio Citarella, Bruno Vincenzi, Silvia Angeletti, Giuseppe Tonini

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Background: Although immune checkpoint inhibitors (ICIs) have changed the treatment paradigm of non–small cell lung cancer (NSCLC), these drugs fail to elicit durable responses in the majority of NSCLC patients. The gut microbiota, able to regulate immune responsiveness, is emerging as a promising, modifiable target to improve ICIs response rates. Since the oral microbiome has been demonstrated to be the primary source of bacterial microbiota in the lungs, we investigated its composition as a potential predictive biomarker to identify and select patients who could benefit from immunotherapy. Methods: Thirty-five patients with stage IV squamous and non-squamous cell NSCLC eligible for an anti-PD-1/PD-L1 as monotherapy were enrolled. Saliva samples were collected from patients prior to the start of treatment, bacterial DNA was extracted using the QIAamp® DNA Microbiome Kit (QIAGEN) and the 16S rRNA gene was sequenced on a MiSeq sequencing instrument (Illumina). Results: NSCLC patients were dichotomized as “Responders” (partial or complete response) and “Non-Responders” (progressive disease), after 12 weeks of treatment, based on RECIST criteria. A prevalence of the phylum Candidatus Saccharibacteria was found in the 10 responders compared to non-responders (abundance 5% vs 1% respectively; p-value = 1.46 x 10-7; False Discovery Rate (FDR) = 1.02 x 10-6). Moreover, a higher prevalence of Saccharibacteria Genera Incertae Sedis genus (belonging to the Candidatus Saccharibacteria phylum) was observed in "responders" (p-value = 6.01 x 10-7 and FDR = 2.46 x 10-5). Finally, the patients who benefit from immunotherapy showed a significant abundance of TM7 Phylum Sp Oral Clone FR058 strain, member of Saccharibacteria Genera Incertae Sedis genus (p-value = 6.13 x 10-7 and FDR=7.66 x 10-5). Conclusions: These preliminary results showed a significant association between oral microbiota and ICIs response in NSCLC patients. In particular, the higher prevalence of Candidatus Saccharibacteria phylum and TM7 Phylum Sp Oral Clone FR058 strain in responders suggests their potential immunomodulatory role. The study is still ongoing and updated data will be presented at the congress.

Keywords: oral microbiota, immune checkpoint inhibitors, non-small cell lung cancer, predictive biomarker

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Authors: Sridhar A. Malkaram, Tamer E. Fandy

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Purpose: 5-Azacytidine (5AC) and decitabine (DC) are DNA hypomethylating agents. We recently demonstrated that both drugs increase the enzymatic activity of the histone deacetylase enzyme SIRT6. Accordingly, we are comparing the changes H3K9 acetylation changes in the whole genome induced by both drugs using leukemia cells. Description of Methods & Materials: Mononuclear cells from the bone marrow of six de-identified naive acute myeloid leukemia (AML) patients were cultured with either 500 nM of DC or 5AC for 72 h followed by ChIP-Seq analysis using a ChIP-validated acetylated-H3K9 (H3K9ac) antibody. Chip-Seq libraries were prepared from treated and untreated cells using SMARTer ThruPLEX DNA- seq kit (Takara Bio, USA) according to the manufacturer’s instructions. Libraries were purified and size-selected with AMPure XP beads at 1:1 (v/v) ratio. All libraries were pooled prior to sequencing on an Illumina HiSeq 1500. The dual-indexed single-read Rapid Run was performed with 1x120 cycles at 5 pM final concentration of the library pool. Sequence reads with average Phred quality < 20, with length < 35bp, PCR duplicates, and those aligning to blacklisted regions of the genome were filtered out using Trim Galore v0.4.4 and cutadapt v1.18. Reads were aligned to the reference human genome (hg38) using Bowtie v2.3.4.1 in end-to-end alignment mode. H3K9ac enriched (peak) regions were identified using diffReps v1.55.4 software using input samples for background correction. The statistical significance of differential peak counts was assessed using a negative binomial test using all individuals as replicates. Data & Results: The data from the six patients showed significant (Padj<0.05) acetylation changes at 925 loci after 5AC treatment versus 182 loci after DC treatment. Both drugs induced H3K9 acetylation changes at different chromosomal regions, including promoters, coding exons, introns, and distal intergenic regions. Ten common genes showed H3K9 acetylation changes by both drugs. Approximately 84% of the genes showed an H3K9 acetylation decrease by 5AC versus 54% only by DC. Figures 1 and 2 show the heatmaps for the top 100 genes and the 99 genes showing H3K9 acetylation decrease after 5AC treatment and DC treatment, respectively. Conclusion: Despite the similarity in hypomethylating activity and chemical structure, the effect of both drugs on H3K9 acetylation change was significantly different. More changes in H3K9 acetylation were observed after 5 AC treatments compared to DC. The impact of these changes on gene expression and the clinical efficacy of these drugs requires further investigation.

