The human gut microbiota plays a major role in maintaining human health and was recently recognized as a promising target for disease prevention and treatment. Many diseases are traceable to microbiota dysbiosis, implicating altered gut microbial ecosystems, or, in many cases, disrupted microbial enzymes carrying out essential physio-biochemical reactions. Thus, the changes of essential microbial enzyme levels may predict human disorders. With the rapid development of high-throughput sequencing technologies, metagenomics analysis has emerged as an important method to explore the microbial communities in the human body, as well as their functionalities. In this study, we analyzed 156 gut metagenomics samples from patients with colorectal cancer (CRC) and adenoma, as well as that from healthy controls. We estimated the abundance of microbial enzymes using the HMP Unified Metabolic Analysis Network method and identified the differentially abundant enzymes between CRCs and controls. We constructed enzymatic association networks using the extended local similarity analysis algorithm. We identified CRC-associated enzymic changes by analyzing the topological features of the enzymatic association networks, including the clustering coefficient, the betweenness centrality, and the closeness centrality of network nodes. The network topology of enzymatic association network exhibited a difference between the healthy and the CRC environments. The ABC (ATP binding cassette) transporter and small subunit ribosomal protein S19 enzymes, had the highest clustering coefficient in the healthy enzymatic networks. In contrast, the Adenosylhomocysteinase enzyme had the highest clustering coefficient in the CRC enzymatic networks. These enzymic and metabolic differences may serve as risk predictors for CRCs and are worthy of further research.

Ischemic stroke is a severe neurodegenerative disease with a high mortality rate. Retinoic acid is a representative metabolite of vitamin A. It has many beneficial effects including anti-inflammatory, anti-apoptotic, and neuroprotective effects. The purpose of this study is to identify specific proteins that are regulated by retinoic acid in ischemic stroke. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia. Retinoic acid (5 mg/kg) or vehicle was injected intraperitoneally into male rats for four days prior to MCAO operation. Neurobehavioral tests were performed 24 hr after MCAO and the cerebral cortex was collected for proteomic study. Retinoic acid alleviates neurobehavioral deficits and histopathological changes caused by MCAO. Furthermore, we identified various proteins that were altered by retinoic acid in MCAO damage. Among these identified proteins, adenosylhomocysteinase, isocitrate dehydrogenase [NAD+] subunit α, glycerol-3-phosphate dehydrogenase, Rab GDP dissociation inhibitor β, and apolipoprotein A1 were down-regulated in MCAO animals with vehicle treatment, whereas retinoic acid treatment alleviated these reductions. However, heat shock protein 60 was up-regulated in MCAO animals with vehicle, while retinoic acid treatment attenuated this increase. The changes in these expressions were confirmed by reverse transcription-PCR. These proteins regulate cell metabolism and mediate stress responses. Our results demonstrated that retinoic acid attenuates the neuronal damage by MCAO and regulates the various protein expressions that are involved in the survival of cells. Thus, we can suggest that retinoic acid exerts neuroprotective effects on focal cerebral ischemia by modulation of specific proteins.

In this study, we have investigated the transcriptome of Ralstonia eutropha H16 during cultivation with gluconate in presence of 3,3'-thiodipropionic acid (TDP) or 3,3'-dithiodipropionic acid (DTDP) during biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptopropionate). Genome-wide transcriptome analyses revealed several genes which were upregulated during cultivation in presence of the above-mentioned compounds. Obtained data strongly suggest that two ABC-type transport system and three probable extracytoplasmic solute receptors mediate the uptake of TDP and DTDP, respectively. In addition, genes encoding the hydrolase S-adenosylhomocysteinase AhcY and the thiol-disulfide interchange proteins DsbA, DsbD, and FrnE were upregulated during cultivation on DTDP and, in case of AhcY and FrnE, on TDP as well. It is assumed that the corresponding enzymes are involved in the cleavage of TDP and DTDP. Several genes of the fatty acid metabolism exhibited increased expression levels: genes encoding two acetyltransferases, a predicted acyltransferase, the acetoacetyl-CoA reductase phaB3, an enoyl-CoA hydratase as well as an acyl dehydratase, an acetyl-CoA synthetase, two acyl-CoA dehydrogenases, the methylmalonyl-CoA mutase encoded by sbm1 and sbm2 and phaY1 were detected. Furthermore, ORF H16_A0217 encoding a hypothetical protein and exhibiting 54% amino acids identical to an acyl-CoA thioesterase from Saccharomonospora viridis was found to be highly upregulated. As the 2-methylcitrate synthase PrpC exhibited a three- to fourfold increased activity in cells grown in presence of TDP or DTDP as compared to gluconate, metabolization of the cleavage products 3MP and 3-hydroxypropionate to propionyl-CoA is proposed.

