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Background: Progression to diabetes mellitus (DM) is variable and the screening time interval not well defined. The American Diabetes Association and US Preventive Services Task Force suggest screening every 3 years, but evidence is limited. The objective of the study was to develop a model to predict the probability of developing DM and suggest a risk-based screening interval. Methods: We included non-diabetic adult patients screened for DM in the Cleveland Clinic Health System if they had at least two measurements of glycated hemoglobin (HbA1c), an initial one less than 6.5% (48 mmol/mol) in 2008, and another between January, 2009 and December, 2013. Cox proportional hazards models were created. The primary outcome was DM defined as HbA1C greater than 6.4% (46 mmol/mol). The optimal rescreening interval was chosen based on the predicted probability of developing DM. Results: Of 5084 participants, 100 (4.4%) of the 2281 patients with normal HbA1c and 772 (27.5%) of the 2803 patients with prediabetes developed DM within 5 years. Factors associated with developing DM included HbA1c (HR per 0.1 units increase 1.20; 95%CI, 1.13–1.27), family history (HR 1.31; 95%CI, 1.13–1.51), smoking (HR 1.18; 95%CI, 1.03–1.35), triglycerides (HR 1.01; 95%CI, 1.00–1.03), alanine aminotransferase (HR 1.07; 95%CI, 1.03–1.11), body mass index (HR 1.06; 95%CI, 1.01–1.11), age (HR 0.95; 95%CI, 0.91–0.99) and high-density lipoproteins (HR 0.93; 95% CI, 0.90–0.95). Five percent of patients in the highest risk tertile developed DM within 8 months, while it took 35 months for 5% of the middle tertile to develop DM. Only 2.4% percent of the patients in the lowest tertile developed DM within 5 years. Conclusion: A risk prediction model employing commonly available data can be used to guide screening intervals. Based on equal intervals for equal risk, patients in the highest risk category could be rescreened after 8 months, while those in the intermediate and lowest risk categories could be rescreened after 3 and 5 years respectively. [ABSTRACT FROM AUTHOR]

Among therapeutic approaches for amyloid-related diseases, attention has recently turned to the use of natural products as effective anti-aggregation compounds. Although a wealth of in vitro and in vivo evidence indicates some common inhibitory activity of these compounds, they don’t generally suggest the same mechanism of action. Here, we show that taxifolin, a ubiquitous bioactive constituent of foods and herbs, inhibits formation of HEWL amyloid fibrils and their related toxicity by causing formation of very large globular, chain-like aggregates. A range of amyloid-specific techniques were employed to characterize this process. We found that taxifolin exerts its effect by binding to HEWL prefibrillar species, rather than by stabilizing the molecule in its native-like state. Furthermore, it’s binding results in diverting the amyloid pathway toward formation of very large globular, chain-like aggregates with low β-sheet content and reduced solvent-exposed hydrophobic patches. ThT fluorescence measurements show that the binding capacity of taxifolin is significantly reduced, upon generation of large protofibrillar aggregates at the end of growth phase. We believe these results may help design promising inhibitors of protein aggregation for amyloid-related diseases. [ABSTRACT FROM AUTHOR]

By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70’s ability to function in a wide range of cellular processes—modulating the Hsp70-substrate interaction cycle. [ABSTRACT FROM AUTHOR]

Methods: We quantified 378 HRMAS 1H NMR spectra of human brain tumours (132 glioblastomas, 101 astrocytomas, 75 meningiomas, 37 oligodendrogliomas and 33 metastases) from the eTumour database and looked for metabolic interactions by metabolite-metabolite correlation analysis (MMCA). Results: All tumour types showed remarkably similar metabolic correlations. Lactate correlated positively with alanine, glutamate with glutamine; creatine + phosphocreatine (tCr) correlated positively with lactate, alanine and choline + phosphocholine + glycerophosphocholine (tCho), and tCho correlated positively with lactate; fatty acids correlated negatively with lactate, glutamate + glutamine (tGlut), tCr and tCho. Oligodendrogliomas had fewer correlations but they still fitted that pattern. Conclusions: Possible explanations include (i) glycolytic tumour cells (the Warburg effect) generating pyruvate which is converted to lactate, alanine, glutamate and then glutamine; (ii) an association between elevated glycolysis and increased choline turnover in membranes; (iii) an increase in the tCr pool to facilitate phosphocreatine-driven glutamate uptake; (iv) lipid signals come from cytosolic lipid droplets in necrotic or pre-necrotic tumour tissue that has lower concentrations of anabolic and catabolic metabolites. Additional metabolite exchanges with host cells may also be involved. If tumours co-opt a standard set of biochemical mechanisms to grow in the brain, then drugs might be developed to disrupt those mechanisms. [ABSTRACT FROM AUTHOR]

The enzyme tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes 3’ trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase ZS, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase ZL, related to tRNase ZS through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase ZS. tRNase ZL consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether) and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: NdomTprox, located at the carboxy end of the amino domain proximal to the linker, and Tflex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F329 and L332) at the carboxy end of NdomTprox show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the Ndom- linker boundary. The Ala substitution for N378 in the Tflex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R382 reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain. [ABSTRACT FROM AUTHOR]

Background: Muscle atrophy has been explained by an anabolic resistance following food intake and an increase of dietary protein intake is recommended. To be optimal, a dietary protein has to be effective not only to initiate but also to prolong a muscle anabolic response in a catabolic state. To our knowledge, whether or not a dairy or a dairy/plant protein blend fulfills these criterions is unknown in a muscle wasting situation. Objective: Our aim was, in a control and a catabolic state, to measure continuously muscle anabolism in term of intensity and duration in response to a meal containing casein (CAS), whey (WHEY) or a whey/ plant protein blend (BLEND) and to evaluate the best protein source to elicit the best post prandial anabolism according to the physio-pathological state. Methods: Adult male Yucatan mini pigs were infused with U-13C-Phenylalanine and fed either CAS, WHEY or BLEND. A catabolic state was induced by a glucocorticoid treatment for 8 days (DEX). Muscle protein synthesis, proteolysis and balance were measured with the hind limb arterio-venous differences technique. Repeated time variance analysis were used to assess significant differences. Results: In a catabolic situation, whey proteins were able to initiate muscle anabolism which remained transient in contrast to the stimulated muscle protein accretion with WHEY, CAS or BLEND in healthy conditions. Despite the same leucine intake compared to WHEY, BLEND did not restore a positive protein balance in DEX animals. Conclusions: Even with WHEY, the duration of the anabolic response was not optimal and has to be improved in a catabolic state. The use of BLEND remained of lower efficiency even at same leucine intake than whey. [ABSTRACT FROM AUTHOR]

Human Respiratory Syncytial Virus (HRSV) is a leading cause of bronchopneumonia in infants and the elderly. To date, knowledge of viral and host protein interactions within HRSV is limited and are critical areas of research. Here, we show that HRSV Matrix (M) protein interacts with the cellular adaptor protein complex 3 specifically via its medium subunit (AP-3Mu3A). This novel protein-protein interaction was first detected via yeast-two hybrid screen and was further confirmed in a mammalian system by immunofluorescence colocalization and co-immunoprecipitation. This novel interaction is further substantiated by the presence of a known tyrosine-based adaptor protein MU subunit sorting signal sequence, YXXФ: where Ф is a bulky hydrophobic residue, which is conserved across the related RSV M proteins. Analysis of point-mutated HRSV M derivatives indicated that AP-3Mu3A- mediated trafficking is contingent on the presence of the tyrosine residue within the YXXL sorting sequence at amino acids 197–200 of the M protein. AP-3Mu3A is up regulated at 24 hours post-infection in infected cells versus mock-infected HEp2 cells. Together, our data suggests that the AP-3 complex plays a critical role in the trafficking of HRSV proteins specifically matrix in epithelial cells. The results of this study add new insights and targets that may lead to the development of potential antivirals and attenuating mutations suitable for candidate vaccines in the future. [ABSTRACT FROM AUTHOR]

