SNP Analysis of TLR4 Promoter and Its Transcriptional Factor Binding Profile in Relevance to Bovine Subclinical Mastitis.

Bovine mastitis is a complex infectious disease that develops in the mammary gland, predominantly caused by a bacterial infection of mammary tissue. Genetic variability of mastitis is well established and depends upon different quantitative trait loci (QTL) related to mastitis resistance or susceptibility. The susceptibility is often attributed to single-nucleotide polymorphisms (SNPs) in the variable cow breed genomes. Several global investigative attempts have resulted in studies mapping mastitis to the variations in the relevant genes. Reports have been attributed to dramatic genetic expression changes in Toll-Like Receptor 4 (TLR4) genes in mastitis-positive cows. However, the mechanism behind this variable genetic expression of TLR4 genes has been studied poorly. The present study aims to investigate SCM through various screening tests like somatic cell count (SCC), electric conductivity (EC), pH, and California mastitis test (CMT) in milk samples. This study also aims to investigate possible mechanisms behind this variable expression of TLR4 by comparative SNP evaluation and transcriptional factor profile mining. So that the important genetic mutations and effects thereof can be exploited in selecting specific breeds with higher mastitis resistance and milk yield. Seventy Holstein Frisian (HF) crossbred dairy cows were selected in the present study. The animals were screened based on various diagnostic tests (SCC, pH, EC, and CMT). Blood samples (5 mL) were collected for extraction of DNA followed by amplification of PPR1 and PPR2 of the promoter region and 5'UTR of the bovine TLR4 gene using specific primers. Sanger's enzymatic DNA sequencing technique sequenced the amplified PCR products. Further, the identification of SNPs was done through various bioinformatic tools used in this study. The findings of the present study revealed that CMT, EC, pH, and SCC could be used for the early detection of subclinical mastitis. In the present study, a significant increase in the EC, pH, and SCC in milk samples of animals affected with SCM was found in comparison to the healthy animals. The present study also revealed 16 SNPs falling in TLR4 promoter and 5' untranslated region (5'UTR) sequences in mastitis-positive genotypes compared to reference genomes. The study also investigates the potential transcriptional factor program deployed in response to variable mastitis development resistance. In the present study, the allelic and genotype frequencies of all SNP variants in the three regions viz., PPR1, PPR2, and 5'UTR, were the same indicating the absence of heterozygous condition at the respective loci. The present study has wide applicability for researchers developing mastitis-resistant breeding programs and the data generated may aid in the selection of better genetic breeds. The transcription factor binding profiles can serve as concrete leads about the studies on bovine mastitis at the molecular level and may also aid global research groups working on transcription factor (TF)-based molecular pathology of mastitis.

Rapid arsenite oxidation by Paenarthrobacter nicotinovorans strain SSBW5: unravelling the role of GlpF, aioAB and aioE genes.

A novel arsenite resistant bacterial strain SSBW5 was isolated from the battery waste site of Corlim, Goa, India. This strain interestingly exhibited rapid arsenite oxidation with an accumulation of 5 mM arsenate within 24 h and a minimum inhibitory concentration (MIC) of 18 mM. The strain SSBW5 was identified as Paenarthrobacter nicotinovorans using 16S rDNA sequence analysis. Fourier-transformed infrared (FTIR) spectroscopy of arsenite-exposed cells revealed the interaction of arsenite with several important functional groups present on the cell surface, possibly involved in the resistance mechanism. Interestingly, the whole genome sequence analysis also clearly elucidated the presence of genes, such as GlpF, aioAB and aioE encoding transporter, arsenite oxidase and oxidoreductase enzyme, respectively, conferring their role in arsenite resistance. Furthermore, this strain also revealed the presence of several other genes conferring resistance to various metals, drugs, antibiotics and disinfectants. Further suggesting the probable direct or indirect involvement of these genes in the detoxification of arsenite thereby increasing its tolerance limit. In addition, clumping of bacterial cells was observed through microscopic analysis which could also be a strategy to reduce arsenite toxicity thus indicating the existence of multiple resistance mechanisms in strain SSBW5. In the present communication, we are reporting for the first time the potential of P. nicotinovorans strain SSBW5 to be used in the bioremediation of arsenite via arsenite oxidation along with other toxic metals and metalloids.

