Authenticity of dried aromatic herbs and herbal powders for the ASU (ayurvedic, siddha, unani) drug formulations is a key of their clinical success. The DNA based authentication is an answer; ...however, extraction of PCR quality DNA from such material is often problematic due to the presence of various co-extracted PCR inhibitors. Here, we report a novel DNA isolation and purification method utilizing cow skim milk that successfully yields PCR quality DNA from the aromatic herbs and dried herbal powders. The improved method presented in this study could be used as an alternative to successfully extract PCR quality DNA from such plant materials. Further, we present a set of robust matK primers which could be used as plant barcoding resource in future studies.
The magnitude of the HIV epidemic in women requires urgent efforts to find effective preventive methods. Even though sex hormones have been described to influence HIV infection in epidemiological ...studies and regulate different immune responses that may affect HIV infection, the direct role that female sex hormones play in altering the susceptibility of target cells to HIV-infection is largely unknown. Here we evaluated the direct effect of 17-β-estradiol (E2) and ethinyl estradiol (EE) in HIV-infection of CD4(+) T-cells and macrophages. Purified CD4(+) T-cells and monocyte-derived macrophages were generated in vitro from peripheral blood and infected with R5 and X4 viruses. Treatment of CD4(+) T-cells and macrophages with E2 prior to viral challenge reduced their susceptibility to HIV infection in a dose-dependent manner. Addition of E2 2 h after viral challenge however did not result in reduced infection. In contrast, EE reduced infection in macrophages to a lesser extent than E2 and had no effect on CD4(+) T-cell infection. Reduction of HIV-infection induced by E2 in CD4(+) T-cells was not due to CCR5 down-regulation, but was an entry-mediated mechanism since infection with VSV-G pseudotyped HIV was not modified by E2. In macrophages, despite the lack of an effect of E2 on CCR5 expression, E2-treatment reduced viral entry 2 h after challenge and increased MIP-1β secretion. These results demonstrate the direct effect of E2 on susceptibility of HIV-target cells to infection and indicate that inhibition of target cell infection involves cell-entry related mechanisms.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Claudin-1 (CLDN1), a tight junction (TJ) protein, has recently been identified as an entry co-receptor for hepatitis C virus (HCV). Ectopic expression of CLDN1 rendered several non-hepatic cell lines ...permissive to HCV infection. However, little is known about the mechanism by which CLDN1 mediates HCV entry. It is believed that an additional entry receptor(s) is required because ectopic expression of CLDN1 in both HeLa and NIH3T3 cells failed to confer susceptibility to viral infection. Here we found that CLDN1 was co-immunoprecipitated with both HCV envelope proteins when expressed in 293T cells. Results from biomolecular fluorescence complementation assay showed that overexpressed CLDN1 also formed complexes with CD81 and low density lipoprotein receptor. Subsequent imaging analysis revealed that CLDN1 was highly enriched at sites of cell-cell contact in permissive cell lines, co-localizing with the TJ marker, ZO-1. However, in both HeLa and NIH3T3 cells the ectopically expressed CLDN1 appeared to reside predominantly in intracellular vesicles. The CLDN1-CD81 complex formed in HeLa cells was also exclusively distributed intracellularly, co-localizing with EEA1, an early endosomal marker. Correspondingly, transepithelial electric resistance, obtained from the naturally susceptible human liver cell line, Huh7, was much higher than that of the HeLa-CLDN1 cell line, suggesting that Huh7 is likely to form functional tight junctions. Finally, the disruption of TJ-enriched CLDN1 by tumor necrosis factor-α treatment markedly reduced the susceptibility of Huh7.5.1 cells to HCV infection. Our results suggest that the specific localization pattern of CLDN1 may be crucial in the regulation of HCV cellular tropism.
