Search results for: sequences of AMR gene

Authors: Rosa Figueroa, Christopher Flores

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Text categorization is the problem of assigning a new document to a set of predetermined categories, on the basis of a training set of free-text data that contains documents whose category membership is known. To train a classification model, it is necessary to extract characteristics in the form of tokens that facilitate the learning and classification process. In text categorization, the feature extraction process involves the use of word sequences also known as N-grams. In general, it is expected that documents belonging to the same category share similar features. The Smith-Waterman (SW) algorithm is a dynamic programming algorithm that performs a local sequence alignment in order to determine similar regions between two strings or protein sequences. This work explores the use of SW algorithm as an alternative to feature extraction in text categorization. The dataset used for this purpose, contains 2,610 annotated documents with the classes Obese/Non-Obese. This dataset was represented in a matrix form using the Bag of Word approach. The score selected to represent the occurrence of the tokens in each document was the term frequency-inverse document frequency (TF-IDF). In order to extract features for classification, four experiments were conducted: the first experiment used SW to extract features, the second one used unigrams (single word), the third one used bigrams (two word sequence) and the last experiment used a combination of unigrams and bigrams to extract features for classification. To test the effectiveness of the extracted feature set for the four experiments, a Support Vector Machine (SVM) classifier was tuned using 20% of the dataset. The remaining 80% of the dataset together with 5-Fold Cross Validation were used to evaluate and compare the performance of the four experiments of feature extraction. Results from the tuning process suggest that SW performs better than the N-gram based feature extraction. These results were confirmed by using the remaining 80% of the dataset, where SW performed the best (accuracy = 97.10%, weighted average F-measure = 97.07%). The second best was obtained by the combination of unigrams-bigrams (accuracy = 96.04, weighted average F-measure = 95.97) closely followed by the bigrams (accuracy = 94.56%, weighted average F-measure = 94.46%) and finally unigrams (accuracy = 92.96%, weighted average F-measure = 92.90%).

Keywords: comorbidities, machine learning, obesity, Smith-Waterman algorithm

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

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

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

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Authors: Minhas Alam, Muhammad Hidayat Rasool, Mohsin Khurshid, Bilal Aslam

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Acinetobacter baumannii is a notorious bacterial pathogen that is an emerging nightmare in clinical settings and is mainly involved in severe nosocomial infections. However, the data related to carbapenem-resistant A. baumannii (CRAB) from veterinary settings is limited, especially in developing countries like Pakistan. To investigate the genetic diversity and molecular basis of carbapenem resistance in Acinetobacter baumannii isolates from Cattle, a total of 1960 samples were collected from cattle from Punjab, Pakistan. The isolates were analyzed by routine microbiological procedures and confirmed by polymerase chain reaction (PCR). The isolates were further screened for antimicrobial susceptibility and the presence of multiple antimicrobial-resistant determinants by PCR. Multilocus sequence typing (MLST) was performed. The results of the current study revealed that the overall prevalence of A. baumannii in cattle was 3.28% (65/1980). Among cattle 27.7% (18/65) were found CRAB strains. The CRAB isolates harbor class D β- lactamases genes, e-g, blaOXA-23 and blaOXA-51, 94.4% (17/18). CRAB isolates carry class B β- lactamases gene blaIMP, and only one isolate carries the blaNDM-1 gene. The MLST results of CRAB isolates from cattle demonstrated 5 STs and one new ST. The commonly found sequence types in CRAB isolates were ST2 (n=10, 55.5%), followed by ST642 (n=5, 27.8%) and ST600 & ST889 (n=1, 5.55%). The presence of CRAB isolates in cattle indicates an alarming situation in Punjab, Pakistan. Immediate control measures should be taken to stop the transmission of CRAB isolates within cattle, to the environment, and to clinical settings.

Keywords: acinetobacter baumannii, carbapenemases, veterinary, drug resistance

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Authors: Katerina Balounova, Jiri Pacha, Peter Ergang, Martin Vodicka, Pavlina Kvapilova

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MicroRNAs are small non-coding RNAs involved in a wide range of physiological processes. Post-transcriptional regulation of gene expression by microRNAs gives the organism a further level of control of the gene-expression program and the disruption of this microRNA regulatory mechanism seems to increase the risk of various pathophysiological conditions including tumorigenesis. To the present day, microRNAs were shown to participate in the mayor signalization pathways leading to tumorigenesis, including proliferation, cell cycle, apoptosis and metastasis formation. In addition, microRNAs have been found to play important roles in the generation and maintenance of circadian clock. These clocks generate circadian rhythms, which participate in a number of regulatory pathways. Disruption of the circadian signals seems to be associated with the development and the progression of tumours including colorectal cancer. We investigated therefore whether the diurnal profiles of miRNAs linked to tumorigenesis and regulation of circadian clock are changed during tumorigenesis. Based on published data we chose 10 microRNAs linked to tumorigenesis or circadian clock (let-7b-5p, miR 1 3p, miR 106b 5p, miR 141 3p, miR 191 5p, miR 20a 5p, miR 25 3p, miR 29a 3p, miR 34a 5p and miR 93 5p) and compared their 24-hr expression profiles in healthy and in chemically induces primary colorectal tumours of 52week-old mice. Using RT-qPCR we proved circadian rhythmicity in let-7b-5p, miR 106b 5p, miR 141 3p, miR 191 5p, miR 20a 5p, miR 25 3p, miR 29a 3p and miR 93 5p in healthy colon but not in tumours. The acrophases of miR 106b 5p, miR 141 3p, miR 191 5p, miR 20a 5p, miR 25 3p and miR 93 5p were reached around CT 24, the acrophases of let-7b-5p and miR-29a-3p were slightly shifted and reached around CT 21. In summary, our results show that circadian regulation of some colonic microRNAs is greatly affected by neoplastic transformation.

