Search results for: high-throughput sequencing

Authors: Vanessa De Carvalho, Chad MacPherson, Julien Tremblay, Julie Champagne, Stephanie-Anne Girard

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Background: The interactions between bacteria and fungi in the human vaginal microbiome are fundamental to the concept of health and disease. The means by which the microbiota and mycobiota interact is still poorly understood and further studies are necessary to properly characterize this complex ecosystem. The aim of this study was to select a DNA extraction method capable of recovering high qualities of fungal and bacterial DNA from a single vaginal swab. Methods: 11 female volunteers ( ≥ 20 to < 55 years old) self-collected vaginal swabs in triplicates. Three commercial extraction kits: Masterpure Yeast Purification kit (Epicenter), PureLink™ Microbiome DNA Purification kit (Invitrogen), and Quick-DNA™ Fecal/Soil Microbe Miniprep kit (Zymo) were evaluated on the ability to recover fungal and bacterial DNA simultaneously. The extraction kits were compared on the basis of recovery, yield, purity, and the community richness of bacterial (16S rRNA - V3-V4 region) and fungal (ITS1) microbiota composition by Illumina MiSeq amplicon sequencing. Results: Recovery of bacterial DNA was achieved with all three kits while fungal DNA was only consistently recovered with Masterpure Yeast Purification kit (yield and purity). Overall, all kits displayed similar microbiota profiles for the top 20 OTUs; however, Quick-DNA™ Fecal/Soil Microbe Miniprep kit (Zymo) showed more species richness than the other two kits. Conclusion: In the present study, Masterpure Yeast purification kit proved to be a good candidate for purification of high quality fungal and bacterial DNA simultaneously. These findings have potential benefits that could be applied in future vaginal microbiome research. Whilst the use of a single extraction method would lessen the burden of multiple swab sampling, decrease laboratory workload and off-set costs associated with multiple DNA extractions, thoughtful consideration must be taken when selecting an extraction kit depending on the desired downstream application.

Keywords: bacterial vaginosis, DNA extraction, microbiota, mycobiota, vagina, vulvovaginal candidiasis, women’s health

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Authors: Sohan Sengupta, Arnab Pramanik, Abhrajyoti Ghosh, Maitree Bhattacharyya

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Newly emerged phyto-pathogens and multi drug resistance have been threating the world for last few decades. Actinomycetes, the most endowed group of microorganisms isolated from unexplored regions of the world may be the ultimate solution to these problems. Thus the aim of this study was to isolate several bioactive actinomycetes strains capable of producing antimicrobial secondary metabolite from Sundarbans, the only mangrove tiger land of the world. Fifty four actinomycetes were isolated and analyzed for antimicrobial activity against fifteen test organisms including three phytopathogens. Nine morphologically distinct and biologically active isolates were subjected to polyphasic identification study. 16s rDNA sequencing indicated eight isolates to reveal maximum similarity to the genus streptomyces, whereas one isolate presented only 93.57% similarity with Streptomyces albogriseolus NRRL B-1305T. Seventy-one carbon sources and twenty-three chemical sources utilization assay revealed their metabolic relatedness. Among these nine isolates three specific strains were found to have notably higher degree of antimicrobial potential effective in a broader range including phyto-pathogenic fungus. PCR base whole genome screen for PKS and NRPS genes, confirmed the occurrence of bio-synthetic gene cluster in some of the isolates for novel antibiotic production. Finally the strain SMS_SU21, which showed antimicrobial activity with MIC value of 0.05 mg ml-1and antioxidant activity with IC50 value of 0.242±0.33 mg ml-1 was detected to be the most potential one. True prospective of this strain was evaluated utilizing GC-MS and the bioactive compound responsible for antimicrobial activity was purified and characterized. Rare bioactive actinomycetes were isolated from unexplored heritage site. Diversity of the biosynthetic gene cluster for antimicrobial compound production has also been evaluated. Antimicrobial compound SU21-C has been identified and purified which is active against a broad range of pathogens.

Keywords: actinomycetes, sundarbans, antimicrobial, pks nrps, phyto-pathogens, GC-MS

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Authors: John Masani Nduko, Joseph Wafula Matofari

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Many spontaneously fermented milk products are produced in Kenya, where they are integral to the human diet and play a central role in enhancing food security and income generation via small-scale enterprises. Fermentation enhances product properties such as taste, aroma, shelf-life, safety, texture, and nutritional value. Some of these products have demonstrated therapeutic and probiotic effects although recent reports have linked some to death, biotoxin infections, and esophageal cancer. These products are mostly processed from poor quality raw materials under unhygienic conditions resulting to inconsistent product quality and limited shelf-lives. Though very popular, research on their processing technologies is low, and none of the products has been produced under controlled conditions using starter cultures. To modernize the processing technologies for these products, our study aims at describing the microbiology and biochemistry of a representative Kenyan spontaneously fermented milk product, Mursik using modern biotechnology (DNA sequencing) and their chemical composition. Moreover, co-creation processes reflecting stakeholders’ experiences on traditional fermented milk production technologies and utilization, ideals and senses of value, which will allow the generation of products based on common ground for rapid progress will be discussed. Knowledge of the value of clean starting raw material will be emphasized, the need for the definition of fermentation parameters highlighted, and standard equipment employment to attain controlled fermentation discussed. This presentation will review the available information regarding traditional fermented milk (Mursik) and highlight our current research work on the application of molecular approaches (metagenomics) for the valorization of Mursik production process through starter culture/ probiotic strains isolation and identification, and quality and safety aspects of the product. The importance of the research and future research areas on the same subject will also be highlighted.

Keywords: lactic acid bacteria, high throughput biotechnology, spontaneous fermentation, Mursik

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Authors: Nadia Naseem, Farwa Batool, Nasir Mehmood, AbdulHannan Nagi

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Background: The incidence and mortality of breast cancer vary significantly in geographically distinct populations. In Pakistan, breast cancer has shown an increase in incidence in young females and is characterized by more aggressive behavior. The tumor suppressor TP53 gene is a crucial genetic factor that plays a significant role in breast carcinogenesis. This study investigated the TP53 mutations in molecular subtypes of both nodes negative and positive breast cancer in young Pakistani patients. Material and Methods: p53, Estrogen Receptor (ER), Progesterone Receptor (PR), Her-2 neu and Ki 67 expressions were analyzed immunohistochemically in a series of 75 node negative (A) and 75 node positive (B) young (aged: 19-40 years) breast cancer patients diagnosed between 2014 to 2017 at two leading hospitals of Punjab, Pakistan. Tumor tissue specimens and peripheral blood samples were examined for TP53 mutations by direct sequencing of the gene (exons 4-9). The relation of TP53 mutations to these markers and clinicopathological data was investigated. Results: Mean age of the patients was 32.4 + 9.1 SD. Invasive breast carcinoma was the most frequent histological variant (A=92%, B=94.6%). Grade 3 carcinoma was the commonest grade (A=72%, B=81.3%). Triple negative cases (ER-, PR-, Her-2) formed most of the molecular subtypes (A=44%, B=50.6%). A total of 17.2% (A: 6.6%, B: 10.6%) patients showed TP53 mutations. Mutations were significantly more frequent in triple negative cases (A: 74.8%, B: 62.2%) compared to HER2-positive patients (P < 0.0001). In the multivariate analysis of the whole patient group, the independent prognosticator were triple negative cases (P=0.021), TP53 overexpression by IHC (P=0.001) and advanced-stage disease (P=0.007). No statistically significant correlation between TP53 mutations and clinicopathological parameters was found (P < 0.05). Conclusions: It is concluded that TP53 mutations are infrequently present in breast carcinoma of young Pakistani population and there was no significant correlation between p53 mutation and early onset disease. Immunohistochemically detected TP53 expression in our resource-constrained to set up can be beneficial in predicting mutations at the younger age in our population.

