Phylogenetic and Functional Diversity of Faecal Microbiome of Pack Animals

The present chapter describes the microbial diversity of faecal microbiomes of pack animals. The sequencing data generated through ion semiconductor sequencing technology were analysed using EBI metagenomics and MG‐RAST server tools. Bacteria were the major domain in all the pack animals. At the phylogenetic level, Firmicutes was the major phylum. Clostridiales was the major order. Ruminococcus flavefaciens was the major species in camel, whereas the top‐most species existing in Equidae family was Streptococcus equinus. Among the 28 major functional categories, protein metabolism functionality was dominant in pack animals. The genes associated with protein processing and modification as well as for protein folding are higher in mules and in camel they are lowest. Central carbohydrate metabolism was the major functional group under carbohydrate metabolism in pack animals. Variation in the amino acids and its derivatives was seen in pack animals. Genes associated with proline and 4‐hydroxy prolines were present in Equidae family only. Clustering using ward with Bray‐Curtis distance matrix for the functional categories showed that donkey and mule are most closely related and clustered with the horse metagenome

Cytotoxic effect of methanolic extracts and partially purified fractions of some medicinal plants used in traditional medication

In this study, the cytotoxic activity of methanolic extracts different parts of seven plant species was checked on NRK-52E (Rat renal proximal tubular cells) using MTT assay. Based on their cytotoxic activities, methanol extract of Vitex negundo (V. negundo) was selected and their partition in hexane, chloroform, ethylacetate, butanol and water was done. Among all fractions, chloroform fraction was most active on NRK-52E cells as determined by MTT assay. In NRK-52E cells induction of apoptosis was checked by analyzing DNA fragmentation by agarose gel electrophoresis. To study the molecular mechanism of apoptosis, expression levels of different genes BCL-2, BCL-Xl, SOD, TGF, Foxo and BAX were assessed using quantitative real-time PCR. Chloroform fraction of Vitex negundo (VnCE) was found to be highly antiproliferative and also showed DNA fragmentation in NRK-52E cells. VnCE showed up regulation of BCL-2, BCL-Xl, SOD, Foxo and BAX genes and down regulation of TGF gene.Cytotoxic activity, Apoptosis, DNA fragmentation assay, MTT assay, Real time PCR assa

Cytotoxic effect of methanolic extracts and partially purified fractions of some medicinal plants used in traditional medication

119-126In this study, the cytotoxic activity of methanolic extracts different parts of seven plant species was checked on NRK-52E (Rat renal proximal tubular cells) using MTT assay. Based on their cytotoxic activities, methanol extract of Vitex negundo (V. negundo) was selected and their partition in hexane, chloroform, ethylacetate, butanol and water was done. Among all fractions, chloroform fraction was most active on NRK-52E cells as determined by MTT assay. In NRK-52E cells induction of apoptosis was checked by analyzing DNA fragmentation by agarose gel electrophoresis. To study the molecular mechanism of apoptosis, expression levels of different genes BCL-2, BCL-Xl, SOD, TGF, Foxo and BAX were assessed using quantitative real-time PCR. Chloroform fraction of Vitex negundo (VnCE) was found to be highly antiproliferative and also showed DNA fragmentation in NRK-52E cells. VnCE showed up regulation of BCL-2, BCL-Xl, SOD, Foxo and BAX genes and down regulation of TGF gene

IDENTIFICATION OF PUTATIVE DRUG TARGETS IN MASTITIS CAUSING STAPHYLOCOCCUS AUREUS BY IN SILICO APPROACH

Objective: In the present study an attempt has been made by the use of a computational approach to investigate putative drug targets in Staphylococcus aureus.Methods: In silico comparative analysis of the metabolic pathways between the pathogen and the Bos taurus was carried out. Further detection of bacterial genes that are non homologous to host, but are essential for the survival of the pathogen represents a promising means of identifying novel drug targets. Metabolic pathways were obtained from the metabolic pathway database Kyoto Encyclopedia of Genes and Genomes (KEGG) and were compared to identify unique pathways present only in the pathogen and absent in the host.Results: We have identified total 1930 proteins, which are non homologous to Bos taurus protein sequences and among them 374 enzymes are found to be essential for survival of the S. aureus according to the database of essential genes (DEG) database. Further, 10 proteins were predicted as cytoplasmic and cell wall associated proteins, which could serve as potential drug target candidates.Conclusion: The identified potential drug targets form a platform for further investigation in discovery of novel therapeutic agents against S. aureus.Â

