Search results for: gene profiling

Authors: Vikash Yadav, Ashish Arora

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Microorganisms have self-defense mechanisms to protect themselves from toxic environments. Phenolic acid decarboxylase(pad) is responsible for the defense against toxicity caused by phenolic acids, converting them into less toxic vinyl derivatives. The transcription of the pad gene is regulated by a negative transcription factor, phenolic acid decarboxylase regulators (PadR), in a substrate-inducible manner. The PadR family members share the conserved DNA-binding features and interact with the operator DNA using a winged helix-turn-helix (wHTH) motif, which contains a three-helix motif and a β-stranded wing. The members of this family function as transcriptional regulators that are involved in various cellular survival processes, such as toxin production, detoxification, multidrug resistance, antibiotic biosynthesis, and carbon catabolism. Rv1176 of Mycobacterium tuberculosis H37Rv has been assigned to the PadR family protein that remains to be structurally and functionally uncharacterized. To reveal the structural mechanism by which Rv1176 could regulates effector-responsive transcription, several experiments were performed, including Electrophoretic Mobility Shift Assay (EMSA) for DNA protein interaction, differential scanning calorimetry (DSC) and Differential Scanning Fluorimetry (DSF) for temperature and ligand-dependent protein stability, Circular Dichroism (CD) spectroscopy for secondary structure analysis. Further, to evaluate the functional role of Rv1176, the intracellular survival of recombinant M. smegmatis was examined in murine macrophage cell line J774A.1 and different stressed conditions like oxidative, pH, and nutritive stress. All these studies demonstrated that Rv1176 could behave as a transcription regulator and its expression in recombinant M. smegmatis increases intracellular survival.

Keywords: EMSA, Mycobacterium tuberculosis, PadR family protein, transcriptional regulator

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Authors: S. Fahiminiya, J. Nadaf, F. Rauch, L. Jerome-Majewska, J. Majewski

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Background: Sequencing of protein coding regions of human genome (Whole Exome Sequencing; WES), has demonstrated a great success in the identification of causal mutations for several rare genetic disorders in human. Generally, most of WES studies have focused on rare variants in coding exons and splicing-sites where missense substitutions lead to the alternation of protein product. Although focusing on this category of variants has revealed the mystery behind many inherited genetic diseases in recent years, a subset of them remained still inconclusive. Here, we present the result of our WES studies where analyzing only rare variants in coding regions was not conclusive but further investigation revealed the involvement of non-coding variants and copy number variations (CNV) in etiology of the diseases. Methods: Whole exome sequencing was performed using our standard protocols at Genome Quebec Innovation Center, Montreal, Canada. All bioinformatics analyses were done using in-house WES pipeline. Results: To date, we successfully identified several disease causing mutations within gene coding regions (e.g. SCARF2: Van den Ende-Gupta syndrome and SNAP29: 22q11.2 deletion syndrome) by using WES. In addition, we showed that variants in non-coding regions and CNV have also important value and should not be ignored and/or filtered out along the way of bioinformatics analysis on WES data. For instance, in patients with osteogenesis imperfecta type V and in patients with glucocorticoid deficiency, we identified variants in 5'UTR, resulting in the production of longer or truncating non-functional proteins. Furthermore, CNVs were identified as the main cause of the diseases in patients with metaphyseal dysplasia with maxillary hypoplasia and brachydactyly and in patients with osteogenesis imperfecta type VII. Conclusions: Our study highlights the importance of considering non-coding variants and CNVs during interpretation of WES data, as they can be the only cause of disease under investigation.

Keywords: whole exome sequencing data, non-coding variants, copy number variations, rare diseases

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Authors: Mohamad Mohsen Homayouni, Niloofar Taghipour, Ahmad Reza Memar, Niloofar Khalaji, Hamed Kiani, Seyyed Javad Seyyed Tabaei

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Background and Objective: Cryptosporidium is a coccidian protozoan parasite; its oocysts in surface water are a global health problem. Due to the low number of parasites in the water resources and the lack of laboratory culture, rapid and sensitive method for detection of the organism in the water resources is necessarily required. We applied modified acid-fast staining and PCR for the detection of the Cryptosporidium spp. and analysed the genotypes in 55 samples collected from surface water. Methods: Over a period of nine months, 55 surface water samples were collected from the five rivers in Tehran, Iran. The samples were filtered by using cellulose acetate membrane filters. By acid fast method, initial identification of Cryptosporidium oocyst were carried out on surface water samples. Then, nested PCR assay was designed for the specific amplification and analysed the genotypes. Results: Modified Ziehl-Neelsen method revealed 5–20 Cryptosporidium oocysts detected per 10 Liter. Five out of the 55 (9.09%) surface water samples were found positive for Cryptosporidium spp. by Ziehl-Neelsen test and seven (12.7%) were found positive by nested PCR. The staining results were consistent with PCR. Seven Cryptosporidium PCR products were successfully sequenced and five gp60 subtypes were detected. Our finding of gp60 gene revealed that all of the positive isolates were Cryptosporidium parvum and belonged to subtype families IIa and IId. Conclusion: Our investigations were showed that collection of water samples were contaminated by Cryptosporidium, with potential hazards for the significant health problem. This study provides the first report on detection and genotyping of Cryptosporidium species from surface water samples in Iran, and its result confirmed the low clinical incidence of this parasite on the community.

Keywords: Cryptosporidium spp., membrane filtration, subtype, surface water, Iran

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Authors: Yanhua Zhao, Yu Gou, Shu Feng, Dongdong Li, Chuanmin Tao