Keywords: DNA methylation, leukemia, decitabine, 5-Azacytidine, epigenetics

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Authors: Fiona Wahr, Sitalakshmi Venkatraman

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Industry employers require new graduates to bring with them a range of knowledge, skills and abilities which mean these new employees can immediately make valuable work contributions. These will be a combination of discipline and professional knowledge, skills and abilities which give graduates the technical capabilities to solve practical problems whilst interacting with a range of stakeholders. Underpinning the development of these disciplines and professional knowledge, skills and abilities, are “enabling” knowledge, skills and abilities which assist students to engage in learning. These are academic and learning skills which are essential to common starting points for both the learning process of students entering the course as well as forming the foundation for the fully developed graduate knowledge, skills and abilities. This paper reports on a project created to introduce and strengthen these enabling skills into the first semester of a Bachelor of Information Technology degree in an Australian polytechnic. The project uses an action research approach in the context of ongoing continuous improvement for the course to enhance the overall learning experience, learning sequencing, graduate outcomes, and most importantly, in the first semester, student engagement and retention. The focus of this is implementing the new curriculum in first semester subjects of the course with the aim of developing the “enabling” learning skills, such as literacy, research and numeracy based knowledge, skills and abilities (KSAs). The approach used for the introduction and embedding of these KSAs, (as both enablers of learning and to underpin graduate attribute development), is presented. Building on previous publications which reported different aspects of this longitudinal study, this paper recaps on the rationale for the curriculum redevelopment and then presents the quantitative findings of entering students’ reading literacy and numeracy knowledge and skills degree as well as their perceived research ability. The paper presents the methodology and findings for this stage of the research. Overall, the cohort exhibits mixed KSA levels in these areas, with a relatively low aggregated score. In addition, the paper describes the considerations for adjusting the design and delivery of the new subjects with a targeted learning experience, in response to the feedback gained through continuous monitoring. Such a strategy is aimed at accommodating the changing learning needs of the students and serves to support them towards achieving the enabling learning goals starting from day one of their higher education studies.

Keywords: enabling skills, student retention, embedded learning support, continuous improvement

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Authors: Gizaw Mamo Gebeyehu, Marianna Pap, Geza Makkai, Tibor Z. Janosi, Shima Rashidian, Tibor A. Rauch

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Sepsis poses a significant global health threat, necessitating extensive exploration of indicators tied to its pathological mechanisms and multi-organ dysfunction. While murine studies have shed light on sepsis, the intricate cellular and molecular landscape in human sepsis remains enigmatic. Exploring the influence of activated monocyte-derived exosomes in sepsis sheds light on a promising pathway for understanding the intricate cellular and molecular mechanisms involved in this condition in humans. In sepsis, exosome-borne mRNA and miRNA orchestrate immune response gene expression in recipient cells. Yet, the specifics of exosome-mediated cell-to-cell communication, especially how mRNA cargoes modulate gene expression in recipient cells, remain poorly understood. This study aims to elucidate the precise molecular pathways through which exosomal mRNA cargo, particularly MAFB, contributes to the developing sepsis-induced molecular aberrations in liver tissues, employing rigorously defined cell culture conditions. THP-1 cells were treated with LPS to induce changes in exosomal RNA profiles. Exosomes were isolated and characterized using microscopy and mass spectrometry. RNA was extracted from exosomes and sequenced. The most abundant exosomal mRNAs were subjected to GO analysis for functional annotation analysis and KEGG database analysis to identify the involved enriched pathways. PCR (Polymerase Chain Reaction), RNA sequencing, and Western blotting were involved to analyze changes in gene expression, protein levels, and signaling pathways within the liver cells( HepG2) after exposure to exosomal MAFB. This study pinpoints exosomal MAFB as a potential key regulator linked to liver cell damage during sepsis, along with associated genes (miR155HG, H3F3A, and possibly JARD2) forming a crucial molecular pathway contributing to liver cell injury, Together, these elements indicate a vital molecular pathway that plays a significant role in the emergence of liver cell injury during sepsis.. These findings suggest the importance of further research on these components for potential therapeutic interventions in managing acute liver damage in sepsis.

Keywords: sepsis, exososome, exosomal MAFB, LPS-induced THP-1 cells, RNA profiles, sepsis-triggered liver injury

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Authors: Olga V. Chervyakova, Elmira T. Tailakova, Vitaliy M. Strochkov, Kulyaisan T. Sultankulova, Nurlan T. Sandybayev, Lev G. Nemchinov, Rosemarie W. Hammond