Keywords: circadian rhythm, colon, colorectal cancer, microRNA, tumorigenesis

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Authors: Salina Saddick, Mohamed Afifi, Osama Abuznadah

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This study was carried out to determine the median lethal concentrations (LC50) of Zinc nanoparticles (ZnNPs) on Oreochromis niloticus and Tilapia zillii. The biochemical and molecular potential effects of ZnNPs (500 and 2000 μg L−1) on the antioxidant system in the brain tissue of O. niloticus and T. zillii were investigated. Four hundred fish were used for acute and sub-acute studies. ZnNP LC50 concentrations were investigated in O. niloticus and T. zillii. The effect of 500 and 2000 μg L−1 ZnNPs on brain antioxidants of O. niloticus and T. zillii was investigated. The result indicated that 69 h LC50 was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. Fish exposed to 500 μg L−1 ZnNPs showed a significant increase in reduced glutathione (GSH), total glutathione (tGSH) levels, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activity and gene expression. On the contrary, malondialdehyde (MDA) levels significantly decreased. Meanwhile, fish exposed to 2000 μg L−1 ZnNPs showed a significant decrease of GSH, tGSH levels, SOD, CAT, GR, GPx and GST activity and gene expression. On the contrary, MDA levels significantly increased. It was concluded that, the 96 h LC50 of ZnNPs was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. ZnNPs in exposure concentrations of 2000 μg/L induced a deleterious effect on the brain antioxidant system of O. nilotica and T. zillii. In contrast, ZnNPs in exposure concentrations of 500 μg L−1 produced an inductive effect on the brain antioxidant system of O. nilotica and T. zillii.

Keywords: ZnNPs, LC50, antioxidants, O. nilotica

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Authors: Chul Lee, Seoae Cho, Erich D. Jarvis, Heebal Kim

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Vocal learning, the ability to imitate vocalizations based on auditory experience, is a homoplastic character state observed in different independent lineages of animals such as songbirds, parrots, hummingbirds and human. It has now become possible to perform genome-wide molecular analyses across vocal learners and vocal non-learners with the recent expansion of avian genome data. It was analyzed the whole genomes of human and 48 avian species including those belonging to the three avian vocal learning lineages, to determine if behavior and neural convergence are associated with molecular convergence in divergent species of vocal learners. Analyses of 8295 orthologous genes across bird species revealed 141 genes with amino acid substitutions specific to vocal learners. Out of these, 25 genes have vocal learner specific genetic homoplasies, and their functions were enriched for learning. Several sites in these genes are estimated under convergent evolution and positive selection. A potential role for a subset of these genes in vocal learning was supported by associations with gene expression profiles in vocal learning brain regions of songbirds and human disease that cause language dysfunctions. The key candidate gene with multiple independent lines of the evidences specific to vocal learners was DRD5. Our findings suggest cAMP-based learning pathway in avian vocal learners, indicating molecular homoplastic changes associated with a complex behavioral trait, vocal learning.

Keywords: amino acid substitutions, convergent evolution, positive selection, vocal learning

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Authors: Seyed Davar Siadat, Samira Tarashi, Abolfazl Fateh, Arfa Moshiri

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Background: The global health issue of tuberculosis (TB) still affects patients in every country. TB control may not be as effective as it should be, especially when resistant strains are involved. In this regard, mycobacterial MTases play a major role in tuberculosis, but the mechanisms underlying their function have yet to be fully deciphered. Methods: Five resistant isolates of M.tb were accumulated. As a reference strain, M.tb H37Rv (ATCC 27249) was used. For this analysis, seven putative mycobacterial MTase genes (Rv0645c, Rv1694, Rv2966c, Rv3919c, Rv2756c, Rv1988, and Rv3263), as well as Rv1392 as SAM synthase, were selected. Comparing mutations and expression levels of MTases in different strains was accomplished by PCR-sequencing and qRT-PCR. The relative expression levels of these genes were calculated using the 2 -ΔΔCt method. Results: The Rv3919c gene (T to G in codon 341) and Rv1392 gene (G to A in codon 97) were the only mutations found in the INHR strain. In all sensitive and resistant isolates, Rv0645c, Rv3263, Rv2756c, and Rv2966c were overexpressed. However, the expression of Rv1988 and Rv3919c decreased in the sensitive strains, whereas the expression of Rv1694 increased. There was also a decreased expression of Rv1392 in the INHR isolate. Conclusion: The presence of mycobacterial MTases as well as resistance to antibiotics were found to be correlated in M.tb strains. Undoubtedly, there are some MTases that are associated with the virulence process. It is necessary to conduct additional studies to fully explore the impact of mycobacterial MTases within specific strains of M.tb to develop novel diagnostic and treatment strategies.

Keywords: mycobacterium tuberculosis, drug resistance, methyltransferases, s-adenosylmethionine

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

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

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

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Authors: Mukta Singh, Ratan Kumar Saha, Himadri Saha, Paramveer Singh

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The present study evaluated the effect of sub-lethal doses of antifungal drug miconazole nitrate (MCZ) on immunological responses including immune-related gene expression and its role as a prophylactic drug against S. parasitica in Labeo rohita fingerlings. Fish were fed with sub lethal doses of MCZ i.e., T1- 6.30 mg MCZ kgBW⁻¹, T2- 12.61 mg MCZ kgBW⁻¹ and T3- 25.22 mg MCZ kgBW⁻¹ and sampling was done at different time intervals for 240 h. Immunological parameters viz. lysozyme activity, oxygen radical production and plasma anti-protease activity showed significant enhancement (p < 0.05) in fish fed with T2 and T3 doses. Significant reduction in plasma protein content was observed in all the dietary groups as compared to control. Expression of immune-relevant genes like TLR-22 and β2-M showed significantly higher expression at six h and 24 h of sampling in both liver and head-kidney. However, these genes showed a down-regulation after 120 h of sampling in both the tissues. Preventive efficacy study showed that single dose of MCZ provides protection against oomycetes up to the fourth day of infection. Significantly higher mortality was observed in control diet-fed fish as compared to fish fed with MCZ medicated diet. Thus, from the study, it can be concluded that the MCZ can act as a potent antifungal agent for preventing oomycetes infection as well as to enhance the immune response.

Keywords: antifungal, immune gene, immunological, miconazole nitrate, prophylactic

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Authors: Mahmoodreza Tahamtan