Arsenite biotransformation and bioaccumulation by Klebsiella pneumoniae strain SSSW7 possessing arsenite oxidase (aioA) gene.

Arsenite oxidizing Klebsiella pneumoniae strain SSSW7 isolated from shipyard waste Goa, India showed a minimum inhibitory concentration of 21 mM in mineral salts medium. The strain possessed a small supercoiled plasmid and PCR amplification of arsenite oxidase gene (aioA) was observed on plasmid as well as chromosomal DNA. It was confirmed that arsenite oxidase enzyme was a periplasmic protein with a 47% increase in arsenite oxidase activity at 1 mM sodium arsenite. Scanning electron microscopy coupled with electron dispersive X-ray spectroscopic (SEM-EDS) analysis of 15 mM arsenite exposed cells revealed long chains of cells with no surface adsorption of arsenic. Transmission electron microscopy combined with electron dispersive X-ray spectroscopic (TEM-EDS) analysis demonstrated plasma membrane disruption, cytoplasmic condensation and periplasmic accumulation of arsenic. The bacterial strain oxidized 10 mM of highly toxic arsenite to less toxic arsenate after 24 h of incubation. Fourier transformed infrared (FTIR) spectroscopy confirmed the interaction of arsenite with functional groups present on the bacterial cell surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of 5 mM arsenite exposed cells demonstrated over-expression of 87 kDa and 14 kDa proteins of two subunits aioA and aioB of heterodimer arsenite oxidase enzyme as compared to control cells. Therefore, this bacterial strain might be employed as a potential candidate for bioremediation of arsenite contaminated environmental sites.

Phosphatase mediated bioprecipitation of lead as pyromorphite by Achromobacter xylosoxidans.

Achromobacter xylosoxidans strain SJ11, tolerating up to 4.0 mM lead nitrate, in a defined minimal medium was isolated from the waste of a battery manufacturing industry, Goa, India. Interestingly, it formed white precipitate on exposure to lead nitrate which was also evident from scanning electron micrograph (SEM). Energy dispersive X-ray spectroscopic analysis revealed the presence of lead (48.5% by weight) along with phosphorus and chlorine in the precipitate. Transmission electron microscopy (TEM) of bacterial cells clearly refuted the possibility of intracellular lead uptake confirming extracellular precipitation as a predominant mechanism of lead resistance in this bacterium. The extracellular precipitate was further identified as pyromorphite [Pb5(PO4)3Cl] by X-ray diffraction analysis. This was also corroborated by fourier transformed infrared spectroscopy (FTIR) indicating a significant involvement of phosphate groups. Atomic absorption spectroscopic analysis clearly demonstrated that 465.8 mg g-1 lead was precipitated by the bacterial cells. There was remarkable increase of 160% in phosphatase activity suggesting it's important role in lead precipitation. This was further substantiated by significant up-regulation of phosphatase, CheZ using LC-MS/MS. Therefore phosphatase mediated extracellular precipitation of lead as pyromorphite by A. xylosoxidans strain SJ11 clearly demonstrated it's potential in bioremediation of lead contaminated environmental sites.

Immulina® mitigates the development of illness when administered during the prodromal period of influenza viral infection in mice (Part 2).

BACKGROUND: Immulina®, a dietary supplement derived from Limnospira (formerly Arthrospira), is being investigated as a potential agent to increase antiviral resilience. In our recently published manuscript, we described the effects of Immulina® on influenza when taken daily, beginning before infection (prophylaxis) or after the onset of clinical symptoms of viral illness (therapeutic). However, the benefit of Immulina® in infected individuals before the manifestation of any symptoms (prodromal) has not been investigated yet. PURPOSE: To evaluate Immulina®'s potential use to increase the host antiviral immune response using a prodromal therapy regime. STUDY DESIGN: The efficacy of Immulina® extract was evaluated in rodents using a prodromal protocol (test material administered prior to the emergence of viral illness symptoms). METHODS: Immulina® (25, 50 and 100 mg/kg body weight) was orally administered to both genders of mice, 2 h following influenza A viral infection, and continued daily for 14 days. RESULTS: Compared to the infected control mice, animals fed Immulina® exhibited statistically significant reduction in the emergence of various physical symptoms of viral-induced illness and decreased viral RNA levels. The effects are likely mediated through the host immune system since the level of various cytokines (IL-6 and IFN-γ) were significantly increased in lung tissue. CONCLUSION: This study, together with our previous paper, indicate that Immulina® was most effective at enhancing immune antiviral resilience if administered before or soon after initial infection. The data generated can be used to guide additional research using human subjects.

