Pyruvate Metabolism Pathways Research Articles

Effects of Dietary Cottonseed Protein Concentrate Levels on Growth Performance, Health Status, Flesh Quality and Intestinal Microbiota of Grass Carp (Ctenopharyngodon idellus).

The aim of this study was to evaluate the nutritional value of cottonseed protein concentrate (CPC) as a single dietary protein source and the optimal protein level for grass carp (Ctenopharyngodon idellus). An 8-week feeding trial was conducted by feeding juvenile grass carp (initial body weight: 4.68 ± 0.01 g) with six experimental diets containing graded levels of protein provided by CPC. The results showed that the optimal CPC level (CPC4) improved the growth performance and health status of grass carp. The optimal dietary protein level was estimated to be 38.61 and 38.66% based on specific growth rate (SGR) and feed efficiency (FE), respectively. The CPC4 group significantly increased the total antioxidant capacity (T-AOC) content and glutathione peroxidase (GSH-Px) activity in the hepatopancreas (p < 0.05). In addition, the CPC4 group increased the muscle T-AOC and glutathione (GSH) content and improved muscle hardness, and the gene expression of MRFs, fgf6a, myhc-7, myhc-1, myhc-4, igf-II, and tor was upregulated while mstn gene expression was downregulated (p < 0.05). Correlation analysis revealed that the optimal dietary CPC level promoted grass carp growth, health, and flesh quality by regulating the relative abundance of intestinal microbes. Furthermore, CPC6 upregulated the ko00480 (Glutathione metabolism) and ko00620 (Pyruvate metabolism) pathways compared to CPC1 (p < 0.05), possibly indicating that low dietary CPC levels adversely affected amino acid metabolism in the intestinal microbiota of grass carp, while a high level of CPC will meet the metabolic needs of the body by increasing the utilization of energy.

Engineering of global transcription factor FruR to redirect the carbon flow in Escherichia coli for enhancing l-phenylalanine biosynthesis

BackgroundThe catabolite repressor/activator protein (FruR) is a global regulatory protein known to control the expression of several genes concerned with carbon utilization and energy metabolism. This study aimed to illustrate effects of the FruR mutant on the L-phenylalanine (L-PHE) producing strain PHE01.ResultsRandom mutagenesis libraries of fruR generated in vitro were first integrated into the chromosome of PHE01 by CRISPR/Cas9 technique, and then the best mutant PHE07 (FruRE173K) was obtained. With this mutant, a final L-PHE concentration of 70.50 ± 1.02 g/L was achieved, which was 23.34% higher than that of PHE01. To better understand the mechanism, both transcriptomes and metabolomes of PHE07 were carried out and compared to that of PHE01. Specifically, the transcript levels of genes involved in gluconeogenesis pathway, pentose phosphate pathway, Krebs cycle, and glyoxylate shunt were up-regulated in the FruRE173K mutant, whereas genes aceEF, acnB, and icd were down-regulated. From the metabolite level, the FruRE173K mutation led to an accumulation of pentose phosphate pathway and Krebs cycle products, whereas the products of pyruvate metabolism pathway: acetyl-CoA and cis-aconic acid, were down-regulated. As a result of the altered metabolic flows, the utilization of carbon sources was improved and the supply of precursors (phosphoenolpyruvate and erythrose 4-phosphate) for L-PHE biosynthesis was increased, which together led to the enhanced production of L-PHE.ConclusionA novel strategy for L-PHE overproduction by modification of the global transcription factor FruR in E. coli was reported. Especially, these findings expand the scope of pathways affected by the fruR regulon and illustrate its importance as a global regulator in L-PHE production.

Efficacy of Acupoint Application on In Vitro Fertilization Outcome in Patients with Polycystic Ovary Syndrome: A UHPLC-MS-Based Metabolomic Study.

