MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo

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Secondary structure analysis of HIV-1-gp41 in solution and adsorbed to aluminum hydroxide by Fourier transform infrared spectroscopy

The formulation of human vaccines often includes adjuvants such as aluminum hydroxide that are added to enhance the immune responses to vaccine antigens. However, these adjuvants may also affect the conformation of antigenic proteins. Such structural modifications could lead to changes in antigenicity such that suboptimal protective immune responses could be generated relative to those induced by the vaccine antigens alone. Here, we used attenuated total reflectance infrared spectroscopy (ATR-FTIR) to compare the secondary structures of recombinant HIV-1-gp41 (gp41) in solution or adsorbed to aluminum hydroxide. The gp41 secondary structure content was 72% alpha-helices and 28% beta-sheets in 5 mM formate buffer p(2)H 2.5, while it was 66% beta-sheets and 34% random coil in acetonitril/(2)H(2)O (95/5:v/v). A fully reversible conformational change of gp41 in acetonitril/(2)H(2)O (95/5:v/v) was observed upon addition of either 35 mM formate p(2)H 2.5 or 0.1% (w/v) detergent (Tween 20, Hecameg, Brij 35 or beta-d-octyl-glucopyranoside). When gp41 was adsorbed to aluminum hydroxide in the presence of 0.1% (w/v) detergent, in either formate or in acetonitril/(2)H(2)O (95/5:v/v) its secondary structure remained stable and was identical to that of gp41 in 5 mM formate buffer p(2)H 2.5. The method described here could be applied for the characterization of gp41 conformers for use in immunological screening of antigens, and more generally to all antigenic proteins adsorbed to aluminum hydroxide.     

Induction of tumor-specific T-cell responses by vaccination with tumor lysate-loaded dendritic cells in colorectal cancer patients with carcinoembryonic-antigen positive tumors

Background Dendritic cells (DCs) are the most effective antigen-presenting cells. In the last decade, the use of DCs for immunotherapy of cancer patients has been vastly increased. High endocytic capacity together with a unique capability of initiating primary T-cell responses have made DCs the most potent candidates for this purpose. Although DC vaccination occasionally leads to tumor regression, clinical efficacy, and immunogenicity of DCs in clinical trials has not been yet clarified. The present study evaluated the safety and effectiveness of tumor-lysate loaded DC vaccines in advanced colorectal cancer (CRC) patients with carcinoembryonic antigen (CEA) positive tumors. Results Six patients HLA-A*0201-positive were vaccinated with autologous DCs loaded with tumor lysates (TL) together with tetanus toxoid antigen, hepatitis B, and influenza matrix peptides. Two additional patients were injected with DCs that were generated from their sibling or parent with one haplotype mismatch. All patients received the vaccines every 2 weeks, with a total of three intra-nodal injections per patient. The results indicated that DC vaccination was safe and well tolerated by the patients. Specific immune responses were detected and in some patients, transient stabilization or even reduction of CEA levels were observed. The injection of haplotype mismatched HLA-A*0201-positive DCs resulted in some enhancement of the anti-tumor response in vitro and led to stabilization/reduction of CEA levels in the serum, compared to the use of autologous DCs. Conclusion Altogether, these results suggest that TL-pulsed DCs may be an effective vaccine method in CRC patients. Elimination of regulatory mechanisms as well as adjustment of the vaccination protocol may improve the efficacy of DC vaccination. An erratum to this article can be found at     

Cd-induced growth reduction in the halophyte <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> is significantly improved by NaCl

The effects of Cd²⁺ and NaCl, applied together or separately, on growth and uptake of Cd²⁺ were determined for the halophyte Sesuvium portulacastrum L. Seedlings were cultivated in the presence of 50 or 100 μmol L⁻¹ Cd²⁺ alone or combined with 100 or 400 mmol L⁻¹ NaCl. Data showed that alone, Cd²⁺ induced chlorosis, necrosis, and inhibited growth. Addition of NaCl to Cd²⁺-containing medium restored growth and alleviated the toxicity, however. NaCl also enhanced the amounts of Cd²⁺ accumulated in the shoots. All Cd²⁺ treatment reduced K⁺ and Ca²⁺ uptake and transport to the shoots. Accumulation of Na⁺ in the shoots was not affected by Cd²⁺, however. Thus S. portulacastrum maintained its halophytic characteristics in the presence of Cd²⁺. We suggest this halophyte could be used for phytoextraction of Cd²⁺ from salt-contaminated sites.     