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The natural history of HBV infection could experience immune tolerant (IT), immune clearance (IC), HBeAg-negative inactive/quienscent carrier (ENQ), and HBeAg-negative hepatitis (ENH). As current biomarkers for discriminating these four phases have some weaknesses, additional serological indicators are needed. Hepatits B core-related antigen (HBcrAg) encoded with precore/core gene contains denatured HBeAg, HBV core antigen (HBcAg) and a 22KDa precore protein (p22cr), which was demonstrated to have a close association with natural history of hepatitis B infection, but no specific cutoff values and diagnostic parameters to evaluate the diagnostic efficacy. This study aimed to clarify the distribution of HBcrAg levels and evaluate its diagnostic performance during the natural history of infection from a Western Chinese perspective. 294 samples collected from treatment-naïve chronic hepatitis B (CHB) patients in different phases (IT=64; IC=72; ENQ=100, and ENH=58). We detected the HBcrAg values and analyzed the relationship between HBcrAg and HBV DNA. HBsAg and other clinical parameters were quantitatively tested. HBcrAg levels of four phases were 9.30 log U/mL, 8.80 log U/mL, 3.00 log U/mL, and 5.10 logU/mL, respectively (p < 0.0001). Receiver operating characteristic curve analysis demonstrated that the area under curves (AUCs) of HBcrAg and quantitative HBsAg at cutoff values of 9.25 log U/mL and 4.355 log IU/mL for distinguishing IT from IC phases were 0.704 and 0.694, with sensitivity 76.39% and 59.72%, specificity 53.13% and 79.69%, respectively. AUCs of HBcrAg and quantitative HBsAg at cutoff values of 4.15 log U/mlmL and 2.395 log IU/mlmL for discriminating between ENQ and ENH phases were 0.931 and 0.653, with sensitivity 87.93% and 84%, specificity 91.38% and 39%, respectively. Therefore, HBcrAg levels varied significantly among four natural phases of HBV infection. It had higher predictive performance than quantitative HBsAg for distinguishing between ENQ-patients and ENH-patients and similar performance with HBsAg for the discrimination between IT and IC phases, which indicated that HBcrAg could be a potential serological marker for CHB.

Keywords: chronic hepatitis B, hepatitis B core-related antigen, hepatitis B surface antigens, hepatitis B virus

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Authors: Roshni Raha, Karthikeyan S.

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The world's largest livestock population resides in India. Existing strategies must be modified to increase the production of livestock and their by-products in order to meet the demands of the growing human population. Even though India leads the world in both milk production and the number of cows, average production is not very healthy and productive. This may be due to the animals' poor nutrition caused by a chronic under-availability of high-quality fodder and feed. This article explores Azolla pinnata to be a promising source to produce high-quality unconventional feed and fodder for effective livestock production and good quality breeding in India. This article is an exploratory study using a literature survey and experimentation analysis. In the realm of agri-biotechnology, azolla sp gained attention for helping farmers achieve sustainability, having minimal land requirements, and serving as a feed element that doesn't compete with human food sources. It has high methionine content, which is a good source of protein. It can be easily digested as the lignin content is low. It has high antioxidants and vitamins like beta carotene, vitamin A, and vitamin B12. Using this concept, the paper aims to investigate and develop a model of using azolla plants as a novel, high-potential feed source to combat the problems of low production and poor quality of animals in India. A representative sample of animal feed is collected where azolla is added. The sample is ground into a fine powder using mortar. PITC (phenylisothiocyanate) is added to derivatize the amino acids. The sample is analyzed using HPLC (High-Performance Liquid Chromatography) to measure the amino acids and monitor the protein content of the sample feed. The amino acid measurements from HPLC are converted to milligrams per gram of protein using the method of amino acid profiling via a set of calculations. The amino acid profile data is then obtained to validate the proximate results of nutrient enhancement of the composition of azolla in the sample. Based on the proximate composition of azolla meal, the enhancement results shown were higher compared to the standard values of normal fodder supplements indicating the feed to be much richer and denser in nutrient supply. Thus azolla fed sample proved to be a promising source for animal fodder. This would in turn lead to higher production and a good breed of animals that would help to meet the economic demands of the growing Indian population. Azolla plants have no side effects and can be considered as safe and effective to be immersed in the animal feed. One area of future research could begin with the upstream scaling strategy of azolla plants in India. This could involve introducing several bioreactor types for its commercial production. Since azolla sp has been proved in this paper as a promising source for high quality animal feed and fodder, large scale production of azolla plants will help to make the process much quicker, more efficient and easily accessible. Labor expenses will also be reduced by employing bioreactors for large-scale manufacturing.

Keywords: azolla, fodder, nutrient, protein

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Authors: Shirin jalili, Hadi Shirzad, Mahasti Modarresi, Samaneh Nabavi, Somayeh Khanjani

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Identifying the source tissue of biological material found at crime scenes can be very informative in a number of cases. Despite their usefulness, current visual, catalytic, enzymatic, and immunologic tests for presumptive and confirmatory tissue identification are applicable only to a subset of samples, might suffer limitations such as low specificity, lack of sensitivity, and are substantially impacted by environmental insults. In addition their results are operator-dependent. Recently the possibility of discriminating body fluids using mRNA expression differences in tissues has been described but lack of long term stability of that Molecule and the need to normalize samples for each individual are limiting factors. The use of DNA should solve these issues because of its long term stability and specificity to each body fluid. Cells in the human body have a unique epigenome, which includes differences in DNA methylation in the promoter of genes. DNA methylation, which occurs at the 5′-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers.The presence or absence of a methyl group on the 5’ carbon of the cytosine pyridine ring in CpG dinucleotide regions called ‘CpG islands’ dictates whether the gene is expressed or silenced in the particular body fluid. Were described methylation patterns at tissue specific differentially methylated regions (tDMRs) to be stable and specific, making them excellent markers for tissue identification. The results demonstrate that methylation-based tissue identification is more than a proof-of-concept. The methodology holds promise as another viable forensic DNA analysis tool for characterization of biological materials.

Keywords: DNA methylation, forensic science, epigenome, tDMRs

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Authors: Sayyed-Javad Ziaolhagh, Mojtaba Hokmabadi

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Introduction: Childhood obesity is a serious medical condition that affects children and adolescents even in the central nervous system. Semaphorins also play a role in the inflammatory process of the nervous system. On the other hand, it has been stated that garlic and stevia extracts following aerobic exercise are effective on immune system inflammation in addition to aerobic activity. Materials and Methods: For 15 weeks, 50 3-week-old male Wistar rats were fed with conventional rodent chow for control and a high-fat diet to induce obesity. Obese rats then were randomly assigned into 7 groups (n=5) based on the Lee index: healthy control (C), obese (OBS), obese + garlic (OBS+GAR), obese + Stevia (OBS+STV), obese + aerobic exercise (OBS+EXE), obese + garlic + aerobic exercise (OBS+GAR+EXE), and obese + stevia + aerobic exercise (OBS+STV+EXE). Training groups completed a progressive aerobic running program (at 8-15 m/min, 5-20 min/day, 5 days/week), and Stevia and garlic extract group (250 mg/kg/day, 5 days/week) were given orally once a day. Real-time PCR was used to determine the levels of Semaphorin 4A, and Plexin D1 gene expressions in the hypothalamus. Fold change analysis with ANOVA was performed for statistical analysis, with a significance threshold of P<0.05. Results: Body weight increased significantly in OBS compared to C (p= 0.013), but was not significantly changed in all treatment rats. Moreover, Semaphorin 4A was significantly increased in obese compared to control group (p= 0.041) and after 8 weeks, stevia extract (p=0.006), aerobic exercise (p=0.012) and garlic extract + aerobic exercise (p=0.008) significantly decreased compared to obese rats. In addition, Plexin D1 genes were also found in the hypothalamus of both obese and control rats but were insignificantly up-regulated when compared with the obese group (p=0.950). Conclusion: High-fat diet caused neuroinflammation by elevation of sema4A in obese rats and stevia, stevia with aerobic and garlic with aerobic could reduce this inflammation in rats. Also, none of them could alter Plexin D1.