Tenofovir (TFV) has been widely used for pre-exposure prophylaxis of HIV-1 infection with mixed results. While the use of TFV in uninfected individuals for prevention of HIV-1 acquisition is actively ...being investigated, the possible consequences of TFV exposure for the HIV-target cells and the mucosal microenvironment are unknown. In the current study, we evaluated the effects of TFV treatment on blood-derived CD4⁺ T cells, monocyte-derived macrophages and dendritic cells (DC). Purified HIV-target cells were treated with different concentrations of TFV (0.001-1.0 mg/ml) for 2 to 24 hr. RNA was isolated and RT-PCR was performed to compare the levels of mRNA expression of nucleotidases and pro-inflammatory cytokine genes (MIP3α, IL-8 and TNFα) in the presence or absence of TFV. We found that TFV increases 5'-ecto-nucleotidase (NT5E) and inhibits mitochondrial nucleotidase (NT5M) gene expression and increases 5' nucleotidase activity in macrophages. We also observed that TFV stimulates the expression and secretion of IL-8 by macrophages, DC, and activated CD4⁺ T cells and increases the expression and secretion of MIP3α by macrophages. In contrast, TFV had no effect on TNFα secretion from macrophages, DC and CD4⁺ T cells. Our results demonstrate that TFV alters innate immune responses in HIV-target cells with potential implications for increased inflammation at mucosal surfaces. As new preventive trials are designed, these findings should provide a foundation for understanding the effects of TFV on HIV-target cells in microbicide trials.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The innate immune responses to virus infection are initiated by either Toll-like receptors (TLR3/7/8/9) or cytoplasmic double-stranded RNA (dsRNA)-recognizing RNA helicases RIG-I and MDA5. To avoid ...causing injury to the host, these signaling pathways must be switched off in time by negative regulators.
Through yeast-two hybrid screening, we found that an ubiquitin-like protein named protein linking integrin-associated protein to cytoskeleton 1(PLIC-1 or Ubiquilin 1) interacted with the Toll/interleukin-1 receptor (TIR) domain of TLR4. Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway. Concomitantly, reduction of endogenous PLIC-1 by short-hairpin interfering RNA (shRNA) enhanced TLR3 activation both in luciferase reporter assays as well as in new castle disease virus (NDV) infected cells. An interaction between PLIC-1 and Trif was confirmed in co-immunoprecipitation (Co-IP) and GST-pull-down assays. Subsequent confocal microscopic analysis revealed that PLIC-1 and Trif colocalized with the autophagosome marker LC3 in punctate subcellular structures. Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner.
Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Poor translatability of animal disease models has hampered the development of new inflammatory bowel disorder (IBD) therapeutics. We describe a preclinical, ex vivo system using freshly obtained and ...well-characterized human colorectal tissue from patients with ulcerative colitis (UC) and healthy control (HC) participants to test potential therapeutics for efficacy and target engagement, using the JAK/STAT inhibitor tofacitinib (TOFA) as a model therapeutic. Colorectal biopsies from HC participants and patients with UC were cultured and stimulated with multiple mitogens ± TOFA. Soluble biomarkers were detected using a 29-analyte multiplex ELISA. Target engagement in CD3
CD4
and CD3
CD8
T-cells was determined by flow cytometry in peripheral blood mononuclear cells (PBMCs) and isolated mucosal mononuclear cells (MMCs) following the activation of STAT1/3 phosphorylation. Data were analyzed using linear mixed-effects modeling,
test, and analysis of variance. Biomarker selection was performed using penalized and Bayesian logistic regression modeling, with results visualized using uniform manifold approximation and projection. Under baseline conditions, 27 of 29 biomarkers from patients with UC were increased versus HC participants. Explant stimulation increased biomarker release magnitude, expanding the dynamic range for efficacy and target engagement studies. Logistic regression analyses identified the most representative UC baseline and stimulated biomarkers. TOFA inhibited biomarkers dependent on JAK/STAT signaling. STAT1/3 phosphorylation in T-cells revealed compartmental differences between PBMCs and MMCs. Immunogen stimulation increases biomarker release in similar patterns for HC participants and patients with UC, while enhancing the dynamic range for pharmacological effects. This work demonstrates the power of ex vivo human colorectal tissue as preclinical tools for evaluating target engagement and downstream effects of new IBD therapeutic agents.
