Folding pathway mediated by an intramolecular chaperone: intrinsically unstructured propeptide modulates stochastic activation of subtilisin

Several secreted proteases are synthesized with N-terminal propeptides that function as intramolecular chaperones (IMCs) and direct the folding of proteases to their native functional states. Using subtilisin E as our model system, we had earlier established that (i) release and degradation of the IMC from its complex with the protease upon completion of folding is the rate-determining step to protease maturation and, (ii) IMC of SbtE is an extremely charged, intrinsically unstructured polypeptide that adopts an alpha-beta structure only in the presence of the protease. Here, we explore the mechanism of IMC release and the intricate relationship between IMC structure and protease activation. We establish that the release of the first IMC from its protease domain is a non-deterministic event that subsequently triggers an activation cascade through trans-proteolysis. By in silico simulation of the protease maturation pathway through application of stochastic algorithms, we further analyze the sub-stages of the release step. Our work shows that modulating the structure of the IMC domain through external solvent conditions can vary both the time and randomness of protease activation. This behavior of the protease can be correlated to varying the release-rebinding equilibrium of IMC, through simulation. Thus, a delicate balance underlies IMC structure, release, and protease activation. Proteases are ubiquitous enzymes crucial for fundamental cellular processes and require deterministic activation mechanisms. Our work on SbtE establishes that through selection of an intrinsically unstructured IMC domain, nature appears to have selected for a viable deterministic handle that controls a fundamentally random event. While this outlines an important mechanism for regulation of protease activation, it also provides a unique approach to maintain industrially viable subtilisins in extremely stable states that can be activated at will.     

The analog of cGAMP,c-di-AMP,activates STING mediated cell death pathway in estrogen-receptor negative breast cancer cells

Apoptosis - Immune adaptor protein like STING/MITA regulate innate immune response and plays a critical role in inflammation in the tumor microenvironment and regulation of metastasis including...     

Design, synthesis and pharmacological evaluation of conformationally restricted N-arylsulfonyl-3-aminoalkoxy indoles as a potential 5-HT6 receptor ligands

A series of novel conformationally restricted N(1)-arylsulfonyl-3-aminoalkoxy indoles were designed and synthesized as 5-HT(6) receptor (5-HT(6)R) ligands. Many of the synthesized compounds have moderate in vitro-binding affinities at 5-HT(6)R. The lead compound 8b (% inhibition?=?97.2 at 1 μM) from this series has good pharmacokinetic profile in male Wister rats and is active in animal model of cognition like Morris water maze. The details of chemistry, SAR, pharmacokinetics and pharmacological data constitute the subject matter of this report.     

Bacillus subtillis RTSBA6 6.00, a new strain isolated from gut of Helicoverpa armigera (Lepidoptera: Noctuidae) produces chymotrypsin-like proteases

Exploring bacterial communities with proteolytic activity from the gut of the Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) insect pests was the purpose of this study. As initial efforts to achieve this goal here we report the isolation of new Bacillus subtillis RTSBA6 6.00 strain from the gut of H. armigera and demonstrated as proteases producer. Zymographic analysis revealed 12 proteolytic bands with apparent molecular weights ranging from 20 to 185 kDa. Although some activity was detected at acidic pH, the major activity was observed at slight alkaline pH (7.8). The optimum temperature was found to be 35 °C with complete loss of activity at 70 °C. All proteases were completely inactivated by PMSF (phenylmethylsulfonyl fluoride) and TPCK (N-tosyl-l-phenylalanine chloromethyl ketone), suggesting that proteases secreted by B. subtillis RTSBA6 6.00 belong to serine proteases class with chymotrypsin-like activity. The occurrence of protease producing bacterial community in the gut of the H. armigera advocates its probable assistance to insect in proteinaceous food digestion and adaptation to protease inhibitors of host plants.     

Severe Human Influenza Infections in Thailand: Oseltamivir Treatment and Risk Factors for Fatal Outcome

Background

Influenza is often not recognized as an important cause of severe or fatal disease in tropical and subtropical countries in Southeast Asia. The extent to which Oseltamivir treatment may protect against a fatal outcome in severe influenza infections is not known. Thailand''s National Avian Influenza Surveillance (NAIS) system affords a unique opportunity to describe the epidemiology of laboratory-confirmed severe and fatal human influenza infections.

