Small Molecule Metabolism Research Articles

Analysis of RHOA mutations and their significance in the proliferation and transcriptome of digestive tract cancer cells.

The ras homolog family member A (RHOA) gene encodes a member of the Rho family of small GTPases and is known to function in reorganization of the actin cytoskeleton, which is associated with regulation of cell shape, attachment and motility. RHOA has been found to be recurrently mutated in gastrointestinal cancer; however, the functional significance of the mutated RHOA protein in digestive tract cancers remains to be uncovered. The aim of the present study was to understand the role of mutant RHOA in the proliferation and transcriptome of digestive tract cancer cells. Mutations of RHOA in one esophageal cancer cell line, OE19, eight gastric cancer cell lines, namely, AGS, GCIY, HGC-27, KATO III, MKN1, MKN45, SNU16 and SNU719, as well as two colon cancer cell lines, CCK-81 and SW948, were determined using Sanger sequencing. The results uncovered several mutations, including p.Arg5Gln and p.Tyr42Cys in CCK-81, p.Arg5Trp and p.Phe39Leu in SNU16, p.Gly17Glu in SW948, p.Tyr42Ser in OE19, p.Ala61Val in SNU719, p.Glu64del in AGS. Wild-type RHOA was identified in GCIY, HGC-27, KATO III, MKN1 and MKN45. Knockdown of RHOA using small interfering RNA attenuated the in vitro proliferation in the three-dimensional culture systems of GCIY, MKN1, OE19 and SW948, whereas no apparent changes were seen in CCK-81, HGC-27 and SNU719. Transcriptome analysis revealed that downregulation of the long non-coding RNA (lnc)-DERA-1 was observed in all tested cell lines following RHOA knockdown in the RHOA-mutated cell lines. Gene Ontology analysis showed that the genes associated with small molecule metabolic process, oxidation-reduction processes, protein kinase activity, transport, and cell junction were commonly downregulated in cells whose proliferation was attenuated by the knockdown of RHOA. These results suggested that certain RHOA mutations may result in upregulation of lnc-DERA-1 and genes associated with cellular metabolism and proliferation in digestive tract cancers.

Systematic Identification and Validation of Housekeeping and Tissue-Specific Genes in Allotetraploid Chenopodium quinoa

Quinoa is a gluten-free food crop that contains all the essential amino acids and vitamins. The selection of proper housekeeping and tissue-specific genes is the crucial prerequisite for gene expression analysis using the common approach, real-time quantitative PCR (RT-qPCR). In this study, we identified 40 novel candidate housekeeping genes by the minimum transcript per million (TPM), coefficient of variation (CV) and maximum fold change (MFC) methods and 19 candidate tissue-specific genes by the co-expression network method based on an RNA-seq dataset that included 53 stem, leaf, flower and seed samples, as well as additional shoot and root samples under different stresses. The expression stability of 12 housekeeping and tissue-specific genes, as well as that of another two traditionally used housekeeping genes, was further evaluated using qPCR and ranked using NormFinder, BestKeeper and the comparative delta-Ct method. The results demonstrated that MIF, RGGA, VATE and UBA2B were ranked as the top four most stable candidate housekeeping genes. qPCR analysis also revealed three leaf-specific genes and five root-specific genes, but no stem-specific gene was identified. Gene Ontology (GO) enrichment analysis identified that housekeeping genes were mainly enriched in the small molecule metabolic process, organonitrogen compound metabolic process, NAD binding and ligase activity. In addition, tissue-specific genes are closely associated with the major functions of a specific tissue. Specifically, GO terms “photosynthesis” and “thylakoid” were most significantly overrepresented in candidate leaf-specific genes. The novel housekeeping and tissue-specific genes in our study will enable better normalization and quantification of transcript levels in quinoa.

