Liposome Damage and Modeling of Fragments of Human Islet Amyloid Polypeptide (IAPP) Support a Two-Step Model of Membrane Destruction

A key factor in the development of Type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide (hIAPP) is believed to play a crucial role in this process by forming small aggregates that disrupt the cellular membrane. During Type II diabetes mellitus, human IAPP (hIAPP) fibrillizes to form amyloid deposits. However, the role of various regions of the 37 amino acid peptide in the process of membrane disruption has yet to be fully elucidated. Therefore, several fragments (10–19, 20–29, 10–29, 1–19) of hIAPP were synthesized and compared to full length hIAPP for their effects on model PC/PS bilayers. These fragments were also modeled using density functional methods and analyzed by circular dichroism, to determine possible correlations between activity and available conformations and charge distribution. Results from dye leakage and Thioflavin T fluorescence assays confirmed that the hIAPP fragments disrupt the lipid bilayer to varying extents, despite their inability to form amyloid fibrils. The longer and more positively charged fragments were most active in the assay (1–19 > 10–29 > 10–19 > 20–29), though none rivaled the activity of the native full length peptide. This may reflect their relative abilities to interact with the negatively charged membrane. Data support a two-step model for membrane disruption: insertion by the N-terminus followed by fibrillization mediated by the middle to C-terminal region.     

Pathotype and Genetic Diversity amongst Indian Isolates of Xanthomonas oryzae pv. oryzae

A number of rice resistance genes, called Xa genes, have been identified that confer resistance against various strains of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. An understanding of pathotype diversity within the target pathogen population is required for identifying the Xa genes that are to be deployed for development of resistant rice cultivars. Among 1024 isolates of Xoo collected from 20 different states of India, 11 major pathotypes were distinguished based on their reaction towards ten Xa genes (Xa1, Xa3, Xa4, xa5, Xa7, xa8, Xa10, Xa11, xa13, Xa21). Isolates belonging to pathotype III showing incompatible interaction towards xa8, xa13 and Xa21 and compatible interaction towards the rest of Xa genes formed the most frequent (41%) and widely distributed pathotype. The vast majority of the assayed Xoo isolates were incompatible with one or more Xa genes. Exceptionally, the isolates of pathotype XI were virulent on all Xa genes, but have restricted distribution. Considering the individual R-genes, Xa21 appeared as the most broadly effective, conferring resistance against 88 % of the isolates, followed in decreasing order by xa13 (84 %), xa8 (64 %), xa5 (30 %), Xa7 (17 %) and Xa4 (14 %). Fifty isolates representing all the eleven pathotypes were analyzed by southern hybridization to determine their genetic relatedness using the IS1112 repeat element of Xoo. Isolates belonging to pathotype XI were the most divergent. The results suggest that one RFLP haplotype that is widely distributed all over India and is represented in strains from five different pathotypes might be an ancestral haplotype. A rice line with xa5, xa13 and Xa21 resistance genes is resistant to all strains, including those belonging to pathotype XI. This three gene combination appears to be the most suitable Xa gene combination to be deployed in Indian rice cultivars.     

Safety, efficacy and anti-inflammatory activity of rho iso-alpha-acids from hops

A defined mixture of rho iso-alpha-acids (RIAA), a modified hop extract, was evaluated for anti-inflammatory efficacy and safety. RIAA inhibited LPS-stimulated PGE(2) formation with >200-fold selectivity of COX-2 (IC(50)=1.3 microg/ml) over COX-1 (IC(50)>289 microg/ml). This occurred only when RIAA was added prior to, but not post, LPS stimulation. Consistent with this observation, RIAA produced no physiologically relevant, direct inhibition of COX-1 or COX-2 peroxidase activity. This suggests that RIAA inhibits inducible but not constitutive COX-2. In support, we found RIAA showed minimal PGE(2) inhibition (IC(50)=21mug/ml) relative to celecoxib (IC(50)=0.024 microg/ml), aspirin (IC(50)=0.52 microg/ml) or ibuprofen (IC(50)=0.57 microg/ml) in the AGS gastric mucosal model, where COX-1 and -2 are expressed constitutively. Taken together these results predict RIAA may have lower potential for gastrointestinal and cardiovascular toxicity observed with COX enzyme inhibitors. Following confirmation of bioavailable RIAA administered orally, gastrointestinal safety was assessed using the fecal calprotectin biomarker in a 14-day human clinical study; RIAA (900 mg/day) produced no change compared to naproxen (1000 mg/day), which increased fecal calprotectin 200%. Cardiovascular safety was addressed by PGI-M measurements where RIAA (1000 mg) did not reduce PGI-M or affect the urinary PGI-M/TXB(2) ratio. Drug interaction potential was evaluated against six major CYPs; of relevance, RIAA inhibited CYP2C9. Toxicity was assessed in a 21-day oral, mouse subchronic toxicity study where no dose dependent histopathological effects were noted. Clinically, RIAA (1000 mg/day) produced a 54% reduction in WOMAC Global scores in a 6-week, open-label trial of human subjects exhibiting knee osteoarthritis.     