Glutathione protects cells against arsenite-induced toxicity

To understand the role of glutathione (GSH) in the protection of cells from arsenite toxicity, we studied the mechanism of apoptotic cell death in cells genetically unable to synthesize GSH (GCS-2 cells). Arsenite stimulated an increase in protein ubiquitination in GCS-2 cells while the wild-type cells were unaffected. Arsenite treatment increased lipid peroxidation and induced ubiquitination of molecular chaperone Hsp90 and impaired its ability to bind cochaperone p50(Cdc-37) and client proteins Plk-1 and Cdk-4 in GCS-2 cells. Treatment with arsenite also partially inhibited proteasome activity in GCS-2 cells. In these cells stably transfected with GFP(u) (a reporter consisting of a short degron fused to the COOH-terminus of GFP), intracellular fluorescence increased, suggesting the accumulation of GFP aggregates. GCS-2 cells underwent apoptosis accompanied by release of cytochrome c into the cytoplasm. Taken together, these data suggest that a possible mechanism of arsenite-induced apoptosis is the accumulation of ubiquitinated proteins and impairment of the protein degradative pathway. Further, protection from arsenite-induced ubiquitination is mediated by GSH and to a lesser extent by available reducing equivalents in the cells.     

Involvement of COX-1 in A3 adenosine receptor-mediated contraction through endothelium in mice aorta

We investigated whether A(3) adenosine receptor (A(3)AR) is involved in endothelium-mediated contraction through cyclooxygenases (COXs) with the use of wild-type (WT) and A(3) knockout (A(3)KO) mice aorta. A(3)AR-selective agonist, Cl-IBMECA, produced a concentration-dependent contraction (EC(50): 2.9 +/- 0.2 x 10(-9) M) in WT mouse aorta with intact endothelium (+E) and negligible effects in A(3)KO +E aorta. At 10(-7) M, contractions produced by Cl-IBMECA were 29% in WT +E, while being insignificant in A(3)KO +E aorta. Cl-IBMECA-induced responses were abolished in endothelium-denuded tissues (-E), in both WT and A(3)KO aorta. A(3)AR gene and protein expression were reduced by 74 and 72% (P < 0.05), respectively, in WT -E compared with WT +E aorta, while being undetected in A(3)KO +E/-E aorta. Indomethacin (nonspecific COXs blocker, 10(-5) M), SC-560 (specific COX-1 blocker, 10(-8) M), SQ 29549 (thromboxane prostanoid receptor antagonist, 10(-6) M), and furegrelate (thromboxane synthase inhibitor, 10(-5) M) inhibited Cl-IBMECA-induced contraction significantly. Cl-IBMECA-induced thromboxane B(2) production was also attenuated significantly by indomethacin, SC-560, and furegrelate in WT +E aorta, while having negligible effects in A(3)KO +E aorta. NS-398 (specific COX-2 blocker) produced negligible inhibition of Cl-IBMECA-induced contraction in both WT +E and A(3)KO +E aorta. Cl-IBMECA-induced increase in COX-1 and thromboxane prostanoid receptor expression were significantly inhibited by MRS1523, a specific A(3)AR antagonist in WT +E aorta. Expression of both A(3)AR and COX-1 was located mostly on endothelium of WT and A(3)KO +E aorta. These results demonstrate for the first time the involvement of COX-1 pathway in A(3)AR-mediated contraction via endothelium.     

