Slow Regulated Release of H2S Inhibits Oxidative Stress Induced Cell Death by Influencing Certain Key Signaling Molecules

Hydrogen sulphide (H₂S) is one of three gaseous signaling molecules after nitric oxide and carbon monoxide. Various H₂S donor compounds have been synthesized to study its physiological function. Among these compounds sodium hydrosulphide (NaHS), a donor of releasing H₂S rapidly have shown to be protective in certain neuronal cell line but several in vivo studies have generated conflicting data. Furthermore several slow releasing H₂S donors have been shown to have positive effects on cells in culture. The intracellular concentration of H₂S and hence its rate of production may be a factor in keeping the balance between its neuroprotective and toxic effects. The present study was undertaken to deduce how a rapid releasing H₂S donor (NaHS) as opposed to a slow releasing donor (ADTOH), affect oxidative stress related intracellular components and survival of RGC-5 cells. It was concluded that when RGC-5 cells are exposed to the toxic effects of glutamate in combination with buthionine sulfoxime (Glu/BSO), ADTOH was more efficacious in inhibiting apoptosis, scavenging reactive oxygen species (ROS), stimulation of glutathione (GSH) and gluthathione-S-transferase (GST). Western blot and qPCR analysis showed ADTOH increased the levels of Nrf2, HO-1, PKCα, p-Akt, Bcl-2 and XIAP but caused a decrease of Nfκβ and xCT greater than NaHS. This study is first to compare the efficacy of two H₂S donor drugs as potential neuroprotectants and demonstrate that slow regulated release of H₂S to cell culture can be more beneficial in inhibiting oxidative stress induced cell death.     

Suitability of tomato leaves for larval development of Potato Tuber Moth,Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

The potato tuber moth (PTM) is an important pest of the potato plant and its tuber. With the expansion of its geographic range, the PTM could be potential threat to the tomato (Solanum lycopersicum L.), a congeneric species of the potato. To assess that risk, we tested larval feeding and development of PTM on leaves of five cultivated tomato varieties namely Moneymaker, Campari, Ailsa Craig, LA3475, E6203 and one wild tomato variety S. pimpinellifolium. PTM larvae accepted all tested plant leaves and developed into adults. Larval development was fastest on the Ailsa Craig variety. Pupae developed fastest on the Moneymaker variety and slowest on LA3475. Host acceptance and survival were highest on Ailsa Craig and lowest on LA3475. The significantly highest male proportion occurred on LA3475 variety. The study showed that PTM could be a potential threat to tomato cultivation which is rapidly increasing in temperate regions owing to climate change.     

Probiotics L. acidophilus and B. clausii Modulate Gut Microbiota in Th1- and Th2-Biased Mice to Ameliorate Salmonella Typhimurium-Induced Diarrhea

Gut microbiota play important role in maintaining health. Probiotics are believed to augment it further. We aimed at comparing effects of probiotics, Lactobacillus acidophilus (LA) and Bacillus clausii (BC) (a) on the gut microbiota abundance and diversity and (b) their contributions to control intestinal dysbiosis and inflammation in Th1- and Th2-biased mice following Salmonella infection. We report how could gut microbiota and the differential immune bias (Th1 or Th2) of the host regulate host responses when challenged with Salmonella typhimurium in the presence and absence of either of the probiotics. LA was found to be effective in ameliorating the microbial dysbiosis and inflammation caused by Salmonella infection, in Th1 (C57BL/6) and Th2 (BALB/c)-biased mouse. BC was able to ameliorate Salmonella-induced dysbiosis and inflammation in Th2 but not in Th1-biased mouse. These results may support probiotics LA as a treatment option in the case of Salmonella infection.

     

Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously “undruggable” proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12–186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target “degradability.”     

Fc gamma RIIB1/SHIP-mediated inhibitory signaling in B cells involves lipid rafts

One type of membrane microdomain, enriched in glycosphingolipids and cholesterol and referred to as lipid rafts, has been implicated in the generation of activating signals triggered by a variety of stimuli. Several laboratories, including ours, have recently demonstrated that the B cell receptor (BCR) inducibly localizes to the rafts upon activation and that functional lipid rafts are important for BCR-mediated "positive" signaling. In the later phases of the immune response, coligation of the BCR and the inhibitory receptor Fc gamma RIIB1 leads to potent inhibition of BCR-induced positive signaling through the recruitment of the inositol phosphatase SHIP to Fc gamma RIIB1. One potential model is that the Fc gamma RIIB1 itself might be excluded from the rafts basally and that destabilization of raft-dependent BCR signaling might be part of the mechanism for the Fc gamma RIIB1-mediated negative regulation. We tested this hypothesis and observed that preventing BCR raft localization is not the mechanism for this inhibition. Surprisingly, a fraction of Fc gamma RIIB1 is constitutively localized in the rafts and increases further after BCR + FcR coligation. SHIP is actively recruited to lipid rafts under negative stimulation conditions, and the majority of Fc gamma RIIB1-SHIP complexes localize to lipid rafts compared with non-raft regions of the plasma membrane. This suggested that this negative feedback loop is also initiated in the lipid rafts. Despite its basal localization to the rafts, Fc gamma RIIB1 did not become phosphorylated after BCR alone cross-linking and did not colocalize with the BCR that moves to rafts upon BCR engagement alone (positive signaling conditions), perhaps suggesting the existence of different subsets of rafts. Taken together, these data suggest that lipid rafts play a role in both the positive signaling via the BCR as well as the inhibitory signaling through Fc gamma RIIB1/SHIP.     

Generation of Marburg virus-like particles by co-expression of glycoprotein and matrix protein

Marburg virus (MARV), the causative agent of a severe hemorrhagic fever, has a characteristic filamentous morphology. Here we report that co-expression of MARV glycoprotein and matrix protein (VP40) in mammalian cells leads to spontaneous budding of filamentous particles strikingly similar to wild-type MARV. In addition, these particles elicit an immune response in BALB/c mice. The generation of non-replicating Marburg virus-like particles (VLPs) should significantly facilitate the research on molecular mechanisms of MARV assembly and release. Furthermore, VLPs may be an excellent vaccine candidate against Marburg infection.