Objectives: The current concepts of human fetal-placental amino acid exchange and metabolism are mainly based on animal-, in vitro- and ex vivo models. We aimed to determine and assess the paired relationships between concentrations and arteriovenous differences of 19 amino acids on the maternal and fetal sides of the human placenta in a large study sample. Methods: This cross-sectional in vivo study included 179 healthy women with uncomplicated term pregnancies. During planned cesarean section, we sampled blood from incoming and outgoing vessels on the maternal (radial artery and uterine vein) and fetal (umbilical vein and artery) sides of the placenta. Amino acid concentrations were measured by liquid chromatography—tandem mass spectrometry. We calculated paired arteriovenous differences and performed Wilcoxon signed-rank tests and Spearman’s correlations. Results: In the umbilical circulation, we observed a positive venoarterial difference (fetal uptake) for 14 amino acids and a negative venoarterial difference (fetal release) for glutamic acid (p<0.001). In the maternal circulation, we observed a positive arteriovenous difference (uteroplacental uptake) for leucine (p = 0.005), isoleucine (p = 0.01), glutamic acid (p<0.001) and arginine (p = 0.04) and a negative arteriovenous difference (uteroplacental release) for tyrosine (p = 0.002), glycine (p = 0.01) and glutamine (p = 0.02). The concentrations in the maternal artery and umbilical vein were correlated for all amino acids except tryptophan, but we observed no correlations between the uteroplacental uptake and the fetal uptake or the umbilical vein concentration. Two amino acids showed a correlation between the maternal artery concentration and the fetal uptake. Conclusions: Our human in vivo study expands the current insight into fetal-placental amino acid exchange, and discloses some differences from what has been previously described in animals. Our findings are consistent with the concept that the fetal supply of amino acids in the human is the result of a dynamic interplay between fetal and placental amino acid metabolism and interconversions. [ABSTRACT FROM AUTHOR]

Ivermectin (IVM) is a widely-used anthelmintic that works by binding to and activating glutamate-gated chloride channel receptors (GluClRs) in nematodes. The resulting chloride flux inhibits the pharyngeal muscle cells and motor neurons of nematodes, causing death by paralysis or starvation. IVM resistance is an emerging problem in many pest species, necessitating the development of novel drugs. However, drug optimisation requires a quantitative understanding of GluClR activation and modulation mechanisms. Here we investigated the biophysical properties of homomeric α (avr-14b) GluClRs from the parasitic nematode, H. contortus, in the presence of glutamate and IVM. The receptor proved to be highly responsive to low nanomolar concentrations of both compounds. Analysis of single receptor activations demonstrated that the GluClR oscillates between multiple functional states upon the binding of either ligand. The G36’A mutation in the third transmembrane domain, which was previously thought to hinder access of IVM to its binding site, was found to decrease the duration of active periods and increase receptor desensitisation. On an ensemble macropatch level the mutation gave rise to enhanced current decay and desensitisation rates. Because these responses were common to both glutamate and IVM, and were observed under conditions where agonist binding sites were likely saturated, we infer that G36’A affects the intrinsic properties of the receptor with no specific effect on IVM binding mechanisms. These unexpected results provide new insights into the activation and modulatory mechanisms of the H. contortus GluClRs and provide a mechanistic framework upon which the actions of drugs can be reliably interpreted. [ABSTRACT FROM AUTHOR]

During chemical transmission, the function of synaptic proteins must be coordinated to efficiently release neurotransmitter. Synaptotagmin 2, the Ca2+ sensor for fast, synchronized neurotransmitter release at the human neuromuscular junction, has recently been implicated in a dominantly inherited congenital myasthenic syndrome associated with a non-progressive motor neuropathy. In one family, a proline residue within the C2B Ca2+-binding pocket of synaptotagmin is replaced by a leucine. The functional significance of this residue has not been investigated previously. Here we show that in silico modeling predicts disruption of the C2B Ca2+-binding pocket, and we examine the in vivo effects of the homologous mutation in Drosophila. When expressed in the absence of native synaptotagmin, this mutation is lethal, demonstrating for the first time that this residue plays a critical role in synaptotagmin function. To achieve expression similar to human patients, the mutation is expressed in flies carrying one copy of the wild type synaptotagmin gene. We now show that Drosophila carrying this mutation developed neurological and behavioral manifestations similar to those of human patients and provide insight into the mechanisms underlying these deficits. Our Drosophila studies support a role for this synaptotagmin point mutation in disease etiology. [ABSTRACT FROM AUTHOR]

The type VI secretion (T6S) system is a molecular device for the delivery of proteins from one cell into another. T6S function depends on the contractile sheath comprised of TssB/VipA and TssC/VipB proteins. We previously reported on a mutant variant of TssB that disrupts T6S-dependent export of the self-identity protein, IdsD, in the bacterium Proteus mirabilis. Here we determined the mechanism underlying that initial observation. We show that T6S-dependent export of multiple self-recognition proteins is abrogated in this mutant strain. We have mapped the mutation, which is a single amino acid change, to a region predicted to be involved in the formation of the TssB-TssC sheath. We have demonstrated that this mutation does indeed inhibit sheath formation, thereby explaining the global disruption of T6S activity. We propose that this mutation could be utilized as an important tool for studying functions and behaviors associated with T6S systems. [ABSTRACT FROM AUTHOR]

Diabetes is an irreversible condition characterized by elevated blood glucose levels. Currently, there are no predictive biomarkers for this disease and the existing ones such as hemoglobin A1c and fasting blood glucose are used only when diabetes symptoms are noticed. The objective of this work was first to explore the potential of leucine and isoleucine amino acids as diabetes type 2 biomarkers using their Raman spectroscopic signatures. Secondly, we wanted to explore whether Raman spectroscopy can be applied in comparative efficacy studies between commercially available anti-diabetic drug pioglitazone and the locally used anti-diabetic herbal extract Momordica spinosa (Gilg.)Chiov. Sprague Dawley (SD) rat’s blood was used and were pipetted onto Raman substrates prepared from conductive silver paste smeared glass slides. Prominent Raman bands associated with glucose (926, 1302, 1125 cm−1), leucine (1106, 1248, 1302, 1395 cm−1) and isolecucine (1108, 1248, 1437 and 1585 cm−1) were observed. The Raman bands centered at 1125 cm−1, 1395 cm−1 and 1437 cm−1 associated respectively to glucose, leucine and isoleucine were chosen as biomarker Raman peaks for diabetes type 2. These Raman bands displayed decreased intensities in blood from diabetic SD rats administered antidiabetic drugs pioglitazone and herbal extract Momordica spinosa (Gilg.)Chiov. The intensity decrease indicated reduced concentration levels of the respective biomarker molecules: glucose (1125 cm−1), leucine (1395 cm−1) and isoleucine (1437 cm−1) in blood. The results displayed the power and potential of Raman spectroscopy in rapid (10 seconds) diabetes and pre-diabetes screening in blood (human or rat’s) with not only glucose acting as a biomarker but also leucine and isoleucine amino-acids where intensities of respectively assigned bands act as references. It also showed that using Raman spectroscopic signatures of the chosen biomarkers, the method can be an alternative for performing comparative efficacy studies between known and new anti-diabetic drugs. Reports on use of Raman spectroscopy in type 2 diabetes mellitus screening with Raman bands associated with leucine and isoleucine molecules acting as reference is rare in literature. The use of Raman spectroscopy in pre-diabetes screening of blood for changes in levels of leucine and isoleucine amino acids is particularly interesting as once elevated levels are noticed, necessary interventions to prevent diabetes development can be initiated. [ABSTRACT FROM AUTHOR]

