Equid herpesvirus type 1 (EHV-1) disrupt actin cytoskeleton during productive infection in equine leukocytes

Equid herpesvirus type 1 (EHV-1) is a prevalent causative agent of equine diseases worldwide. After primary replication in the respiratory epithelium the virus disseminates systemically through a peripheral blood mononuclear cell (PBMC)-associated viraemia. EHV-1 is the only alphaherpes-virus known so far which is capable of establishing latent infection not only in neurons but also in immune system cells (mainly in lymphocytes and macrophages). Since leukocytes are not the target cells for viral replication but are used to transport EHV-1 to the internal organs, the question remains how the virus avoids the immune response and whether it could potentially be associated with virus-induced cytoskeletal rearrangements. Therefore, the aim of this study was to investigate the progress of EHV-1 replication in leukocytes stimulated by phytohemagglutinin and the impact of EHV-1 infection on the actin cytoskeleton. Using the real-time PCR method we evaluated the quantity of viral DNA from samples collected at indicated time points post infection. In order to examine possible changes in actin cytoskeleton organization due to EHV-1 infection, we performed immunofluorescent staining using TRITC-phalloidin conjugate. The results showed that EHV-1 replicates in leukocytes at a restricted level but with the accompaniment of chromatin degradation. Simultaneously, infection with EHV-1 caused disruption of the actin cytoskeleton; this was particularly apparent in further stages of infection. Disruption of the actin cytoskeleton may lead to the limited release of the virus from the cells, but may be also beneficial for the virus, since at the same time it potentially impairs the immune function of leukocytes

A Global Federated Real-World Data and Analytics Platform for Research

Objective This article describes a scalable, performant, sustainable global network of electronic health record data for biomedical and clinical research. Materials and Methods TriNetX has created a technology platform characterized by a conservative security and governance model that facilitates collaboration and cooperation between industry participants, such as pharmaceutical companies and contract research organizations, and academic and community-based healthcare organizations (HCOs). HCOs participate on the network in return for access to a suite of analytics capabilities, large networks of de-identified data, and more sponsored trial opportunities. Industry participants provide the financial resources to support, expand, and improve the technology platform in return for access to network data, which provides increased efficiencies in clinical trial design and deployment. Results TriNetX is a growing global network, expanding from 55 HCOs and 7 countries in 2017 to over 220 HCOs and 30 countries in 2022. Over 19 000 sponsored clinical trial opportunities have been initiated through the TriNetX network. There have been over 350 peer-reviewed scientific publications based on the network’s data. Conclusions The continued growth of the TriNetX network and its yield of clinical trial collaborations and published studies indicates that this academic-industry structure is a safe, proven, sustainable path for building and maintaining research-centric data networks

Drug Resistance Mutations in Drug-Naive HIV Type 1 Subtype C-Infected Individuals from Rural Malawi

In this preliminary study we show that in 2008, 3 years after antiretroviral therapy was introduced into the Karonga District, Malawi, a greater than expected number of drug-naive individuals have been infected with HIV-1 subtype C virus harboring major and minor drug resistance mutations (DRMs). From a sample size of 40 reverse transcriptase (RT) consensus sequences from drug-naive individuals we found five showing NRTI and four showing NNRTI mutations with one individual showing both. From 29 protease consensus sequences, again from drug-naive individuals, we found evidence of minor DRMs in three. Additional major and minor DRMs were found in clonal sequences from a number of individuals that were not present in the original consensus sequences. This clearly illustrates the importance of sequencing multiple HIV-1 variants from individuals to fully assess drug resistance

Drug resistance mutations in drug-naive hiv type 1 subtype c-infected individuals from rural malawi

In this preliminary study we show that in 2008, 3 years after antiretroviral therapy was introduced into the Karonga District, Malawi, a greater than expected number of drug-naive individuals have been infected with HIV-1 subtype C virus harboring major and minor drug resistance mutations (DRMs). From a sample size of 40 reverse transcriptase (RT) consensus sequences from drug-naive individuals we found five showing NRTI and four showing NNRTI mutations with one individual showing both. From 29 protease consensus sequences, again from drug-naive individuals, we found evidence of minor DRMs in three. Additional major and minor DRMs were found in clonal sequences from a number of individuals that were not present in the original consensus sequences. This clearly illustrates the importance of sequencing multiple HIV-1 variants from individuals to fully assess drug resistance

CpdA stimulation of the Hsp70 gene promoter occurs via a GR-dependent mechanism.

