Anchoring of Surface Proteins to the Cell Wall of Staphylococcus aureus: sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH2-Gly3 substrates

Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. We find that purified recombinant sortase hydrolyzed peptides bearing an LPXTG motif at the peptide bond between threonine and glycine. In the presence of NH2-Gly3, sortase catalyzed exclusively a transpeptidation reaction, linking the carboxyl group of threonine to the amino group of NH2-Gly3. In the presence of amino group donors the rate of sortase mediated cleavage at the LPXTG motif was increased. Hydrolysis and transpeptidation required the sulfhydryl of cysteine 184, suggesting that sortase catalyzed the transpeptidation reaction of surface protein anchoring via the formation of a thioester acyl-enzyme intermediate

An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein.

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro-molecular scale to the induction of mutant-like oligomerisation in a wild-type protein. Using the common, pathogenic Glu342Lys (Z) variant of α1-antitrypsin as antigen - whose native state is susceptible to the formation of a proto-oligomeric intermediate - we have produced a mAb (5E3) that increases the rate of oligomerisation of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognises a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerisation intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerisation by either the pathogenic Glu342Lys mutation or the binding of mAb5E3 occurs without affecting energetic barrier to polymerisation. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the Glu342Lys mutation, by facilitating the conformational interchange between these two states

Opioid suppression of conditioned anticipatory brain responses to breathlessness

© 2017 The Authors Opioid painkillers are a promising treatment for chronic breathlessness, but are associated with potentially fatal side effects. In the treatment of breathlessness, their mechanisms of action are unclear. A better understanding might help to identify safer alternatives. Learned associations between previously neutral stimuli (e.g. stairs) and repeated breathlessness induce an anticipatory threat response that may worsen breathlessness, contributing to the downward spiral of decline seen in clinical populations. As opioids are known to influence associative learning, we hypothesized that they may interfere with the brain processes underlying a conditioned anticipatory response to breathlessness in relevant brain areas, including the amygdala and the hippocampus. Healthy volunteers viewed visual cues (neutral stimuli) immediately before induction of experimental breathlessness with inspiratory resistive loading. Thus, an association was formed between the cue and breathlessness. Subsequently, this paradigm was repeated in two identical neuroimaging sessions with intravenous infusions of either low-dose remifentanil (0.7 ng/ml target-controlled infusion) or saline (randomised). During saline infusion, breathlessness anticipation activated the right anterior insula and the adjacent operculum. Breathlessness was associated with activity in a network including the insula, operculum, dorsolateral prefrontal cortex, anterior cingulate cortex and the primary sensory and motor cortices. Remifentanil reduced breathlessness unpleasantness but not breathlessness intensity. Remifentanil depressed anticipatory activity in the amygdala and the hippocampus that correlated with reductions in breathlessness unpleasantness. During breathlessness, remifentanil decreased activity in the anterior insula, anterior cingulate cortex and sensory motor cortices. Remifentanil-induced reduction in breathlessness unpleasantness was associated with increased activity in the rostral anterior cingulate cortex and nucleus accumbens, components of the endogenous opioid system known to decrease the perception of aversive stimuli. These findings suggest that in addition to effects on brainstem respiratory control, opioids palliate breathlessness through an interplay of altered associative learning mechanisms. These mechanisms provide potential targets for novel ways to develop and assess treatments for chronic breathlessness

Using particle size analysis to determine the hydrophobicity and suspension of fungal conidia with particular relevance to formulation of biopesticide

Fungal formulations are vital for effective biopesticide development. Good formulations help to optimise field efficacy while poor formulations result in product failure. This study aimed to produce a hydrophobicity test that would be appropriate for fungal conidia produced to a commercial quality and determine relative hydrophobicity of fungi from four different genera by using laser diffraction. A particle size analyser was used to determine the hydrophobicity of: three Metarhizium acridum samples, M. anisopliae, Beauveria bassiana, Trichoderma stromaticum, T. harzianum, T. viride and Alternaria eichhorniae conidia, by suspending the conidia in three different liquids: Shellsol T (a mineral oil), water and 0.05 % Tween 80. Hydrophobicity was determined by the size of the particles formed in each of the liquids. All the Metarhizium samples were the most hydrophobic followed by B. bassiana and A. eichhorniae. The Trichoderma samples were the least hydrophobic. As a comparison a phase exclusion assay and a salt-mediated aggregation and sedimentation (SAS) test were performed. It was not possible to get a reliable reading for the B. bassiana, A. eichhorniae and T. viride samples using the phase exclusion assay. The addition of salt in the SAS test did not affect the rate of sedimentation. It was hypothesised that conidia size affected the results of the SAS test that made A. eichhorniae the most hydrophobic conidia. Particle size analysis was a more accurate test for comparing fungi from difference genera compared to the SAS test and phase exclusion assay. PSA was also used to test three emulsions and demonstrated that different formulations had an effect on particle size

Antagonism of Aspergillus terreus to Sclerotinia sclerotiorum.

