Search results for: amplicon sequencing

Authors: Neha P. Amin, Maliha Zainib, Sean Parker, Malcolm Mattes

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Purpose/Objectives: Combination immunotherapy (IT) and radiation therapy (RT) is an actively growing field of clinical investigation due to promising findings of synergistic effects from immune-mediated mechanisms observed in preclinical studies and clinical data from case reports of abscopal effects. While there are many ongoing trials of combined IT-RT, there are still limited data on toxicity and outcome optimization regarding RT dose, fractionation, and sequencing of RT with IT. Nivolumab (NIVO), an anti-PD-1 monoclonal antibody, has been rapidly adopted in the clinic over the past 2 years, resulting in more patients being considered for concurrent RT-NIVO. Knowledge about the toxicity profile of combined RT-NIVO is important for both the patient and physician when making educated treatment decisions. The acute toxicity profile of concurrent RT-NIVO was analyzed in this study. Materials/Methods: A retrospective review of all consecutive patients who received NIVO from 1/2015 to 5/2017 at 4 separate centers within two separate institutions was performed. Those patients who completed a course of RT from 1 day prior to initial NIVO infusion through 1 month after last NIVO infusion were considered to have received concurrent therapy and included in the subsequent analysis. Descriptive statistics are reported for patient/tumor/treatment characteristics and observed acute toxicities within 3 months of RT completion. Results: Among 261 patients who received NIVO, 46 (17.6%) received concurrent RT to 67 different sites. The median f/u was 3.3 (.1-19.8) months, and 11/46 (24%) were still alive at last analysis. The most common histology, RT prescription, and treatment site included non-small cell lung cancer (23/46, 50%), 30 Gy in 10 fractions (16/67, 24%), and central thorax/abdomen (26/67, 39%), respectively. 79% (53/67) of irradiated sites were treated with 3D-conformal technique and palliative dose-fractionation. Grade 3, 4, and 5 toxicities were experienced by 11, 1, and 2 patients, respectively. However all grade 4 and 5 toxicities were outside of the irradiated area and attributed to the NIVO alone, and only 4/11 (36%) of the grade 3 toxicities were attributed to the RT-NIVO. The irradiated site in these cases included the brain [2/10 (20%)] and central thorax/abdomen [2/19 (10.5%)], including one unexpected grade 3 pancreatitides following stereotactic body RT to the left adrenal gland. Conclusions: Concurrent RT-NIVO is generally well tolerated, though with potentially increased rates of severe toxicity when irradiating the lung, abdomen, or brain. Pending more definitive data, we recommend counseling patients on the potentially increased rates of side effects from combined immunotherapy and radiotherapy to these locations. Future prospective trials assessing fractionation and sequencing of RT with IT will help inform combined therapy recommendations.

Keywords: combined immunotherapy and radiation, immunotherapy, Nivolumab, toxicity of concurrent immunotherapy and radiation

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Authors: M. F. F. Ab Rashid, A. N. Mohd Rose, N. M. Z. Nik Mohamed, W. S. Wan Harun, S. A. Che Ghani

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Traveling salesman problem with precedence constraint (TSPPC) is one of the most complex problems in combinatorial optimization. The existing algorithms to solve TSPPC cost large computational time to find the optimal solution. The purpose of this paper is to present an efficient genetic algorithm that guarantees optimal solution with less number of generations and iterations time. Unlike the existing algorithm that generates priority factor as chromosome, the proposed algorithm directly generates sequence of solution as chromosome. As a result, the proposed algorithm is capable of generating optimal solution with smaller number of generations and iteration time compare to existing algorithm.

Keywords: traveling salesman problem, sequencing, genetic algorithm, precedence constraint

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Authors: Paula I. Buonfiglio, Carlos David Bruque, Lucia Salatino, Vanesa Lotersztein, Sebastián Menazzi, Paola Plazas, Ana Belén Elgoyhen, Viviana Dalamón

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Hearing loss (HL) is the most prevalent sensorineural disorder affecting about 10% of the global population, with more than half due to genetic causes. About 1 in 500-1000 newborns present congenital HL. Most of the patients are non-syndromic with an autosomal recessive mode of inheritance. To date, more than 100 genes are related to HL. Therefore, the Whole-exome sequencing (WES) technique has become a cost-effective alternative approach for molecular diagnosis. Nevertheless, new challenges arise from the detection of novel variants, in particular missense changes, which can lead to a spectrum of genotype-to-phenotype correlations, which is not always straightforward. In this work, we aimed to identify the genetic causes of HL in isolated and familial cases by designing a multistep approach to analyze target genes related to hearing impairment. Moreover, we performed in silico and in vivo analyses in order to further study the effect of some of the novel variants identified in the hair cell function using the zebrafish model. A total of 650 patients were studied by Sanger Sequencing and Gap-PCR in GJB2 and GJB6 genes, respectively, diagnosing 15.5% of sporadic cases and 36% of familial ones. Overall, 50 different sequence variants were detected. Fifty of the undiagnosed patients with moderate HL were tested for deletions in STRC gene by Multiplex ligation-dependent probe amplification technique (MLPA), leading to 6% of diagnosis. After this initial screening, 50 families were selected to be analyzed by WES, achieving diagnosis in 44% of them. Half of the identified variants were novel. A missense variant in MYO6 gene detected in a family with postlingual HL was selected to be further analyzed. A protein modeling with AlphaFold2 software was performed, proving its pathogenic effect. In order to functionally validate this novel variant, a knockdown phenotype rescue assay in zebrafish was carried out. Injection of wild-type MYO6 mRNA in embryos rescued the phenotype, whereas using the mutant MYO6 mRNA (carrying c.2782C>A variant) had no effect. These results strongly suggest the deleterious effect of this variant on the mobility of stereocilia in zebrafish neuromasts, and hence on the auditory system. In the present work, we demonstrated that our algorithm is suitable for the sequential multigenic approach to HL in our cohort. These results highlight the importance of a combined strategy in order to identify candidate variants as well as the in silico and in vivo studies to analyze and prove their pathogenicity and accomplish a better understanding of the mechanisms underlying the physiopathology of the hearing impairment.