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Background: Malignant surface epithelial ovarian tumors (SEOT) account for approximately 90% of primary ovarian cancer. Wilms tumor gene (WT1) product was defined as a tumor suppressor gene, but today it is considered capable of performing oncogenic functions. There seems to be differences in WT1 expression patterns among SEOT subtypes. We evaluate the immunohistochemical expression of WT1 protein among different histologic subtypes of SEOT. Materials and Methods: Immunohistochemistry for WT1 was done on 35 serous cystadenocarcinomas, 9 borderline serous tumors, 3 mucinous cystadenocarcinomas, 10 borderline mucinous tumors, 7 endometrioid ovarian carcinomas, 3 clear cell carcinomas, 1 malignant Brenner tumor, 2 metastatic adenocarcinomas, and 6 endometrial adenocarcinomas. A tumor was considered negative if < 1% of tumor cells were stained.Positive reactions were graded as follows:1+,1%-24%; 2+,25%-49%; 3+,50%-74%; 4+,75%-100%. Results: Of the 35 cases of ovarian serous cystadenocarcinoma, 30(85.7%) were diffusely positive (3+,4+),4 showed reactivity of < 50% of the tumor cells (1+,2+), and one were negative. All 9 borderline serous tumors showed immunoreactivity with WT1. All the mucinous tumors(n:13), endometrioid carcinomas (n: 7), clear cell carcinomas (n: 3), metastatic adenocarcinomas (n: 2) and primary endometrial carcinomas (n:6) were negative. The single malignant Brenner tumor showed a positive reaction for WT1(4+) Conclusion: WT1 is a good marker to distinguish primary ovarian serous carcinomas from other surface epithelial tumors (especially endometrioid subtype) and metastatic carcinomas (especially endometrial serous carcinoma), other than malignant mesothelioma. We cannot rely to the degree of expression inorder to separate high grade borderline serous tumors from low grade ones.

Keywords: WT1, ovary, epithelial tumors, malignant

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Authors: M. Al Zallouha, Y. Landkocz, J. Brunet, R. Cousin, J. M. Halket, E. Genty, P. J. Martin, A. Verdin, D. Courcot, S. Siffert, P. Shirali, S. Billet

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Toluene is one of the most used Volatile Organic Compounds (VOCs) in the industry. Amongst VOCs, Benzene, Toluene, Ethylbenzene and Xylenes (BTEX) emitted into the atmosphere have a major and direct impact on human health. It is, therefore, necessary to minimize emissions directly at source. Catalytic oxidation is an industrial technique which provides remediation efficiency in the treatment of these organic compounds. However, during operation, the catalysts can release some compounds, called byproducts, more toxic than the original VOCs. The catalytic oxidation of a gas stream containing 1000ppm of toluene on Pd/α-Al2O3 can release a few ppm of benzene, according to the operating temperature of the catalyst. The development of new catalysts must, therefore, include chemical and toxicological validation phases. In this project, A549 human lung cells were exposed in air/liquid interface (Vitrocell®) to gas mixtures derived from the oxidation of toluene with a catalyst of Pd/α-Al2O3. Both exposure concentrations (i.e. 10 and 100% of catalytic emission) resulted in increased gene expression of Xenobiotics Metabolising Enzymes (XME) (CYP2E1 CYP2S1, CYP1A1, CYP1B1, EPHX1, and NQO1). Some of these XMEs are known to be induced by polycyclic organic compounds conventionally not searched during the development of catalysts for VOCs degradation. The increase in gene expression suggests the presence of undetected compounds whose toxicity must be assessed before the adoption of new catalyst. This enhances the relevance of toxicological validation of such systems before scaling-up and marketing.

Keywords: BTEX toxicity, air/liquid interface cell exposure, Vitrocell®, catalytic oxidation

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Authors: S. Hannah Elizabeth, A. Panneerselvam

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Objective: The main objective was to evaluate the degradative properties of Bacillus cereus sp TUHP2 isolated from TNT-Polluted soils in the Vellore District, Tamil Nadu, India. Methods: Among the 3 bacterial genera isolated from different soil samples, one potent TNT degrading strain Bacillus cereus sp TUHP2 was identified. The morphological, physiological and the biochemical properties of the strain Bacillus cereus sp TUHP2 was confirmed by conventional methods and genotypic characterization was carried out using 16S r-DNA partial gene amplification and sequencing. The broken down by products of DNT in the extract was determined by Gas Chromatogram- Mass spectrometry (GC-MS). Supernatant samples from the broth studied at 24 h interval were analyzed by HPLC analysis and the effect on various nutritional and environmental factors were analysed and optimized for the isolate. Results: Out of three isolates one strain TUHP2 were found to have potent efficiency to degrade TNT and revealed the genus Bacillus. 16S rDNA gene sequence analysis showed highest homology (98%) with Bacillus cereus and was assigned as Bacillus cereus sp TUHP2. Based on the energy of the predicted models, the secondary structure predicted by MFE showed the more stable structure with a minimum energy. Products of TNT Transformation showed colour change in the medium during cultivation. TNT derivates such as 2HADNT and 4HADNT were detected by HPLC chromatogram and 2ADNT, 4ADNT by GC/MS analysis. Conclusion: Hence this study presents the clear evidence for the biodegradation process of TNT by strain Bacillus cereus sp TUHP2.

Keywords: bioremediation, biodegradation, biotransformation, sequencing

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Authors: Pei-Ying Lo, Jing-Hao Ciou, Kai-Cheng Yang, Jia-Huei Zheng, Shih-Hsiang Huang, Kuen-Chan Lee, Er-Chieh Cho

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As-produced CNTs are insoluble in all organic solvents and aqueous solutions have imposed limitations to the use of CNTs. Therefore, how to debundle carbon nanotubes and to modify them for further uses is an important issue. There are several methods for the dispersion of CNTs in water using covalent attachment of hydrophilic groups to the surface of tubes. These methods, however, alter the electronic structure of the nanotubes by disrupting the network of sp2 hybridized carbons. In order to keep the nanotubes’ intrinsic mechanical and electrical properties intact, non-covalent interactions are increasingly being explored as an alternative route for dispersion. Apart from conventional surfactants such as sodium dodecylsulfate (SDS) or sodium dodecylbenzenesulfonate (SDBS) which are highly effective in dispersing CNTs, biopolymers have received much attention as dispersing agents due to the anticipated biocompatibility of the dispersed CNTs. Also, The pyrenyl group is known to interact strongly with the basal plane of graphene via π-stacking. In this study, a highly re-dispersible biopolymer is reported for the synthesis of pyrene-modified poly-L-lysine (PBPL) and poly(D-Glu, D-Lys) (PGLP). To provide the evidence of the safety of the PBPL/CNT & PGLP/CNT materials we use in this study, H1299 and HCT116 cells were incubated with PBPL/CNT & PGLP/CNT materials for toxicity analysis, MTS assays. The results from MTS assays indicated that no significant cellular toxicity was shown in H1299 and HCT116 cells. Furthermore, the fluorescence marker fluorescein isothiocyanate (FITC) was added to PBPL & PGLP dispersions. From the fluorescent measurements showed that the chemical functionalisation of the PBPL/CNT & PGLP/CNT conjugates with the fluorescence marker were successful. The fluorescent PBPL/CNT & PGLP/CNT conjugates could find application in medical imaging. In the next step, the GFP gene is immobilized onto PBPL/CNT conjugates by introducing electrostatic interaction. GFP-transfected cells that emitted fluorescence were imaged and counted under a fluorescence microscope. Due to the unique biocompatibility of PBPL modified CNTs, the GFP gene could be transported into H1299 cells without using antibodies. The applicability of such soluble and chemically functionalised polypeptide/CNT conjugates in biomedicine is currently investigated. We expect that this polypeptide/CNT system will be a safe and multi-functional nanomedical delivery platform and contribute to future medical therapy.