Objective To explore the clinical effect of acupoint application on the outcome of in vitro fertilization-embryo transfer (IVF-ET) in patients with polycystic ovary syndrome (PCOS) of the phlegm-dampness type and elucidate its possible mechanism of action from the perspective of follicular fluid metabolomics. Methods A total of 90 patients undergoing IVF-ET due to infertility were selected and divided into three groups: the treatment group (PCOS with acupoint application, n = 30), the control group (PCOS without acupoint application, n = 30), and the normal group (non-PCOS, n = 30). All patients received a gonadotropin-releasing hormone agonist (GnRH-a) long protocol for controlled ovarian hyperstimulation (COH). Among them, the treatment group was also given the acupoint application from the day of pituitary downregulation to the day of the human chorionic gonadotrophin (hCG) trigger. Ultrahigh-performance liquid chromatography connected with quadrupole time-of-flight mass spectrometry (UHPLC-MS) was adopted for untargeted metabolomic analysis of follicular fluid collected from the three groups of patients on the day of oocyte pick-up (OPU). The significantly differential metabolites were screened using univariate and multivariate statistical analysis, and the related metabolic pathways were identified by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Results Metabolomic analysis showed that the treatment group's follicular fluid samples were aggregated with the normal group and separated from the control group. A total of 34 significantly differential metabolites were found in the follicular fluid of patients with phlegm-dampness PCOS and normal people. With the intervention of acupoint application, seven metabolites (pseudouridine, phenol, 2-oxoadipic acid, 9R,10S-EpOME, DL-lactate, nicotinamide, and DL-indole-3-lactic acid) were all downregulated, mainly involving the pathways of pyruvate metabolism, nicotinate and nicotinamide metabolism, protein digestion and absorption, biosynthesis of amino acids, and pyrimidine metabolism. Conclusions Acupoint application can effectively improve the clinical symptoms and the outcome of IVF-ET treatment in patients with PCOS of the phlegm-dampness type, and its mechanism of action may be related to the regulation of the pathways of pyruvate metabolism, nicotinate and nicotinamide metabolism, protein digestion and absorption, biosynthesis of amino acids, and pyrimidine metabolism.

Multi-Omics, an Integrated Approach to Identify Novel Blood Biomarkers of Alzheimer's Disease.

The metabolomic and proteomic basis of mild cognitive impairment (MCI) and Alzheimer’s disease (AD) is poorly understood, and the relationships between systemic abnormalities in metabolism and AD/MCI pathogenesis is unclear. This study compared the metabolomic and proteomic signature of plasma from cognitively normal (CN) and dementia patients diagnosed with MCI or AD, to identify specific cellular pathways and new biomarkers altered with the progression of the disease. We analysed 80 plasma samples from individuals with MCI or AD, as well as age- and gender-matched CN individuals, by utilising mass spectrometry methods and data analyses that included combined pathway analysis and model predictions. Several proteins clearly identified AD from the MCI and CN groups and included plasma actins, mannan-binding lectin serine protease 1, serum amyloid A2, fibronectin and extracellular matrix protein 1 and Keratin 9. The integrated pathway analysis showed various metabolic pathways were affected in AD, such as the arginine, alanine, aspartate, glutamate and pyruvate metabolism pathways. Therefore, our multi-omics approach identified novel plasma biomarkers for the MCI and AD groups, identified changes in metabolic processes, and may form the basis of a biomarker panel for stratifying dementia participants in future clinical trials.

RNase III coding genes modulate the cross-kingdom biofilm of Streptococcus mutans and Candida albicans.

Streptococcus mutans constantly coexists with Candida albicans in plaque biofilms of early childhood caries (ECC). The progression of ECC can be influenced by the interactions between S. mutans and C. albicans through exopolysaccharides (EPS). Our previous studies have shown that rnc, the gene encoding ribonuclease III (RNase III), is implicated in the cariogenicity of S. mutans by regulating EPS metabolism. The DCR1 gene in C. albicans encodes the sole functional RNase III and is capable of producing non-coding RNAs. However, whether rnc or DCR1 can regulate the structure or cariogenic virulence of the cross-kingdom biofilm of S. mutans and C. albicans is not yet well understood. By using gene disruption or overexpression assays, this study aims to investigate the roles of rnc and DCR1 in modulating the biological characteristics of dual-species biofilms of S. mutans and C. albicans and to reveal the molecular mechanism of regulation. The morphology, biomass, EPS content, and lactic acid production of the dual-species biofilm were assessed. Quantitative real-time polymerase chain reaction (qRT-PCR) and transcriptomic profiling were performed to unravel the alteration of C. albicans virulence. We found that both rnc and DCR1 could regulate the biological traits of cross-kingdom biofilms. The rnc gene prominently contributed to the formation of dual-species biofilms by positively modulating the extracellular polysaccharide synthesis, leading to increased biomass, biofilm roughness, and acid production. Changes in the microecological system probably impacted the virulence as well as polysaccharide or pyruvate metabolism pathways of C. albicans, which facilitated the assembly of a cariogenic cross-kingdom biofilm and the generation of an augmented acidic milieu. These results may provide an avenue for exploring new targets for the effective prevention and treatment of ECC.