Keywords: diagnosis, genetics, hearing loss, in silico analysis, in vivo analysis, WES, zebrafish

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Authors: Atef Ibrahim, Hamed Elsimary, Abdullah Aljumah, Fayez Gebali

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This paper presents a novel semi-systolic array architecture for an optimized parallel sequence alignment algorithm. This architecture has the advantage that it can be modified to be reused for multiple pass processing in order to increase the number of processing elements that can be packed into a single FPGA and to increase the number of sequences that can be aligned in parallel in a single FPGA. This resolves the potential problem of many FPGA resources left unused for designs that have large values of short read length. When using the previously published conventional hardware design. FPGA implementation results show that, for large values of short read lengths (M>128), the proposed design has a slightly higher speed up and FPGA utilization over the the conventional one.

Keywords: bioinformatics, genome sequence alignment, re-sequencing applications, systolic array

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Authors: Fitri Rahmi Fadhilah, Edhyana Sahiratmadja, Ani Melani Maskoen, Ratu Safitri, Supartini Syarif, Herman Susanto

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Cervical cancer is the second most common cancer in women after breast cancer. In Indonesia, the incidence of cervical cancer cases is estimated at 25-40 per 100,000 women per year. Human papillomavirus (HPV) infection is a major cause of cervical cancer, and HPV-16 is the most common genotype that infects the cervical tissue. The major late protein L1 may be associated with infectivity and pathogenicity and its variation can be used to classify HPV isolates. The aim of this study was to determine the phylogenetic tree of HPV 16 L1 gene from cervical cancer patient isolates in Bandung. After confirming HPV-16 by Linear Array Genotyping Test, L1 gene was amplified using specific primers and subject for sequencing. Phylogenetic analysis revealed that HPV 16 from Bandung was in the subgroup of Asia and East Asia, showing the close host-agent relationship among the Asian type.

Keywords: L1 HPV 16, cervical cancer, bandung, phylogenetic

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Authors: Fan Gao, Lior Pachter

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The primary tool currently used to pre-process 10X Chromium single-cell ATAC-seq data is Cell Ranger, which can take very long to run on standard datasets. To facilitate rapid pre-processing that enables reproducible workflows, we present a suite of tools called scATAK for pre-processing single-cell ATAC-seq data that is 15 to 18 times faster than Cell Ranger on mouse and human samples. Our tool can also calculate chromatin interaction potential matrices, and generate open chromatin signal and interaction traces for cell groups. We use scATAK tool to explore the chromatin regulatory landscape of a healthy adult human brain and unveil cell-type specific features, and show that it provides a convenient and computational efficient approach for pre-processing single-cell ATAC-seq data.

Keywords: single-cell, ATAC-seq, bioinformatics, open chromatin landscape, chromatin interactome

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Authors: Prajakta M. Wazat, D. N. Raut

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The study consists of a fast moving consumer goods (FMCG) beverage company wherein a portion of the supply chain which deals with outbound logistics is taken for improvement in order to reduce its logistics cost by using critical path method (CPM) method. Logistics is a major portion of the supply chain where customers are not willing to pay as it adds cost to product without adding value. In this study, it is necessary to ensure that products are delivered to clients at the right time while preserving high-quality standards from the beginning to the end of the supply chain. CPM is a logical sequencing method where in the most efficient route is achieved by arranging the series of events. CPM enables to identify a critical factor in order to minimize the delays and interruption by providing a feasible solution.

Keywords: FMCG, supply chain, outbound logistics, vehicle turnaround time, critical path method, cost reduction

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Authors: Vishnu Pratap Singh Kirar, Madhuri Saxena

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Advancement in sequence data generation technologies is churning out voluminous omics data and posing a massive challenge to annotate the biological functional features. With so much data available, the use of machine learning methods and tools to make novel inferences has become obvious. Machine learning methods have been successfully applied to a lot of disciplines, including computational biology and bioinformatics. Researchers in computational biology are interested to develop novel machine learning frameworks to classify the huge amounts of biological data. In this proposal, it plan to employ novel machine learning approaches to aid the understanding of how apparently innocuous mutations (in intergenic DNA and at synonymous sites) cause diseases. We are also interested in discovering novel functional sites in the genome and mutations in which can affect a phenotype of interest.

Keywords: genome wide association studies (GWAS), next generation sequencing (NGS), deep learning, omics

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Authors: Felice Elefant, Akanksha Bhatnaghar, Keegan Krick, Elizabeth Heller