Oleic Acid and Insulin as Key Characteristics of T2D Promote Colorectal Cancer Deterioration in Xenograft Mice Revealed by Functional Metabolomics.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers with high mortality worldwide. Type 2 diabetes mellitus (T2D), known as a risk factor of CRC, can promote the deterioration of CRC, but the underlying mechanism is elusive. In this study, we aimed to reveal the relationship between CRC and T2D from the perspective of small-molecule metabolism. First, a list of common dysregulated metabolites in CRC and T2D was obtained by retrieving existing metabolomics publications. Among these metabolites, oleic acid (OA) was found to be able to promote the proliferation and migration of colon carcinoma cell HCT116. Further experiments proved that insulin could significantly strengthen this promotion and showed a synergistic effect with OA. Mechanism study found that OA and insulin acted synergistically through the extracellular signal-regulated kinase (ERK)1/2/c-Myc/cyclin D1 pathway. In addition, the combination of ERK1/2 inhibitor SCH772984 and cyclin-dependent kinase (CDK)4/6 inhibitor palbociclib showed a remarkable inhibitory effect on tumor growth in vivo. Taken together, the current study found that OA plays an important role in CRC development by using a functional metabolomics approach. More importantly, insulin and OA were confirmed to synergistically promote the deterioration of CRC in vitro and in vivo via ERK1/2/c-Myc/cyclin D1 pathway. Our findings may shed light on CRC treatment among the T2D population.

Integrated Analysis of Hepatic miRNA and mRNA Expression Profiles in the Spontaneous Reversal Process of Liver Fibrosis.

Liver fibrosis results from the imbalance between extracellular matrix (ECM) production and degradation, which is a common pathological consequence of various chronic liver diseases. Although many miRNAs have been reported in liver fibrosis progression, miRNA-mRNA interactions in its reversal process remain to be elucidated. In the current study, we performed an integrated analysis of miRNA and mRNA expression profiles in the mouse model with the spontaneous reversal potency of liver fibrosis. A total of 102 miRNA and 2,845 mRNAs showed significant differential expression in reversal mice compared to fibrotic mice. Moreover, 3,769 putative negatively correlated miRNA-mRNA pairs were revealed to be potentially implicated in the biological function regulation of small molecule metabolism and ECM organization. By integrating miRNA-mRNA regulatory networks, mmu-miR-1843a-5p, mmu-miR-193a-5p, mmu-miR-194-2-3p, and mmu-miR-30c-2-3p were identified as lysyl oxidases-specific miRNAs that were correlated with fibrosis reversal. Our results provided potential candidate targets for the treatment of liver fibrosis.

The MpsAB Bicarbonate Transporter Is Superior to Carbonic Anhydrase in Biofilm-Forming Bacteria with Limited CO2 Diffusion.

ABSTRACTCO2 and bicarbonate are required for carboxylation reactions, which are essential in most bacteria. To provide the cells with sufficient CO2, there exist two dissolved inorganic carbon supply (DICS) systems: the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA). Recently, it has been shown that MpsAB is a bicarbonate transporter that is present not only in photo- and autotrophic bacteria, but also in a diverse range of nonautotrophic microorganisms. Since the two systems rarely coexist in a species but are interchangeable, we investigated what advantages the one system might have over the other. Using the genus Staphylococcus as a model, we deleted the CA gene can in Staphylococcus carnosus and mpsABC genes in Staphylococcus aureus. Deletion of the respective gene in one or the other species led to growth inhibition that could only be reversed by CO2 supplementation. While the S. carnosus Δcan mutant could be fully complemented with mpsABC, the S. aureus ΔmpsABC mutant was only partially complemented by can, suggesting that MpsAB outperforms CA. Interestingly, we provide evidence that mucus biofilm formation such as that involving polysaccharide intercellular adhesin (PIA) impedes the diffusion of CO2 into cells, making MpsAB more advantageous in biofilm-producing strains or species. Coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. In conclusion, the distribution of MpsAB or CA in bacteria does not appear to be random as expression of bicarbonate transporters provides an advantage where diffusion of CO2 is impeded.IMPORTANCE CO2 and bicarbonate are required for carboxylation reactions in central metabolism and biosynthesis of small molecules in all bacteria. This is achieved by two different systems for dissolved inorganic carbon supply (DICS): these are the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA), but both rarely coexist in a given species. Here, we compared both systems and demonstrate that the distribution of MpsAB and/or CA within the phylum Firmicutes is apparently not random. The bicarbonate transporter MpsAB has an advantage in species where CO2 diffusion is hampered—for instance, in mucus- and biofilm-forming bacteria. However, coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. Given the clinical relevance of Staphylococcus in the medical environment, such findings contribute to the understanding of bacterial metabolism and thus are crucial for exploration of potential targets for antimicrobials. The knowledge gained here as exemplified by staphylococcal species could be extended to other pathogenic bacteria.