Rapid and extensive rhizome development is a desirable trait for perennial grass growth and adaptation to environmental stresses. The objective of this study was to determine proteomic changes and associated metabolic pathways of gibberellin (GA) -regulation of rhizome elongation in two perennial grass species differing in rhizome development. Plants of a short-rhizome bunch-type tall fescue (TF; Festuca arundinacea; 'BR') and an extensive rhizomatous Kentucky bluegrass (KB; Poa pratensis; 'Baron') were treated with 10 μM GA3 in hydroponic culture in growth chambers. The average rhizome length in KB was significantly longer than that in TF regardless of GA3 treatment, and increased significantly with GA3 treatment, to a greater extent than that in TF. Comparative proteomic analysis using two-dimensional electrophoresis and mass spectrometry was performed to further investigate proteins and associated metabolic pathways imparting increased rhizome elongation by GA. A total of 37 and 38 differentially expressed proteins in response to GA3 treatment were identified in TF and KB plants, respectively, which were mainly involved in photosynthesis, energy and amino acid metabolism, protein synthesis, defense and cell development processes. Accelerated rhizome elongation in KB by GA could be mainly associated with the increased abundance of proteins involved in energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and ATP synthase), amino acid metabolism (S-adenosylmethionine and adenosylhomocysteinase), protein synthesis (HSP90, elongation factor Tu and eukaryotic translation initiation factor 5A), cell-wall development (cell dividion cycle protein, alpha tubulin-2A and actin), and signal transduction (calreticulin). These proteins could be used as candidate proteins for further analysis of molecular mechanisms controlling rhizome growth.

Clara cell protein (CC16) is an anti-inflammatory protein, which is expressed in the airway epithelium. It is involved in the development of airway inflammatory diseases, including chronic obstructive pulmonary disease and asthma. However, the exact molecular mechanism underlying its anti‑inflammatory action remains to be fully elucidated. The aim of the present study was to define the protein profiles of the anti‑inflammatory effect of CC16 in lipopolysaccharide (LPS)‑treated rat tracheal epithelial (RTE) cells using shotgun proteomics. Protein extracts were obtained from control RTE cells, RTE cells treated with LPS and RTE cells treated with LPS and recombinant CC16 (rCC16). Subsequent label‑free quantification and bioinformatics analyses identified 12 proteins that were differentially expressed in the three treatment groups as a cluster of five distinct groups according to their molecular functions. Five of the twelve proteins were revealed to be associated with the cytoskeleton: Matrix metalloproteinase‑9, myosin heavy chain 10, actin‑related protein‑3 homolog, elongation factor 1‑α‑1 (EF‑1‑α‑1), and acidic ribosomal phosphoprotein P0. Five of the twelve proteins were associated with cellular proliferation: DNA‑dependent protein kinase catalytic subunit, EF‑1‑α‑1, tyrosine 3‑monooxygenase, caspase recruitment domain (CARD) protein 12 and adenosylhomocysteinase (SAHH) 3. Three proteins were associated with gene regulation: EF‑1‑α‑1, SAHH 3 and acidic ribosomal phosphoprotein P0. Three proteins were associated with inflammation: Tyrosine 3‑monooxygenase, CARD protein 12 and statin‑related protein. ATPase (H+‑transporting, V1 subunit A, isoform 1) was revealed to be associated with energy metabolism, and uridine diphosphate glycosyltransferase 1 family polypeptide A8 with drug metabolism and detoxification. The identified proteins were further validated using reverse transcription‑quantitative polymerase chain reaction. These protein profiles, and their interacting protein network, may facilitate the elucidation of the molecular mechanisms underlying the anti‑inflammatory effects of CC16.