Prophylactic and therapeutic mouse models for evaluating immunologic resilience to infection with influenza virus by Immulina® (Part 1).

BACKGROUND: Illness resulting from influenza is a global health problem that has significant adverse socioeconomic impact. Although various strategies such as flu vaccination have beneficial effects, the risk of this illness has not been eliminated. The use of botanicals may provide a complementary approach by enhancement of the host antiviral immune response. PURPOSE: Generate preclinical data using rodent models to determine the most effective utility of a Limnospira (formerly Arthrospira)-derived oral supplement (Immulina®) for enhancing host immunity to improve antiviral resilience. STUDY DESIGN: Two non-lethal mouse models (prophylactic and therapeutic) were used to evaluate the impact of Immulina® on increasing host resilience against experimental influenza infection. METHODS: Mice were fed Immulina® only for the 2 weeks prior to viral infection (prophylactic regime) or starting 3 days post-viral infection (at the onset of symptoms, therapeutic design). Three doses of Immulina® were evaluated in each model using both female and male mice. RESULTS: Significant protective effect of Immulina® against viral illness was observed in the prophylactic model (improved clinical scores, less body weight loss, decreased lung/body weight ratio, lower lung viral load, and increased lung IFN-γ and IL-6). Substantially less (minimal) protective effect was observed in the therapeutic model. CONCLUSION: This study demonstrates that Immulina® exerts a protective effect against influenza illness when administered using a prophylactic regime and may not be effective if given after the onset of symptoms. The results will help to optimally design future clinical trials.

Isolation and identification of Aeromonas caviae strain KS-1 as TBTC- and lead-resistant estuarine bacteria.

Tributyltin chloride (TBTC)- and lead-resistant estuarine bacterium from Mandovi estuary, Goa, India was isolated and identified as Aeromonas caviae strain KS-1 based on biochemical characteristics and FAME analysis. It tolerates TBTC and lead up to 1.0 and 1.4 mM, respectively, in the minimal salt medium (MSM) supplemented with 0.4 % glucose. Scanning electron microscopy clearly revealed a unique morphological pattern in the form of long inter-connected chains of bacterial cells on exposure to 1 mM TBTC, whereas cells remained unaltered in presence of 1.4 mM Pb(NO3)2 but significant biosorption of lead (8 %) on the cell surface of this isolate was clearly revealed by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. SDS-PAGE analysis of whole-cell proteins of this lead-resistant isolate interestingly demonstrated three lead-induced proteins with molecular mass of 15.7, 16.9 and 32.4 kDa, respectively, when bacterial cells were grown under the stress of 1.4 mM Pb (NO3)2. This clearly demonstrated their possible involvement exclusively in lead resistance. A. caviae strain KS-1 also showed tolerance to several other heavy metals, viz. zinc, cadmium, copper and mercury. Therefore, we can employ this TBTC and lead-resistant bacterial isolate for lead bioremediation and also for biomonitoring TBTC from lead and TBTC contaminated environment.

Immunogenetic Role of IL17A Polymorphism in the Pathogenesis of Recurrent Miscarriage.

Interleukin-17A (IL17A) is a proinflammatory cytokine and is assumed to play an important role in fetal rejection. In order to evaluate the potential role of IL17A polymorphism in the pathogenesis of recurrent miscarriage (RM), serum IL17A levels were estimated by ELISA. Single-nucleotide polymorphism was assessed by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) using gene-specific primers and the EcoNI restriction enzyme. Serum IL17A levels were nonsignificantly (p > 0.5) low in RM patients compared with the control group. IL17A gene amplification by PCR yielded the undigested product of 815 bp, and its digestion with EcoNI enzyme produced 815, 529, 286, and 270 bp fragments for the GG genotype; 529, 286, and 270 bp fragments for the GA genotype; and 529 and 286 bp fragments for the AA genotype. The genotype frequency between the RM and control groups exhibited a significant difference (p = 0.001), whereas no significant difference was observed between allele frequencies in the two groups (p = 0.0954). These data suggest that the IL17A gene polymorphism exhibits no significant effect on IL17A gene expression. However, it significantly decreases and increases RM risk in the homozygous and recessive models, suggesting its potential pregnancy-protecting and -harming roles in the AA and GA + GG genotypes, respectively.