Methodology/Principal Findings

During January 2004 through December 2006, 11,641 notifications to the NAIS were investigated in 73 of 76 Thai provinces. Clinical and demographic data and respiratory swab specimens were collected and tested by PCR for influenza. Using the NAIS database, we identified all patients with laboratory confirmed human influenza (A/H3N2, A/H1N1 and Type B) infection. A retrospective medical record review was conducted on all fatal cases with laboratory confirmed influenza and from a sample of hospitalized cases in 28 provinces. The association of underlying risk factors, Oseltamivir treatment and risk of a fatal outcome were examined. Human influenza infections were identified in 2,075 (18%) cases. Twenty-two (1%) deaths occurred including seven deaths in children less than ten years of age. Thirty-five percent of hospitalized human influenza infections had chest X-ray confirmed pneumonia. Current or former smoking; advanced age, hypertension and underlying cardiovascular, pulmonary or endocrine disease were associated with a fatal outcome from human influenza infection. Treatment with Oseltamivir was statistically associated with survival with a crude OR of .11 (95% CI: 0.04–0.30) and .13 (95% CI: 0.04–0.40) after controlling for age.

Conclusions

Severe and fatal human influenza infections were commonly identified in the NAIS designed to identify avian A/H5N1 cases. Treatment with Oseltamivir is associated with survival in hospitalized human influenza pneumonia patients.     

Immunogenicity and safety of a respiratory syncytial virus fusion protein (RSV F) nanoparticle vaccine in older adults

Background

A preventative strategy for Respiratory Syncytial Virus (RSV) infection constitutes an under-recognized unmet medical need among older adults. Four formulations of a novel recombinant RSV F nanoparticle vaccine (60 or 90 μg RSV F protein, with or without aluminum phosphate adjuvant) administered concurrently with a licensed inactivated trivalent influenza vaccine (TIV) in older adult subjects were evaluated for safety and immunogenicity in this randomized, observer-blinded study.

Results

A total of 220 healthy males and females?≥?60 years of age, without symptomatic cardiopulmonary disease, were vaccinated concurrently with TIV and RSV F vaccine or placebo. All vaccine formulations produced an acceptable safety profile, with no vaccine-related serious adverse events or evidence of systemic toxicity. Vaccine-induced immune responses were rapid, rising as early as 7 days post-vaccination; and were comparable in all formulations in terms of magnitude, with maximal levels attained within 28 (unadjuvanted) or 56 (adjuvanted) days post-vaccination. Peak anti-F protein IgG antibody levels rose 3.6- to 5.6-fold, with an adjuvant effect observed at the 60 μg dose, and a dose-effect observed between the unadjuvanted 60 and 90 μg regimens. The anti-F response persisted through 12 months post-vaccination. Palivizumab-competitive antibodies were below quantifiable levels (<33 μg/mL) at day 0. The rise of antibodies with specificity for Site II peptide, and the palivizumab-competitive binding activity, denoting antibodies binding at, or in proximity to, antigenic Site II on the F protein, closely paralleled the anti-F response. However, a larger proportion of antibodies in adjuvanted vaccine recipients bound to the Site II peptide at high avidity. Day 0 neutralizing antibodies were high in all subjects and rose 1.3- to 1.7-fold in response to vaccination. Importantly, the RSV F vaccine co-administered with TIV did not impact the serum hemagglutination inhibition antibody responses to a standard-dose TIV, and TIV did not impact the immune response to the RSV F vaccine.

Conclusions

RSV F protein nanoparticle vaccine induced increases in measures of functional immunity to RSV in older adults and demonstrated an acceptable safety profile. Adjuvanted formulations provided additional immunogenicity benefit as compared to increasing antigen dose alone. This trial was registered with ClinicalTrials.gov number NCT01709019.

     

Design, synthesis and preliminary screening of novel 3-(2-N,N-dimethylaminoethylthio) indole derivatives as potential 5-HT(6) receptor ligands

The synthesis and potential 5-hydroxytryptamine(6) receptor (5-HT6R) antagonist activity of a novel series of N-arylsulfonyl-3-(2-N,N-dimethylaminoethylthio) indoles has been reported. The molecular modeling, synthesis and in-vitro radioligand binding data of this series are discussed. The present article describes 37 derivatives of the title series. It was observed that the increased side-chain length with the insertion of a sulfur atom did not lead to the loss of binding affinity of these compounds, although the affinities were reduced. The compounds exhibited moderate affinity and selectivity to human 5-HT6 receptors.