Keywords: sema 4A, plexin D1, garlic, stevia

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Authors: Viktoriia Burkina, Vladimir Zlabek, Galia Zamaratskaia

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Cytochromes P450 (CYP) are important family of enzymes in Phase I metabolism. Environmental pollutants often act as inducers of the gene expression and activities CYP1A1 and CYP3A-like isoforms in fish. The activities are generally measured in the fish liver or gills, and less is known about tissue distribution of expression. In present study, the CYP1A1 and CYP3A-like activities were measured in rainbow trout liver, gill, intestine, heart, brain and gonads. The activities of CYP1A1 and CYP3A-like proteins were estimated as the rates of 7-ethoxyresorufin-O-deethylation (EROD) and benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylation (BFCOD), respectively. The CYP1A1 and CYP3A-like activities were detectable in all investigated fish tissues, with the highest activity in hepatic tissue followed by heart > brain > gill > intestine > gonads. To confirm the presence of CYP1A1 in different tissues, EROD activity was measured in presence of the selective inhibitors ellipticine (CYP1A1), ketoconazole (CYP3A), 8-methoxypsoralen (human CYP2A) and diallyl sulphide (CYP2E1). It was found that ellipticine, ketoconazole and 8-methoxypsoralen inhibited hepatic EROD activity by 88-98%. Ellipticine inhibited gill, intestine, and gonad EROD activity by 50%. In conclusion, EROD and BFCOD activities were detected in rainbow trout liver, gill, intestine, heart, brain and gonads. Further studies are needed to fully identify all CYP450 isoforms responsible for these activities. Acknowledgement: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - projects „CENAKVA “(No. CZ.1.05/2.1.00/01.0024), “CENAKVA Center Development “(No. CZ.1.05/2.1.00/19.0380), “CENAKVA II “(No. LO1205 under the NPU I program), and "Development of USB - International mobility (No. CZ.02.2.69/0.0/0.0/16_027/0008364).

Keywords: BFCOD, EROD, fish, phase I metabolism, selective inhibitors

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Authors: Zuzana Tatarkova, Ivana Pilchova, Michal Cibulka, Martin Kolisek, Peter Racay, Peter Kaplan

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Introduction: Normal functioning of mitochondria is crucial for cardiac performance. Mitochondria undergo mitophagy and biogenesis, and mitochondrial proteins are subject to extensive post-translational modifications. The state of mitochondrial homeostasis reflects overall cellular fitness and longevity. Perturbed mitochondria produce less ATP, release greater amounts of reactive molecules, and are more prone to apoptosis. Therefore mitochondrial turnover is an integral aspect of quality control in which dysfunctional mitochondria are selectively eliminated through mitophagy. Currently, the progressive deterioration of physiological functions is seen as accumulation of modified/damaged proteins with limiting regenerative ability and disturbance of such affected protein-protein communication throughout aging in myocardial cells. Methodologies: For our study was used immunohistochemistry, biochemical methods: spectrophotometry, western blotting, immunodetection as well as more sophisticated 2D electrophoresis and mass spectrometry for evaluation protein-protein interactions and specific post-translational modification. Results and Discussion: Mitochondrial stress response to reactive species was evaluated as electron transport chain (ETC) complexes, redox-active molecules, and their possible communication. Protein-protein interactions revealed a strong linkage between age and ETC protein subunits. Redox state was strongly affected in senescent mitochondria with shift in favor of more pro-oxidizing condition within cardiomyocytes. Acute myocardial ischemia and ischemia-reperfusion (IR) injury affected ETC complexes I, II and IV with no change in complex III. Ischemia induced decrease in total antioxidant capacity, MnSOD, GSH and catalase activity with recovery in some extent during reperfusion. While MnSOD protein content was higher in IR group, activity returned to 95% of control. Nitric oxide is one of the biological molecules that can out compete MnSOD for superoxide and produce peroxynitrite. This process is faster than dismutation and led to the 10-fold higher production of nitrotyrosine after IR injury in adult with higher protection in senescent ones. 2D protein profiling revealed 140 mitochondrial proteins, 12 of them with significant changes after IR injury and 36 individual nitrotyrosine-modified proteins further identified by mass spectrometry. Linking these two groups, 5 proteins were altered after IR as well as nitrated, but only one showed massive nitration per lowering content of protein after IR injury in adult. Conclusions: Senescent cells have greater proportion of protein content, which might be modulated by several post-translational modifications. If these protein modifications are connected to functional consequences and protein-protein interactions are revealed, link may lead to the solution. Assume all together, dysfunctional proteostasis can play a causative role and restoration of protein homeostasis machinery is protective against aging and possibly age-related disorders. This work was supported by the project VEGA 1/0018/18 and by project 'Competence Center for Research and Development in the field of Diagnostics and Therapy of Oncological diseases', ITMS: 26220220153, co-financed from EU sources.

Keywords: aging heart, mitochondria, proteomics, redox state

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Authors: Benjaporn Buranrat, Nootchanat Mairuae

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Cratoxylum formosum (Jack) Dyer (CF) has been used for the traditional medicines in South East Asian and Thailand. Normally, northeast Thai vegetables have proven cytotoxic to many cancer cells. Therefore, the present study aims to explore the molecular mechanisms underlying CF-induced cancer cell death and apoptosis on breast and liver cancer cells. The cytotoxicity and antiproliferative effects of CF on the human breast MCF-7 and liver HepG2 cancer cell lines were evaluated using sulforhodamine B assay and colony formation assay. Cell migration assay was measured using wound healing assay. The apoptosis induction mechanisms were investigated through reactive oxygen species formation, caspase 3 activity, and JC-1 activity. Gene expression by real-time PCR and apoptosis related protein levels by Western blot analysis. CF induced MCF-7 and HepG2 cell death by time- and dose-dependent manner. Furthermore, CF had the greater cytotoxic potency on MCF-7 more than HepG2 cells with IC50 values of 85.70+4.52 μM and 219.03±9.96 μM respectively, at 24 h. Treatment with CF also caused a dose-dependent decrease in colony forming ability and cell migration, especially on MCF-7 cells. CF induced ROS formation, increased caspase 3 activities, and decreased the mitochondrial membrane potential, and causing apoptotic body production and DNA fragmentation. CF significantly decreased expression of the cell cycle regulatory protein RAC1 and downstream proteins, cdk6. Additionally, CF enhanced p21 and reduced cyclin D1 protein levels. CF leaf extract induced cell death, apoptosis, antimigration in both of MCF-7 and HepG2 cells. CF could be useful for developing to anticancer drug candidate for breast and liver cancer therapy.

