Search results for: gene profiling

Authors: Mohammadjavad Sotoudeheian

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COVID-19, which began in December 2019, uses the angiotensin-converting enzyme 2 (ACE2) receptor to enter and spread through the cells. ACE2 mRNA is present in almost every organ, including nasopharynx, lung, as well as the brain. Ports of entry of SARS-CoV-2 into the central nervous system (CNS) may include arterial circulation, while viremia is remarkable. However, it is imperious to develop neurological symptoms evaluation CSF analysis in patients with COVID-19, but theoretically, ACE2 receptors are expressed in cerebellar cells and may be a target for SARS-CoV-2 infection in the brain. Recent evidence agrees that SARS-CoV-2 can impact the brain through direct and indirect injury. Two biomarkers for CNS injury, glial fibrillary acidic protein (GFAP) and neurofilament light chain (NFL) detected in the plasma of patients with COVID-19. NFL, an axonal protein expressed in neurons, is related to axonal neurodegeneration, and GFAP is over-expressed in CNS inflammation. GFAP cytoplasmic accumulation causes Schwan cells to misfunction, so affects myelin generation, reduces neuroskeletal support over NfLs during CNS inflammation, and leads to axonal degeneration. Interleukin-6 (IL-6), which extensively over-express due to interleukin storm during COVID-19 inflammation, regulates gene expression, as well as GFAP through STAT molecular pathway. IL-6 also impresses the phosphoinositide 3-kinase (PI3K)/STAT/smads pathway. The PI3K/ protein kinase B (Akt) pathway is the main modulator upstream of the mammalian target of rapamycin (mTOR), and alterations in this pathway are common in neurodegenerative diseases. Most neurodegenerative diseases show a disruption of autophagic function and display an abnormal increase in protein aggregation that promotes cellular death. Therefore, induction of autophagy has been recommended as a rational approach to help neurons clear abnormal protein aggregates and survive. The mTOR is a major regulator of the autophagic process and is regulated by cellular stressors. The mTORC1 pathway and mTORC2, as complementary and important elements in mTORC1 signaling, have become relevant in the regulation of the autophagic process and cellular survival through the extracellular signal-regulated kinase (ERK) pathway.

Keywords: mTORC1, COVID-19, PI3K, autophagy, neurodegeneration

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Authors: Xiaoyu Sun

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In recent years, the rapid development of Artificial Intelligence (AI) has extensively promoted medicine, pharmaceutical, and other related fields. The medical research and development of artificial intelligence by scientific and commercial organizations are on the fast track. The ethics review is one of the critical procedures of registration to get the products approved and launched. However, the SOPs for ethics review is not enough to guide the healthy and rapid development of artificial intelligence in healthcare in China. Ethical Review Measures for Biomedical Research Involving Human Beings was enacted by the National Health Commission of the People's Republic of China (NHC) on December 1st, 2016. However, from a legislative design perspective, it was neither updated timely nor in line with the trends of AI international development. Therefore, it was great that NHC published a consultation paper on the updated version on March 16th, 2021. Based on the most updated laws and regulations in the States and EU, and in-depth-interviewed 11 subject matter experts in China, including lawmakers, regulators, and key members of ethics review committees, heads of Regulatory Affairs in SaMD industry, and data scientists, several suggestions were proposed on top of the updated version. Although the new version indicated that the Ethics Review Committees need to be created by National, Provincial and individual institute levels, the review authorities of different levels were not clarified. The suggestion is that the precise scope of review authorities for each level should be identified based on Risk Analysis and Management Model, such as the complicated leading technology, gene editing, should be reviewed by National Ethics Review Committees, it will be the job of individual institute Ethics Review Committees to review and approve the clinical study with less risk such as an innovative cream to treat acne. Furthermore, to standardize the research and development of artificial intelligence in healthcare in the age of AI, more clear guidance should be given to data security in the layers of data, algorithm, and application in the process of ethics review. In addition, transparency and responsibility, as two of six principles in the Rome Call for AI Ethics, could be further strengthened in the updated version. It is the shared goal among all countries to manage well and develop AI to benefit human beings. Learned from the other countries who have more learning and experience, China could be one of the most advanced countries in artificial intelligence in healthcare.

Keywords: biomedical research involving human beings, data security, ethics committees, ethical review, medical artificial intelligence

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Authors: Gileade P. Freitas, Helena B. Lopes, Alann T. P. Souza, Paula G. F. P. Oliveira, Adriana L. G. Almeida, Paulo G. Coelho, Marcio M. Beloti, Adalberto L. Rosa

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Bone tissue presents great capacity to regenerate when injured by trauma, infectious processes, or neoplasia. However, the extent of injury may exceed the inherent tissue regeneration capability demanding some kind of additional intervention. In this scenario, cell therapy has emerged as a promising alternative to treat challenging bone defects. This study aimed at evaluating the effect of local injection of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) on bone regeneration of rat calvaria defects. BM-MSCs and AT-MSCs were isolated and characterized by expression of surface markers; cell viability was evaluated after injection through a 21G needle. Defects of 5 mm in diameter were created in calvaria and after two weeks a single injection of BM-MSCs, AT-MSCs or vehicle-PBS without cells (Control) was carried out. Cells were tracked by bioluminescence and at 4 weeks post-injection bone formation was evaluated by micro-computed tomography (μCT) and histology, nanoindentation, and through gene expression of bone remodeling markers. The data were evaluated by one-way analysis of variance (p≤0.05). BM-MSCs and AT-MSCs presented characteristics of mesenchymal stem cells, kept viability after passing through a 21G needle and remained in the defects until day 14. In general, injection of both BM-MSCs and AT-MSCs resulted in higher bone formation compared to Control. Additionally, this bone tissue displayed elastic modulus and hardness similar to the pristine calvaria bone. The expression of all evaluated genes involved in bone formation was upregulated in bone tissue formed by BM-MSCs compared to AT-MSCs while genes involved in bone resorption were upregulated in AT-MSCs-formed bone. We show that cell therapy based on the local injection of BM-MSCs or AT-MSCs is effective in delivering viable cells that displayed local engraftment and induced a significant improvement in bone healing. Despite differences in the molecular cues observed between BM-MSCs and AT-MSCs, both cells were capable of forming bone tissue at comparable amounts and properties. These findings may drive cell therapy approaches toward the complete bone regeneration of challenging sites.

Keywords: cell therapy, mesenchymal stem cells, bone repair, cell culture

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Authors: Javed Mohammed

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Right now, bio-medical infrastructure lags well behind the curve. Our healthcare system is dispersed and disjointed; medical records are a bit of a mess; and we do not yet have the capacity to store and process the crazy amounts of data coming our way from widespread whole-genome sequencing. And then there are privacy issues. Despite these infrastructure challenges, some researchers are plunging into bio medical Big Data now, in hopes of extracting new and actionable knowledge. They are doing delving into molecular-level data to discover bio markers that help classify patients based on their response to existing treatments; and pushing their results out to physicians in novel and creative ways. Computer scientists and bio medical researchers are able to transform data into models and simulations that will enable scientists for the first time to gain a profound under-standing of the deepest biological functions. Solving biological problems may require High-Performance Computing HPC due either to the massive parallel computation required to solve a particular problem or to algorithmic complexity that may range from difficult to intractable. Many problems involve seemingly well-behaved polynomial time algorithms (such as all-to-all comparisons) but have massive computational requirements due to the large data sets that must be analyzed. High-throughput techniques for DNA sequencing and analysis of gene expression have led to exponential growth in the amount of publicly available genomic data. With the increased availability of genomic data traditional database approaches are no longer sufficient for rapidly performing life science queries involving the fusion of data types. Computing systems are now so powerful it is possible for researchers to consider modeling the folding of a protein or even the simulation of an entire human body. This research paper emphasizes the computational biology's growing need for high-performance computing and Big Data. It illustrates this article’s indispensability in meeting the scientific and engineering challenges of the twenty-first century, and how Protein Folding (the structure and function of proteins) and Phylogeny Reconstruction (evolutionary history of a group of genes) can use HPC that provides sufficient capability for evaluating or solving more limited but meaningful instances. This article also indicates solutions to optimization problems, and benefits Big Data and Computational Biology. The article illustrates the Current State-of-the-Art and Future-Generation Biology of HPC Computing with Big Data.