Incidence of Active Pulmonary Tuberculosis in Patients with Coincident Filarial and/or Intestinal Helminth Infections Followed Longitudinally in South India

Background

Filarial (and other helminth) infections are known to modulate mycobacteria-specific pro-inflammatory cytokine responses necessary for maintaining latency in tuberculosis (TB). We sought to address whether helminth co-infection alters progression to active pulmonary TB in a co-endemic area of South India.

Methods/Principal Findings

Incidence of active pulmonary TB was assessed in 5096 subjects from five villages among helminth-infected (hel⁺) and –uninfected (hel⁻) groups. Baseline stool examinations, circulating filarial antigen, and tuberculin skin testing (PPD) were performed along with chest radiographs, sputum microscopy, and culture. During three follow-up visits each 2.5 years, patients were assessed using PPD tests and questionnaires and—for those with potential symptoms of TB—sputum microscopy and culture. Of the 5096 subjects, 1923 were found to be hel⁺ and 3173 were hel⁻. Follow up interval stool examination could not be performed. In each group, 21 developed active TB over the course of the study. After adjusting for sex, age, BCG vaccination status, and PPD positivity, no difference was seen in active TB incidence between hel⁺ and hel⁻ groups either at baseline (relative risk (RR) 1.60; 95% confidence interval (CI): 0.69, 3.71, P = 0·27), or when followed prospectively (RR 1.24; 95% CI: 0.48, 3.18, P = 0·66).

Conclusions/Significance

Our findings suggest that, despite the immunomodulatory effects of helminth infection, baseline co-morbid infection with these parasites had little effect on the clinical progression from latent to active pulmonary TB.     

Nanobiopolymer for direct targeting and inhibition of EGFR expression in triple negative breast cancer

Treatment options for triple negative breast cancer (TNBC) are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR) therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid) (PMLA) nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb) 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR) antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON) to inhibit EGFR synthesis. The nanobioconjugates variants were: (1) P (BioPolymer) with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR), and (2) P with AON and 2C5 (P/AON/2C5). Controls included (3) P with 2C5 but without AON (P/2C5), (4) PBS, and (5) P with PEG and leucine ester (LOEt) for endosomal escape (P/mPEG/LOEt). Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging) and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting. In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1) [P = 0.03 vs. controls; P<0.05 vs. nanobioconjugate variant (2)]. Lead nanobioconjugate (1) also showed stronger inhibition of EGFR expression and Akt phosphorylation than other treatments. Treatment of TNBC with the new nanobioconjugate results in tumor growth arrest by inhibiting EGFR and its downstream signaling intermediate, phosphorylated Akt. The nanobioconjugate represents a new generation of nanodrugs for treatment of TNBC.     

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.     

Sequence and structural analysis of fibronectin‐binding protein reveals importance of multiple intrinsic disordered tandem repeats

The location of certain amino acid sequences like repeats along the polypeptide chain is very important in the context of forming the overall shape of the protein molecule which in fact determines its function. In gram‐positive bacteria, fibronectin‐binding protein (FnBP) is one such repeat containing protein, and it is a cell wall‐attached protein responsible for various acute infections in human. Several studies on sequence, structure, and function of fibronectin‐binding regions of FnBPs were reported; however, no detailed study was carried out on the full‐length protein sequence. In the present study, we have made a thorough sequence and structure analysis on FnBP_A of Staphylococcus aureus and explored the presence of dual ligand‐binding ability of fibrinogen (fg)‐binding region and its molecular recognition processes. Multiple sequence alignment and protein‐protein docking analysis reveal the regions which are likely involved in dual ligand binding. Further analysis of docking of FnBP_A fg‐binding region and fn N‐terminal modules suggests that if the latter binds to the fg‐binding region of FnBP_A, it would inhibit the subsequent binding of fg because of steric hindrance. The sequence analysis further suggests that the abundance of disorder promoting residue glutamic acid and dual personality (both order/disorder promoting) residue threonine in tandem repeats of FnBP_A and B proteins possibly would help the molecule to undergo a conformational change while binding with fn by β‐zipper mechanism. The segment‐based power spectral analysis was carried out which helps to understand the distribution of hydrophobic residues along the sequence particularly in intrinsic disordered tandem repeats. The results presented here will help to understand the role of internal repeats and intrinsic disorder in the molecular recognition process of a pathogenic cell surface protein.