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Context: The severity of Alzheimer’s Disease (AD) progression involves an interplay of genetics, age, and environmental factors orchestrated by histone acetyltransferase (HAT) mediated neuroepigenetic mechanisms. While disruption of Tip60 HAT action in neural gene control is implicated in AD, alternative mechanisms underlying Tip60 function remain unexplored. Altered RNA splicing has recently been highlighted as a widespread hallmark in the AD transcriptome that is implicated in the disease. Research Aim: The aim of this study was to identify a novel RNA binding/splicing function for Tip60 in human hippocampus and impaired in brains from AD fly models and AD patients. Methodology/Analysis: The authors used RNA immunoprecipitation using RNA isolated from 200 pooled wild type Drosophila brains for each of the 3 biological replicates. To identify Tip60’s RNA targets, they performed genome sequencing (DNB-SequencingTM technology, BGI genomics) on 3 replicates for Input RNA and RNA IPs by Tip60. Findings: The authors' transcriptomic analysis of RNA bound to Tip60 by Tip60-RNA immunoprecipitation (RIP) revealed Tip60 RNA targets enriched for critical neuronal processes implicated in AD. Remarkably, 79% of Tip60’s RNA targets overlap with its chromatin gene targets, supporting a model by which Tip60 orchestrates bi-level transcriptional regulation at both the chromatin and RNA level, a function unprecedented for any HAT to date. Since RNA splicing occurs co-transcriptionally and splicing defects are implicated in AD, the authors investigated whether Tip60-RNA targeting modulates splicing decisions and if this function is altered in AD. Replicate multivariate analysis of transcript splicing (rMATS) analysis of RNA-Seq data sets from wild-type and AD fly brains revealed a multitude of mammalian-like AS defects. Strikingly, over half of these altered RNAs were bonafide Tip60-RNA targets enriched for in the AD-gene curated database, with some AS alterations prevented against by increasing Tip60 in fly brain. Importantly, human orthologs of several Tip60-modulated spliced genes in Drosophila are well characterized aberrantly spliced genes in human AD brains, implicating disruption of Tip60’s splicing function in AD pathogenesis. Theoretical Importance: The authors' findings support a novel RNA interaction and splicing regulatory function for Tip60 that may underlie AS impairments that hallmark AD etiology. Data Collection: The authors collected data from RNA immunoprecipitation experiments using RNA isolated from 200 pooled wild type Drosophila brains for each of the 3 biological replicates. They also performed genome sequencing (DNBSequencingTM technology, BGI genomics) on 3 replicates for Input RNA and RNA IPs by Tip60. Questions: The question addressed by this study was whether Tip60 has a novel RNA binding/splicing function in human hippocampus and whether this function is impaired in brains from AD fly models and AD patients. Conclusions: The authors' findings support a novel RNA interaction and splicing regulatory function for Tip60 that may underlie AS impairments that hallmark AD etiology.

Keywords: Alzheimer's disease, cognition, aging, neuroepigenetics

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Authors: Katherine Madero Valencia, Carlos Jaramillo, Elida Dueñas, Carlos Torres, María Del Pilar Delgado

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Asthma, described as a chronic inflammatory condition of the airways, courses accompanied by episodes known as exacerbations, characterized by a worsening of symptoms. Among the triggers, some allergen-irritative and infectious agents are found, including Chlamydophila pneumoniae which seems to play an increasingly important role. In this paper a PCR was used to detect C. pneumoniae in order to estimate the frequency of infections caused by this agent in pediatric patients with asthma exacerbations. C. pneumoniae distribution throughout the study period was also evaluated. 175 nasopharyngeal aspirates from children with asthma exacerbations were analyzed by PCR and sequencing. A global prevalence of C. pneumoniae of 53.71% was obtained. This study highlights a high circulation of C. pneumoniae during the study period, in children of all ages and especially in children under 5 years old. Molecular tests applied permit a rapid detection and improved our knowledge about these infections in children with asthma.

Keywords: Chlamydophila pneumoniae, detection, molecular techniques, pediatric asthma

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Authors: Fatimazahrae Elbakri, Azeddine Ibrahimi, Meryem Lemrani, Dris Belghyti

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Leishmaniasis represents a major public health problem because of the number of cases recorded each year and the wide distribution of the disease. It is a parasitic disease of flagellated protozoa transmitted by the bite of certain species of sandfly, causing a spectrum of clinical pathology in humans ranging from disfiguring skin lesions to fatal visceral leishmaniasis. Cutaneous leishmaniasis due to Leishmania major is a polymorphic disease; in fact, the infection can be asymptomatic, localized, or disseminated. The objective of this work is to determine the genomic diversity that contributes to clinical variability by trying to identify the variation in chromosome number and to extract SNPs and SNPs and InDels; it is based on four sequences (WGS) of Leishmania major available on NCBI in Fastq form, from three countries: Tunisia, Algeria, and Israel, the analysis is set up from a pipeline to facilitate the discovery of genetic diversity, in particular SNP and chromosomal somy.

Keywords: Leshmania major, cutaneous Leishmania, NGS, genomic, somy, variant calling

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Authors: Mariia A. Parfenenko, Ilya S. Dantsev, Sergei V. Bochenkov, Natalia V. Vinogradova, Olga S. Groznova, Victoria Yu. Voinova

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Introduction: Autism is the most common neurodevelopmental disorder. Autism is characterized by difficulties in social interaction and adherence to stereotypic behavioral patterns and frequently co-occurs with epilepsy, intellectual disabilities, connective tissue disorders, and other conditions. CHD8 codes for chromodomain-helicase-DNA-binding protein 8 - a chromatin remodeler that regulates cellular proliferation and neurodevelopment in embryogenesis. CHD8 is one of the genes most frequently involved in autism. Patients and methods: 2 unrelated male patients, P3 and P12, aged 3 and 12 years old, underwent whole genome sequencing, which determined that they both had different likely pathogenic variants, both previously undescribed in literature. Sanger sequencing later determined that P12 inherited the variant from his affected mother. Results: P3 and P12 presented with autism, a developmental delay, ataxia, sleep disorders, overgrowth, and macrocephaly, as well as other clinical features typically present in patients with causative variants in CHD8. The mother of P12 also has autistic traits, as well as ataxia, hypotonia, sleep disorders, and other symptoms. However, P3 and P12 also have different cardiac abnormalities. P3 had signs of a repolarization disorder: a flattened T wave in the III and aVF derivations and a negative T wave in the V1-V2 derivations. He also had structural valve anomalies with associated regurgitation, local contractility impairment of the left ventricular, and diastolic dysfunction of the right ventricle. Meanwhile, P12 had Wolff-Parkinson-White syndrome and underwent radiofrequency ablation at the age of 2 years. At the time of observation, P12 had mild sinus arrhythmia and an incomplete right bundle branch block, as well as arterial hypertension. Discussion: Cardiac abnormalities were not previously reported in patients with causative variants in CHD8. The underlying mechanism for the formation of those abnormalities is currently unknown. However, the two hypotheses are either a disordered interaction with CHD7 – another chromodomain remodeler known to be directly involved in the cardiophenotype of CHARGE syndrome – a rare condition characterized by coloboma, heart defects and growth abnormalities, or the disrupted functioning of CHD8 as an A-Kinase Anchoring Protein, which are known to modulate cardiac function. Conclusion: We observed 2 unrelated autistic males with likely pathogenic variants in CHD8 that presented with typical symptoms of CHD8-related neurodevelopmental disorder, as well as cardiac abnormalities. Cardiac abnormalities have, until now, been considered uncharacteristic for patients with causative variants in CHD8. Further accumulation of data, including experimental evidence of the involvement of CHD8 in heart formation, will elucidate the mechanism underlying the cardiophenotype of those patients. Acknowledgements: Molecular genetic testing of the patients was made possible by the Charity Fund for medical and social genetic aid projects «Life Genome.»