PREDICTION OF FUNCTIONAL, STRUCTURAL AND STABILITY CHANGES IN PMM2 GENE ASSOCIATED WITH NEPHROTIC SYNDROME USING COMPUTATIONAL ANALYSIS

Objective: Nephrotic syndrome defines as a disorder with a group of symptoms like proteinuria, hypoalbuminemia, hyperlipidemia, and edema. PMM2 encodes phosphomannosemutase protein enzyme involved in the synthesis of N-glycan. Methods: Different Insilico analysis tools: SIFT, PolyPhen, PROVEAN, SNPandGO, MetaSNP, PhDSNP, MutPred, I-Mutant, STRUM, PROCHECK-Ramachandran, COACH and ConSurf, were used to check the effect of nsSNP on protein structure and function. Results: The genetic polymorphism in the PMM2 gene was retrieved from NCBI ClinVar and UniProtKB. Total 20 SNPs were predicted most significant and responsible for disease-causing and decrease protein stability. Conclusion: This study helps to discover disease-causing deleterious SNPs with different computational tools and gives information about potent SNPs

Positioning canine induced pluripotent stem cells (iPSCs) in the reprogramming landscape of naïve or primed state in comparison to mouse and human iPSCs

Aims: Deriving canine-induced pluripotent stem cells (ciPSCs) have paved the way for developing novel cell-based disease models and transplantation therapies in the dog. Though ciPSCs have been derived in the presence of Leukemia inhibitory factor (LIF) as well in the presence of basic fibroblast growth factor (bFGF), the positioning of ciPSCs in the naïve or the primed state of pluripotency remains elusive. This study aims to understand whether canine iPSCs belong to naïve or prime state in comparison to mouse (m) iPSCs and human (h) iPSCs.// Main methods: In the present study, we derived ciPSCs in presence of LIF and compared their state of pluripotency with that of miPSCs and hiPSCs by culturing them in the presence of LIF, bFGF, and LIF + bFGF. Gene expression level at transcript level was performed by RT-PCR and qRT-PCR and at the protein level was analysed by immunofluorescence. We also attempted to understand the pluripotency state using lipid body analysis by bodipy staining and blue fluorescence emission.// Key findings: In contrast to miPSCs, the naïve pluripotent stem cells, ciPSCs showed the expression of FGF5 similar to that of primed pluripotent stem cell, hiPSCs. Compared to miPSCs, ciPSCs cultured in presence of LIF showed enhanced expression of primed pluripotent marker FGF5, similar to hiPSCs cultured in presence of bFGF. Upon culturing in hiPSC culture condition, ciPSCs showed enhanced expression of core pluripotency genes compared to miPSCs cultured in similar condition. However, ciPSCs expressed naïve pluripotent marker SSEA1 similar to miPSCs and lacked the expression of primed state marker SSEA4 unlike hiPSCs. Interestingly, for the first time, we demonstrate the ciPSC pluripotency using lipid body analysis wherein ciPSCs showed enhanced bodipy staining and blue fluorescence emission, reflecting the primed state of pluripotency. ciPSCs expressed higher levels of fatty acid synthase (FASN), the enzyme involved in the synthesis of palmitate, similar to that of hiPSCs and higher than that of miPSCs. As ciPSCs exhibit characteristic properties of both naïve and primed pluripotent state, it probably represents a unique intermediary state of pluripotency that is distinct from that of mice and human pluripotent stem cells.// Significance: Elucidating the pluripotent state of ciPSCs assists in better understanding of the reprogramming events and development in different species. The study would provide a footprint of species-specific differences involved in reprogramming and the potential implication of iPSCs as a tool to analyse evolution