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Sheep pox is a highly contagious infection that OIE regards to be one of the most dangerous animal diseases. It causes enormous economic losses because of death and slaughter of infected animals, lower productivity, cost of veterinary and sanitary as well as quarantine measures. To control spread of sheep pox infection the attenuated vaccines are widely used in the Republic of Kazakhstan and other Former Soviet Union countries. In spite of high efficiency of live vaccines, the possible presence of the residual virulence, potential genetic instability restricts their use in disease-free areas that leads to necessity to exploit new approaches in vaccine development involving recombinant DNA technology. Vaccines on the basis of recombinant proteins are the newest generation of prophylactic preparations. The main advantage of these vaccines is their low reactogenicity and this fact makes them widely used in medical and veterinary practice for vaccination of humans and farm animals. The objective of the study is to produce recombinant immunogenic proteins for development of the high-performance means for sheep pox prophylaxis. The SPV proteins were chosen for their homology with the known immunogenic vaccinia virus proteins. Assay of nucleotide and amino acid sequences of the target SPV protein genes. It has been shown that four proteins SPPV060 (ortholog L1), SPPV074 (ortholog H3), SPPV122 (ortholog A33) and SPPV141 (ortholog B5) possess transmembrane domains at N- or C-terminus while in amino acid sequences of SPPV095 (ortholog А 4) and SPPV117 (ortholog А 27) proteins these domains were absent. On the basis of these findings the primers were constructed. Target genes were amplified and subsequently cloned into the expression vector рЕТ26b(+) or рЕТ28b(+). Six constructions (pSPPV060ΔТМ, pSPPV074ΔТМ, pSPPV095, pSPPV117, pSPPV122ΔТМ and pSPPV141ΔТМ) were obtained for expression of the SPV genes under control of T7 promoter in Escherichia coli. To purify and detect recombinant proteins the amino acid sequences were modified by adding six histidine molecules at C-terminus. Induction of gene expression by IPTG was resulted in production of the proteins with molecular weights corresponding to the estimated values for SPPV060, SPPV074, SPPV095, SPPV117, SPPV122 and SPPV141, i.e. 22, 30, 20, 19, 17 and 22 kDa respectively. Optimal protocol of expression for each gene that ensures high yield of the recombinant protein was identified. Assay of cellular lysates by western blotting confirmed expression of the target proteins. Recombinant proteins bind specifically with antibodies to polyhistidine. Moreover all produced proteins are specifically recognized by the serum from experimentally SPV-infected sheep. The recombinant proteins SPPV060, SPPV074, SPPV117, SPPV122 and SPPV141 were also shown to induce formation of antibodies with virus-neutralizing activity. The results of the research will help to develop a new-generation high-performance means for specific sheep pox prophylaxis that is one of key moments in animal health protection. The research was conducted under the International project ISTC # K-1704 “Development of methods to construct recombinant prophylactic means for sheep pox with use of transgenic plants” and under the Grant Project RK MES G.2015/0115RK01983 "Recombinant vaccine for sheep pox prophylaxis".

Keywords: prophylactic preparation, recombinant protein, sheep pox virus, subunit vaccine

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Authors: Maierhaba Mijiti

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Objective：The aim is to understand the relationship between the expression level of the long-non-coding RNA LINC AC008063 and the clinicopathological parameters of patients with head and neck squamous cell carcinoma (HNSCC), and to clarify the biological function of LINC AC008063 in HNSCC cells. Moreover, it provides a potential biomarker for the diagnosis, treatment, and prognosis evaluation of HNSCC. Methods: The expression level of LINC AC008063 in the HNSCC was analyzed using transcriptome sequencing data from the TCGA (The cancer genome atlas) database. The expression levels of LINC AC008063 in human embryonic lung diploid cells 2BS, human immortalized keratinocytes HACAT, HNSCC cell lines CAL-27, Detroit562, AMC-HN-8, FD-LSC-1, FaDu and WSU-HN30 were determined by real-time quantitative PCR (qPCR). RNAi (RNA interference) was introduced for LINC AC008063 knockdown in HNSCC cell lines, the localization and abundance analysis of LINC AC008063 was determined by RT-qPCR, and the biological functions were examined by CCK-8, clone formation, flow cytometry, transwell invasion and migration assays, Seahorse assay. Results: LINC AC008063 was upregulated in HNSCC tissue (P<0.001), and verified b CCK-8, clone formation, flow cytometry, transwell invasion and migration assays, Seahorse assayy qPCR in HNSCC cell lines. The survival analysis revealed that the overall survival rate (OS) of patients with high LINC AC008063 expression group was significantly lower than that in the LINC AC008063 expression group, the median survival times for the two groups were 33.10 months and 61.27 months, respectively (P=0.002). The clinical correlation analysis revealed that its expression was positively correlated with the age of patients with HNSCC (P<0.001) and positively correlated with pathological state (T3+T4＞T1+T2, P=0.03). The RT-qPCR results showed that LINC AC008063 was mainly enriched in cytoplasm (P=0.01). Knockdown of LINC AC008063 inhibited proliferation, colony formation, migration and invasion; the glycolytic capacity was significantly decreased in HNSCC cell lines (P＜0.05). Conclusion: High level of LINC AC008063 was associated with the malignant progression of HNSCC as well as promoting the important biological functions of proliferation, colony formation, migration and invasion; in particular, the glycolytic capacity was decreased in HNSCC cells. Therefore, LINC AC008063 may serve as a potential biomarker for HNSCC and a distinct molecular target to inhibit glycolysis.

Keywords: head and neck squamous cell carcinoma, oncogene, long non-coding RNA, LINC AC008063, invasion and metastasis

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Authors: Anushka Naidoo, Veron Ramsuran, Maxwell Chirehwa, Paolo Denti, Kogieleum Naidoo, Helen McIlleron, Nonhlanhla Yende-Zuma, Ravesh Singh, Sinaye Ngcapu, Nesri Padayatachi