Keywords: immunohistochemistry (IHC), invasive breast carcinoma (IBC), Pakistan, TP53

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Authors: Zeina Bourhane, Anders Lanzen, Christine Cagnon, Olfa Ben Said, Cristiana Cravo-Laureau, Robert Duran

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The anthropogenic pressure in coastal areas increases dramatically with the exploitation of environmental resources. Biomonitoring coastal areas are crucial to determine the impact of pollutants on bacterial communities in soils and sediments since they provide important ecosystem services. However, relevant biomonitoring tools allowing fast determination of the ecological status are yet to be defined. Microbial ecology approaches provide useful information for developing such microbial monitoring tools reporting on the effect of environmental stressors. Chemical and microbial molecular approaches were combined in order to determine microbial bioindicators for assessing the ecological status of soil and river ecosystems around the Ichkeul Lake (Tunisia), an area highly impacted by human activities. Samples were collected along soil/river/lake continuums in three stations around the Ichkeul Lake influenced by different human activities at two seasons (summer and winter). Contaminant pressure indexes (PI), including PAHs (Polycyclic aromatic hydrocarbons), alkanes, and OCPs (Organochlorine pesticides) contents, showed significant differences in the contamination level between the stations with seasonal variation. Bacterial communities were characterized by 16S ribosomal RNAs (rRNA) gene metabarcoding. Although microgAMBI indexes, determined from the sequencing data, were in accordance with contaminant contents, they were not sufficient to fully explain the PI. Therefore, further microbial indicators are still to be defined. The comparison of bacterial communities revealed the specific microbial assemblage for soil, river, and lake sediments, which were significantly correlated with contaminant contents and PI. Such observation offers the possibility to define a relevant set of bioindicators for reporting the effects of human activities on the microbial community structure. Such bioindicators might constitute useful monitoring tools for the management of microbial communities in coastal areas.

Keywords: bacterial communities, biomonitoring, contamination, human impacts, microbial bioindicators

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Authors: A. Berraf-Tebbal, Z. Bouznad, , A.J.L. Phillips

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Phaeomoniella is a fungus genus in the mitosporic ascomycota which includes Phaeomoniella chlamydospora specie associated with two declining diseases on grapevine (Vitis vinifera) namely Petri disease and esca. Recent studies have shown that several Phaeomoniella species also cause disease on many other woody crops, such as forest trees and woody ornamentals. Two new species, Phaeomoniella zymoides and Phaeomoniella pinifoliorum H.B. Lee, J.Y. Park, R.C. Summerbell et H.S. Jung, were isolated from the needle surface of Pinus densiflora Sieb. et Zucc. in Korea. The identification of species in Phaeomoniella genus can be a difficult task if based solely on morphological and cultural characters. In this respect, the application of molecular methods, particularly PCR-based techniques, may provide an important contribution. MSP-PCR (microsatellite primed-PCR) fingerprinting has proven useful in the molecular typing of fungal strains. The high discriminatory potential of this method is particularly useful when dealing with closely related or cryptic species. In the present study, the application of PCR fingerprinting was performed using the micro satellite primer M13 for the purpose of species identification and strain typing of 84 Phaeomoniella -like isolates collected from grapevines with typical symptoms of dieback. The bands produced by MSP-PCR profiles divided the strains into 3 clusters and 5 singletons with a reproducibility level of 80%. Representative isolates from each group and, when possible, isolates from Eutypa dieback and esca symptoms were selected for sequencing of the ITS region. The ITS sequences for the 16 isolates selected from the MSP-PCR profiles were combined and aligned with sequences of 18 isolates retrieved from GenBank, representing a selection of all known Phaeomoniella species. DNA sequences were compared with those available in GenBank using Neighbor-joining (NJ) and Maximum-parsimony (MP) analyses. The phylogenetic trees of the ITS region revealed that the Phaeomoniella isolates clustered with Phaeomoniella chlamydospora reference sequences with a bootstrap support of 100 %. The complexity of the pathosystems vine-trunk diseases shows clearly the need to identify unambiguously the fungal component in order to allow a better understanding of the etiology of these diseases and justify the establishment of control strategies against these fungal agents.

Keywords: Genotyping, MSP-PCR, ITS, phylogeny, trunk diseases

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Authors: Anukwu John Uchenn, Nwamba, Helen Ogochukwu, Achikanu Cosmas, Chiaha Emiliana

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The continuous decline in biodiversity is worrisome. This decline is predominant in fish population. Although taxonomy has been an age-long field of science, there are still undiscovered members of species and new species are waiting to be uncovered. The failure of traditional taxonomic method to address this issue has resulted to the adoption of a molecular approach-DNA barcoding. It was proposed that DNA barcoding using mitochondrion cytochrome oxidase subunit I (COI) gene has the capability to serve as a barcode for fishes. The aim of this study was to use DNA barcoding in the identification of fish species in Imo River, Rivers State. A total of eight (8) fish samples were collected and used for this study. Quick DNA Miniprep Plus kit (D4068, Zymo Research) was used for the DNA extraction which was followed by PCR amplification and sequencing. BLAST result shows correlation between the sequence queried and the biological sequences with the NCBI database. The names of the samples, percentage ID, predicted organisms and GenBank Accession numbers were clearly identified. A total of 16 sequences (all > 600bp) belonging to 7 species, 7 genera, 7 families and 4 orders were validated and submitted to the NCBI database. Each nucleotide peak was represented by a single colour with various percentage occurrences. Four (50%) out of the 8 original samples analyzed corresponded with the predicted organisms from BLAST result. Pairwise sequence alignment showed different consensus positions and a total of 11 mutations found in Chrysichthys nigrodigitatus (n=4), Oreochromis niloticus (n=2) and Clarias gariepinus (n=5). The whole mutations were substitution (transition and transversion) with no deletion and insertion. However, the transition mutation (n=6) was more in number compared to the transversion (n=5) mutation. There were a total of 834 positions in the final dataset. This work will facilitate more research in other keys areas such as identification of mislabeled fish products, illegal trading of endangered species and effective tracking of fish biodiversity.

Keywords: DNA barcoding, Imo river, phylogenetic tree, pairwise DNA alignment

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Authors: Irena Maus, Katharina Gabriella Cibis, Andreas Bremges, Yvonne Stolze, Geizecler Tomazetto, Daniel Wibberg, Helmut König, Alfred Pühler, Andreas Schlüter

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Within the agricultural sector, the production of biogas from organic substrates represents an economically attractive technology to generate bioenergy. Complex consortia of microorganisms are responsible for biomass decomposition and biogas production. Recently, species belonging to the phylum Thermotogae were detected in thermophilic biogas-production plants utilizing renewable primary products for biomethanation. To analyze adaptive genome features of representative Thermotogae strains, Defluviitoga tunisiensis L3 was isolated from a rural thermophilic biogas plant (54°C) and completely sequenced on an Illumina MiSeq system. Sequencing and assembly of the D. tunisiensis L3 genome yielded a circular chromosome with a size of 2,053,097 bp and a mean GC content of 31.38%. Functional annotation of the complete genome sequence revealed that the thermophilic strain L3 encodes several genes predicted to facilitate growth of this microorganism on arabinose, galactose, maltose, mannose, fructose, raffinose, ribose, cellobiose, lactose, xylose, xylan, lactate and mannitol. Acetate, hydrogen (H2) and carbon dioxide (CO2) are supposed to be end products of the fermentation process. The latter gene products are metabolites for methanogenic archaea, the key players in the final step of the anaerobic digestion process. To determine the degree of relatedness of dominant biogas community members within selected digester systems to D. tunisiensis L3, metagenome sequences from corresponding communities were mapped on the L3 genome. These fragment recruitments revealed that metagenome reads originating from a thermophilic biogas plant covered 95% of D. tunisiensis L3 genome sequence. In conclusion, availability of the D. tunisiensis L3 genome sequence and insights into its metabolic capabilities provide the basis for biotechnological exploitation of genome features involved in thermophilic fermentation processes utilizing renewable primary products.