Keywords: autism spectrum disorders, chromodomain-helicase-DNA-binding protein 8, neurodevelopmental disorder, cardio phenotype

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Authors: António Inês, Ana Guimarães, José Maria, Vânia Laranjo, Armando Venâncio, Luís Abrunhosa

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Lactic acid bacteria (LAB) play a key role in the biopreservation of a wide range of fermented food products, such as yogurt, cheese, fermented milks, meat, fish, vegetables (sauerkraut, olives and pickles), certain beer brands, wines and silage, allowing their safe consumption, which gave to these bacteria a GRAS (Generally Recognised as Safe) status. Besides that, the use of LAB in food and feed is a promising strategy to reduce the exposure to dietary mycotoxins, improving their shelf life and reducing health risks, given the unique mycotoxin decontaminating characteristic of some LAB. Mycotoxins present carcinogenic, mutagenic, teratogenic, neurotoxic and immunosuppressive effects over animals and Humans, being the most important ochratoxin A (OTA), aflatoxins (AFB1), trichothecenes, zearalenone (ZEA), fumonisin (FUM) and patulin. In a previous work of our group it was observed OTA biodegradation by some strains of Pediococcus parvulus isolated from Douro wines. So, the aim of this study was to enlarge the screening of the biodetoxification over more mycotoxins besides OTA, including AFB1, and ZEA. This ability was checked in a collection of LAB isolated from vegetable (wine, olives, fruits and silage) and animal (milk and dairy products, sausages) sources. All LAB strains were characterized phenotypically (Gram, catalase) and genotypically. Molecular characterisation of all LAB strains was performed using genomic fingerprinting by MSP-PCR with (GTG)5 and csM13 primers. The identification of the isolates was confirmed by 16S rDNA sequencing. To study the ability of LAB strains to degrade OTA, AFB1 and ZEA, a MRS broth medium was supplemented with 2.0 μg/mL of each mycotoxin. For each strain, 2 mL of MRS supplemented with the mycotoxins was inoculated in triplicate with 109 CFU/mL. The culture media and bacterial cells were extracted by the addition of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v) to the culture tubes. A 2 mL sample was then collected and filtered into a clean 2 mL vial using PP filters with 0.45 μm pores. The samples were preserved at 4 °C until HPLC analysis. Among LAB tested, 10 strains isolated from milk were able to eliminate AFB1, belonging to Lactobacillus casei (7), Lb. paracasei (1), Lb. plantarum (1) and 1 to Leuconostoc mesenteroides. Two strains of Enterococcus faecium and one of Ec. faecalis from sausage eliminated ZEA. Concerning to strains of vegetal origin, one Lb. plantarum isolated from elderberry fruit, one Lb. buchnerii and one Lb. parafarraginis both isolated from silage eliminated ZEA. Other 2 strains of Lb. plantarum from silage were able to degrade both ZEA and OTA, and 1 Lb. buchnerii showed activity over AFB1. These enzymatic activities were also verified genotypically through specific gene PCR and posteriorly confirmed by sequencing analysis. In conclusion, due the ability of some strains of LAB isolated from different sources to eliminate OTA, AFB1 and ZEA one can recognize their potential biotechnological application to reduce the health hazards associated with these mycotoxins. They may be suitable as silage inoculants or as feed additives or even in food industry.

Keywords: bio-detoxification, lactic acid bacteria, mycotoxins, food and feed

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Authors: Rugang Tian

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In this study, we generated whole-genome sequence (WGS) data from110 native cattle. Together with whole-genome sequences from world-wide cattle populations, we estimated the genetic diversity and population genetic structure of different cattle populations. Our findings revealed clustering of cattle groups in line with their geographic locations. We identified noticeable genetic diversity between indigenous cattle breeds and commercial populations. Among all studied cattle groups, lower genetic diversity measures were found in commercial populations, however, high genetic diversity were detected in some local cattle, particularly in Rashoki and Mongolian breeds. Our search for potential genomic regions under selection in native cattle revealed several candidate genes related with immune response and cold shock protein on multiple chromosomes such as TRPM8, NMUR1, PRKAA2, SMTNL2 and OXR1 that are involved in energy metabolism and metabolic homeostasis.

Keywords: cattle, whole-genome, population structure, adaptation

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Authors: Ajay Kumar Rana

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Predicting human behaviour, discerning monozygotic twins or left over remnant tissues/fluids of a single human source remains a big challenge in forensic science. Recent advances in the field of DNA methylations which are broadly chemical hallmarks in response to environmental factors can certainly help to identify and discriminate various single-source DNA samples collected from the crime scenes. In this review, cytosine methylation of DNA has been methodologically discussed with its broad applications in many challenging forensic issues like body fluid identification, race/ethnicity identification, monozygotic twins dilemma, addiction or behavioural prediction, age prediction, or even authenticity of the human DNA. With the advent of next-generation sequencing techniques, blooming of DNA methylation datasets and together with standard molecular protocols, the prospect of investigating and solving the above issues and extracting the exact nature of the truth for reconstructing the crime scene events would be undoubtedly helpful in defending and solving the critical crime cases.