Keywords: cratoxylum formosum (jack) dyer, breast cancer, liver cancer, cell death

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Authors: Debit Datta, Jeffrey J. Coleman, Scott A. Enebak, Lori G. Eckhardt

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Loblolly pine (Pinus taeda) is a leading productive timber species in the southeastern USA. Over the past three years, an emerging threat is expressed by successive needle defoliation followed by stunted growth and tree mortality in loblolly pine plantations. Considering economic significance, it has now become a rising concern among landowners, forest managers, and forest health state cooperators. However, the symptoms of the disease were perplexed somewhat with root disease(s) and recurrently attributed to invasive Phytophthora species due to the similarity of disease nature and devastation. Therefore, the study investigated the potential causal agent of this disease and characterized the fungi associated with loblolly pine needle defoliation in the southeastern USA. Besides, 70 trees were selected at seven long-term monitoring plots at Chatom, Alabama, to monitor and record the annual disease incidence and severity. Based on colony morphology and ITS-rDNA sequence data, a total of 28 species of fungi representing 17 families have been recovered from diseased loblolly pine needles. The native brown-spot pathogen, Lecanosticta acicola, was the species most frequently recovered from unhealthy loblolly pine needles in combination with some other common needle cast and rust pathogen(s). Identification was confirmed using morphological similarity and amplification of translation elongation factor 1-alpha gene region of interest. Tagged trees were consistently found chlorotic and defoliated from 2019 to 2020. The current emergence of the brown-spot pathogen causing loblolly pine mortality necessitates the investigation of the role of changing climatic conditions, which might be associated with increased pathogen pressure to loblolly pines in the southeastern USA.

Keywords: brown-spot needle blight, loblolly pine, needle defoliation, plantation forestry

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Authors: Wasim Sajjad, Manzoor Ahmad, Sundas Qadir, Muhammad Rafiq, Fariha Hasan, Richard Tehan, Kerry L. McPhail, Aamer Ali Shah

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Aim: This study aims to investigate the possible radiation protective role of a compatible solute in the tolerance of radio-halophilic bacterium against stresses, like desiccation and exposure to ionizing radiation. Methods and Results: Nine different radio-resistant bacteria were isolated from desert soil, where strain WMA-LM19 was chosen for detailed studies on the basis of its high tolerance for ultraviolet radiation among all these isolates. 16S rRNA gene sequencing indicated that the bacterium was closely related to Stenotrophomonas sp. (KT008383). A bacterial milking strategy was applied for extraction of intracellular compatible solutes in 70% (v/v) ethanol, which were purified by high-performance liquid chromatography (HPLC). The compound was characterized as ectoine by 1H and 13C nuclear magnetic resonance (NMR), and mass spectrometry (MS). Ectoine demonstrated more efficient preventive activity (54.80%) to erythrocyte membranes and also inhibited oxidative damage to proteins and lipids in comparison to the standard ascorbic acid. Furthermore, a high level of ectoine-mediated protection of bovine serum albumin against ionizing radiation (1500-2000 Jm-2) was observed, as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Conclusion: The results indicated that ectoine can be used as a potential mitigator and radio-protective agent to overcome radiation- and salinity-mediated oxidative damage in extreme environments. Significance and Impact of the Study: This study shows that ectoine from radio-halophiles can be used as a potential source in topical creams as sunscreen. The investigation of ectoine as UV protectant also changes the prospective that radiation resistance is specific only to molecular adaptation.

Keywords: ectoine, anti-oxidant, stenotrophomonas sp., ultraviolet radiation

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Authors: Adriana Haulica

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Powered by Machine Learning, Precise medicine is tailored by now to use genetic and molecular profiling, with the aim of optimizing the therapeutic benefits for cohorts of patients. As the majority of Machine Language algorithms come from heuristics, the outputs have contextual validity. This is not very restrictive in the sense that medicine itself is not an exact science. Meanwhile, the progress made in Molecular Biology, Bioinformatics, Computational Biology, and Precise Medicine, correlated with the huge amount of human biology data and the increase in computational power, opens new healthcare challenges. A more accurate diagnosis is needed along with real-time treatments by processing as much as possible from the available information. The purpose of this paper is to present a deeper vision for the future of Artificial Intelligence in Precise medicine. In fact, actual Machine Learning algorithms use standard mathematical knowledge, mostly Euclidian metrics and standard computation rules. The loss of information arising from the classical methods prevents obtaining 100% evidence on the diagnosis process. To overcome these problems, we introduce MEDICOMPILLS, a new architectural concept tool of information processing in Precise medicine that delivers diagnosis and therapy advice. This tool processes poly-field digital resources: global knowledge related to biomedicine in a direct or indirect manner but also technical databases, Natural Language Processing algorithms, and strong class optimization functions. As the name suggests, the heart of this tool is a compiler. The approach is completely new, tailored for omics and clinical data. Firstly, the intrinsic biological intuition is different from the well-known “a needle in a haystack” approach usually used when Machine Learning algorithms have to process differential genomic or molecular data to find biomarkers. Also, even if the input is seized from various types of data, the working engine inside the MEDICOMPILLS does not search for patterns as an integrative tool. This approach deciphers the biological meaning of input data up to the metabolic and physiologic mechanisms, based on a compiler with grammars issued from bio-algebra-inspired mathematics. It translates input data into bio-semantic units with the help of contextual information iteratively until Bio-Logical operations can be performed on the base of the “common denominator “rule. The rigorousness of MEDICOMPILLS comes from the structure of the contextual information on functions, built to be analogous to mathematical “proofs”. The major impact of this architecture is expressed by the high accuracy of the diagnosis. Detected as a multiple conditions diagnostic, constituted by some main diseases along with unhealthy biological states, this format is highly suitable for therapy proposal and disease prevention. The use of MEDICOMPILLS architecture is highly beneficial for the healthcare industry. The expectation is to generate a strategic trend in Precise medicine, making medicine more like an exact science and reducing the considerable risk of errors in diagnostics and therapies. The tool can be used by pharmaceutical laboratories for the discovery of new cures. It will also contribute to better design of clinical trials and speed them up.