Transcriptome analysis of the production enhancement mechanism of antimicrobial lipopeptides of Streptomyces bikiniensis HD-087 by co-culture with Magnaporthe oryzae Guy11

The lipopeptides produced by Streptomyces bikiniensis have a significant inhibitory effect on Magnaporthe oryzae, but the low yield limits its application. In this study, the anti-M. oryzae activity of the broth of S. bikiniensis HD-087 co-cultured with M. oryzae Guy11 mycelium has risen by 41.22% compared with pure culture, and under induction conditions of adding Guy11-inducer (cell-free supernatant of M. oryzae Guy11), the activity of strain HD-087 improved 61.76%. The result proved that the enhancement effect of Guy11 on the antimicrobial activity of HD-087 was mainly related to metabolites but mycelium cells. Under optimum induction conditions, NRPS gene expression levels of HD-087 were significantly increased by induction with Guy11-inducer, the biomass of HD-087 had no significant change, but crude extract of lipopeptide (CEL) production was 107.4% higher than pure culture, and TLC result under acid hydrolysis showed that the induced culture has one component more than pure culture. To clarify the regulation mechanism of improving lipopeptide production of HD-087 with Guy11-inducer, transcriptomic analysis was performed using RNAseq to compare the induced culture and pure culture. In the induced culture, 943 genes were up-regulated, while 590 genes were down-regulated in DEGs (differentially expressed genes). KEGG results showed that the expression of genes related to amino acid synthesis, fatty acid metabolism, TCA cycle and pyruvate metabolism pathway were significantly increased. The increased expression of genes related to these metabolic pathways provided sufficient precursors for lipopeptide synthesis. Accordingly, key enzyme genes responsible for the synthesis of lipopeptides Srf and NRPS was significantly increased. Quorum sensing related genes OppA and MppA were significantly up-regulated, and then ComP was activated and promoted lipopeptide synthesis. These results provided a scientific basis for using M. oryzae to induce the increase of the production of Streptomyces lipopeptides, and also laid a foundation for further exploring the co-culture mechanisms among different genera.

Formation mechanisms of ethyl acetate and organic acids in Kluyveromyces marxianus L1-1 in Chinese acid rice soup

This study aims to explore the formation mechanism of ethyl acetate and organic acids in acid rice soup (rice-acid soup) inoculated with Kluyveromyces marxianus L1-1 through the complementary analysis of transcriptome and proteome. The quantity of K. marxianus L1-1 varied significantly in the fermentation process of rice-acid soup and the first and third days were the two key turning points in the growth phase of K. marxianus L1-1. Importantly, the concentrations of ethyl acetate, ethanol, acetic acid, and L-lactic acid increased from day 1 to day 3. At least 4231 genes and 2937 proteins were identified and 610 differentially expressed proteins were annotated to 30 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways based on the analysis results of transcriptome and proteome. The key genes and proteins including up-regulated alcohol dehydrogenase family, alcohol O-acetyltransferase, acetyl-CoA C-acetyltransferase, acyl-coenzyme A thioester hydrolase, and down-regulated aldehyde dehydrogenase family were involved in glycolysis/gluconeogenesis pathways, starch and sucrose metabolism pathways, amino sugar and nucleotide sugar metabolism pathways, tricarboxylic acid (TCA) cycle, and pyruvate metabolism pathways, thus promoting the formation of ethyl acetate, organic acids, alcohols, and other esters. Our results revealed the formation mechanisms of ethyl acetate and organic acids in rice-acid soup inoculated with K. marxianus L1-1.

Using AI-Based Evolutionary Algorithms to Elucidate Adult Brain Tumor (Glioma) Etiology Associated with IDH1 for Therapeutic Target Identification.