Keywords: DNA methylation, differentially methylated regions, human identification, forensics

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Authors: Mohamed M. Bumadian, D. James Gilmour

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Three bacterial species were isolated from the River Wye (Derbyshire, England) and identified using 16S rRNA gene sequencing as Serratia proteamaculans, Aeromonas hydrophila and Klebsiella pneumoniae. Respiration rates of the strains were measured in order to determine the metabolic activity under salt stress. The highest respiration rates of all three strains were found at 0.17 M and 0.5 M NaCl and then the respiration rate decreased with increasing concentrations of NaCl. In addition, the effect of increasing concentrations of NaCl on malate dehydrogenase activity was determined using cell-free extracts of the three strains. Malate dehydrogenase activity was stimulated at NaCl concentrations up to 0.5 M, and a small level of activity remained even at 3.5 M NaCl. The pH optimum of the malate dehydrogenase in cell-free extracts of all strains was higher than pH 7.5.

Keywords: fresh water, halotolerant pathogenic bacteria, 16S rRNA gene, cell-free extracts, respiration rates, malate dehydrogenase

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Authors: Yassir AbdelRazig

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Evolutionary optimization methods such as genetic algorithms have been used extensively for the construction site layout problem. More recently, ant colony optimization algorithms, which are evolutionary methods based on the foraging behavior of ants, have been successfully applied to benchmark combinatorial optimization problems. This paper proposes a formulation of the site layout problem in terms of a sequencing problem that is suitable for solution using an ant colony optimization algorithm. In the construction industry, site layout is a very important planning problem. The objective of site layout is to position temporary facilities both geographically and at the correct time such that the construction work can be performed satisfactorily with minimal costs and improved safety and working environment. During the last decade, evolutionary methods such as genetic algorithms have been used extensively for the construction site layout problem. This paper proposes an ant colony optimization model for construction site layout. A simple case study for a highway project is utilized to illustrate the application of the model.

Keywords: ant colony, construction site layout, optimization, genetic algorithms

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Authors: Sumeet Rai, Anuj Tyagi, B. T. Naveen Kumar, Shubhkaramjeet Kaur, Niraj K. Singh

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Since their discovery, indiscriminate use of antibiotics in human, veterinary and aquaculture systems has resulted in global emergence/spread of multidrug-resistant bacterial pathogens. Thus, the need for alternative approaches to control bacterial infections has become utmost important. High selectivity/specificity of bacteriophages (phages) permits the targeting of specific bacteria without affecting the desirable flora. In this study, a lytic phage (Ahp1) specific to Aeromonas hydrophila subsp. hydrophila was isolated from finfish aquaculture pond. The host range of Ahp1 range was tested against 10 isolates of A. hydrophila, 7 isolates of A. veronii, 25 Vibrio cholerae isolates, 4 V. parahaemolyticus isolates and one isolate each of V. harveyi and Salmonella enterica collected previously. Except the host A. hydrophila subsp. hydrophila strain, no lytic activity against any other bacterial was detected. During the adsorption rate and one-step growth curve analysis, 69.7% of phage particles were able to get adsorbed on host cell followed by the release of 93 ± 6 phage progenies per host cell after a latent period of ~30 min. Phage nucleic acid was extracted by column purification methods. After determining the nature of phage nucleic acid as dsDNA, phage genome was subjected to next-generation sequencing by generating paired-end (PE, 2 x 300bp) reads on Illumina MiSeq system. De novo assembly of sequencing reads generated circular phage genome of 42,439 bp with G+C content of 58.95%. During open read frame (ORF) prediction and annotation, 22 ORFs (out of 49 total predicted ORFs) were functionally annotated and rest encoded for hypothetical proteins. Proteins involved in major functions such as phage structure formation and packaging, DNA replication and repair, DNA transcription and host cell lysis were encoded by the phage genome. The complete genome sequence of Ahp1 along with gene annotation was submitted to NCBI GenBank (accession number MF683623). Stability of Ahp1 preparations at storage temperatures of 4 °C, 30 °C, and 40 °C was studied over a period of 9 months. At 40 °C storage, phage counts declined by 4 log units within one month; with a total loss of viability after 2 months. At 30 °C temperature, phage preparation was stable for < 5 months. On the other hand, phage counts decreased by only 2 log units over a period of 9 during storage at 4 °C. As some of the phages have also been reported as glycerol sensitive, the stability of Ahp1 preparations in (0%, 15%, 30% and 45%) glycerol stocks were also studied during storage at -80 °C over a period of 9 months. The phage counts decreased only by 2 log units during storage, and no significant difference in phage counts was observed at different concentrations of glycerol. The Ahp1 phage discovered in our study had a very narrow host range and it may be useful for phage typing applications. Moreover, the endolysin and holin genes in Ahp1 genome could be ideal candidates for recombinant cloning and expression of antimicrobial proteins.

Keywords: Aeromonas hydrophila, endolysin, phage, narrow host range

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Authors: Imad H. Hameed, Mohammad A. Jebor, Ammera J. Omer

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The aim of this research is to study the mitochonderial coding region by using the Sanger sequencing technique and establish the degree of variation characteristic of a fragment. FTA® Technology (FTA™ paper DNA extraction) utilized to extract DNA. Portion of coding region encompassing positions 11719 –12384 amplified in accordance with the Anderson reference sequence. PCR products purified by EZ-10 spin column then sequenced and Detected by using the ABI 3730xL DNA Analyzer. Five new polymorphic positions 11741, 11756, 11878, 11887 and 12133 are described may be suitable sources for identification purpose in future. The calculated value D= 0.95 and RMP=0.048 of the genetic diversity should be understood as high in the context of coding function of the analysed DNA fragment. The relatively high gene diversity and a relatively low random match probability were observed in Iraq population. The obtained data can be used to identify the variable nucleotide positions characterized by frequent occurrence which is most promising for various identifications.