Keywords: high performance, big data, parallel computation, molecular data, computational biology

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Authors: Pierre-Louis Lucas, Corinne Loutelier-Bourhis, Narimane Mati-Baouche, Philippe Chan Tchi-Song, Patrice Lerouge, Elodie Mathieu-Rivet, Muriel Bardor

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N-glycosylation is a post-translational modification taking place in the Endoplasmic Reticulum and the Golgi apparatus where defined glycan features are added on protein in a very specific sequence Asn-X-Thr/Ser/Cys were X can be any amino acid except proline. Because it is well-established that those N-glycans play a critical role in protein biological activity, protein half-life and that a different N-glycan structure may induce an immune response, they are very important in Biopharmaceuticals which are mainly glycoproteins bearing N-glycans. From now, most of the biopharmaceuticals are produced by mammalian cells like Chinese Hamster Ovary cells (CHO) for their N-glycosylation similar to the human, but due to the high production costs, several other species are investigated as the possible alternative system. In this purpose, the green microalgae Chlamydomonas reinhardtii was investigated as the potential production system for Biopharmaceuticals. This choice was influenced by the facts that C. reinhardtii is a well-study microalgae which is growing fast with a lot of molecular biology tools available. This organism is also producing N-glycan on its endogenous proteins. However, the analysis of the N-glycan structure of this microalgae has revealed some differences as compared to the human. Rather than in Human where the glycans are processed by key enzymes called N-acetylglucosaminyltransferase I and II (GnTI and GnTII) adding GlcNAc residue to form a GlcNAc₂Man₃GlcNAc₂ core N-glycan, C. reinhardtii lacks those two enzymes and possess a GnTI independent glycosylation pathway. Moreover, some enzymes like xylosyltransferases and methyltransferases not present in human are supposed to act on the glycans of C. reinhardtii. Furthermore, the recent structural study by mass spectrometry shows that the N-glycosylation precursor supposed to be conserved in almost all eukaryotic cells results in a linear Man₅GlcNAc₂ rather than a branched one in C. reinhardtii. In this work, we will discuss the new released MS information upon C. reinhardtii N-glycan structure and their impact on our attempt to modify the glycan in a Human manner. Two strategies will be discussed. The first one consisted in the study of Xylosyltransferase insertional mutants from the CLIP library in order to remove xyloses from the N-glycans. The second will go further in the humanization by transforming the microalgae with the exogenous gene from Toxoplasma gondii having an activity similar to GnTI and GnTII with the aim to synthesize GlcNAc₂Man₃GlcNAc₂ in C. reinhardtii.

Keywords: Chlamydomonas reinhardtii, N-glycosylation, glycosyltransferase, mass spectrometry, humanization

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Authors: Ljiljana Stojković, Manja Zec, Maja Zivkovic, Maja Bundalo, Marija Glibetić, Dragan Alavantić, Aleksandra Stankovic

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Cardiovascular disease (CVD) is associated with alterations in DNA methylation, the latter modulated by dietary polyphenols. The present pilot study (part of the original clinical study registered as NCT02800967 at www.clinicaltrials.gov) aimed to investigate the impact of 4-week daily consumption of polyphenol-rich Aronia melanocarpa juice on Long Interspersed Nucleotide Element-1 (LINE-1) methylation in peripheral blood leukocytes, in subjects (n=34, age of 41.1±6.6 years) at moderate CVD risk, including an increased body mass index, central obesity, high normal blood pressure and/or dyslipidemia. The goal was also to examine whether factors known to affect DNA methylation, such as folate intake levels, MTHFR C677T gene variant, as well as the anthropometric and metabolic parameters, modulated the LINE-1 methylation levels upon consumption of polyphenol-rich Aronia juice. The experimental analysis of LINE-1 methylation was done by the MethyLight method. MTHFR C677T genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism method. Folate intake was assessed by processing the data from the food frequency questionnaire and repeated 24-hour dietary recalls. Serum lipid profile was determined by using Roche Diagnostics kits. The statistical analyses were performed using the Statistica software package. In women, after vs. before the treatment period, a significant decrease in LINE-1 methylation levels was observed (97.54±1.50% vs. 98.39±0.86%, respectively; P=0.01). The change (after vs. before treatment) in LINE-1 methylation correlated directly with MTHFR 677T allele presence, average daily folate intake and the change in serum low-density lipoprotein cholesterol, while inversely with the change in serum triacylglycerols (R=0.72, R2=0.52, adjusted R2=0.36, P=0.03). The current results imply potential cardioprotective effects of habitual polyphenol-rich Aronia juice consumption achieved through the modifications of DNA methylation pattern in subjects at CVD risk, which should be further confirmed. Hence, the precision nutrition-driven modulations of DNA methylation may become targets for new approaches in the prevention and treatment of CVD.

Keywords: Aronia melanocarpa, cardiovascular risk, LINE-1, methylation, peripheral blood leukocytes, polyphenol

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Authors: Lethukuthula Ngobese, Abin Gupthar, Patrick Govender

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The ability of yeast cell wall-derived mannoproteins (glycoproteins) to positively contribute to oenological properties has been a key factor that stimulates research initiatives into these industrially important glycoproteins. In addition, and from a fundamental research perspective, yeast cell wall glycoproteins are involved in a wide range of biological interactions. To date, and to the best of our knowledge, our understanding of the fine molecular structure of these mannoproteins is fairly limited. Generally, the amino acid sequences of their protein moieties have been established from structural and functional analysis of the genomic sequence of these yeasts whilst far less information is available on the glycosyl moieties of these mannoproteins. A novel strategy was devised in this study that entails the genetic engineering of yeast strains that over-express and release cell wall-associated glycoproteins into the liquid growth medium. To this end, the Flo1p mannoprotein was overexpressed in Saccharomyces cerevisiae laboratory strains bearing a specific deletion in KNR4 and GPI7 genes involved in cell wall biosynthesis that have been previously shown to extracellularly hyper-secrete cell wall-associated glycoproteins. A polymerase chain reaction (PCR) -based cloning strategy was employed to generate transgenic yeast strains in which the native cell wall FLO1 glycoprotein-encoding gene is brought under transcriptional control of the constitutive PGK1 promoter. The modified Helm’s flocculation assay was employed to assess flocculation intensities of a Flo1p over-expressing wild type and deletion mutant as an indirect measure of their abilities to release the desired mannoprotein. The flocculation intensities of the transformed strains were assessed and all the strains showed similar intensities (>98% flocculation). To assess if mannoproteins were released into the growth medium, the supernatant of each strain was subjected to the BCA protein assay and the transformed Δknr4 strain showed a considerable increase in protein levels. This study has the potential to produce mannoproteins in sufficient quantities that may be employed in future investigations to understand their molecular structures and mechanisms of interaction to the benefit of both fundamental and industrial applications.