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Background: Despite efforts to introduce new drugs and shorter drug regimens for drug-susceptible tuberculosis (TB), the standard first-line treatment has not changed in over 50 years. Rifampicin, isoniazid, and pyrazinamide are critical components of the current standard treatment regimens. Some studies suggest that microbiologic failure and acquired drug resistance are primarily driven by low drug concentrations that result from pharmacokinetic (PK) variability independent of adherence to treatment. Wide between-patient pharmacokinetic variability for rifampin, isoniazid, and pyrazinamide has been reported in prior studies. There may be several reasons for this variability. However, genetic variability in genes coding for drug metabolizing and transporter enzymes have been shown to be a contributing factor for variable tuberculosis drug exposures. Objective: We describe the pharmacokinetics of first-line TB drugs rifampicin, isoniazid, and pyrazinamide and assess the effect of genetic variability in relevant selected drug metabolizing and transporter enzymes on pharmacokinetic parameters of isoniazid and rifampicin. Methods: We conducted the randomized-controlled Improving retreatment success TB trial in Durban, South Africa. The drug regimen included rifampicin, isoniazid, and pyrazinamide. Drug concentrations were measured in plasma, and concentration-time data were analysed using nonlinear-mixed-effects models to quantify the effects of relevant covariates and single nucleotide polymorphisms (SNP’s) of drug metabolizing and transporter genes on rifampicin, isoniazid and pyrazinamide exposure. A total of 25 SNP’s: four NAT2 (used to determine acetylator status), four SLCO1B1, three Pregnane X receptor (NR1), six ABCB1 and eight UGT1A, were selected for analysis in this study. Genotypes were determined for each of the SNP’s using a TaqMan® Genotyping OpenArray™. Results: Among fifty-eight patients studied; 41 (70.7%) were male, 97% black African, 42 (72.4%) HIV co-infected and 40 (95%) on efavirenz-based ART. Median weight, fat-free mass (FFM), and age at baseline were 56.9 kg (interquartile range, IQR: 51.1-65.2), 46.8 kg (IQR: 42.5-50.3) and 37 years (IQR: 31-42), respectively. The pharmacokinetics of rifampicin and pyrazinamide was best described using one-compartment models with first-order absorption and elimination, while for isoniazid two-compartment disposition was used. The median (interquartile range: IQR) AUC (h·mg/L) and Cmax (mg/L) for rifampicin, isoniazid, and pyrazinamide were; 25.62 (23.01-28.53) and 4.85 (4.36-5.40), 10.62 (9.20-12.25) and 2.79 (2.61-2.97), 345.74 (312.03-383.10) and 28.06 (25.01-31.52), respectively. Eighteen percent of patients were classified as rapid acetylators, and 34% and 43% as slow and intermediate acetylators, respectively. Rapid and intermediate acetylator status based on NAT 2 genotype resulted in 2.3 and 1.6 times higher isoniazid clearance than slow acetylators. We found no effects of the SLCO1B1 genotypes on rifampicin pharmacokinetics. Conclusion: Plasma concentrations of rifampicin, isoniazid, and pyrazinamide were low overall in our patients. Isoniazid clearance was high overall and as expected higher in rapid and intermediate acetylators resulting in lower drug exposures. In contrast to reports from previous South African or Ugandan studies, we did not find any effects of the SLCO1B1 or other genotypes tested on rifampicin PK. However, our findings are in keeping with more recent studies from Malawi and India emphasizing the need for geographically diverse and adequately powered studies. The clinical relevance of the low tuberculosis drug concentrations warrants further investigation.

Keywords: rifampicin, isoniazid pharmacokinetics, genetics, NAT2, SLCO1B1, tuberculosis

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Authors: Kakali Roy, Sahana P. Raju, Subhra Dhar, Sandipan Dhar

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Introduction: Xeroderma pigmentosa (XP) is a rare inherited (autosomal recessive) disease resulting from impairment in DNA repair that involves recognition and repair of ultraviolet radiation (UVR) induced DNA damage in the nucleotide excision repair pathway. Which results in increased photosensitivity, UVR induced damage to skin and eye, increased susceptibility of skin and ocular cancer, and progressive neurodegeneration in some patients. XP is present worldwide, with higher incidence in areas having frequent consanguinity. Being extremely rare, there is limited literature on XP and associated complications. Here, the clinico-pathological experience (spectrum of clinical presentation, histopathological findings of malignant skin lesions, and progression) of managing 8 cases of XP is presented. Methodology: A retrospective study was conducted in a pediatric tertiary care hospital in eastern India during a ten-year period from 2013 to 2022. A clinical diagnosis was made based on severe sun burn or premature photo-aging and/or onset of cutaneous malignancies at early age (1st decade) in background of consanguinity and autosomal recessive inheritance pattern in family. Results: The mean age of presentation was 1.2 years (range of 7month-3years), while three children presented during their infancy. Male to female ratio was 5:3, and all were born of consanguineous marriage. They presented with dermatological manifestations (100%) followed by ophthalmic (75%) and/or neurological symptoms (25%). Patients had normal skin at birth but soon developed extreme sensitivity to UVR in the form of exaggerated sun tanning, burning, and blistering on minimal sun exposure, followed by abnormal skin pigmentation like freckles and lentiginosis. Subsequently, over time there was progressive xerosis, atrophy, wrinkling, and poikiloderma. Six patients had varied degree of ocular involvement, while three of them had severe manifestation, including madarosis, tylosis, ectropion, Lagopthalmos, Pthysis bulbi, clouding and scarring of the cornea with complete or partial loss of vision, and ophthalmic malignancies. 50% (n=4) cases had skin and ocular pre-malignant (actinic keratosis) and malignant lesions, including melanoma and non melanoma skin cancer (NMSC) like squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) in their early childhood. One patient had simultaneous occurrence of multiple malignancies together (SCC, BCC, and melanoma). Subnormal intelligence was noticed as neurological feature, and none had sensory neural hearing loss, microcephaly, neuroregression, or neurdeficit. All the patients had been being managed by a multidisciplinary team of pediatricians, dermatologists, ophthalmologists, neurologists and psychiatrists. Conclusion: Although till date there is no complete cure for XP and the disease is ultimately fatal. But increased awareness, early diagnosis followed by persistent vigorous protection from UVR, and regular screening for early detection of malignancies along with psychological support can drastically improve patients’ quality of life and life expectancy. Further research is required on formulating optimal management of XP, specifically the role and possibilities of gene therapy in XP.

