Paternal undernutrition and overnutrition modify semen composition and preimplantation embryo developmental kinetics in mice.

BACKGROUND: The importance of parental diet in relation to eventual offspring health is increasing in prominence due to the increased frequency of parents of reproductive age consuming poor diets. Whilst maternal health and offspring outcome have been studied in some detail, the paternal impacts are not as well understood. A father's poor nutritional status has been shown to have negative consequences on foetal growth and development and ultimately impact the long-term adult health of the offspring. In this study, we examined sperm- and seminal vesicle fluid-mediated mechanisms of preimplantation embryo development alterations in response to sub-optimal paternal diets. RESULTS: Male mice were fed a diet to model either under (low-protein diet (LPD)) or over (high-fat/sugar 'Western' diet (WD)) nutrition, LPD or WD supplemented with methyl donors or a control diet (CD) before mating with age-matched females. Male metabolic health was influenced by WD and MD-WD, with significant changes in multiple serum lipid classes and hepatic 1-carbon metabolites. Sperm RNA sequencing revealed significant changes to mRNA profiles in all groups when compared to CD (LPD: 32, MD-LPD: 17, WD: 53, MD-WD: 35 transcripts). Separate analysis of the seminal vesicle fluid proteome revealed a significant number of differentially expressed proteins in all groups (LPD: 13, MD-LPD: 27, WD: 24, MD-WD: 19) when compared to control. Following mating, in vitro time-lapse imaging of preimplantation embryos revealed a significant increase in the timing of development in all experimental groups when compared to CD embryos. Finally, qPCR analysis of uterine tissue at the time of implantation identified perturbed expression of Cd14 and Ptgs1 following mating with WD-fed males. CONCLUSIONS: Our current study shows that paternal nutritional status has the potential to influence male metabolic and reproductive health, impacting on embryonic development and the maternal reproductive tract. This study highlights potential direct (sperm-mediated) and indirect (seminal vesicle fluid-mediated) pathways in which a father's poor diet could shape the long-term health of his offspring.

Probing the Mechanism of Action of Bis(phenolato) Amine (ONO Donor Set) Titanium(IV) Anticancer Agents.

The need for anticancer therapies that overcome metallodrug resistance while minimizing adverse toxicities is targeted, herein, using titanium coordination complexes. Octahedral titanium(IV) trans,mer-[Ti{R1N(CH2-2-MeO-4-R1-C6H2)2}2] [R1 = Et, allyl, n-Pr, CHO, F, CH2(morpholino), the latter from the formyl derivative; R2 = Me, Et; not all combinations] are attained from Mannich reactions of commercial 2-methoxyphenols (27-74% overall yield, 2 steps). These crystalline (four X-ray structures) Ti(IV)-complexes are active against MCF-7, HCT-116, HT-29, PANC-1, and MDA-MB-468 cancer cell lines (GI50 = 0.5-38 µM). Their activity and cancer selectivity (vs nontumor MRC-5 cells) typically exceeds that of cisplatin (up to 16-fold). Proteomic analysis (in MCF-7) supported by other studies (G2/M cell cycle arrest, ROS generation, γH2AX production, caspase activation, annexin positivity, western blot, and kinase screens in MCF-7 and HCT-116) suggest apoptosis elicited by more than one mechanism of action. Comparison of these data to the modes of action proposed for salan Ti(IV) complexes is made.

Xylo-oligosaccharide-based prebiotics upregulate the proteins of the Sus-like system in caecal Bacteroidetes of the chicken: evidence of stimbiotic mechanism.