Using colorectal biopsy material from healthy volunteers and patients with clinically defined IBD supports translational research by informing the evaluation of therapeutic efficacy and target engagement for the development of new therapeutic entities. Combining experimental readouts from intact and dissociated tissue enhances our understanding of the tissue-resident immune system that contribute to disease pathology. Bayesian logistic regression modeling is an effective tool for predicting ex vivo explant biomarker release patterns.
Abstract We examined the antiviral activity of ADAR1 against HIV-1. Our results indicated that ADAR1 in a transfection system inhibited production of viral proteins and infectious HIV-1 in various ...cell lines including 293T, HeLa, Jurkat T and primary CD4 + T cells, and was active against a number of X4 and R5 HIV-1 of different clades. Further analysis showed that ADAR1 inhibited viral protein synthesis without any effect on viral RNA synthesis. Mutational analysis showed that ADAR1 introduced most of the A-to-G mutations in the rev RNA, in the region of RNA encoding for Rev Response Element (RRE) binding domain and in env RNA. These mutations inhibited the binding of rev to the RRE and inhibited transport of primary transcripts like gag, pol and env from nucleus to cytoplasm resulting in inhibition of viral protein synthesis without any effect on viral RNA synthesis. Furthermore, ADAR1 induced mutations in the env gene inhibited viral infectivity.
There are limited data on the immunological responsiveness of healthy intestinal tissue when it is cultured and stimulated ex vivo. Such an ex vivo model has the potential to be a valuable tool in ...understanding disease pathogenesis and as a preclinical tool for the assessment of candidate therapeutic agents used to treat inflammatory bowel disease (IBD).
We undertook a comprehensive study to evaluate ex vivo immunological responses of intestinal tissue and isolated mucosal mononuclear cells (MMC) to a broad range of stimuli.
Colorectal biopsies (explants) were obtained from healthy participants by flexible sigmoidoscopy and were placed either directly into culture or digested to isolate MMC prior to placement in culture. Explants or MMC were treated with polyinosinic:polycytidylic acid (Poly IC), phytohemagglutinin (PHA), lipopolysacccharides from E Coli (LPS), anti-CD3/CD28 antibodies, or IL-1β/TNF-α for 24 h. Supernatants were assayed for 40 inflammatory biomarkers using multiplexed enzyme-linked immunosorbent assay (ELISA). The isolated MMCs were further characterized using twelve color flow cytometry.
Explants have greater weight adjusted constitutive expression of inflammatory biomarkers than MMCs. Biomarker responses varied as a function of immunogen and use of intact tissue or isolated cells. PHA applied to intact explants was the most effective agent in inducing biomarker changes. Stimulation induced activated and memory cellular phenotypes in both explants and MMCs.
The breadth and magnitude of responses from intact and enzymatically digested intestinal tissue explants stimulated with exogenous immunogens are complex and vary by tissue form and treatment. Overall, PHA stimulation of intact explants produced the most robust responses in normal human colorectal tissue. This system could potentially serve as a preliminary model of the disease state, suitable for small scale screening of new therapeutic agents prior to using IBD patient derived tissue.
► MOF CuBTC was used for CO2–N2 separation by Vacuum Swing Adsorption (VSA). ► A six step single column heavy reflux VSA cycle was evaluated for MOF and Zeolite X. ► The CO2 separation performance of ...the MOF material was compared with Zeolite X. ► The CO2 purity achieved with Zeolite X was more than MOF CuBTC. ► However, CO2 recoveries obtained with MOF were more than Zeolite.
CO2–N2 separation by single column Vacuum Swing Adsorption was compared between a commercially available MOF (CuBTC) and a Zeolite X based adsorbent. A six-step heavy reflux VSA cycle configuration was employed in this study. For each adsorbent effect of different VSA parameters such as adsorption time, CO2 rinse time, evacuation time and evacuation with N2 purge time on CO2–N2 separation performance were studied. Results indicate that at low pressures, performance of Zeolite in terms of CO2 purity obtained was better than MOF for all the parameters studied. However, CO2 recoveries obtained with MOF were consistently more than obtained with Zeolite, which clearly indicates better regenerability of MOF.
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