aOvarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication. Molecular & Cellular

IntroductionThis study was aimed at deciphering the secretome of adipose-derived mesenchymal stromal cells (ADSCs) cultured in standard and hypoxic conditions to reveal proteins, which may be responsible for regenerative action of these cells.MethodsHuman ADSCs were isolated from 10 healthy donors and cultured for 3–4 passages. Cells were serum deprived and cell purity was assessed using multiple cell surface markers. Conditioned media was collected and analyzed using LC-MS with a focus on characterizing secreted proteins.ResultsPurity of the ADSC assessed as CD90+/CD73+/CD105+/CD45-/CD31- cells was greater than 99 % and viability was greater than 97 %. More than 600 secreted proteins were detected in conditioned media of ADSCs. Of these 100 proteins were common to all cultures and included key molecules involved in tissue regeneration such as collagens and collagen maturation enzymes, matrix metalloproteases, matricellular proteins, macrophage-colony stimulating factor and pigment epithelium derived factor. Common set of proteins also included molecules, which contribute to regenerative processes but were not previously associated with ADSCs. These included olfactomedin-like 3, follistatin-like 1 and prosaposin. In addition, ADSCs from the different subjects secreted proteins, which were variable between different cultures. These included proteins with neurotrophic activities, which were not previously associated with ADSCs, such as mesencephalic astrocyte-derived neurotrophic factor, meteorin and neuron derived neurotrophic factor. Hypoxia resulted in secretion of 6 proteins, the most prominent included EGF-like repeats and discoidin I-like domains 3, adrenomedullin and ribonuclease 4 of RNase A family. It also caused the disappearance of 8 proteins, including regulator of osteogenic differentiation cartilage-associated protein.ConclusionsHuman ADSCs with CD90+/CD73+/CD105+/CD45-/CD31-/PDGFRβ+/NG2+/CD146+(−) immunophenotype secrete a large array of proteins, the most represented group is comprised of extracellular matrix components. Number of secreted proteins is largely unaffected by prolonged hypoxia. Variability in the secretion of several proteins from cultured ADSCs of individual subjects suggests that these cells exist as a heterogeneous population containing functionally distinct subtypes, which differ in numbers between donors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0209-8) contains supplementary material, which is available to authorized users.

Numerous studies are devoted to the intestinal microbiota and intercellular communication maintaining homeostasis. In this regard, vesicles secreted by bacteria represent one of the most popular topics for research. For example, the outer membrane vesicles (OMVs) of Bacteroides fragilis play an important nutritional role with respect to other microorganisms and promote anti-inflammatory effects on immune cells. However, toxigenic B. fragilis (ETBF) contributes to bowel disease, even causing colon cancer. If nontoxigenic B. fragilis (NTBF) vesicles exert a beneficial effect on the intestine, it is likely that ETBF vesicles can be utilized for potential pathogenic implementation. To confirm this possibility, we performed comparative proteomic HPLC-MS/MS analysis of vesicles isolated from ETBF and NTBF. Furthermore, we performed, for the first time, HPLC-MS/MS and GS-MS comparative metabolomic analysis for the vesicles isolated from both strains with subsequent reconstruction of the vesicle metabolic pathways. We utilized fluxomic experiments to validate the reconstructed biochemical reaction activities and finally observed considerable difference in the vesicle proteome and metabolome profiles. Compared with NTBF OMVs, metabolic activity of ETBF OMVs provides their similarity to micro reactors that are likely to be used for long-term persistence and implementing pathogenic potential in the host.

The oldest prokaryotic photoautotrophic organisms, cyanobacteria, produce many different metabolites. Among them is the water-soluble neurotoxic non-protein amino acid beta-N-methylamino-L-alanine (BMAA), whose biological functions in cyanobacterial metabolism are of fundamental scientific and practical interest. An early BMAA inhibitory effect on nitrogen fixation and heterocyst differentiation was shown in strains of diazotrophic cyanobacteria Nostoc sp. PCC 7120, Nostoc punctiforme PCC 73102 (ATCC 29133), and Nostoc sp. strain 8963 under conditions of nitrogen starvation. Herein, we present a comprehensive proteomic study of Nostoc (also called Anabaena) sp. PCC 7120 in the heterocyst formation stage affecting by BMAA treatment under nitrogen starvation conditions. BMAA disturbs proteins involved in nitrogen and carbon metabolic pathways, which are tightly co-regulated in cyanobacteria cells. The presented evidence shows that exogenous BMAA affects a key nitrogen regulatory protein, PII (GlnB), and some of its protein partners, as well as glutamyl-tRNA synthetase gltX and other proteins that are involved in protein synthesis, heterocyst differentiation, and nitrogen metabolism. By taking into account the important regulatory role of PII, it becomes clear that BMAA has a severe negative impact on the carbon and nitrogen metabolism of starving Nostoc sp. PCC 7120 cells. BMAA disturbs carbon fixation and the carbon dioxide concentrating mechanism, photosynthesis, and amino acid metabolism. Stress response proteins and DNA repair enzymes are upregulated in the presence of BMAA, clearly indicating severe intracellular stress. This is the first proteomic study of the effects of BMAA on diazotrophic starving cyanobacteria cells, allowing a deeper insight into the regulation of the intracellular metabolism of cyanobacteria by this non-protein amino acid.

Genomes contain millions of short (<100 codons) open reading frames (sORFs), which are usually dismissed during gene annotation. Nevertheless, peptides encoded by such sORFs can play important biological roles, and their impact on cellular processes has long been underestimated. Here, we analyzed approximately 70,000 transcribed sORFs in the model plant Physcomitrella patens (moss). Several distinct classes of sORFs that differ in terms of their position on transcripts and the level of evolutionary conservation are present in the moss genome. Over 5000 sORFs were conserved in at least one of 10 plant species examined. Mass spectrometry analysis of proteomic and peptidomic data sets suggested that tens of sORFs located on distinct parts of mRNAs and long noncoding RNAs (lncRNAs) are translated, including conserved sORFs. Translational analysis of the sORFs and main ORFs at a single locus suggested the existence of genes that code for multiple proteins and peptides with tissue-specific expression. Functional analysis of four lncRNA-encoded peptides showed that sORFs-encoded peptides are involved in regulation of growth and differentiation in moss. Knocking out lncRNA-encoded peptides resulted in a decrease of moss growth. In contrast, the overexpression of these peptides resulted in a diverse range of phenotypic effects. Our results thus open new avenues for discovering novel, biologically active peptides in the plant kingdom.

Several studies have described functional peptides encoded in RNA that are considered to be noncoding. Telomerase RNA together with telomerase reverse transcriptase and regulatory proteins make up the telomerase complex, the major component of the telomere length-maintaining machinery. In contrast to protein subunits, telomerase RNA is expressed constitutively in most somatic cells where telomerase reverse transcriptase is absent. We show here that the transcript of human telomerase RNA codes a 121 amino acid protein (hTERP). The existence of hTERP was shown by immunoblotting, immunofluorescence microscopy and mass spectroscopy. Gain-of-function and loss-of-function experiments showed that hTERP protects cells from drug-induced apoptosis and participates in the processing of autophagosome. We suggest that hTERP regulates crosstalk between autophagy and apoptosis and is involved in cellular adaptation under stress conditions.

Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.

Background Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. Results We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. Conclusions Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.