<p>(A) A549 cells were transfected with siControl or siRNA targeting GR (siGR). Total RNA or total protein extracts were prepared 48h post-transfection. In the left panel, purified mRNA was subjected to RT-qPCR detecting GR gene expression levels, normalized to housekeeping controls cyclophilin and 28S. For the siControl-transfected sample, signal was set at 100%. Data from SiGR-transfected cells were recalculated accordingly. Statistical analysis (unpaired t-test) was performed to show significant difference between siControl and siGR conditions (*** p<0.001). In the right panel, total cell lysates were subjected to Western blot analysis to detect GR protein, with NF-κB p65 as a loading control. (B) In parallel with (A) A549 cells were transfected with siControl or siGR. 41h post transfection, cells were induced with Solv or CpdA (10µM) for 8h. The derived purified mRNA was subjected to RT-qPCR detecting HSPA1A gene expression levels and specific results were normalized to housekeeping controls cyclophilin and 28S. The condition Solv (siControl) was set as 1 to allow ratio comparisons. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed for selected pair wise comparisons (ns not significant; * p<0.05). This experiment is representative for 2 independent experiments. (C) and (D) A549 cells, serum-starved for48h in 0% DMEM, were treated with Solv, Dex (1µM), CpdA (10 µM) for 2h, or exposed to a 43°C heat shock (HS) for 1h. Total cell extracts were subjected to a ChIP assay targeting GR. Ensuing, qPCR signal of immunoprecipitated HSPA1A and GILZ gene promoter fragments is presented relative to input data. Binding to rabbit IgG represents aspecific binding. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to show significant difference with the Solv condition (ns not significant; *** p<0.001). This experiment is representative for 2 independent experiments.</p

Both Compound A and heat shock diminish NF-κB-driven gene expression and NF-κB activation.

<p>(A) A549 cells, starved for 48h, were pretreated for 1.5h with solvent (Solv), DEX (1µM), CpdA (10µM) or subjected to heat shock treatment (1h at 43°C and 30′ recovery at 37°C), ensued with TNF (2000IU/ml) for 5.5h. Isolated total RNA was subjected to RT-qPCR assaying IL8 mRNA levels, normalized to cyclophilin household gene mRNA levels. The TNF condition was set at 100 and results were recalculated accordingly. These results are representative of 2 independent experiments. Statistical analysis (ANOVA and Tukey multiple comparison post test) were performed for selected pair-wise comparisons. (B) A549 cells, starved for 48h, were pretreated for 2h with solvent (Solv), DEX (1µM) or CpdA (10µM), after which TNF(2000IU/ml) was added as indicated. Western blot analysis of total cells lysates detects IκBα protein, with NF-κB p65 as loading control. This figure is representative for 2 independent experiments. (C) A549 cells, starved for 48h, were untreated or heat-shocked at 43°C for 2h. Ensuing, cells were treated by TNF (2000IU/ml) for the indicated times. Total cell lysates were analyzed as in (B), with tubulin as loading control. Results were obtained on 2 separate blots in one experiment. (D) A549 cells, starved for 48h in Optimem, were pretreated for 2h with Solv, DEX (1µM), CpdA (10µM) or 2h heat shock (HS) at 43°C. Subsequently, TNF (2000IU/ml) was added for 30′, where indicated. After washing, fixation, and permeabilization, indirect immunofluorescence detects endogenous NF-κB p65. DAPI staining indicates the nuclei. Additionally, we present overlays. This figure is representative for 2 independent experiments. (E) ImageJ integrated density analysis of the TNF-treated conditions in (D) allows statistical analysis (Mann-Whitney U test) and we show the P-value of comparisons to the Solv/TNF condition. These results are representative of 2 independent experiments.</p

Compound A augments Hsp70 gene expression.

