A high-throughput turbidometric assay for screening inhibitors of Leishmania major protein disulfide isomerase

The use of a high-throughput technique to perform a pilot screen for Leishmania major protein disulfide isomerase (LmPDI) inhibitors identification is reported. In eukaryotic cells, protein disulfide isomerase (PDI) plays a crucial role in protein folding by catalyzing the rearrangement of disulfide bonds in substrate proteins following their synthesis. LmPDI displays similar domain structure organization and functional properties to other PDI family members and is involved in Leishmania virulence. The authors used a method based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol. The screen of a small library of 1920 compounds was performed in a 384-well format and led to the identification of 27 compounds with inhibitory activity against LmPDI. The authors further tested the cytotoxicity of these compounds using Jurkat cells as well as their effect on Leishmania donovani amastigotes using high-content analysis. Results show hexachlorophene and a mixture of theaflavin monogallates inhibit Leishmania multiplication in infected macrophages derived from THP-1 cells, although the inhibitory effect on LmPDI enzymatic activity does not necessarily correlate with the antileishmanial activity.     

Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol

Mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3α-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD⁺-dependent 3α-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3α-hydroxysteroids and bile acids using NAD⁺ as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3α-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD⁺-dependent type of cytosolic 3α-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3α-HSD resulted in a switch in favor of NADP⁺ specificity, suggesting their key roles in coenzyme specificity.     

Optimization of methods for peripheral blood mononuclear cells isolation and expansion of human gamma delta T cells

Human Vg9/Vδ2 T cells (γδ T cells) are immune surveillance cells both in innate and adaptive immunity and are a possible target for anticancer therapies, which can induce immune responses in a variety of cancers. Small non-peptide antigens such as zoledronate can do activation and expansion of T cells in vitro. It is evident that for adoptive cancer therapies, large numbers of functional cells are needed into cancer patients. Hence, optimization of methods needs to be carried out for the efficient expansion of these T cells. Standardization of peripheral blood mononuclear cells (PBMCs) isolation was devised. Cytokines (interleukin 2 (IL-2) and interleukin 15 (IL-15)) and zoledronate were also standardized for different concentrations. It was found that an increased number of PBMCs were recovered when washing was done at 1100 revolution per minute (rpm). Significantly high expansion fold was (2524 ± 787 expansion fold) achieved when stimulation of PBMCs was done with 1 µM of zoledronate and both cytokines IL-2 and IL-15 supported the expansion and survival of cells at the concentrations of 100 IU/ml and 10 ng/ml respectively. 14-day cultures showed highly pure (91.6 ± 5.1%) and live (96.5 ± 2.5%) expanded γδ T cells. This study aimed to standardize an easy to manipulate technique for the expansion of γδ T cells, giving a higher yield.     

Guillain-Barré syndrome as a cause of acute flaccid paralysis in Iraqi children: a result of 15 years of nation-wide study

Background

Guillain-Barré syndrome (GBS) is the most common cause of acute flaccid paralysis (AFP) in the post-poliomyelitis eradication era. This is the first study done to identify the epidemiology, clinical features, and outcome of GBS in Iraqi children over 15 years.

Methods

The surveillance database about AFP cases?<?15 years reported during January 1997-December 2011 was used.

Results

GBS represented 52.5% of AFP cases, with an incidence of 1.33 case/100,000 population?<?15 years/year. There was a higher incidence in the Southern provinces, age group 1–4 years, males, and outside the capital city of province, with no significant seasonal variations (p?=?.22). Survival probability after the 1 year of onset for those with respiratory muscle involvement was .76 (95% CI: .60-.86), versus .97 (95% Cl: .96-.98) for those who did not develop it (p?<?.001); and .97 (95% CI: .96-.98) for those living inside the capital city, versus .94 (.93-.95) for those living outside (p?=?.001). Cumulative incidence of residual paralysis for patients living inside the capital city was .21 (95% CI: .18-.24), versus .27 (95% CI: .25-.29) for those living outside (p?<?.001).