The present study was conducted to investigate the stimbiotic mechanism of xylo-oligosaccharide (XOS) in degrading the complex polysaccharides by the caecal bacteria of the chicken, by applying a proteomic approach. A total of 800 as-hatched Ross 308 broiler chicks were equally divided into 4 experimental pens (200 chicks per pen) at a commercial poultry barn, allocating 2 pens per treatment. Birds were fed ad libitum with 2 dietary treatments; CON (without XOS) and XOS (with 0.1g XOS/kg diet) from d 0 to 35. From each pen, 60 Individual birds were weighed weekly whereas caecal content was obtained from 5 birds cervically dislocated on d 35. The caecal bacteria were lysed and their proteins were quantified using label-free quantitative proteomic mass spectrometry. The results showed that XOS significantly increased (P < 0.05) bird weight on d 7, 14, 21, and 28, and body weight gain on d 7, 14, 21, and 35 compared to CON. However, no difference (P > 0.05) in body weight gain was observed from d 0 to 35 between CON and XOS. The proteomic analysis of caecal bacteria revealed that 29 proteins were expressed differently between the CON and the XOS group. Out of 29, 20 proteins were significantly increased in the XOS group compared to CON and 9 of those proteins belonged to the starch-utilizing system (Sus)-like system of the gram-negative Bacteroidetes. Bacteroides thetaiotaomicron (Bt) is a significant constituent of the human gut microbiota, known for its remarkable ability to hydrolyze most glycosidic bonds of polysaccharides. This microorganism possesses a 5-protein complex in its outer membrane, named the starch utilization system (Sus), responsible for adhering to, breaking down, and transporting starch into the cell. Sus serves as an exemplar system for numerous polysaccharide utilization loci that target glycans found in Bt and other members of the Bacteroidetes phylum. The proteins of the Sus-like system are involved in the degradation of complex polysaccharides and transportation of the oligosaccharides into the periplasm of the caecal bacteria where they are further broken down into smaller units. These smaller units are then transported into the cytoplasm of the cell where they are utilized in metabolic pathways leading to potential generation of short-chain fatty acids, thus improving the nutritive value of residual feed. In conclusion, XOS supplementation upregulates the expression of the proteins of the Sus-like system indicating its role as a stimbiotic.

Differential expression of genes controlling lymphocyte differentiation and migration in two distinct endotypes of type 1 diabetes.

AIMS: Morphological studies of pancreas samples obtained from young people with recent-onset type 1 diabetes have revealed distinct patterns of immune cell infiltration of the pancreatic islets suggestive of two age-associated type 1 diabetes endotypes that differ by inflammatory responses and rates of disease progression. The objective of this study was to investigate whether these proposed disease endotypes are associated with pathological differences in immune cell activation and cytokine secretion by applying multiplexed gene expression analysis to pancreatic tissue from recent-onset type 1 diabetes cases. METHODS: RNA was extracted from samples of fixed, paraffin-embedded pancreas tissue from type 1 diabetes cases characterised by endotype and from controls without diabetes. Expression levels of 750 genes associated with autoimmune inflammation were determined by hybridisation to a panel of capture and reporter probes and these were counted as a measure of gene expression. Normalised counts were analysed for differences in expression between 29 type 1 diabetes cases and 7 controls without diabetes, and between the two type 1 diabetes endotypes. RESULTS: Ten inflammation-associated genes, including INS, were significantly under-expressed in both endotypes and 48 genes were more highly expressed. A different set of 13 genes associated with the development, activation and migration of lymphocytes was uniquely overexpressed in the pancreas of people developing diabetes at younger age. CONCLUSIONS: The results provide evidence that histologically defined type 1 diabetes endotypes differ in their immunopathology and identify inflammatory pathways specifically involved in disease developing at a young age, essential for a better understanding of disease heterogeneity.

PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response.

The ADP-ribosyltransferase, PARP1 enzymatically generates and applies the post-translational modification, ADP-Ribose (ADPR). PARP1 roles in genome maintenance are well described, but recent work highlights roles in many fundamental processes including cellular identity and energy homeostasis. Herein, we show in both mouse and human skeletal muscle cells that PARP1-mediated PARylation is a regulator of the myogenic program and the muscle transcriptional response to steroid hormones. Chemical PARP1 modulation impacts the expression of major myocellular proteins, including troponins, key in dictating muscle contractile force. Whilst PARP1 in absence of DNA damage is often assumed to be basally inactive, we show PARylation to be acutely sensitive to extracellular glucose concentrations and the steroid hormone class, glucocorticoids which exert considerable authority over muscle tissue mass. Specifically, we find during myogenesis, a transient and significant rise in PAR. This early-stage differentiation event, if blocked with PARP1 inhibition, reduced the abundance of important muscle proteins in the fully differentiated myotubes. This suggests that PAR targets during early-stage differentiation are central to the proper development of the muscle contractile unit. We also show that reduced PARP1 in myoblasts impacts a variety of metabolic pathways in line with the recorded actions of glucocorticoids. Currently, as both regulators of myogenesis and muscle mass loss, glucocorticoids represent a clinical conundrum. Our work goes on to identify that PARP1 influences transcriptional activation by glucocorticoids of a subset of genes critical to human skeletal muscle pathology. These genes may therefore signify a regulatory battery of targets through which selective glucocorticoid modulation could be achieved. Collectively, our data provide clear links between PARP1-mediated PARylation and skeletal muscle homeostatic mechanisms crucial to tissue mass maintenance and endocrine response.