From a clinical point of view, easily obtainable and useful markers of a particular pathological status are required for appropriate diagnosis and treatment. Analysis of the proteomic profile of saliva may allow for the selection of potential marker of preterm delivery in humans. Saliva samples were collected from 12 patients diagnosed with threatened preterm delivery and 10 controls with uncomplicated pregnancies at the same gestational age. Samples were analysed using 2D electrophoresis. Based on statistical analysis, spots of interest were selected and collected from gels. Subsequently, spots were decoloured and proteins were identified by mass spectrometry using the matrix assisted laser desorption ionization‑time of flight technique. The results of identification were compared with the Swiss‑Prot database. A total of 1,393 spots were detected in the present study with 59 significantly different between control and preterm samples. Increased intensity of staining of 32 spots was observed in the premature delivery group compared with control patients and 27 spots were stained more intensely in the control group compared with the premature delivery group. A total of nine spots, which were significantly different between examined samples were identified and three of them exhibited increased intensity of staining in premature delivery group compared with controls, including dedicator of cytokinesis protein 1, metallothionein‑2, guanylyl cyclase‑activating protein 1. The six remaining spots included, epithelial‑stromal interaction protein 1, serum albumin, tyrosine‑tRNA ligase, cytoplasmic, protein chibby homolog 3, leukemia inhibitory factor receptor and adenosylhomocysteinase 3, and exhibited increased intensity of staining in healthy controls compared with premature delivery group. Further studies with an increased number of patients and identification of the complete protein profile are required to confirm the results of the present study and applicability of saliva as a source of disease biomarkers.

Somatic embryogenesis is the process by which bipolar structures with no vascular connection with the surrounding tissue are formed from a single or a group of vegetative cells, and in conifers it can be divided into five different steps: initiation, proliferation, maturation, germination and acclimatization. Somatic embryogenesis has long been used as a model to study the mechanisms regulating stress response in plants, and recent research carried out in our laboratory has demonstrated that high temperatures during initial stages of conifer somatic embryogenesis modify subsequent phases of the process, as well as the behavior of the resulting plants ex vitro. The development of high-throughput techniques has facilitated the study of the molecular response of plants to numerous stress factors. Proteomics offers a reliable image of the cell status and is known to be extremely susceptible to environmental changes. In this study, the proteome of radiata pine somatic embryos was analyzed by LC-MS after the application of high temperatures during initiation of embryonal masses [(23°C, control; 40°C (4 h); 60°C (5 min)]. At the same time, the content of specific soluble sugars and sugar alcohols was analyzed by HPLC. Results confirmed a significant decrease in the initiation rate of embryonal masses under 40°C treatments (from 44 to 30.5%) and an increasing tendency in the production of somatic embryos (from 121.87 to 170.83 somatic embryos per gram of embryogenic tissue). Besides, heat provoked a long-term readjustment of the protein synthesis machinery: a great number of structural constituents of ribosomes were increased under high temperatures, together with the down-regulation of the enzyme methionine-tRNA ligase. Heat led to higher contents of heat shock proteins and chaperones, transmembrane transport proteins, proteins related with post-transcriptional regulation (ARGONAUTE 1D) and enzymes involved in the synthesis of fatty acids, specific compatible sugars (myo-inositol) and cell-wall carbohydrates. On the other hand, the protein adenosylhomocysteinase and enzymes linked with the glycolytic pathway, nitrogen assimilation and oxidative stress response were found at lower levels.

