Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ_0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ_0461 (rMHJ_0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ_0461 resides on the surface of M. hyopneumoniae. rMHJ_0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ_0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ_0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae.

A vital ingredient for engineering bone tissue, in the culture dish, is the use of recombinant matrix and growth proteins to help accelerate the growth of cultivated tissues into clinically acceptable quantities. The skeletal organic matrices of calcifying marine invertebrates are an untouched potential source of such growth inducing proteins. They have the advantage of being ready-made and retain the native state of the original protein. Striking evidence shows that skeleton building bone morphogenic protein-2/4 (BMP) and transforming growth factor beta (TGF-β) exist within various marine invertebrates such as, corals. Best practice mariculture and the latest innovations in long-term marine invertebrate cell cultivation can be implemented to ensure that these proteins are produced sustainably and supplied continuously. This also guarantees that coral reef habitats are not damaged during the collection of specimens. Potential proteins for bone repair, either extracted from the skeleton or derived from cultivated tissues, can be identified, evaluated and retrieved using chromatography, cell assays and proteomic methods. Due to the current evidence for bone matrix protein analogues in marine invertebrates, together with the methods established for their production and retrieval there is a genuine prospect that they can be used to regenerate living bone for potential clinical use.

Background: Adipose derived stem cells (ADSCs) are acquired from abdominal liposuction yielding a thousand fold more stem cells per millilitre than those from bone marrow. A large research void exists as to whether ADSCs are capable of transdermal differentiation toward neuronal phenotypes. Previous studies have investigated the use of chemical cocktails with varying inconclusive results. Methods: Human ADSCs were treated with a chemical stimulant, beta-mercaptoethanol, to direct them toward a neuronal-like lineage within 24 hours. Quantitative proteomics using iTRAQ was then performed to ascertain protein abundance differences between ADSCs, beta-mercaptoethanol treated ADSCs and a glioblastoma cell line. Results: The soluble proteome of ADSCs differentiated for 12 hours and 24 hours was significantly different from basal ADSCs and control cells, expressing a number of remodeling, neuroprotective and neuroproliferative proteins. However toward the later time point presented stress and shock related proteins were observed to be up regulated with a large down regulation of structural proteins. Cytokine profiles support a large cellular remodeling shift as well indicating cellular distress. Conclusion: The earlier time point indicates an initiation of differentiation. At the latter time point there is a vast loss of cell population during treatment. At 24 hours drastically decreased cytokine profiles and overexpression of stress proteins reveal that exposure to beta-mercaptoethanol beyond 24 hours may not be suitable for clinical application as our results indicate that the cells are in trauma whilst producing neuronal-like morphologies. The shorter treatment time is promising, indicating a reducing agent has fast acting potential to initiate neuronal differentiation of ADSCs.

BackgroundEnterotoxigenic Escherichia coli (ETEC) are a major economic threat to pig production globally, with serogroups O8, O9, O45, O101, O138, O139, O141, O149 and O157 implicated as the leading diarrhoeal pathogens affecting pigs below four weeks of age. A multiple antimicrobial resistant ETEC O157 (O157 SvETEC) representative of O157 isolates from a pig farm in New South Wales, Australia that experienced repeated bouts of pre- and post-weaning diarrhoea resulting in multiple fatalities was characterized here. Enterohaemorrhagic E. coli (EHEC) O157:H7 cause both sporadic and widespread outbreaks of foodborne disease, predominantly have a ruminant origin and belong to the ST11 clonal complex. Here, for the first time, we conducted comparative genomic analyses of two epidemiologically-unrelated porcine, disease-causing ETEC O157; E. coli O157 SvETEC and E. coli O157:K88 734/3, and examined their phylogenetic relationship with EHEC O157:H7.ResultsO157 SvETEC and O157:K88 734/3 belong to a novel sequence type (ST4245) that comprises part of the ST23 complex and are genetically distinct from EHEC O157. Comparative phylogenetic analysis using PhyloSift shows that E. coli O157 SvETEC and E. coli O157:K88 734/3 group into a single clade and are most similar to the extraintestinal avian pathogenic Escherichia coli (APEC) isolate O78 that clusters within the ST23 complex. Genome content was highly similar between E. coli O157 SvETEC, O157:K88 734/3 and APEC O78, with variability predominantly limited to laterally acquired elements, including prophages, plasmids and antimicrobial resistance gene loci. Putative ETEC virulence factors, including the toxins STb and LT and the K88 (F4) adhesin, were conserved between O157 SvETEC and O157:K88 734/3. The O157 SvETEC isolate also encoded the heat stable enterotoxin STa and a second allele of STb, whilst a prophage within O157:K88 734/3 encoded the serum survival gene bor. Both isolates harbor a large repertoire of antibiotic resistance genes but their association with mobile elements remains undetermined.ConclusionsWe present an analysis of the first draft genome sequences of two epidemiologically-unrelated, pathogenic ETEC O157. E. coli O157 SvETEC and E. coli O157:K88 734/3 belong to the ST23 complex and are phylogenetically distinct to EHEC O157 lineages that reside within the ST11 complex.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1382-y) contains supplementary material, which is available to authorized users.

Regenerative medicine is a rapidly expanding area in research and clinical applications. Therapies involving the use of small molecule chemicals aim to simplify the creation of specific drugs for clinical applications. Adult mesenchymal stem cells have recently shown the capacity to differentiate into several cell types applicable for regenerative medicine (specifically neural cells, using chemicals). Valproic acid was an ideal candidate due to its clinical stability. It has been implicated in the induction of neural differentiation; however, the mechanism and the downstream events were not known. In this study, we showed that using valproic acid on adult mesenchymal stem cells induced neural differentiation within 24 h by upregulating the expression of suppressor of cytokine signaling 5 (SOCS5) and Fibroblast growth factor 21 (FGF21), without increasing the potential death rate of the cells. Through this, the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is downregulated, and the mitogen-activated protein kinase (MAPK) cascade is activated. The bioinformatics analyses revealed the expression of several neuro-specific proteins as well as a range of functional and structural proteins involved in the formation and development of the neural cells.

Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O′Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single ‘spots’ in a polyacrylamide gel, allowing the quantitation of changes in a proteoform′s abundance to ascertain changes in an organism′s phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the ‘Top-Down’. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O’Farrell’s paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism′s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer.

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Ion substitution Human stem cells Bone biomaterial Calcium phosphate Dicalcium phosphate dihydrate, DCPD (CaHP0 4 •2H 2 O), brushite, is an important calcium phosphate compound encountered in mineralized tissues and used in medicine, especially in bone cernent for mulations. However, the use of DCPD as direct implantable biomaterial has not received dedicated attention. In addition, the possibility to dope DCPD with biologically active ions to modulate its per formances was not systematically explored. We have investigated in depth the doping of DCPD with Mg 2+ , sr2 + , Zn 2+ , Cu 2+ , and Ag + ions. Clear modifications in terms of chemical composition, particl e size, pore distribution, crystal morphology, and affinity for water were pointed out. Then, the samples were cultured with human adipose derived stem cells to explore cytotoxicity and proliferation. Various be haviors were noticed dependent on the incorporated metal ions. Such DCPD compounds associated with bioactive metal ions, and particularly Ag + and Zn 2+ , appear promising as a new family of reactive ma terials for use, as such or in combination, in bone related applications.