Small extracellular vesicles released from germinated kiwi pollen (pollensomes) present characteristics similar to mammalian exosomes and carry a plant homolog of ALIX.

Introduction: In the last decade, it has been discovered that allergen-bearing extracellular nanovesicles, termed "pollensomes", are released by pollen during germination. These extracellular vesicles (EVs) may play an important role in pollen-pistil interaction during fertilization, stabilizing the secreted bioactive molecules and allowing long-distance signaling. However, the molecular composition and the biological role of these EVs are still unclear. The present study had two main aims: (I) to clarify whether pollen germination is needed to release pollensomes, or if they can be secreted also in high humidity conditions; and (II) to investigate the molecular features of pollensomes following the most recent guidelines for EVs isolation and identification. Methods: To do so, pollensomes were isolated from hydrated and germinated kiwi (Actinidia chinensis Planch.) pollen, and characterized using imaging techniques, immunoblotting, and proteomics. Results: These analyses revealed that only germinated kiwi pollen released detectable concentrations of nanoparticles compatible with small EVs for shape and protein content. Moreover, a plant homolog of ALIX, which is a well-recognized and accepted marker of small EVs and exosomes in mammals, was found in pollensomes. Discussion: The presence of this protein, along with other proteins involved in endocytosis, is consistent with the hypothesis that pollensomes could comprehend a prominent subpopulation of plant exosome-like vesicles.

Multi-Omic Analysis of Two Common P53 Mutations: Proteins Regulated by Mutated P53 as Potential Targets for Immunotherapy.

The p53 protein is mutated in more than 50% of human cancers. Mutated p53 proteins not only lose their normal function but often acquire novel oncogenic functions, a phenomenon termed mutant p53 gain-of-function. Mutant p53 has been shown to affect the transcription of a range of genes, as well as protein-protein interactions with transcription factors and other effectors; however, no one has intensively investigated and identified these proteins, or their MHC presented epitopes, from the viewpoint of their ability to act as targets for immunotherapeutic interventions. We investigated the molecular changes that occurred after the TP53 null osteosarcoma cells, SaOS-2, were transfected with one of two conformational p53-mutants, either R175H or R273H. We then examined the phenotypic and functional changes using macroscopic observations, proliferation, gene expression and proteomics alongside immunopeptidome profiling of peptide antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. We identified several candidate proteins in both TP53 mutant cell lines with differential expression when compared to the TP53 null vector control, SaOS-V. Quantitative SWATH proteomics combined with immune-peptidome analysis of the class-I eluted peptides identified several epitopes presented on pMHC and in silico analysis shortlisted which antigens were expressed in a range of cancerous but not adjacent healthy tissues. Out of all the candidates, KLC1 and TOP2A showed high levels of expression in every tumor type examined. From these proteins, three A2 and four pan HLA-A epitopes were identified in both R175H and R273H from TOP2A. We have now provided a short list of future immunotherapy targets for the treatment of cancers harboring mutated TP53.

Extracellular transglutaminase-2, nude or associated with astrocytic extracellular vesicles, modulates neuronal calcium homeostasis.

We have uncovered a novel role for astrocytes-derived extracellular vesicles (EVs) in controlling intraneuronal Ca2+ concentration ([Ca2+]i) and identified transglutaminase-2 (TG2) as a surface-cargo of astrocytes-derived EVs. Incubation of hippocampal neurons with primed astrocyte-derived EVs have led to an increase in [Ca2+]i, unlike EVs from TG2-knockout astrocytes. Exposure of neurons or brain slices to extracellular TG2 promoted a [Ca2+]i rise, which was reversible upon TG2 removal and was dependent on Ca2+ influx through the plasma membrane. Patch-clamp and calcium imaging recordings revealed TG2-dependent neuronal membrane depolarization and activation of inward currents, due to the Na+/Ca2+-exchanger (NCX) operating in the reverse mode and indirect activation of L-type VOCCs, as indicated by VOCCs/NCX pharmacological inhibitors. A subunit of Na+/K+-ATPase was selected by comparative proteomics and identified as being functionally inhibited by extracellular TG2, implicating Na+/K+-ATPase inhibition in NCX reverse mode-switching leading to Ca2+ influx and higher basal [Ca2+]i. These data suggest that reactive astrocytes control intraneuronal [Ca2+]i through release of EVs with TG2 as responsible cargo, which could have a significant impact on synaptic activity in brain inflammation.