Adult brain tumors (glioma) represent a cancer of unmet need where standard-of-care is non-curative; thus, new therapies are urgently needed. It is unclear whether isocitrate dehydrogenases (IDH1/2) when not mutated have any role in gliomagenesis or tumor growth. Nevertheless, IDH1 is overexpressed in glioblastoma (GBM), which could impact upon cellular metabolism and epigenetic reprogramming. This study characterizes IDH1 expression and associated genes and pathways. A novel biomarker discovery pipeline using artificial intelligence (evolutionary algorithms) was employed to analyze IDH-wildtype adult gliomas from the TCGA LGG-GBM cohort. Ninety genes whose expression correlated with IDH1 expression were identified from: (1) All gliomas, (2) primary GBM, and (3) recurrent GBM tumors. Genes were overrepresented in ubiquitin-mediated proteolysis, focal adhesion, mTOR signaling, and pyruvate metabolism pathways. Other non-enriched pathways included O-glycan biosynthesis, notch signaling, and signaling regulating stem cell pluripotency (PCGF3). Potential prognostic (TSPYL2, JAKMIP1, CIT, TMTC1) and two diagnostic (MINK1, PLEKHM3) biomarkers were downregulated in GBM. Their gene expression and methylation were negatively and positively correlated with IDH1 expression, respectively. Two diagnostic biomarkers (BZW1, RCF2) showed the opposite trend. Prognostic genes were not impacted by high frequencies of molecular alterations and only one (TMTC1) could be validated in another cohort. Genes with mechanistic links to IDH1 were involved in brain neuronal development, cell proliferation, cytokinesis, and O-mannosylation as well as tumor suppression and anaplerosis. Results highlight metabolic vulnerabilities and therapeutic targets for use in future clinical trials.

Blood-based untargeted metabolomics in relapsing-remitting multiple sclerosis revealed the testable therapeutic target.

Metabolic aberrations impact the pathogenesis of multiple sclerosis (MS) and possibly can provide clues for new treatment strategies. Using untargeted metabolomics, we measured serum metabolites from 35 patients with relapsing-remitting multiple sclerosis (RRMS) and 14 healthy age-matched controls. Of 632 known metabolites detected, 60 were significantly altered in RRMS. Bioinformatics analysis identified an altered metabotype in patients with RRMS, represented by four changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these four pathways is the glycolysis pathway. Real-time bioenergetic analysis of the patient-derived peripheral blood mononuclear cells showed enhanced glycolysis, supporting the altered metabolic state of immune cells. Experimental autoimmune encephalomyelitis mice treated with the glycolytic inhibitor 2-deoxy-D-glucose ameliorated the disease progression and inhibited the disease pathology significantly by promoting the antiinflammatory phenotype of monocytes/macrophage in the central nervous system. Our study provided a proof of principle for how a blood-based metabolomic approach using patient samples could lead to the identification of a therapeutic target for developing potential therapy.

A study on bisphenol S induced nephrotoxicity and assessment of altered downstream kidney metabolites using gas chromatography–mass spectrometry based metabolomics

The global use of bisphenol S (BPS) has now been significantly increased for commensurate utilization as a substitute for BPA for its regulatory concerns. Though, previous reports indicated that BPS been also appeared as a toxic congener comparable to BPA. In the present study, we determined nephrotoxicity condition induced due to BPS exposure. Results indicated that BPS significantly promoted histopathological disturbance in the kidney, and altered the levels of biomarkers of kidney damage in serum and urine samples of Wistar rats. It is also indicated that BPS altered the expression of kidney damage biomarkers associated with glomerular and tubular injury. Additionally, we determined the perturbation of kidney metabolites in the underlying pathophysiological response of kidney injury due to BPS exposure. Gas chromatography–mass spectrometry based untargeted metabolomics exhibited 20 significantly perturbed metabolites. Moreover, metabolic pathway analysis revealed significant disturbance in the TCA cycle and pyruvate metabolism pathways.

The Effects of Alcohol Drinking on Oral Microbiota in the Chinese Population.