Supply of acetyl-CoA to N<sub>2</sub>-fixing bacteroids: insights from the mutational and proteomic analyses of <em>Sinorhizobium meliloti</em>

Rhizobia represent a diverse group of gram-negative bacteria capable of fixing atmospheric nitrogen in symbiosis with leguminous plants. Mechanisms of symbiotic efficiency are important to study not only to reveal the “fine tuning” of the host–symbiont supra-organismal genetic system emergence, but also to develop agriculture with minimal environmental risks. In this paper we demonstrate that among seven genes whose inactivation by Tn5 insertions results in an increased efficiency of rhizobia (Sinorhizobium meliloti) symbiosis with alfalfa (Eff++ phenotype), six genes are involved in the metabolism of small molecules. One of them (SMc04399) encodes for acetate-CoA transferase catalyzing the formation of acetyl-CoA from acyl-CoA. Since acetyl-CoA is required for operation of the Krebs cycle, providing ATP for symbiotic N2 fixation, we suggest that a significant portion of this coenzyme utilized by bacteroids is provided by the plant cell supporting the energy-consuming nitrogenase reaction. Proteomic data analysis allow us to reveal the lability of enzymatic pathways which are involved in bacteroids in the production and catabolism of acetyl-CoA and which should be modified to obtain the Eff++ phenotype. This phenotype was developed also after inactivation of NoeB protein which is involved in the host-specific nodulation and is characterized by an elevated production in wild type S. meliloti bacteroids, suggesting a multifunctional role of noeB in the symbiosis operation.

Study on mechanism of Bushen Culuan Formula in treatment of polycystic ovary syndrome based on network pharmacology and molecular docking

This study used network pharmacology and molecular docking to study the mechanism of Bushen Culuan Formula in the treatment of infertility caused by polycystic ovary syndrome(PCOS). The active ingredients and potential drug targets of Bushen Cu-luan Decoction were obtained by searching the Traditional Chinese Medicine System Pharmacology(TCMSP) database, and the targets of PCOS by searching GeneCards. After the drug targets and disease targets were corrected by Uniprot, the intersection genes were obtained. STRING database and Cytoscape 3.7.2 were used for protein-protein interaction(PPI) analysis of the intersection genes. The ClueGO plug-in of Cytoscape 3.7.2 was employed to perform gene ontology(GO) enrichment and KEGG pathway enrichment for the intersection genes. Finally, molecular docking of the key active ingredients with the targets of Bushen Culuan Formula was performed using AutoDockVina and MGLtools. A total of 136 active ingredients and 314 drug targets of the decoction were obtained from TCMSP, and 136 disease targets from GeneCards. Finally, 49 drug-disease intersection genes were obtained. GO enrichment found that the genes were mainly involved in the regulation of muscle cell apoptosis, positive regulation of small molecule metabolism, core promoter binding, RNA polymerase Ⅱ regulation of pri-miRNA transcription, negative regulation of transmembrane transport and other biological functions. The enriched KEGG pathways mainly included MAPK, PI3 K-Akt, p53, and HIF-1 signaling pathways. The results of molecular docking showed that quercetin and PTGS2 can bind stably and interact through amino acid residues THR206, TRP387, ASN382, etc. This study preliminarily reveals the multi-component, multi-target, and multi-pathway mechanism of Bushen Culuan Formula in the treatment of PCOS-related infertility, which provides a basis for further research.

Proteomic Identification of Bombyx mori Organelles Using the Engineered Ascorbate Peroxidase APEX and Development of Silkworm Organelle Proteome Database (SilkOrganPDB).

