Levels of Alpha-Toxin Correlate with Distinct Phenotypic Response Profiles of Blood Mononuclear Cells and with agr Background of Community-Associated Staphylococcus aureus Isolates

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of alpha-toxin than did the proliferative supernatants. Addition of alpha-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, delta-toxin or phenol soluble modulin alpha-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.

Exclusive Detection of Sub-Nanomolar Levels of Palladium(II) in Water: An Excellent Probe for Multiple Applications

A new colorimetric probe has been developed for the detection and estimation of Pd-II at sub-nanomolar concentrations. The probe consisted of rhodamine (signaling unit), which was linked with a bis-picolyl moiety (binding site) through a phenyl ring. Pd-II induced opening of the spirolactam ring of the probe with the generation of a prominent pink color. The excellent selectivity of the probe towards Pd-II over Pd-0 or Rh-II ensured its potential utility for the detection of residual palladium contamination in pharma-ceutical drugs and in Pd-catalyzed reactions. The probe showed a ``turn-on'' (bright yellow) fluorescence upon the addition of Pd-II, which made it suitable for the detection of Pd contaminants in mammalian cells.

Optical detection of submicromolar levels of nitro explosives by a submicron sized metal-organic phosphor material

Two isomorphous submicron sized metal-organic network compounds, Y-2(PDA)(3)(H2O)1]center dot 2H(2)O (PDA = 1,4-phenylenediacetate), 1 and Y1.8Tb0.2(PDA)(3)(H2O)1]center dot 2H(2)O, Tb@1 have been synthesized by employing solvent assisted liquid grinding followed by heating at 180 degrees C for 1' min and washing with water. Single crystal X-ray data of bulk 1 confirmed a three dimensional porous structure. The structure and morphology of 1 and Tb@1 were systematically characterized by PXRD, TGA, DSC, IR, SEM and EDX analysis. Dehydrated Tb@1 Tb@1'] shows a high intense visible green emission upon exposure to UV light. The green emission of Tb@1' was used for the detection of nitro explosives, such as 2,4,6-trinitrophenol (TNP), 1,3-dinitro benzene (DNB), 2,4-dinitro toluene (DNT), nitro benzene (NB), and 4-nitro toluene (NT) in acetonitrile. The results show that the emission intensity of dehydrated Tb@1' can be quenched by all the nitro analytes used in the present work. Remarkably, Tb@1' exhibited a high efficiency for TNP, DNB and DNT detection with K-SV K-SV = quenching constant based on linear Stern-Volmer plot] values of 70 920, 44 000 and 35 430 M-1, respectively, which are the highest values amongst known metal-organic materials. Using this material submicromolar level (equivalent to 0.18 ppm), a detection of nitro explosives has been achieved.

Identifying functionally important cis-peptide containing segments in proteins and their utility in molecular function annotation

Cis-peptide embedded segments are rare in proteins but often highlight their important role in molecular function when they do occur. The high evolutionary conservation of these segments illustrates this observation almost universally, although no attempt has been made to systematically use this information for the purpose of function annotation. In the present study, we demonstrate how geometric clustering and level-specific Gene Ontology molecular-function terms (also known as annotations) can be used in a statistically significant manner to identify cis-embedded segments in a protein linked to its molecular function. The present study identifies novel cis-peptide fragments, which are subsequently used for fragment-based function annotation. Annotation recall benchmarks interpreted using the receiver-operator characteristic plot returned an area-under-curve >0.9, corroborating the utility of the annotation method. In addition, we identified cis-peptide fragments occurring in conjunction with functionally important trans-peptide fragments, providing additional insights into molecular function. We further illustrate the applicability of our method in function annotation where homology-based annotation transfer is not possible. The findings of the present study add to the repertoire of function annotation approaches and also facilitate engineering, design and allied studies around the cis-peptide neighborhood of proteins.

Return levels of hydrologic droughts under climate change

Developments in the statistical extreme value theory, which allow non-stationary modeling of changes in the frequency and severity of extremes, are explored to analyze changes in return levels of droughts for the Colorado River. The transient future return levels (conditional quantiles) derived from regional drought projections using appropriate extreme value models, are compared with those from observed naturalized streamflows. The time of detection is computed as the time at which significant differences exist between the observed and future extreme drought levels, accounting for the uncertainties in their estimates. Projections from multiple climate model-scenario combinations are considered; no uniform pattern of changes in drought quantiles is observed across all the projections. While some projections indicate shifting to another stationary regime, for many projections which are found to be non-stationary, detection of change in tail quantiles of droughts occurs within the 21st century with no unanimity in the time of detection. Earlier detection is observed in droughts levels of higher probability of exceedance. (C) 2014 Elsevier Ltd. All rights reserved.