The Transglutaminase-2 Interactome in the APP23 Mouse Model of Alzheimer's Disease.

Amyloid-beta (Aß) deposition in the brain is closely linked with the development of Alzheimer's disease (AD). Unfortunately, therapies specifically targeting Aß deposition have failed to reach their primary clinical endpoints, emphasizing the need to broaden the search strategy for alternative targets/mechanisms. Transglutaminase-2 (TG2) catalyzes post-translational modifications, is present in AD lesions and interacts with AD-associated proteins. However, an unbiased overview of TG2 interactors is lacking in both control and AD brain. Here we aimed to identify these interactors using a crossbreed of the AD-mimicking APP23 mouse model with wild type and TG2 knock-out (TG2-/-) mice. We found that absence of TG2 had no (statistically) significant effect on Aß pathology, soluble brain levels of Aß1-40 and Aß1-42, and mRNA levels of TG family members compared to APP23 mice at 18 months of age. Quantitative proteomics and network analysis revealed a large cluster of TG2 interactors involved in synaptic transmission/assembly and cell adhesion in the APP23 brain typical of AD. Comparative proteomics of wild type and TG2-/- brains revealed a TG2-linked pathological proteome consistent with alterations in both pathways. Our data show that TG2 deletion leads to considerable network alterations consistent with a TG2 role in (dys)regulation of synaptic transmission and cell adhesion in APP23 brains.

Cold Exposure Drives Weight Gain and Adiposity following Chronic Suppression of Brown Adipose Tissue.

Therapeutic activation of thermogenic brown adipose tissue (BAT) may be feasible to prevent, or treat, cardiometabolic disease. However, rodents are commonly housed below thermoneutrality (~20 °C) which can modulate their metabolism and physiology including the hyperactivation of brown (BAT) and beige white adipose tissue. We housed animals at thermoneutrality from weaning to chronically supress BAT, mimic human physiology and explore the efficacy of chronic, mild cold exposure (20 °C) and ß3-adrenoreceptor agonism (YM-178) under these conditions. Using metabolic phenotyping and exploratory proteomics we show that transfer from 28 °C to 20 °C drives weight gain and a 125% increase in subcutaneous fat mass, an effect not seen with YM-178 administration, thus suggesting a direct effect of a cool ambient temperature in promoting weight gain and further adiposity in obese rats. Following chronic suppression of BAT, uncoupling protein 1 mRNA was undetectable in the subcutaneous inguinal white adipose tissue (IWAT) in all groups. Using exploratory adipose tissue proteomics, we reveal novel gene ontology terms associated with cold-induced weight gain in BAT and IWAT whilst Reactome pathway analysis highlights the regulation of mitotic (i.e., G2/M transition) and metabolism of amino acids and derivatives pathways. Conversely, YM-178 had minimal metabolic-related effects but modified pathways involved in proteolysis (i.e., eukaryotic translation initiation) and RNA surveillance across both tissues. Taken together these findings are indicative of a novel mechanism whereby animals increase body weight and fat mass following chronic suppression of adaptive thermogenesis from weaning. In addition, treatment with a B3-adrenoreceptor agonist did not improve metabolic health in obese animals raised at thermoneutrality.

MiR-423-5p prevents MALAT1-mediated proliferation and metastasis in prostate cancer.