Keywords: bio-semantic units, multiple conditions diagnosis, NLP, omics

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Authors: K. Cartelier, D. Aime, V. Vernoud, J. Buitink, J. M. Prosperi, K. Gallardo, C. Le Signor

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Legumes can produce protein-rich seeds without nitrogen fertilizer through root symbiosis with nitrogen-fixing rhizobia. Rich in lysine, these proteins are used for human nutrition and animal feed. However, the instability of seed protein yield and quality due to environmental fluctuations limits the wider use of legumes such as pea. Breeding efforts are needed to optimize and stabilize seed nutritional value, which requires to identify the genetic determinism of seed protein plasticity in response to the environment. Towards this goal, we have studied the plasticity of protein content and composition of seeds from a collection of 200 Medicago truncatula ecotypes grown under four controlled conditions (optimal, drought, and winter/spring sowing). A quantitative analysis of one-dimensional protein profiles of these mature seeds was performed and plasticity indices were calculated from each abundant protein band. Genome-Wide Association Studies (GWAS) from these data identified major GWAS hotspots, from which a list of candidate genes was obtained. A Gene Ontology Enrichment Analysis revealed an over-representation of genes involved in several amino acid metabolic pathways. This led us to propose that environmental variations are likely to modulate amino acid balance, thus impacting seed protein composition. The selection of candidate genes for controlling the plasticity of seed protein composition was refined using transcriptomics data from developing Medicago truncatula seeds. The pea orthologs of key genes were identified for functional studies by mean of TILLING (Targeting Induced Local Lesions in Genomes) lines in this crop. We will present how this study highlighted mechanisms that could govern seed protein plasticity, providing new cues towards the stabilization of legume seed quality.

Keywords: GWAS, Medicago truncatula, plasticity, seed, storage proteins

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Authors: Mahesh M. Patil, K. A. Anu-Appaiah

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Overweight and obesity is a serious medical problem, increasing in prevalence, and affecting millions worldwide. Investigators have been trying from decades to articulate the burden of obesity and related risk factors. To answer this problem, we suggest a new therapeutic anti-obesity compounds from Garcinia xanthochymus fruit. However, there is little published scientific information on non-hydroxycitric acid Garcinia species. Our findings include biochemical characterization of the fruit; in vivo toxicity and bio-efficacy study of G. xanthochymus in high fat diet wistar rat model. We observed that Garcinia pericarp is a rich source of organic acids, polyphenols, mono- (40.63%) and poly-unsaturated fatty acids (16.45%; omega-3: 10.02%). Toxicological studies have showed that Garcinia is safe and had no observed adverse effect level up to 400 mg/kg/day. Body weight and food intake was significantly (P<0.05) reduced in oral gavage treated rats (sonicated Garcinia powder) in 13 weeks. Subcutaneous fat was significantly (P<0.05) reduced in Garcinia treated rats. Hepatocytes significantly (p<0.05) overexpressed sterol regulatory element binding protein 2, liver X receptor- α, liver X receptor- β, lipoprotein lipase and monoacylglycerol lipase. Fatty acid binding protein 1 and peroxisome proliferator activated receptor- α were down regulated as assessed by real time qPCR. Currently our research is focused on the adipocyte obesity related gene expressions, effect of Garcinia on 3T3-adipocyte cell lines and high fat diet induced mice model. This in vivo pre-clinical data suggests that G. xanthochymus may have clinical utility for the treatment of obesity. However, further studies are required to establish its potency.

Keywords: Garcinia xanthochymus, anti-obesity, high fat diet, real time qPCR

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Authors: Shangqing Song, Bin Xu, Yajun Cheng, Zhong Wang

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miRNAs derived from exosomes exist in a body fluid such as urine were regarded as potential biomarkers for various human cancers diagnosis and prognosis, as mature miRNAs can be steadily preserved by exosomes. However, its potential value in clear cell renal cell carcinoma (ccRCC) diagnosis and prognosis remains unclear. In the present study, differentially expressed miRNAs from urinal exosomes were identified by next-generation sequencing (NGS) technology. The 16 differentially expressed miRNAs were identified between ccRCC patients and healthy donors. To explore the specific diagnosis biomarker of ccRCC, we validated these urinary exosomes from 70 early-stage renal cancer patients, 30 healthy people and other urinary system cancers, including 30 early-stage prostate cancer patients and 30 early-stage bladder cancer patients by qRT-PCR. The results showed that urinary exosome miR-30c-5p could be stably amplified and meanwhile the expression of miR-30c-5p has no significant difference between other urinary system cancers and healthy control, however, expression level of miR-30c-5p in urinary exosomal of ccRCC patients was lower than healthy people and receiver operation characterization (ROC) curve showed that the area under the curve (AUC) values was 0.8192 (95% confidence interval was 0.7388-0.8996, P= 0.0000). In addition, up-regulating miR-30c-5p expression could inhibit renal cell carcinoma cells growth. Lastly, HSP5A was found as a direct target gene of miR-30c-5p. HSP5A depletion reversed the promoting effect of ccRCC growth casued by miR-30c-5p inhibitor, respectively. In conclusion, this study demonstrated that urinary exosomal miR-30c-5p is readily accessible as diagnosis biomarker of early-stage ccRCC, and miR-30c-5p might modulate the expression of HSPA5, which correlated with the progression of ccRCC.

