Identification of humoral immune responses in protein microarrays using DNA microarray data analysis techniques

MOTIVATION: We present a study of antigen expression signals from a newly developed high-throughput protein microarray technique. These signals are a measure of antibody-antigen binding activity and provide a basis for understanding humoral immune responses to various infectious agents and supporting vaccine and diagnostic development. RESULTS: We investigate the characteristics of these expression profiles and show that noise models, normalization, variance estimation and differential expression analysis techniques developed in the context of DNA microarray analysis can be adapted and applied to these protein arrays. Using a high-dimensional dataset containing measurements of expression profiles of antibody reactivity against each protein (295 antigens and 9 controls) in 42 malaria (Plasmodium falciparum) protein arrays derived from 22 donors with various clinical presentations of malaria, we present a methodology for the analysis and identification of significantly expressed antigens targeted by immune responses for individual sera, groups of sera and across stages of infection. We also conduct a short study highlighting the top immunoreactive antigens where we identify three novel high priority antigens for future evaluation. AVAILABILITY: All software programs (in R) used for the analysis described in this paper are freely available for academic purposes at www.igb.uci.edu/servers/servers.html.     

Tracking factors modulating cytoplasmic incompatibilities in the mosquito Culex pipiens

Wolbachia are maternally inherited endosymbiotic bacteria that infect many arthropod species and may induce cytoplasmic incompatibility (CI), resulting in abortive embryonic development. One Wolbachia host, Culex pipiens complex mosquitoes, displays high levels of variability in both CI crossing types (cytotypes) and DNA markers. We report here an analysis of 14 mosquito strains, containing 13 Wolbachia variants, and with 13 different cytotypes. Cytotypes were Wolbachia-dependent, as antibiotic treatment rendered all strains tested compatible. Cytotype distributions were independent of geographical distance between sampling sites and host subspecies, suggesting that Wolbachia does not promote a reproductive isolation depending on these parameters. Backcross analysis demonstrated a mild restoring effect of the nuclear genome, indicating that CI is mostly cytoplasmically determined for some crosses. No correlation was found between the phenotypic and genotypic variability of 16 WO prophage and transposon markers, except for the WO prophage Gp15 gene, which encodes a protein similar to a bacterial virulence factor. However, Gp15 is partially correlated with CI expression, suggesting that it could be just linked to a CI gene.     

The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1

This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx K_t at pH 5.5 for H247A was ↓8.4-fold. Although wild type (WT)-PCFT K_i values varied among the folates, K_i values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His²⁴⁷ to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser¹⁷². The folic acid influx K_t for S172A-PCFT was decreased similar to H247A. His²⁸¹ faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ∼12-fold↑ in the folic acid influx K_t. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx K_t was unchanged, but the K_t decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His²⁴⁷ to Ser¹⁷² interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His²⁸¹ residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.     

VitAL: Viterbi Algorithm for de novo Peptide Design

Background

Drug design against proteins to cure various diseases has been studied for several years. Numerous design techniques were discovered for small organic molecules for specific protein targets. The specificity, toxicity and selectivity of small molecules are hard problems to solve. The use of peptide drugs enables a partial solution to the toxicity problem. There has been a wide interest in peptide design, but the design techniques of a specific and selective peptide inhibitor against a protein target have not yet been established.

Methodology/Principal Findings

A novel de novo peptide design approach is developed to block activities of disease related protein targets. No prior training, based on known peptides, is necessary. The method sequentially generates the peptide by docking its residues pair by pair along a chosen path on a protein. The binding site on the protein is determined via the coarse grained Gaussian Network Model. A binding path is determined. The best fitting peptide is constructed by generating all possible peptide pairs at each point along the path and determining the binding energies between these pairs and the specific location on the protein using AutoDock. The Markov based partition function for all possible choices of the peptides along the path is generated by a matrix multiplication scheme. The best fitting peptide for the given surface is obtained by a Hidden Markov model using Viterbi decoding. The suitability of the conformations of the peptides that result upon binding on the surface are included in the algorithm by considering the intrinsic Ramachandran potentials.

Conclusions/Significance

The model is tested on known protein-peptide inhibitor complexes. The present algorithm predicts peptides that have better binding energies than those of the existing ones. Finally, a heptapeptide is designed for a protein that has excellent binding affinity according to AutoDock results.     

Cell membranes: the lipid perspective

Although cell membranes are packed with proteins mingling with lipids, remarkably little is known about how proteins interact with lipids to carry out their function. Novel analytical tools are revealing the astounding diversity of lipids in membranes. The issue is now to understand the cellular functions of this complexity. In this Perspective, we focus on the interface of integral transmembrane proteins and membrane lipids in eukaryotic cells. Clarifying how proteins and lipids interact with each other will be important for unraveling membrane protein structure and function. Progress toward this goal will be promoted by increasing overlap between different fields that have so far operated without much crosstalk.     