Keywords: childhood malignancies, dermato-pathological findings, eastern India, Xeroderma pigmentosa

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Authors: Boutheina Ben Abdelmoumen Mardassi, Salim Chibani, Safa Boujemaa, Amaury Vaysse, Julien Guglielmini, Elhem Yacoub

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Background and aim: Mycoplasma hominis (MH) is a pathogenic bacterium belonging to the Mollicutes class. It causes a wide range of gynecological infections and infertility among adults. Recently, we have explored for the first time the phylodistribution of Tunisian M. hominis clinical strains using an expanded MLST. We have demonstrated their distinction into two pure lineages, which each corresponding to a specific pathotype: genital infections and infertility. The aim of this project is to gain further insight into the evolutionary dynamics and the specific genetic factors that distinguish MH pathotypes Methods: Whole genome sequencing of Mycoplasma hominis clinical strains was performed using illumina Miseq. Denovo assembly was performed using a publicly available in-house pipeline. We used prokka to annotate the genomes, panaroo to generate the gene presence matrix and Jolytree to establish the phylogenetic tree. We used treeWAS to identify genetic loci associated with the pathothype of interest from the presence matrix and phylogenetic tree. Results: Our results revealed a clear categorization of the 62 MH clinical strains into two distinct genetic lineages, with each corresponding to a specific pathotype.; gynecological infections and infertility[AV1] . Genome annotation showed that GC content is ranging between 26 and 27%, which is a known characteristic of Mycoplasma genome. Housekeeping genes belonging to the core genome are highly conserved among our strains. TreeWas identified 4 virulence genes associated with the pathotype gynecological infection. encoding for asparagine--tRNA ligase, restriction endonuclease subunit S, Eco47II restriction endonuclease, and transcription regulator XRE (involved in tolerance to oxidative stress). Five genes have been identified that have a statistical association with infertility, tow lipoprotein, one hypothetical protein, a glycosyl transferase involved in capsule synthesis, and pyruvate kinase involved in biofilm formation. All strains harbored an efflux pomp that belongs to the family of multidrug resistance ABC transporter, which confers resistance to a wide range of antibiotics. Indeed many adhesion factors and lipoproteins (p120, p120', p60, p80, Vaa) have been checked and confirmed in our strains with a relatively 99 % to 96 % conserved domain and hypervariable domain that represent 1 to 4 % of the reference sequence extracted from gene bank. Conclusion: In summary, this study led to the identification of specific genetic loci associated with distinct pathotypes in M hominis.

Keywords: mycoplasma hominis, infertility, gynecological infections, virulence genes, antibiotic resistance

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Authors: Sugandha Asthana, Geetika Vajpayee, Pratibha Kumari, Shanthy Sundaram

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An economically important disease of wheat in North Western region of India is Karnal Bunt caused by smut fungus Tilletia indica. This fungal pathogen spreads by air, soil and seed borne sporodia at the time of flowering, which ultimately leads to partial bunting of wheat kernels with fishy odor and taste to wheat flour. It has very serious effects due to quarantine measures which have to be applied for grain exports. Chemical fungicides such as mercurial compounds and Propiconazole applied to the control of Karnal bunt have been only partially successful. Considering the harmful effects of chemical fungicides on man as well as environment, many countries are developing biological control as the superior substitute to chemical control. Repeated use of fungicides can be responsible for the development of resistance in fungal pathogens against certain chemical compounds. The present investigation is based on the isolation and evaluation of antifungal properties of some isolated (from natural manure) and commercial bacterial strains against Tilletia indica. Total 23 bacterial isolates were obtained and antagonistic activity of all isolates and commercial bacterial strains (Bacillus subtilis MTCC8601, Bacillus pumilus MTCC 8743, Pseudomonas aeruginosa) were tested against T. indica by dual culture plate assay (pour plate and streak plate). Test for the production of antifungal volatile organic compounds (VOCs) by antagonistic bacteria was done by sealed plate method. Amongst all s1, s3, s5, and B. subtilis showed more than 80% inhibition. Production of extracellular hydrolytic enzymes such as protease, beta 1, 4 glucanase, HCN and ammonia was studied for confirmation of antifungal activity. s1, s3, s5 and B. subtilis were found to be the best for protease activity and s5 and B. subtilis for beta 1, 4 glucanase activity. Bacillus subtilis was significantly effective for HCN whereas s3, s5 and Bacillus subtilis for ammonia production. Isolates were identified as Pseudomonas aeruginosa (s1) and B. licheniformis (s3, s5) by various biochemical assays and confirmed by16s rRNA sequencing. Use of microorganisms or their secretions as biocontrol agents to avoid plant diseases is ecologically safe and may offer long term of protection to crop. The above study reports the promising effects of these strains in better pathogen free crop production and quality maintenance as well as prevention of the excessive use of synthetic fungicides.

