Isolation, purification, and characterization of pregnancy-specific protein B from elk and moose placenta.

Pregnancy-specific protein B (PSPB) was isolated, purified, and partially characterized from elk and moose placenta. The procedure, which was monitored by bovine PSPB (bPSPB) RIA, included homogenization and extraction in aqueous solution, acidic and ammonium sulfate precipitation, and ion exchange, gel filtration, and affinity chromatographies. The estimated molecular sizes of moose PSPB (mPSPB) were 58 kDa and 31 kDa, and of elk PSPB (ePSPB) were 57 kDa, 45 kDa, and 31 kDa by SDS-PAGE. The isoelectric points of mPSPB were 4.8, 6.6, and 6.7, and of ePSPB were 4.8, 4.9, 6.1, and 6.2 as determined by isoelectric focusing and two-dimensional gel electrophoresis. The carbohydrate contents of mPSPB and ePSPB were approximately 3.15% and 4.98%, respectively. Although ePSPB and mPSPB were recognized by anti-bPSPB in an Ouchterlony double immunodiffusion test, they were found to share identical epitopes and partial identities compared to bPSPB. After treatment at different temperatures (20-60 degrees C) for 1 h, the immunoreactivities of ePSPB and mPSPB in serum were very stable. Only ePSPB in serum treated at 60 degrees C lost some immunoreactivity. After alteration of serum pH (pH 3-11) for 2 h, the immunoreactivities of ePSPB and mPSPB became lower at pH 3 and 4, and remained stable from pH 5 to 11. These data show that moose and elk PSPB have properties similar to those of bovine and ovine PSPB.     

Investigation of Inflammation and Tissue Patterning in the Gut Using a Spatially Explicit General-Purpose Model of Enteric Tissue (SEGMEnT)

The mucosa of the intestinal tract represents a finely tuned system where tissue structure strongly influences, and is turn influenced by, its function as both an absorptive surface and a defensive barrier. Mucosal architecture and histology plays a key role in the diagnosis, characterization and pathophysiology of a host of gastrointestinal diseases. Inflammation is a significant factor in the pathogenesis in many gastrointestinal diseases, and is perhaps the most clinically significant control factor governing the maintenance of the mucosal architecture by morphogenic pathways. We propose that appropriate characterization of the role of inflammation as a controller of enteric mucosal tissue patterning requires understanding the underlying cellular and molecular dynamics that determine the epithelial crypt-villus architecture across a range of conditions from health to disease. Towards this end we have developed the Spatially Explicit General-purpose Model of Enteric Tissue (SEGMEnT) to dynamically represent existing knowledge of the behavior of enteric epithelial tissue as influenced by inflammation with the ability to generate a variety of pathophysiological processes within a common platform and from a common knowledge base. In addition to reproducing healthy ileal mucosal dynamics as well as a series of morphogen knock-out/inhibition experiments, SEGMEnT provides insight into a range of clinically relevant cellular-molecular mechanisms, such as a putative role for Phosphotase and tensin homolog/phosphoinositide 3-kinase (PTEN/PI3K) as a key point of crosstalk between inflammation and morphogenesis, the protective role of enterocyte sloughing in enteric ischemia-reperfusion and chronic low level inflammation as a driver for colonic metaplasia. These results suggest that SEGMEnT can serve as an integrating platform for the study of inflammation in gastrointestinal disease.     

Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.     

Pentacycloundecane lactam vs lactone norstatine type protease HIV inhibitors: binding energy calculations and DFT study

Background

Novel pentacycloundecane (PCU)-lactone-CO-EAIS peptide inhibitors were designed, synthesized, and evaluated against wild-type C-South African (C-SA) HIV-1 protease. Three compounds are reported herein, two of which displayed IC₅₀ values of less than 1.00 μM. A comparative MM-PB(GB)SA binding free energy of solvation values of PCU-lactam and lactone models and their enantiomers as well as the PCU-lactam-NH-EAIS and lactone-CO-EAIS peptide inhibitors and their corresponding diastereomers complexed with South African HIV protease (C-SA) was performed. This will enable us to rationalize the considerable difference between inhibitory concentration (IC₅₀) of PCU-lactam-NH-EAIS and PCU-lactone-CO-EAIS peptides.