Keywords: genome sequence, thermophilic biogas plant, Thermotogae, Defluviitoga tunisiensis

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Authors: S. Sreeja, Radhakrishnan R. Nair, Noble Gracious, Sreeja S. Nair, M. Radhakrishna Pillai

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Background: Tacrolimus is a potent immunosuppressant clinically used for the long term treatment of antirejection of transplanted organs in liver and kidney transplant recipients though dose optimization is poorly managed. However, So far no study has been carried out on the South Indian kidney transplant patients. The objective of this study is to evaluate the potential influence of a functional polymorphism in CYP3A5*3 gene on tacrolimus physiological availability/dose ratio in South Indian renal transplant patients. Materials and Methods: Twenty five renal transplant recipients receiving tacrolimus were enrolled in this study. Their body weight, drug dosage, and therapeutic concentration of Tacrolimus were observed. All patients were on standard immunosuppressive regime of Tacrolimus-Mycophenolate mofetil along with steroids on a starting dose of Tac 0.1 mg/kg/day. CYP3A5 genotyping was performed by PCR followed with RFLP. Conformation of RFLP analysis and variation in the nucleotide sequence of CYP3A5*3 gene were determined by direct sequencing using a validated automated generic analyzer. Results: A significant association was found between tacrolimus per dose/kg/d and CYP3A5 gene (A6986G) polymorphism in the study population. The CYP3A5 *1/*1, *1/*3 and *3/*3 genotypes were detected in 5 (20 %), 5 (20 %) and 15 (60 %) of the 25 graft recipients, respectively. CYP3A5*3 genotypes were found to be a good predictor of tacrolimus Concentration/Dose ratio in kidney transplant recipients. Significantly higher L/D was observed among non-expressors 9.483 ng/mL(4.5- 14.1) as compared with the expressors 5.154 ng/mL (4.42-6.5 ) of CYP3A5. Acute rejection episodes were significantly higher for CYP3A5*1 homozygotes compared to patients with CYP3A5*1/*3 and CYP3A5*3/*3 genotypes (40 % versus 20 % and 13 %, respectively ). The dose normalized TAC concentration (ng/ml/mg/kg) was significantly lower in patients having CYP3A5*1/*3 polymorphism. Conclusion: This is the first study to extensively determine the effect of CYP3A5*3 genetic polymorphism on tacrolimus pharmacokinetics in South Indian renal transplant recipients and also shows that majority of our patients carry mutant allele A6986G in CYP3A5*3 gene. Identification of CYP3A5 polymorphism prior to transplantation could contribute to evaluate the appropriate initial dosage of tacrolimus for each patient.

Keywords: kidney transplant patients, CYP3A5 genotype, tacrolimus, RFLP

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Authors: Rofida Gamal, Mostafa Mohammed, Mariam Adel, Marwa Gamal, Marwa kamal, Ayat Saber, Maha Mamdouh, Amira Emad, Mai Ramadan

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Lynch Syndrome is an inherited genetic condition associated with an increased risk of colorectal and other cancers. Detecting Lynch Syndrome in individuals is crucial for early intervention and preventive measures. This study proposes a computational pipeline for Lynch Syndrome detection by integrating alignment, variant calling, and annotation. The pipeline leverages popular tools such as FastQC, Trimmomatic, BWA, bcftools, and ANNOVAR to process the input FASTQ file, perform quality trimming, align reads to the reference genome, call variants, and annotate them. It is believed that the computational pipeline was applied to a dataset of Lynch Syndrome cases, and its performance was evaluated. It is believed that the quality check step ensured the integrity of the sequencing data, while the trimming process is thought to have removed low-quality bases and adaptors. In the alignment step, it is believed that the reads were accurately mapped to the reference genome, and the subsequent variant calling step is believed to have identified potential genetic variants. The annotation step is believed to have provided functional insights into the detected variants, including their effects on known Lynch Syndrome-associated genes. The results obtained from the pipeline revealed Lynch Syndrome-related positions in the genome, providing valuable information for further investigation and clinical decision-making. The pipeline's effectiveness was demonstrated through its ability to streamline the analysis workflow and identify potential genetic markers associated with Lynch Syndrome. It is believed that the computational pipeline presents a comprehensive and efficient approach to Lynch Syndrome detection, contributing to early diagnosis and intervention. The modularity and flexibility of the pipeline are believed to enable customization and adaptation to various datasets and research settings. Further optimization and validation are believed to be necessary to enhance performance and applicability across diverse populations.

Keywords: Lynch Syndrome, computational pipeline, alignment, variant calling, annotation, genetic markers

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Authors: Nefzi Ahlem, Aydi Ben Abdallah Rania, Jabnoun-Khiareddine Hayfa, Ammar Nawaim, Mejda Daami-Remadi

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Fusarium Crown and Root Rot (FCRR) caused by Fusarium oxysporum f. sp. radicis-lycopersici (FORL) is a serious tomato (Solanum lycopersicum L.) disease in Tunisia. Its management is very difficult due to the long survival of its resting structures and to the luck of genetic resistance. In this work, we explored the wild Solanaceae species Withania somnifera, growing in the Tunisian Centre-East, as a potential source of biocontrol agents effective in FCRR suppression and tomato growth promotion. Seven fungal isolates were shown able to colonize tomato roots, crowns, and stems. Used as conidial suspensions or cell-free culture filtrates, all tested fungal treatments significantly enhanced tomato growth parameters by 21.5-90.3% over FORL-free control and by 27.6-93.5% over pathogen-inoculated control. All treatments significantly decreased the leaf and root damage index by 28.5-92.8 and the vascular browning extent 9.7-86.4% over FORL-inoculated and untreated control. The highest disease suppression ability (decrease by 86.4-92.8% in FCRR severity) over pathogen-inoculated control and by 81.3-88.8 over hymexazol-treated control) was expressed by I6 based treatments. This endophytic fungus was morphologically characterized and identified using rDNA sequencing gene as Fusarium sp. I6 (MG835371). This fungus was shown able to reduce FORL radial growth by 58.5–83.2% using its conidial suspension or cell-free culture filtrate. Fusarium sp. I6 showed chitinolytic, proteolytic and amylase activities. The current study clearly demonstrated that Fusarium sp. (I6) is a promising biocontrol candidate for suppressing FCRR severity and promoting tomato growth. Further investigations are required for elucidating its mechanism of action involved in disease suppression and plant growth promotion.

Keywords: antifungal activity, associated fungi, Fusarium oxysporum f. sp. radicis-lycopersici, Withania somnifera, tomato growth

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Authors: Fouad Chebib, Marie Hogan, Ziad El-Zoghby, Maria Irazabal, Sarah Senum, Christina Heyer, Charles Madsen, Emilie Cornec-Le Gall, Atta Behfar, Barbara Ehrlich, Peter Harris, Vicente Torres

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Background: Mutations in PKD1 and PKD2, the genes encoding the proteins polycystin-1 (PC1) and polycystin-2 (PC2) cause autosomal dominant polycystic kidney disease (ADPKD). ADPKD is a systemic disease associated with several extrarenal manifestations. Animal models have suggested an important role for the polycystins in cardiovascular function. The aim of the current study is to evaluate the association of various cardiomyopathies in a large cohort of patients with ADPKD. Methods: Clinical data was retrieved from medical records for all patients with ADPKD and cardiomyopathies (n=159). Genetic analysis was performed on available DNA by direct sequencing. Results: Among the 58 patients included in this case series, 39 patients had idiopathic dilated cardiomyopathy (IDCM), 17 had hypertrophic obstructive cardiomyopathy (HOCM), and 2 had left ventricular noncompaction (LVNC). The mean age at cardiomyopathy diagnosis was 53.3, 59.9 and 53.5 years in IDCM, HOCM and LVNC patients respectively. The median left ventricular ejection fraction at initial diagnosis of IDCM was 25%. Average basal septal thickness was 19.9 mm in patients with HOCM. Genetic data was available in 19, 8 and 2 cases of IDCM, HOCM, and LVNC respectively. PKD1 mutations were detected in 47.4%, 62.5% and 100% of IDCM, HOCM and LVNC cases. PKD2 mutations were detected only in IDCM cases and were overrepresented (36.8%) relative to the expected frequency in ADPKD (~15%). The prevalence of IDCM, HOCM, and LVNC in our ADPKD clinical cohort was 1:17, 1:39 and 1:333 respectively. When compared to the general population, IDCM and HOCM was approximately 10-fold more prevalent in patients with ADPKD. Conclusions: In summary, we suggest that PKD1 or PKD2 mutations may predispose to idiopathic dilated or hypertrophic cardiomyopathy. There is a trend for patients with PKD2 mutations to develop the former and for patients with PKD1 mutations to develop the latter. Predisposition to various cardiomyopathies may be another extrarenal manifestation of ADPKD.