BACKGROUND: The long non-coding RNA (lncRNA), MALAT1, plays a key role in the development of different cancers, and its expression is associated with worse prognosis in patients. However, its mechanism of action and its regulation are not well known in prostate cancer (PCa). A general mechanism of action of lncRNAs is their interaction with other epigenetic regulators including microRNAs (miRNAs). METHODS: Using lentiviral stable miRNA transfection together with cell biology functional assays and gene expression/target analysis, we investigated the interaction between MALAT1 and miR-423-5p, defined as a target with in silico prediction analysis, in PCa. RESULTS: Through bioinformatic analysis of data available from TCGA, we have found that MALAT1 expression correlates with high Gleason grade, metastasis occurrence, and reduced survival in PCa patients. These findings were validated on a TMA of PCa showing a significant correlation between MALAT1 expression with both stage and grading. We report that, in PCa cells, MALAT1 expression and activity is regulated by miR-423-5p that binds MALAT1, downregulates its expression and inhibits its activity in promoting proliferation, migration, and invasion. Using NanoString analysis, we unraveled downstream cell pathways that were affected by miR-423-5p expression and MALAT1 downregulation and identified several alterations in genes that are involved in metastatic response and angiogenic pathways. In addition, we showed that the overexpression of miR-423-5p increases survival and decreases metastases formation in a xenograft mouse model. CONCLUSIONS: We provide evidence on the role of MALAT1 in PCa tumorigenesis and progression. Also, we identify a direct interaction between miR-423-5p and MALAT1, which results in the suppression of MALAT1 action in PCa.

A Proteomic Approach Reveals That miR-423-5p Modulates Glucidic and Amino Acid Metabolism in Prostate Cancer Cells.

Recently, we have demonstrated that miR-423-5p modulates the growth and metastases of prostate cancer (PCa) cells both in vitro and in vivo. Here, we have studied the effects of miR-423-5p on the proteomic profile in order to identify its intracellular targets and the affected pathways. Applying a quantitative proteomic approach, we analyzed the effects on the protein expression profile of miR-423-5p-transduced PCa cells. Moreover, a computational analysis of predicted targets of miR-423-5p was carried out by using several target prediction tools. Proteomic analysis showed that 63 proteins were differentially expressed in miR-423-5-p-transfected LNCaP cells if compared to controls. Pathway enrichment analysis revealed that stable overexpression of miR-423-5p in LNCaP PCa cells induced inhibition of glycolysis and the metabolism of several amino acids and a parallel downregulation of proteins involved in transcription and hypoxia, the immune response through Th17-derived cytokines, inflammation via amphorin signaling, and ion transport. Moreover, upregulated proteins were related to the S phase of cell cycle, chromatin modifications, apoptosis, blood coagulation, and calcium transport. We identified seven proteins commonly represented in miR-423-5p targets and differentially expressed proteins (DEPs) and analyzed their expression and influence on the survival of PCa patients from publicly accessible datasets. Overall, our findings suggest that miR-423-5p induces alterations in glucose and amino acid metabolism in PCa cells paralleled by modulation of several tumor-associated processes.

Carnosine protects stimulus-secretion coupling through prevention of protein carbonyl adduction events in cells under metabolic stress.

Type 2 diabetes is characterised by failure to control glucose homeostasis, with numerous diabetic complications attributable to the resulting exposure of cells and tissues to chronic elevated concentrations of glucose and fatty acids. This, in part, results from formation of advanced glycation and advanced lipidation end-products that are able to modify protein, lipid, or DNA structure, and disrupt normal cellular function. Herein we used mass spectrometry to identify proteins modified by two such adduction events in serum of individuals with obesity, type 2 diabetes, and gestational diabetes, along with similar analyses of human and mouse skeletal muscle cells and mouse pancreatic islets exposed to glucolipotoxic stress. We also report that carnosine, a histidine containing dipeptide, prevented 65-90% of 4-hydroxynonenal and 3-nitrotyrosine adduction events, and that this in turn preserved mitochondrial function and protected stimulus-secretion coupling in cells exposed to metabolic stress. Carnosine therefore offers significant therapeutic potential against metabolic diseases.

Homocitrullination of lysine residues mediated by myeloid-derived suppressor cells in the tumor environment is a target for cancer immunotherapy.