<p>(A) A549 cells were left untreated or were induced with heat shock (HS) at 43°C for 2h either or not followed by 4h recovery time (Rec) at 37°C. Isolated total RNA was subjected to RT-qPCR for the detection of HSPA1A, normalized to cyclophilin housekeeping control. The non-induced condition was set as 1 to allow ratio comparisons. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed for selected pair wise comparisons (ns not significant; *p<0.05; ** p<0.01). These results are representative of three independent experiments. (B) A549 cells, treated with solvent (Solv) or CpdA (10µM), were either incubated at 37°C for 6 hours or subjected to the following temperature protocol : 2 hours at 37°C (pre-induction), followed by 2 hours at 43°C heat shock (HS) and lastly 2 hours at 37°C (recovery; Rec). Isolated total RNA was subjected to RT-PCR for the detection of HSPA1A and control GAPDH gene expression levels. The displayed bands were detected from one single gel. The respective bands were quantified using ImageJ software and normalized to GAPDH control expression levels. Solv was set as 1 and all other conditions were recalculated relative to this condition and expressed as relative mRNA expression level. The figure is representative for 2 independent experiments. (C) Eight week old female BALB/c mice were injected intraperitoneally with either PBS as a control or CpdA dissolved in PBS (5mg/kg or 10mg/kg) and 24h later total skin samples were resected and their respective mRNA samples were subjected to RT-qPCR analysis assaying for HSPA1A gene expression levels and normalized to RPL13a, HMBS and ACTB housekeeping controls. The non-induced condition was set as 1 to allow ratio comparisons. Statistical analysis (Mann Whitney-U-test) was performed for selected pair wise comparisons (ns not significant; ** p<0.01).</p

Compound A increases Hsp70 promoter activity dose-dependently and transiently.

<p>(A) L929sA cells, stably transfected with a mHsp70i-luc reporter gene construct, were left untreated (Untr) or were stimulated with heat shock at 43°C for 2h, followed by a recovery period of 2h at 37°C (HS+Rec). Normalized luciferase levels were presented as relative reporter gene activity with the condition ‘Untr’ set as 1. Statistical analysis (unpaired t-test) was performed. This figure is representative for 6 independent experiments. (B) L929sA cells, stably transfected with mHsp70i-luc, were induced for 8h with various concentrations of CpdA, as indicated. All samples were controlled to a similar amount of solvent. This figure is representative for 4 independent experiments. (C) L929sA cells, stably transfected with mHsp70i-luc, were treated with solvent (Solv) or CpdA (10µM) for 6h, 24h or 48h. Data were presented as relative reporter gene activity with the Solv condition set as 1. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to explore if Solv differs from CpdA treatment for each respective induction time. (D) A549 cells, were treated with solvent or CpdA 10µM for the indicated time period. Total cellular mRNA was subjected to RT-qPCR detecting gene expression levels for HSPA1A, normalized using housekeeping 36B4 and β-actin mRNA levels. Four independent experiments with slightly varying time kinetics all show comparable results. (E) PC-3 cells were starved for 48h in 0% DMEM, after which these cells were left untreated or treated with Compound A (10µM), as indicated. Purified mRNA was subjected to RT-qPCR detecting HSPA1A gene expression levels and specific results were normalized to housekeeping controls cyclophilin, GAPDH and 36B4. (B)(D)(E) Solv condition was set as 1 and results recalculated accordingly. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to compare with Solv (ns not significant; ** p<0.01; *** p<0.001).</p

The glucocorticoid receptor interacts with Hsp70 and Hsp90.

<p>HEK293T cells were transiently co-transfected with the MAPPIT GRα-bait plasmid, the STAT3-responsive rPAP1-luci reporter plasmid and prey-plasmids as indicated. Empty prey is used as a negative control. Twenty-four hours after transfection cells were stimulated with leptin (100ng/ml) and leptin in combination with DEX (1µM) or CpdA (10µM) for another 24h or were left untreated (NS). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (ratio stimulated/untreated).</p