Directing peptide conformation with centrally positioned pre-organized dipeptide segments: studies of a 12-residue helix and beta-hairpin

Secondary structure formation in oligopeptides can be induced by short nucleating segments with a high propensity to form hydrogen bonded turn conformations. Type I/III turns facilitate helical folding while type II'/I' turns favour hairpin formation. This principle is experimentally verified by studies of two designed dodecapeptides, Boc-Val-Phe-Leu-Phe-Val-Aib-Aib-Val-Phe-Leu-Phe-Val-OMe 1 and Boc-Val-Phe-Leu-Phe-Val- (D) Pro- (L) Pro-Val-Phe-Leu-Phe-Val-OMe 2. The N- and C-terminal flanking pentapeptide sequences in both cases are identical. Peptide 1 adopts a largely alpha-helical conformation in crystals, with a small 3(10) helical segment at the N-terminus. The overall helical fold is maintained in methanol solution as evidenced by NMR studies. Peptide 2 adopts an antiparallel beta-hairpin conformation stabilized by 6 interstrand hydrogen bonds. Key nuclear Overhauser effects (NOEs) provide evidence for the antiparallel beta-hairpin structure. Aromatic proton chemical shifts provide a clear distinction between the conformation of peptides 1 (helical) and 2 (beta-hairpin). The proximity of facing aromatic residues positioned at non-hydrogen bonding positions in the hairpin results in extensively ring current shifted proton resonances in peptide 2.

Remotely triggered micro-shock wave responsive drug delivery system for resolving diabetic wound infection and controlling blood sugar levels

A novel, micro-shock wave responsive spermidine and dextran sulfate microparticle was developed. Almost 90% of the drug release was observed when the particles were exposed to micro-shock waves 5 times. Micro-shock waves served two purposes; of releasing the antibiotic from the system and perhaps disrupting the S. aureus biofilm in the skin infection model. A combination of shock waves with ciprofloxacin loaded microparticles could completely cure the S. aureus infection lesion in a diabetic mouse model. As a proof of concept insulin release was triggered using micro-shock waves in diabetic mice to reduce the blood glucose level. Insulin release could be triggered for at least 3 days by exposing subcutaneously injected insulin loaded particles.

The peptide NCbz-Val-Tyr-OMe and aromatic pi-pi interactions

The peptide N-benzyloxycarbonyl-L-valyl-L-tyrosine methyl ester or NCbz-Val-Tyr-OMe (where NCbz is N-benzyloxycarbonyl and OMe indicates the methyl ester), C23H28N2O6, has an extended backbone conformation. The aromatic rings of the Tyr residue and the NCbz group are involved in various attractive intra- and intermolecular aromatic - interactions which stabilize the conformation and packing in the crystal structure, in addition to NH...O and OH...O hydrogen bonds. The aromatic - interactions include parallel-displaced, perpendicular T-shaped, perpendicular L-shaped and inclined orientations.

Effects of hydrogen bonding on amide-proton chemical shift anisotropy in a proline-containing model peptide

Longitudinal relaxation due to cross-correlation between dipolar ((HN-1H alpha)-H-1) and amide-proton chemical shift anisotropy (H-1(N) CSA) has been measured in a model tripeptide Piv-(L)Pro-(L)Pro-(L)Phe-OMe. The peptide bond across diproline segment is known to undergo cis/trans isomerization and only in the cis form does the lone Phe amide-proton become involved in intramolecular hydrogen bonding. The strength of the cross correlated relaxation interference is found to be significantly different between cis and trans forms, and this difference is shown as an influence of intramolecular hydrogen bonding on the amide-proton CSA. (C) 2015 Elsevier B.V. All rights reserved.