Keywords: coding region, Iraq, mitochondrial DNA, polymorphic positions, sanger technique

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Authors: Leo Nnamdi Ozurumba-Dwight

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Messenger ribooxynucleic acid (mRNA) molecules are compositional, protein-based. These proteins, encoding mRNA molecules (which collectively connote the transcriptome), when analyzed by RNA sequencing (RNAseq), unveils the nature of gene expression in the RNA. The obtained gene expression provides clues of cellular traits and their dynamics in presentations. These can be studied in relation to function and responses. RNAseq is a practical concept in Genomics as it enables detection and quantitative analysis of mRNA molecules. Single cell and spatial transcriptomics both present varying avenues for expositions in genomic characteristics of single cells and pooled cells in disease conditions such as cancer, auto-immune diseases, hematopoietic based diseases, among others, from investigated biological tissue samples. Single cell transcriptomics helps conduct a direct assessment of each building unit of tissues (the cell) during diagnosis and molecular gene expressional studies. A typical technique to achieve this is through the use of a single-cell RNA sequencer (scRNAseq), which helps in conducting high throughput genomic expressional studies. However, this technique generates expressional gene data for several cells which lack presentations on the cells’ positional coordinates within the tissue. As science is developmental, the use of complimentary pre-established tissue reference maps using molecular and bioinformatics techniques has innovatively sprung-forth and is now used to resolve this set back to produce both levels of data in one shot of scRNAseq analysis. This is an emerging conceptual approach in methodology for integrative and progressively dependable transcriptomics analysis. This can support in-situ fashioned analysis for better understanding of tissue functional organization, unveil new biomarkers for early-stage detection of diseases, biomarkers for therapeutic targets in drug development, and exposit nature of cell-to-cell interactions. Also, these are vital genomic signatures and characterizations of clinical applications. Over the past decades, RNAseq has generated a wide array of information that is igniting bespoke breakthroughs and innovations in Biomedicine. On the other side, spatial transcriptomics is tissue level based and utilized to study biological specimens having heterogeneous features. It exposits the gross identity of investigated mammalian tissues, which can then be used to study cell differentiation, track cell line trajectory patterns and behavior, and regulatory homeostasis in disease states. Also, it requires referenced positional analysis to make up of genomic signatures that will be sassed from the single cells in the tissue sample. Given these two presented approaches to RNA transcriptomics study in varying quantities of cell lines, with avenues for appropriate resolutions, both approaches have made the study of gene expression from mRNA molecules interesting, progressive, developmental, and helping to tackle health challenges head-on.

Keywords: transcriptomics, RNA sequencing, single cell, spatial, gene expression.

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Authors: Z. Chekini, P. Afsharian, F. Ramezanali, A. A. Akhlaghi, R. Aflatoonian

Abstract:

Macrophage migration inhibitory factor (MIF) is a key pro-inflammatory cytokine that involves in pathophysiological events of endometriosis. We aimed to evaluate the association between mRNA expression levels and polymorphisms of MIF in endometriosis. Seventy endometriosis patients and 70 volunteer fertile women were recruited. RFLP was applied to determine -173G/C polymorphism. ORF polymorphisms and -794(CATT)5-8 were detected by sequencing. Q-PCR was used for expression study of 14 ectopic tissues of patients. Homozygote of CATT5 was observed only in controls. The CATT5/G haplotype related to controls (p=0.094, OR=0.61). Expression level of MIF with -794(CATT)6,7/-173GC was significantly more than the other haplotypes (p=0.00). We identified four SNPs including: +254rs2096525 (p=0.843), +626rs33958703 (p=0.029), +656rs2070766 (p=0.703) and +509rs182012324 (p=1.00). In conclusion, increased repeat of CATT and presence of C allele in promoter of MIF were significantly associated with mRNA level in patients. It seems that +509rs182012324 and +626rs33958703 SNPs were significantly correlated with susceptibility to endometriosis.

Keywords: endometriosis, haplotype, macrophage migration inhibitory factor, polymorphism

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Authors: Gyeong-Sung Kim, In-soo Ahn, Yong Cho

Abstract:

SBR (Sequencing Batch Reactor) process is usually applied to membrane water treatment plants to treat its concentrated wastewater. The role of SBR process is to remove COD (Chemical Oxygen Demand) and NH3 from wastewater before discharging it outside of the water treatment plant using microorganism. Microorganism’s nitrification capability is influenced by water temperature because the nitrification rate of the concentrated wastewater becomes ‘zero’ as water temperature approach 0℃. Heating system is necessary to operate SBR in winter season even though the operating cost increase sharply. The operating cost of SBR at ‘D’ RO water treatment plant in Korea was 51.8 times higher in winter (October to March) compare to summer (April to September) season in 2014. Otherwise the effluent water temperature maintained around 8℃ constantly in winter. This study focuses on application heat pump system to recover the thermal energy from the effluent water of ‘D’ RO plant so that the operating cost will be reduced.

Keywords: water treatment, water thermal energy, energy saving, RO, SBR

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Authors: H. Boulahyaoui, S. Alaoui Amine, C. Loutfi, H. El Annaz, N. Touil, El M. El Fahim, S. Mrani

Abstract:

Three Moroccan children fully vaccinated with Rotarix™ have been hospitalized for Rotavirus Gastroenteritis (RVGE) in the pediatric division of the Farabi Hospital, Oujda. Rotavirus G/P genotypes could not be typed because of their delayed crossing threshold (Ct) resolute with a group A rotavirus (RVA) real time RT-PCR. These strains were adapted to cell culture. All viruses replicated and caused extensive cytopathic effects after four or five passages in MA104 cell lines. Significant improvements have been obtained in the amount of viral particles. Each virus multiplied to a high titer (7.5 TCID50/ml). VP7 and VP4 partial gene sequencing revealed distinct genotypes compared to the Rotarix(®) vaccine strain. Two strains were of G2P[4] genotype whereas the third was G9P[8] genotype. Virus isolation while labor intensive, is recommended as a second test, especially when higher sensitivity for conventional RVA genotyping RT-PCR is needed. VP7 antigenic similarities between these strains and Rotarix were determined.