Keywords: autosomal dominant polycystic kidney (ADPKD), polycystic kidney disease, cardiovascular, cardiomyopathy, idiopathic dilated cardiomyopathy, hypertrophic cardiomyopathy, left ventricular noncompaction

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Authors: Vaibhav D. Bhatt, Anju P. Kunjadia, D. S. Nauriyal, Bhumika J. Joshi, Chaitanya G. Joshi

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Metagenomic analysis of milk samples collected from local cattle breed, kankrej (Bos indicus), Gir (Bos indicus) and Crossbred (Bos indicus X Bos taurus) cattle harbouring subclinical mastitis was carried out by next-generation sequencing (NGS) 454 GS-FLX technology. Around 56 different species including members of Enterobacteriales, Pseudomonadales, Bacillales and Lactobacillales with varying abundance were detected in infected milk. The interesting presence of bacteriophages against Staphylococcus aureus, Escherichia coli, Enterobacter and Yersinia species were observed, especially Enterobacteria and E. coli phages (0∙32%) in Kankrej, Enterobacteria and Staphylococcus phages (1∙05%) in Gir and Staphylococcus phages (2∙32%) in crossbred cattle. NGS findings suggest that phages may be involved in imparting natural resistance of the cattle against pathogens. Further infected milk samples were subjected for bacterial isolation. Fourteen different isolates were identified, and DNA was extracted. Genes (Tet-K, Msr-A, and Mec-A) providing antibiotic resistance to the bacteria were screened by Polymerase Chain Reaction and results were validated with traditional antibiotic assay. Total 3 bacteriophages were isolated from nearby environment of the cattle farm. The efficacy of phages was checked against multi-drug resistant bacteria, identified by PCR. In-vivo study was carried out for phage therapy in mammary glands of female rats “Wister albino”. Mammary glands were infused with MDR isolates for 3 consecutive days. Recovery was observed in infected rats after intramammary infusion of sterile phage suspension. From day 4th onwards, level of C-reactive protein was significant increases up to day 12th . However, significant reduction was observed between days 12th to 18th post treatment. Bacteriophages have significant potential as antibacterial agents and their ability to replicate exponentially within their hosts and their specificity, make them ideal candidates for more sustainable mastitis control.

Keywords: bacteriophages, c-reactive protein, mastitis, metagenomic analysis

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Authors: Mukolwe D. Lubembe, Odongo O. David, Githaka Naftali, Kanduma Esther, Marinda Oosthuizen, Kgomotso P. Sibeko

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Theileriosis is a tick-borne disease of cattle caused by an apicomplexan protozoan parasite of the genus Theileria. In eastern and southern Africa, Theileria infections in cattle are caused by the species Theileria parva whose natural reservoir is the African buffalo (Syncerus caffer). Currently, East Coast Fever (ECF) caused by the cattle-derived Theileria parva is still a major problem in eastern Africa and some parts of southern Africa but not in South Africa following its eradication in the 1950s. However, Corridor disease (CD) caused by the buffalo-derived Theileria parva still remains a concern in South Africa. The diversity of Theileria parva in South Africa in comparison to other affected countries is poorly defined yet its known to be the survival strategy of this parasite. We assessed the antigenic diversity of Theileria parva isolates from Buffalo and cattle at the wildlife-livestock interface comparing samples from South Africa, Zimbabwe, Kenya, Tanzania, and Uganda. Antigenic epitopes of eight schizont antigen genes (Tp1, Tp3, Tp4, Tp5, Tp6, Tp7, Tp8 and Tp10) were amplified by PCR from genomic DNA extracted from blood samples collected from cattle and buffalo at the wildlife-livestock interface. Amplicons were purified and then sequenced on NGS platform. Full length open reading frames (ORFs) of two schizont antigen genes (Tp2 and Tp9) and one sporozoite antigen gene, p67 were also amplified from genomic DNA. Amplicons were then purified and cloned for sequencing. Analysis was based on sequence differences in the genes. Preliminary results show an extensively diverse population of Theileria parva circulating in buffalo and cattle populations at the wildlife-livestock interface. Diversity of the antigen genes contributes to the evasion of the immune system of the host by Theileria parva. This possess a concern in that, some of the Theileria parva populations may re-assort and become adapted to cattle to cause a form of theileriosis that is as fatal as ECF in areas where ECF was eradicated or is absent

Keywords: Theileria parva, east coast fever, corridor diseases, antigen genes, diversity

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Authors: Saima Saleem, Zubair Abbasi, Abdul Hameed, Mansoor Ahmed Khan, Navid Rashid Qureshi, Abid Azhar

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Oral Squamous Cell Carcinoma (OSCC) is the leading cause of death in the developing countries like Pakistan. This problem aggravates because of the excessive use of available chewing products. In spite of widespread information on their use and purported legislations against their use the Pakistani markets are classical examples of selling chewable carcinogenic mutagens. Reported studies indicated that these products are rich in reactive oxygen species (ROS) and polyphenols. TP53 gene is involved in the suppression of tumor. It has been reported that somatic mutations caused by TP53 gene are the foundation of the cancer. This study aims to find the loss of TP53 functions due to mutation/polymorphism caused by genomic alteration and interaction with tobacco and its related ingredients. Total 260 tissues and blood specimens were collected from OSCC patients and compared with age and sex matched controls. Mutations in exons 2-11 of TP53 were examined by PCR-SSCP. Samples showing mobility shift were directly sequenced. Two mutations were found in exon 4 at nucleotide position 108 and 215 and one in exon 7 at nucleotide position 719 of the coding sequences in patient’s tumor samples. These results show that substitution of proline with arginine at codon 72 and serine with threonine at codon 240 of p53 protein. These polymorphic changes, found in tumor samples of OSCC, could be involved in loss of heterozygocity and apoptotic activity in the binding domain of TP53. The model of the mutated TP53 gene elaborated a nonfunctional unfolded p53 protein, suggesting an important role of these mutations in p53 protein inactivation and malfunction. This nonfunctional 3D model also indicates that exogenous tobacco related carcinogens may act as DNA-damaging agents affecting the structure of DNA. The interpretations could be helpful in establishing the pathways responsible for tumor formation in OSCC patients.

