Prophylactic or therapeutic administration of Agaricus blazei Murill is effective in treatment of murine visceral leishmaniasis

The present study aimed to investigate the in vitro antileishmanial activity of five fractions obtained from Agaricus blazei water extract (AbM), namely, Fab1, Fab2, Fab3, Fab4, and Fab5; and use the selected leishmanicidal fraction to treat BALB/c mice infected with Leishmania chagasi. A curve dose-titration was performed to obtain the concentration to be test in infected animals. In this context, Fab5 fraction and AbM were used in the doses of 20 and 100mg/kg/day, respectively, with the product been administered once a day. The effect induced by a chemo-prophylactic regimen, based on the administration Fab5 fraction and AbM 5days before infection, and maintained for an additional 20days post-infection was compared to a therapeutic regimen, in which the compounds were administered from 0 to 20days of infection. Control animals were either treated with amphotericin B deoxycholate (AmpB) or received distilled water. All groups were followed up for 10weeks post-infection, when parasitological and immunological parameters were analyzed. The Fab5 presented the best results of in vitro leishmanicidal activity. In the in vivo experiments, the use of Fab5 or AbM, as compared to control groups, resulted in significant reduced parasite burdens in the liver, spleen, and draining lymph nodes of the infected animals, as compared to control groups. A Type 1 immune response was observed in the Fab5 or AbM treated animals. No significant toxicity was observed. The chemo-prophylactic regimen proved to be more effective to induce theses responses. In this context, the data presented in this study showed the potential of the purified Fab5 fraction of AbM as a therapeutic alternative to treat visceral leishmaniasis. In addition, it can be postulated that this fraction can be also employed in a chemo-prophylactic regimen associated or not with other therapeutic products.     

Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F～P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F～P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.     

SLC33A1/AT-1 Protein Regulates the Induction of Autophagy Downstream of IRE1/XBP1 Pathway

One of the main functions of the unfolded protein response is to ensure disposal of large protein aggregates that accumulate in the lumen of the endoplasmic reticulum (ER) whereas avoiding, at least under nonlethal levels of ER stress, cell death. When tightly controlled, autophagy-dependent ER-associated degradation (ERAD(II)) allows the cell to recover from the transient accumulation of protein aggregates; however, when unchecked, it can be detrimental and cause autophagic cell death/type 2 cell death. Here we show that IRE1/XBP1 controls the induction of autophagy/ERAD(II) during the unfolded protein response by activating the ER membrane transporter SLC33A1/AT-1, which ensures continuous supply of acetyl-CoA into the lumen of the ER. Failure to induce AT-1 leads to widespread autophagic cell death. Mechanistically, the regulation of the autophagic process involves N(ε)-lysine acetylation of Atg9A.     

Novel mechanistic link between focal adhesion remodeling and glucose-stimulated insulin secretion

Actin cytoskeleton remodeling is well known to be positively involved in glucose-stimulated pancreatic β cell insulin secretion. We have observed glucose-stimulated focal adhesion remodeling at the β cell surface and have shown this to be crucial for glucose-stimulated insulin secretion. However, the mechanistic link between such remodeling and the insulin secretory machinery remained unknown and was the major aim of this study. MIN6B1 cells, a previously validated model of primary β cell function, were used for all experiments. Total internal reflection fluorescence microscopy revealed the glucose-responsive co-localization of focal adhesion kinase (FAK) and paxillin with integrin β1 at the basal cell surface after short term stimulation. In addition, blockade of the interaction between β1 integrins and the extracellular matrix with an anti-β1 integrin antibody (Ha2/5) inhibited short term glucose-induced phosphorylation of FAK (Tyr-397), paxillin (Tyr-118), and ERK1/2 (Thr-202/Tyr-204). Pharmacological inhibition of FAK activity blocked glucose-induced actin cytoskeleton remodeling and glucose-induced disruption of the F-actin/SNAP-25 association at the plasma membrane as well as the distribution of insulin granules to regions in close proximity to the plasma membrane. Furthermore, FAK inhibition also completely blocked short term glucose-induced activation of the Akt/AS160 signaling pathway. In conclusion, these results indicate 1) that glucose-induced activation of FAK, paxillin, and ERK1/2 is mediated by β1 integrin intracellular signaling, 2) a mechanism whereby FAK mediates glucose-induced actin cytoskeleton remodeling, hence allowing docking and fusion of insulin granules to the plasma membrane, and 3) a possible functional role for the Akt/AS160 signaling pathway in the FAK-mediated regulation of glucose-stimulated insulin secretion.     

Rearing of the fruit fly parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae) on X-ray irradiated larvae of Ceratitis capitata (Diptera: Tephritidae)

We evaluated the effects of X-ray irradiation on larvae of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), with the aim of finding a treatment that prevented adult fly emergence, yet did not adversely affect larval quality as rearing hosts for the parasitoid Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Ichneumonidae). Two experiments were carried out. In the first experiment, small numbers of larvae were held in Petri dishes with and without small amounts of rearing media and then irradiated with doses of X-rays ranging from 0 to 8333.6 R (equivalent to 80 Gy). In the second experiment, higher numbers of larvae were held in a manner resembling mass-rearing conditions, and were then irradiated with X-rays ranging from 0 to 10,417 R (equivalent to 100 Gy). In both experiments, the only factor that significantly affected fly emergence was irradiation of larvae. Fly emergence decreased markedly as the irradiation dose increased, and complete suppression of fly emergence was achieved at 6250.2 R (equivalent to 60 Gy) when larvae were irradiated in small batches with or without rearing media. Irradiation also affected the fertility of those flies that did emerge following treatment. In the second experiment, we found the parasitoids reared from irradiated larvae produced a higher parasitism rate and a higher number of female offspring than did parasitoids reared from control (non-irradiated) larvae. Mean fecundity of F1 parasitoids reared from irradiated larvae were affected positively by irradiation only at the 8333.6 R (80 Gy) dose. Our results show that X-ray irradiation can be used to inhibit fruit fly adult emergence and that irradiated larvae are at least as good a rearing substrate as non-irradiated larvae. Future studies should focus on the adjustment of our findings to a mass-rearing scale.     