Keywords: carbon nanotube, nanotoxicology, GFP transfection, polypeptide/CNT hybrids

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Authors: Ratneev Kaur, Tajinder Kaur, Anupam Kaur

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Introduction: Polycystic Ovary Syndrome (PCOS) is a heterogenous disorder of endocrine system among women of reproductive age. PCOS is characterized by hyperandrogenism, anovulation, polycystic ovaries, hirsutism, obesity, and hyperinsulinemia. Several pathways are implicated in its etiology including the metabolic pathway of steroid hormone synthesis regulatory pathways. PCOS is an androgen excess disorder, genes operating in steroidogenesis may alter pathogenesis of PCOS. The cytochrome P450scc is a cholesterol side chain cleavage enzyme coded by CYP11A1 gene and catalyzes conversion of cholesterol to pregnenolone, the initial and rate-limiting step in steroid hormone synthesis. It is postulated that polymorphisms in this gene may play an important role in the regulation of CYP11A1 expression and leading to increased or decreased androgen production. The present study will be the first study from north India to best of our knowledge, to analyse the association of CYP11A1 (rs11632698) polymorphism in women suffering from PCOS. Methodology: The present study was approved by ethical committee of Guru Nanak Dev University in consistent with declaration of Helsinki. A total of 300 samples (150 PCOS cases and 150 controls) were recruited from Hartej hospital, for the present study. Venous blood sample (3ml) was withdrawn from women diagnosed with PCOS by doctor, according to Rotterdam 2003 criteria and from healthy age matched controls only after informed consent and detailed filled proforma. For molecular genetics analysis, blood was stored in EDTA vials. After DNA isolation by organic method, PCR-RFLP approach was used for genotyping and association analysis of rs11632698 polymorphism. Statistical analysis was done to check for significance of selected polymorphism with PCOS. Results: In 150 PCOS cases, the frequency of AA, AG and GG genotype was found to be 48%, 35%, and 13% compared to 62%, 27% and 8% in 150 controls. The major allele (A) and minor allele (G) frequency was 68% and 32% in cases and 78% and 22% in controls. Minor allele frequency was higher in cases as compared to controls, as well as the distribution of genotype was observed to be statistically significant (ᵡ²=6.525, p=0.038). Odds ratio in dominant, co-dominant and recessive models observed was 1.81 (p=0.013), 1.54 (p=0.012) and 1.77 (p=0.132) respectively. Conclusion: The present study showed statistically significant association of rs11632698 with PCOS (p=0.038) in North Indian women.

Keywords: polycystic ovary syndrome, CYP11A1, rs11632698, hyperandrogenism

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Authors: Samira Khalaji, , Fenneke Klein Jan, Kay-E. Gottschalk, Eugenia Makrantonaki, Karin Scharffetter-Kochanek

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Biological aging is a multi-dimensional process that takes place over a whole range of scales from the nanoscopic alterations within individual cells, over transformations in tissues and organs and to changes of the whole organism. On the single cell level, aging involves mutation of genes, differences in gene expression levels as well as altered posttranslational modifications of proteins. A variety of proteins is affected, including proteins of the cell cytoskeleton and migration machinery. Previous work quantified the expression of cytoskeleton proteins on the gene and protein levels in senescent and young fibroblasts. Their results show that senescent skin fibroblasts have an upregulated expression of the intermediate filament (IF) protein vimentin in contrast to actin and tubulin, which are downregulated. IFs play an important role in providing mechanical stability of cells. However, the mechanical properties of IFs depending on cellular senescence or age of the donor has not been studied so far. Hence, we employed passive microrheology on primary human dermal fibroblasts from female donors with age of 28 years (young) and 86 years (old) as model of in vivo aging and human normal dermal fibroblast from 11-year old male with CPD 17-35 (young) and CPD 58-59 (senescence) as a model of in vitro replicative senescence. In contrast to the expectations, our primary results show no significant differences in the viscoelastic properties of fibroblasts depending on age of the donor or cellular replicative senescence.

Keywords: aging, cytoskeleton, fibroblast, mechanical properties

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Authors: Renata Checiches, Thierry Coche, Nicolas F. Delahaye, Albert Linder, Fernando Ulloa Montoya, Olivier Gruselle, Karen Langfeld, An de Creus, Bart Spiessens, Vincent G. Brichard, Jamila Louahed, Frédéric F. Lehmann

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Background: The RNA-expression levels of cancer-testis antigens MAGE A3 and PRAME were determined in resected tissue from patients with primary non-small-cell lung cancer (NSCLC) and related to clinical outcome. EGFR, KRAS and BRAF mutation status was determined in a subset to investigate associations with MAGE A3 and PRAME expression. Methods: We conducted a single-centre, uncontrolled, retrospective study of 1260 tissue-bank samples from stage IA-III resected NSCLC. The prognostic value of antigen expression (qRT-PCR) was determined by hazard-ratio and Kaplan-Meier curves. Results: Thirty-seven percent (314/844) of tumours expressed MAGE-A3, 66% (723/1092) expressed PRAME and 31% (239/839) expressed both. Respective frequencies in squamous-cell tumours and adenocarcinomas were 43%/30% for MAGE A3 and 80%/44% for PRAME. No correlation with stage, tumour size or patient age was found. Overall, no prognostic value was identified for either antigen. A trend to poorer overall survival was associated with MAGE-A3 in stage IIIB and with PRAME in stage IB. EGFR and KRAS mutations were found in 10.1% (28/311) and 33.8% (97/311) of tumours, respectively. EGFR (but not KRAS) mutation status was negatively associated with PRAME expression. Conclusion: No clear prognostic value for either PRAME or MAGE A3 was observed in the overall population, although some observed trends may warrant further investigation.

Keywords: MAGE A3, PRAME, cancer-testis gene, NSCLC, survival, EGFR

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Authors: M. Hamdi, R. Rhouma, S. Belghith

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Generating random numbers are mainly used to create secret keys or random sequences. It can be carried out by various techniques. In this paper we present a very simple and efficient pseudo-random number generator (PRNG) based on chaotic maps and S-Box tables. This technique adopted two main operations one to generate chaotic values using two logistic maps and the second to transform them into binary words using random S-Box tables. The simulation analysis indicates that our PRNG possessing excellent statistical and cryptographic properties.