Alpha 1-antitrypsin (A1AT) is a serine protease inhibitor that mainly inhibits neutrophil elastase in the lungs. A variant of A1AT at the P1 position with methionine 358 to arginine (A1AT-Pittsburgh) is a rapid inhibitor of thrombin with greatly diminished anti-elastase activity. The P2 residue (position 357) of A1AT-Pittsburgh has been shown to play an important role in interactions with thrombin and kallikrein, but the role of P2 residue in wild-type A1AT has largely been unraveled. Here, we investigated the effects of P2 proline substitutions in wild-type A1AT on interactions with porcine pancreatic elastase (PPE) and human neutrophil elastase (HNE). The mutant A1AT proteins (P357A, P357D, P357K, P357L, P357N, P357S, and P357W) were less efficient than the wild-type A1AT at inhibiting PPE and HNE. Among the mutants, P357D did not form a complex with PPE, whereas P357L, P357N, and P357W showed significantly reduced complex formation with PPE. Surprisingly, mass spectrometry analysis revealed that P357D had two cleavage sites after the P9 alanine and the P3 isoleucine residues. Our results indicate that the size and negative charge of the R group of the P2 residue influence the interaction with elastases. Specifically, the negative charge at the P2 residue is disfavored and the resulting conformational changes in the reactive center loop upon interaction with PPE lead to cleavage at new sites. Overall, the results of this study demonstrate a previously unknown role for P2 residue in determining inhibitory specificity of A1AT. [ABSTRACT FROM AUTHOR]

Using protein counts sampled from single cell proteomics distributions to constrain fluxes through a genome-scale model of metabolism, Population flux balance analysis (Population FBA) successfully described metabolic heterogeneity in a population of independent Escherichia coli cells growing in a defined medium. We extend the methodology to account for correlations in protein expression arising from the co-regulation of genes and apply it to study the growth of independent Saccharomyces cerevisiae cells in two different growth media. We find the partitioning of flux between fermentation and respiration predicted by our model agrees with recent 13C fluxomics experiments, and that our model largely recovers the Crabtree effect (the experimentally known bias among certain yeast species toward fermentation with the production of ethanol even in the presence of oxygen), while FBA without proteomics constraints predicts respirative metabolism almost exclusively. The comparisons to the 13C study showed improvement upon inclusion of the correlations and motivated a technique to systematically identify inconsistent kinetic parameters in the literature. The minor secretion fluxes for glycerol and acetate are underestimated by our method, which indicate a need for further refinements to the metabolic model. For yeast cells grown in synthetic defined (SD) medium, the calculated broad distribution of growth rates matches experimental observations from single cell studies, and we characterize several metabolic phenotypes within our modeled populations that make use of diverse pathways. Fast growing yeast cells are predicted to perform significant amount of respiration, use serine-glycine cycle and produce ethanol in mitochondria as opposed to slow growing cells. We use a genetic algorithm to determine the proteomics constraints necessary to reproduce the growth rate distributions seen experimentally. We find that a core set of 51 constraints are essential but that additional constraints are still necessary to recover the observed growth rate distribution in SD medium. [ABSTRACT FROM AUTHOR]

Aminopeptidase A (APA) is a membrane-bound zinc metalloprotease cleaving, in the brain, the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the control of blood pressure in hypertensive animals. Using a refined APA structure derived from the human APA crystal structure, we docked the specific and selective APA inhibitor, EC33 in the presence of Ca2+. We report the presence in the S1 subsite of Arg-887 (Arg-878 in mouse APA), the guanidinium moiety of which established an interaction with the electronegative sulfonate group of EC33. Mutagenic replacement of Arg-878 with an alanine or a lysine residue decreased the affinity of the recombinant enzymes for the acidic substrate, α-L-glutamyl-β-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA for the acidic substrate, the mutated enzymes hydrolyzing more efficiently basic and neutral substrates, although the addition of Ca2+ partially restored the acidic substrate specificity. The analysis of the 3D models of the Arg-878 mutated APAs revealed a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis. These findings demonstrate the key role of Arg-878 together with Ca2 + in APA substrate specificity for N-terminal acidic amino acid residues by ensuring the optimal positioning of acidic substrates during catalysis. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis is an in vitro system employed for plant propagation and the study of embryo development. Nitrogen is essential for plant growth and development and, hence, the production of healthy embryos during somatic embryogenesis. Glutamine has been shown to increase plant biomass in many in vitro applications, including somatic embryogenesis. However, several aspects of nitrogen nutrition during somatic embryogenesis remain unclear. Therefore, we investigated the uptake and assimilation of nitrogen in Norway spruce pro-embryogenic masses to elucidate some of these aspects. In our study, addition of glutamine had a more positive effect on growth than inorganic nitrogen. The nitrogen uptake appeared to be regulated, with a strong preference for glutamine; 67% of the assimilated nitrogen in the free amino acid pool originated from glutamine-nitrogen. Glutamine addition also relieved the apparently limited metabolism (as evidenced by the low concentration of free amino acids) of pro-embryogenic masses grown on inorganic nitrogen only. The unusually high alanine concentration in the presence of glutamine, suggests that alanine biosynthesis was involved in alleviating these constraints. These findings inspire further studies of nitrogen nutrition during the somatic embryogenesis process; identifying the mechanism(s) that govern glutamine enhancement of pro-embryogenic masses growth is especially important in this regard. [ABSTRACT FROM AUTHOR]

The metabolism of living systems involves many enzymes that play key roles as catalysts and are essential to biological function. Searching ligands with the ability to modulate enzyme activities is central to diagnosis and therapeutics. Peptides represent a promising class of potential enzyme modulators due to the large chemical diversity, and well-established methods for library synthesis. Peptides and their derivatives are found to play critical roles in modulating enzymes and mediating cellular uptakes, which are increasingly valuable in therapeutics. We present a methodology that uses molecular dynamics (MD) and point-variant screening to identify short peptide motifs that are critical for inhibiting β-galactosidase (β-Gal). MD was used to simulate the conformations of peptides and to suggest short motifs that were most populated in simulated conformations. The function of the simulated motifs was further validated by the experimental point-variant screening as critical segments for inhibiting the enzyme. Based on the validated motifs, we eventually identified a 7-mer short peptide for inhibiting an enzyme with low μM IC50. The advantage of our methodology is the relatively simplified simulation that is informative enough to identify the critical sequence of a peptide inhibitor, with a precision comparable to truncation and alanine scanning experiments. Our combined experimental and computational approach does not rely on a detailed understanding of mechanistic and structural details. The MD simulation suggests the populated motifs that are consistent with the results of the experimental alanine and truncation scanning. This approach appears to be applicable to both natural and artificial peptides. With more discovered short motifs in the future, they could be exploited for modulating biocatalysis, and developing new medicine. [ABSTRACT FROM AUTHOR]

Allergy to peanuts has become a common and severe problem, especially in westernized countries. In this study, we evaluated the target and epitope specificity of the capture and detection mouse monoclonal antibodies (mAbs) used in a commercial peanut allergen detection platform. We first identified the target of these antibodies as Ara h 3 and then used an overlapping peptide array of Ara h 3 to determine the antibody-binding epitopes. Further amino acids critical for the binding via alanine substitutions at individual amino acid residues within the epitope were mapped. Finally, inhibition ELISA and inhibition immunoblotting using a recombinant Ara h 3 protein were performed to confirm these results. Surprisingly, the capture and detection mAbs showed identical binding characteristics and were presumed to represent two isolates of the same clone, a notion supported by both isoelectric focusing electrophoresis and Liquid chromatography–mass spectrometry experiments. The simultaneous binding of a pair of identical mAbs to an individual allergen such as Ara h3 is attributed to the multivalency of the analyte and has implications for developing diagnostic assays for additional multimeric allergens. [ABSTRACT FROM AUTHOR]

Understanding the pathophysiology of Alzheimer disease has relied upon the use of amyloid peptides from a variety of sources, but most predominantly synthetic peptides produced using t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. These synthetic methods can lead to minor impurities which can have profound effects on the biological activity of amyloid peptides. Here we used a combination of cytotoxicity assays, fibrillation assays and high resolution mass spectrometry (MS) to identify impurities in synthetic amyloid preparations that inhibit both cytotoxicity and aggregation. We identify the Aβ42Δ39 species as the major peptide contaminant responsible for limiting both cytotoxicity and fibrillation of the amyloid peptide. In addition, we demonstrate that the presence of this minor impurity inhibits the formation of a stable Aβ42 dimer observable by MS in very pure peptide samples. These results highlight the critical importance of purity and provenance of amyloid peptides in Alzheimer’s research in particular, and biological research in general. [ABSTRACT FROM AUTHOR]