Mycobacterium tuberculosis has diminished capacity to counteract redox stress induced by elevated levels of endogenous superoxide

Mycobacterium tuberculosis (Mtb) has evolved protective and detoxification mechanisms to maintain cytoplasmic redox balance in response to exogenous oxidative stress encountered inside host phagocytes. In contrast, little is known about the dynamic response of this pathogen to endogenous oxidative stress generated within Mtb. Using a noninvasive and specific biosensor of cytoplasmic redox state of Mtb, we for first time discovered a surprisingly high sensitivity of this pathogen to perturbation in redox homeostasis induced by elevated endogenous reactive oxygen species (ROS). We synthesized a series of hydroquinone-based small molecule ROS generators and found that ATD-3169 permeated mycobacteria to reliably enhance endogenous ROS including superoxide radicals. When Mtb strains including multidrug-resistant (MDR) and extensively drug-resistant (XDR) patient isolates were exposed to this compound, a dose-dependent, long-lasting, and irreversible oxidative shift in intramycobacterial redox potential was detected. Dynamic redox potential measurements revealed that Mtb had diminished capacity to restore cytoplasmic redox balance in comparison with Mycobacterium smegmatis (Msm), a fast growing nonpathogenic mycobacterial species. Accordingly, Mtb strains were extremely susceptible to inhibition by ATD-3169 but not Msm, suggesting a functional linkage between dynamic redox changes and survival. Microarray analysis showed major realignment of pathways involved in redox homeostasis, central metabolism, DNA repair, and cell wall lipid biosynthesis in response to ATD-3169, all consistent with enhanced endogenous ROS contributing to lethality induced by this compound. This work provides empirical evidence that the cytoplasmic redox poise of Mtb is uniquely sensitive to manipulation in steady-state endogenous ROS levels, thus revealing the importance of targeting intramycobacterial redox metabolism for controlling TB infection. (C) 2015 The Authors. Published by Elsevier Inc.

Deciphering complex patterns of class-I HLA-peptide cross-reactivity via hierarchical grouping

T-cell responses in humans are initiated by the binding of a peptide antigen to a human leukocyte antigen (HLA) molecule. The peptide-HLA complex then recruits an appropriate T cell, leading to cell-mediated immunity. More than 2000 HLA class-I alleles are known in humans, and they vary only in their peptide-binding grooves. The polymorphism they exhibit enables them to bind a wide range of peptide antigens from diverse sources. HLA molecules and peptides present a complex molecular recognition pattern, as many peptides bind to a given allele and a given peptide can be recognized by many alleles. A powerful grouping scheme that not only provides an insightful classification, but is also capable of dissecting the physicochemical basis of recognition specificity is necessary to address this complexity. We present a hierarchical classification of 2010 class-I alleles by using a systematic divisive clustering method. All-pair distances of alleles were obtained by comparing binding pockets in the structural models. By varying the similarity thresholds, a multilevel classification was obtained, with 7 supergroups, each further subclassifying to yield 72 groups. An independent clustering performed based only on similarities in their epitope pools correlated highly with pocket-based clustering. Physicochemical feature combinations that best explain the basis of clustering are identified. Mutual information calculated for the set of peptide ligands enables identification of binding site residues contributing to peptide specificity. The grouping of HLA molecules achieved here will be useful for rational vaccine design, understanding disease susceptibilities and predicting risk of organ transplants.

A sleep-inducing peptide from the venom of the Indian cone snail Conus araneosus

The marine snail Conus araneosus has unusual significance due to its confined distribution to coastal regions of southeast India and Sri Lanka. Due to its relative scarceness, this species has been poorly studied. In this work, we characterized the venom of C. araneosus to identify new venom peptides. We identified 14 novel compounds. We determined amino acid sequences from chemically-modified and unmodified crude venom using liquid chromatography-electrospray ionization mass spectrometry and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Ten sequences showed six Cys residues arranged in a pattern that is most commonly associated with the M-superfamily of conotoxins. Four other sequences had four Cys residues in a pattern that is most commonly associated with the T-superfamily of conotoxins. The post-translationally modified residue (pyroglutamate) was determined at the N-terminus of two sequences, ar3h and ar3i respectively. In addition, two sequences, ar3g and ar3h were C-terminally amidated. At a dose of 2 nmol, peptide ar3j elicited sleep when injected intraperitoneally into mice. To our knowledge, this is the first report of a peptide from a molluscivorous cone snail with sleep-inducing effects in mice. The novel peptides characterized herein extend the repertoire of unique peptides derived from cone snails and may add value to the therapeutic promise of conotoxins. (C) 2015 Elsevier Ltd. All rights reserved.