Keywords: Random Numbers, Chaotic map, S-box, cryptography, statistical tests

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

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

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

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Authors: Yakup Ulusu, Numan Eczacıoğlu, İsa Gökçe, Helen Waller, Jeremy H. Lakey

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Besides having the appropriate amino acid sequence to perform the function of proteins, it is important to have correct conformation after this sequence to process. To consist of this conformation depends on the amino acid sequence at the primary structure, hydrophobic interaction, chaperones and enzymes in charge of folding etc. Misfolded proteins are not functional and tend to be aggregated. Cysteine originating disulfide cross-links make stable this conformation of functional proteins. When two of the cysteine amino acids come side by side, disulfide bond is established that forms a cystine bridge. Due to this feature cysteine plays an important role on the formation of three-dimensional structure of many proteins. There are two cysteine amino acids (C44, C69) in the Tol-A-III protein. Unlike protein disulfide bonds from within his own, any non-specific cystine bridge causes a change in the three dimensional structure of the protein. Proteins can be expressed in various host cells as directly or fusion (chimeric). As a result of overproduction of the recombinant proteins, aggregation of insoluble proteins in the host cell can occur by forming a crystal structure called inclusion body. In general fusion proteins are produced for provide affinity tags to make proteins more soluble and production of some toxic proteins via fusion protein expression system like pTolT. Proteins can be modified by using a site-directed mutagenesis. By this way, creation of non-specific disulfide crosslinks can be prevented at fusion protein expression system via the present cysteine replaced by another amino acid such as serine, glycine or etc. To do this, we need; a DNA molecule that contains the gene that encodes for the target protein, required primers for mutation to be designed according to site directed mutagenesis reaction. This study was aimed to be replaced cysteine encoding codon TGT with serine encoding codon AGT. For this sense and reverse primers designed (given below) and used site-directed mutagenesis reaction. Several new copy of the template plasmid DNA has been formed with above mentioned mutagenic primers via polymerase chain reaction (PCR). PCR product consists of both the master template DNA (wild type) and the new DNA sequences containing mutations. Dpn-l endonuclease restriction enzyme which is specific for methylated DNA and cuts them to the elimination of the master template DNA. E. coli cells obtained after transformation were incubated LB medium with antibiotic. After purification of plasmid DNA from E. coli, the presence of the mutation was determined by DNA sequence analysis. Developed this new plasmid is called PtolT-δ.

Keywords: site directed mutagenesis, Escherichia coli, pTolT, protein expression

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Authors: Aizati N. A. Daud, Jorieke E. H. Bergman, Wilhelmina S. Kerstjens-Frederikse, Pieter Van Der Vlies, Eelko Hak, Rolf M. F. Berger, Henk Groen, Bob Wilffert

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Prenatal use of SRIs was previously associated with Congenital Heart Anomalies (CHA). The aim of the study is to explore whether pharmacogenetics plays a role in this teratogenicity using a gene-environment interaction study. A total of 33 case-mother dyads and 2 mother-only (children deceased) registered in EUROCAT Northern Netherlands were included in a case-only study. Five case-mother dyads and two mothers-only were exposed to SRIs (paroxetine=3, fluoxetine=2, venlafaxine=1, paroxetine and venlafaxine=1) in the first trimester of pregnancy. The remaining 28 case-mother dyads were not exposed to SRIs. Ten genes that encode the enzymes or proteins important in determining fetal exposure to SRIs or its mechanism of action were selected: CYPs (CYP1A2, CYP2C9, CYP2C19, CYP2D6), ABCB1 (placental P-glycoprotein), SLC6A4 (serotonin transporter) and serotonin receptor genes (HTR1A, HTR1B, HTR2A, and HTR3B). All included subjects were genotyped for 58 genetic variations in these ten genes. Logistic regression analyses were performed to determine the interaction odds ratio (OR) between genetic variations and SRIs exposure on the risk of CHA. Due to low phenotype frequencies of CYP450 poor metabolizers among exposed cases, the OR cannot be calculated. For ABCB1, there was no indication of changes in the risk of CHA with any of the ABCB1 SNPs in the children and their mothers. Several genetic variations of the serotonin transporter and receptors (SLC6A4 5-HTTLPR and 5-HTTVNTR, HTR1A rs1364043, HTR1B rs6296 & rs6298, HTR3B rs1176744) were associated with an increased risk of CHA, but with too limited sample size to reach statistical significance. For SLC6A4 genetic variations, the mean genetic scores of the exposed case-mothers tended to be higher than the unexposed mothers (2.5 ± 0.8 and 1.88 ± 0.7, respectively; p=0.061). For SNPs of the serotonin receptors, the mean genetic score for exposed cases (children) tended to be higher than the unexposed cases (3.4 ± 2.2, and 1.9 ± 1.6, respectively; p=0.065). This study might be among the first to explore the potential gene-environment interaction between pharmacogenetic determinants and SRIs use on the risk of CHA. With small sample sizes, it was not possible to find a significant interaction. However, there were indications for a role of serotonin receptor polymorphisms in fetuses exposed to SRIs on fetal risk of CHA which warrants further investigation.

Keywords: gene-environment interaction, heart defects, pharmacogenetics, serotonin reuptake inhibitors, teratogenicity

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Authors: Antonela Matana, Dubravka Brdar, Vesela Torlak, Marijana Popovic, Ivana Gunjaca, Ozren Polasek, Vesna Boraska Perica, Maja Barbalic, Ante Punda, Caroline Hayward, Tatijana Zemunik