Keywords: antagonistic, antifungal, biocontrol, Karnal bunt

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Authors: Danish Laidin, Peter Gostomski, Aaron Marshall, Carlo Carere

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Groundwater contamination of nitrate (NO3-) is becoming more prevalent in regions of intensive and extensive agricultural activities. Household nitrate removal involves using ion exchange membranes and reverse osmosis (RO) systems, whereas industrial nitrate removal may use organic carbon substrates (e.g. methanol) for heterotrophic microbial denitrification. However, these approaches both require high capital investment and operating costs. In this study, denitrification was demonstrated using bio-electrochemical systems (BESs) inoculated from sediments and microbial enrichment cultures. The BES reactors were operated continuously as microbial electrolytic cells (MECs) with a poised potential of -0.7V and -1.1V vs Ag/AgCl. Three parallel MECs were inoculated using hydrogen-driven denitrifying enrichments, stream sediments, and biofilm harvested from a denitrifying biotrickling filter, respectively. These reactors were continuously operated for over a year as various operating conditions were investigated to determine the optimal conditions for electroactive denitrification. The mass loading rate of nitrate was varied between 10 – 70 mg NO3-/d, and the maximum observed nitrate removal rate was 22 mg NO3- /(cm2∙d) with a current of 2.1 mA. For volumetric load experiments, the dilution rate of 1 mM NO3- feed was varied between 0.01 – 0.1 hr-1 to achieve a nitrate loading rate similar to the mass loading rate experiments. Under these conditions, the maximum rate of denitrification observed was 15.8 mg NO3- /(cm2∙d) with a current of 1.7mA. Hydrogen (H2) was supplied intermittently to investigate the hydrogenotrophic potential of the denitrifying biofilm electrodes. H2 supplementation at 0.1 mL/min resulted in an increase of nitrate removal from 0.3 mg NO3- /(cm2∙d) to 3.4 mg NO3- /(cm2∙d) in the hydrogenotrophically subcultured reactor but had no impact on the reactors which exhibited direct electron transfer properties. Results from this study depict the denitrification performance of the immobilized biofilm electrodes, either by direct electron transfer or hydrogen-driven denitrification, and the contribution of the planktonic cells present in the growth medium. Other results will include the microbial community analysis via 16s rDNA amplicon sequencing, varying the effect of poising cathodic potential from 0.7V to 1.3V vs Ag/AgCl, investigating the potential of using in-situ electrochemically produced hydrogen for autotrophic denitrification and adjusting the conductivity of the feed solution to mimic groundwater conditions. These findings highlight the overall performance of sediment inoculated MECs in removing nitrate and will be used for the future development of sustainable solutions for the treatment of nitrate polluted groundwater.

Keywords: bio-electrochemical systems, groundwater, electroactive denitrification, microbial electrolytic cell

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Authors: Masoud Sheidai, Fahimeh Koohdar, Hashem Sharifi

Abstract:

Juglans regia (L.) commonly known as Persian walnut of the genus Juglans L. (Juglandaceae) is one of the most important cultivated plant species due to its high-quality wood and edible nuts. The genetic diversity analysis is essential for conservation and management of tree species. Persian walnut is native from South-Eastern Europe to North-Western China through Tibet, Nepal, Northern India, Pakistan, and Iran. The species like Persian walnut, which has a wide range of geographical distribution, should harbor extensive genetic variability to adapt to environmental fluctuations they face. We aimed to study the population genetic structure of seven Persian walnut populations including three wild and four cultivated populations by using ISSR (Inter simple sequence repeats) and SRAP (Sequence related amplified polymorphism) molecular markers. We also aimed to compare the genetic variability revealed by ISSR neutral multilocus marker and rDNA ITS sequences. The studied populations differed in morphological features as the samples in each population were clustered together and were separate from the other populations. Three wild populations studied were placed close to each other. The mantel test after 5000 times permutation performed between geographical distance and morphological distance in Persian walnut populations produced significant correlation (r = 0.48, P = 0.002). Therefore, as the populations become farther apart, they become more divergent in morphological features. ISSR analysis produced 47 bands/ loci, while we obtained 15 SRAP bands. Gst and other differentiation statistics determined for these loci revealed that most of the ISSR and SRAP loci have very good discrimination power and can differentiate the studied populations. AMOVA performed for these loci produced a significant difference (< 0.05) supporting the above-said result. AMOVA produced significant genetic difference based on ISSR data among the studied populations (PhiPT = 0.52, P = 0.001). AMOVA revealed that 53% of the total variability is due to among population genetic difference, while 47% is due to within population genetic variability. The results showed that both multilocus molecular markers and ITS sequences can differentiate Persian walnut populations. The studied populations differed genetically and showed isolation by distance (IBD). ITS sequence based MP and Bayesian phylogenetic trees revealed that Iranian walnut cultivars form a distinct clade separated from the cultivars studied from elsewhere. Almost all clades obtained have high bootstrap value. The results indicated that a combination of multilpcus and sequencing molecular markers can be used in genetic differentiation of Persian walnut.

