Identification of Novel Allelic Variants of Integrin Beta 2 (ITGB2) Gene and Screening for Bubaline Leukocyte Adhesion Deficiency Syndrome in Indian Water Buffaloes (Bubalus bubalis)

A fragment of 570 bp corresponding to exon 5 and 6 of integrin beta 2 (ITGB2) gene was amplified for screening D128G mutation in one hundred and fifty two buffaloes (Bubalus bubalis) which causes bovine leukocyte adhesion deficiency syndrome (BLAD) in cattle, as well as to ascertain polymorphism. TaqI PCR-RFLP revealed no such mutation thus indicating the absence of bubaline leukocyte adhesion deficiency (BuLAD) allele in animals under study. However, the polymorphism studies using MspI restriction enzyme revealed two genotypic patterns viz. AA pattern (bands of 293, 141, 105, and 31 bp) and BB pattern (bands of 293, 105, 77, 64, and 31 bp). The sequences of A and B alleles were submitted to the GenBank (EU853307 and AY821799).     

Pervaporation Aided Esterification of Carboxylic Acids with Ethanol Catalyzed by Porcine pancreatic Lipase

The results of pervaporation-coupled esterification of various carboxylic acids with ethanol catalyzed by Porcine pancreatic lipase are reported. The effect of lipase and substrate concentrations has been studied and the advantage of pervaporation on the equilibrium conversion has been deduced. The kinetics of reaction were analyzed with a three-parameter model which coupled the effect of pervaporation. The intrinsic kinetic constants for all the reactions were estimated and correlated with the carbon number, an indicator of hydrophobicity of the acids. It was found that the rate constant increases with decrease in carbon number. The experimental concentration profiles were simulated from the model for all the reactions and the model prediction was found to be reasonably good. The water permeability was also correlated well with acid hydrophobicity. The pervaporation coupled reaction efficiency, as represented by the reaction time for equilibrium conversion, was found to bear a profound relation to membrane surface area per unit volume of the reaction mixture (A/V). The time for equilibrium conversion was found to decrease with an increase in A/V value, reaching a minimum and then increasing with a further increase of A/V. A probable explanation has been postulated for such an observation.     

Synthesis and Biological Activity of 6-Hydroxyguanidino-and 6-Hydroxyureidopurine And Their Ribonucleosides

Abstract

N ⁶ ?(1-hydroxyguanidino)purine IIa, and its 9-β-D-ribonucleoside derivative IIb were prepared by reacting at room temperature 6-hydroxyadenine Ia and 6-hydroxyadenosine Ib, with 1-guanyl-3,5-dimethylpyrazole nitrate in DMF. Refluxing IIa and IIb in 95% ethanol gave N⁶?(1-hydroxyureido)purine and its ribonucleoside derivative respectively; the latter compound was also obtained by refluxing Ib with 1-guanyl-3,5-dimethylpyrazole nitrate in ethanol. The two base analogs were inactive against L1210 cells in vitro, but the nucleoside derivatives inhibited the growth of these cells by 50% at 5 × 10 ^-6 and 6 × 10^?7 M respectively. Compound IIb, at 200 mg/kg/day × 5, increased the life span of L1210-bearing DBA/2N mice by 57%. Cytofluorometric determinations showed that IIb inhibited cell growth in the G₂ phase of the cell cycle. also found to inhibit adenosine deaminase activity with a K_i = 3.47 μM.     

Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.     

The Contribution of Non-Conventional T Cells and NK Cells in the Mycobacterial-Specific IFNγ Response in Bacille Calmette-Guérin (BCG)-Immunized Infants

Background

The Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is given to >120 million infants each year worldwide. Most studies investigating the immune response to BCG have focused on adaptive immunity. However the importance of TCR-gamma/delta (γδ) T cells and NK cells in the mycobacterial-specific immune response is of increasing interest.

Methods

Participants in four age-groups were BCG-immunized. Ten weeks later, in vitro BCG-stimulated blood was analyzed for NK and T cell markers, and intracellular IFNgamma (IFNγ) by flow cytometry. Total functional IFNγ response was calculated using integrated median fluorescence intensity (iMFI).

Results

In infants and children, CD4 and CD4-CD8- (double-negative (DN)) T cells were the main IFNγ-expressing cells representing 43-56% and 27-37% of total CD3+ IFNγ+ T cells respectively. The iMFI was higher in DN T cells compared to CD4 T cells in all age groups, with the greatest differences seen in infants immunized at birth (p=0.002) or 2 months of age (p<0.0001). When NK cells were included in the analysis, they accounted for the majority of total IFNγ-expressing cells and, together with DN Vδ2 γδ T cells, had the highest iMFI in infants immunized at birth or 2 months of age.

Conclusion

In addition to CD4 T cells, NK cells and DN T cells, including Vδ2 γδ T cells, are the key populations producing IFNγ in response to BCG immunization in infants and children. This suggests that innate immunity and unconventional T cells play a greater role in the mycobacterial immune response than previously recognized and should be considered in the design and assessment of novel tuberculosis vaccines.     