The dysbiosis of oral microbiota is linked to numerous diseases and is associated with personal lifestyles, such as alcohol drinking. However, there is inadequate data to study the effect of alcohol drinking on oral microbiota from the Chinese population. Here, we profiled the oral microbiota of 150 healthy subjects in the Chinese population by 16S rRNA gene sequencing. The results showed that drinkers had significantly higher alpha diversity than non-drinkers. A significant difference in overall microbiota composition was observed between non-drinkers and drinkers. Additionally, using DESeq analysis, we found genus Prevotella and Moryella, and species Prevotella melaninogenica and Prevotella tannerae were significantly enriched in drinkers; meanwhile, the genus Lautropia, Haemophilus and Porphyromonas, and species Haemophilus parainfluenzae were significantly depleted in drinkers. PICRUSt analysis showed that significantly different genera were mainly related to metabolism pathways. The oxygen-independent pathways, including galactose, fructose and mannose metabolism pathways, were enriched in drinkers and positively associated with genera enriched in drinkers; while the pyruvate metabolism pathway, an aerobic metabolism pathway, was decreased in drinkers and negatively associated with genera enriched in drinkers. Our results suggested that alcohol drinking may affect health by altering oral microbial composition and potentially affecting microbial functional pathways. These findings may have implications for better understanding the potential role those oral bacteria play in alcohol-related diseases.

Unveiling a Novel Role of Cdc42 in Pyruvate Metabolism Pathway to Mediate Insecticidal Activity of Beauveria bassiana.

The small GTPase Cdc42 acts as a molecular switch essential for cell cycles and polar growth in model yeast, but has not been explored in Beaurveria bassiana, an insect-pathogenic fungus serving as a main source of fungal formulations against arthropod pests. Here, we show the indispensability of Cdc42 for fungal insecticidal activity. Deletion of cdc42 in B. bassiana resulted in a great loss of virulence to Galleria mellonella, a model insect, via normal cuticle infection as well as defects in conidial germination, radial growth, aerial conidiation, and conidial tolerance to heat and UVB irradiation. The deleted mutant’s hyphae formed fewer or more septa and produced unicellular blastospores with disturbed cell cycles under submerged-culture conditions. Transcriptomic analysis revealed differential expression of 746 genes and dysregulation of pyruvate metabolism and related pathways, which were validated by marked changes in intracellular pyruvate content, ATP content, related enzyme activities, and in extracellular beauvericin content and Pr1 protease activity vital for fungal virulence. These findings uncover a novel role for Cdc42 in the pathways of pyruvate metabolism and the pyruvate-involved tricarboxylic acid cycle (TCA cycle) and a linkage of the novel role with its indispensability for the biological control potential of B. bassiana against arthropod pests.

Proteomics Reveal the Effect of Exogenous Electrons on Electroactive Escherichia coli.

Microbial cells utilizing electricity to produce high-value fuels and chemicals are the foundation of the biocathodic bioelectrochemical system. However, molecular mechanisms of electron transfer and utilization have not been elucidated. In this work, Escherichia coli engineered by introducing the Mtr pathway from Shewanella oneidensis exhibited stronger electrochemical activity than control and could utilize exogenous electrons to stimulate metabolite profiles and boost succinate production in the bioelectrochemical system. Proteomic analysis and real-time PCR were performed to investigate the effect of exogenous electrons on electroactive E. coli. Bioinformatics analysis suggested that the proteins of molecular function associated with oxidoreductase activity, 4 iron, 4 sulfur([4Fe-4S]) cluster binding, iron-sulfur cluster binding, and metal cluster binding were positively affected by exogenous electrons. Moreover, mapping to the Kyoto Encyclopedia of Genes and Genomes pathway database showed that the up-regulated proteins were mainly involved in metabolic pathways of tricarboxylic acid cycle, pyruvate metabolism, and nitrogen metabolism pathway, providing support for the metabolic balance of microbial cells shifting toward reduced end-products due to electron utilization. Using a biochemical method, the ompF-overexpressed strain was employed to investigate the function of the channel protein. These findings provided a theoretical basis for further improving electron transfer and utilization efficiency, and contributed to the potential applications of the bioelectrochemical system.