Silkworm Bombyx mori is an economically important insect and a lepidopteran model. Organelle proteome is vital to understanding gene functions; however, it remains to be identified in silkworm. Here, using the engineered ascorbate peroxidase APEX, we constructed transgenic B. mori embryo cells (BmE) expressing APEX-NLS, COX4-APEX, APEX-Rev, and APEX-KDEL in nucleus, mitochondrial matrix (MM), cytosol, and endoplasmic reticulum (ER), and isolated the biotin-labeled proteins using streptavidin-affinity purification, respectively. The isolated proteins were determined using LC-MS/MS and annotated by searching B. mori genomes downloaded from GenBank, SilkBase, SilkDB 2.0, and SilkDB 3.0, resulting in 842, 495, 311, and 445 organelle proteins identified, respectively. We mapped the 296 MM proteins annotated in the GenBank data to mitochondrial protein databases of the fly, human, and mouse, and found that 140 (47%) proteins are homologous to 80 fly proteins, and 65 (22%) proteins match to 31 and 29 human and mouse proteins, respectively. Protein orthology was predicted in multiple insects using OrthoMCL, producing 460 families containing 839 proteins we identified. Out of 460 families, 363 were highly conserved and found in all insects, leaving only three proteins without orthology in other insects, indicating that the identified proteins are highly conserved and probably play important roles in insects. A gene ontology enrichment analysis by clusterProfiler revealed that the nucleus proteins significantly enriched in cellular component terms of nucleus and nucleolus, the MM proteins markedly enriched in molecular function terms of nucleotide binding, and the cytosol proteins mainly enriched in biological process terms of small molecule metabolism. To facilitate the usage and analysis of our data, we developed an open-access database, Silkworm Organelle Proteome Database (SilkOrganPDB), which provides multiple modules for searching, browsing, downloading, and analyzing these proteins, including BLAST, HMMER, Organelle Proteins, Protein Locations, Sequences, Gene Ontology, Homologs, and Phylogeny. In summary, our work revealed the protein composition of silkworm BmE organelles and provided a database resource helpful for understanding the functions and evolution of these proteins.

Multiple nutritional phenotypes of fission yeast mutants defective in genes encoding essential mitochondrial proteins

Mitochondria are essential for regulation of cellular respiration, energy production, small molecule metabolism, anti-oxidation and cell ageing, among other things. While the mitochondrial genome contains a small number of protein-coding genes, the great majority of mitochondrial proteins are encoded by chromosomal genes. In the fission yeast Schizosaccharomyces pombe, 770 proteins encoded by chromosomal genes are located in mitochondria. Of these, 195 proteins, many of which are implicated in translation and transport, are absolutely essential for viability. We isolated and characterized eight temperature-sensitive (ts) strains with mutations in essential mitochondrial proteins. Interestingly, they are also sensitive to limited nutrition (glucose and/or nitrogen), producing low-glucose-sensitive and 'super-housekeeping' phenotypes. They fail to produce colonies under low-glucose conditions at the permissive temperature or lose cell viability under nitrogen starvation at the restrictive temperature. The majority of these ts mitochondrial mutations may cause defects of gene expression in the mitochondrial genome. mrp4 and mrp17 are defective in mitochondrial ribosomal proteins. ppr3 is defective in rRNA expression, and trz2 and vrs2 are defective in tRNA maturation. This study promises potentially large dividends because mitochondrial quiescent functions are vital for human brain and muscle, and also for longevity.

Proteomic analysis reveals that naturally produced citral can significantly disturb physiological and metabolic processes in the rice blast fungus Magnaporthe oryzae

Rice blast (Magnaporthe oryzae), a major fungal disease in rice producing areas all over the world as well as in China, seriously affects the safety of rice production. Citral, a mixture of Z/E and trans isomers, is a natural acycloid monoterpene compound with good bacteriostatic effect on rice blast. To further investigate the underlying molecular mechanism, a comparative proteomics analysis was conducted between citral-treated and non-treated M. oryzae spores through two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Our analysis identified 1600–1800 proteins from M. oryzae ZB15, of which 147 were differentially expressed in 100 μg/mL citral-treated samples relative to the control group. Among these differentially expressed proteins (DEPs), 40 proteins showed significantly different expression. GO enrichment and NCBI conserved domains database analysis showed that the main groups of the cellular component were cytoplasm (23.33%), and the major molecular function categories were ion binding (31.37%), and the major categories of biological processes included small molecule metabolic process (22.22%) and transport (13.89%). Further analysis found that down-regulated proteins included the tubulin α chain, ATP synthase subunit β and malate dehydrogenase, while the tubulin β, enolase were upregulated. These DEPs could possibly limit the availability of energy required for many cellular processes and result in various physiological adaptions of M. oryzae. This study represents the first proteomic analysis of M. oryzae treated by citral and will help to uncover the mode-of-action of this biologically active compound against M. oryzae. These findings have practical implications with respect to the use of citral for fungal disease control.