All organisms have evolved pathways to respond to different forms of cellular stress. The Gcn2 kinase is best known as a regulator of translation initiation in response to starvation for amino acids. Work in budding yeast has showed that the molecular mechanism of GCN2 activation involves the binding of uncharged tRNAs, which results in a conformational change and GCN2 activation. This pathway requires GCN1, which ensures delivery of the uncharged tRNA onto GCN2. However, Gcn2 is activated by a number of other stresses which do not obviously involve accumulation of uncharged tRNAs, raising the question how Gcn2 is activated under these conditions. Here we investigate the requirement for ongoing translation and tRNA binding for Gcn2 activation after different stresses in fission yeast. We find that mutating the tRNA-binding site on Gcn2 or deleting Gcn1 abolishes Gcn2 activation under all the investigated conditions. These results suggest that tRNA binding to Gcn2 is required for Gcn2 activation not only in response to starvation but also after UV irradiation and oxidative stress. [ABSTRACT FROM AUTHOR]

The plastid genomes of different plant species exhibit significant variation, thereby providing valuable markers for exploring evolutionary relationships and population genetics. Glycine soja (wild soybean) is recognized as the wild ancestor of cultivated soybean (G. max), representing a valuable genetic resource for soybean breeding programmes. In the present study, the complete plastid genome of G. soja was sequenced using Illumina paired-end sequencing and then compared it for the first time with previously reported plastid genome sequences from nine other Glycine species. The G. soja plastid genome was 152,224 bp in length and possessed a typical quadripartite structure, consisting of a pair of inverted repeats (IRa/IRb; 25,574 bp) separated by small (178,963 bp) and large (83,181 bp) single-copy regions, with a 51-kb inversion in the large single-copy region. The genome encoded 134 genes, including 87 protein-coding genes, eight ribosomal RNA genes, and 39 transfer RNA genes, and possessed 204 randomly distributed microsatellites, including 15 forward, 25 tandem, and 34 palindromic repeats. Whole-plastid genome comparisons revealed an overall high degree of sequence similarity between G. max and G. gracilis and some divergence in the intergenic spacers of other species. Greater numbers of indels and SNP substitutions were observed compared with G. cyrtoloba. The sequence of the accD gene from G. soja was highly divergent from those of the other species except for G. max and G. gracilis. Phylogenomic analyses of the complete plastid genomes and 76 shared genes yielded an identical topology and indicated that G. soja is closely related to G. max and G. gracilis. The complete G. soja genome sequenced in the present study is a valuable resource for investigating the population and evolutionary genetics of Glycine species and can be used to identify related species. [ABSTRACT FROM AUTHOR]

Nicotinamidases are amidohydrolases that convert nicotinamide into nicotinic acid, contributing to NAD+ homeostasis in most organisms. In order to increase the number of nicotinamidases described to date, this manuscript characterizes a nicotinamidase obtained from a metagenomic library fosmid clone (JFF054_F02) obtained from a geothermal water stream microbial mat community in a Japanese epithermal mine. The enzyme showed an optimum temperature of 90°C, making it the first hyperthermophilic bacterial nicotinamidase to be characterized, since the phylogenetic analysis of this fosmid clone placed it in a clade of uncultured geothermal bacteria. The enzyme, named as UbNic, not only showed an alkaline optimum pH, but also a biphasic pH dependence of its kcat, with a maximum at pH 9.5–10.0. The two pKa values obtained were 4.2 and 8.6 for pKes1 and pKes2, respectively. These results suggest a possible flexible catalytic mechanism for nicotinamidases, which reconciles the two previously proposed mechanisms. In addition, the enzyme showed a high catalytic efficiency, not only toward nicotinamide, but also toward other nicotinamide analogs. Its mutational analysis showed that a tryptophan (W83) is needed in one of the faces of the active site to maintain low Km values toward all the substrates tested. Furthermore, UbNic proved to contain a Fe2+ ion in its metal binding site, and was revealed to belong to a new nicotinamidase subgroup. All these characteristics, together with its high pH- and thermal stability, distinguish UbNic from previously described nicotinamidases, and suggest that a wide diversity of enzymes remains to be discovered in extreme environments. [ABSTRACT FROM AUTHOR]

Neisseria gonorrhoeae is the causative organism of gonorrhoea, a sexually transmitted disease that globally accounts for an estimated 80 to 100 million new infections per year. Increasing resistances to all common antibiotics used for N. gonorrhoeae treatment pose the risk of an untreatable disease. Further knowledge of ways of infection and host immune response are needed to understand the pathogen-host interaction and to discover new treatment alternatives against this disease. Therefore, detailed information about immunogenic proteins and their properties like epitope sites could advance further research in this area. In this work, we investigated immunogenic proteins of N. gonorrhoeae for linear epitopes by microarrays. Dominant linear epitopes were identified for eleven of the nineteen investigated proteins with three polyclonal rabbit antibodies from different immunisations. Identified linear epitopes were further examined for non-specific binding with antibodies to Escherichia coli and the closely related pathogen Neisseria meningitidis. On top of that, amino acids crucial for the antibody epitope binding were detected by microarray based alanine scans. [ABSTRACT FROM AUTHOR]

The collagen binding integrin α2β1 plays a crucial role in hemostasis, fibrosis, and cancer progression amongst others. It is specifically inhibited by rhodocetin (RC), a C-type lectin-related protein (CLRP) found in Malayan pit viper (Calloselasma rhodostoma) venom. The structure of RC alone reveals a heterotetramer arranged as an αβ and γδ subunit in a cruciform shape. RC specifically binds to the collagen binding A-domain of the integrin α2 subunit, thereby blocking collagen-induced platelet aggregation. However, until now, the molecular basis for this interaction has remained unclear. Here, we present the molecular structure of the RCγδ-α2A complex solved to 3.0 Å resolution. Our findings show that RC undergoes a dramatic structural reorganization upon binding to α2β1 integrin. Besides the release of the nonbinding RCαβ tandem, the RCγ subunit interacts with loop 2 of the α2A domain as result of a dramatic conformational change. The RCδ subunit contacts the integrin α2A domain in the “closed” conformation through its helix C. Combined with epitope-mapped antibodies, conformationally locked α2A domain mutants, point mutations within the α2A loop 2, and chemical modifications of the purified toxin protein, this molecular structure of RCγδ-α2A complex explains the inhibitory mechanism and specificity of RC for α2β1 integrin. [ABSTRACT FROM AUTHOR]

Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. C. difficile spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca2+ during C. difficile spore germination and provide direct evidence that intestinal Ca2+ coordinates with bile salts to stimulate germination. Endogenous Ca2+ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca2+. However, environmental Ca2+ replaces glycine as a co-germinant and circumvents the need for endogenous Ca2+ fluxes. Cd630_32980 is dispensable for colonization in a murine model of C. difficile infection and ex vivo germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca2+ present within the gastrointestinal tract plays a critical role in C. difficile germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (e.g., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI. [ABSTRACT FROM AUTHOR]

The postsynaptic adhesion proteins Neuroligins (NLs) are essential for proper synapse function, and their alterations are associated with a variety of neurodevelopmental disorders. It is increasingly clear that each NL isoform occupies specific subsets of synapses and is able to regulate the function of discrete networks. Studies of NL2 and NL4 in the retina in particular have contributed towards uncovering their role in inhibitory synapse function. In this study we show that NL3 is also predominantly expressed at inhibitory postsynapses in the retinal inner plexiform layer (IPL), where it colocalizes with both GABAA- and glycinergic receptor clusters in a 3:2 ratio. In the NL3 deletion-mutant (knockout or KO) mouse, we uncovered a dramatic reduction of the number of GABAAα2-subunit containing GABAA receptor clusters at the IPL. Retinal activity was thereafter assessed in KO and wild-type (WT) littermates by multi-electrode-array recordings of the output cells of retina, the retinal ganglion cells (RGCs). RGCs in the NL3 KO showed reduced spontaneous activity and an altered response to white noise stimulation. Moreover, upon application of light flashes, the proportion of cells firing at light offset (OFF RGCs) was significantly lower in the NL3 KO compared to WT littermates, whereas the relative number of cells firing at light onset (ON RGCs) increased. Interestingly, although GABAAα2-bearing receptors have been related to direction-selective circuits of the retina, features of direction selective-retinal ganglion cells recorded remained unperturbed in the NL3 KO. Together our data underscore the importance of NL3 for the integrity of specific GABAAergic retinal circuits and identifies NL3 as an important regulator of retinal activity. [ABSTRACT FROM AUTHOR]