Dhole pack size variation: Assessing the effect of Prey availability and Apex predator

In multipredator systems, group sizes of social carnivores are shaped by the asymmetric intraguild interactions. Subordinate social carnivores experience low recruitment rates as an outcome of predation pressure. In South and Southeast Asia, the Tiger (Panthera tigris), Dhole (Cuon alpinus), and Leopard (Panthera pardus) form a widely distributed sympatric guild of large carnivores, wherein tigers are the apex predators followed by dhole and leopard. In this study, we attempted to understand the variation in pack size of a social carnivore, the dhole, at two neighboring sites in the Central Indian landscape. We further evaluated local‐scale patterns of variation in pack size at a larger scale by doing a distribution‐wide assessment across the dhole ranging countries. At the local scale, we found an inverse relationship between the density of tiger and pack size of dhole while accounting for variability in resources and habitat heterogeneity. Larger dhole packs (16.8 ± 3.1) were observed at the site where the tiger density was low (0.46/100 km²), whereas a smaller pack size (6.4 ± 1.3) was observed in the site with high tiger density (5.36/100 km²). Our results for the distribution‐wide assessment were concordant with local‐scale results, showing a negative association of pack size with the tiger densities (effect size −0.77) and a positive association with the prey abundance (effect size 0.64). The study advances our understanding to answer the age‐old question of “what drives the pack size of social predators in a multipredator system?” This study also highlights the importance of understanding demographic responses of subordinate predator for varying competitor densities, often helpful in making informed decisions for conservation and management strategies such as population recovery and translocation of species.     

Cross-amplification of ungulate microsatellite markers in the endemic Indian antelope or blackbuck (Antilope cervicapra) for population monitoring and conservation genetics studies in south Asia

The Indian antelope or blackbuck (Antilope cervicapra) is endemic to the Indian subcontinent, inhabiting scrublands and dry grasslands. Most of the blackbuck populations are small, isolated, and threatened by habitat fragmentation and degradation. Management of such disjunct populations requires genetic characterization, which is critical for assessing hazards of stochastic events and inbreeding. Addressing the scarcity of such information on the blackbuck, we describe a novel panel of microsatellite markers that could be used to monitor blackbuck demography and population genetic parameters using non-invasive faecal sampling. We screened microsatellites (n?=?40) that had been reported to amplify in bovid and cervid species using faecal samples of the blackbuck collected from Kaimoor Wildlife Sanctuary, Uttar Pradesh, India and its vicinities. We selected 12 markers for amplification using faecal DNA extracts (n?=?140) in three multiplex reactions. We observed a mean amplification success rate of 72.4% across loci (92.1–25.7%) with high allele diversity (mean number of alleles/locus?=?8.67?±?1.03). Mean genotyping error rates across the markers were low to moderate (allelic drop-out rate?=?0.09; false allele rate?=?0.11). The proportions of first- and second-order relatives in the study population were 0.69% and 6.21%, respectively. Based on amplification success, genotyping error rates and the probability of identity (P_ID), we suggest (i) a panel of five microsatellite markers (cumulative P_ID?=?1.24?×?10^–5) for individual identification and population monitoring and (ii) seven additional markers for conservation genetics studies. This study provides essential tools capable of augmenting blackbuck conservation strategies at the landscape level, integral to protecting the scrubland-grassland ecosystem.

     

Pluripotency state regulates cytoneme selectivity and self-organization of embryonic stem cells

To coordinate cell fate with changes in spatial organization, stem cells (SCs) require specific and adaptable systems of signal exchange and cell-to-cell communication. Pluripotent embryonic stem cells (ESCs) use cytonemes to pair with trophoblast stem cells (TSCs) and form synthetic embryonic structures in a Wnt-dependent manner. How these interactions vary with pluripotency states remains elusive. Here we show that ESC transition to an early primed ESC (pESC) state reduces their pairing with TSCs and impairs synthetic embryogenesis. pESCs can activate the Wnt/β-catenin pathway in response to soluble Wnt ligands, but their cytonemes form unspecific and unstable interactions with localized Wnt sources. This is due to an impaired crosstalk between Wnt and glutamate receptor activity and reduced generation of Ca²⁺ transients on the cytonemes upon Wnt source contact. Induced iGluR activation can partially restore cytoneme function in pESCs, while transient overexpression of E-cadherin improves pESC–TSC pairing. Our results illustrate how changes in pluripotency state alter the mechanisms SCs use to self-organize.