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Parathyroid hormone (PTH) plays a critical role in the regulation of bone mineral metabolism and calcium homeostasis. Higher PTH levels are associated with heart failure, hypertension, coronary artery disease, cardiovascular mortality and poorer bone health. A twin study estimated that 60% of the variation in PTH concentrations is genetically determined. Only one GWAS of PTH concentration has been reported to date. Identified loci explained 4.5% of the variance in circulating PTH, suggesting that additional genetic variants remain undiscovered. Therefore, the aim of this study was to identify novel genetic variants associated with PTH levels in a general population. We have performed a GWAS meta-analysis on 2596 individuals originating from three Croatian cohorts: City of Split and the Islands of Korčula and Vis, within a large-scale project of “10,001 Dalmatians”. A total of 7 411 206 variants, imputed using the 1000 Genomes reference panel, with minor allele frequency ≥ 1% and Rsq ≥ 0.5 were analyzed for the association. GWAS within each data set was performed under an additive model, controlling for age, gender and relatedness. Meta-analysis was conducted using the inverse-variance fixed-effects method. Furthermore, to identify sex-specific effects, we have conducted GWAS meta-analyses analyzing males and females separately. In addition, we have performed biological pathway analysis. Four SNPs, representing one locus, reached genome-wide significance. The most significant SNP was rs11099476 on chromosome 4 (P=1.15x10-8), which explained 1.14 % of the variance in PTH. The SNP is located near the protein-coding gene RASGEF1B. Additionally, we detected suggestive association with SNPs, rs77178854 located on chromosome 2 in the DPP10 gene (P=2.46x10-7) and rs481121 located on chromosome 1 (P=3.58x10-7) near the GRIK1 gene. One of the top hits detected in the main meta-analysis, intron variant rs77178854 located within DPP10 gene, reached genome-wide significance in females (P=2.21x10-9). No single locus was identified in the meta-analysis in males. Fifteen biological pathways were functionally enriched at a P<0.01, including muscle contraction, ion homeostasis and cardiac conduction as the most significant pathways. RASGEF1B is the guanine nucleotide exchange factor, known to be associated with height, bone density, and hip. DPP10 encodes a membrane protein that is a member of the serine proteases family, which binds specific voltage-gated potassium channels and alters their expression and biophysical properties. In conclusion, we identified 2 novel loci associated with PTH levels in a general population, providing us with further insights into the genetics of this complex trait.

Keywords: general population, genome-wide association analysis, parathyroid hormone, single nucleotide polymorphisms.

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Authors: Vasundhara Arora, Anupam Jhobta, Suresh Thakur, Sanjiv Sharma

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Aims and Objectives: Intracranial tuberculosis (TB) is one of the most devastating manifestations of TB and a challenging public health issue of considerable importance and magnitude world over. This study elaborates on the imaging spectrum of neurotuberculosis on magnetic resonance imaging (MRI) in 29 clinically suspected cases from a tertiary care hospital. Materials and Methods: The prospective hospital based evaluation of MR imaging features of neuro-tuberculosis in 29 clinically suspected cases was carried out in Department of Radio-diagnosis, Indira Gandhi Medical Hospital from July 2017 to August 2018. MR Images were obtained on a 1.5 T Magnetom Avanto machine and were analyzed to identify any abnormal meningeal enhancement or parenchymal lesions. Microbiological and Biochemical CSF analysis was performed in radio-logically suspected cases and the results were compared with the imaging data. Clinical follow up of the patients started on anti-tuberculous treatment was done to evaluate the response to treatment and clinical outcome. Results: Age range of patients in the study was between 1 year to 73 years. The mean age of presentation was 11.5 years. No significant difference in the distribution of cerebral tuberculosis was noted among the two genders. Imaging findings of neuro-tuberculosis obtained were varied and non specific ranging from lepto-meningeal enhancement, cerebritis to space occupying lesions such as tuberculomas and tubercular abscesses. Complications presenting as hydrocephalus (n= 7) and infarcts (n=9) was noted in few of these patients. 29 patients showed radiological suspicion of CNS tuberculosis with meningitis alone observed in 11 cases, tuberculomas alone were observed in 4 cases, meningitis with parenchymal tuberculomas in 11 cases. Tubercular abscess and cerebritis were observed in one case each. Tuberculous arachnoiditis was noted in one patient. Gene expert positivity was obtained in 11 out of 29 radiologically suspected patients; none of the patients showed culture positivity. Meningeal form of the disease alone showed higher positivity rate of gene Xpert (n=5) followed by combination of meningeal and parenchymal forms of disease (n=4). The parenchymal manifestation of disease alone showed least positivity rates (n= 3) with gene xpert testing. All 29 patients were started on anti tubercular treatment based on radiological suspicion of the disease with clinical improvement observed in 27 treated patients. Conclusions: In our study, higher incidence of neuro- tuberculosis was noted in paediatric population with predominance of the meningeal form of the disease. Gene Xpert positivity obtained was low due to paucibacillary nature of cerebrospinal fluid (CSF) with even lower positivity of CSF samples in parenchymal form of the manifestation. MRI showed high accuracy in detecting CNS lesions in neuro-tuberculosis. Hence, it can be concluded that MRI plays a crucial role in the diagnosis because of its inherent sensitivity and specificity and is an indispensible imaging modality. It caters to the need of early diagnosis owing to poor sensitivity of microbiological tests more so in the parenchymal manifestation of the disease.

Keywords: neurotuberculosis, tubercular abscess, tuberculoma, tuberculous meningitis

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Authors: Tiisetso Mpai, Sanjay K. Jaiswal, Felix D. Dakora

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The South African Cape fynbos biome is a global biodiversity hotspot. This biome contains a diversity of endemic shrub legumes, including Polhillia, Wiborgia, and Wiborgiella species, which are important for ecotourism as well as for improving soil fertility status. This is due to their proven N₂-fixing abilities when in association with compatible soil bacteria. In fact, Polhillia, Wiborgia, and Wiborgiella species have been reported to derive over 61% of their needed nitrogen through biological nitrogen fixation and to exhibit acid and alkaline phosphatase activity in their rhizospheres. Thus, their interactions with soil microbes may explain their survival mechanisms under the continued summer droughts and acidic, nutrient-poor soils in this region. However, information regarding their rhizosphere microbiome is still unavailable, yet it is important for Fynbos biodiversity management. Therefore, the aim of this study was to assess the microbial community structures associated with rhizosphere soils of Polhillia pallens, Polhillia brevicalyx, Wiborgia obcordata, Wiborgia sericea, and Wiborgiella sessilifolia growing at different locations of the South African Cape fynbos, during the wet and dry seasons. The hypothesis is that the microbial communities in these legume rhizospheres are the same type and are not affected by the growing season due to the restricted habitat of these wild fynbos legumes. To obtain the results, DNA was extracted from 0.5 g of each rhizosphere soil using PowerSoil™ DNA Isolation Kit, and sequences were obtained using the 16S rDNA Miseq Illumina technology. The results showed that in both seasons, bacteria were the most abundant microbial taxa in the rhizosphere soils of all five legume species, with Actinobacteria showing the highest number of sequences (about 30%). However, over 19.91% of the inhabitants in all five legume rhizospheres were unclassified. In terms of genera, Mycobacterium and Conexibacter were common in rhizosphere soils of all legumes in both seasons except for W. obcordata soils sampled during the dry season, which had Dehalogenimonas as the major inhabitant (6.08%). In conclusion, plant species and season were found to be the main drivers of microbial community structure in Cape fynbos, with the wet season being more dominant in shaping microbial diversity relative to the dry season. Wiborgia obcordata had a greater influence on microbial community structure than the other four legume species.