Label-free quantitative proteomics analysis of Humulus scandens (Lour.) Merr. leaves treated by an odor compound of Periploca sepium Bunge

The odor compound from Periploca sepium Bunge, 2-hydroxy-4-methoxy-benzaldehyde (HMB), is an allelochemical agent and is one of the least investigated isomers of vanillin. In this study, we used label-free quantitative proteomics analysis technology to investigate the effect of HMB on the protein expression of Humulus scandens (Lour.) Merr. leaves in July 2019 on Guiyang. A total of 269 proteins of 624 identified proteins were differentially expressed, among which 21.18% of the proteins were up-regulated and 32.71% down-regulated. These proteins were classified into 11 cell components and more than 20% of differentially expressed proteins were located in cell membrane and chloroplast. Functional classification analysis showed that 12 molecular functions were altered upon HMB treatment, and the ratio of catalytic activity was the highest (19.53%). At least 12 biological functions were affected, which involved small molecule metabolic processes, organic substance metabolic processes, gene expression, and photosynthesis. Our data provide resources and insights into the biochemical mechanism by which HMB kills weeds.

Clinical and Translational Considerations for Understanding Depression and Anxiety in Patients with Inflammatory Bowel Disease.

Depression and anxiety are comorbidities of inflammatory bowel disease (IBD). Though previous studies have proposed a relationship between anxiety, depression, and IBD, causality and directionality are largely unknown. Current and future research in these areas is aimed at exploring the biological underpinnings of this relationship, specifically pertaining to small molecule metabolism, such as tryptophan. Tryptophan is acquired through the diet and is the precursor to several vital bioactive metabolites including the hormone melatonin, the neurotransmitter serotonin, and vitamin B3. In this review, we discuss previous findings relating mental health comorbidities with IBD and underline ongoing research of tryptophan catabolite analysis.

Chiral Analysis of Lactate during Direct Contact Coculture by Single-Cell On-Probe Enzymatic Dehydrogenation Derivatization: Unraveling Metabolic Changes Caused by d-Lactate.

In vitro noncontact cell-based coculture models are frequently employed to study cell-to-cell communication. However, these models cannot accurately represent the complexity of in vivo signaling. d-Lactate is an unusual metabolite produced and released by cancer cells. The characterization of d-lactate is challenging as it shares the same mass but has much lower amounts compared with l-lactate. Herein, d-α-hydroxy acids were specifically recognized and dehydrogenated by d-α-hydroxy acid dehydrogenase. The dehydrogenation products were rapidly quaternized for enhancement of mass signals. An on-probe enzymatic dehydrogenation-derivatization method was proposed for chiral analysis of α-hydroxy acids at the single-cell level. It is a promising amplification methodology and affords over 3 orders of magnitude signal enhancement. Furthermore, direct contact coculture models were used to precisely mimic the tumor microenvironment and explore the communication between cancer and normal cells. Single-cell mass spectrometry (SCMS) was further applied to easily sample cell extracts and study the differences of the aspects of small molecule metabolism in cocultured cells. On the basis of direct contact coculture SCMS, several differential small molecule metabolites and differences of oxidative stress between cocultured and monocultured normal cells were successfully detected. Additionally, d-lactate was discovered as a valuable differential metabolite with application of the two developed methods. It may account for the cancer-associated metabolic behavior of normal cells. These changes could be relieved after d-lactate metabolism-related drug treatment. This discovery may promote the investigation of d-lactate metabolism, which may provide a novel direction for cancer therapy.

Rapid toxin sequestration modifies poison frog physiology.

Poison frogs sequester chemical defenses from their diet of leaf litter arthropods for defense against predation. Little is known about the physiological adaptations that confer this unusual bioaccumulation ability. We conducted an alkaloid-feeding experiment with the Diablito poison frog (Oophaga sylvatica) to determine how quickly alkaloids are accumulated and how toxins modify frog physiology using quantitative proteomics. Diablito frogs rapidly accumulated the alkaloid decahydroquinoline within 4 days, and dietary alkaloid exposure altered protein abundance in the intestines, liver and skin. Many proteins that increased in abundance with decahydroquinoline accumulation are plasma glycoproteins, including the complement system and the toxin-binding protein saxiphilin. Other protein classes that change in abundance with decahydroquinoline accumulation are membrane proteins involved in small molecule transport and metabolism. Overall, this work shows that poison frogs can rapidly accumulate alkaloids, which alter carrier protein abundance, initiate an immune response, and alter small molecule transport and metabolism dynamics across tissues.