Keywords: TP53 mutation/polymorphism, OSCC, PCR-SSCP, direct DNA sequencing, 3D structure

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Authors: Khemakhem Nahed, Hammami Fatma, Trabelsi Houaida, Neji Sourour, Sellami Hayet, Makni Fattouma, Turki Hamida, Ayadi Ali

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Dermatophytoses are considered a public health problem and represent 10% of dermatological consultations in our region. Their epidemiology is influenced by various factors, such as lifestyle, human migration patterns, changes in the environment and the host relationship. The understanding of epidemiology has a major impact on their prevention and treatment. The aim of the study is to determine the prevalence pattern of aetiological agents and to describe the clinical characteristics of dermatophytoses between 1999 and 2019. Out of 65 059 subjects suspected to have superficial mycoses, 36 220 (55.67%) were affected with dermatophytoses. The mean age was 40.1 years (range: 10 days to 99 years). The sex ratio was 0.8. Our patients were from urban regions in 80.9% of cases. The most common type of infection was onychomycosis (42.64%), followed by tinea pedis (20.8%), intertrigo (18.3%), tinea corporis (8.48%) and tinea capitis (7.87%). The most isolated dermatophyte was Trichophyton rubrum (76.5%), followed by T. mentagrophytes complex (6.3%), Microsporum canis (5.8%), T. violaceum (5.3%), T. verrucosum (0.83%) and Epidermophyton floccosum (0.3%). Zoophilic agents have become more prevalent and their frequency has been increased from 6.46% in 1999 to 13% in 2019. It is interesting to note that M. canis has been on the rise since 2010 and it was the first etiological agent of tinea capitis (48%), while infections caused by T. violaceum continued to decrease from 1999 (16.2%) to 2019 (4.7%). Other dermatophytes have been rarely isolated: T. tonsurans (9 cases), T. schoenleinii (3 cases), T. soudanense (2 cases), M. fulvum (1 case), M. audouinii (1 case) and M. ferrugineum (2 cases).T. mentagrophytes var. quinckeanum was isolated from an inflammatory tinea capitis lesion in an a-3-year-old girl. T. mentagrophytes var. erinacei was isolated from the first case of tinea manuum, in-a-10-year-old girl. The same fungus was isolated from the hair and scales of the hedgehog. Our study showed significant changes in the dermatophytes spectrum in our region. The prevalence of zoophilic species increased in recent years due to people's behavioral changes with the adoption of pets and animal husbandry in urban settings. Molecular methods are often crucial that help us to refine the identification strains of dermatophytes and to identify their origin of the contamination.

Keywords: dermatophytoses, PCR-sequencing, spectrum, Sfax, Tunisia

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Authors: Valeria Arcucci, Musarat Ishaq, Steven A. Stacker, Greg J. Goodall, Marc G. Achen

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Metastasis is the lethal aspect of cancer for most patients. Remodelling of lymphatic vessels associated with a tumour is a key initial step in metastasis because it facilitates the entry of cancer cells into the lymphatic vasculature and their spread to lymph nodes and distant organs. Although it is clear that vascular endothelial growth factors (VEGFs), such as VEGF-C and VEGF-D, are key drivers of lymphatic remodelling, the means by which many signaling pathways in endothelial cells are coordinately regulated to drive growth and remodelling of lymphatics in cancer is not understood. We seek to understand the broader molecular mechanisms that control cancer metastasis, and are focusing on microRNAs, which coordinately regulate signaling pathways involved in complex biological responses in health and disease. Here, using small RNA sequencing, we found that a specific microRNA, miR-132, is upregulated in expression in lymphatic endothelial cells (LECs) in response to the lymphangiogenic growth factors. Interestingly, ectopic expression of miR-132 in LECs in vitro stimulated proliferation and tube formation of these cells. Moreover, miR-132 is expressed in lymphatic vessels of a subset of human breast tumours which were previously found to express high levels of VEGF-D by immunohistochemical analysis on tumour tissue microarrays. In order to dissect the complexity of regulation by miR-132 in lymphatic biology, we performed Argonaute HITS-CLIP, which led us to identify the miR-132-mRNA interactome in LECs. We found that this microRNA in LECs is involved in the control of many different pathways mainly involved in cell proliferation and regulation of the extracellular matrix and cell-cell junctions. We are now exploring the functional significance of miR-132 targets in the biology of LECs using biochemical techniques, functional in vitro cell assays and in vivo lymphangiogenesis assays. This project will ultimately define the molecular regulation of lymphatic remodelling by miR-132, and thereby identify potential therapeutic targets for drugs designed to restrict the growth and remodelling of tumour lymphatics resulting in metastatic spread.

Keywords: argonaute HITS-CLIP, cancer, lymphatic remodelling, miR-132, VEGF

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Authors: Vivian A. Panes, Raymond John S. Rebong, Miel Q. Diaz

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Moringa oleifera Lam. is known mainly for its high nutritional value and medicinal properties contributing to its popular reputation as a 'miracle plant' in the tropical climates where it usually grows. The main objective of this study is to discover the genes and gene products involved in abiotic stress-induced activity that may impact the M. oleifera Lam. mature seeds as well as their corresponding functions. In this study, RNA-sequencing and de novo transcriptome assembly were performed using two assemblers, Trinity and Oases, which produced 177,417 and 120,818 contigs respectively. These transcripts were then subjected to various bioinformatics tools such as Blast2GO, UniProt, KEGG, and COG for gene annotation and the analysis of relevant metabolic pathways. Furthermore, FPKM analysis was performed to identify gene expression levels. The sequences were filtered according to the 'response to stress' GO term since this study dealt with stress response. Clustered Orthologous Groups (COG) showed that the highest frequencies of stress response gene functions were those of cytoskeleton which make up approximately 14% and 23% of stress-related sequences under Trinity and Oases respectively, recombination, repair and replication at 11% and 14% respectively, carbohydrate transport and metabolism at 23% and 9% respectively and defense mechanisms 16% and 12% respectively. KEGG pathway analysis determined the most abundant stress-response genes in the phenylpropanoid biosynthesis at counts of 187 and 166 pathways for Oases and Trinity respectively, purine metabolism at 123 and 230 pathways, and biosynthesis of antibiotics at 105 and 102. Unique and cumulative GO term counts revealed that majority of the stress response genes belonged to the category of cellular response to stress at cumulative counts of 1,487 to 2,187 for Oases and Trinity respectively, defense response at 754 and 1,255, and response to heat at 213 and 208, response to water deprivation at 229 and 228, and oxidative stress at 508 and 488. Lastly, FPKM was used to determine the levels of expression of each stress response gene. The most upregulated gene encodes for thiamine thiazole synthase chloroplastic-like enzyme which plays a significant role in DNA damage tolerance. Data analysis implies that M. oleifera stress response genes are directed towards the effects of climate change more than other stresses indicating the potential of M. oleifera for cultivation in harsh environments because it is resistant to climate change, pathogens, and foreign invaders.

Keywords: stress response, genes, Moringa oleifera, transcriptomics

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Authors: Guna Raj Dhungana, Roshan Nepal, Apshara Parajuli, , Archana Maharjan, Shyam K. Mishra, Pramod Aryal, Rajani Malla

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Introduction: Klebsiella pneumoniae is a well-known opportunistic human pathogen, primarily causing healthcare-associated infections. The global emergence of carbapenemase-producing K. pneumoniaeis a major public health burden, which is often extensively multidrug resistant.Thus, because of the difficulty to treat these ‘superbug’ and menace and some term as ‘apocalypse’ of post antibiotics era, an alternative approach to controlling this pathogen is prudent and one of the approaches is phage mediated control and/or treatment. Objective: In this study, we aimed to isolate novel bacteriophage against carbapenemase-producing K. pneumoniaeand characterize for potential use inphage therapy. Material and Methods: Twenty lytic phages were isolated from river water using double layer agar assay and purified. Biological features, physiochemical characters, burst size, host specificity and activity spectrum of phages were determined. One most potent phage: Phage TU_Kle10O was selected and characterized by electron microscopy. Whole genome sequences of the phage were analyzed for presence/absence of virulent factors, and other lysin genes. Results: Novel phage TU_Kle10O showed multiple host range within own genus and did not induce any BIM up to 5th generation of host’s life cycle. Electron microscopy confirmed that the phage was tailed and belonged to Caudovirales family. Next generation sequencing revealed its genome to be 166.2 Kb. bioinformatical analysis further confirmed that the phage genome ‘did not’ contain any ‘bacterial genes’ within phage genome, which ruled out the concern for transfer of virulent genes. Specific 'lysin’ enzyme was identified phages which could be used as 'antibiotics'. Conclusion: Extensively multidrug resistant bacteria like carbapenemase-producing K. pneumoniaecould be treated efficiently by phages.Absence of ‘virulent’ genes of bacterial origin and presence of lysin proteins within phage genome makes phages an excellent candidate for therapeutics.