Keywords: clear cell renal cell carcinoma, exosome, HSP5A, miR-30c-5p

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Authors: Ahmad Rasyadan Arshad, A. Rahman A. Jamal, N. Mohamed Ibrahim, Nor Azian Abdul Murad

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Introduction: Parkinson’s disease (PD) is a complex and asymptomatic disease where patients are usually diagnosed at late stage where about 70% of the dopaminergic neurons are lost. Therefore, identification of molecular biomarkers is crucial for early diagnosis of PD. MicroRNA (miRNA) is a short nucleotide non-coding small RNA which regulates the gene expression in post-translational process. The involvement of these miRNAs in neurodegenerative diseases includes maintenance of neuronal development, necrosis, mitochondrial dysfunction and oxidative stress. Thus, miRNA could be a potential biomarkers for diagnosis of PD. Objective: This study aim to identify the miRNA involved in Late Onset PD (LOPD) and Early Onset PD (EOPD) compared to the controls. Methods: This is a case-control study involved PD patients in the Chancellor Tunku Muhriz Hospital at the UKM Medical Centre. miRNA samples were extracted using miRNeasy serum/plasma kit from Qiagen. The quality of miRNA extracted was determined using Agilent RNA 6000 Nano kit in the Bioanalyzer. miRNA expression was performed using GeneChip miRNA 4.0 chip from Affymetrix. Microarray was performed in EOPD (n= 7), LOPD (n=9) and healthy control (n=11). Expression Console and Transcriptomic Analyses Console were used to analyze the microarray data. Result: miR-129-5p was significantly downregulated in EOPD compared to LOPD with -4.2 fold change (p = <0.050. miR-301a-3p was upregulated in EOPD compared to healthy control (fold = 10.3, p = <0.05). In LOPD versus healthy control, miR-486-3p (fold = 15.28, p = <0.05), miR-29c-3p (fold = 12.21, p = <0.05) and miR-301a-3p (fold = 10.01, p =< 0.05) were upregulated. Conclusion: Several miRNA have been identified to be differentially expressed in EOPD compared to LOPD and PD versus control. These miRNAs could serve as the potential biomarkers for early diagnosis of PD. However, these miRNAs need to be validated in a larger sample size.

Keywords: early onset PD, late onset PD, microRNA (miRNA), microarray

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Authors: Rohit Kothari, Muneer Mohamed, Vivekanandh K., Sunmeet Sandhu, Preema Sinha, Anuj Bhatnagar

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Introduction: Hyper-IgE syndrome is a rare primary immunodeficiency syndrome characterised by triad of severe atopic dermatitis, recurrent pulmonary infections, and recurrent staphylococcal skin infections. The diagnosis requires a high degree of suspicion, typical clinical features, and not mere rise in serum-IgE levels, which may be seen in multiple conditions. Genetic studies are not always possible in a resource poor setting. This study highlights various presentations of Hyper-IgE syndrome in skin of color children. Case-series: Our study had six children of Hyper-IgE syndrome aged twomonths to tenyears. All had onset in first ten months of life except one with a late-onset at two years. All had recurrent eczematoid rash, which responded poorly to conventional treatment, secondary infection, multiple episodes of hospitalisation for pulmonary infection, and raised serum IgE levels. One case had occasional vesicles, bullae, and crusted plaques over both the extremities. Genetic study was possible in only one of them who was found to have pathogenic homozygous deletions of exon-15 to 18 in DOCK8 gene following which he underwent bone marrow transplant (BMT), however, succumbed to lower respiratory tract infection two months after BMT and rest of them received multiple courses of antibiotics, oral/ topical steroids, and cyclosporine intermittently with variable response. Discussion: Our study highlights various characteristics, presentation, and management of this rare syndrome in children. Knowledge of these manifestations in skin of color will facilitate early identification and contribute to optimal care of the patients as representative data on the same is limited in literature.

Keywords: absolute eosinophil count, atopic dermatitis, eczematous rash, hyper-immunoglobulin E syndrome, pulmonary infection, serum IgE, skin of color

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Authors: Yan Li, Qingxiang Meng, Bo Zhou, Zhenming Zhou

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The objective of this study was to compare the effects of ensiled mulberry leaves (EML), and sun-dried mulberry fruit pomace (SMFP) on fecal bacterial communities in Simmental crossbred finishing steers fed the following 3 diets: a standard TMR diet, standard diet containing EML and standard diet containing SMFP, and the diets had similar protein and energy levels. Bacterial communities in the fecal content were analyzed using Illumina Miseq sequencing of the V4 region of the 16S rRNA gene amplification. Quantitative real-time PCR was used to detect the selected bacterial species in the feces. Most of the sequences were assigned to phyla Firmicutes (56.67%) and Bacteroidetes(35.90%), followed by Proteobacteria(1.86%), Verrucomicrobia(1.80%) and Tenericutes(1.37%). And the predominant genera included the 5-7N15 (5.91%), CF231 (2.49%), Oscillospira (2.33%), Paludibacter (1.23%) and Akkermansia(1.11%). As for the treatments, no significant differences were observed in Firmicutes (p = 0.28), Bacteroidetes (p = 0.63), Proteobacteria (p = 0.46), Verrucomicrobia (p = 0.17) and Tenericutes (p = 0.75). On the genus level, classified genera with high abundance (more than 0.1%) mainly came from two phyla: Bacteroidetes and Firmicutes. Also no differences were observed in most genera level, 5-7N15 (p = 0.21), CF231 (p = 0.62), Oscillospira (p = 0.9), Paludibacter (p = 0.33) and Akkermansia (p = 0.37), except that rc4-4 were lower in the CON and SMFP groups compared to the EML animals (p = 0.02). Additionally, there were no differences in richness estimate and diversity indices (p > 0.16), and treatments had no significant effect on most selected bacterial species in the fecal (p > 0.06), except that Ruminococcus albus were higher in the EML group (p < 0.01) and Streptococcus bovis were lower in the CON group (p < 0.01). In conclusion, diets supplemented with EML and SMFP have little influence on fecal bacterial community composition in finishing steers.

Keywords: fecal bacteria community composition, sequencing, ensiled mulberry leaves (EML), sun-dried mulberry fruit pomace (SMFP)

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Authors: Salma H. Abu Hafsa, A. Mendonca, B. Brehm-Stecher, A. A. Hassan, S. A. Ibrahim

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Enterococci are important inhabitants of the animal intestine and are widely used in probiotic products. A probiotic strain is expected to possess several desirable properties in order to exert beneficial effects. Therefore, the objective of this study was to isolate and characterize strains of Enterococcus sp. from chicken cecal and fecal samples to determine potential probiotic properties. Enterococci were isolated from thirty one chicken cecal and fecal samples collected from a local farm. In vitro studies were performed to assess antibacterial activity (using agar well diffusion and cell free supernatant broth technique against Salmonella enterica serotype Enteritidis), susceptibility to antibiotics (amoxycillin, cotrimoxazole, chloramphenicol, cefuroxime, ceftriaxone, ciprofloxacin, and nalidixic acid), survival in acidic conditions, resistance to bile salts, and their survival during simulated gastric juice conditions at pH 2.5. Isolates were identified using biochemical and molecular assays (API 50 CHL, and API ZYM kits followed by 16S rDNA gene sequence analysis). Two strains were identified, of which, Enteroccocus faecium was capable of inhibiting the growth of S. enteritidis and was susceptible to a wide range of antibiotics. In addition, the isolated strain exhibited significant resistance under highly acidic conditions (pH=2.5) for 8 hours and survived well in bile salt at 0.2% for 24 hours and showing ability to survive in the presence of simulated gastric juice at pH 2.5. Based on these results, the E. faecium isolate fulfills some of the criteria to be considered as a probiotic strain and therefore, could be used as a feed additive with good potential for controlling S. enteritidis in chickens. However, in vivo studies are needed to determine the safety of the strain.

