Epitope analysis of peanut allergen Ara h1 with oligoclonal IgM antibody from human B-lymphoblastoid cells

To analyze epitopes of peanut allergen Ara h1, Epstein-Barr virus-transformed human peripheral oligoclonal B-cells were cultured to obtain antibodies to Ara h1. The combined reaction pattern with six oligoclonal antibodies showed there were six antibody binding areas named a to f in Ara h1. We found the novel antibody binding area named “area c” (171–230aa).     

Development and use of quantitative competitive PCR assays for relative quantifying rumen anaerobic fungal populations in both in vitro and in vivo systems

This paper describes the use of a quantitative competitive polymerase chain reaction (QC-PCR) assay; using PCR primers to the rRNA locus of rumen fungi and a standard-control DNA including design and validation. In order to test the efficiency of this method for quantifying anaerobic rumen fungi, it has been attempted to evaluate this method in in vitro conditions by comparing with an assay based on measuring cell wall chitin. The changes in fungal growth have been studied when they are grown in in vitro on either untreated (US) or sodium hydroxide treated wheat straw (TS). Results showed that rumen fungi growth was significantly higher in treated samples compared with untreated during the 12 d incubation (P < 0.05) and plotting the chitin assay's results against the competitive PCR's showed high positive correlation (R² ≥ 0.87). The low mean values of the coefficients of variance in repeatability in the QC-PCR method against the chitin assay demonstrated more reliability of this new approach. And finally, the efficiency of this method was investigated in in vivo conditions. Samples of rumen fluid were collected from four fistulated Holstein steers which were fed four different diets (basal diet, high starch, high sucrose and starch plus sucrose) in rotation. The results of QC-PCR showed that addition of these non-structural carbohydrates to the basal diets caused a significant decrease in rumen anaerobic fungi biomass. The QC-PCR method appears to be a reliable and can be used for rumen samples.     

Change in subcellular localization of overexpressed vaccine peptide in rice endosperm cell that is caused by suppression of endogenous seed storage proteins

Rice-based peptide vaccine based on T cell epitopes acts as an ideal oral tolerogen for the treatment of type 1 allergic diseases. To improve production yields of oral tolerogen against Japanese cedar pollen allergy, hybrid peptide comprising seven predominant human T cell epitopes (7Crp) derived from Japanese cedar pollen allergens, Cry j 1 and Cry j 2, was produced in transgenic rice seed by expression of its codon optimized gene under the control of the endosperm-specific 26 kD globulin (Glb-1) promoter containing its signal peptide and the simultaneous suppression of endogenous seed storage proteins (SSPs) by RNA interference. Accumulation level of 7Crp peptide produced as a secretory protein was remarkably enhanced by suppression of both the 13–14 kDa prolamins and GluA and GluB glutelins as compared to those under suppression of either of them or in wild type rice. When these SSPs were down-regulated, the 7Crp peptide was observed to be localized in ER lumen as well as ER derived PBs (PB-Is). Especially, accumulation as self-aggregates in ER lumen increased by reduction of the endogenous 13–14 kDa prolamins. It is interesting to note that the absence of C terminal KDEL ER retention signal from the 7Crp peptide resulted in higher level accumulation (116 µg/grain) than that containing the KDEL.     

Estimating mutant microsatellite allele frequencies in somatic cells by small-pool PCR

Identifying microsatellite instability (MSI) by partitioning DNA into multiple small pools containing only single genome amounts of DNA results in trapping both progenitor and low-frequency mutant alleles into pools where they can be identified and counted following PCR. Statistical approaches determining both the frequencies and the significant differences between frequencies of these Poisson-distributed alleles are presented. Results indicate a level of sensitivity and quantification not possible by standard PCR methods. Using material from colon cancer patients with high levels of MSI in their tumors, we also present the molecular and robotic methods for carrying out such studies. Validation experiments indicated mutants detectable at frequencies >0.03 above background. Frequencies obtained in tumor tissue (>0.25) met the expectations of the approach. Significant levels of MSI were detected in the constitutive tissue of the patient carrying a germ-line mutation for mismatch repair, suggesting both mechanistic and clinical applications of the procedure.     

The Effect of Mustard Gas on Salivary Trace Metals (Zn,Mn, Cu,Mg, Mo,Sr, Cd,Ca, Pb,Rb)

We have determined and compared trace metals concentration in saliva taken from chemical warfare injures who were under the exposure of mustard gas and healthy subjects by means of inductively coupled plasma optical emission spectroscopy (ICP-OES) for the first time. The influence of preliminary operations on the accuracy of ICP-OES analysis, blood contamination, the number of restored teeth in the mouth, salivary flow rate, and daily variations in trace metals concentration in saliva were also considered. Unstimulated saliva was collected at 10:00–11:00 a.m. from 45 subjects in three equal groups. The first group was composed of 15 healthy subjects (group 1); the second group consisted of 15 subjects who, upon chemical warfare injuries, did not use Salbutamol spray, which they would have normally used on a regular basis (group 2); and the third group contained the same number of patients as the second group, but they had taken their regular medicine (Salbutamol spray; group 3). Our results showed that the concentration of Cu in saliva was significantly increased in the chemical warfare injures compared to healthy subjects, as follows: healthy subjects 15.3± 5.45(p.p.b.), patients (group 2) 45.77±13.65, and patients (Salbutamol spray; group 3) 29 ±8.51 (P <0.02). In contrast, zinc was significantly decreased in the patients, as follows: healthy subjects 37 ± 9.03(p.p.b.), patients (group 2) 12.2 ± 3.56, and patients (Salbutamol spray; group 3) 20.6 ±10.01 (P < 0.01). It is important to note that direct dilution of saliva samples with ultrapure nitric acid showed the optimum ICP-OES outputs.     