Conclusions

The incidence, age and gender distribution, and seasonality of GBS among Iraqi children is similar to those reported from other previous studies. It is the most important cause of AFP, especially in those between the age of 1 to 4 years living in rural areas.

     

Constraint-based, homology model of the extracellular domain of the epithelial Na+ channel α subunit reveals a mechanism of channel activation by proteases

The epithelial Na(+) channel (ENaC) mediates Na(+) transport across high resistance epithelia. This channel is assembled from three homologous subunits with the majority of the protein's mass found in the extracellular domains. Acid-sensing ion channel 1 (ASIC1) is homologous to ENaC, but a key functional domain is highly divergent. Here we present molecular models of the extracellular region of α ENaC based on a large data set of mutations that attenuate inhibitory peptide binding in combination with comparative modeling based on the resolved structure of ASIC1. The models successfully rationalized the data from the peptide binding screen. We engineered new mutants that had not been tested based on the models and successfully predict sites where mutations affected peptide binding. Thus, we were able to confirm the overall general fold of our structural models. Further analysis suggested that the α subunit-derived inhibitory peptide affects channel gating by constraining motions within two major domains in the extracellular region, the thumb and finger domains.     

Bayesian Estimation Using Ranked Set Sampling

Bayesian Estimation of the parameter of a distribution is considered using Ranked set sampling (RSS). It is shown that for at least one RSS plan, the Bayes estimator has smaller Bayes risk than the Bayes estimator using simple random sampling (SRS). Furthermore, for exponential family with conjugate prior, the Bayes estimator of the mean using balanced RSS dominates, in terms of its Bayes risk, the Bayes estimator of the mean using SRS. This procedure is used to estimate the average Milk yield of four hundreds and two sheep. The empirical efficiency supports the theoretical findings.     

Probing the inhibitor selectivity pocket of human 20α-hydroxysteroid dehydrogenase (AKR1C1) with X-ray crystallography and site-directed mutagenesis

Human 20α-hydroxysteroid dehydrogenase (AKR1C1) is an important drug target due to its role in the development of lung and endometrial cancers, premature birth and neuronal disorders. We report the crystal structure of AKR1C1 complexed with the first structure-based designed inhibitor 3-chloro-5-phenylsalicylic acid (K_i = 0.86 nM) bound in the active site. The binding of 3-chloro-5-phenylsalicylic acid to AKR1C1 resulted in a conformational change in the side chain of Phe311 to accommodate the bulky phenyl ring substituent at the 5-position of the inhibitor. The contributions of the nonconserved residues Leu54, Leu306, Leu308 and Phe311 to the binding were further investigated by site-directed mutagenesis, and the effects of the mutations on the K_i value were determined. The Leu54Val and Leu306Ala mutations resulted in 6- and 81-fold increases, respectively, in K_i values compared to the wild-type enzyme, while the remaining mutations had little or no effects.     

Base of the thumb domain modulates epithelial sodium channel gating

The activity of the epithelial sodium channel (ENaC) is modulated by multiple external factors, including proteases, cations, anions and shear stress. The resolved crystal structure of acid-sensing ion channel 1 (ASIC1), a structurally related ion channel, and mutagenesis studies suggest that the large extracellular region is involved in recognizing external signals that regulate channel gating. The thumb domain in the extracellular region of ASIC1 has a cylinder-like structure with a loop at its base that is in proximity to the tract connecting the extracellular region to the transmembrane domains. This loop has been proposed to have a role in transmitting proton-induced conformational changes within the extracellular region to the gate. We examined whether loops at the base of the thumb domains within ENaC subunits have a similar role in transmitting conformational changes induced by external Na(+) and shear stress. Mutations at selected sites within this loop in each of the subunits altered channel responses to both external Na(+) and shear stress. The most robust changes were observed at the site adjacent to a conserved Tyr residue. In the context of channels that have a low open probability due to retention of an inhibitory tract, mutations in the loop activated channels in a subunit-specific manner. Our data suggest that this loop has a role in modulating channel gating in response to external stimuli, and are consistent with the hypothesis that external signals trigger movements within the extracellular regions of ENaC subunits that are transmitted to the channel gate.