Effects of notoginseng leaf triterpenes on small molecule metabolism after cerebral ischemia/reperfusion injury assessed using MALDI-MS imaging.

BackgroundNotoginseng leaf triterpenes (PNGL) is believed to have neuroprotective effects via the inhibition of inflammatory response and neuronal apoptosis. However, its mechanisms underlying the anti-ischemia/reperfusion (I/R) injury effects on the regulation of small molecule metabolism in rat brain remains unclear. The purpose of this study was thus to explore the mechanisms of PNGL on the regulation of small molecule metabolism in rat brain after I/R injury using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI).MethodsAs a model of in vivo cerebral I/R injury, male Sprague-Dawley (SD) rats were established with a middle cerebral artery occlusion/reperfusion (MCAO/R) model after PNGL administration with 40 mg·kg-1 through intraperitoneal injection (i.p.) for 7 days. We assessed the neurological behavior, regional cerebral blood flow (r CBF), neuron injury, and spatial distribution of metabolic small molecules.ResultsOur in vivo results suggested that PNGL increased cerebral blood flow and relieved neurological dysfunction. Furthermore, using MALDI-MSI, we demonstrated that PNGL regulated 16 endogenous small molecules implicated in metabolic networks including tricarboxylic acid (TCA) cycle, adenosine triphosphate (ATP) metabolism, malate-aspartate shuttle, metal ions, and antioxidants underwent noticeable changes after reperfusion for 24 h.ConclusionsPNGL is a novel cerebrovascular agent that can improve cerebral blood flow and attenuate adverse neurological disorders. The mechanisms are closely correlated with relative metabolic pathways, which offers insight into exploring new mechanisms in PNGL for the treatment of cerebral I/R injury.

The pleiotropic effects of prebiotic galacto-oligosaccharides on the aging gut

BackgroundPrebiotic galacto-oligosaccharides (GOS) have an extensively demonstrated beneficial impact on intestinal health. In this study, we determined the impact of GOS diets on hallmarks of gut aging: microbiome dysbiosis, inflammation, and intestinal barrier defects (“leaky gut”). We also evaluated if short-term GOS feeding influenced how the aging gut responded to antibiotic challenges in a mouse model of Clostridioides difficile infection. Finally, we assessed if colonic organoids could reproduce the GOS responder—non-responder phenotypes observed in vivo.ResultsOld animals had a distinct microbiome characterized by increased ratios of non-saccharolytic versus saccharolytic bacteria and, correspondingly, a lower abundance of β-galactosidases compared to young animals. GOS reduced the overall diversity, increased the abundance of specific saccharolytic bacteria (species of Bacteroides and Lactobacillus), increased the abundance of β-galactosidases in young and old animals, and increased the non-saccharolytic organisms; however, a robust, homogeneous bifidogenic effect was not observed. GOS reduced age-associated increased intestinal permeability and increased MUC2 expression and mucus thickness in old mice. Clyndamicin reduced the abundance Bifidobacterium while increasing Akkermansia, Clostridium, Coprococcus, Bacillus, Bacteroides, and Ruminococcus in old mice. The antibiotics were more impactful than GOS on modulating serum markers of inflammation. Higher serum levels of IL-17 and IL-6 were observed in control and GOS diets in the antibiotic groups, and within those groups, levels of IL-6 were higher in the GOS groups, regardless of age, and higher in the old compared to young animals in the control diet groups. RTqPCR revealed significantly increased gene expression of TNFα in distal colon tissue of old mice, which was decreased by the GOS diet. Colon transcriptomics analysis of mice fed GOS showed increased expression of genes involved in small-molecule metabolic processes and specifically the respirasome in old animals, which could indicate an increased oxidative metabolism and energetic efficiency. In young mice, GOS induced the expression of binding-related genes. The galectin gene Lgals1, a β-galactosyl-binding lectin that bridges molecules by their sugar moieties and is an important modulator of the immune response, and the PI3K-Akt and ECM-receptor interaction pathways were also induced in young mice. Stools from mice exhibiting variable bifidogenic response to GOS injected into colon organoids in the presence of prebiotics reproduced the response and non-response phenotypes observed in vivo suggesting that the composition and functionality of the microbiota are the main contributors to the phenotype.ConclusionsDietary GOS modulated homeostasis of the aging gut by promoting changes in microbiome composition and host gene expression, which was translated into decreased intestinal permeability and increased mucus production. Age was a determining factor on how prebiotics impacted the microbiome and expression of intestinal epithelial cells, especially apparent from the induction of galectin-1 in young but not old mice.7Hn5CXANwfeha3RgfUXqAuVideo abstract

