Keratinase Production by Endophytic Bacteria Aneurinibacillus aneurinilyticus VRCS-4 Isolated from Xerophytic Plant Opuntia ficus - indica (Prickly pear)

Bacterial endophytes colonize an ecological niche which is unexplored site makes them suitable to produce pharmacologically active substances with vast biotechnological potential therefore, xerophytes were chosen to isolate the endophytes. In the present study forty endophytic bacterial isolates were isolated from xerophytic plants grown near poultry farms and feather dumping sites. Of them eight isolates showed zone of hydrolysis and the maximum zone of hydrolyisis of 36mm was with VRCS-4 on skimmed milk agar. This isolate exhibited efficient feather degradation and was identified as Aneurinibacillus aneurinilyticus based on its morphological, biochemical test and molecular sequencing method. The isolate was deposited in NCBI with an accession number MW227423.The isolate showed maximum en-zyme activity of 140.24U/ml at 72h, pH 7.5 and 40º C at 140 rpm. Chicken feather 1% (w/v) used as a sole source of carbon and nitrogen. Feather deg-radation by A.aneurinilyticus VRCS-4 showed 90% degradation in feather meal broth. Ours appears to be the first report on keratinase production by endophytic bacteria from xerophytic plant (Opuntia ficus -indica).

Complete Genomic Sequencing of Canine Distemper Virus With Nanopore Technology During an Epizootic Event

Canine distemper virus (CDV) endangers a wide range of wild animal populations and can cross species barriers, representing a significant conservational and animal health risk around the globe. During spring to autumn 2021, according to our current estimates a minimum of 50 wild live red foxes (Vulpes vulpes) died of CDV in Hungary, with CDV lesions. Oral, nasal and rectal swab samples were RT-PCR screened for Canine Distemper Virus from red fox carcasses. To investigate in more detail the origins of these CDV strains, 19 complete genomes were sequenced with a pan-genotype CDV-specific amplicon-based sequencing method developed by our laboratory and optimized for Oxford Nanopore Technologies platform. Phylogenetic analysis of the complete genomic sequences and separately the hemagglutinin gene sequences revealed the role of the Europe lineage of CDV as a causative agent for the current epizootic. Here we highlight the growing importance of fast developing rapid sequencing technologies to aid rapid response activities during epidemics or epizootic events. We also emphasize the urgent need for improved surveillance of CDV, considering the epizootic capability of enzootic strains as reported in the current study. For such future efforts, we provide a novel NGS protocol, which facilitates future genomic surveillance studies.

Investigation of genetic relationships within three Miscanthus species using SNP markers identified with SLAF-seq

Background Miscanthus, which is a leading dedicated-energy grass in Europe and in parts of Asia, is expected to play a key role in the development of the future bioeconomy. However, due to its complex genetic background, it is difficult to investigate phylogenetic relationships in this genus. Here, we investigated 50 Miscanthus germplasms: 1 female parent (M. lutarioriparius), 30 candidate male parents (M. lutarioriparius, M. sinensis, and M. sacchariflorus), and 19 offspring. We used high-throughput Specific-Locus Amplified Fragment sequencing (SLAF-seq) to identify informative single nucleotide polymorphisms (SNPs) in all germplasms. Results We identified 257,889 SLAF tags, of which 87,162 were polymorphic. Each tag was 264–364 bp long. The obtained 724,773 population SNPs were used to investigate genetic relationships within three species of Miscanthus. We constructed a phylogenetic tree of the 50 germplasms using the obtained SNPs and grouped them into two clades: one clade comprised of M. sinensis alone and the other one included the offspring, M. lutarioriparius, and M. sacchariflorus. Genetic cluster analysis had revealed that M. lutarioriparius germplasm C3 was the most likely male parent of the offspring. Conclusions As a high-throughput sequencing method, SLAF-seq can be used to identify informative SNPs in Miscanthus germplasms and to rapidly characterize genetic relationships within this genus. Our results will support the development of breeding programs with the focus on utilizing Miscanthus cultivars with elite biomass- or fiber-production potential for the developing bioeconomy.

