Identification of Novel Antimicrobial Compounds Targeting Mycobacterium tuberculosis S-Adenosyl-L-Homocysteine Hydrolase Using Dual Hierarchical In Silico Structure-Based Drug Screening.

The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis (M. tuberculosis) has become a major medical problem. S-adenosyl-L-homocysteine hydrolase (MtSAHH) was selected as the target protein for the identification of novel anti-TB drugs. Dual hierarchical in silico Structure-Based Drug Screening was performed using a 3D compound structure library (with over 150 thousand synthetic chemicals) to identify compounds that bind to MtSAHH's active site. In vitro experiments were conducted to verify whether the nine compounds selected as new drug candidates exhibited growth-inhibitory effects against mycobacteria. Eight of the nine compounds that were predicted by dual hierarchical screening showed growth-inhibitory effects against Mycobacterium smegmatis (M. smegmatis), a model organism for M. tuberculosis. Compound 7 showed the strongest antibacterial activity, with an IC50 value of 30.2 µM. Compound 7 did not inhibit the growth of Gram-negative bacteria or exert toxic effects on human cells. Molecular dynamics simulations of 40 ns using the MtSAHH-Compound 7 complex structure suggested that Compound 7 interacts stably with the MtSAHH active site. These in silico and in vitro results suggested that Compound 7 is a promising lead compound for the development of new anti-TB drugs.

Sulfur assimilation using gaseous carbonyl sulfide by the soil fungus Trichoderma harzianum.

Fungi have the capacity to assimilate a diverse range of both inorganic and organic sulfur compounds. It has been recognized that all sulfur sources taken up by fungi are in soluble forms. In this study, we present evidence that fungi can utilize gaseous carbonyl sulfide (COS) for the assimilation of a sulfur compound. We found that the filamentous fungus Trichoderma harzianum strain THIF08, which has constitutively high COS-degrading activity, was able to grow with COS as the sole sulfur source. Cultivation with 34S-labeled COS revealed that sulfur atom from COS was incorporated into intracellular metabolites such as glutathione and ergothioneine. COS degradation by strain THIF08, in which as much of the moisture derived from the agar medium as possible was removed, indicated that gaseous COS was taken up directly into the cell. Escherichia coli transformed with a COS hydrolase (COSase) gene, which is clade D of the ß-class carbonic anhydrase subfamily enzyme with high specificity for COS but low activity for CO2 hydration, showed that the COSase is involved in COS assimilation. Comparison of sulfur metabolites of strain THIF08 revealed a higher relative abundance of reduced sulfur compounds under the COS-supplemented condition than the sulfate-supplemented condition, suggesting that sulfur assimilation is more energetically efficient with COS than with sulfate because there is no redox change of sulfur. Phylogenetic analysis of the genes encoding COSase, which are distributed in a wide range of fungal taxa, suggests that the common ancestor of Ascomycota, Basidiomycota, and Mucoromycota acquired COSase at about 790-670 Ma.IMPORTANCEThe biological assimilation of gaseous CO2 and N2 involves essential processes known as carbon fixation and nitrogen fixation, respectively. In this study, we found that the fungus Trichoderma harzianum strain THIF08 can grow with gaseous carbonyl sulfide (COS), the most abundant and ubiquitous gaseous sulfur compound, as a sulfur source. When the fungus grew in these conditions, COS was assimilated into sulfur metabolites, and the key enzyme of this assimilation process is COS hydrolase (COSase), which specifically degrades COS. Moreover, the pathway was more energy efficient than the typical sulfate assimilation pathway. COSase genes are widely distributed in Ascomycota, Basidiomycota, and Mucoromycota and also occur in some Chytridiomycota, indicating that COS assimilation is widespread in fungi. Phylogenetic analysis of these genes revealed that the acquisition of COSase in filamentous fungi was estimated to have occurred at about 790-670 Ma, around the time that filamentous fungi transitioned to a terrestrial environment.

Streptococcal arginine deiminase system defences macrophage bactericidal effect mediated by XRE family protein XtrSs.