Keywords: glycoproteins, genetic engineering, flocculation, over-expression

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Authors: M. M. Essa, S. Subash, M. Akbar, S. Al-Adawi, A. Al-Asmi, G. J. Guillemein

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Objective: Transgenic (tg) mice which contain an amyloid precursor protein (APP) gene mutation, develop extracellular amyloid beta (Aβ) deposition in the brain, and severe memory and behavioural deficits with age. These mice serve as an important animal model for testing the efficacy of novel drug candidates for the treatment and management of symptoms of Alzheimer's disease (AD). Several reports have suggested that oxidative stress is the underlying cause of Aβ neurotoxicity in AD. Pomegranates contain very high levels of antioxidants and several medicinal properties that may be useful for improving the quality of life in AD patients. In this study, we investigated the effect of dietary supplementation of Omani pomegranate extract on the memory, anxiety and learning skills along with inflammation in an AD mouse model containing the double Swedish APP mutation (APPsw/Tg2576). Methods: The experimental groups of APP-transgenic mice from the age of 4 months were fed custom-mix diets (pellets) containing 4% pomegranate. We assessed spatial memory and learning ability, psychomotor coordination, and anxiety-related behavior in Tg and wild-type mice at the age of 4-5 months and 18-19 months using the Morris water maze test, rota rod test, elevated plus maze test, and open field test. Further, inflammatory parameters also analysed. Results: APPsw/Tg2576 mice that were fed a standard chow diet without pomegranates showed significant memory deficits, increased anxiety-related behavior, and severe impairment in spatial learning ability, position discrimination learning ability and motor coordination along with increased inflammation compared to the wild type mice on the same diet, at the age of 18-19 months In contrast, APPsw/Tg2576 mice that were fed a diet containing 4% pomegranates showed a significant improvements in memory, learning, locomotor function, and anxiety with reduced inflammatory markers compared to APPsw/Tg2576 mice fed the standard chow diet. Conclusion: Our results suggest that dietary supplementation with pomegranates may slow the progression of cognitive and behavioural impairments in AD. The exact mechanism is still unclear and further extensive research needed.

Keywords: Alzheimer's disease, pomegranates, oman, cognitive decline, memory loss, anxiety, inflammation

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Authors: Rosa Tsegaye Aga, Xuan Jiang, Pavel Vazquez Faci, Siqing Liu, Simon Rayner, Endalkachew Alemu, Markos Abebe

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Tuberculosis (TB) is a major cause of disease globally. In most cases, TB is treatable and curable, but only with the proper treatment. There is a time when drug-resistant TB occurs when bacteria become resistant to the drugs that are used to treat TB. Current strategies to identify drug-resistant TB bacteria are laboratory-based, and it takes a longer time to identify the drug-resistant bacteria and treat the patient accordingly. But machine learning (ML) and data science approaches can offer new approaches to the problem. In this study, we propose to develop an ML-based model to predict the antibiotic resistance phenotypes of TB isolates in minutes and give the right treatment to the patient immediately. The study has been using the whole genome sequence (WGS) of TB isolates as training data that have been extracted from the NCBI repository and contain different countries’ samples to build the ML models. The reason that different countries’ samples have been included is to generalize the large group of TB isolates from different regions in the world. This supports the model to train different behaviors of the TB bacteria and makes the model robust. The model training has been considering three pieces of information that have been extracted from the WGS data to train the model. These are all variants that have been found within the candidate genes (F1), predetermined resistance-associated variants (F2), and only resistance-associated gene information for the particular drug. Two major datasets have been constructed using these three information. F1 and F2 information have been considered as two independent datasets, and the third information is used as a class to label the two datasets. Five machine learning algorithms have been considered to train the model. These are Support Vector Machine (SVM), Random forest (RF), Logistic regression (LR), Gradient Boosting, and Ada boost algorithms. The models have been trained on the datasets F1, F2, and F1F2 that is the F1 and the F2 dataset merged. Additionally, an ensemble approach has been used to train the model. The ensemble approach has been considered to run F1 and F2 datasets on gradient boosting algorithm and use the output as one dataset that is called F1F2 ensemble dataset and train a model using this dataset on the five algorithms. As the experiment shows, the ensemble approach model that has been trained on the Gradient Boosting algorithm outperformed the rest of the models. In conclusion, this study suggests the ensemble approach, that is, the RF + Gradient boosting model, to predict the antibiotic resistance phenotypes of TB isolates by outperforming the rest of the models.

Keywords: machine learning, MTB, WGS, drug resistant TB

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Authors: Y. Landkocz, P. Gosset, A. Héliot, C. Corbière, C. Vendeville, V. Keravec, S. Billet, A. Verdin, C. Monteil, D. Préterre, J-P. Morin, F. Sichel, T. Douki, P. J. Martin

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Air Pollution produced by automobile traffic is one of the main sources of pollutants in urban atmosphere and is largely due to exhausts of the diesel engine powered vehicles. The International Agency for Research on Cancer, which is part of the World Health Organization, classified in 2012 diesel engine exhaust as carcinogenic to humans (Group 1), based on sufficient evidence that exposure is associated with an increased risk for lung cancer. Amongst the strategies aimed at limiting exhausts in order to take into consideration the health impact of automobile pollution, filtration of the emissions and use of biofuels are developed, but their toxicological impact is largely unknown. Diesel exhausts are indeed complex mixtures of toxic substances difficult to study from a toxicological point of view, due to both the necessary characterization of the pollutants, sampling difficulties, potential synergy between the compounds and the wide variety of biological effects. Here, we studied the potential indirect genotoxicity of emission of Diesel engines through on-line exposure of rats in inhalation chambers to a subchronic high but realistic dose. Following exposure to standard gasoil +/- rapeseed methyl ester either upstream or downstream of a particle filter or control treatment, rats have been sacrificed and their lungs collected. The following indirect genotoxic parameters have been measured: (i) telomerase activity and telomeres length associated with rTERT and rTERC gene expression by RT-qPCR on frozen lungs, (ii) γH2AX quantification, representing double-strand DNA breaks, by immunohistochemistry on formalin fixed-paraffin embedded (FFPE) lung samples. These preliminary results will be then associated with global cellular response analyzed by pan-genomic microarrays, monitoring of oxidative stress and the quantification of primary DNA lesions in order to identify biological markers associated with a potential pro-carcinogenic response of diesel or biodiesel, with or without filters, in a relevant system of in vivo exposition.

Keywords: diesel exhaust exposed rats, γH2AX, indirect genotoxicity, lung carcinogenicity, telomerase activity, telomeres length

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Authors: Vipin Kumar Verma, Neeta Sehgal, Om Prakash

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Cyprinus carpio has a wide spread occurrence in the lakes and rivers of Europe and Asia. Heavy losses in natural environment due to anthropogenic activities, including pollution as well as pathogenic diseases have landed this fish in IUCN red list of vulnerable species. The significance of a suitable diet in preserving the health status of fish is widely recognized. In present study, artificial feed supplemented with leaves of two weed plants, Eichhornia crassipes and Ricinus communis were evaluated for their role on the fish immune system. To achieve this objective fish were acclimatized to laboratory conditions (25 ± 1 °C; 12 L: 12D) for 10 days prior to start of experiment and divided into 4 groups: non-challenged (negative control= A), challenged [positive control (B) and experimental (C & D)]. Group A, B were fed with non-supplemented feed while group C & D were fed with feed supplemented with 5% Eichhornia crassipes and 5% Ricinus communis respectively. Supplemented feeds were evaluated for their effect on growth, health, immune system and disease resistance in fish when challenged with Vibrio harveyi. Fingerlings of C. carpio (weight, 2.0±0.5 g) were exposed with fresh overnight culture of V. harveyi through bath immunization (concentration 2 Χ 105) for 2 hours on 10 days interval for 40 days. The growth was monitored through increase in their relative weight. The rate of mortality due to bacterial infection as well as due to effect of feed was recorded accordingly. Immune response of fish was analyzed through differential leucocyte count, percentage phagocytosis and phagocytic index. The effect of V. harveyi on fish organs were examined through histo-pathological examination of internal organs like spleen, liver and kidney. The change in the immune response was also observed through gene expression analysis. The antioxidant potential of plant extracts was measured through DPPH and FRAP assay and amount of total phenols and flavonoids were calculates through biochemical analysis. The chemical composition of plant’s methanol extracts was determined by GC-MS analysis, which showed presence of various secondary metabolites and other compounds. Investigation revealed immuno-modulatory effect of plants, when supplemented with the artificial feed of fish.