Keywords: acid tolerance, antimicrobial activity, Enterococcus faecium, probiotic

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Authors: Ananya Govindarajan

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Tetrahymena thermophila is a single-cell, eukaryotic organism that belongs to the Protozoa Kingdom. Tetrahymena thermophila is often used in signal transduction pathway studies because of its ability to model sensory input and the effects of environmental conditions such as chemicals and temperature. The recently discovered G37 chemorepellent receptor showed increased responsiveness to all chemorepellents. Investigating the mutant G37 Tetrahymena gene in various test solutions, including ferric chloride, ferrous sulfate, hydrogen peroxide, tetrazolium blue, potassium chloride, and dithiothreitol were performed to determine the role of oxidants and reducing agents with the mutant and wild-type cells (CU427) to assess the role of the receptor. Behavioral assays and recordings processed by ImageJ indicated that ferric chloride, hydrogen peroxide, and tetrazolium blue yielded little to no chemorepellent responses from G37 cells (<20% ARs). CU427 cells were over-responsive based on the mean percent of cells (>50% ARs). Reducing agents elicited chemorepellent responses from both G37 and CU427, in addition to potassium chloride. Cell responses were classified as over-responsive (>50% ARs). Dithiothreitol yielded unexpected results as G37 (37.0% ARs) and CU427 (38.1% ARs) had relatively similar responses and were only responsive and not over-responsive to the reducing agent test chemical solution. Ultimately, this indicates that the G37 receptor is more interactive with molecules that are reducing agents or non-oxidant compounds; G37 may be unable to sense and respond to oxidants effectively, further elucidating the pathways of the G37 strain and nature of this receptor. Results also indicate that the CSF most likely contained an oxidant, like ferric chloride. This research can be further applied to neuronal influences and how specific compounds may affect human neurons individually and their excitability as the responses model action potentials and membrane potential.

Keywords: tetrahymena thermophila, signal transduction, chemosensory, oxidant, reducing agent

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Authors: Suneeta Gireesh Panicker

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Aim: Aminopeptidase P (APP) is an enzyme that has specificity for proline, can specifically cleave Xaa-Proline peptides and is a metallo-aminopeptidase. The bonds nearby to the imino acid proline are tough to cleave by many peptidases, but APP can specifically break peptide bonds engaged with proline. Membrane-bound form and a cytosolic form are the two forms in which this enzyme exists. The exact physiological function of APP remains unclear and hence the present work attempts to determine it. Methods: In the present study, the expression pattern of cytosolic Aminopeptidase P (DAP) was determined in all the embryonic stages and larval stages of wild-type Drosophila by using polyclonal monospecific antibodies. To show the presence of DAP RNA in embryonic and larval stages, RNA in situ hybridization was performed. DAP promoter-LacZ fusion reporter gene vector was used to construct transgenic embryos to study the regulation pattern of DAP. To study the DAP expression profile, a transgenic fly consisting of a DAP promoter with β-gal and GFP reporter genes in front of it was constructed. Results: DAP protein expression was observed in neuroectodermal cells, posterior midgut primordium, proctodeum, ventral neuroblast and primordial stomatogastric nervous system. It was observed in the ventral cord and midgut in stage 12. The completely developed embryos showed the intense occurrence of it in the ventral cord and gut region. The eye-antennal disc, wing disc and leg disc also showed the presence of DAP protein. LacZ expression in transgenic embryos also showed the same pattern. Conclusion: Similar to various known multiple-functional proteins, DAP could be one with different functions at different stages and in different cells. Data presented here designates DAP functions in the early embryonic and imaginal dics differentiation and development, suggesting that it may be required for the metabolism of proteins like neuropeptides and tachykinins.

Keywords: aminopeptidase P, in situ hybridization, transgenic fly, embryonic stages

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Authors: Ahmed Elbeltagy, Francesca Bertolini, Damarius Fleming, Angelica Van Goor, Chris Ashwell, Carl Schmidt, Donald Kugonza, Susan Lamont, Max Rothschild

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The major factor in shaping genomic variation of the African indigenous rural chicken is likely natural selection drives the development genetic footprints in the chicken genomes. To investigate such a hypothesis of a selection footprint, a total of 292 birds were randomly sampled from three indigenous ecotypes from East Africa (Uganda, Rwanda) and North Africa (Egypt) and two registered Egyptian breeds (Fayoumi and Dandarawi), and from the synthetic Kuroiler breed. Samples were genotyped using the Affymetrix 600K Axiom® Array. A total of 526,652 SNPs were utilized in the downstream analysis after quality control measures. The intra-population runs of homozygosity (ROH) that were consensuses in > 50% of individuals of an ecotype or > 75% of a breed were studied. To identify inter-population differentiation due to genetic structure, FST was calculated for North- vs. East- African populations in addition to population-pairwise combinations for overlapping windows (500Kb with an overlap of 250Kb). A total of 28,563 ROH were determined and were classified into three length categories. ROH and Fst detected sweeps were identified on several autosomes. Several genes in these regions are likely to be related to adaptation to local environmental stresses that include high altitude, diseases resistance, poor nutrition, oxidative and heat stresses and were linked to gene ontology terms (GO) related to immune response, oxygen consumption and heme binding, carbohydrate metabolism, oxidation-reduction, and behavior. Results indicated a possible effect of natural selection forces on shaping genomic structure for adaptation to local environmental stresses.