Keywords: esacpe-vaccine, Morocco, Rotarix, G2P[4], G9P[8]

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Authors: Nhat-To Huynh, Chen-Fu Chien

Abstract:

Textile batch dyeing scheduling problem is complicated which includes batch formation, batch assignment on machines, batch sequencing with sequence-dependent setup time. Most manufacturers schedule their orders manually that are time consuming and inefficient. More power methods are needed to improve the solution. Motivated by the real needs, this study aims to propose approaches in which genetic algorithm is developed with multi-subpopulation and hybridised with estimation of distribution algorithm to solve the constructed problem for minimising the makespan. A heuristic algorithm is designed and embedded into the proposed algorithms to improve the ability to get out of the local optima. In addition, an empirical study is conducted in a textile company in Taiwan to validate the proposed approaches. The results have showed that proposed approaches are more efficient than simulated annealing algorithm.

Keywords: estimation of distribution algorithm, genetic algorithm, multi-subpopulation, scheduling, textile dyeing

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Authors: Berengere Vire, Nicolas Salvetat, Yoann Lannay, Guillaume Marcellin, Siem Van Der Laan, Franck Molina, Dinah Weissmann

Abstract:

Predicting suicidal behaviors is one of the most complex challenges of daily psychiatric practices. Today, suicide risk prediction using biological tools is not validated and is only based on subjective clinical reports of the at-risk individual. Therefore, there is a great need to identify biomarkers that would allow early identification of individuals at risk of suicide. Alterations of adenosine-to-inosine (A-to-I) RNA editing of neurotransmitter receptors and other proteins have been shown to be involved in etiology of different psychiatric disorders and linked to suicidal behavior. RNA editing is a co- or post-transcriptional process leading to a site-specific alteration in RNA sequences. It plays an important role in the epi transcriptomic regulation of RNA metabolism. On postmortem human brain tissue (prefrontal cortex) of depressed suicide victims, Alcediag found specific alterations of RNA editing activity on the mRNA coding for the serotonin 2C receptor (5-HT2cR). Additionally, an increase in expression levels of ADARs, the RNA editing enzymes, and modifications of RNA editing profiles of prime targets, such as phosphodiesterase 8A (PDE8A) mRNA, have also been observed. Interestingly, the PDE8A gene is located on chromosome 15q25.3, a genomic region that has recurrently been associated with the early-onset major depressive disorder (MDD). In the current study, we examined whether modifications in RNA editing profile of prime targets allow identifying disease-relevant blood biomarkers and evaluating suicide risk in patients. To address this question, we performed a clinical study to identify an RNA editing signature in blood of depressed patients with and without the history of suicide attempts. Patient’s samples were drawn in PAXgene tubes and analyzed on Alcediag’s proprietary RNA editing platform using next generation sequencing technology. In addition, gene expression analysis by quantitative PCR was performed. We generated a multivariate algorithm comprising various selected biomarkers to detect patients with a high risk to attempt suicide. We evaluated the diagnostic performance using the relative proportion of PDE8A mRNA editing at different sites and/or isoforms as well as the expression of PDE8A and the ADARs. The significance of these biomarkers for suicidality was evaluated using the area under the receiver-operating characteristic curve (AUC). The generated algorithm comprising the biomarkers was found to have strong diagnostic performances with high specificity and sensitivity. In conclusion, we developed tools to measure disease-specific biomarkers in blood samples of patients for identifying individuals at the greatest risk for future suicide attempts. This technology not only fosters patient management but is also suitable to predict the risk of drug-induced psychiatric side effects such as iatrogenic increase of suicidal ideas/behaviors.

Keywords: blood biomarker, next-generation-sequencing, RNA editing, suicide

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Authors: Lesley Northrop, Justin DeGrazia, Jessica Greenwood

Abstract:

ASPIRA Labs now offers an en-suit and ready-to-implement technology transfer solution to enable labs and hospitals that lack the resources to build it themselves to offer in-house genetic testing. This unique platform employs a patented Molecular Inversion Probe (MIP) technology that combines the specificity of a hybrid capture protocol with the ease of an amplicon-based protocol and utilizes an advanced bioinformatics analysis pipeline based on machine learning. To demonstrate its efficacy, two independent genetic tests were validated on this technology transfer platform: expanded carrier screening (ECS) and hereditary cancer testing (HC). The analytical performance of ECS and HC was validated separately in a blinded manner for calling three different types of variants: SNVs, short indels (typically, <50 bp), and large indels/CNVs defined as multi-exonic del/dup events. The reference set was constructed using samples from Coriell Institute, an external clinical genetic testing laboratory, Maine Molecular Quality Controls Inc. (MMQCI), SeraCare and GIAB Consortium. Overall, the analytical performance showed a sensitivity and specificity of >99.4% for both ECS and HC in detecting SNVs. For indels, both tests reported specificity of 100%, and ECS demonstrated a sensitivity of 100%, whereas HC exhibited a sensitivity of 96.5%. The bioinformatics pipeline also correctly called all reference CNV events resulting in a sensitivity of 100% for both tests. No additional calls were made in the HC panel, leading to a perfect performance (specificity and F-measure of 100%). In the carrier panel, however, three additional positive calls were made outside the reference set. Two of these calls were confirmed using an orthogonal method and were re-classified as true positives leaving only one false positive. The pipeline also correctly identified all challenging carrier statuses, such as positive cases for spinal muscular atrophy and alpha-thalassemia, resulting in 100% sensitivity. After confirmation of additional positive calls via long-range PCR and MLPA, specificity for such cases was estimated at 99%. These performance metrics demonstrate that this tech-transfer solution can be confidently internalized by clinical labs and hospitals to offer mainstream ECS and HC as part of their test catalog, substantially increasing access to quality germline genetic testing for labs of all sizes and resources levels.