Identification of differential protein expression and putative drug target in metacyclic stage of Leishmania major and Leishmania tropica: A quantitative proteomics and computational view

Cutaneous leishmaniasis (CL) is an infectious disease that commonly caused by Leishmania (L.) major and L.tropica. Recently there has been a growing interest in proteomics analysis on Leishmania for drug target discovery. Therefore, we aimed to distinguish proteins which might be characteristic for each of the species from those shared by both to the detection of drug targets, which may become helpful for designing new drugs for CL. To identify differences in protein profiles of L. major and L. tropica, we conducted a Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) analysis. Totally 67 differentially expressed proteins (DEPs) (fold change> 2 and p < 0.05) were identified between species. Of these, 42 and 25 proteins were up-regulated in L. major and L. tropica, respectively. Several enriched GO terms were identified via biological process of up-regulated proteins. Furthermore, the small molecule metabolic process and translation were detected as significant biological processes for up-regulated proteins in L. major, while translation was identified for L. tropica. Also, KEGG analysis has revealed glycolysis/gluconeogenesis and translation as the top pathways in the proteins up-regulated in L. major and L. tropica, respectively. Finally glycosomal malate dehydrogenase was identified as putative drug target using network and homology analyses. The DEPs between the species are essential in host-pathogen interactions and parasite survival in the macrophage. Furthermore, L. major and L. tropica possibly uses different pathogenicity mechanisms that leads to anthroponotic or zoonotic CL. Our results may help in the drug discovery and chemotherapeutic interventions.

GENETIC POLYMOPRHISMS MAY DIFFERENTIATE BETWEEN COMPLICATED PEDIATRIC SMALL BOWEL VERSUS COLONIC CROHN’S DISEASE

Abstract Objectives About 9% of pediatric Crohn’s disease (CD) patients experience complications within 3 years from diagnosis in spite of anti-Tumor Necrosis Factor-alpha (anti-TNF) biologic treatment. Within 5 years, such complications may require surgical interventions in up to 20% of cases. Due to the relatively rare nature of these complicated pediatric CD cases within large CD cohorts, traditional genome wide association studies (GWAS) rarely capture this patient population. Our aim was to identify critical genetic associations of anti-TNF refractory complicated pediatric CD subtypes. Methods The study was approved by BCM IRB. Included patients had developed CD between the ages of 6 and 18. The Paris classification was used to place patients in the following categories: 1. Anti-TNF refractory small bowel CD (RSB-CD): L4b; L4b/B2; or L4b/B2B3 who had or were imminent to have small bowel resection. 2.. Anti-TNF refractory colonic CD (RC-CD): L2 requiring ileal diversion; or L2/B2; or L2/B2B3 who had or were considered to receive colectomy or partial colonic resection. Whole-exome sequencing (WES) was performed from peripheral blood derived DNA. PLINK (https://www.cog-genomics.org/plink2/) was used to identify enriched single nucleotide polymorphisms (SNPs). The level of significance for exome wide differentiation was relaxed to p&amp;lt;0.01. The potential deleteriousness of single nucleotide variants was determined by CADD (https://cadd.gs.washington.edu/). Gene Ontology enRIchment anaLysis and visuaLizAtion tool (GORILLA) was used to compare gene lists associated with colonic enriched SNPs versus ileal enriched SNPs (http://geneontology.org/). Results In eight RSB-CD and 11 RC-CD cases, age at presentation, gender, ethnicity, and need for surgery (75% and 55%, respectively) did not significantly differ between the groups. There were 269 genes associated with 277 SNPs that had significantly (p&amp;lt;0.01) different allele variation between colonic vs ileal CD. Of these, 170 genes associated with 116 distinct SNPs had a CADD score of &amp;gt;10. Out of the top five SNP candidate associated genes, four (4/5 = 80%: EFNA3, STEAP1B, DSG1, and CYP4F2) have been already linked with Crohn’s disease and/or colitis. GORILLA based investigation indicated that colonic CD associated genes were enriched (p&amp;lt;0.001) in biologic function relevant for lipid metabolic process (GO:0006629), small molecule metabolic process (GO:0044281), and organic substance metabolic process (GO:0071704) compared to ileal CD associated genes. Conclusions We have uncovered candidate genetic risk loci, which may differentially influence pediatric CD towards developing primarily small intestinal or colonic complications. The discovered loci hold significant potential as novel genetic modifiers, which may set the basis for risk assessment, prevention and personalized treatment against complicated pediatric CD.