Smokeless Tobacco Extract (STE)-Induced Toxicity in Mammalian Cells is Mediated by the Disruption of Cellular Microtubule Network: A Key Mechanism of Cytotoxicity

Smokeless tobacco usage is a growing public health problem worldwide. The molecular mechanism(s) underlying smokeless tobacco associated tissue damage remain largely unidentified. In the present study we have tried to explore the effects of aqueous extract of smokeless tobacco (STE) on tubulin-microtubule, the major cytoskeleton protein that maintains cells morphology and participates in cell division. Exposure to STE resulted in dose-dependent cytotoxicity in a variety of mammalian transformed cell lines such as human lung epithelial cells A549, human liver epithelial cells HepG2, and mouse squamous epithelial cells HCC7, as well as non-tumorogenic human peripheral blood mononuclear cells PBMC. Cellular morphology of STE-treated cells was altered and the associated disruption of microtubule network indicates that STE targets tubulin-microtubule system in both cell lines. Furthermore it was also observed that STE-treatment resulted in the selective degradation of cellular tubulin, whereas actin remains unaltered. In vitro, polymerization of purified tubulin was inhibited by STE with the IC₅₀ value∼150 µg/ml and this is associated with the loss of reactive cysteine residues of tubulin. Application of thiol-based antioxidant N-acetyl cysteine (NAC) significantly abrogates STE-mediated microtubule damage and associated cytotoxicity in both A549 and HepG2 cells. These results suggest that microtubule damage is one of the key mechanisms of STE-induced cytotoxity in mammalian cells.     

Identification of Key Contributory Factors Responsible for Vascular Dysfunction in Idiopathic Recurrent Spontaneous Miscarriage

Poor endometrial perfusion during implantation window is reported to be one of the possible causes of idiopathic recurrent spontaneous miscarriage (IRSM). We have tested the hypothesis that certain angiogenic and vasoactive factors are associated with vascular dysfunction during implantation window in IRSM and, therefore, could play a contributory role in making the endometrium unreceptive in these women. This is a prospective case-controlled study carried out on 66 women with IRSM and age and BMI matched 50 fertile women serving as controls. Endometrial expression of pro-inflammatory (IL-1β, TNF-α, IFN-γ, TGF-β1), anti-inflammatory (IL-4, -10), angiogenesis-associated cytokines (IL-2, -6, -8), angiogenic and vasoactive factors including prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), nitric oxide (NO) and adrenomedullin (ADM) were measured during implantation window by ELISA. Subendometrial blood flow (SEBF) was assessed by color Doppler ultrasonography. Multivariate analysis was used to identify the significant factor(s) responsible for vascular dysfunction in IRSM women during window of implantation and further correlated with vascular dysfunction. Endometrial expression of pro-inflammatory cytokines and PGE2 were up-regulated and anti-inflammatory and angiogenesis-associated cytokines down-regulated in IRSM women as compared with controls. Further, the angiogenic and vasoactive factors including VEGF, eNOS, NO and ADM were found to be down-regulated and SEBF grossly affected in these women. Multivariate analysis identified IL-10, followed by VEGF and eNOS as the major factors contributing towards vascular dysfunction in IRSM women. Moreover, these factors strongly correlated with blood flow impairment. This study provides an understanding that IL-10, VEGF and eNOS are the principal key components having a contributory role in endometrial vascular dysfunction in women with IRSM. Down-regulation of these factors is also associated with impaired endometrial perfusion which possibly makes the endometrium unreceptive that may eventually cause early pregnancy loss.     

Microbiota Dynamics in Patients Treated with Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembraneous colitis and is responsible for a large and increasing fraction of hospital-acquired infections. Fecal microbiota transplantation (FMT) is an alternate treatment option for recurrent C. difficile infection (RCDI) refractory to antibiotic therapy. It has recently been discussed favorably in the clinical and scientific communities and is receiving increasing public attention. However, short- and long-term health consequences of FMT remain a concern, as the effects of the transplanted microbiota on the patient remain unknown. To shed light on microbial events associated with RCDI and treatment by FMT, we performed fecal microbiota analysis by 16S rRNA gene amplicon pyrosequencing of 14 pairs of healthy donors and RCDI patients treated successfully by FMT. Post-FMT patient and healthy donor samples collected up to one year after FMT were studied longitudinally, including one post-FMT patient with antibiotic-associated relapse three months after FMT. This analysis allowed us not only to confirm prior reports that RCDI is associated with reduced diversity and compositional changes in the fecal microbiota, but also to characterize previously undocumented post-FMT microbiota dynamics. Members of the Streptococcaceae, Enterococcaceae, or Enterobacteriaceae were significantly increased and putative butyrate producers, such as Lachnospiraceae and Ruminococcaceae were significantly reduced in samples from RCDI patients before FMT as compared to post-FMT patient and healthy donor samples. RCDI patient samples showed more case-specific variations than post-FMT patient and healthy donor samples. However, none of the bacterial groups were invariably associated with RCDI or successful treatment by FMT. Overall microbiota compositions in post-FMT patients, specifically abundances of the above-mentioned Firmicutes, continued to change for at least 16 weeks after FMT, suggesting that full microbiota recovery from RCDI may take much longer than expected based on the disappearance of diarrheal symptoms immediately after FMT.     

Association of Weather and Anthropogenic Factors for Transmission of Japanese Encephalitis in an Endemic Area of India

Weather and anthropogenic factors are important determinants for Japanese encephalitis (JE) transmission. During 2008–2010, an increasing trend of JE was observed in Dibrugarh district of Northeast India. The JE cases were found to be clustered between June to October in each year. Monthly minimum temperature and rainfall were significantly associated with JE transmission at 1 and 2 months lagged. However, the relationship was more prominent at a lag of 1 month than that of two. Regression analysis suggested that rainfall, minimum and maximum temperature, and relative humidity at 6:00 h are significant predictors (P < 0.05) of quarterly occurrence of JE cases. Additional anthropogenic risk factors including the conditions such as pig sty/cattle shed around and lower part of the houses and proximity of rice field to the dwelling houses (P < 0.05) were also found to be predictors for JE occurrence. Meteorological and anthropogenic risk factors can be used to forecast JE outbreaks in Assam which in turn can help the local health authorities to protect communities in JE prone areas.