Integrating In Silico and In Vitro Analysis of Peptide Binding Affinity to HLA-Cw*0102: A Bioinformatic Approach to the Prediction of New Epitopes

Background

Predictive models of peptide-Major Histocompatibility Complex (MHC) binding affinity are important components of modern computational immunovaccinology. Here, we describe the development and deployment of a reliable peptide-binding prediction method for a previously poorly-characterized human MHC class I allele, HLA-Cw*0102.

Methodology/Findings

Using an in-house, flow cytometry-based MHC stabilization assay we generated novel peptide binding data, from which we derived a precise two-dimensional quantitative structure-activity relationship (2D-QSAR) binding model. This allowed us to explore the peptide specificity of HLA-Cw*0102 molecule in detail. We used this model to design peptides optimized for HLA-Cw*0102-binding. Experimental analysis showed these peptides to have high binding affinities for the HLA-Cw*0102 molecule. As a functional validation of our approach, we also predicted HLA-Cw*0102-binding peptides within the HIV-1 genome, identifying a set of potent binding peptides. The most affine of these binding peptides was subsequently determined to be an epitope recognized in a subset of HLA-Cw*0102-positive individuals chronically infected with HIV-1.

Conclusions/Significance

A functionally-validated in silico-in vitro approach to the reliable and efficient prediction of peptide binding to a previously uncharacterized human MHC allele HLA-Cw*0102 was developed. This technique is generally applicable to all T cell epitope identification problems in immunology and vaccinology.     

Pediatricians’ Own Weight: Self‐perception,Misclassification, and Ease of Counseling

Objectives: Pediatricians underdiagnose overweight and feel ineffective at counseling. Given the relationship between physicians’ health and health habits and counseling behaviors, we sought to determine the 1) percentage of pediatricians who are overweight; 2) accuracy of pediatricians’ own weight status classification; and 3) relationship between weight self‐perception and perceived ease of obesity counseling. Research Methods and Procedures: This study was a cross‐sectional, mail survey of North Carolina pediatricians that queried about their weight status and ease of counseling. Accuracy of pediatricians’ self‐classification of weight status was compared with BMIs derived from self‐reported height and weight. Using logistic regression, controlling for potential confounding variables, we examined the association between weight perception and ease of counseling. Results: The unadjusted response rate was 62%, and the adjusted response rate was 71% (n = 355). Nearly one‐half (49%) of overweight pediatricians did not identify themselves as such. Men had greater adjusted odds of misclassifying overweight than women [odds ratio (OR), 3.61; 95% confidence interval (CI) = 1.81, 7.21]. Self‐classified “thin” pediatricians had nearly six times the odds of reporting more counseling difficulty as a result of their weight than “average” weight pediatricians (OR = 5.69; 95% CI = 2.30, 14.1), and self‐identified “overweight” pediatricians reported nearly four times as great counseling difficulty as “average” weight physicians (OR = 3.84; 95% CI = 1.11, 13.3), after adjustment for self‐reported BMI weight status and other potential confounders. Discussion: The roles that physician weight misclassification and self‐perception potentially play in influencing rates of obesity counseling warrant further research.     