The arginine deiminase system (ADS) has been identified in various bacteria and functions to supplement energy production and enhance biological adaptability. The current understanding of the regulatory mechanism of ADS and its effect on bacterial pathogenesis is still limited. Here, we found that the XRE family transcriptional regulator XtrSs negatively affected Streptococcus suis virulence and significantly repressed ADS transcription when the bacteria were incubated in blood. Electrophoretic mobility shift (EMSA) and lacZ fusion assays further showed that XtrSs directly bind to the promoter of ArgR, an acknowledged positive regulator of bacterial ADS, to repress ArgR transcription. Moreover, we provided compelling evidence that S. suis could utilize arginine via ADS to adapt to acid stress, while ΔxtrSs enhanced this acid resistance by upregulating the ADS operon. Moreover, whole ADS-knockout S. suis increased arginine and antimicrobial NO in the infected macrophage cells, decreased intracellular survival, and even caused significant attenuation of bacterial virulence in a mouse infection model, while ΔxtrSs consistently presented the opposite results. Our experiments identified a novel ADS regulatory mechanism in S. suis, whereby XtrSs regulated ADS to modulate NO content in macrophages, promoting S. suis intracellular survival. Meanwhile, our findings provide a new perspective on how Streptococci evade the host's innate immune system.

Purification and characterization of L-arginine deiminase from Penicillium chrysogenum.

L-arginine deiminase (ADI, EC 3.5.3.6) hydrolyzes arginine to ammonia and citrulline which is a natural supplement in health care. ADI was purified from Penicillium chrysogenum using 85% ammonium sulfate, DEAE-cellulose and Sephadex G200. ADI was purified 17.2-fold and 4.6% yield with a specific activity of 50 Umg- 1 protein. The molecular weight was 49 kDa. ADI expressed maximum activity at 40oC and an optimum pH of 6.0. ADI thermostability was investigated and the values of both t0.5 and D were determined. Kd increased by temperature and the Z value was 38oC. ATP, ADP and AMP activated ADI up to 0.6 mM. Cysteine and dithiothreitol activated ADI up to 60 µmol whereas the activation by thioglycolate and reduced glutathione (GSH) prolonged to 80 µmol. EDTA, α,α-dipyridyl, and o-phenanthroline inactivated ADI indicating that ADI is a metalloenzyme. N-ethylmaleimide (NEM), N-bromosuccinimide (NBS), butanedione (BD), dansyl chloride (DC), diethylpyrocarbonate (DEPC) and N-acetyl-imidazole (NAI) inhibited ADI activity indicating the necessity of sulfhydryl, tryptophanyl, arginyl, lysyl, histidyl and tyrosyl groups, respectively for ADI catalysis. The obtained results show that ADI from P. chrysogenum could be a potential candidate for industrial and biotechnological applications.

Folic acid blocks ferroptosis induced by cerebral ischemia and reperfusion through regulating folate hydrolase transcriptional adaptive program.

Cerebral ischemia-reperfusion (I/R) injury is notably linked with folic acid (FA) deficiency. The aim of our investigation was to explore the effects and underlying mechanisms by which FA mitigates I/R, specifically through regulating the GCPII transcriptional adaptive program. Initially, we discovered that following cerebral I/R, levels of FA, methionine synthase (MTR), and methylenetetrahydrofolate reductase (MTHFR) were decreased, while GCPII expression was elevated. Secondly, administering FA could mitigate cognitive impairment and neuronal damage induced by I/R. Thirdly, the mechanism of FA supplementation involved suppressing the transcriptional factor Sp1, subsequently inhibiting GCPII transcription, reducing Glu content, obstructing cellular ferroptosis, and alleviating cerebral I/R injury. In summary, our data demonstrate that FA affords protection against cerebral I/R injury by inhibiting the GCPII transcriptional adaptive response. These findings unveil that targeting GCPII might be a viable therapeutic strategy for cerebral I/R.

Unleashing synergistic potential of microbially enhanced anaerobic co-digestion of petroleum refinery biosludge and yard waste: Impact of nutrient balance and microbial diversity.

Petroleum refinery sludge, an egregious solid residue generated from the wastewater treatment plants poses an environmental hazard owing to its intricate hydrocarbon composition, necessitating competent treatment for secure disposal. The study proposes a green solution through anaerobic co-digestion of nitrogen-rich petroleum refinery sludge (PS) with carbon-rich yard waste (YW), balancing the nutrients and moisture content for efficient microbial proliferation. Using Central Composite Design-Response Surface Methodology, 1 L batch experiments were conducted with varying carbon/nitrogen (C/N) ratios and pH to achieve maximum biogas yield within 50 days of co-digestion. However, the sluggish biogas recovery (40%) indicated a slow rate-limiting hydrolysis, necessitating pretreatment. Feedstock incubation with Bacillus subtilis IH1 strain, isolated from the microbially-enriched PS, at 108 colony forming units (CFU) per mL for 5 days maximized the soluble chemical oxygen demand and volatile fatty acids by 2.2 and 1.4 folds respectively compared to untreated feedstock. Scale-up Bacillus subtilis aided co-digestion studies further augmented biogas by 76% against untreated monodigestion of PS with significant total petroleum hydrocarbons, emulsions, and lignocellulosic degradation. Further identification of major organic pollutants in the batch digestate revealed significant degradation of the toxic organic hydrocarbon pollutants apotheosizing the efficacy of the synergistic sustainable technique for the management of PS. ENVIRONMENTAL IMPLICATION: The effluent treatment plants (ETPs) of petroleum refining industries generate sludge which is a complex mixture of petroleum hydrocarbons, oil-water (O/W) emulsions and heavy metals. These petroleum hydrocarbon constituents can be linear/cyclic alkanes, polyaromatics, resins and asphaltenes, whose intricate composition is reportedly carcinogenic, cytogenic and mutagenic, classifying it as hazardous waste. Biological treatment of these sludge through anaerobic digestion leads to utilization of petroleum hydrocarbons with subsequent energy recovery. Co-digestion of these sludge with competent co-substrates leads to nutrient balance, diverse microbial proliferation and toxicant dilution. Microbially aided co-digestion further augments methane rendering a digestate with utmost pollutant degradation.