Keywords: immuno-modulation, gc-ms, Cyprinus carpio, Eichhornia crassipes, Ricinus communis

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

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Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system can facilitate targeted genome editing in organisms. Dual or single guide RNA (gRNA) can program the Cas9 nuclease to cut target DNA in particular areas; thus, introducing concise mutations either via error-prone non-homologous end-joining repairing or via incorporating foreign DNAs by homologous recombination between donor DNA and target area. In spite of high demand of such promising technology, developing a well-organized procedure in order for reliable mining of potential target sites for gRNAs in large genomic data is still challenging. Hence, we aimed to perform high-throughput detection of target sites by specific PAMs for not only common Streptococcus pyogenes (SpCas9) but also for Neisseria meningitides (NmCas9) CRISPR-Cas systems. Previous research confirmed the successful application of such RNA-guided Cas9 orthologs for effective gene targeting and subsequently genome manipulation. However, Cas9 orthologs need their particular PAM sequence for DNA cleavage activity. Activity levels are based on the sequence of the protospacer and specific combinations of favorable PAM bases. Therefore, based on the specific length and sequence of PAM followed by a constant length of the target site for the two orthogonals of Cas9 protein, we created a reliable procedure to explore possible gRNA sequences. To mine CRISPR target sites, four different searching modes of sgRNA binding to target DNA strand were applied. These searching modes are as follows i) coding strand searching, ii) anti-coding strand searching, iii) both strand searching, and iv) paired-gRNA searching. Finally, a complete list of all potential gRNAs along with their locations, strands, and PAMs sequence orientation can be provided for both SpCas9 as well as another potential Cas9 ortholog (NmCas9). The artificial design of potential gRNAs in a genome of interest can accelerate functional genomic studies. Consequently, the application of such novel genome editing tool (CRISPR/Cas technology) will enhance by presenting increased versatility and efficiency.

Keywords: CRISPR/Cas9 genome editing, gRNA mining, SpCas9, NmCas9

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Authors: Eriko Ochiai, Fumiko Satoh, Keiko Miyashita, Yu Kakimoto, Motoki Osawa

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Sudden and unexpected deaths from unknown causes occur in infants and youths. Recently, molecular links between a part of these deaths and several genetic diseases are examined in the postmortem. For instance, hereditary long QT syndrome and Burgada syndrome are occasionally fatal through critical ventricular tachyarrhythmia. There are a large number of target genes responsible for such diseases, the conventional analysis using the Sanger’s method has been laborious. In this report, we attempted to analyze sudden deaths comprehensively using the next generation sequencing (NGS) technique. Multiplex PCR to subject’s DNA was performed using Ion AmpliSeq Library Kits 2.0 and Ion AmpliSeq Inherited Disease Panel (Life Technologies). After the library was constructed by emulsion PCR, the amplicons were sequenced 500 flows on Ion Personal Genome Machine System (Life Technologies) according to the manufacture instruction. SNPs and indels were analyzed to the sequence reads that were mapped on hg19 of reference sequences. This project has been approved by the ethical committee of Tokai University School of Medicine. As a representative case, the molecular analysis to a 40 years old male who received a diagnosis of Brugada syndrome demonstrated a total of 584 SNPs or indels. Non-synonymous and frameshift nucleotide substitutions were selected in the coding region of heart disease related genes of ANK2, AKAP9, CACNA1C, DSC2, KCNQ1, MYLK, SCN1B, and STARD3. In particular, c.629T-C transition in exon 3 of the SCN1B gene, resulting in a leu210-to-pro (L210P) substitution is predicted “damaging” by the SIFT program. Because the mutation has not been reported, it was unclear if the substitution was pathogenic. Sudden death that failed in determining the cause of death constitutes one of the most important unsolved subjects in forensic pathology. The Ion AmpliSeq Inherited Disease Panel can amplify the exons of 328 genes at one time. We realized the difficulty in selection of the true source from a number of candidates, but postmortem genetic testing using NGS analysis deserves of a diagnostic to date. We now extend this analysis to SIDS suspected subjects and young sudden death victims.

Keywords: postmortem genetic testing, sudden death, SIDS, next generation sequencing

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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: Shalini TV, Gangadharan GG, Sriranjini S Jaideep, ASN Seshasayee, Awadhesh Pandit

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Introduction: Prakriti (body-mind constitution of an individual) is a conventional, customized and unique understanding of which is essential for the personalized medicine described in Ayurveda, Indian System of Medicine. Based on the Doshas( functional, bio humoral unit in the body), individuals are categorized into three major Prakriti- Vata, Pitta, and Kapha. The human gut microbiome hosts plenty of highly diverse and metabolically active microorganisms, mainly dominated by the bacteria, which are known to influence the physiology of an individual. Few researches have shown the correlation between the Prakriti and the biochemical parameters. In this study, an attempt was made to explore any correlation between the Prakriti (phenotype of an individual) with the Genetic makeup of the gut microbiome in healthy individuals. Materials and methods: 270 multi-ethnic, healthy volunteers of both sex with the age group between 18 to 40 years, with no history of antibiotics in the last 6 months were recruited into three groups of Vata, Pitta, and Kapha. The Prakriti of the individual was determined using Ayusoft, a software designed by CDAC, Pune, India. The volunteers were subjected to initial screening for the assessment of their height, weight, Body Mass Index, Vital signs and Blood investigations to ensure they are healthy. The stool and saliva samples of the recruited volunteers were collected as per the standard operating procedure developed, and the bacterial DNA was isolated using Qiagen kits. The extracted DNA was subjected to 16s rRNA sequencing using the Illumina kits. The sequencing libraries are targeting the variable V3 and V4 regions of the 16s rRNA gene. Paired sequencing was done on the MiSeq system and data were analyzed using the CLC Genomics workbench 11. Results: The 16s rRNA sequencing of the V3 and V4 regions showed a diverse pattern in both the oral and stool microbial DNA. The study did not reveal any specific pattern of bacterial flora amongst the Prakriti. All the p-values were more than the effective alpha values for all OTUs in both the buccal cavity and stool samples. Therefore, there was no observed significant enrichment of an OTU in the patient samples from either the buccal cavity or stool samples. Conclusion: In healthy volunteers of multi-ethnicity, due to the influence of the various factors, the correlation between the Prakriti and the gut microbiome was not seen.