Results

The PCU-lactam model exhibited more negative calculated binding free energies of solvation than the PCU-lactone model. The same trend was observed for the PCU-peptide inhibitors, which correspond to the experimental activities for the PCU-lactam-NH-EAIS peptide (IC₅₀ = 0.076 μM) and the PCU-lactone-CO-EAIS peptide inhibitors (IC₅₀ = 0.850 μM). Furthermore, a density functional theory (DFT) study on the natural atomic charges of the nitrogen and oxygen atoms of the three PCU-lactam, PCU-lactim and PCU-lactone models were performed using natural bond orbital (NBO) analysis. Electrostatic potential maps were also used to visualize the electron density around electron-rich regions. The asymmetry parameter (η) and quadrupole coupling constant (χ) values of the nitrogen and oxygen nuclei of the model compounds were calculated at the same level of theory. Electronic molecular properties including polarizability and electric dipole moments were also calculated and compared. The Gibbs theoretical free solvation energies of solvation (∆G_solv) were also considered.

Conclusions

A general trend is observed that the lactam species appears to have a larger negative charge distribution around the heteroatoms, larger quadrupole constant, dipole moment and better solvation energy, in comparison to the PCU-lactone model. It can be argued that these characteristics will ensure better eletronic interaction between the lactam and the receptor, corresponding to the observed HIV protease activities in terms of experimental IC₅₀ data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0115-5) contains supplementary material, which is available to authorized users.     

Prediction of Severe Disease in Children with Diarrhea in a Resource-Limited Setting

Objective

To investigate the accuracy of three clinical scales for predicting severe disease (severe dehydration or death) in children with diarrhea in a resource-limited setting.

Methods

Participants included 178 children admitted to three Rwandan hospitals with diarrhea. A local physician or nurse assessed each child on arrival using the World Health Organization (WHO) severe dehydration scale and the Centers for Disease Control (CDC) scale. Children were weighed on arrival and daily until they achieved a stable weight, with a 10% increase between admission weight and stable weight considered severe dehydration. The Clinical Dehydration Scale was then constructed post-hoc using the data collected for the other two scales. Receiver Operator Characteristic (ROC) curves were constructed for each scale compared to the composite outcome of severe dehydration or death.

Results

The WHO severe dehydration scale, CDC scale, and Clinical Dehydration Scale had areas under the ROC curves (AUCs) of 0.72 (95% CI 0.60, 0.85), 0.73 (95% CI 0.62, 0.84), and 0.80 (95% CI 0.71, 0.89), respectively, in the full cohort. Only the Clinical Dehydration Scale was a significant predictor of severe disease when used in infants, with an AUC of 0.77 (95% CI 0.61, 0.93), and when used by nurses, with an AUC of 0.78 (95% CI 0.63, 0.93).

Conclusions

While all three scales were moderate predictors of severe disease in children with diarrhea, scale accuracy varied based on provider training and age of the child. Future research should focus on developing or validating clinical tools that can be used accurately by nurses and other less-skilled providers to assess all children with diarrhea in resource-limited settings.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

Development of enzyme-immunoassays based on egg yolk antibodies for the detection of mycotoxins

Enzyme-linked immunosorbent assays (ELISA) proved to be a fast and simple method for the detection of mycotoxins and other undesired contaminants in food and feed. The present study is focused on the optimisation and exploitation of the egg yolk antibody technology in order to develop competitive ELISAs for the detection of mycotoxins in cereals. Due to its importance as one of the most relevant Fusarium mycotoxins, the trichothecene deoxynivalenol (DON) was selected as representative. Chickens were immunised with different protein conjugates performing varying booster intervals. The antibodies were isolated by the poly(ethylene glycol) precipitation method according to Polson. By use of these antibodies an indirect competitive ELISA was developed for the detection of DON. First investigations of naturally contaminated wheat samples showed a good correspondence with results obtained by GC-ECD when calibration in blank wheat extracts was performed.