Nanomicelle curcumin-induced testicular toxicity: Implications for altered mitochondrial biogenesis and mitophagy following redox imbalance.

The purpose of this study was to examine the effects of nano-micelle curcumin (NMC)-induced redox imbalance on mitochondrial biogenesis and mitophagy. For this purpose, 24 mature male Wistar rats were divided into control and NMC-received groups (7.5, 15, and 30 mg/kg) groups. After 48 days, the Nrf1, Nrf2, and SOD (Cu/Zn) expression levels, as well as GSH/GSSG, NADP+ /NADPH relative balances (elements involved in redox homeostasis) were analyzed. Moreover, to explore the effect of NMC on mitochondrial biogenesis, the expression levels of Mfn1, Mfn2, OPA1, Fis1, and Drp1 were investigated. Finally, the expression levels of Parkin/PARK and PINK (genes involved in mitochondrial quality control), as well as LC3-I/II (mitophagy marker), were analyzed. Observations showed that NMC, dose-dependently, altered GSH/GSSG, NADP+ /NADPH relative balances, suppressed SOD expression and diminished its biochemical level, and repressed Nrf1 and Nrf2 expression levels. Moreover, it could change the Mfn1, Mfn2, OPA1, Fis1, and Drp1 expression pattern and stimulate the Parkin/PARK and PINK as well as LC3-I/II expression levels, dose-dependently. In conclusion, chronic and high-dose NMC is able to suppress the redox capacity by down-regulating the Nrf1 and Nrf2 expression. Finally, at high-dose levels, it is able to trigger mitophagy signaling in the testicles.

In Silico Discovery of Potential Inhibitors Targeting the RNA Binding Loop of ADAR2 and 5-HT2CR from Traditional Chinese Natural Compounds.

Adenosine deaminase acting on RNA 2 (ADAR2) is an important enzyme involved in RNA editing processes, particularly in the conversion of adenosine to inosine in RNA molecules. Dysregulation of ADAR2 activity has been implicated in various diseases, including neurological disorders (including schizophrenia), inflammatory disorders, viral infections, and cancers. Therefore, targeting ADAR2 with small molecules presents a promising therapeutic strategy for modulating RNA editing and potentially treating associated pathologies. However, there are limited compounds that effectively inhibit ADAR2 reactions. This study therefore employed computational approaches to virtually screen natural compounds from the traditional Chinese medicine (TCM) library. The shortlisted compounds demonstrated a stronger binding affinity to the ADAR2 (<-9.5 kcal/mol) than the known inhibitor, 8-azanebularine (-6.8 kcal/mol). The topmost compounds were also observed to possess high binding affinity towards 5-HT2CR with binding energies ranging from -7.8 to -12.9 kcal/mol. Further subjecting the top ADAR2-ligand complexes to molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations revealed that five potential hit compounds comprising ZINC000014637370, ZINC000085593577, ZINC000042890265, ZINC000039183320, and ZINC000101100339 had favorable binding free energies of -174.911, -137.369, -117.236, -67.023, and -64.913 kJ/mol, respectively, with the human ADAR2 protein. Residues Lys350, Cys377, Glu396, Cys451, Arg455, Ser486, Gln488, and Arg510 were also predicted to be crucial in ligand recognition and binding. This finding will provide valuable insights into the molecular interactions between ADAR2 and small molecules, aiding in the design of future ADAR2 inhibitors with potential therapeutic applications. The potential lead compounds were also profiled to have insignificant toxicities. A structural similarity search via DrugBank revealed that ZINC000039183320 and ZINC000014637370 were similar to naringin and naringenin, which are known adenosine deaminase (ADA) inhibitors. These potential novel ADAR2 inhibitors identified herein may be beneficial in treating several neurological disorders, cancers, viral infections, and inflammatory disorders caused by ADAR2 after experimental validation.