BACKGROUND: Homocitrullination is the post-translational modification of lysine that is recognized by T cells. METHODS: This study identified homocitrullinated peptides from aldolase, enolase, cytokeratin and binding immunoglobulin protein and used human leukocyte antigen (HLA) transgenic mice to assess immunogenicity by enzyme-linked immunosorbent spot assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma expressing constitutive or interferon γ (IFNγ)-inducible major histocompatibility complex class II (MHC-II) as represented by most human tumors. To determine the mechanism behind the therapy, immune cell infiltrates were analyzed using flow cytometry and therapy studies in the presence of myeloperoxidase (MPO) inhibitor and T-cell depletion performed. We assessed the T-cell repertoire to homocitrullinated peptides in patients with cancer and healthy donors using flow cytometry. RESULTS: Homocitrulline (Hcit) peptide vaccination stimulated strong CD4 T-cell responses and induced significant antitumor therapy in an established tumor model. The antitumor response was dependent on CD4 T cells and the effect was driven mainly via direct tumor recognition, as responses were only observed if the tumors were induced to express MHC-II. In vitro proliferation assays show that healthy donors and patients with cancer have an oligoclonal CD4 T-cell repertoire recognizing homocitrullinated peptides. Inhibition of cyanate generation, which mediates homocitrullination, by MPO inhibition reduced tumor therapy by the vaccine induced T cells (p=0.0018). Analysis of the tumor microenvironment (TME) suggested that myeloid-derived suppressor cells (MDSCs) were a potential source of MPO. The selected B16 melanoma model showed MDSC infiltration and was appropriate to see if the Hcit vaccine could overcome the immunosuppression associated with MDSCs. The vaccine was very effective (90% survival) as the induced CD4 T cells directly targeted the homocitrullinated tumor and likely reversed the immunosuppressive environment. CONCLUSION: We propose that MPO, potentially produced by MDSCs, catalyzes the buildup of cyanate in the TME which diffuses into tumor cells causing homocitrullination of cytoplasmic proteins which are degraded and, in the presence of IFNγ, presented by MHC-II for direct CD4 T-cell recognition. Homocitrullinated proteins are a new target for cancer vaccines and may be particularly effective against tumors containing high levels of MPO expressing MDSCs.

Supramolecular Chromatographic Separation of C60 and C70 Fullerenes: Flash Column Chromatography vs. High Pressure Liquid Chromatography.

A silica-bound C-butylpyrogallol[4]arene chromatographic stationary phase was prepared and characterised by thermogravimetric analysis, scanning electron microscopy, NMR and mass spectrometry. The chromatographic performance was investigated by using C60 and C70 fullerenes in reverse phase mode via flash column and high-pressure liquid chromatography (HPLC). The resulting new stationary phase was observed to demonstrate size-selective molecular recognition as postulated from our in-silico studies. The silica-bound C-butylpyrogallol[4]arene flash and HPLC stationary phases were able to separate a C60- and C70-fullerene mixture more effectively than an RP-C18 stationary phase. The presence of toluene in the mobile phase plays a significant role in achieving symmetrical peaks in flash column chromatography.

Multiomic analysis of stretched osteocytes reveals processes and signalling linked to bone regeneration and cancer.

Exercise is a non-pharmacological intervention that can enhance bone regeneration and improve the management of bone conditions like osteoporosis or metastatic bone cancer. Therefore, it is gaining increasing importance in an emerging area of regenerative medicine-regenerative rehabilitation (RR). Osteocytes are mechanosensitive and secretory bone cells that orchestrate bone anabolism and hence postulated to be an attractive target of regenerative exercise interventions. However, the human osteocyte signalling pathways and processes evoked upon exercise remain to be fully identified. Making use of a computer-controlled bioreactor that mimics exercise and the latest omics approaches, RNA sequencing (RNA-seq) and tandem liquid chromatography-mass spectrometry (LC-MS), we mapped the transcriptome and secretome of mechanically stretched human osteocytic cells. We discovered that a single bout of cyclic stretch activated network processes and signalling pathways likely to modulate bone regeneration and cancer. Furthermore, a comparison between the transcriptome and secretome of stretched human and mouse osteocytic cells revealed dissimilar results, despite both species sharing evolutionarily conserved signalling pathways. These findings suggest that osteocytes can be targeted by exercise-driven RR protocols aiming to modulate bone regeneration or metastatic bone cancer.

Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation.

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-dependent enzyme tissue transglutaminase (TG2). At 1-10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs.

Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines.