Anticancer Effects of Valproic Acid on Esophageal Stem-like Carcinoma Cells

Valproic acid (VPA) is a histone deacetylase inhibitor that has been an object of interest to clinicians for its promising potency in cancer therapy, as it induces apoptosis and differentiation, and enhances of chemotherapy sensitivity. Esophageal squamous cell carcinoma (ESCC) is a malignant disease with growing incidence and low survival rate. Due to limited information on VPA activity in ESCC cells, we aimed to determine effects of VPA on chemotherapy responsiveness and expression of malignant markers in ESCC stem-like cells. Upon coadministration of non-toxic VPA + cisplatin (DDP), paclitaxel and 5-fluorouracil, viability of KYSE30 cells was assessed, and induced apoptosis was evaluated by DAPI staining, DNA laddering and flow cytometry. In addition, real time RT-PCR was performed to study changes in the expression of P21, CD44 and BMI-1 upon treatments. MTT test demonstrated that VPA significantly (P < 0.05) increased toxicity of DDP, which was confirmed by DNA laddering, flow cytometry analysis and significant (P < 0.05) overexpression of P21. Moreover, real time RT-PCR results indicated significant (P < 0.05) down regulation of CD44 and BMI-1 after VPA administration. Present attempt provided evidence, for the first time, that VPA not only improved responsiveness of esophageal stem-like cancer cells to DDP, also negatively regulated cancer stem cells markers in these cells.     

Role of non-coding variants in cardiovascular disease

Cardiovascular diseases (CVDs) constitute one of the significant causes of death worldwide. Different pathological states are linked to CVDs, which despite interventions and treatments, still have poor prognoses. The genetic component, as a beneficial tool in the risk stratification of CVD development, plays a role in the pathogenesis of this group of diseases. The emergence of genome-wide association studies (GWAS) have led to the identification of non-coding parts associated with cardiovascular traits and disorders. Variants located in functional non-coding regions, including promoters/enhancers, introns, miRNAs and 5′/3′ UTRs, account for 90% of all identified single-nucleotide polymorphisms associated with CVDs. Here, for the first time, we conducted a comprehensive review on the reported non-coding variants for different CVDs, including hypercholesterolemia, cardiomyopathies, congenital heart diseases, thoracic aortic aneurysms/dissections and coronary artery diseases. Additionally, we present the most commonly reported genes involved in each CVD. In total, 1469 non-coding variants constitute most reports on familial hypercholesterolemia, hypertrophic cardiomyopathy and dilated cardiomyopathy. The application and identification of non-coding variants are beneficial for the genetic diagnosis and better therapeutic management of CVDs.     

The predictive performance of short-linear motif features in the prediction of calmodulin-binding proteins

Background

The prediction of calmodulin-binding (CaM-binding) proteins plays a very important role in the fields of biology and biochemistry, because the calmodulin protein binds and regulates a multitude of protein targets affecting different cellular processes. Computational methods that can accurately identify CaM-binding proteins and CaM-binding domains would accelerate research in calcium signaling and calmodulin function. Short-linear motifs (SLiMs), on the other hand, have been effectively used as features for analyzing protein-protein interactions, though their properties have not been utilized in the prediction of CaM-binding proteins.

Results

We propose a new method for the prediction of CaM-binding proteins based on both the total and average scores of known and new SLiMs in protein sequences using a new scoring method called sliding window scoring (SWS) as features for the prediction module. A dataset of 194 manually curated human CaM-binding proteins and 193 mitochondrial proteins have been obtained and used for testing the proposed model. The motif generation tool, Multiple EM for Motif Elucidation (MEME), has been used to obtain new motifs from each of the positive and negative datasets individually (the SM approach) and from the combined negative and positive datasets (the CM approach). Moreover, the wrapper criterion with random forest for feature selection (FS) has been applied followed by classification using different algorithms such as k-nearest neighbors (k-NN), support vector machines (SVM), naive Bayes (NB) and random forest (RF).

Conclusions

Our proposed method shows very good prediction results and demonstrates how information contained in SLiMs is highly relevant in predicting CaM-binding proteins. Further, three new CaM-binding motifs have been computationally selected and biologically validated in this study, and which can be used for predicting CaM-binding proteins.

     

A review of anaerobic digestion bio-kinetics

Anaerobic digestion is the key to sustainable wastewater management and bioenergy production. Kinetics plays an important role in the design of bioreactors, processes, and process scale-up in anaerobic digestion. This article focuses on a state-of-the-art literature review on the experimental kinetic studies of conventional anaerobic bioreactors and anaerobic membrane bioreactors. Various kinetic models that were used to fit the experimental data and derive the kinetic parameters are summarized and discussed in the literature. The values of the maximum specific growth rate µ_max, half saturation constant K_s, decay co-efficient k_d, sludge yield Y, and methane yield Y_CH4 from experimental studies are summarized for each model. This paper can serve as an updated comprehensive source of anaerobic bio-kinetic studies and digester design.