Keywords: 16S rDNA, Cape fynbos, endemic legumes, microbiome, rhizosphere

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Authors: Bhuvanesh Baniya

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Alzheimer disease is illnesses that are responsible for neuronal cell death and resulting in lifelong cognitive problems. Due to their unclear mechanism, there are no effective drugs available for the treatment. For a long time, herbal drugs have been used as a role model in the field of the drug discovery process. Holy basil in the Indian medicinal system (Ayurveda) is used for several neuronal disorders like insomnia and memory loss for decades. This study aims to identify active components of holy basil as potential inhibitors for the treatment of Alzheimer disease. To fulfill this objective, the Network pharmacology approach, gene ontology, pharmacokinetics analysis, molecular docking, and molecular dynamics simulation (MDS) studies were performed. A total of 7 active components in holy basil, 12 predicted neurodegenerative targets of holy basil, and 8063 Alzheimer-related targets were identified from different databases. The network analysis showed that the top ten targets APP, EGFR, MAPK1, ESR1, HSPA4, PRKCD, MAPK3, ABL1, JUN, and GSK3B were found as significant target related to Alzheimer disease. On the basis of gene ontology and topology analysis results, APP was found as a significant target related to Alzheimer’s disease pathways. Further, the molecular docking results to found that various compounds showed the best binding affinities. Further, MDS top results suggested could be used as potential inhibitors against APP protein and could be useful for the treatment of Alzheimer’s disease.

Keywords: holy basil, network pharmacology, neurodegeneration, active phytochemicals, molecular docking and simulation

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Authors: Farideh K. Khosroushahi, Serkan Erdal, Mucip Geni̇şel

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Soil salinity is one of the major abiotic stress factors that restricts arable land and reduces crop productivity worldwide. High salt concentration adversely affects plant growth and development inducing water deficit, ionic toxicity, nutrient imbalance, and lead to oxidative stress. Although the stimulating role of mammalian sex hormones on various biological and biochemical processes under normal and stress condition have been proven, there is no study regarding with these hormone's effect on modulation of the alternative respiration pathway and AOX gene expression. In this study, changes in alternative respiration pathway in leaves of maize seedlings under salinity and the possible modulating effect of estrogen on these changes were investigated. Maize seedlings were grown in a hydroponic media for 11 days and then were exposed to salt stress for 3 days after being sprayed estrogen. The data obtained from oxygen consumption revealed that salt stress elevated cellular respiration value in the leaves. In addition, a marked increase was observed at alternative respiration level in salt-stressed seedlings. Compared to salt application alone, supplementation with estrogen resulted in a significant rise in alternative oxidase (AOX) activities. Similarly, while salt stress caused to rise in expressions of AOX gene compared to control seedlings, estrogen application resulted in further activation of these genes’ expression compared to stressed-seedlings alone. These data revealed that mitigating role of estrogen against the detrimental effects of salt stress is linked to modulation of alternative respiration pathway.

Keywords: alternative oxidase, estrogen, Ssalt stress, AOX, maize

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Authors: A. Bhandari, S. Mukherjee

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Two frames in a Hilbert space are called woven or weaving if all possible merge combinations between them generate frames of the Hilbert space with uniform frame bounds. Weaving frames are powerful tools in wireless sensor networks which require distributed data processing. Considering the practical applications, this article deals with finite woven frames. We provide methods of constructing finite woven frames, in particular, bounded linear operators are used to construct woven frames from a given frame. Several examples are discussed. We also introduce the notion of woven frame sequences and characterize them through the concepts of gaps and angles between spaces.

Keywords: frames, woven frames, gap, angle

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Authors: V. Cesaroni, C. Girometta, A. Bernicchia, M. Brusoni, F. Corana, R. M. Baiguera, C. M. Cusaro, M. L. Guglielminetti, B. Mannucci, H. Kawagishi, C. Perini, A. M. Picco, P. Rossi, E. Salerni, E. Savino

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Fungi of the genus Hericium contain various compounds with antibacterial activity, cytotoxic effect on cancer cells and bioactive molecules. Some of the active metabolites stimulate the synthesis of the Nerve Growth Factor (NGF). Recently, the effect of dietary supplement based on Hericium erinaceus on recognition memory and on hippocampal mossy fiber-CA3 neurotransmission was published. The aim of this study was to investigate the presence of Hericium species on Italian territory in order to isolate the strains for further studies and applications. The first step was to collect Hericium sporophores in Tuscany: H. alpestre Pers., H. coralloides (Scop.) Pers. and H. erinaceus (Bull.) Pers. were the species present. The strains of H. alpestre (H.a.1), H. coralloides (H.c.1) and H. erinaceus (H.e.1 & H.e.2) have been isolated in pure culture and preserved in the collection of the University of Pavia (MicUNIPV). The DNA sequences obtained from the strains were compared to other sequences found in international databases. Therefore, it was possible to construct a phylogenetic tree that highlights the clear separation in clades of the sequences and the molecular identification of our strains with the species of Hericium considered. The second step was to cultivate indoor and outdoor H. erinaceus in order to obtain as many sporophores as possible for further chemical analysis. All the procedures for H. erinaceus cultivation have been followed. Among the available recipes for indoor H. erinaceus cultivation, it was used a substrate formulation contained 70% oak sawdust, 20% rice bran, 10% wheat straw, 1% CaCO3 and 1% sucrose. The bioactive compounds present in the mycelia and in the sporophores of H. erinaceus were chemically analyzed in collaboration with the Centro Grandi Strumenti of the University of Pavia using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). The materials to be analyzed were previously freeze-dried and then extracted with an alcoholic procedure. Preliminary chromatographic analysis revealed the presence of potentially bioactive and structurally different secondary metabolites such as polysaccharides, erinacins, ericenones, steroids and other terpenoids. Ericenones C and D (in sporophores) and erinacin A (in mycelium) have been identified by comparison with the respective standards. These molecules are known to have effects on the Central Nervous System (CNS) cells, which is the main objective of our studies. Thanks to the high sensitivity in the detection of bioactive compounds of H. erinaceus, it will be possible to use the To obtain lyophilized mycelium and the respective culture broth, 4 small pieces (about 5 mm2) of the respective H.e.1 or H.c.1 strains, taken from the margin of growing cultures (MEA), were inoculated into 1 liter of 2% ME (malt extract, Biokar Diagnostics). The static liquid cultures were kept at 24 °C in the dark chamber and fungi grew for one month. 10 replicates for each strain have been done. The method proposed as an analytical screening protocol to determine the optimal growth conditions of the fungus and to improve the production chain of H. erinaceus. These results encourage to carry out chemical analyzes also on H. alpestre and H. coralloides in order to evaluate the presence of bioactive compounds in these two species.