Green house gas emissions due to concrete manufacture

Background, Aim and Scope The issues of environmental impacts of concrete have become important since many major infrastructure owners are now requiring environmentally sustainable design (ESD). The carbon dioxide (CO₂) emissions are often used as a rating tool to compare the environmental impact of different construction materials in ESD. Currently, the designers are forced to make estimates of CO₂ emissions for concrete in ESD based on conjecture rather than data. The aim of this study was to provide hard data collected from a number of quarries and concrete manufacturing plants so that accurate estimates can be made for concretes in ESD. Materials and Methods This paper presents the results of a research project aimed to quantify the CO₂ emissions associated with the manufacture and placement of concrete. The life cycle inventory data was collected from two coarse aggregates quarries, one fine aggregates quarry, six concrete batching plants and several other sources. The results are presented in terms of equivalent CO₂ emissions. The potential of fly ash and ground granulated blast furnace slag (GGBFS) to reduce the emissions due to concrete was investigated. A case study of a building is also presented. Results Portland cement was found to be the primary source of CO₂ emissions generated by typical commercially produced concrete mixes, being responsible for 74% to 81% of total CO₂ emissions. The next major source of CO₂ emissions in concrete was found to be coarse aggregates, being responsible for 13% to 20% of total CO₂ emissions. The majority contribution of CO₂ emissions in coarse aggregates production was found to from electricity, typically about 80%. Blasting, excavation, hauling and transport comprise less than 25%. While the explosives had very high emission factors per unit mass, they contribute very small amounts (<0.25%) to coarse aggregate production, since only small quantities are used. Production of a tonne of fine aggregates was found to generate 30% to 40% of the emissions generated by the production of a tonne of coarse aggregates. Fine aggregates generate less equivalent CO₂ since they are only graded, not crushed. Diesel and electricity were found to contribute almost equally to the CO₂ emissions due to fine aggregates production. Emission contributions due to admixtures were found to be negligible. Concrete batching, transport and placement activities were all found to contribute very small amounts of CO₂ to total concrete emissions. Discussion The CO₂ emissions generated by typical normal strength concrete mixes using Portland cement as the only binder were found to range between 0.29 and 0.32 t CO₂-e/m³. GGBFS was found to be capable of reducing concrete CO₂ emissions by 22% in typical concrete mixes. Fly ash was found to be capable of reducing concrete CO₂ emissions by 13% to 15% in typical concrete mixes. Conclusions The results presented are based on typical concrete manufacturing and placement methods in Australia. The data presented in this paper can be utilized to compare green house gas emissions due to concrete with those associated with alternative construction materials. Recommendations and Perspectives The various rating schemes used to compare alternative construction materials should use models such as the one presented in this paper, based on hard data so that reliable comparisons can be made. A case study is presented in this paper demonstrating how the results may be utilized. ESS-Submission Editor: Dr. Stefanie Hellweg (stefanie.hellweg@ifu.baug.ethz.ch)     

Annexin A1 modulates macula densa function by inhibiting cyclooxygenase 2

Annexin A1 (ANXA1) exerts anti-inflammatory effects through multiple mechanisms including inhibition of prostaglandin synthesis. Once secreted, ANXA1 can bind to G protein-coupled formyl peptide receptors (Fpr) and activate diverse cellular signaling pathways. ANXA1 is known to be expressed in cells of the juxtaglomerular apparatus, but its relation to the expression of cyclooxygenase 2 (COX-2) in thick ascending limb and macula densa cells has not been elucidated. We hypothesized that ANXA1 regulates the biosynthesis of COX-2. ANXA1 abundance in rat kidney macula densa was extensively colocalized with COX-2 (95%). Furosemide, an established stimulus for COX-2 induction, caused enhanced expression of both ANXA1 and COX-2 with maintained colocalization (99%). In ANXA1-deficient mice, COX-2-positive cells were more numerous than in control mice (+107%; normalized to glomerular number; P < 0.05) and renin expression was increased (+566%; normalized to glomerular number; P < 0.05). Cultured macula densa cells transfected with full-length rat ANXA1 revealed downregulation of COX-2 mRNA (-59%; P < 0.05). Similarly, treatment with dexamethasone suppressed COX-2 mRNA in the cells (-49%; P < 0.05), while inducing ANXA1 mRNA (+56%; P < 0.05) and ANXA1 protein secretion. Inhibition of the ANXA-1 receptor Fpr1 with cyclosporin H blunted the effect of dexamethasone on COX-2 expression. These data show that ANXA1 exerts an inhibitory effect on COX-2 expression in the macula densa. ANXA1 may be a novel intrinsic modulator of renal juxtaglomerular regulation by inhibition of PGE(2) synthesis.     