Heterodera glycines, the soybean cyst nematode, is the most economically important plant-parasitic nematode on soybean production in the U.S. The objectives of this study were to evaluate the potential of plant growth-promoting rhizobacteria (PGPR) strains for mortality of H. glycines J2 in vitro and for reducing nematode population density on soybean in greenhouse, microplot, and field trials. The major group causing mortality to H. glycines in vitro was the genus Bacillus that consisted of 92.6% of the total 663 PGPR strains evaluated. The subsequent greenhouse, microplot, and field trials indicated that B. velezensis strain Bve2 consistently reduced H. glycines cyst population density at 60 DAP. Bacillus mojavensis strain Bmo3 suppressed H. glycines cyst and total H. glycines population density under greenhouse conditions. Bacillus safensis strain Bsa27 and Mixture 1 (Bve2 + Bal13) reduced H. glycines cyst population density at 60 DAP in the field trials. Bacillus subtilis subsp. subtilis strains Bsssu2 and Bsssu3, and B. velezensis strain Bve12 increased early soybean growth including plant height and plant biomass in the greenhouse trials. Bacillus altitudinis strain Bal13 increased early plant growth on soybean in the greenhouse and microplot trials. Mixture 2 (Abamectin + Bve2 + Bal13) increased early plant growth in the microplot trials at 60 DAP, and also enhanced soybean yield at harvest in the field trials. These results demonstrated that individual PGPR strains and mixtures can reduce H. glycines population density in the greenhouse, microplot, and field conditions, and increased yield of soybean. [ABSTRACT FROM AUTHOR]

Background: Secondary bacterial infections from snakebites contribute to the high complication rates that can lead to permanent function loss and disabilities. Although common in endemic areas, routine empirical prophylactic use of antibiotics aiming to prevent secondary infection lacks a clearly defined policy. The aim of this work was to estimate the efficacy of amoxicillin clavulanate for reducing the secondary infection incidence in patients bitten by Bothrops snakes, and, secondarily, identify risk factors for secondary infections from snakebites in the Western Brazilian Amazon. Methods and findings: This was an open-label, two-arm individually randomized superiority trial to prevent secondary infection from Bothrops snakebites. The antibiotic chosen for this clinical trial was oral amoxicillin clavulanate per seven days compared to no intervention. A total of 345 patients were assessed for eligibility in the study period. From this total, 187 accomplished the inclusion criteria and were randomized, 93 in the interventional group and 94 in the untreated control group. All randomized participants completed the 7 days follow-up period. Enzyme immunoassay confirmed Bothrops envenoming diagnosis in all participants. Primary outcome was defined as secondary infection (abscess and/or cellulitis) until day 7 after admission. Secondary infection incidence until 7 days after admission was 35.5% in the intervention group and 44.1% in the control group [RR = 0.80 (95%CI = 0.56 to 1.15; p = 0.235)]. Survival analysis demonstrated that the time from patient admission to the onset of secondary infection was not different between amoxicillin clavulanate treated and control group (Log-rank = 2.23; p = 0.789).Secondary infections incidence in 7 days of follow-up was independently associated to fibrinogen >400 mg/dL [AOR = 4.78 (95%CI = 2.17 to 10.55; p<0.001)], alanine transaminase >44 IU/L [AOR = 2.52 (95%CI = 1.06 to 5.98; p = 0.037)], C-reactive protein >6.5 mg/L [AOR = 2.98 (95%CI = 1.40 to 6.35; p = 0.005)], moderate pain [AOR = 24.30 (95%CI = 4.69 to 125.84; p<0.001)] and moderate snakebites [AOR = 2.43 (95%CI = 1.07 to 5.50; p = 0.034)]. Conclusions/Significance: Preemptive amoxicillin clavulanate was not effective for preventing secondary infections from Bothrops snakebites. Laboratorial markers, such as high fibrinogen, alanine transaminase and C-reactive protein levels, and severity clinical grading of snakebites, may help to accurately diagnose secondary infections. Trial registration: Brazilian Clinical Trials Registry (ReBec): ; UTN Number: . [ABSTRACT FROM AUTHOR]

Diacylglycerol acyltransferases (DGATs) have a crucial role in the biosynthesis of triacylglycerol (TG), the major storage form of metabolic energy in eukaryotic organisms. Even though DGAT2, one of two distinct DGATs, has a vital role in TG biosynthesis, little is known about the regulation of DGAT2 activity. In this study, we examined the role of cysteine and its oxidation in the enzymatic activity of human DGAT2 in vitro. Human DGAT2 activity was considerably inhibited not only by thiol-modifying reagents (NEM and IA) but also by ROS-related chemicals (H2O2 and β-lapachone), while human DGAT1 and GPAT1 were little affected. Particularly, ROS-related chemicals concomitantly induced intermolecular disulfide crosslinking of human DGAT2. Both the oxidative inactivation and disulfide crosslinking were almost completely reversed by the treatment with DTT, a disulfide-reducing agent. These results clearly demonstrated the significant role of ROS-induced intermolecular crosslinking in the inactivation of human DGAT2 and also suggested DGAT2 as a redox-sensitive regulator in TG biosynthesis. [ABSTRACT FROM AUTHOR]

α-Ketoisocaproate (KIC) is used widely in the pharmaceutical and nutraceutical industries. In previous studies, we achieved a one-step biosynthesis of KIC from -leucine, using an Escherichia coli whole-cell biocatalyst expressing an -amino acid deaminase (-AAD) from Proteus vulgaris. Herein, we report the fine-tuning of -AAD gene expression in E. coli BL21 (DE3) at the transcriptional and translational levels to improve the KIC titer. By optimizing the plasmid origin with different copy numbers, modulating messenger RNA structure downstream of the initiation codon, and designing the sequences at the ribosome binding site, we increased biocatalyst activity to 31.77%, 24.89%, and 30.20%, respectively, above that achieved with BL21/pet28a-lad. The highest KIC titers reached 76.47 g·L-1, 80.29 g·L-1, and 81.41 g·L-1, respectively. Additionally, the integration of these three engineering strategies achieved an even higher KIC production of 86.55 g·L-1 and a higher -leucine conversion rate of 94.25%. The enzyme-engineering strategies proposed herein may be generally applicable to the construction of other biocatalysts. [ABSTRACT FROM AUTHOR]

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities. [ABSTRACT FROM AUTHOR]

Vibrio cholerae is a diarrheal pathogen that induces accumulation of lipid droplets in enterocytes, leading to lethal infection of the model host Drosophila melanogaster. Through untargeted lipidomics, we provide evidence that this process is the product of a host phospholipid degradation cascade that induces lipid droplet coalescence in enterocytes. This infection-induced cascade is inhibited by mutation of the V. cholerae glycine cleavage system due to intestinal accumulation of methionine sulfoxide (MetO), and both dietary supplementation with MetO and enterocyte knock-down of host methionine sulfoxide reductase A (MsrA) yield increased resistance to infection. MsrA converts both free and protein-associated MetO to methionine. These findings support a model in which dietary MetO competitively inhibits repair of host proteins by MsrA. Bacterial virulence strategies depend on functional host proteins. We propose a novel virulence paradigm in which an intestinal pathogen ensures the repair of host proteins essential for pathogenesis through consumption of dietary MetO. [ABSTRACT FROM AUTHOR]