Keywords: Hericium species, Hercium erinaceus bioactive compounds, medicinal mushrooms, mushroom cultivation

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Authors: Rozeena Shaikh, Syed M Shahid, Jamil Ahmad, Qaisar Mansoor, Muhammad Ismail, Abid Azhar

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Introduction: Diabetes mellitus (DM) is a prevalent non-communicable disease worldwide. In most high-income countries as well as middle-income and low- income countries. DM is among the top causes of deaths. DM may lead to many vascular complications like hypertension, nephropathy, retinopathy, neuropathy, and foot. Diabetic nephropathy (DN) characterized by persistent albuminuria is a leading cause of end stage renal failure (ESRF). Pathogenesis of diabetic nephropathy is implicated by the polymorphisms in genes encoding the components of reninangiotensin- aldosteron system (RAAS) which include angiotensinogen (AGT), angiotensin-II receptor and particularly angiotensin converting enzyme (ACE) gene. Method: Study subjects include 110 control, 110 patients with DM without hypertension, 110 patients with DM with hypertension and 110 patients with DN. Blood samples were collected for Biochemical analysis and PCR and sequencing for the specific region of both genes. Results: The frequency of DD genotype and D allele of ACE (I/D) was significantly (p<0.05) high in DM normotensive, DM hypertensive and DN patients when compared to control. The ACE G2350A genotypes and allele frequencies were significantly different (p<0.05) in DM hypertensive patients as compared to control and DN, while no difference was observed between DM normotensive and DN when compared to control. The genotypes and alleles of AGT (M268T) polymorphism were significantly different (p<0.05) in DM normotensive, DM hypertensive and DN when compared to control. Conclusion: The DD genotype and D allele of ACE (I/D), GG genotype and G allele of ACE (G2350A) and the TT genotype and T allele of AGT (M268T) polymorphism have shown a significant difference in genotype and allele frequencies between controls and patients.

Keywords: genetic variations, ACE, AGT, diabetes mellitus, diabetic nephropathy, Pakistan

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Authors: L. Lamola, E. Manolas, A. Krause

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Background: The incidence of childhood cancer incidence is increasing gradually in low-middle income countries, such as South Africa. Globally, there is an extensive range of familial- and hereditary-cancer syndromes, where underlying germline variants increase the likelihood of developing cancer in childhood. Next-Generation Sequencing (NGS) technologies have been key in determining the occurrence and genetic contribution of germline variants to paediatric cancer development. We aimed to design and evaluate a candidate gene panel specific to inherited cancer-predisposing genes to provide a comprehensive insight into the contribution of germline variants to childhood cancer. Methods: 32 paediatric patients (aged 0-18 years) diagnosed with a malignant tumour were recruited, and biological samples were obtained. After quality control, DNA was sequenced using an ion Ampliseq 50 candidate gene panel design and Ion Torrent S5 technologies. Sequencing variants were called using Ion Torrent Suite software and were subsequently annotated using Ion Reporter and Ensembl's VEP. High priority variants were manually analysed using tools such as MutationTaster, SIFT-INDEL and VarSome. Putative identified candidates were validated via Sanger Sequencing. Results: The patients studied had a variety of cancers, the most common being nephroblastoma (13), followed by osteosarcoma (4) and astrocytoma (3). We identified 10 pathogenic / likely pathogenic variants in 10 patients, most of which were novel. Conclusions: According to the literature, we expected ~10% of our patient population to harbour pathogenic or likely pathogenic germline variants, however, we reported about 3 times (~30%) more than we expected. Majority of the identified variants are novel; this may be because this is the first study of its kind in an understudied South African population.

Keywords: Africa, genetics, germline-variants, paediatric-cancer

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Authors: Abiodun Olayinka, Daniel Dzidzienyo, Pangirayi Tongoona, Samuel Offei, Edwige Gaby Nkouaya Mbanjo, Chiedozie Egesi, Ismail Yusuf Rabbi

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Cassava (Manihot esculenta Crantz) is a major source of starch for various industrial applications. However, the traditional cultivation and harvesting methods of cassava are labour-intensive and inefficient, limiting the supply of fresh cassava roots for industrial starch production. To achieve improved productivity and quality of fresh cassava roots through mechanized cultivation, cassava cultivars with compact plant architecture and moderate plant height are needed. Plant architecture-related traits, such as plant height, harvest index, stem diameter, branching angle, and lodging tolerance, are critical for crop productivity and suitability for mechanized cultivation. However, the genetics of cassava plant architecture remain poorly understood. This study aimed to identify the genetic bases of the relationships between plant architecture traits and productivity-related traits, particularly starch content. A panel of 453 clones developed at the International Institute of Tropical Agriculture, Nigeria, was genotyped and phenotyped for 18 plant architecture and productivity-related traits at four locations in Nigeria. A genome-wide association study (GWAS) was conducted using the phenotypic data from a panel of 453 clones and 61,238 high-quality Diversity Arrays Technology sequencing (DArTseq) derived Single Nucleotide Polymorphism (SNP) markers that are evenly distributed across the cassava genome. Five significant associations between ten SNPs and three plant architecture component traits were identified through GWAS. We found five SNPs on chromosomes 6 and 16 that were significantly associated with shoot weight, harvest index, and total yield through genome-wide association mapping. We also discovered an essential candidate gene that is co-located with peak SNPs linked to these traits in M. esculenta. A review of the cassava reference genome v7.1 revealed that the SNP on chromosome 6 is in proximity to Manes.06G101600.1, a gene that regulates endodermal differentiation and root development in plants. The findings of this study provide insights into the genetic basis of plant architecture and yield in cassava. Cassava breeders could leverage this knowledge to optimize plant architecture and yield in cassava through marker-assisted selection and targeted manipulation of the candidate gene.

Keywords: Manihot esculenta Crantz, plant architecture, DArtseq, SNP markers, genome-wide association study

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