Protein molecular responses of field-collected oysters Crassostrea hongkongensis with greatly varying Cu and Zn body burdens

The oyster Crassostrea hongkongensis is an ideal biomonitor due to its widespread distribution along the coast of Southern China and the ability to hyperaccumulate metals including Cu and Zn. In this study, we conducted the first investigation of the molecular responses to metal hyperaccumulation based on quantitative shotgun proteomics technique and genome information. Gill tissue of oysters collected from the uncontaminated environment (Site 1, 59.6 μg/g and 670 μg/g dry weight for Cu and Zn) displayed significant protein profile differentiation compared to those from a moderately contaminated (Site 2, 1,465 μg/g and 10,170 μg/g for Cu and Zn) and a severely contaminated environment (Site 3, 3,899 μg/g and 39,170 μg/g for Cu and Zn). There were 626 proteins identified to be differentially expressed at Site 3 but only 247 proteins at Site 2. Oysters from a moderately contaminated estuary (Site 2) displayed fewer effects as compared to oysters under severe contamination, with fluctuated small molecule metabolism and enhanced translation process. At Site 3, the induction of reactive oxygen species (ROS) was the main toxicity under the extremely high level of metal stress, which resulted in protein damage. Additionally, the impaired structure of cytoskeleton and modified membrane tracking process at Site 3 oysters led to the blockage or less efficient protein or macromolecule distribution within cells. Nonetheless, proteomic analysis in this study revealed that oysters could partly alleviate the adverse metal effects by boosting the translation process, enhancing the ability to recycle the misfolded proteins, and enhancing the potential to eliminate the excess ROS. Our study demonstrated an adaptive potential of oysters at the protein level to survive under conditions of metal hyper-accumulation.

Evolutionary Rescue of an Environmental Pseudomonas otitidis in Response to Anthropogenic Perturbation.

Anthropogenic perturbations introduce novel selective pressures to natural environments, impacting the genomic variability of organisms and thus altering the evolutionary trajectory of populations. Water overexploitation for agricultural purposes and defective policies in Cuatro Cienegas, Coahuila, Mexico, have strongly impacted its water reservoir, pushing entire hydrological systems to the brink of extinction along with their native populations. Here, we studied the effects of continuous water overexploitation on an environmental aquatic lineage of Pseudomonas otitidis over a 13-year period which encompasses three desiccation events. By comparing the genomes of a population sample from 2003 (original state) and 2015 (perturbed state), we analyzed the demographic history and evolutionary response to perturbation of this lineage. Through coalescent simulations, we obtained a demographic model of contraction-expansion-contraction which points to the occurrence of an evolutionary rescue event. Loss of genomic and nucleotide variation alongside an increment in mean and variance of Tajima’s D, characteristic of sudden population expansions, support this observation. In addition, a significant increase in recombination rate (R/θ) was observed, pointing to horizontal gene transfer playing a role in population recovery. Furthermore, the gain of phosphorylation, DNA recombination, small-molecule metabolism and transport and loss of biosynthetic and regulatory genes suggest a functional shift in response to the environmental perturbation. Despite subsequent sampling events in the studied site, no pseudomonad was found until the lagoon completely dried in 2017. We speculate about the causes of P. otitidis final decline or possible extinction. Overall our results are evidence of adaptive responses at the genomic level of bacterial populations in a heavily exploited aquifer.