Unique Associations of DNA Methylation Regions With 24-Hour Blood Pressure Phenotypes in Blacks

Background: Epigenetic marks (eg, DNA methylation) may capture the effect of gene-environment interactions. DNA methylation is involved in blood pressure (BP) regulation and hypertension development; however, no studies have evaluated its relationship with 24-hour BP phenotypes (daytime, nighttime, and 24-hour average BPs). Methods: We examined the association of whole blood DNA methylation with 24-hour BP phenotypes and clinic BPs in a discovery cohort of 281 Blacks using reduced representation bisulfite sequencing. We developed a deep and region-specific methylation sequencing method, Bisulfite ULtrapLEx Targeted Sequencing and utilized it to validate our findings in a separate validation cohort (n=117). Results: Analysis of 38 215 DNA methylation regions (MRs), derived from 1 549 368 CpG sites across the genome, identified up to 72 regions that were significantly associated with 24-hour BP phenotypes. No MR was significantly associated with clinic BP. Two to 3 MRs were significantly associated with various 24-hour BP phenotypes after adjustment for age, sex, and body mass index. Together, these MRs explained up to 16.5% of the variance of 24-hour average BP, while age, sex, and BMI explained up to 11.0% of the variance. Analysis of one of the MRs in an independent cohort using Bisulfite ULtrapLEx Targeted Sequencing confirmed its association with 24-hour average BP phenotype. Conclusions: We identified several MRs that explain a substantial portion of variances in 24-hour BP phenotypes, which might be excellent markers of cumulative effect of factors influencing 24-hour BP levels. The Bisulfite ULtrapLEx Targeted Sequencing workflow has potential to be suitable for clinical testing and population screenings on a large scale.

Caving Impacts on Cave Microbial Diversity: Case Study of Morca Cave, Turkey

Some microorganisms identified in cave ecosystems have been reported to play a permanent and significant role for maintaininglife in such environments. Human entrance into caves can induce some changes on cave physic-ochemical parameters which altimately affects the living organisms. In this regard, for the first time, Morca Cave was explored to evaluate the impacts that human activities may have on the microbial diversity of the cave in a limited period of time. During this expedition at a depth of 1040 m, a camp was established for four days. Before the installation and at the end of the camp, sediments and surface samples were taken from different points of the camp area and the area around it. Sequencing of 16s rRNA of each sample was performed using the next generation sequencing method. The profile of the microbial diversity before the camping reaveled that Thermoplasmata dominated the archaea group and Gamma- and Alpha-proteobacteria were the most dominant bacterial group. After the camp, a decrease in the microbial diversity especially the previously mentioned classes strains is observed at the most of the sampled areas. The results also showed that Bacilli strains significantly increased after the camp and increase of Bacteroidia strains is observed at the most active sampled areas. This present study therefore highlights how microbial diversity inside a closed cave can respond to the human activities within a short period. Furthermore, it may constitute a solid basis to support efforts targeted at improving technics for cave management and expedition for the conservation of cave nature.

Strategies for Reducing Ruminant Methane Emissions

The paper studies the effect of additional administration of ultrafine particles on the cattle rumen microbiome composition. The in vitro method was used using the ANKOM Daisy II incubator according to a specialized method. Microflora analysis was performed using MiSeq (Illumina, USA) by a new generation sequencing method with a MiSeq reagent kit. After a detailed analysis of the structure and composition of the microbial community in the contents of the rumen sampled for different diets, it was found that no significant differences were observed in the bacterial communities, with the exception of a slight shift in the Bacteroidetes/Firmicutes ratio. However, we observed numerical differences in the abundance of some representatives, namely, with additional inclusion of Fe and Cr2O3, decrease in the abundance of the methane-forming species Methanobrevibacter, Methanobacterium, Methanosphaera, and Methnaomicrobium was noted regarding the control.