Keywords: bacteriophage, Klebsiella pneumoniae, MDR, phage therapy, carbapenemase,

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Authors: Liaqat Ali, Hubert E. Blum, Türkân Sakinc

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Enterococcus faecium is an emerging multidrug-resistant nosocomial pathogen increased dramatically worldwide and causing bacteremia, endocarditis, urinary tract and surgical site infections in immunocomprised patients. The capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system are also involved in hindering leukocyte killing of enterococci. The gene cluster (enterococcal polysaccharide antigen) of E. faecalis encoding homologues of many genes involved in polysaccharide biosynthesis. We identified two putative loci with 22 kb and 19 kb which contained 11 genes encoding for glycosyltransferases (GTFs); this was confirmed by using genome comparison of already sequenced strains that has no homology to known capsule genes and the epa-locus. The polysaccharide-conjugate vaccines have rapidly emerged as a suitable strategy to combat different pathogenic bacteria, therefore, we investigated a polysaccharide biosynthesis CapD protein in E. faecium contains 336 amino acids and had putative function for N-linked glycosylation. The deletion/knock-out capD mutant was constructed and complemented by homologues recombination method and confirmed by using PCR and sequencing. For further characterization and functional analysis, in-vitro cell culture and in-vivo a mouse infection models were used. Our ΔcapD mutant shows a strong hydrophobicity and all strains exhibited biofilm production. Subsequently, the opsonic activity was tested in an opsonophagocytic assay which shows increased in mutant compared complemented and wild type strains but more than two fold decreased in colonization and adherence was seen on surface of uroepithelial cells. However, a significant higher bacterial colonialization was observed in capD mutant during animal bacteremia infection. Unlike other polysaccharides biosynthesis proteins, CapD does not seems to be a major virulence factor in enterococci but further experiments and attention is needed to clarify its function, exact mechanism and involvement in pathogenesis of enteroccocal nosocomial infections eventually to develop a vaccine/ or targeted therapy.

Keywords: E. faecium, pathogenesis, polysaccharides, biofilm formation

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Authors: Karol Rodriguez-Peña, Martha L. Macias-Rubalcava, Leticia Rocha-Zavaleta, Sergio Sanchez

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A bioassay-guided study for anti-cancer compounds from endophytes of the Mexican medicinal plant Amphipteryygium adstringens resulted in the isolation of a streptomycete capable of producing a group of compounds with high cytotoxic activity. Microorganisms from surface sterilized samples of various sections of the plant were isolated and all the actinomycetes found were evaluated for their potential to produce compounds with cytotoxic activity against cancer cell lines MCF7 (breast cancer) and HeLa (cervical cancer) as well as the non-tumoural cell line HaCaT (keratinocyte). The most active microorganism was picked for further evaluation. The identification of the microorganism was carried out by 16S rDNA gene sequencing, finding the closest proximity to Streptomyces scabrisporus, but with the additional characteristic that the strain isolated in this study was capable of producing colorful compounds never described for this species. Crude extracts of dichloromethane and ethyl acetate showed IC50 values of 0.29 and 0.96 μg/mL for MCF7, 0.51 and 1.98 μg/mL for HeLa and 0.96 and 2.7 μg/mL for HaCaT. Scaling the fermentation to 10 L in a bioreactor generated 1 g of total crude extract, which was fractionated by silica gel open column to yield 14 fractions. Nine of the fractions showed cytotoxic activity. Fraction 4 was chosen for subsequent purification because of its high activity against cancerous cell lines, lower activity against keratinocytes. HPLC-UV-MS/ESI was used for the evaluation of this fraction, finding at least 10 different compounds with high values of m/z (≈588). Purification of the compounds was carried out by preparative thin-layer chromatography. The prevalent compound was Steffimycin B, a molecule known for its antibiotic and cytotoxic activities and also for its low solubility in aqueous solutions. Along with steffimycin B, another five compounds belonging to the steffimycin family were isolated and at this moment their structures are being elucidated, some of which display better solubility in water: an attractive property for the pharmaceutical industry. As a conclusion to this study, the isolation of endophytes resulted in the discovery of a strain capable of producing compounds with high cytotoxic activity that need to be studied for their possible utilization.

Keywords: amphipterygium adstringens, cytotoxicity, streptomyces scabrisporus, steffimycin

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Authors: Muhammad Tanveer Altaf, Mehmet Bedir, Waqas Liaqat, Gönül Cömertpay, Volkan Çatalkaya, Celaluddin Barutçular, Nergiz Çoban, Ibrahim Cerit, Muhammad Azhar Nadeem, Tolga Karaköy, Faheem Shehzad Baloch

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Food production needs to be almost double by 2050 in order to feed around 9 billion people around the Globe. Plant production mostly relies on fertilizers, which also have one of the main roles in environmental pollution. In addition to this, climatic conditions are unpredictable, and the earth is expected to face severe drought conditions in the future. Therefore, water and fertilizers, especially nitrogen are considered as main constraints for future food security. To face these challenges, developing integrative approaches for germplasm characterization and selecting the resilient genotypes performing under limiting conditions is very crucial for effective breeding to meet the food requirement under climatic change scenarios. This study is part of the European Research Area Network (ERANET) project for the characterization of the diversity panel of 172 sorghum accessions and six hybrids as control cultivars under limiting (+N/-H2O, -N/+H2O) and non-limiting conditions (+N+H2O). This study was planned to characterize the sorghum diversity in relation to resource Use Efficiency (RUE), with special attention on harnessing the interaction between genotype and environment (GxE) from a physiological and agronomic perspective. Experiments were conducted at Adana, a Mediterranean climate, with augmented design, and data on various agronomic and physiological parameters were recorded. Plentiful diversity was observed in the sorghum diversity panel and significant variations were seen among the limiting water and nitrogen conditions in comparison with the control experiment. Potential genotypes with the best performance are identified under limiting conditions. Whole genome resequencing was performed for whole germplasm under investigation for diversity analysis. GWAS analysis will be performed using genotypic and phenotypic data and linked markers will be identified. The results of this study will show the adaptation and improvement of sorghum under climate change conditions for future food security.

Keywords: germplasm, sorghum, drought, nitrogen, resources use efficiency, sequencing

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Authors: Ricardo de Oliveira Orsi, Aline Astolfi, Daniel Diego Mendes, Isabella Cristina de Castro Lippi, Jaine da Luz Scheffer, Yan Souza Lima, Juliana Lunardi, Giovanna do Padro Ribeiro, Samir Moura Kadri

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NECTAR Brazilian group (Center of Education, Science, and Technology in Rational Beekeeping) conducted studies on the pesticides honey bees effects using the transcriptome sequencing (RNA-Seq) analyzes for gene expression studies. In this way, we analyzed the effects of Pyraclostrobin and Fipronil on the honey bees with 21 old-days (forager) in laboratory conditions. For this, frames containing sealed brood were removed from the beehives and maintenance on the stove (32°C and 75% humidity) until the bees were born. So, newly emerged workers were marked on the pronotum with a non-toxic pen and reintroduced into their original hives. After 21 days, 120 marked bees were collected with an entomological forces and immediately stored in Petri dishes, perforated to ensure ventilation, and kept fasted for 3 hours. These honeybees were exposed to food contaminated or not with the sublethal dose of Pyraclostrobin (850 ppb/bee) or Fipronil (2.5 ppb/bee). After four hours of exposure, 15 bees from each treatment were referred to transcriptome analysis. Total RNA analysis was extracted from the brain pools (03 brains per pool) using the TRIzol® reagent protocol according to the manufacturer's instructions. cDNA libraries were constructed, and the FASTQC program was used to check adapter content and assess the quality of raw reads. Differential expression analysis was performed with the DESeq2 package. Genes that had an adjusted value of less than 0.05 were considered to be significantly up-regulated. Regarding the Pyraclostrobin, alterations were observed in the pattern of 17 gene related to of antioxidant system, cellular respiration, glucose metabolism, and regulation of juvenile hormone and the hormone insulin. Glyphosate altered the 10 gene related to the digestive system, exoskeleton composition, vitamin E transport, and antioxidant system. The results indicate that the necessity of studies using the sublethal doses to evaluate the pesticides uses and risks on crops and its effects on the honey bees.