Keywords: clinical genetics, genetic testing, molecular genetics, technology transfer

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Authors: Foad Alzoughool

Abstract:

Conflicting studies on the association between the rs5219 polymorphism and type 2 diabetes, some studies have confirmed a strong relationship between this variant and type2 diabetes, on the other hand, many studies denied the existence of this association. This study aimed to provide evidence about whether the rs5219 polymorphism has or hasn't a role as a risk factor for diabetes and meta-analysis to investigate the role of the control age group in the association. Genotyping of the rs5219 polymorphism was performed in a cohort of 266 healthy elderly subjects with a mean age (60.2 ± 5.1) with no history of diabetes (HbA1c < 6%) using standard Sanger sequencing methods. Lys/Lys alleles were detected in 20 persons (7.5%), Lys/Glu alleles in 96 persons (36.1%), and Glu/Glu in 150 persons (56.4%). The genotype distribution was consistent with Hardy–Weinberg equilibrium (P =0.7). Meta-analysis notably indicates no association between rs5219 polymorphism and type 2 diabetes in all studies used the younger age of the control group compared to the patient's age. In conclusion, our study sheds light on the importance of age factor among the control group recruited in case-control studies.

Keywords: Type 2 diabetes, rs5219 polymorphism, E23K, KCNJ11 gene

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Authors: Biswabandhu Bankura, Srikanta Guria, Madhusudan Das

Abstract:

Purpose: Thyroid peroxidase (TPO) is the key enzyme in the biosynthesis of thyroid hormones. We aimed to identify the spectrum of mutations in the TPO gene leading to hypothyroidism in the population of West Bengal to establish the genetic etiology of the disease. Methods: 200 hypothyroid patients (case) and their corresponding sex and age matched 200 normal individuals (control) were screened depending on their clinical manifestations. Genomic DNA was isolated from peripheral blood samples and TPO gene (Exon 7 to Exon 14) was amplified by PCR. The PCR products were subjected to sequencing to identify mutations. Results: Single nucleotide changes such as Glu 641 Lys, Asp 668 Asn, Thr 725 Pro, Asp 620 Asn, Ser 398 Thr, and Ala 373 Ser were found. Changes in the TPO were assayed in vitro to compare mutant and wild-type activities. Five mutants were enzymatically inactive in the guaiacol and iodide assays. This is a strong indication that the mutations are present at crucial positions of the TPO gene, resulting in inactivated TPO. Key Findings: The results of this study may help to develop a genetic screening protocol for goiter and hypothyroidism in the population of West Bengal.

Keywords: thyroid peroxidase, hypothyroidism, mutation, in vitro assay, transfection

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Authors: Biswabandhu Bankura, Srikanta Guria, Madhusudan Das

Abstract:

Purpose: Thyroid peroxidase (TPO) is the key enzyme in the biosynthesis of thyroid hormones. We aimed to identify the spectrum of mutations in the TPO gene leading to hypothyroidism in the population of West Bengal to establish the genetic etiology of the disease. Methods: 200 hypothyroid patients (case) and their corresponding sex and age matched 200 normal individuals (control) were screened depending on their clinical manifestations. Genomic DNA was isolated from peripheral blood samples and TPO gene (Exon 7 to Exon 14) was amplified by PCR. The PCR products were subjected to sequencing to identify mutations. Results: Single nucleotide changes such as Glu 641 Lys, Asp 668 Asn, Thr 725 Pro, Asp 620 Asn, Ser 398 Thr, and Ala 373 Ser were found. Changes in the TPO were assayed in vitro to compare mutant and wild-type activities. Five mutants were enzymatically inactive in the guaiacol and iodide assays. This is a strong indication that the mutations are present at crucial positions of the TPO gene, resulting in inactivated TPO. Key Findings: The results of this study may help to develop a genetic screening protocol for goiter and hypothyroidism in the population of West Bengal.

Keywords: thyroid peroxidase, hypothyroidism, mutation, in vitro assay, transfection

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Authors: Emma J. Dale, Christina D. Buesching, Kevin R. Theis, David W. Macdonald

Abstract:

This study investigates the oral and intestinal microbiomes of wild-living European badgers (Meles meles) and will relate inter-individual differences to social contact networks, somatic and reproductive fitness, varying susceptibility to bovine tuberculous (bTB) and to the olfactory advertisement. Badgers are an interesting model for this research, as they have great variation in body condition, despite living in complex social networks and having access to the same resources. This variation in somatic fitness, in turn, affects breeding success, particularly in females. We postulate that microbiota have a central role to play in determining the successfulness of an individual. Our preliminary results, characterising the microbiota of individual badgers, indicate unique compositions of microbiota communities within social groups of badgers. This basal information will inform further questions related to the extent microbiota influence fitness. Hitherto, the potential role of microbiota has not been considered in determining host condition, but also other key fitness variables, namely; communication and resistance to disease. Badgers deposit their faeces in communal latrines, which play an important role in olfactory communication. Odour profiles of anal and subcaudal gland secretions are highly individual-specific and encode information about group-membership and fitness-relevant parameters, and their chemical composition is strongly dependent on symbiotic microbiota. As badgers sniff/ lick (using their Vomeronasal organ) and over-mark faecal deposits of conspecifics, these microbial communities can be expected to vary with social contact networks. However, this is particularly important in the context of bTB, where badgers are assumed to transmit bTB to cattle as well as conspecifics. Interestingly, we have found that some individuals are more susceptible to bTB than are others. As acquired immunity and thus potential susceptibility to infectious diseases are known to depend also on symbiotic microbiota in other members of the mustelids, a role of particularly oral microbiota can currently not be ruled out as a potential explanation for inter-individual differences in infection susceptibility of bTB in badgers. Tri annually badgers are caught in the context of a long-term population study that began in 1987. As all badgers receive an individual tattoo upon first capture, age, natal as well as previous and current social group-membership and other life history parameters are known for all animals. Swabs (subcaudal ‘scent gland’, anal, genital, nose, mouth and ear) and fecal samples will be taken from all individuals, stored at -80oC until processing. Microbial samples will be processed and identified at Wayne State University’s Theis (Host-Microbe Interactions) Lab, using High Throughput Sequencing (16S rRNA-encoding gene amplification and sequencing). Acknowledgments: Gas-Chromatography/ Mass-spectrometry (in the context of olfactory communication) analyses will be performed through an established collaboration with Dr. Veronica Tinnesand at Telemark University, Norway.

Keywords: communication, energetics, fitness, free-ranging animals, immunology

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