Keywords: genetic diversity, population, molecular markers, genetic difference

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Authors: Habtamu Abera Goshu, Ping Yan

Abstract:

Copy number variation (CNV) is a significant marker of the genetic and phenotypic diversity among individuals that accounts for complex quantitative traits of phenotype and diseases via modulating gene dosage, position effects, alteration of downstream pathways, modification of chromosome structure, and position within the nucleus and disrupting coding regions in the genome. Associating copy number variations (CNVs) with growth and gene expression are a powerful approach for identifying genomic characteristics that contribute to phenotypic and genotypic variation. A previous study using next-generation sequencing illustrated that the choline kinase beta (CHKB), Krüpple-like factor 6 (KLF6), glypican 1(GPC1), and cholinergic receptor muscarinic 3 (CHRM3) genes reside within copy number variable regions (CNVRs) of yak populations that overlap with quantitative trait loci (QTLs) of meat quality and growth. As a result, this research aimed to determine the association of CNVs of the KLF6, CHKB, GPC1, and CHRM3 genes with growth traits in the Datong yak breed. The association between the CNV types of the KLF6, CHKB, GPC1, and CHRM3 genes and the growth traits in the Datong yak breed was determined by one-way analysis of variance (ANOVA) using SPSS software. The CNV types were classified as a loss (a copy number of 0 or 1), gain (a copy number >2), and normal (a copy number of 2) relative to the reference gene, BTF3 in the 387 individuals of Datong yak. These results indicated that the normal CNV types of the CHKB and GPC1 genes were significantly (P<0.05) associated with high body length, height and weight, and chest girth in six-month-old and five-year-old Datong yaks. On the other hand, the loss CNV types of the KLF6 gene is significantly (P<0.05) associated with body weight and length and chest girth at six-month-old and five-year-old Datong yaks. In the contrary, the gain CNV type of the CHRM3 gene is highly (P<0.05) associated with body weight, length, height, and chest girth in six-month-old and five-year-old. This work provides the first observation of the biological role of CNVs of the CHKB, KLF6, GPC1, and CHRM3 genes in the Datong yak breed and might, therefore, provide a novel opportunity to utilize data on CNVs in designing molecular markers for the selection of animal breeding programs for larger populations of various yak breeds. Therefore, we hypothesized that this study provided inclusive information on the application of CNVs of the CHKB, KLF6, GPC1, and CHRM3 genes in growth traits in Datong yaks and its possible function in bovine species.

Keywords: Copy number variation, growth traits, yak, genes

Procedia PDF Downloads 174

Authors: Roger WUMBA

Abstract:

The objective of this study was to determine the prevalence of intestinal parasites, with special emphasis on microsporidia and Cryptosporidium, as well as their association with human immunodeficiency virus (HIV) symptoms, risk factors, and other digestive parasites. We also wish to determine the molecular biology definitions of the species and genotypes of microsporidia and Cryptosporidium in HIV patients. In this cross-sectional study, carried out in Kinshasa, Democratic Republic of the Congo, stool samples were collected from 242 HIV patients (87 men and 155 women) with referred symptoms and risk factors for opportunistic intestinal parasites. The analysis of feces specimen were performed using Ziehl–Neelsen stainings, real-time polymerase chain reaction (PCR), immunofluorescence indirect monoclonal antibody, nested PCR-restriction fragment length polymorphism, and PCR amplification and sequencing. Odds ratio (OR) and 95% confidence intervals were used to quantify the risk. Of the 242 HIV patients, 7.8%, 0.4%, 5.4%, 0.4%, 2%, 10.6%, and 2.8% had Enterocytozoon bieneusi, Encephalitozoon intestinalis, Cryptosporidium spp., Isospora belli, pathogenic intestinal protozoa, nonpathogenic intestinal protozoa, and helminths, respectively. We found five genotypes of E. bieneusi: two older, NIA1 and D, and three new, KIN1, KIN2, and KIN3. Only 0.4% and 1.6% had Cryptosporidium parvum and Cryptosporidium hominis, respectively. Of the patients, 36.4%, 34.3%, 31%, and 39% had asthenia, diarrhea, a CD4 count of ,100 cells/mm³, and no antiretroviral therapy (ART), respectively. The majority of those with opportunistic intestinal parasites and C. hominis, and all with C. parvum and new E. bieneusi genotypes, had diarrhea, low CD4+ counts of ,100 cells/mm³, and no ART. There was a significant association between Entamoeba coli, Kaposi sarcoma, herpes zoster, chronic diarrhea, and asthenia, and the presence of 28 cases with opportunistic intestinal parasites. Rural areas, public toilets, and exposure to farm pigs were the univariate risk factors present in the 28 cases with opportunistic intestinal parasites. In logistic regression analysis, a CD4 count of ,100 cells/mm³ (OR = 4.60; 95% CI 1.70–12.20; P = 0.002), no ART (OR = 5.00; 95% CI 1.90–13.20; P , 0.001), and exposure to surface water (OR = 2.90; 95% CI 1.01–8.40; P = 0.048) were identified as the significant and independent determinants for the presence of opportunistic intestinal parasites. E. bieneusi and Cryptosporidium are becoming more prevalent in Kinshasa, Congo. Based on the findings, we recommend epidemiology surveillance and prevention by means of hygiene, the emphasis of sensitive PCR methods, and treating opportunistic intestinal parasites that may be acquired through fecal–oral transmission, surface water, normal immunity, rural area-based person–person and animal–human nfection, and transmission of HIV. Therapy, including ART and treatment with fumagillin, is needed.