Methods: Alanine scan of insulin receptor (IR)-B exon 11 and site-directed mutagenesis of amino acid 718 in human IR-A and IR-B were performed. Ligand affinities to wild type and mutated receptors were studied by displacement of radioactive insulin in binding assay on secreted soluble midi receptors or solubilized semi-purified full length receptors stably expressed in Baby Hamster Kidney cells. Phosphorylation of IR in response to insulin, IGF1 and IGF2 was measured using ELISA. Results: Insulin, insulin detemir and insulin glargine maximally showed two fold differences in affinity for human IR-A and IR-B, but IGF1 and IGF2 had up to 10 fold preference for IR-A. Alanine scan of exon 11 revealed that position 718 is important for low IGF1 affinity to IR-B. Mutational analysis of amino acid residue 718 in IR-A and IR-B demonstrated that charge is important for IGF1 and IGF2 affinity but not important for insulin affinity. The affinity of IGF1 and IGF2 for the mutant IR-A P718K was comparable to the wild type IR-B whereas the affinity of IGF1 and IGF2 for the mutant IR-B K718P was comparable to the wild type IR-A. Changes in affinity were also reflected in the IR activation pattern. Conclusion: Mutating position 718 in human IR-B to the proline found at position 718 in human IR-A increased IGF1 and IGF2 affinity to a level comparable to IR-A and mutating position 718 in IR-A to the lysine found at position 718 in IR-B decreased IGF1 and IGF2 affinity to a level comparable to IR-B, whereas a negatively charged glutamate did not. These changes in the affinities were also reflected in the IR phosphorylation pattern, meaning that position 718 is important for both affinity and activation of the receptor. It should be emphasized that none of the mutations affected insulin affinity, indicating that the mutations did not alter the overall receptor structure and that the effect is ligand specific. [ABSTRACT FROM AUTHOR]

Background: Retrospective analyses suggest anaesthetic-analgesics technique during cancer surgery may affect recurrence/metastasis. This could involve direct effects of anaesthetic-analgesic drugs on cancer cells. While μ-opioid receptor over-expression in lung tumours is associated with greater metastasis, other anaesthetic-analgesic receptor targets in cancer recurrence/metastasis remain unexplored. Therefore, we evaluated the association between genetic expression of anaesthetic-analgesic receptor targets and recurrence/metastasis, using a repository of breast cancer gene expression and matching clinical data. Methods: A list of 23 genes encoding for the most prominent anaesthetic-analgesic receptor targets was compiled. This was processed through BreastMark- an algorithm integrating gene expression data from ~17,000 samples and clinical data from >4,500 breast cancer samples. Gene expression data was dichotomized using disease-free survival (survival without recurrence) and distant disease-free survival (survival without metastasis) as end points. Hazard ratios were calculated by Cox-regression analysis. Enrichment for prognostic markers was determined by randomly choosing 23-member gene lists from all available genes, calculating how often >5 significant markers were observed and adjusting p-values for multiple testing. This was repeated 10,000 times and an empirical p-value calculated. Results: Of 23 selected genes, 9 were significantly associated with altered rates of metastasis and 4 with recurrence on univariate analysis. Adjusting for multiple testing, 5 of these 9 genes remained significantly associated with metastasis, non with recurrence. This ratio of genes (5/23) was not significantly enriched for markers of metastasis (p = 0.07). Conclusion: Several anaesthetic-analgesic receptor genes were associated with metastatic spread in breast cancer. Overall there was no significant enrichment in prognostic markers of metastasis, although a trend was observed. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) toxicity affects numerous metabolic processes in plants. In the presence of Cd, plants accumulate specific amino acids which may be beneficial to developing Cd tolerance. Our study aimed to characterize the changes in the metabolism of selected free amino acids that are associated with Cd tolerance, and investigate the levels of selected microelements in order to relate these changes to the adaptation strategies of two metallophytes—Noccaea caerulescens (Redlschlag, Austria) and Noccaea praecox (Mežica, Slovenia). The plants were exposed to Cd contamination (90 mg Cd/kg soil) for 120 days in a pot experiment. Our results showed higher Cd accumulation in N. praecox compared to N. caerulescens. Cadmium contamination reduced the zinc and nickel levels in both species and a mixed effect was determined for copper and manganese content. Differences in free amino acid metabolism were observed between the two metallophytes growing under Cd-free and Cd-loaded conditions. Under Cd-free conditions, aromatic amino acids (phenylalanine, tryptophan and tyrosine) and branched-chain amino acids (leucine, isoleucine and valine) were accumulated more in the leaves of N. praecox than in N. caerulescens. Cd stress increased the content of these amino acids in both species but this increase was significant only in N. caerulescens leaves. Marked differences in the responses of the two species to Cd stress were shown for alanine, phenylalanine, threonine and sarcosine. Cadmium contamination also induced an increase of threonine as alanine and sarcosine decrease, which was larger in N. caerulescens than in N. praecox. All these factors contribute to the higher adaptation of N. praecox to Cd stress. [ABSTRACT FROM AUTHOR]

Background: Intracellular HCV-RNA reduction is a proposed mechanism of action of direct-acting antivirals (DAAs), alternative to hepatocytes elimination by pegylated-interferon plus ribavirin (PR). We modeled ALT and HCV-RNA kinetics in cirrhotic patients treated with currently-used all-DAA combinations to evaluate their mode of action and cytotoxicity compared with telaprevir (TVR)+PR. Study design: Mathematical modeling of ALT and HCV-RNA kinetics was performed in 111 HCV-1 cirrhotic patients, 81 treated with all-DAA regimens and 30 with TVR+PR. Kinetic-models and Cox-analysis were used to assess determinants of ALT-decay and normalization. Results: HCV-RNA kinetics was biphasic, reflecting a mean effectiveness in blocking viral production >99.8%. The first-phase of viral-decline was faster in patients receiving NS5A-inhibitors compared to TVR+PR or sofosbuvir+simeprevir (p<0.001), reflecting higher efficacy in blocking assembly/secretion. The second-phase, noted δ and attributed to infected-cell loss, was faster in patients receiving TVR+PR or sofosbuvir+simeprevir compared to NS5A-inhibitors (0.27 vs 0.21 d-1, respectively, p = 0.0012). In contrast the rate of ALT-normalization, noted λ, was slower in patients receiving TVR+PR or sofosbuvir+simeprevir compared to NS5A-inhibitors (0.17 vs 0.27 d-1, respectively, p<0.001). There was no significant association between the second-phase of viral-decline and ALT normalization rate and, for a given level of viral reduction, ALT-normalization was more profound in patients receiving DAA, and NS5A in particular, than TVR+PR. Conclusions: Our data support a process of HCV-clearance by all-DAA regimens potentiated by NS5A-inhibitor, and less relying upon hepatocyte death than IFN-containing regimens. This may underline a process of “cell-cure” by DAAs, leading to a fast improvement of liver homeostasis. [ABSTRACT FROM AUTHOR]

RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP) and P34 binds to 5S ribosomal RNA (rRNA) and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine these domains’ interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP) 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP. [ABSTRACT FROM AUTHOR]

We describe the methodology and results from our validation study of the fully automated antibody structure prediction tool available in the BIOVIA (formerly Accelrys) protein modeling suite. Extending our previous study, we have validated the automated approach using a larger and more diverse data set (157 unique antibody Fv domains versus 11 in the previous study). In the current study, we explore the effect of varying several parameter settings in order to better understand their influence on the resulting model quality. Specifically, we investigated the dependence on different methods of framework model construction, antibody numbering schemes (Chothia, IMGT, Honegger and Kabat), the influence of compatibility of loop templates using canonical type filtering, wider exploration of model solution space, and others. Our results show that our recently introduced Top5 framework modeling method results in a small but significant improvement in model quality whereas the effect of other parameters is not significant. Our analysis provides improved guidelines of best practices for using our protocol to build antibody structures. We also identify some limitations of the current computational model which will enhance proper evaluation of model quality by users and suggests possible future enhancements. [ABSTRACT FROM AUTHOR]