Profile analysis of circRNAs in human THP-1 derived macrophages infected with intracellular Staphylococcus aureus

BackgroundIntracellular Staphylococcus aureus (S. aureus) infection is generally persistent, recurrent and difficult to treat due to the poor availability of antibiotics within macrophages cells and the lack of ideal diagnostic markers. Circular RNAs (circRNAs), with covalently closed circular structures, exists in the serum stably and is not easily degraded by nucleases. Besides, circRNAs play a pivotal in the eukaryotic regulation of genes expression and served as biomarkers in variety disease including microbial infections. However, the function of host circRNAs in intracellular S. aureus infection remains largely unclear. MethodsIn this study, the circRNAs expression profile was investigated by RNA sequencing technology in both S. aureus-infected THP-1 derived macrophages and mock control cells. The differentially expressed circRNAs (DE circRNAs) with a fold-change >1.5 (p < 0.05) are analyzed using functional pathway clustering prediction. Then, RT-qPCR was performed to verify the top 2 up-regulated circRNAs in the THP-1 cell and human serum samples so as to evaluate the value of circRNAs for S. aureus diagnosis. ResultsAn intracellular survival THP-1 derived macrophages model of S. aureus infection was established. A total of 5,299 circRNAs were identified in human THP-1 derived macrophages infected with intracellular S. aureus. There were 61 DE circRNAs with a fold-change >1.5 (p < 0.05) after S. aureus infection. Among them, 22 circRNAs were up-regulated while 39 circRNAs down-regulated. GO and KEGG pathway analysis demonstrated that DE circRNAs were enriched in the processes such as Neurotrophin, Pyruvate metabolism and Notch signaling pathway. Moreover, hsa_circ_0000311 and chr13:43500472–43544806-(novel) were verified to be significantly upregulated in THP-1 derived macrophages and human serum samples between two groups. Finally, the networks of circRNA-miRNA-mRNA based on these two circRNAs were constructed respectively. ConclusionOur study provides the first profile analysis of host circRNAs involved in intracellular S. aureus infection, which may serve as biomarkers for S. aureus diagnosis and contribute to the understanding of S. aureus evasion mechanisms.

Quantitative Proteomics Analysis Expands the Roles of Lysine β-Hydroxybutyrylation Pathway in Response to Environmental β-Hydroxybutyrate.

Lysine β-hydroxybutyrylation (Kbhb) is a newly identified protein posttranslational modification (PTM) derived from β-hydroxybutyrate (BHB), a product of ketone body metabolism in liver. BHB could serve as an energy source and play a role in the suppression of oxidative stress. The plasma concentration of BHB could increase up to 20 mM during starvation and in pathological conditions. Despite the progress, how the cells derived from extrahepatic tissues respond to elevated environmental BHB remains largely unknown. Given that BHB can significantly drive Kbhb, we characterized the BHB-induced lysine β-hydroxybutyrylome and acetylome by quantitative proteomics. A total of 840 unique Kbhb sites on 429 proteins were identified, with 42 sites on 39 proteins increased by more than 50% in response to BHB. The results showed that the upregulated Kbhb induced by BHB was involved in aminoacyl-tRNA biosynthesis, 2-oxocarboxylic acid metabolism, citrate cycle, glycolysis/gluconeogenesis, and pyruvate metabolism pathways. Moreover, some BHB-induced Kbhb substrates were significantly involved in diseases such as cancer. Taken together, we investigate the dynamics of lysine β-hydroxybutyrylome and acetylome induced by environmental BHB, which reveals the roles of Kbhb in regulating various biological processes and expands the biological functions of BHB.

In vitro anti-Helicobacter pylori activity of Syzygium aromaticum and the preliminary mechanism of action