Human Borna disease virus infection in Australia: serological markers of infection in multi-transfused patients

Borna disease virus (BDV) causes neurological disease in horses, however, there is no consensus as to the extent or significance of human infection. BDV antigen levels in plasma (BDVpAg) and anti-BDV were measured by ELISAs. Confirmation was by Western blot (WB), immunofluorescence assay (IFA) or BDV-peptide-epitope ELISA. For 42 volunteers psychiatrically-defined as non-depressed (82 samples) neither BDVpAg nor anti-BDV was detected. For 104 patients with diagnosed depression (290 samples) 1 was BDVpAg positive and 5 anti-BDV positive, one epitope-e8 positive and 4 IFA positive, with 96% concordance for repeat samples. No BDVpAg was detected in 214 pregnant women, 2 were anti-BDV positive, one WB-confirmed (p24/p40). For 219 donors 2 were BDVpAg positive with anti-BDV detected in 5 (2.3%) one IFA 1:10, another IFA 1:40/epitope-e8 positive. In multitransfused patients, 3/168 were BDV pAg positive, with 14/168 anti-BDV positive, 1 epitope-e8 positive, 2 WB positive and 1 IFA 1:10. In BDVpAg positive multi-transfused patients there was an elevated risk of transaminitis. In one case, a patient BDV-negative prior to transfusion was BDVpAg positive for several months posttransfusion (associated with transaminitis). These data provide serological evidence, supported by confirmatory assays and repeat-sample concordance, of BDV infection in Australia, particularly in multi-transfused patients.     

Distribution and symptoms of epiphyte infection in major carrageenophyte-producing farms

High density commercial farming of carrageenophyte Kappaphycus alvarezii is often plagued with “ice-ice” disease and epiphyte infection, which eventually leads to reduced production and in some cases collapse of crop. Epiphyte outbreak has been occurring regularly in major carrageenophyte farms in the Philippines, Indonesia, Malaysia and Tanzania. Infected materials from these countries were studied to establish baseline information on the epiphyte’s identity, density, symptoms and secondary infection on the host seaweed. The causative organism was identified as Neosiphonia apiculata (Hollenberg) Masuda et Kogame, based on its morphological features. Epiphyte density on host seaweed materials decreased in the following order: the Philippines (88.5 epi cm⁻²), Tanzania (69.0 epi cm⁻²), Indonesia (56.5 epi cm⁻²) and Malaysia (42.0 epi cm⁻²). Initial symptoms were the presence of tiny black spots, indicating the embedded tetrasporeling in seaweed cortex layer. Vegetative form emerged after 2 weeks measuring less than 0.5 mm in length with a density of less than 25.0 epi cm⁻². Upon maturation, infected seaweed takes on a “hairy” appearance with “goose-bumps” like cortical swellings. The epiphyte appears as a solitary plant with multiple secondary rhizoids or as multiple epiphytes appearing from a single cortical opening. At the end of infection, the epiphytes left dark pits on the cortical swelling, and the carrageenophytes are infected by opportunistic bacteria. Bacterial enumeration of healthy and infected seaweed materials showed an increase of more than 300% in total bacterial count on infected materials dominated by Alteromonas sp., Flavobacterium sp. and Vibrio sp.     

Identification of several species of phospholipase inhibitory protein(s) by radioimmunoassay for lipomodulin

Radioimmunoassay of lipomodulin has been developed using a monoclonal anti-lipomodulin antibody and ¹²⁵I-labelled lipomodulin. Lipomodulin activity was measured in peritoneal lavage fluids obtained from rats injected with dexamethasone by radioimmunoassay and by enzymatic assay with phospholipase A₂. Three species of immunoreactive substances with Mr= 40,000, 30,000 and 16,000 were found. While two species of Mr= 40,000 and 30,000 had phospholipase inhibitory activities, the species of Mr= 16,000 could inhibit phospholipase A₂ only after dephosphorylation by alkaline phosphatase treatment.