Keywords: gut microbiome, ayurveda Prakriti, sequencing, multi-ethnic urban population

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Authors: Sandisiwe Mangali, Graeme Bradley

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Genome is complete set of the organism's hereditary information encoded as either deoxyribonucleic acid or ribonucleic acid in most viruses. The three different types of genomes are nuclear, mitochondrial and the plastid genome and their sequences which are uncovered by genome sequencing are known as an archive for all genetic information and enable researchers to understand the composition of a genome, regulation of gene expression and also provide information on how the whole genome works. These sequences enable researchers to explore the population structure, genetic variations, and recent demographic events in threatened species. Particularly, genome sequencing refers to a process of figuring out the exact arrangement of the basic nucleotide bases of a genome and the process through which all the afore-mentioned genomes are sequenced is referred to as whole or complete genome sequencing. Gelidium pristoides is South African endemic Rhodophyta species which has been harvested in the Eastern Cape since the 1950s for its high economic value which is one motivation for its sequencing. Its endemism further motivates its sequencing for conservation biology as endemic species are more vulnerable to anthropogenic activities endangering a species. As sequencing, mapping and annotating the Gelidium pristoides genome is the aim of this study. To accomplish this aim, the genomic DNA was extracted and quantified using the Nucleospin Plank Kit, Qubit 2.0 and Nanodrop. Thereafter, the Ion Plus Fragment Library was used for preparation of a 600bp library which was then sequenced through the Ion S5 sequencing platform for two runs. The produced reads were then quality-controlled and assembled through the SPAdes assembler with default parameters and the genome assembly was quality assessed through the QUAST software. From this assembly, the plastid and the mitochondrial genomes were then sampled out using Gelidiales organellar genomes as search queries and ordered according to them using the Geneious software. The Qubit and the Nanodrop instruments revealed an A260/A280 and A230/A260 values of 1.81 and 1.52 respectively. A total of 30792074 reads were obtained and produced a total of 94140 contigs with resulted into a sequence length of 217.06 Mbp with N50 value of 3072 bp and GC content of 41.72%. A total length of 179281bp and 25734 bp was obtained for plastid and mitochondrial respectively. Genomic data allows a clear understanding of the genomic constituent of an organism and is valuable as foundation information for studies of individual genes and resolving the evolutionary relationships between organisms including Rhodophytes and other seaweeds.

Keywords: Gelidium pristoides, genome, genome sequencing and assembly, Ion S5 sequencing platform

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Authors: Hamed Ahari, Behrouz Akbari Adreghani, Vadood Razavilar, Amirali Anvar, Sima Moradi, Hourieh Shalchi

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Considering the ever increasing population and industrialization of the developmental trend of humankind's life, we are no longer able to detect the toxins produced in food products using the traditional techniques. This is due to the fact that the isolation time for food products is not cost-effective and even in most of the cases, the precision in the practical techniques like the bacterial cultivation and other techniques suffer from operator errors or the errors of the mixtures used. Hence with the advent of nanotechnology, the design of selective and smart sensors is one of the greatest industrial revelations of the quality control of food products that in few minutes time, and with a very high precision can identify the volume and toxicity of the bacteria. Methods and Materials: In this technique, based on the bacterial antibody connection to nanoparticle, a sensor was used. In this part of the research, as the basis for absorption for the recognition of bacterial toxin, medium sized silica nanoparticles of 10 nanometer in form of solid powder were utilized with Notrino brand. Then the suspension produced from agent-linked nanosilica which was connected to bacterial antibody was positioned near the samples of distilled water, which were contaminated with Staphylococcus aureus bacterial toxin with the density of 10-3, so that in case any toxin exists in the sample, a connection between toxin antigen and antibody would be formed. Finally, the light absorption related to the connection of antigen to the particle attached antibody was measured using spectrophotometry. The gene of 23S rRNA that is conserved in all Staphylococcus spp., also used as control. The accuracy of the test was monitored by using serial dilution (l0-6) of overnight cell culture of Staphylococcus spp., bacteria (OD600: 0.02 = 107 cell). It showed that the sensitivity of PCR is 10 bacteria per ml of cells within few hours. Result: The results indicate that the sensor detects up to 10-4 density. Additionally, the sensitivity of the sensors was examined after 60 days, the sensor by the 56 days had confirmatory results and started to decrease after those time periods. Conclusions: Comparing practical nano biosensory to conventional methods like that culture and biotechnology methods(such as polymerase chain reaction) is accuracy, sensitiveness and being unique. In the other way, they reduce the time from the hours to the 30 minutes.

Keywords: exotoxin, nanobiosensor, recognition, Staphylococcus aureus

Procedia PDF Downloads 362

Authors: Kamran Jawed, Anu Jose Mattam, Zia Fatma, Saima Wajid, Malik Z. Abdin, Syed Shams Yazdani

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Worldwide demand of natural and sustainable fuels and chemicals have encouraged researchers to develop microbial platform for synthesis of short chain fatty acids as they are useful precursors to replace petroleum-based fuels and chemicals. In this study, we evaluated the role of fatty acid synthesis and β-oxidation cycle of Escherichia coli to produce butyric acid, a 4-carbon short chain fatty acid, with the help of three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron. We found that E. coli strain transformed with gene for TesBT and grown in presence of 8 g/L glucose produced maximum butyric acid titer at 1.46 g/L, followed by that of TesBF at 0.85 g/L and TesAT at 0.12 g/L, indicating that these thioesterases were efficiently converting short chain fatty acyl-ACP intermediate of fatty acid synthesis pathway into the corresponding acid. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. Deletion of genes for fatty acyl-CoA synthetase and acyl-CoA dehydrogenase, which are involved in initiating the fatty acid degradation cycle, and overexpression of FadR, which is a dual transcriptional regulator and exerts negative control over fatty acid degradation pathway, reduced up to 30% of butyric acid titer. This observation suggested that β-oxidation pathway is working synergistically with fatty acid synthesis pathway in production of butyric acid. Moreover, accelerating the fatty acid elongation cycle by overexpressing acetyl-CoA carboxyltransferase (Acc) and 3-hydroxy-acyl-ACP dehydratase (FabZ) or by deleting FabR, the transcription suppressor of elongation, did not improve the butyric acid titer, rather favored the long chain fatty acid production. Finally, a balance between cell growth and butyric acid production was achieved with the use of phosphorous limited growth medium and 14.3 g/L butyric acid, and 17.5 g/L total free fatty acids (FFAs) titer was achieved during fed-batch cultivation. We have engineered an E. coli strain which utilizes the intermediate of both fatty acid synthesis and degradation pathway, i.e. butyryl-ACP and -CoA, to produce butyric acid from glucose. The strategy used in this study resulted in highest reported titers of butyric acid and FFAs in engineered E. coli.