Osteogenesis imperfecta (OI) is a heterogeneous group of disorders of connective tissue, caused mainly by mutations in the collagen I genes (COL1A1 and COL1A2). Dentinogenesis imperfecta (DGI) and other dental aberrations are common features of OI. We investigated the association between collagen I mutations and DGI, taurodontism, and retention of permanent second molars in a retrospective cohort of 152 unrelated children and adolescents with OI. The clinical examination included radiographic evaluations. Teeth from 81 individuals were available for histopathological evaluation. COL1A1/2 mutations were found in 104 individuals by nucleotide sequencing. DGI was diagnosed clinically and radiographically in 29% of the individuals (44/152) and through isolated histological findings in another 19% (29/152). In the individuals with a COL1A1 mutation, 70% (7/10) of those with a glycine substitution located C-terminal of p.Gly305 exhibited DGI in both dentitions while no individual (0/7) with a mutation N-terminal of this point exhibited DGI in either dentition (p = 0.01). In the individuals with a COL1A2 mutation, 80% (8/10) of those with a glycine substitution located C terminal of p.Gly211 exhibited DGI in both dentitions while no individual (0/5) with a mutation N-terminal of this point (p = 0.007) exhibited DGI in either dentition. DGI was restricted to the deciduous dentition in 20 individuals. Seventeen had missense mutations where glycine to serine was the most prevalent substitution (53%). Taurodontism occurred in 18% and retention of permanent second molars in 31% of the adolescents. Dental aberrations are strongly associated with qualitatively changed collagen I. The varying expressivity of DGI is related to the location of the collagen I mutation. Genotype information may be helpful in identifying individuals with OI who have an increased risk of dental aberrations. [ABSTRACT FROM AUTHOR]

In this work, SrCrxFe12-xO19 (x = 0.0, 0.5, 1.0, 1.5) nanostructures were successfully synthesized by sol-gel auto-combustion method, and different aminoacids were used as green reductants. Various analysis results show that SrCrxFe12-xO19 nanoparticles synthesized successfully.The present study shows that SrCrxFe12-xO19 nanoparticle could be used as adsorbent for the desulfurization of liquid fuels. Increasing of nanoparticles concentration was caused to increase the adsorption rate of sulfur contents of fuel. The adsorption rate of sulfur contents of fuel in various concentrations 4.5, 9.5, and 18.5 g. L -1 of SrCrxFe12-xO19 nanoparticles in solution was estimated about 39, 50, and 62% for 30 min, respectively. The results of catalytic tests reveals that SrCrxFe12-xO19 nanoparticles have the potential to be used as a new kind of semiconductor catalysts for the desulfurization of liquid fuels. Magnetic property of the final sample was measured at room temperature by a vibration sample magnetometer (VSM) and shown that the intrinsic coercivity of product is about 6000 Oe and it exhibits characteristics of single magnetic domains (Mr/ Ms = 0.53). [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) undergo phosphorylation at several intracellular residues by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers arrestin recruitment and receptor desensitization. The exact sites of phosphorylation and their function remained largely unknown for the human β1-adrenoceptor (ADRB1), a key GPCR in adrenergic signal transduction and the target of widely used drugs such as β-blockers. The present study aimed to identify the intracellular phosphorylation sites in the ADRB1 and to delineate their function. The human ADRB1 was expressed in HEK293 cells and its phosphorylation pattern was determined by mass spectrometric analysis before and after stimulation with a receptor agonist. We identified a total of eight phosphorylation sites in the receptor’s third intracellular loop and C-terminus. Analyzing the functional relevance of individual sites using phosphosite-deficient receptor mutants we found phosphorylation of the ADRB1 at Ser461/Ser462 in the distal part of the C-terminus to determine β-arrestin2 recruitment and receptor internalization. Our data reveal the phosphorylation pattern of the human ADRB1 and the site that mediates recruitment of β-arrestin2. [ABSTRACT FROM AUTHOR]

Leucine (Leu), one of the three branch chain amino acids, acts as a signaling molecule in the regulation of overall amino acid (AA) and protein metabolism. Leucine is also considered to be a potent stimulus for the secretion of insulin from pancreatice β-cells. Our objective was to study the effects of a duodenal bolus infusion of Leu on insulin and glucagon secretion, on plasma AA concentrations, and to do a metabolomic profiling of dairy cows as compared to infusions with either glucose or saline. Six duodenum-fistulated Holstein cows were studied in a replicated 3 × 3 Latin square design with 3 periods of 7 days, in which the treatments were applied at the end of each period. The treatments were duodenal bolus infusions of Leu (DIL; 0.15 g/kg body weight), glucose (DIG; at Leu equimolar dosage) or saline (SAL). On the day of infusion, the treatments were duodenally infused after 5 h of fasting. Blood samples were collected at -15, 0, 10, 20, 30, 40, 50, 60, 75, 90, 120, 180, 210, 240 and 300 min relative to the start of infusion. Blood plasma was assayed for concentrations of insulin, glucagon, glucose and AA. The metabolome was also characterized in selected plasma samples (i.e. from 0, 50, and 120 min relative to the infusion). Body weight, feed intake, milk yield and milk composition were recorded throughout the experiment. The Leu infusion resulted in significant increases of Leu in plasma reaching 20 and 15-fold greater values than that in DIG and SAL, respectively. The elevation of plasma Leu concentrations after the infusion led to a significant decrease (P<0.05) in the plasma concentrations of isoleucine, valine, glycine, and alanine. In addition, the mean concentrations of lysine, methionine, phenylalanine, proline, serine, taurine, threonine, and asparagine across all time-points in plasma of DIL cows were reduced (P<0.05) compared with the other groups. In contrast to the working hypothesis about an insulinotropic effect of Leu, the circulating concentrations of insulin were not affected by Leu. In DIG, insulin and glucose concentrations peaked at 30–40 and 40–50 min after the infusion, respectively. Insulin concentrations were greater (P<0.05) from 30–40 min in DIG than DIL and SAL, and glucose was elevated in DIG over DIL and SAL from 30–75 min and 40–50 min, respectively. Multivariate metabolomics data analysis (principal component analysis and partial least squares discriminant analysis) revealed a clear separation when the DIL cows were compared with the DIG and SAL cows at 50 and 120 min after the infusion. By using this analysis, several metabolites, mainly acylcarnitines, methionine sulfoxide and components from the kynurenine pathway were identified as the most relevant for separating the treatment groups. These results suggest that Leu regulates the plasma concentrations of branched-chain AA, and other AA, apparently by stimulating their influx into the cells from the circulation. A single-dose duodenal infusion of Leu did not elicit an apparent insulin response, but affected multiple intermediary metabolic pathways including AA and energy metabolism by mechanisms yet to be elucidated. [ABSTRACT FROM AUTHOR]

Current through the σ-pore was first detected in hKv1.3_V388C channels, where the V388C mutation in hKv1.3 channels opened a new pathway (σ-pore) behind the central α-pore. Typical for this mutant channel was inward current at potentials more negative than -100 mV when the central α-pore was closed. The α-pore blockers such as TEA+ and peptide toxins (CTX, MTX) could not reduce current through the σ-pore of hKv1.3_V388C channels. This new pathway would proceed in parallel to the α-pore in the S6-S6 interface gap. To see whether this phenomenon is restricted to hKv1.3 channels we mutated hKv1.2 at the homologue position (hKv1.2_V370C). By overexpression of hKv1.2_V370C mutant channels in COS-7 cells we could show typical σ-currents. The electrophysiological properties of the σ-pore in hKv1.3_V388C and hKv1.2_V370C mutant channels were similar. The σ-pore of hKv1.2_V370C channels was most permeable to Na+ and Li+ whereas Cl- and protons did not influence current through the σ-pore. Tetraethylammonium (TEA+), charybdotoxin (CTX) and maurotoxin (MTX), known α-pore blockers, could not reduce current through the σ-pore of hKv1.2_V370C channels. Taken together we conclude that the observation of σ-pore currents is not restricted to Kv1.3 potassium channels but can also be observed in a closely related potassium channel. This finding could have implications in the treatment of different ion channel diseases linked to mutations of the respective channels in regions close to homologue position investigated by us. [ABSTRACT FROM AUTHOR]