Spinal cord injury (SCI) is a specific type of damage to the central nervous system causing temporary or permanent changes in its function. The present aimed to identify the genetic changes in neuroplasticity following SCI in rats. The GSE52763 microarray dataset, which included 15 samples [3 sham (1 week), 4 injury only (1 week), 4 injury only (3 weeks), 4 injury + treadmill (3 weeks)] was downloaded from the Gene Expression Omnibus database. An empirical Bayes linear regression model in limma package was used to identify the differentially expressed genes (DEGs) in injury vs. sham and treadmill vs. non‑treadmill comparison groups. Subsequently, time series and enrichment analyses were performed using pheatmap and clusterProfile packages, respectively. Additionally, protein‑protein interaction (PPI) and transcription factor (TF)‑microRNA (miRNA)‑target regulatory networks were constructed using Cytoscape software. In total, 159 and 105 DEGs were identified in injury vs. sham groups and treadmill vs. non‑treadmill groups, respectively. There were 40 genes in cluster 1 that presented increased expression levels in the injury (1 week/3 weeks) groups compared with the sham group, and decreased expression levels in the injury + treadmill group compared with the injury only groups; conversely, 52 genes in cluster 2 exhibited decreased expression levels in the injury (1 week/3 weeks) groups compared with the sham group, and increased expression levels in the injury + treadmill group compared with the injury only groups. Enrichment analysis indicated that clusters 1 and 2 were associated with immune response and signal transduction, respectively. Furthermore, microtubule associated protein 1B, phosphofurin acidic cluster sorting protein 2 and adenosylhomocysteinase‑like 1 exhibited the highest degrees in the regulatory network, and were regulated by miRNAs including miR‑34A, miR‑34B, miR‑34C and miR‑449. These miRNAs and their target genes may serve important roles in neuroplasticity following traumatic SCI in rats. Nevertheless, additional in‑depth studies are required to confirm these data.

It has been observed that the consumption of litchi often causes symptoms characterized by itching or sore throat, gum swelling, oral cavity ulcers and even fever and inflammation, which significantly impair the quality of life of a large population. Using the RAW264.7 cell line, a step-by-step strategy was used to screen for the components in litchi fruits that elicited adverse reactions. The adverse reaction fractions were identified by mass spectrometry and analyzed using the SMART program, and a sequence alignment of the homologous proteins was performed. MTT tests were used to determine the cytotoxicity of a litchi protein extract in RAW264.7 macrophages, and real-time PCR was applied to analyze the expression of inflammatory genes in the RAW264.7 cells treated with lipopolysaccharide or the litchi protein extract. The results showed that the litchi water-soluble protein extract could increase the production of the pro-inflammatory mediators IL-1β, iNOS and COX-2, and the anti-inflammatory mediator HO-1 in the RAW264.7 cell line. The 14-3-3-like proteins GF14 lambda, GF14 omega and GF14 upsilon were likely the candidate proteins that caused the adverse effects.

Histone methylation levels, which are determined by the action of both histone demethylases and methyltransferases, impact multiple biological processes by affecting gene expression activity. Methionine metabolism generates the major methyl donor S-adenosylmethionine (SAM) for histone methylation. The functions of methionine metabolic enzymes in regulating biological processes as well as the interaction between the methionine pathway and histone methylation, however, are still not fully understood. Here, we report that reduced levels of some enzymes involved in methionine metabolism and histone demethylases lead to lethality as well as wing development and cell proliferation defects in Drosophila melanogaster. Additionally, disruption of methionine metabolism can directly affect histone methylation levels. Reduction of little imaginal discs (LID) histone demethylase, but not lysine-specific demethylase 2 (KDM2) demethylase, is able to counter the effects on histone methylation due to reduction of SAM synthetase (SAM-S). Taken together, these results reveal an essential role of key enzymes that control methionine metabolism and histone methylation. Additionally, these findings are an indication of a strong connection between metabolism and epigenetics.

Ferroptosis is a newly characterized form of regulated cell death mediated by iron-dependent accumulation of lipid reactive oxygen species and holds great potential for cancer therapy. However, the molecular mechanisms underlying ferroptosis remain largely elusive. In this study, we define an integrative role of DJ-1 in ferroptosis. Inhibition of DJ-1 potently enhances the sensitivity of tumor cells to ferroptosis inducers both in vitro and in vivo. Metabolic analysis and metabolite rescue assay reveal that DJ-1 depletion inhibits the transsulfuration pathway by disrupting the formation of the S-adenosyl homocysteine hydrolase tetramer and impairing its activity. Consequently, more ferroptosis is induced when homocysteine generation is decreased, which might be the only source of glutathione biosynthesis when cystine uptake is blocked. Thus, our findings show that DJ-1 determines the response of cancer cells to ferroptosis, and highlight a candidate therapeutic target to potentially improve the effect of ferroptosis-based antitumor therapy.