BACKGROUND: Chondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. We hypothesize that chondrocytes adjust their physiology to the inflammatory microenvironment by modulating the expression of cell surface proteins, collectively referred to as the 'surfaceome'. Therefore, the aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1ß) and tumour necrosis factor-alpha (TNF-α). We employed combined methodology that we recently developed for investigating the surfaceome in stem cells. Membrane proteins were isolated using an aminooxy-biotinylation technique and analysed by mass spectrometry using high throughput shotgun proteomics. Selected proteins were validated by western blotting. RESULTS: Amongst the 431 unique cell surface proteins identified, a high percentage of low-abundance proteins, such as ion channels, receptors and transporter molecules were detected. Data are available via ProteomeXchange with identifier PXD014773. A high number of proteins exhibited different expression patterns following chondrocyte stimulation with pro-inflammatory cytokines. Low density lipoprotein related protein 1 (LPR-1), thrombospondin-1 (TSP-1), voltage dependent anion channel (VDAC) 1-2 and annexin A1 were considered to be of special interest and were analysed further by western blotting. CONCLUSIONS: Our results provide, for the first time, a repository for proteomic data on differentially expressed low-abundance membrane proteins on the surface of chondrocytes in response to pro-inflammatory stimuli.

Exercise Training in Obese Rats Does Not Induce Browning at Thermoneutrality and Induces a Muscle-Like Signature in Brown Adipose Tissue.

Aim: Exercise training elicits diverse effects on brown (BAT) and white adipose tissue (WAT) physiology in rodents housed below their thermoneutral zone (i.e., 28-32°C). In these conditions, BAT is chronically hyperactive and, unlike human residence, closer to thermoneutrality. Therefore, we set out to determine the effects of exercise training in obese animals at 28°C (i.e., thermoneutrality) on BAT and WAT in its basal (i.e., inactive) state. Methods: Sprague-Dawley rats (n = 12) were housed at thermoneutrality from 3 weeks of age and fed a high-fat diet. At 12 weeks of age half these animals were randomized to 4-weeks of swim-training (1 h/day, 5 days per week). Following a metabolic assessment interscapular and perivascular BAT and inguinal (I)WAT were taken for analysis of thermogenic genes and the proteome. Results: Exercise attenuated weight gain but did not affect total fat mass or thermogenic gene expression. Proteomics revealed an impact of exercise training on 2-oxoglutarate metabolic process, mitochondrial respiratory chain complex IV, carbon metabolism, and oxidative phosphorylation. This was accompanied by an upregulation of multiple proteins involved in skeletal muscle physiology in BAT and an upregulation of muscle specific markers (i.e., Myod1, CkM, Mb, and MyoG). UCP1 mRNA was undetectable in IWAT with proteomics highlighting changes to DNA binding, the positive regulation of apoptosis, HIF-1 signaling and cytokine-cytokine receptor interaction. Conclusion: Exercise training reduced weight gain in obese animals at thermoneutrality and is accompanied by an oxidative signature in BAT which is accompanied by a muscle-like signature rather than induction of thermogenic genes. This may represent a new, UCP1-independent pathway through which BAT physiology is regulated by exercise training.

ß2-Adrenergic Signalling Promotes Cell Migration by Upregulating Expression of the Metastasis-Associated Molecule LYPD3.

Metastasis is associated with poor prognosis in breast cancer. Although some studies suggest beta-blockers increase survival by delaying metastasis, others have been discordant. This study provides both insights into the anomalous findings and identifies potential biomarkers that may be treatment targets. Cell line models of basal-type and oestrogen receptor-positive breast cancer were profiled for basal levels of adrenoceptor gene/protein expression, and ß2-adrenoceptor mediated cell behaviour including migration, invasion, adhesion, and survival in response to adrenoceptor agonist/antagonist treatment. Protein profiling and histology identified biomarkers and drug targets. Baseline levels of adrenoceptor gene expression are higher in basal-type rather than oestrogen receptor-positive cancer cells. Norepinephrine (NE) treatment increased invasive capacity in all cell lines but did not increase proliferation/survival. Protein profiling revealed the upregulation of the pro-metastatic gene Ly6/PLAUR Domain-Containing Protein 3 (LYPD3) in norepinephrine-treated MDA-MB-468 cells. Histology confirmed selective LYPD3 expression in primary and metastatic breast tumour samples. These findings demonstrate that basal-type cancer cells show a more aggressive adrenoceptor-ß2-activated phenotype in the resting and stimulated state, which is attenuated by adrenoceptor-ß2 inhibition. This study also highlights the first association between ADRß2 signalling and LYPD3; its knockdown significantly reduced the basal and norepinephrine-induced activity of MCF-7 cells in vitro. The regulation of ADRß2 signalling by LYPD3 and its metastasis promoting activities, reveal LYPD3 as a promising therapeutic target in the treatment of breast and other cancers.