Ethnopharmacological relevanceThe dried flower bud of Syzygium aromaticum (L.) Merr. & L.M Perry (S. aromaticum) (Myrtaceae), also known as clove, was used in Traditional Chinese Medicine (TCM) to aid gastrointestinal function and treat stomach disorders including vomiting, flatulence and nausea. And it is a food homology medicine which is a promising candidate for H. pylori treatment. H. pylori is a Gram-negative bacterium that infects approximately 50% of the human population worldwide, which is closely related to multiple gastric diseases, including gastric cancer. However, there are still no sufficient studies on the anti-H. pylori activity of S. aromaticum, especially for the mechanism of action. Aim of studyThis study aimed to study the antibacterial activities of S. aromaticum extracts on both antibiotic-sensitive and -resistant H. pylori strains, and to explore the underlying mechanisms of action. Materials and methodsThe S. aromaticum extracts were obtained by heat reflux extraction and lyophilized to powder form. The phytochemical analyses were performed by High-performance liquid chromatography (HPLC) and UPLC-electrospray ionization mass spectrometry (ESI-MS). In vitro anti-H. pylori activity was evaluated by broth microdilution method. Mechanism of action studies included morphological observation using electron microscopy, determination of expression of virulence genes by reverse transcription quantitative polymerase chain reaction (RT-qPCR), genes expression profile identification by transcriptomic analysis, and exploration of anti-H. pylori infection mechanisms by network pharmacology analysis and western blotting validation. ResultsThe S. aromaticum extracts, aqueous extract (AE) and 75% hydroalcoholic extract (HE), exerted significant antibacterial activities against both antibiotic-sensitive and -resistant H. pylori strains with MICs of 160∼320 μg/ml, without developing drug resistance. Among them, AE was bactericide to all the tested strains with MBCs of less than 4MIC, while HE was merely bacteriostatic to most of the tested strains with MBCs of 2MIC∼16MIC. Besides, they showed no antagonistic effects in combination with clarithromycin, metronidazole, levofloxacin, and amoxicillin. Additionally, these extracts altered the morphology and ultrastructure and down-regulated the virulence genes expression of H. pylori. And transcriptomic analysis showed that they regulated genes expression of multiple H. pylori biological processes, including tricarboxylic acid cycle (TAC) and pyruvate metabolic pathways. Furthermore, these extracts combated the abnormal activation of PI3K-Akt and MAPK signaling pathways caused by H. pylori infection. ConclusionsOverall, the present study firstly analyzed the chemical compositions of S. aromaticum extracts, and then confirmed their activities on both antibiotic-sensitive and -resistant H. pylori strains. In addition, the mechanisms of action of S. aromaticum extracts against H. pylori were found to be destroying the bacterial structure, down-regulating the expression of virulence genes, and interfering TAC and pyruvate metabolic pathways. Finally, S. aromaticum extracts were found to combated the abnormal activation of PI3K-Akt and MAPK signaling pathways to treat H. pylori infection. This study should accelerate further research and application of S. aromaticum against H. pylori infection.

Deep Metabolomic Profiling Reveals Alterations in Fatty Acid Synthesis and Ketone Body Degradations in Spermatozoa and Seminal Plasma of Astheno-Oligozoospermic Bulls.

Male fertility is extremely important in dairy animals because semen from a single bull is used to inseminate several thousand females. Asthenozoospermia (reduced sperm motility) and oligozoospermia (reduced sperm concentration) are the two important reasons cited for idiopathic infertility in crossbred bulls; however, the etiology remains elusive. In this study, using a non-targeted liquid chromatography with tandem mass spectrometry-based approach, we carried out a deep metabolomic analysis of spermatozoa and seminal plasma derived from normozoospermic and astheno-oligozoospermic bulls. Using bioinformatics tools, alterations in metabolites and metabolic pathways between normozoospermia and astheno-oligozoospermia were elucidated. A total of 299 and 167 metabolites in spermatozoa and 183 and 147 metabolites in seminal plasma were detected in astheno-oligozoospermic and normozoospermic bulls, respectively. Among the mapped metabolites, 75 sperm metabolites were common to both the groups, whereas 166 and 50 sperm metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Similarly, 86 metabolites were common to both the groups, whereas 45 and 37 seminal plasma metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Among the differentially expressed metabolites, 62 sperm metabolites and 56 seminal plasma metabolites were significantly dysregulated in astheno-oligozoospermic bulls. In spermatozoa, selenocysteine, deoxyuridine triphosphate, and nitroprusside showed significant enrichment in astheno-oligozoospermic bulls. In seminal plasma, malonic acid, 5-diphosphoinositol pentakisphosphate, D-cysteine, and nicotinamide adenine dinucleotide phosphate were significantly upregulated, whereas tetradecanoyl-CoA was significantly downregulated in the astheno-oligozoospermia. Spermatozoa from astheno-oligozoospermic bulls showed alterations in the metabolism of fatty acid and fatty acid elongation in mitochondria pathways, whereas seminal plasma from astheno-oligozoospermic bulls showed alterations in synthesis and degradation of ketone bodies, pyruvate metabolism, and inositol phosphate metabolism pathways. The present study revealed vital information related to semen metabolomic differences between astheno-oligozoospermic and normospermic crossbred breeding bulls. It is inferred that fatty acid synthesis and ketone body degradations are altered in the spermatozoa and seminal plasma of astheno-oligozoospermic crossbred bulls. These results open up new avenues for further research, and current findings can be applied for the modulation of identified pathways to restore sperm motility and concentration in astheno-oligozoospermic bulls.