The molecular mechanisms underlying vascular inflammation and associated inflammatory vascular diseases are not well defined. Here we show that endothelial intracellular adenosine and its key regulator adenosine kinase (ADK) play important roles in vascular inflammation. Pro-inflammatory stimuli lead to endothelial inflammation by increasing endothelial ADK expression, reducing the level of intracellular adenosine in endothelial cells, and activating the transmethylation pathway through increasing the association of ADK with S-adenosylhomocysteine (SAH) hydrolase (SAHH). Increasing intracellular adenosine by genetic ADK knockdown or exogenous adenosine reduces activation of the transmethylation pathway and attenuates the endothelial inflammatory response. In addition, loss of endothelial ADK in mice leads to reduced atherosclerosis and affords protection against ischemia/reperfusion injury of the cerebral cortex. Taken together, these results demonstrate that intracellular adenosine, which is controlled by the key molecular regulator ADK, influences endothelial inflammation and vascular inflammatory diseases.The molecular mechanisms underlying vascular inflammation are unclear. Here the authors show that pro-inflammatory stimuli lead to endothelial inflammation by increasing adenosine kinase expression, and that its knockdown in endothelial cells inhibits atherosclerosis and cerebral ischemic injury in mice.

DNA methylation is essential for mammalian development and physiology. Here we report that the developmentally regulated H19 lncRNA binds to and inhibits S-adenosylhomocysteine hydrolase (SAHH), the only mammalian enzyme capable of hydrolysing S-adenosylhomocysteine (SAH). SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases that methylate diverse cellular components, including DNA, RNA, proteins, lipids and neurotransmitters. We show that H19 knockdown activates SAHH, leading to increased DNMT3B-mediated methylation of an lncRNA-encoding gene Nctc1 within the Igf2-H19-Nctc1 locus. Genome-wide methylation profiling reveals methylation changes at numerous gene loci consistent with SAHH modulation by H19. Our results uncover an unanticipated regulatory circuit involving broad epigenetic alterations by a single abundantly expressed lncRNA that may underlie gene methylation dynamics of development and diseases and suggest that this mode of regulation may extend to other cellular components.

Class-I phosphatidylinositol 3-kinase (PI3KI) converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 comprises two fatty-acid chains that embed in lipid-bilayer membranes, joined by glycerol to inositol triphosphate. Proteins with domains that specifically bind that head-group (e.g. pleckstrin-homology [PH] domains) are thus tethered to the inner plasma-membrane surface where they have an enhanced likelihood of interaction with other PIP3-bound proteins, in particular other components of their signaling pathways. Null alleles of the C. elegans age-1 gene, encoding the catalytic subunit of PI3KI, lack any detectable class-I PI3K activity and so cannot form PIP3. These mutant worms survive almost 10-fold longer than the longest-lived normal control, and are highly resistant to a variety of stresses including oxidative and electrophilic challenges. Traits associated with age-1 mutation are widely believed to be mediated through AKT-1, which requires PIP3 for both tethering and activation. Active AKT complex phosphorylates and thereby inactivates the DAF-16/FOXO transcription factor. However, extensive evidence indicates that pleiotropic effects of age-1-null mutations, including extreme longevity, cannot be explained by insulin like-receptor/AKT/FOXO signaling alone, suggesting involvement of other PIP3-binding proteins. We used ligand-affinity capture to identify membrane-bound proteins downstream of PI3KI that preferentially bind PIP3. Computer modeling supports a subset of candidate proteins predicted to directly bind PIP3 in preference to PIP2, and functional testing by RNAi knockdown confirmed candidates that partially mediate the stress-survival, aggregation-reducing and longevity benefits of PI3KI disruption. PIP3-specific candidate sets are highly enriched for proteins previously reported to affect translation, stress responses, lifespan, proteostasis, and lipid transport.