A Cross Sectional Sampling Reveals Novel Coronaviruses in Bat Populations of Georgia

Mammal-associated coronaviruses have a long evolutionary history across global bat populations, which makes them prone to be the most likely ancestral origins of coronavirus-associated epidemics and pandemics globally. Limited coronavirus research has occurred at the junction of Europe and Asia, thereby investigations in Georgia are critical to complete the coronavirus diversity map in the region. We conducted a cross-sectional coronavirus survey in bat populations at eight locations of Georgia, from July to October of 2014. We tested 188 anal swab samples, remains of previous pathogen discovery studies, for the presence of coronaviruses using end-point pan-coronavirus RT-PCR assays. Samples positive for a 440 bp amplicon were Sanger sequenced to infer coronavirus subgenus or species through phylogenetic reconstructions. Overall, we found a 24.5% positive rate, with 10.1% for Alphacoronavirus and 14.4% for Betacoronavirus. Albeit R. euryale, R. ferrumequinum, M. blythii and M. emarginatus were found infected with both CoV genera, we could not rule out CoV co-infection due to limitation of the sequencing method used and sample availability. Based on phylogenetic inferences and genetic distances at nucleotide and amino acid levels, we found one putative new subgenus and three new species of Alphacoronavirus, and two new species of Betacoronavirus.

Unknown Mutation Detection via Restriction Hybridization Method Instead of Using Next Generation Sequencing Method

In biology mutation is a change in the nucleotide sequences of the DNA of an organism Mainly there are three types of mutation: point mutation, deletion and insertions. Once the mutation has been defined allele specific oligonucleotide hybridization, amplification, heteroduplex formation method referred to as a diagnostic method some advance technique like CRISPR cas9 system is using for selected mutagenesis. Using restriction method system we can detect a mutation. Let’s say you have a DNA sample with fluorescent labeled from patient and you want to make sure that gene you are interested is in healthy gene. We can design different short fragment sequences to scan through DNA or find specific gene or mutation. The sequences scan the DNA if the sequences does not find targeted gene it does not bind to it its means that no fluorescence color appears under UV-light each different short fragment sequences is label with different colors. If the different short fragments sequence does not bind to the DNA or specific gene or area this means that there will be no color appear under UV light this part or gene will be separated from the DNA by using Restriction enzyme to do a Sanger sequencing gel electrophoresis. Result of the Sanger sequencing will provide the information about sequence of unknown part or gene of the DNA this method is easier and cost economic method instead of Next generation sequencing method [1-7].

The Risk of Prostate Cancer Associated With Common Mutations of AR Gene in Iranian Population: a Perspective Study of Personalized Treatment

Background: Prostate cancer (PC) is one of the most common cancers among men. Genetic predisposition is emerging as a risk factor for PC development. The Androgen receptor (AR) gene is associated with the development and prognosis of PC. Understanding the AR mutations is very important in the precision treatment of PC-resistant patients to androgen deprivation therapy. In this study, we investigate any association between common AR mutations with the risk of PC.Methods and results: In this case-control study, blood samples were collected from 121 radical prostatectomy (RP) patients who were pathologically diagnosed with PC and 120 benign prostate hyperplasia (BPH) subjects as a control group. The targeted area of the AR gene was amplified by PCR and confirmed by the Sanger sequencing method. The target area of the AR gene screened for 124 alterations in intron 7, 44 mutations in exon 8, and 52 variants in the 3'UTR region. rs113528927 DelIns AC>ACACACCAC had the most frequent mutant alleles between case and control groups, but this genotype distribution among the two recruited groups was not significant. Only one mutation, c.2644C>A, was observed in exon 8 in BPH subjects, and six alterations were detected in 3'UTR.Conclusions: For the first time in the Iranian population, AR common mutations were screened in PC patients, and our results indicate no relationship with the risk of PC, which means that other potential molecular risk factors may be engaged for PC in our population.