An Aspergillus terreus strain showed in vitro antagonistic activity against tha plant pathogens Sclerotinia sclerotiorum (Lib.) de Bary.The interacton between A. terreus and sclerotia revealed that the mycoparasite sporulated abundantly on the sclerotial surface. Cell breakdown due to host cell wall disruption was observed in inner rind cells, by a scanning electron microscopy

Development of a rapid, reliable and quantitative method: "SPOTi" for testing antifungal efficacy

A reference method for the antimicrobial susceptibility testing of common fungal pathogens such as dermatophytes, is currently lacking. In this study, we report the successful adaptation of solid agar-based spot culture growth inhibition assay (SPOTi) for dermatophytes, currently being used as a gold-standard in the anti-tubercular drug discovery field. The fungal-SPOTi assay correlated with the disc-diffusion method, and is validated using mycelial plugs. We propose the fungal-SPOTi as a high-throughput alternative to the disc-diffusion and broth micro-dilution anti-fungal assays to screen novel anti-fungals

Ethanol Induced Disordering of Pancreatic Acinar Cell Endoplasmic Reticulum: An ER Stress/Defective Unfolded Protein Response Model.

Background & aimsHeavy alcohol drinking is associated with pancreatitis, whereas moderate intake lowers the risk. Mice fed ethanol long term show no pancreas damage unless adaptive/protective responses mediating proteostasis are disrupted. Pancreatic acini synthesize digestive enzymes (largely serine hydrolases) in the endoplasmic reticulum (ER), where perturbations (eg, alcohol consumption) activate adaptive unfolded protein responses orchestrated by spliced X-box binding protein 1 (XBP1). Here, we examined ethanol-induced early structural changes in pancreatic ER proteins.MethodsWild-type and Xbp1+/- mice were fed control and ethanol diets, then tissues were homogenized and fractionated. ER proteins were labeled with a cysteine-reactive probe, isotope-coded affinity tag to obtain a novel pancreatic redox ER proteome. Specific labeling of active serine hydrolases in ER with fluorophosphonate desthiobiotin also was characterized proteomically. Protein structural perturbation by redox changes was evaluated further in molecular dynamic simulations.ResultsEthanol feeding and Xbp1 genetic inhibition altered ER redox balance and destabilized key proteins. Proteomic data and molecular dynamic simulations of Carboxyl ester lipase (Cel), a unique serine hydrolase active within ER, showed an uncoupled disulfide bond involving Cel Cys266, Cel dimerization, ER retention, and complex formation in ethanol-fed, XBP1-deficient mice.ConclusionsResults documented in ethanol-fed mice lacking sufficient spliced XBP1 illustrate consequences of ER stress extended by preventing unfolded protein response from fully restoring pancreatic acinar cell proteostasis during ethanol-induced redox challenge. In this model, orderly protein folding and transport to the secretory pathway were disrupted, and abundant molecules including Cel with perturbed structures were retained in ER, promoting ER stress-related pancreas pathology

An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states

The pathogenic exon 1 HTT protein is produced by incomplete splicing in Huntington’s disease patients

We have previously shown that exon 1 of the huntingtin gene does not always splice to exon 2 resulting in the production of a small polyadenylated mRNA (HTTexon1) that encodes the highly pathogenic exon 1 HTT protein. The level of this read-through product is proportional to CAG repeat length and is present in all knock-in mouse models of Huntington’s disease (HD) with CAG lengths of 50 and above and in the YAC128 and BACHD mouse models, both of which express a copy of the human HTT gene. We have now developed specific protocols for the quantitative analysis of the transcript levels of HTTexon1 in human tissue and applied these to a series of fibroblast lines and post-mortem brain samples from individuals with either adult-onset or juvenile-onset HD. We found that the HTTexon1 mRNA is present in fibroblasts from juvenile HD patients and can also be readily detected in the sensory motor cortex, hippocampus and cerebellum of post-mortem brains from HD individuals, particularly in those with early onset disease. This finding will have important implications for strategies to lower mutant HTT levels in patients and the design of future therapeutics