Isolation and Characterization of Flavonoid Naringenin and Evaluation of Cytotoxic and Biological Efficacy of Water Lilly (Nymphaea mexicana Zucc.).

Despite its limited exploration, Nymphaea mexicana Zucc. can be beneficial if pharmacology, isolation, and biological evaluation are given attention. It is an aquatic species that belongs to the family Nymphaeaceae. The thrust area of the work was the extraction, isolation, and biological evaluation of different extracts of the N. mexicana Zucc. plant. The primary goal of this research was to assess the antimicrobial, antioxidant, and anticancer activities of the extracts and to isolate the target naringenin compound. Comparative FT IR analysis of different extracts of this plant revealed the presence of functional groups of plant secondary metabolites, including polyphenols, flavonoids, terpenoids, esters, amines, glycosides, alkanes, alkaloids, fatty acids, and alcohols. Moderate free radical scavenging potential has been achieved for the various extracts via reducing power and DPPH assays. While cytotoxic activity was evaluated by colorimetric and lactate dehydrogenase cell viability tests on potent cancer cell lines. Lung adenocarcinoma epithelial cells (A-549), and breast cells (MC-7) were treated with MeOH extract. The antimicrobial activity against bacterial strains was evaluated using Gram-positive and -negative cultures, where maximum and minimum inhibition zones were recorded for different strains, including 1.6-25.6 µg/mL for Streptococcus aureus, using the agar well diffusion method. In addition, the anti-inflammatory activity of different extracts of N. mexicana Zucc. was evaluated in a nitrite radical scavenging assay with high concentrations of secondary metabolites, which are important against human pathogens and other diseases.

Rapid and efficient method to extract metagenomic DNA from estuarine sediments.

Metagenomic DNA from sediments of selective estuaries of Goa, India was extracted using a simple, fast, efficient and environment friendly method. The recovery of pure metagenomic DNA from our method was significantly high as compared to other well-known methods since the concentration of recovered metagenomic DNA ranged from 1185.1 to 4579.7 µg/g of sediment. The purity of metagenomic DNA was also considerably high as the ratio of absorbance at 260 and 280 nm ranged from 1.88 to 1.94. Therefore, the recovered metagenomic DNA was directly used to perform various molecular biology experiments viz. restriction digestion, PCR amplification, cloning and metagenomic library construction. This clearly proved that our protocol for metagenomic DNA extraction using silica gel efficiently removed the contaminants and prevented shearing of the metagenomic DNA. Thus, this modified method can be used to recover pure metagenomic DNA from various estuarine sediments in a rapid, efficient and eco-friendly manner.

Metallothionein assisted periplasmic lead sequestration as lead sulfite by Providencia vermicola strain SJ2A.

Lead resistant Providencia vermicola strain SJ2A was isolated from the waste of a battery manufacturing industry which could tolerate upto 3.0mM lead nitrate in the minimal medium. Interestingly, this isolate showed presence of a plasmid borne metallothionein gene, bmtA that matched significantly (96%) with that of Pseudomonas aeruginosa. Scanning electron micrographs of bacterial cells exposed to lead revealed a unique alteration in the cell morphology from rods to long inter-connected filaments. On the other hand, electron dispersive X-ray spectroscopy (EDX) clearly indicated no significant lead adsorption therefore, we speculated intracellular sequestration in this bacterial strain. Transmission electron micrographs of the bacterial cells exposed to lead evidently demonstrated periplasmic sequestration of lead which was also supported by Fourier transformed infrared spectroscopic (FTIR) analysis. The bacterium internalised 155.12mg Pb2+/g biomass as determined by atomic absorption spectroscopy. Subsequently, the accumulated lead was identified as lead sulfite by X-ray diffraction studies. Therefore P. vermicola strain SJ2A has potential to bioremediate lead contaminated environmental sites.