Keywords: butenoic acid, butyric acid, Escherichia coli, fed-batch fermentation, short chain fatty acids, thioesterase

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Authors: David Taillis, Oussama Becissa, Julien Gabaston, Jean-Michel Merillon, Tristan Richard, Stephanie Cluzet

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Nowadays, there is a strong pressure to reduce the phytosanitary inputs of synthetic chemistry in vineyards. It is, therefore, necessary to find viable alternatives in order to protect the vine against its major diseases. For this purpose, we suggest the use of a plant extract enriched in antimicrobial compounds. Being produced from vine trunks and roots, which are co-products of wine production, the extract produced is part of a circular economy. The antimicrobial molecules present in this plant material are polyphenols and, more particularly, stilbenes, which are derived from a common base, the resveratrol unit, and that are well known vine phytoalexins. The stilbenoids were extracted from trunks and roots (30/70, w/w) by a double extraction with ethyl acetate followed by enrichment by liquid-liquid extraction. The produced extract was characterized by UHPLC-MS, then its antimicrobial activities were tested on Plasmopara viticola and Botrytis cinerea in the laboratory and/or in greenhouse and in vineyard. The major compounds were purified, and their antimicrobial activity was evaluated on B. cinerea. Moreover, after its spraying, the effect of the stilbene extract on the plant defence status was evaluated by analysis of defence gene expression. UHPLC-MS analysis revealed that the extract contains 50% stilbenes with resveratrol, ε-viniferin and r-viniferin as major compounds. The extract showed antimicrobial activities on P. viticola with IC₅₀ and IC₁₀₀ respectively of 90 and 300 mg/L in the laboratory. In addition, it inhibited 40% of downy mildew development in greenhouse. However, probably because of the sensitivity of stilbenes to the environment, such as UV degradation, no activity has been observed in vineyard towards P. viticola development. For B. cinerea, the extract IC50 was 123 mg/L, with resveratrol and ε-viniferin being the most active stilbenes (IC₅₀ of 88 and 142 mg/L, respectively). The analysis of the expression of defence genes revealed that the extract can induce the expression of some defence genes 24, 48, and 72 hours after treatment, meaning that the extract has a defence-stimulating effect at least for the first three days after treatment. In conclusion, we produced a plant extract enriched in stilbenes with antimicrobial properties against two major grapevine pathogenic agents P. viticola and B. cinerea. In addition, we showed that this extract displayed eliciting activity of plant defences. This extract can therefore represent, after formulation development, a viable eco-friendly alternative for vineyard protection. Subsequently, the effect of the stilbenoid extract on primary metabolism will be evaluated by quantitative NMR.

Keywords: antimicrobial, bioprotection, grapevine, Plasmopara viticola, stilbene

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Authors: Zeynep Yazgan, Gökhan Aygün, Reyhan Çalışkan

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Objective: Invasive fungal infections are a serious threat in terms of morbidity and mortality, especially in immunocompromised patients. The most frequently isolated agents are Aspergillus genus fungi, and sensitivity to azoles, which are the first choice in treatment, decreases. In our study, we aimed to investigate the use of the agar plate screening method as a fast, easy, and practical method in determining azole resistance in Aspergillus spp. species. Methods: Our study was conducted with 125 Aspergillus spp. isolates produced from various clinical samples. Aspergillus spp. isolates were identified by conventional methods and azole resistance was determined by gradient test and agar plate screening method. Broth microdilution method was applied to resistant isolates, and CypA-L98H and CypA-M220 mutations in the cyp51A gene were investigated. Results: In our study, 55 A. fumigatus complex (44%), 42 A. flavus (33.6%), 6 A. terreus (5%), 4 A. niger (3%) and 18 Aspergillus spp. (14%) were identified. With the gradient test method, resistance to VOR and POS was detected in 1 (1.8%) of A.fumigatus isolates, and resistance to ITR was detected in 3 (5.45%). With the agar plate method, 1 of the A.fumigatus isolates (1.8%) had VOR, ITR, POS, 1 of the A.terreus isolates (16.7%) had VOR, 1 of the A.niger isolates (25%) had ITR. Resistance to VOR and POS was detected in 2 Aspergillus spp. isolates (11%), and resistance to ITR was detected in 1 (5.6%). Sensitivity and specificity were determined as 100% for VOR and POS in A. fumigatus species, 33.3% and 100% for ITR, respectively, 100% for ITR in A. flavus species, and 100% for ITR and POS in A. terreus species. By broth microdilution method in 7 isolates in which resistance was detected by gradient test and/or agar plate screening method; 1 A.fumigatus resistant to ITR, VOR, POS, 2 A.fumigatus resistant to ITR, 2 Aspergillus spp. ITR, VOR, POS MICs were determined as 2µg/ml and 8µg/ml, 8µg/ml and >32µg/ml, 0.5µg/ml and 4µg/ml, respectively. CypA-L98H mutations were detected in 5 of these isolates, CypA-M220 mutations were detected in 6, and no mutation was detected in 1. CypA-L98H and CypA-M220 mutations were detected in 1 isolate for which resistance was not detected. Conclusion: The need for rapid antifungal susceptibility screening tests is increasing in the treatment of aspergillosis. Although the sensitivity of the agar plate method was determined to be 33.3% for A.fumigatus ITR in our study, its sensitivity and specificity were determined to be 100% for ITR, VOR, and POS in other species. The low sensitivity value detected for A.fumigatus showed that agar plate drug concentrations should be updated in accordance with the latest regulations of EUCAST guidelines. The CypA-L98H and CypA-M220 mutations detected in our study suggested that the distribution of azole resistance-related mutations in different regions in our country should be investigated. In conclusion, it is thought that the agar plate method, which can be easily applied to detect azole resistance, is a fast and practical method in routine use and can contribute to both the determination of effective treatment strategies and the generation of epidemiological data.

Keywords: Aspergillus, agar plate, azole resistance, cyp51A, cypA-L98H, cypA-M220

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Authors: Valencia Fernandes, Dharmendra Kumar Khatri, Shashi Bala Singh

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Background and Aim: Epigenetics are the inaudible signatures of several pathological processes in the brain. This study understands the influence of DNA methylation, a major epigenetic modification, in the prefrontal cortex and hippocampus of the diabetic brain and its notable effect on the cellular chaperones and synaptic proteins. Method: Chronic high fat diet and STZ-induced diabetic mice were studied for cognitive dysfunction, and global DNA methylation, as well as DNA methyltransferase (DNMT) activity, were assessed. Further, the cellular chaperones and synaptic proteins were examined using DNMT inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC)-via intracerebroventricular injection. Moreover, % methylation of these synaptic proteins were also studied so as to correlate its epigenetic involvement. Computationally, its interaction with the DNMT enzyme were also studied using bioinformatic tools. Histological studies for morphological alterations and neuronal degeneration were also studied. Neurogenesis, a characteristic marker for new learning and memory formation, was also assessed via the BrdU staining. Finally, the most important behavioral studies, including the Morris water maze, Y maze, passive avoidance, and Novel object recognition test, were performed to study its cognitive functions. Results: Altered global DNA methylation and increased levels of DNMTs within the nucleus were confirmed in the cortex and hippocampus of the diseased mice, suggesting hypermethylation at a genetic level. Treatment with AzadC, a global DNA demethylating agent, ameliorated the protein and gene expression of the cellular chaperones and synaptic fidelity. Furthermore, the methylation analysis profile showed hypermethylation of the hsf1 protein, a master regulator for chaperones and thus, confirmed the epigenetic involvement in the diseased brain. Morphological improvements and decreased neurodegeneration, along with enhanced neurogenesis in the treatment group, suggest that epigenetic modulations do participate in learning and memory. This is supported by the improved behavioral test battery seen in the treatment group. Conclusion: DNA methylation could possibly accord in dysregulating the memory-associated proteins at chronic stages in type 2 diabetes. This could suggest a substantial contribution to the underlying pathophysiology of several metabolic syndromes like insulin resistance, obesity and also participate in transitioning this damage centrally, such as cognitive dysfunction.