Keywords: beekeeping, honey bees, pesticides, transcriptome

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Authors: Luz Hurtado, Rodrigo Rojas, Jaime Romero, Christopher Concha

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The culture of the marine fish red cusk eel Genypterus chilensis is currently considered a priority for Chilean aquaculture which is a Chilean native species of high gastronomic demand and market value. The microbiota was analyzed in terms of diversity and structure using massive Illumina sequencing. The analysis of alpha diversity was performed in samples of G. chilensis larvae of 6, 18 and 32 dph (days post-hatching) and it was observed that there were significant differences (P = 0.05) between the days of culture for the Chao1 index, being the larvae of 18 dph the one with the highest index followed by the larvae of 6 dph, The lowest value for this index was presented in larvae of 32 dph. There were no significant differences in larvae between the days of culture for the Shannon (P=0.0857) and Simpson (P=0.0714) indices. In general, the larvae of G. chilensis have high rates of diversity. When analyzing the beta diversity, a differentiation between the bacterial communities is observed depending on the day of the culture of the larvae. Considering the PCoA elaborated from the unweighted UniFrac statistic, the explained variance was 46.2% (PC1 29.2% and PC2 17.0%) and in the case of the PCoA elaborated with the weighted UniFrac statistic; the explained variance was 65.5% (PC1 41.8% and PC2 23.7%) these differences were significant based on the Permanova statistical analysis (P= 0.002 and 0.037 respectively). When analyzing the taxonomic composition of the microbiota of the larvae in the different days of culture it was observed that at the phyla level the most abundant in the larvae of 6 dph were Proteobacteria (57%) Verrucomicrobia (24%) and Firmicutes (14%), for the larvae of 18 dph the predominant phyla were Proteobacteria (90%), Dependientiae (5%), Actinobacteria (2%) and Plactomyces (2%), for the larvae of 32 dph the phyla that presented the highest relative abundance were Proteobacteria (57%), Firmicutes (29%), Verrucomicrobia (5%) and Actinobacteria (5%), when comparing the larvae between the days it was observed that the phylum Proteobacteria was the most abundant in the samples of larvae of 6, 18 and 32 dph being the larvae of 18 dph those that present the highest relative abundance, the larvae of 6 dph were those that presented the highest relative abundance for the phylum Verrucomicrobia and in the larvae of 32 dph was observed greater abundance of the phylum Firmicutes compared to the other days of larval culture. At the level of genera, those with the highest relative abundance in larvae of 6 dph were Rubritalea (30%), Psychrobacter (28%), staphylococcus (17%) and Ralstonia (10%), for the larvae of 18 dph the genera with the highest abundance were Psychrobacter (47%), Litoreibacter (13%), Nautella (9%) and Cohesibacter (8%), for the larvae of 32 dph the most abundant genera were Alloiococcus (25%), Dialister (14%), Neptunomonas (13%) and Piscirickettsia (11%). When observing the taxonomic composition of the larvae between the days of larval culture, it is observed that there are differences between them.

Keywords: microbiota, diversity, G. Chilensis, larvae

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Authors: Noura El-Ahmady El-Naggar

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L-asparaginase is an important enzyme as therapeutic agents used in combination therapy with other drugs in the treatment of acute lymphoblastic leukemia in children. L-asparaginase producing actinomycete strain, NEAE-K, was isolated from soil sample and identified on the basis of morphological, cultural, physiological and biochemical properties, together with 16S rDNA sequence as new subsp. Streptomyces rochei subsp. chromatogenes NEAE-K and sequencing product (1532 bp) was deposited in the GenBank database under accession number KJ200343. The study was conducted to screen parameters affecting the production of L-asparaginase by Streptomyces rochei subsp. chromatogenes NEAE-K on solid state fermentation using Plackett–Burman experimental design. Sixteen different independent variables including incubation time, moisture content, inoculum size, temperature, pH, soybean meal+ wheat bran, dextrose, fructose, L-asparagine, yeast extract, KNO3, K2HPO4, MgSO4.7H2O, NaCl, FeSO4. 7H2O, CaCl2, and three dummy variables were screened in Plackett–Burman experimental design of 20 trials. The most significant independent variables affecting enzyme production (dextrose, L-asparagine and K2HPO4) were further optimized by the central composite design. As a result, a medium of the following formula is the optimum for producing an extracellular L-asparaginase by Streptomyces rochei subsp. chromatogenes NEAE-K from solid state fermentation: g/L (soybean meal+ wheat bran 15, dextrose 3, fructose 4, L-asparagine 8, yeast extract 2, KNO3 1, K2HPO4 2, MgSO4.7H2O 0.5, NaCl 0.1, FeSO4. 7H2O 0.02, CaCl2 0.01), incubation time 7 days, moisture content 50%, inoculum size 3 mL, temperature 30°C, pH 8.5.

Keywords: streptomyces rochei subsp. chromatogenes neae-k, 16s rrna, identification, solid state fermentation, l-asparaginase production, plackett-burman design, central composite design

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Authors: S. Pathak, R. Lazarus

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A 25-year-old male HIV patient (CD4 cells 20/µL and HIV viral load 14200000 copies/ml) with a past medical history of duodenal ulcer, pneumocystis carinii pneumonia, oesophageal candidiasis presented with fever and a seizure to hospital. The only recent travel had been a religious pilgrimage from Singapore to Malaysia 5 days prior; during the trip he sustained skin abrasions. The patient had recently started highly active antiretroviral therapy 2 months prior. Clinical examination was unremarkable other than a temperature of 38.8°C and perianal warts. Laboratory tests showed a leukocyte count 12.5x109 cells/L, haemoglobin 9.4 g/dL, normal biochemistry and a C-reactive protein 121 mg/L. CT head and MRI head were unremarkable and cerebrospinal fluid analysis performed after a delay (due to technical difficulties) of 11 days was unremarkable. Blood cultures (three sets) taken on admission showed Gram-negative rods in the anaerobic bottles only at the end of incubation with culture result confirmed by molecular sequencing showing Helicobacter cinaedi. The patient was treated empirically with ceftriaxone for seven days and this was converted to oral co-amoxiclav for a further seven days after the blood cultures became positive. A Transthoracic echocardiogram was unremarkable. The patient made a full recovery. Helicobacter cinaedi is a gram-negative anaerobic fastidious organism affecting patients with comorbidity. Infection may manifest as cellulitius, colitis or as in this case as bloodstream infection – the latter is often attributed to faeco-oral infection. Laboratory identification requires prolonged culture. Therapeutic options may be limited by resistance to macrolides and fluoroquinolones. The likely pathogen inoculation routes in the case described include gastrointestinal translocation due to proctitis at the site of perianal warts, or breach of the skin via abrasions occurring during the pilgrimage. Such organisms are increasing in prevalence as our patient population ages and patients have multiple comorbidities including HIV. It may be necessary in patients with unexplained fever to prolong incubation of sterile sites including blood in order to identify this unusual fastidious organism.