Synthesis of enantiomerically pure perhydro-1,4-diazepine-2,5-dione and 1,4-piperazine-2,5-dione derivatives exhibiting potent activity as apoptosis inhibitors

Apoptosis is the process of programmed cell death and plays a fundamental role in several human diseases. We have previously reported the synthesis of the perhydro-1,4-diazepine-2,5-dione and 1,4-piperazine-2,5-dione derivatives as racemic mixtures. Compounds 1 and 2 showed a potent in vitro and in cellular extracts antiapoptotic activity. In view that the chiral discrimination has been an issue in the development and use of pharmaceutical drugs, the present contribution reports the synthesis of enantiopure peptidomimetics 1 and 2. The biological evaluation of these enantiomers as apoptosis inhibitors is also reported.     

Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins

Bacillus thuringiensis subs. israelensis produces at least three Cry toxins (Cry4Aa, Cry4Ba, and Cry11Aa) that are active against Aedes aegypti larvae. Previous work characterized a GPI-anchored alkaline phosphatase (ALP1) as a Cry11Aa binding molecule from the gut of A. aegypti larvae. We show here that Cry4Ba binds ALP1, and that the binding and toxicity of Cry4Ba mutants located in loop 2 of domain II is correlated. Also, we analyzed the contribution of ALP1 toward the toxicity of Cry4Ba and Cry11Aa toxins by silencing the expression of this protein though RNAi. Efficient silencing of ALP1 was demonstrated by real-time quantitative PCR (qPCR) and Western blot. ALP1 silenced larvae showed tolerance to both Cry4Ba and Cry11Aa although the silenced larvae were more tolerant to Cry11Aa in comparison to Cry4Ba. Our results demonstrate that ALP1 is a functional receptor that plays an important role in the toxicity of the Cry4Ba and Cry11Aa proteins.     

Manipulation of the Anoxic Metabolism in Escherichia coli by ArcB Deletion Variants in the ArcBA Two-Component System

Bioprocesses conducted under conditions with restricted O₂ supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative anaerobe Escherichia coli has elaborate sensing and signal transduction mechanisms for redox control in response to the availability of O₂ and other electron acceptors. The ArcBA two-component system consists of ArcB, a membrane-associated sensor kinase, and ArcA, the cognate response regulator. The tripartite hybrid kinase ArcB possesses a transmembrane, a PAS, a primary transmitter (H1), a receiver (D1), and a phosphotransfer (H2) domain. Metabolic fluxes were compared under anoxic conditions in a wild-type E. coli strain, its ΔarcB derivative, and two partial arcB deletion mutants in which ArcB lacked either the H1 domain or the PAS-H1-D1 domains. These analyses revealed that elimination of different segments in ArcB determines a distinctive distribution of d-glucose catabolic fluxes, different from that observed in the ΔarcB background. Metabolite profiles, enzyme activity levels, and gene expression patterns were also investigated in these strains. Relevant alterations were observed at the P-enol-pyruvate/pyruvate and acetyl coenzyme A metabolic nodes, and the formation of reduced fermentation metabolites, such as succinate, d-lactate, and ethanol, was favored in the mutant strains to different extents compared to the wild-type strain. These phenotypic traits were associated with altered levels of the enzymatic activities operating at these nodes, as well as with elevated NADH/NAD⁺ ratios. Thus, targeted modification of global regulators to obtain different metabolic flux distributions under anoxic conditions is emerging as an attractive tool for metabolic engineering purposes.     

Abundance, dynamics, and biogeographic distribution of seven polycyclic aromatic hydrocarbon dioxygenase gene variants in coastal sediments of Patagonia

Novel polycyclic aromatic hydrocarbon dioxygenase gene variants were present in abundances similar to or higher than those of phnA1 from Cycloclasticus spp. at a chronically polluted subantarctic coastal marine environment in Patagonia. These novel gene variants were detected over a 6-year time span and were also present in sediments from temperate Patagonian sites.     

Colloidal systems for drug delivery: from design to therapy

Nanomedicine, or medicine using nanometric devices, has emerged in the past decade as an exhilarating domain that can help to solve a number of problems linked to unsatisfactory therapeutic responses of so-called 'old drugs'. This dissatisfaction stems from inadequate biodistribution after a drug's application, which leads to a limited therapeutic response but also to numerous side effects to healthy organs. The biodistribution of drugs encapsulated in a nanoobject that will act as a vector can be modified to tune its therapeutic efficacy. This review provides a general overview of existing colloidal nanovectors: liposomes, polymeric micelles, polymeric vesicles, polymeric nanoparticles (NPs), and dendrimers. We describe their characteristics, advantages and drawbacks, and discuss their use in the treatment of various diseases.