A neonatal case of left ventricular noncompaction associated with trisomy 18

A neonatal case of left ventricular non-compaction associated with trisomy 18: Left ventricular noncompaction (LVNC) is a rare congenital cardiomyopathy and exact etiology is still unknown. Trisomy 18 is the second most common autosomal trisomy in live-born infants. LVNC has been described in association with other dysmorphic features, association with trisomy 18 has not been reported previously in a neonate. LVNC broadens the cardiac anomalies associated with trisomy 18.     

The efficacy of Pelargonium sidoides in the treatment of upper respiratory tract infections in children with transient hypogammaglobulinemia of infancy

Transient hypogammaglobulinemia of infancy (THI), defined as prolongation of physiological hypogammaglobulinemia normally seen between the initial 3rd and 6th months of life, is one of the most common immune deficiencies of childhood. Recurrent upper respiratory tract infections (URTI) are rather common in this group of patients, and generally, antibiotic treatment is the usual choice, although viruses involved in most cases. Pelargonium sidoides extract a herbal drug with known immunmodulator, antiviral and antibacterial effects. In this randomized, placebo controlled, prospective, monocentric pilot study, 14 of 28 patients with a diagnosed THI, were given Pelargonium sidoides, while 14 were given placebo during the period of URTI. Before and after the treatment period of one week, complete blood count, prothrombin time, activated prothromboplastin time, serum alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transpeptidase, total and direct bilirubin levels were measured. Mothers were asked to fill in a questionnaire for the recovery of the clinical symptoms during the treatment. The results were evaluated and compared in both group to assess the effect of Pelargonium sidoides. As a conclusion, the Pelargonium sidoides group showed increased appetite. The Pelargonium sidoides were found to beneficial for the nasal congestion, recovery of daily and nocturnal cough but not found be significant. Further studies with large number of participants are necessary to highlight the effect of Pelargonium sidoides in children with transient hypogammaglobulinemia of infancy.     

Normobaric hyperoxia inhibits NADPH oxidase-mediated matrix metalloproteinase-9 induction in cerebral microvessels in experimental stroke

Matrix metalloproteinase-9 (MMP-9) and NADPH oxidase contribute to blood-brain barrier (BBB) disruption after ischemic stroke. We have previously shown that normobaric hyperoxia (NBO) treatment reduces MMP-9 and oxygen free radical generation in ischemic brain. In this study, we tested the hypothesis that NBO protects the BBB through inhibiting NADPH oxidase-mediated MMP-9 induction in transient focal cerebral ischemia. Male Sprague-Dawley rats (n = 69) were given NBO (95% O2) or normoxia (21% O2) during 90-min filament occlusion of the middle cerebral artery. Cerebral microvessels were isolated for analyzing MMP-9 and NADPH oxidase. BBB damage was non-invasively quantified with magnetic resonance imaging. In normoxic rats, both NADPH oxidase catalytic subunit gp91(phox) and MMP-9 expression were up-regulated in ischemic hemispheric microvessels after 90-min middle cerebral artery occlusion with 22.5 h reperfusion. Inhibition of NADPH oxidase with apocynin reduced the MMP-9 increase, indicating a causal link between NADPH oxidase-derived superoxide and MMP-9 induction. NBO treatment inhibited gp91(phox) expression, NADPH oxidase activity, and MMP-9 induction, which led to significantly less BBB damage and brain edema in the ischemic brain. These results suggest that gp91(phox) containing NADPH oxidase plays an important role in MMP-9 induction in ischemic BBB microvasculature, and that NBO treatment may attenuate MMP-9 induction and brain edema through inhibiting NADPH oxidase after transient cerebral ischemia.     

Relationships among Iron,Protein Oxidation and Lipid Peroxidation Levels in Rats with Alcohol-induced Acute Pancreatitis

It has been previously shown that alcohol induces the damage of pancreatic parenchyma tissue, but the mechanism of this damage is still poorly understood. Assuming that oxygen radical damage may be the involved, we measured markers of oxidative damage in pancreatic tissue, blood serum, plasma, and whole blood of rats with early-stage alcohol-induced acute pancreatitis. Thirty-eight male Wistar rats were divided into three groups: the control group (group 1), the acute pancreatitis group 1 day (group 2), and 3 days (group 3) after the injection of ethyl alcohol into the common biliary duct, respectively. The levels of Fe in tissue and serum, whole blood viscosity, plasma viscosity, fibrinogen and homocysteine (Hcy) levels, erythrocyte and plasma malondialdehyde (MDA), and tissue and plasma protein carbonyl levels were found to be significantly higher in groups 2 and 3 than in group 1. However, the levels of reduced glutathione (GSH) in tissue and erythrocytes were significantly lower in groups 2 and 3 than in group 1. These results suggest that elevated Fe levels in serum and pancreatic tissue in rats with early-stage alcohol-induced acute pancreatitis is associated with various hemorheological changes and with oxidative damage of the pancreas.