Characterization of the 3'UTR of the BTD gene and identification of regulatory elements and microRNAs.

Reduced biotinidase activity is associated with a spectrum of deficiency ranging from total deficiency to heterozygous levels, a finding that is not always explained by the pathogenic variants observed in the BTD gene. The investigation of miRNAs, regulatory elements and variants in the 3’UTR region may present relevance in understanding the genotype-phenotype association. The aims of the study were to characterize the regulatory elements of the 3’UTR of the BTD gene and identify variants and miRNAs which may explain the discrepancies observed between genotype and biochemical phenotype. We evaluated 92 individuals with reduced biotinidase activity (level of heterozygotes = 33, borderline = 35, partial DB = 20 or total DB= 4) with previously determined BTD genotype. The 3’UTR of the BTD gene was Sanger sequenced. In silico analysis was performed to identify miRNAs and regulatory elements. No variants were found in the 3’UTR. We found 97 possible miRNAs associated with the BTD gene, 49 predicted miRNAs involved in the alanine, biotin, citrate and pyruvate metabolic pathways and 5 genes involved in biotin metabolism. Six AU-rich elements were found. Our data suggest variants in the 3'UTR of BTD do not explain the genotype-phenotype discrepancies found in Brazilian individuals with reduced biotinidase.

Exploring differentially expressed genes related to metabolism by RNA-Seq in porcine embryonic fibroblast after insulin treatment.

Insulin regulates glucose homeostasis and has important effects on metabolism, cell growth, and differentiation. Depending on the cell type and physiological context, insulin signal has specific pathways and biological outcomes in different tissues and cells. For studying the signal pathway of insulin on glycolipid metabolism in porcine embryonic fibroblast (PEF), we used high-throughput sequencing to monitor gene expression patterns regulated by insulin. The goal of our research was to see how insulin affected glucose and lipid metabolism in PEFs. We cultured the PEFs with the addition of insulin and sampled them at 0, 48, and 72 h for RNA-Seq analysis in triplicate for each time point. At 48 and 72 h, 801 and 1,176 genes were differentially expressed, respectively. Of these, 272 up-regulated genes and 264 down-regulated genes were common to both time points. Gene Ontology analysis was used to annotate the functions of the differentially expressed genes (DEGs), the biological processes related to lipid metabolism and cell cycle were dominant. And the DEGs were significantly enriched in interleukin-17 signaling pathway, phosphatidylinositol-3-kinase-protein kinase B signaling pathway, pyruvate metabolism, and others pathways related to lipid metabolism by Kyoto Encyclopedia of Genes and Genomes enrichment analysis. These results elucidate the transcriptomic response to insulin in PEF. The genes and pathways involved in the transcriptome mechanisms provide useful information for further research into the complicated molecular processes of insulin in PEF.

Network Pharmacology Integrated Molecular Docking to Explore the Mechanism of Blister Beetle Therapy for Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) is one of the major causes of cancer death in the world. Studies show that the effective anticancer component in blister beetles is cantharidin, which can improve chemotherapy efficacy, median survival, and prognosis of LUAD. However, the antitumor mechanism of blister beetles has not been fully clarified. This study aimed to identify the key targets of the treatment of LUAD by blister beetles based on the principle of network pharmacology. An integrated approach including network pharmacology and a molecular docking technique was conducted, which mainly comprises target prediction, weighted gene correlation network analysis (WGCNA) analysis, network construction, gene ontology, and pathway enrichment analysis. 35 key targets were obtained and significantly associated with response to external stimuli, collagen binding, cyclin binding, organic acid binding, pyruvate metabolism, glycolysis, and amino acid biosynthesis pathways. Both LASSO regression and the RF model had a high predictive ability, and 9 candidate genes were screened, among which BIRC5 and PLK1 were the key targets for the treatment of LUAD by using blister beetles and showed significant survival significance. Cantharidin exerts its antitumor effects through 8 targets in 32 pathways, while BIRC5 and PLK1 have obvious survival significance.