Keywords: epigenetics, cognition, chaperones, DNA methylation

Procedia PDF Downloads 171

Authors: M. M. Essa, S. Subash, M. Akbar, S. Al-Adawi, A. Al-Asmi, G. J. Guillemein

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Transgenic (tg) mice which contain an amyloid precursor protein (APP) gene mutation, develop extracellular amyloid beta (Aβ) deposition in the brain, and severe memory and behavioral deficits with age. These mice serve as an important animal model for testing the efficacy of novel drug candidates for the treatment and management of symptoms of Alzheimer's disease (AD). Several reports have suggested that oxidative stress is the underlying cause of Aβ neurotoxicity in AD. Date palm fruits contain very high levels of antioxidants and several medicinal properties that may be useful for improving the quality of life in AD patients. In this study, we investigated the effect of dietary supplementation of Omani date palm fruits on the memory, anxiety and learning skills along with inflammation in an AD mouse model containing the double Swedish APP mutation (APPsw/Tg2576). The experimental groups of APP-transgenic mice from the age of 4 months were fed custom-mix diets (pellets) containing 2% and 4% Date palm fruits. We assessed spatial memory and learning ability, psychomotor coordination, and anxiety-related behavior in Tg and wild-type mice at the age of 4-5 months and 18-19 months using the Morris water maze test, rota rod test, elevated plus maze test, and open field test. Further, inflammatory parameters also analyzed. APPsw/Tg2576 mice that were fed a standard chow diet without dates showed significant memory deficits, increased anxiety-related behavior, and severe impairment in spatial learning ability, position discrimination learning ability and motor coordination along with increased inflammation compared to the wild type mice on the same diet, at the age of 18-19 months In contrast, PPsw/Tg2576 mice that were fed a diet containing 2% and 4% dates showed a significant improvements in memory, learning, locomotor function, and anxiety with reduced inflammatory markers compared to APPsw/Tg2576 mice fed the standard chow diet. Our results suggest that dietary supplementation with dates may slow the progression of cognitive and behavioral impairments in AD. The exact mechanism is still unclear and further extensive research needed.

Keywords: Alzheimer's disease, date palm fruits, Oman, cognitive decline, memory loss, anxiety, inflammation

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Authors: Konstantin S. Vainutis, Mark E. Andreev, Anastasia N. Voronova, Mikhail Yu. Shchelkanov

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Introduction: The trematodes from the family Cyclocoelidae (cyclocoelids) belong to the superfamily Echinostomatoidea infecting air sacs and trachea of wild birds. At present, the family Cyclocoelidae comprises nine valid genera in three subfamilies: Cyclocoelinae (type taxon), Haematotrephinae, and Typhlocoelinae. To our best knowledge, in this study, molecular genetic methods were used for the first time for studying cyclocoelids from the Russian Far East. Here we provide the data on the morphology and phylogeny of cyclocoelids from gadwall from the Russian Far East. The morphological and genetic data obtained for cyclocoelids indicated the necessity to revise the previously proposed classification within the family Cyclocoelidae. Objectives: The first objective was performing the morphological study of cyclocoelids found in M. strepera from the Russian Far East. The second objective is to reconstruct the phylogenetic relationships of the studied trematodes with other cyclocoelids using the 28S gene. Material and methods: During the field studies in the Khasansky district of the Primorsky region, 21 cyclocoelids were recovered from the air sacs of a single gadwall Mareca strepera. Seven samples of cyclocoelids were overstained in alum carmine, dehydrated in a graded ethanol series, cleared in clove oil, and mounted in Canada balsam. Genomic DNA was extracted from four cyclocoelids using the alkaline lysis method HotShot. The 28S rDNA fragment was amplified using the forward primer Digl2 and the reverse primer 1500R. Results: According to morphological features (ovary intratesticular, forming a triangle with the testes), the studied worms belong to the subfamily Cyclocoelinae Stossich, 1902. In particular, the highest morphological similarity was observed in relation to the trematodes of the genus Cyclocoelum Brandes, 1892 – genital pores are pharyngeal. However, the genetic analysis has shown significant discrepancies between the trematodes studied regarding the genus Cyclocoelum. On the phylogenetic tree, these trematodes took the sister position in relation to the genus Morishitium (previously considered in the subfamily Szidatitrematinae). Conclusion: Based on the results of the morphological and genetic studies, cyclocoelids isolated from Mareca strepera are suggested to be described in the previously unknown genus and differentiated from the type genus Cyclocoelum of the type subfamily Cyclocoelinae. Considering the available molecular data, including described cyclocoelids, the family Cyclocoelidae comprises ten valid genera in the three subfamilies mentioned above.

Keywords: new species, trematoda, phylogeny, cyclocoelidae

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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: Michal Pravenec, Miroslava Simakova, Jan Silhavy

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Brown adipose tissue (BAT) plays an important role in lipid and glucose metabolism in rodents and possibly also in humans. Recently, using systems genetics approach in the BAT from BXH/HXB recombinant inbred strains, derived from the SHR (spontaneously hypertensive rat) and BN (Brown Norway) progenitors, we identified Cd36 (fatty acid translocase) as the hub gene of co-expression module associated with BAT relative weight and function. An important aspect of BAT biology is to better understand the mechanisms regulating the uptake and utilization of fatty acids and glucose. Accordingly, BAT function in the SHR that harbors mutant nonfunctional Cd36 variant (hereafter referred to as SHR-Cd36⁻/⁻) was compared with SHR transgenic line expressing wild type Cd36 under control of a universal promoter (hereafter referred to as SHR-Cd36⁺/⁺). BAT was incubated in media containing insulin and 14C-U-glucose alone or 14C-U-glucose together with palmitate. Incorporation of glucose into BAT lipids was significantly higher in SHR-Cd36⁺/⁺ versus SHR-Cd36⁻/⁻ rats when incubation media contained glucose alone (SHR-Cd36⁻/⁻ 591 ± 75 vs. SHR-Cd36⁺/⁺ 1036 ± 135 nmol/gl./2h; P < 0.005). Adding palmitate into incubation media had no effect in SHR-Cd36⁻/⁻ rats but significantly reduced glucose incorporation into BAT lipids in SHR-Cd36⁺/⁺ (SHR-Cd36⁻/⁻ 543 ± 55 vs. SHR-Cd36⁺/⁺ 766 ± 75 nmol/gl./2h; P < 0.05 denotes significant Cd36 x palmitate interaction determined by two-way ANOVA). This Cd36-dependent reduced glucose uptake in SHR-Cd36⁺/⁺ BAT was likely secondary to increased palmitate incorporation and utilization due to the presence of wild type Cd36 fatty acid translocase in transgenic rats. This possibility is supported by increased incorporation of 14C-U-palmitate into BAT lipids in the presence of both palmitate and glucose in incubation media (palmitate alone: SHR-Cd36⁻/⁻ 870 ± 21 vs. SHR-Cd36⁺/⁺ 899 ± 42; glucose+palmitate: SHR-Cd36⁻/⁻ 899 ± 47 vs. SHR-Cd36⁺/⁺ 1460 ± 111 nmol/palm./2h; P < 0.05 denotes significant Cd36 x glucose interaction determined by two-way ANOVA). It is possible that addition of glucose into the incubation media increased palmitate incorporation into BAT lipids in SHR-Cd36⁺/⁺ rats because of glucose availability for glycerol phosphate production and increased triglyceride synthesis. These changes in glucose and palmitate incorporation into BAT lipids were associated with significant differential expression of Irs1, Irs2, Slc2a4 and Foxo1 genes involved in insulin signaling and glucose metabolism only in SHR-Cd36⁺/⁺ rats which suggests Cd36-dependent effects on insulin action. In conclusion, these results provide compelling evidence that Cd36 plays an important role in BAT insulin signaling and energy substrate utilization.