Isolation of chitinolytic Clostridium sp. NCR from Mehsani buffalo rumen, its genomic analysis and potential role in rumen

AbstractGenomic analysis of Clostridium sp. NCR, an anaerobic Gram positive bacterium which was isolated from rumen fluid of Mehsani breed of buffalo revealed presence of various environmental gene tags (EGTs) involved in pathways for utilizing a wide range of substrates. Here we report the sequence of this rumen isolate, its whole genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession number JQHY00000000. The genome comprises of a 3.62-Mb draft genome with a G+C content of 28.10%, which encodes a total of 3126 proteins. Functional analysis provides information about the microbe's role in maintaining host homeostasis and its fiber degradation potential

Polimorfizam u području 3’UTR gena Slc11a1 u indijskih pasmina goveda.

The solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1 (Slc11a1) also called natural resistance-associated macrophage protein 1 gene (Nramp1) is a member of the large family of metal ion-transport proteins. It encodes a divalent cation (Fe+ & Mn+) transporter that localizes in the phagolysosome membrane in macrophages. Slc11a1 gene plays a critical role in innate immunity favoring bacterial killing by macrophages in addition to its influence on adaptative immunity. Polymorphism at the 3’ untranslated region (3’UTR) of Slc11a1 gene is associated with natural resistance against brucellosis in cattle. Such polymorphisms are associated with variations in the number of GT repeats. This study aimed to discover polymorphism in the 3’UTR region of the Slc11a1 gene in the Indian cattle breeds Nimari and Kenkatha. Polymerase Chain Reaction - Single Strand Conformation Polymorphism (PCR-SSCP) of 440 bp amplicon of Slc11a1 gene revealed three common SSCP patterns in these breeds, which was also confirmed by detecting point mutation in sequences of these patterns. The study will augment the information available and be useful in further studies to determine the role of the Slc11a1 gene in disease resistance and for the selection of brucellosis resistant animals.Među proteinima prijenosnicima metalnih iona, familija 11 i njezin član 1 (Slc11a1) zauzimaju značajno mjesto zbog činjenice da se povezuju s prirodnom imunošću. Na temelju toga za Slc11a1 učestalo se rabi i naziv gen za makrofagni protein 1 (Nramp1). Gen kodira protein-prijenosnik dvovalentnog kationa (Fe+ i Mn+) koji je smješten u fagolizosomu membrane makrofaga. Gen Slc11a1 ima važnu ulogu u nespecifičnoj imunosti, prvenstveno pri ubijanju bakterija makrofagima, no pretpostavlja se njegova dodatna uloga u specifičnoj imunosti. Polimorfizam u 3’ nekodirajućoj regiji (3’UTR) gena Slc11a1 osniva se na različitom broju GT ponavljanja i dovodi u vezu s prirodnom otpornošću goveda prema brucelozi. Ovim istraživanjem želi se utvrditi polimorfizam u 3’UTR regiji gena Slc11a1 u indijskih pasmina goveda, nimari i kenkatha. Primjenom lančane reakcije polimerazom odnosno analizom polimorfizma jednolančane konformacije amplikona s 440 baznih parova gena Slc11a1, u promatranih pasmina utvrđene su tri varijante jednolančane konformacije. Navedeno je također potvrđeno opažanjem točkastih mutacija u sekvencijama tih varijanata. Istraživanje će pridonijeti količini informacija iz predmetnog područja te biti korisno za buduća istraživanja koja imaju zadatak utvrditi ulogu gena Slc11a1 u otpornost na bolesti kao i u odabiranju životinja otpornih na brucelozu

Polimorfizam u području 3’UTR gena Slc11a1 u indijskih pasmina goveda.

The solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1 (Slc11a1) also called natural resistance-associated macrophage protein 1 gene (Nramp1) is a member of the large family of metal ion-transport proteins. It encodes a divalent cation (Fe+ & Mn+) transporter that localizes in the phagolysosome membrane in macrophages. Slc11a1 gene plays a critical role in innate immunity favoring bacterial killing by macrophages in addition to its influence on adaptative immunity. Polymorphism at the 3’ untranslated region (3’UTR) of Slc11a1 gene is associated with natural resistance against brucellosis in cattle. Such polymorphisms are associated with variations in the number of GT repeats. This study aimed to discover polymorphism in the 3’UTR region of the Slc11a1 gene in the Indian cattle breeds Nimari and Kenkatha. Polymerase Chain Reaction - Single Strand Conformation Polymorphism (PCR-SSCP) of 440 bp amplicon of Slc11a1 gene revealed three common SSCP patterns in these breeds, which was also confirmed by detecting point mutation in sequences of these patterns. The study will augment the information available and be useful in further studies to determine the role of the Slc11a1 gene in disease resistance and for the selection of brucellosis resistant animals.Među proteinima prijenosnicima metalnih iona, familija 11 i njezin član 1 (Slc11a1) zauzimaju značajno mjesto zbog činjenice da se povezuju s prirodnom imunošću. Na temelju toga za Slc11a1 učestalo se rabi i naziv gen za makrofagni protein 1 (Nramp1). Gen kodira protein-prijenosnik dvovalentnog kationa (Fe+ i Mn+) koji je smješten u fagolizosomu membrane makrofaga. Gen Slc11a1 ima važnu ulogu u nespecifičnoj imunosti, prvenstveno pri ubijanju bakterija makrofagima, no pretpostavlja se njegova dodatna uloga u specifičnoj imunosti. Polimorfizam u 3’ nekodirajućoj regiji (3’UTR) gena Slc11a1 osniva se na različitom broju GT ponavljanja i dovodi u vezu s prirodnom otpornošću goveda prema brucelozi. Ovim istraživanjem želi se utvrditi polimorfizam u 3’UTR regiji gena Slc11a1 u indijskih pasmina goveda, nimari i kenkatha. Primjenom lančane reakcije polimerazom odnosno analizom polimorfizma jednolančane konformacije amplikona s 440 baznih parova gena Slc11a1, u promatranih pasmina utvrđene su tri varijante jednolančane konformacije. Navedeno je također potvrđeno opažanjem točkastih mutacija u sekvencijama tih varijanata. Istraživanje će pridonijeti količini informacija iz predmetnog područja te biti korisno za buduća istraživanja koja imaju zadatak utvrditi ulogu gena Slc11a1 u otpornost na bolesti kao i u odabiranju životinja otpornih na brucelozu

Habitat provision is a major driver of native bird communities in restored urban forests

Urbanization, and the drastic loss of habitat it entails, poses a major threat to global avian biodiversity. Ecological restoration of urban forests is therefore increasingly vital for native bird conservation, but control of invasive predators may also be needed to sustain native bird populations in cities where species invasions have been particularly severe. We evaluated restoration success by investigating changes in native bird communities along a restoration chronosequence of 25 restored urban forests representing 72 years of forest development, which we compared to two target reference systems and a control system. We hypothesized that total species richness and relative abundance of native forest birds would increase with the age of restoration planting. We further hypothesized that relative abundance of rats, possums and cats would negatively impact native birds, while amount of native forest in the surrounding landscape would have a positive effect. We used structural equation modelling (SEM) to investigate the relative influence of forest structure (complexity index, tree height, canopy openness, basal area, species richness and density), landscape attributes (patch area, perimeter length, landscape composition within three buffer zones, distance to the nearest road and water source) and invasive mammalian predator indices of relative abundance on total species richness and relative abundance of native forest birds. Species richness increased with age of restoration planting, with community composition progressing towards that found in target reference systems. SEM revealed that years restored was a direct driver of bird species richness but an indirect driver of abundance, which was directly driven by canopy openness. Contrary to our predictions, invasive mammals had no significant effect on native bird species richness or abundance. Our results demonstrate that provision and improvement of habitat quantity and quality through restoration is the vital first step to re-establishing native forest bird communities in cities