Background: Indoor residual spraying (IRS) with DDT has been the primary strategy for control of the visceral leishmaniasis (VL) vector Phlebotomus argentipes in India but efficacy may be compromised by resistance. Synthetic pyrethroids are now being introduced for IRS, but with a shared target site, the para voltage-gated sodium channel (VGSC), mutations affecting both insecticide classes could provide cross-resistance and represent a threat to sustainable IRS-based disease control. Methodology/Principal findings: A region of the Vgsc gene was sequenced in P. argentipes from the VL hotspot of Bihar, India. Two knockdown resistance (kdr) mutations were detected at codon 1014 (L1014F and L1014S), each common in mosquitoes, but previously unknown in phlebotomines. Both kdr mutations appear largely recessive, but as homozygotes (especially 1014F/F) or as 1014F/S heterozygotes exert a strong effect on DDT resistance, and significantly predict survivorship to class II pyrethroids in short-duration bioassays. The mutations are present at high frequency in wild P. argentipes populations from Bihar, with 1014F significantly more common in higher VL areas. Conclusions/Significance: The Vgsc mutations detected appear to be a primary mechanism underlying DDT resistance in P. argentipes and a contributory factor in reduced pyrethroid susceptibility, suggesting a potential impact if P. argentipes are subjected to suboptimal levels of pyrethroid exposure, or additional resistance mechanisms evolve. The assays to detect kdr frequency changes provide a sensitive, high-throughput monitoring tool to detecting spatial and temporal variation in resistance in P. argentipes. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the interobserver reproducibility of two-dimensional shear wave elastography (2D-SWE) in measuring liver stiffness (LS) and to investigate factors related to liver 2D-SWE. Materials and methods: A prospective study was conducted between August 2011 and August 2012 in rheumatoid arthritis patients who had been treated with methotrexate. Interobserver reproducibility of 2D-SWE was evaluated, and the relationship between interobserver difference in LS and related factors was analyzed using linear regression analyses. We considered age, sex, alanine transaminase, cholesterol, body mass index (BMI), and waist circumference as clinical factors, and the mean value of standard deviation (SDM), its difference between two examiners, mean diameter of the regions of interest (ROIM), and its difference in the elasticity map as investigation factors. The cut-off value for significant factors to predict interobserver discrepancies in LS-based fibrosis stage was also inspected. Results: In total, 176 patients were enrolled. The intraclass correlation coefficient between the two examiners was 0.784. In the univariate analysis, SDM and ROIM were independently associated with interobserver differences in LS as well as BMI, waist circumference, and the difference of ROI, but SDM and ROIM were the only ones significantly related in multivariate analysis (p<0.001 and p = 0.021, respectively). The best cut-off value for SDM in predicting interobserver discrepancy in LS-based fibrosis stage was 1.4. Conclusions: Interobserver reproducibility of 2D-SWE for measuring LS was good and SDM was the most significantly associated factor with interobserver differences in LS and interobserver discordance in LS-based fibrosis stage. [ABSTRACT FROM AUTHOR]

The RNA-binding protein Hu antigen R (HuR) binds to AU-rich elements (ARE) in the 3’-untranslated region (UTR) of target mRNAs. The HuR-ARE interactions stabilize many oncogenic mRNAs that play important roles in tumorigenesis. Thus, small molecules that interfere with the HuR-ARE interaction could potentially inhibit cancer cell growth and progression. Using a fluorescence polarization (FP) competition assay, we identified the compound azaphilone-9 (AZA-9) derived from the fungal natural product asperbenzaldehyde, binds to HuR and inhibits HuR-ARE interaction (IC50 ~1.2 μM). Results from surface plasmon resonance (SPR) verified the direct binding of AZA-9 to HuR. NMR methods mapped the RNA-binding interface of HuR and identified the involvement of critical RNA-binding residues in binding of AZA-9. Computational docking was then used to propose a likely binding site for AZA-9 in the RNA-binding cleft of HuR. Our results show that AZA-9 blocks key RNA-binding residues of HuR and disrupts HuR-RNA interactions in vitro. This knowledge is needed in developing more potent AZA-9 derivatives that could lead to new cancer therapy. [ABSTRACT FROM AUTHOR]

Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Ǻ and 2.75 Ǻ respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs). [ABSTRACT FROM AUTHOR]

The soybean aphid (Aphis glycines) is one of the main insect pests of soybean (Glycine max) worldwide. Genomics approaches have provided important data on transcriptome changes, both in the insect and in the plant, in response to the plant-aphid interaction. However, the difficulties to transform soybean and to rear soybean aphid on artificial media have hindered our ability to systematically test the function of genes identified by those analyses as mediators of plant resistance to the insect. An efficient approach to produce transgenic soybean material is the production of transformed hairy roots using Agrobacterium rhizogenes; however, soybean aphids colonize leaves or stems and thus this approach has not been utilized. Here, we developed a hairy root system that allowed effective aphid feeding. We show that this system supports aphid performance similar to that observed in leaves. The use of hairy roots to study plant resistance is validated by experiments showing that roots generated from cotyledons of resistant lines carrying the Rag1 or Rag2 resistance genes are also resistant to aphid feeding, while related susceptible lines are not. Our results demonstrate that hairy roots are a good system to study soybean aphid-soybean interactions, providing a quick and effective method that could be used for functional analysis of the resistance response to this insect. [ABSTRACT FROM AUTHOR]

We present a coarse-grained simulation model that is capable of simulating the minute-timescale dynamics of protein translocation and membrane integration via the Sec translocon, while retaining sufficient chemical and structural detail to capture many of the sequence-specific interactions that drive these processes. The model includes accurate geometric representations of the ribosome and Sec translocon, obtained directly from experimental structures, and interactions parameterized from nearly 200 μs of residue-based coarse-grained molecular dynamics simulations. A protocol for mapping amino-acid sequences to coarse-grained beads enables the direct simulation of trajectories for the co-translational insertion of arbitrary polypeptide sequences into the Sec translocon. The model reproduces experimentally observed features of membrane protein integration, including the efficiency with which polypeptide domains integrate into the membrane, the variation in integration efficiency upon single amino-acid mutations, and the orientation of transmembrane domains. The central advantage of the model is that it connects sequence-level protein features to biological observables and timescales, enabling direct simulation for the mechanistic analysis of co-translational integration and for the engineering of membrane proteins with enhanced membrane integration efficiency. [ABSTRACT FROM AUTHOR]

Background: We recently reported that tranexamic acid (TXA) evokes pain in rats by inhibiting γ-aminobutyric acid and glycine receptors on neurons in the spinal dorsal horn. Although TXA is commonly used to reduce perioperative blood loss during various surgeries, its potential to induce intraoperative nociception, thereby increasing the need for more analgesics during surgery, has not been investigated. Therefore, this study aimed to investigate whether TXA evokes pain and increases the need for a higher infusion rate of remifentanil in patients undergoing surgery for adolescent idiopathic scoliosis (AIS). Methods: Data were collected from patients with AIS who underwent posterior spinal fusion surgery from January 2008 to December 2015. All surgical procedures were performed under total intravenous anesthesia with propofol and remifentanil, by the same team of orthopedic surgeons and anesthesiologists at a single institution. Patients in the TXA group were administered TXA (loading and maintenance doses, 1000 mg and 100 mg/h) whereas those in the control group were not. Our primary outcome was the infusion rate of the intraoperative opioid analgesic remifentanil. Results: The final analysis was based on data collected from 33 and 30 patients in the control and TXA groups, respectively. No differences were observed in the demographic data or the hemodynamic parameters between the two groups of patients. In the TXA group, the durations of surgery and anesthesia were shorter, intravascular fluid volume and total blood loss were lower, and the doses of fentanyl and ketamine administered were higher than they were in the control group (P < 0.05 for all). The mean infusion rate of intraoperative remifentanil was significantly higher in the TXA group than in the control group (control group: 0.23 ± 0.04 μg/kg/min; TXA group: 0.28 ± 0.12 μg/kg/min; P = 0.014). Conclusions: Patients who received TXA during the AIS surgery required a higher infusion rate of remifentanil, indicating that TXA evoked pain during the surgery. [ABSTRACT FROM AUTHOR]