Keywords: brown adipose tissue, Cd36, energy substrate utilization, insulin signaling, spontaneously hypertensive rat

Procedia PDF Downloads 117

Authors: Ran Vir Singh, Anuj Chauhan, Subhodh Kumar, Rajesh Rathore, Satish Kumar, B Gopi, Sushil Kumar, Tarun Kumar, Ramji Yadav, Donna Phangchopi, Shoor Vir Singh

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Paratuberculosis caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic granulomatous enteritis affecting ruminants. It is responsible for significant economic losses in livestock industry worldwide. This organism is also of public health concern due to an unconfirmed link to Crohn’s disease. Susceptibility to paratuberculosis has been suggested to have genetic component with low to moderate heritability. Number of SNPs in various candidates genes have been observed to be affecting the susceptibility toward paratuberculosis. The objective of this study was to explore the association of various SNPs in the candidate genes and QTL region with MAP. A total of 117 SNPs from SLC11A1, IFNG, CARD15, TLR2, TLR4, CLEC7A, CD209, SP110, ANKARA2, PGLYRP1 and one QTL were selected for study. A total of 1222 cattle from various organized herds, gauhsalas and farmer herds were screened for MAP infection by Johnin intradermal skin test, AGID, serum ELISA, fecal microscopy, fecal culture and IS900 blood PCR. Based on the results of these tests, a case and control population of 200 and 183 respectively was established for study. A total of 117 SNPs from 10 candidate genes and one QTL were selected and validated/tested in our case and control population by PCR-RFLP technique. Data was analyzed using SAS 9.3 software. Statistical analysis revealed that, 107 out of 117 SNPs were not significantly associated with occurrence of MAP. Only SNP rs55617172 of TLR2, rs8193046 and rs8193060 of TLR4, rs110353594 and rs41654445 of CLEC7A, rs208814257of CD209, rs41933863 of ANKRA2, two loci {SLC11A1(53C/G)} and {IFNG (185 G/r) } and SNP rs41945014 in QTL region was significantly associated with MAP. Six SNP from 10 significant SNPs viz., rs110353594 and rs41654445 from CLEC7A, rs8193046 and rs8193060 from TLR4, rs109453173 from SLC11A1 rs208814257 from CD209 were validated in new case and control population. Out of these only one SNP rs8193046 of TLR4 gene was found significantly associated with occurrence of MAP in cattle. ODD ratio indicates that animals with AG genotype were more susceptible to MAP and this finding is in accordance with the earlier report. Hence it reaffirms that AG genotype can serve as a reliable genetic marker for indentifying more susceptible cattle in future selection against MAP infection in cattle.

Keywords: SNP, candidate genes, paratuberculosis, cattle

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Authors: Sridhar A. Malkaram, Tamer E. Fandy

Abstract:

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: W. Mohammedsaeed, A. J. Mcbain, S. M. Cruickshank, C. A. O’Neill

Abstract:

Many studies have demonstrated the importance of probiotics and their potential therapeutic effects within the gut. Recently, the possible therapeutic effects of probiotics in other tissues have also begun to be investigated. Comparatively few studies have evaluated the use of topical probiotics in relation to the skin. In this study, we have conducted preliminary investigations into whether a well-known probiotic, Lactobacillus rhamnosus GG (LGG), can increase the rate of re-epithelialization in a model wound. Full-thickness skin was obtained from individuals undergoing elective cosmetic surgery. This skin was wounded using excisional punch and cultured using a serum-free medium, either in the presence or absence of L. rhamnosus GG lysate. Histological staining of the sections was performed with Haematoxylin& Eosin E to quantify “epithelial tongue length”. This is the length of the new epithelial ‘tongue’ that grows and covers the exposed dermis at the inner wound edges. The length of the new epithelial ‘tongue’ was compared in untreated section and section treated with and L. rhamnosus GG made using108CFU/ml bacterial cells. L. rhamnosus GG lysate enhanced significantly the re-epithelialisation of treated wounds compared with that of untreated wounds (P=0.005, n=3). Tongue length, at day 1 was 7.55μm 0.15, at day 3 it was 18.5μm 0.25 and at day 7 was 22.9μm 0.35. These results can be compared with untreated cultures in which tongue length was 3.25μm 0.35, day 3 was 9.65μm 0.25 and day 7 was 13.5μm 0.15 post-wounding. In ex-vivo proliferation and migration cells were measured by determining the expression of nuclear proliferation marker Ki-67 and the expression of Phosphorylated cortactin respectively demonstrated that L. rhamnosus GG significantly increased NHEK proliferation and migration rates relative to controls. However, the dominant mechanism was migration because in ex-vivo skin treated with the L. rhamnosus GG up-regulated the gene expression of the chemokine receptor and ligands CXCR2 and CXCL2 comparing with controls (P=0.02, P=0.03 respectively, n=3). High levels of CXCL2/CXCL2 have already been implicated in multiple aspects of stimulation of wound healing through activation of keratinocyte migration. These data demonstrate that lysates from Lactobacillus rhamnosus GG increase re-epithelialization by stimulation of keratinocyte migration. The current study identifies the partial mechanism that contribute to stimulating the wound-healing process ex vivo in response to L. rhamnosus GG lysate is an increase in the production of CXCL2/ CXCR2 in ex vivo models. The use of probiotic lysates potentially offers new options to develop treatments that could improve wound healing.

Keywords: Lactobacillus rhamnosus GG, wounds, migration, lysate

Procedia PDF Downloads 305

Authors: Dalal Alghawas, Jetty Lee, Kaisa Poutanen, Hani El-Nezami

Abstract:

Oat β-glucan a water soluble non starch linear polysaccharide has been approved as a cholesterol lowering agent by various food safety administrations and is commonly used to reduce the risk of heart disease. The molecular weight of oat β-glucan can vary depending on the extraction and fractionation methods. It is not clear whether the molecular weight has a significant impact at reducing the acceleration of atherosclerosis. The aim of this study was to investigate three different oat β-glucan fractionations on the development of atherosclerosis in vivo. With special focus on plaque stability and the intestinal barrier function. To test this, ApoE-/- female mice were fed a high fat diet supplemented with oat bran, high molecular weight (HMW) oat β-glucan fractionate and low molecular weight (LMW) oat β-glucan fractionate for 16 weeks. Atherosclerosis risk markers were measured in the plasma, heart and aortic tree. Plaque size was measured in the aortic root and aortic tree. ICAM-1, VCAM-1, E-Selectin, P-Selectin, protein levels were assessed from the aortic tree to determine plaque stability at 16 weeks. The expression of p22phox at the aortic root was evaluated to study the NADPH oxidase complex involved in nitric oxide bioavailability and vascular elasticity. The tight junction proteins E-cadherin and beta-catenin from western blot analyses were analysed as an intestinal barrier function test. Plasma LPS, intestinal D-lactate levels and hepatic FMO gene expression were carried out to confirm whether the compromised intestinal barrier lead to endotoxemia. The oat bran and HMW oat β-glucan diet groups were more effective than the LMW β-glucan diet group at reducing the plaque size and showed marked improvements in plaque stability. The intestinal barrier was compromised for all the experimental groups however the endotoxemia levels were higher in the LMW β-glucan diet group. The oat bran and HMW oat β-glucan diet groups were more effective at attenuating the development of atherosclerosis. Reasons for this could be due to the LMW oat β-glucan diet group’s low viscosity in the gut and the inability to block the reabsorption of cholesterol. Furthermore the low viscosity may allow more bacterial endotoxin translocation through the impaired intestinal barrier. In future food technologists should carefully consider how to incorporate LMW oat β-glucan as a health promoting food.

Keywords: Atherosclerosis, beta glucan, endotoxemia, intestinal barrier function

Procedia PDF Downloads 392