Keywords: African Chicken, runs of homozygosity, FST, selection footprints

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Authors: Abul B. M. M. K. Islam, Mandar Dave, Roderick V. Jensen, Ashok R. Amin

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A systems approach was applied to characterize differentially expressed transcripts, bioinformatics pathways, and proteins and prostaglandins (PGs) from lung fibroblasts procured from wild-type (WT), COX-1-/- and COX-2-/- mice to understand system level control mechanism. Bioinformatics analysis of COX-2 and COX-1 ablated cells induced COX-1 and COX-2 specific signature respectively, which significantly overlapped with an 'IL-1β induced inflammatory signature'. This defined novel cross-talk signals that orchestrated coordinated activation of pathways of sterile inflammation sensed by cellular stress. The overlapping signals showed significant over-representation of shared pathways for interferon y and immune responses, T cell functions, NOD, and toll-like receptor signaling. Gene Ontology Biological Process (GOBP) and pathway enrichment analysis specifically showed an increase in mRNA expression associated with: (a) organ development and homeostasis in COX-1-/- cells and (b) oxidative stress and response, spliceosomes and proteasomes activity, mTOR and p53 signaling in COX-2-/- cells. COX-1 and COX-2 showed signs of functional pathways committed to cell cycle and DNA replication at the genomics level. As compared to WT, metabolomics analysis revealed a significant increase in COX-1 mRNA and synthesis of basal levels of eicosanoids (PGE2, PGD2, TXB2, LTB4, PGF1α, and PGF2α) in COX-2 ablated cells and increase in synthesis of PGE2, and PGF1α in COX-1 null cells. There was a compensation of PGE2 and PGF1α in COX-1-/- and COX-2-/- cells. Collectively, these results support a broader, differential and collaborative regulation of both COX-1 and COX-2 pathways at the metabolic, signaling, and genomics levels in cellular homeostasis and sterile inflammation induced by cellular stress.

Keywords: cyclooxygenases, inflammation, lung fibroblasts, systemic

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Authors: Duaa Mughal, Syeda Areeba Nadeem, Shakil Ahmed, Ishtiaq Ahmed Khan

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The identification of porcine DNA in Halal food items is critical to ensuring compliance with dietary restrictions and religious beliefs. In Islam, Porcine is prohibited as clearly mentioned in Quran (Surah Al-Baqrah, Ayat 173). The purpose of this study was to compare two DNA extraction procedures for detecting 0.001% of porcine DNA in processed Halal food sample mixtures containing chicken, camel, veal, turkey and goat meat using the TaqMan Real-Time PCR technology. In this research, two different commercial kit protocols were compared. The processed sample mixtures were prepared by spiking known concentration of porcine DNA to non-porcine food matrices. Afterwards, TaqMan Real-Time PCR technique was used to target a particular porcine gene from the extracted DNA samples, which was quantified after extraction. The results of the amplification were evaluated for sensitivity, specificity, and reproducibility. The results of the study demonstrated that two DNA extraction techniques can detect 0.01% of porcine DNA in mixture of Halal food samples. However, as compared to the alternative approach, Eurofins| GeneScan GeneSpin DNA Isolation kit showed more effective sensitivity and specificity. Furthermore, the commercial kit-based approach showed great repeatability with minimal variance across repeats. Quantification of DNA was done by using fluorometric assay. In conclusion, the comparison of DNA extraction methods for detecting porcine DNA in Halal food sample mixes using the TaqMan Real-Time PCR technology reveals that the commercial kit-based approach outperforms the other methods in terms of sensitivity, specificity, and repeatability. This research helps to promote the development of reliable and standardized techniques for detecting porcine DNA in Halal food items, religious conformity and assuring nutritional.

Keywords: real time PCR (qPCR), DNA extraction, porcine DNA, halal food authentication, religious conformity

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Authors: Zewdneh Zana Zate

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The study focuses on the evolutionary and ecological genomics of both wild and cultivated Coffea arabica L. at its center of origin, Ethiopia, aiming to uncover how this vital species may withstand future climate changes. Utilizing bioclimatic models, we project the future distribution of Arabica under varied climate scenarios for 2050 and 2080, identifying potential conservation zones and immediate risk areas. Through whole-genome resequencing of accessions from Ethiopian gene banks, this research assesses genetic diversity and divergence between wild and cultivated populations. It explores relationships, demographic histories, and potential hybridization events among Coffea arabica accessions to better understand the species' origins and its connection to parental species. This genomic analysis also seeks to detect signs of natural or artificial selection across populations. Integrating these genomic discoveries with ecological data, the study evaluates the current and future ecological and genomic vulnerabilities of wild Coffea arabica, emphasizing necessary adaptations for survival. We have identified key genomic regions linked to environmental stress tolerance, which could be crucial for breeding more resilient Arabica varieties. Additionally, our ecological modeling predicted a contraction of suitable habitats, urging immediate conservation actions in identified key areas. This research not only elucidates the evolutionary history and adaptive strategies of Arabica but also informs conservation priorities and breeding strategies to enhance resilience to climate change. By synthesizing genomic and ecological insights, we provide a robust framework for developing effective management strategies aimed at sustaining Coffea arabica, a species of profound global importance, in its native habitat under evolving climatic conditions.

Keywords: coffea arabica, climate change adaptation, conservation strategies, genomic resilience

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

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

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

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

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

Keywords: emerging pathogens, Iran, juglans regia, MLSA

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Authors: Eirini Konstanta, Cedric Gouedard, Aggeliki Delimitsou, Stefania Patera, Samuel Murray

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Introduction: Quality reference DNA standards (QRS) for molecular testing by next-generation sequencing (NGS) are essential for accurate quantitation of copy number variations (CNV) for germline and variant allelic frequencies (VAF) for somatic analyses. Objectives: Presently, several molecular analytics for oncology patients are reliant upon quantitative metrics. Test validation and standardisation are also reliant upon the availability of surrogate control materials allowing for understanding test LOD (limit of detection), sensitivity, specificity. We have developed a dual calibration platform allowing for QRS pairs to be included in analysed DNA samples, allowing for accurate quantitation of CNV and VAF metrics within and between patient samples. Methods: QRS™ blocks up to 500nt were designed for common NGS panel targets incorporating ≥ 2 identification tags (IDTDNA.com). These were analysed upon spiking into gDNA, somatic, and ctDNA using a proprietary CalSuite™ platform adaptable to common LIMS. Results: We demonstrate QRS™ calibration reproducibility spiked to 5–25% at ± 2.5% in gDNA and ctDNA. Furthermore, we demonstrate CNV and VAF within and between samples (gDNA and ctDNA) with the same reproducibility (± 2.5%) in a clinical sample of lung cancer and HBOC (EGFR and BRCA1, respectively). CNV analytics was performed with similar accuracy using a single pair of QRS calibrators when using multiple single targeted sequencing controls. Conclusion: Dual paired QRS™ calibrators allow for accurate and reproducible quantitative analyses of CNV, VAF, intrinsic sample allele measurement, inter and intra-sample measure not only simplifying NGS analytics but allowing for monitoring clinically relevant biomarker VAF across patient ctDNA samples with improved accuracy.

Keywords: calibrator, CNV, gene copy number, VAF

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

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

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

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