Keywords: fastidious, Helicobacter cinaedi, HIV, immunocompromised

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Authors: Jean Paul M. Milambo

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Purpose: Aromatase inhibitors (AIs) are indicated in the treatment of hormone-receptive breast cancer in postmenopausal women in various settings. Studies have shown cardiovascular events in some developed countries. To date the data is sparce for evidence-based recommendations in African clinical settings due to lack of cancer registries, capacity building and surveillance systems. Therefore, this study was conducted to assess the feasibility of HyBeacon® probe genotyping adjunctive to standard care for timely prediction and diagnosis of Aromatase inhibitors (AIs) associated adverse events in breast cancer survivors in Africa. Methods: Cross sectional study was conducted to assess the knowledge of POCT among six African countries using online survey and telephonically contacted. Incremental cost effectiveness ratio (ICER) was calculated, using diagnostic accuracy study. This was based on mathematical modeling. Results: One hundred twenty-six participants were considered for analysis (mean age = 61 years; SD = 7.11 years; 95%CI: 60-62 years). Comparison of genotyping from HyBeacon® probe technology to Sanger sequencing showed that sensitivity was reported at 99% (95% CI: 94.55% to 99.97%), specificity at 89.44% (95% CI: 87.25 to 91.38%), PPV at 51% (95%: 43.77 to 58.26%), and NPV at 99.88% (95% CI: 99.31 to 100.00%). Based on the mathematical model, the assumptions revealed that ICER was R7 044.55. Conclusion: POCT using HyBeacon® probe genotyping for AI-associated adverse events maybe cost effective in many African clinical settings. Integration of preventive measures for early detection and prevention guided by different subtype of breast cancer diagnosis with specific clinical, biomedical and genetic screenings may improve cancer survivorship. Feasibility of POCT was demonstrated but the implementation could be achieved by improving the integration of POCT within primary health cares, referral cancer hospitals with capacity building activities at different level of health systems. This finding is pertinent for a future envisioned implementation and global scale-up of POCT-based initiative as part of risk communication strategies with clear management pathways.

Keywords: breast cancer, diagnosis, point of care, South Africa, aromatase inhibitors

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Authors: Sajewicz-Krukowska Joanna, Domańska-Blicharz Katarzyna, Tarasiuk Karolina, Marzec-Kotarska Barbara

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Astroviral infections pose a significant problem in the poultry industry, leading to multiple adverse effects such as decreased egg production, breeding disorders, poor weight gain, and even increased mortality. Commonly observed chicken astrovirus (CAstV) was recently reported to be responsible for "white chicks syndrome" associated with increased embryo/chick mortality. The CAstV-mediated pathogenesis in chicken occurs due to complex interactions between the infectious pathogen and the immune system. Many aspects of CAstV-chicken interactions remain unclear, and there is no information available regarding gene expression changes in the chicken's spleen in response to CAstV infection. We aimed to investigate the molecular background triggered by CAstV infection. Ten 21-day-old SPF White Leghorn chickens were divided into two groups of 5 birds each. One group was inoculated with CAstV, and the other was used as the negative control. On 4th dpi, spleen samples were collected and immediately frozen at -70°C for RNA isolation. We analysed transcriptional profiles of the chickens' spleens at the 4th day following infection using RNA-seq to establish differentially expressed genes (DEGs). The RNA-seq findings were verified by quantitative real-time PCR (qRT-PCR). A total of 31959 transcripts were identified in response to CAstV infection. Eventually 45 DEGs (p-value<0.05; Log2Foldchange>1)were recognized in the spleen after CAstV infection (26 upregulated DEGs and 19 downregulated DEGs). qRT-PCR performed on 4 genes (IFIT5, OASL, RASD1, DDX60) confirmed RNAseq results. Top differentially expressed genes belonged to novel putative IFN-induced CAstV restriction factors. Most of the DEGs were associated with RIG-I–like signalling pathway or, more generally, with an innate antiviral response(upregulated: BLEC3, CMPK2, IFIT5, OASL, DDX60, IFI6, and downregulated: SPIK5, SELENOP, HSPA2, TMEM158, RASD1, YWHAB). The study provided a global analysis of host transcriptional changes that occur during CAstV infection in vivo and proved the cell cycle in the spleen and immune signalling in chickens were predominantly affected upon CAstV infection.

Keywords: chicken astrovirus, CastV, RNA-seq, transcriptome, spleen

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

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Pinus taeda, commonly known as loblolly pine, is a crucial timber species native to the southeastern USA. An emerging problem has been encountered for the past few years, which is better to be known as loblolly pine needle defoliation (LPND), which is threatening the ecological health of southeastern forests and economic vitality of the region’s timber industry. Currently, more than 1000 hectares of loblolly plantations in Alabama are affected with similar symptoms and have created concern among southeast landowners and forest managers. However, it is still uncertain whether LPND results from one or the combination of several fungal pathogens. Therefore, the objectives of the study were to identify and characterize the fungi associated with LPND in the southeastern USA and document the damage being done to loblolly pine as a result of repeated defoliation. Identification of fungi was confirmed using classical morphological methods (microscopic examination of the infected needles), conventional and species-specific priming (SSPP) PCR, and ITS sequencing. To date, 17 species of fungi, either cultured from pine needles or formed fruiting bodies on pine needles, were identified based on morphology and genetic sequence data. Among them, brown-spot pathogen Lecanostica acicola has been frequently recovered from pine needles in both spring and summer. Moreover, Ophistomatoid fungi such as Leptographium procerum, L. terebrantis are associated with pine decline have also been recovered from root samples of the infected stands. Trees have been increasingly and repeatedly chlorotic and defoliated from 2019 to 2020. Based on morphological observations and molecular data, emerging loblolly pine needle defoliation is due in larger part to the brown-spot pathogen L. acoicola followed by pine decline pathogens L. procerum and L. terebrantis. Root pathogens were suspected to emerge later, and their cumulative effects contribute to the widespread mortality of the trees. It is more likely that longer wet spring and warmer temperatures are favorable to disease development and may be important in the disease ecology of LPND. Therefore, the outbreak of the disease is assumed to be expanded over a large geographical area in a changing climatic condition.

Keywords: brown-spot fungi, emerging disease, defoliation, loblolly pine

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Authors: Satwik Majumder, Dongyun Jung, Jennifer Ronholm, Saji George

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Bovine mastitis is the most common infectious disease in dairy cattle, with major economic implications for the dairy industry worldwide. Continuous monitoring for the emergence of antimicrobial resistance (AMR) among bacterial isolates from dairy farms is vital not only for animal husbandry but also for public health. In this study, the prevalence of AMR in 113 Escherichia coli isolates from cases of bovine clinical mastitis in Canada was investigated. Kirby-Bauer disk diffusion test with 18 antibiotics and microdilution method with three heavy metals (copper, zinc, and silver) was performed to determine the antibiotic and heavy-metal susceptibility. Resistant strains were assessed for efflux and ß-lactamase activities besides assessing biofilm formation and hemolysis. Whole-genome sequences for each of the isolates were examined to detect the presence of genes corresponding to the observed AMR and virulence factors. Phenotypic analysis revealed that 32 isolates were resistant to one or more antibiotics, and 107 showed resistance against at least one heavy metal. Quinolones and silver were the most efficient against the tested isolates. Among the AMR isolates, AcrAB-TolC efflux activity and ß-lactamase enzyme activities were detected in 13 and 14 isolates, respectively. All isolates produced biofilm but with different capacities, and 33 isolates showed α-hemolysin activity. A positive correlation (Pearson r = +0.89) between efflux pump activity and quantity of biofilm was observed. Genes associated with aggregation, adhesion, cyclic di-GMP, quorum sensing were detected in the AMR isolates, corroborating phenotype observations. This investigation showed the prevalence of AMR in E. coli isolates from bovine clinical mastitis. The results also suggest the inadequacy of antimicrobials with a single mode of action to curtail AMR bacteria with multiple mechanisms of resistance and virulence factors. Therefore, it calls for combinatorial therapy for the effective management of AMR infections in dairy farms and combats its potential transmission to the food supply chain through milk and dairy products.

Keywords: antimicrobial resistance, E. coli, bovine mastitis, antibiotics, heavy-metals, efflux pump, ß-lactamase enzyme, biofilm, whole-genome sequencing

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