Keywords: diarrhea, enterocytozoon bieneusi, cryptosporidium hominis, cryptosporidium parvum, risk factors, africans

Procedia PDF Downloads 126

Authors: Alsu F. Saifitdinova, Alexey S. Komissarov, Svetlana A. Galkina, Elena I. Koshel, Maria M. Kulak, Stephen J. O'Brien, Elena R. Gaginskaya

Abstract:

The mystery of sex determination is one of the most ancient and still not solved until the end so far. In many species, sex determination is genetic and often accompanied by the presence of dimorphic sex chromosomes in the karyotype. Genomic sequencing gave the information about the gene content of sex chromosomes which allowed to reveal their origin from ordinary autosomes and to trace their evolutionary history. Female-specific W chromosome in birds as well as mammalian male-specific Y chromosome is characterized by the degeneration of gene content and the accumulation of repetitive DNA. Tandem repeats complicate the analysis of genomic data. Despite the best efforts chicken W chromosome assembly includes only 1.2 Mb from expected 55 Mb. Supplementing the information on the sex chromosome composition not only helps to complete the assembly of genomes but also moves us in the direction of understanding of the sex-determination systems evolution. A whole-genome survey to the assembly Gallus_gallus WASHUC 2.60 was applied for repeats search in assembled genome and performed search and assembly of high copy number repeats in unassembled reads of SRR867748 short reads datasets. For cytogenetic analysis conventional methods of fluorescent in situ hybridization was used for previously cloned W specific satellites and specifically designed directly labeled synthetic oligonucleotide DNA probe was used for bioinformatically identified repetitive sequence. Hybridization was performed with mitotic chicken chromosomes and manually isolated giant meiotic lampbrush chromosomes from growing oocytes. A novel chicken W specific satellite (GGAAA)n which is not co-localizes with any previously described classes of W specific repeats was identified and mapped with high resolution. In the composition of autosomes this repeat units was found as a part of upstream regions of gonad specific protein coding sequences. These findings may contribute to the understanding of the role of tandem repeats in sex specific differentiation regulation in birds and sex chromosome evolution. This work was supported by the postdoctoral fellowships from St. Petersburg State University (#1.50.1623.2013 and #1.50.1043.2014), the grant for Leading Scientific Schools (#3553.2014.4) and the grant from Russian foundation for basic researches (#15-04-05684). The equipment and software of Research Resource Center “Chromas” and Theodosius Dobzhansky Center for Genome Bioinformatics of Saint Petersburg State University were used.

Keywords: birds, lampbrush chromosomes, sex chromosomes, tandem repeats

Procedia PDF Downloads 389

Authors: Christofas Stergianos, Jason Atkin, Herve Morvan

Abstract:

As air traffic increases, more airports are interested in utilizing optimization methods. Many processes happen in parallel at an airport, and complex models are needed in order to have a reliable solution that can be implemented for ground movement operations. The ground movement for aircraft in an airport, allocating a path to each aircraft to follow in order to reach their destination (e.g. runway or gate), is one process that could be optimized. The Quickest Path Problem with Time Windows (QPPTW) algorithm has been developed to provide a conflict-free routing of vehicles and has been applied to routing aircraft around an airport. It was subsequently modified to increase the accuracy for airport applications. These modifications take into consideration specific characteristics of the problem, such as: the pushback process, which considers the extra time that is needed for pushing back an aircraft and turning its engines on; stand holding where any waiting should be allocated to the stand; and runway sequencing, where the sequence of the aircraft that take off is optimized and has to be respected. QPPTW involves searching for the quickest path by expanding the search in all directions, similarly to Dijkstra’s algorithm. Finding a way to direct the expansion can potentially assist the search and achieve a better performance. We have further modified the QPPTW algorithm to use a heuristic approach in order to guide the search. This new algorithm is based on the A-star search method but estimates the remaining time (instead of distance) in order to assess how far the target is. It is important to consider the remaining time that it is needed to reach the target, so that delays that are caused by other aircraft can be part of the optimization method. All of the other characteristics are still considered and time windows are still used in order to route multiple aircraft rather than a single aircraft. In this way the quickest path is found for each aircraft while taking into account the movements of the previously routed aircraft. After running experiments using a week of real aircraft data from Zurich Airport, the new algorithm (A-star QPPTW) was found to route aircraft much more quickly, being especially fast in routing the departing aircraft where pushback delays are significant. On average A-star QPPTW could route a full day (755 to 837 aircraft movements) 56% faster than the original algorithm. In total the routing of a full week of aircraft took only 12 seconds with the new algorithm, 15 seconds faster than the original algorithm. For real time application, the algorithm needs to be very fast, and this speed increase will allow us to add additional features and complexity, allowing further integration with other processes in airports and leading to more optimized and environmentally friendly airports.

Keywords: a-star search, airport operations, ground movement optimization, routing and scheduling

Procedia PDF Downloads 231