Plant Polyphenols as Inhibitors of Hydrolases are Regulators of Digestion.

BACKGROUND: There are numerous reports on the use of polyphenol-containing foods and various medicinal plant preparations for the prophylaxis and therapy of metabolic diseases, such as metabolic syndrome and diabetes mellitus, respectively. One unifying aspect to the effect of these natural compounds is their ability to inhibit digestive enzymes, which is the focus of this review. SUMMARY: Polyphenols inhibit nonspecifically hydrolytic enzymes included in the digestion process, e.g., amylases, proteases, lipases. By that, the digestion process is protracted with different consequences as result of the incomplete absorption of monosaccharides, fatty acids, and amino acids as well as for the enhanced availability of substrates for the microbiome in ileum and colon. The resulting postprandial blood concentration of monosaccharides, fatty, and amino acids is lowered and by that different metabolic pathways proceed more slowly. As another positive result, polyphenols can also modulate the intestinal microbiome and thus mediate additional beneficial health effects. KEY MESSAGES: Many medicinal plants possess a broad spectrum of different polyphenols, thereby mediating the nonspecific inhibition of all hydrolytic enzyme activities in the gastrointestinal digestive process. As a consequence of the slowing down of digestive processes, risk factors for the development of metabolic disorders are reduced and the health of the patients with metabolic syndrome improves.Es gibt zahlreiche Berichte über die Verwendung von polyphenolhaltigen Lebensmitteln und verschiedenen Arzneilpflanzenpräparaten zur Prophylaxe und Therapie von Stoffwechselkrankheiten wie dem metabolischen Syndrom und Diabetes mellitus. Ein übergreifender Aspekt der Wirkung dieser Naturstoffe ist ihre Fähigkeit, Verdauungsenzyme zu hemmen, was im Mittelpunkt dieser Übersicht steht.Polyphenole hemmen unspezifisch hydrolytische Enzyme, die am Verdauungsprozess beteiligt sind, z.B. Amylasen, Proteasen, Lipasen. Dadurch wird der Verdauungsprozess verzögert, was sich in einer unvollständigen Resorption von Monosacchariden, Fettsäuren und Aminosäuren sowie in einer erhöhten Verfügbarkeit von Substraten für das Mikrobiom im Ileum und Kolon äußert. Dadurch wird die postprandiale Blutkonzentration von Monosacchariden, Fettsäuren und Aminosäuren gesenkt und verschiedene Stoffwechselwege laufen langsamer ab. Ein weiteres positives Ergebnis ist, dass Polyphenole auch das intestinale Mikrobiom modulieren können und damit zusätzliche positive Gesundheitseffekte vermitteln.Viele Arzneipflanzen verfügen über ein breites Spektrum verschiedener Polyphenole, die eine unspezifische Hemmung aller hydrolytischen Enzymaktivitäten im gastrointestinalen Verdauungsprozess bewirken. Durch die Verlangsamung der Verdauungsprozesse werden Risikofaktoren für die Entwicklung von Stoffwechselstörungen reduziert und der Gesundheitszustand von Patienten mit metabolischem Syndrom verbessert.

Arginine deiminase produced by lactic acid bacteria as a potent anti-cancer drug.

Bacterial-based cancer immunotherapy has recently gained widespread attention due to its exceptional mechanism of rich pathogen-associated molecular patterns in anti-cancer immune responses. Contrary to conventional cancer therapies such as surgery, chemotherapy, radiation and phototherapy, bacteria-based cancer immunotherapy has the unique ability to suppress cancer by selectively accumulating and growing in tumours. In the view of this, several bacterial strains are being used for the treatment of cancer. Of which, lactic acid bacteria are a powerful, albeit still inadequately understood bacteria that possess a wide source of bioactive chemicals. Lactic acid bacteria metabolites, such as bacteriocins, short-chain fatty acids, exopolysaccharides show antitumour property. Amino acid pathways, which have lately been focussed as a new strategy to cancer therapy, are key element of the adaptability and dysregulation of metabolic pathways identified in proliferation of tumour cells. Arginine metabolism, in particular, has been shown to be critical for cancer therapy. As a result, better understanding of arginine metabolism in LAB and cancer cells could lead to new cancer therapeutic targets. This review will outline current advances in the interaction of arginine metabolism with cancer therapy and propose an arginine deiminase expression system to combat cancer more effectively.

Anaerobic purinolytic enzymes enable dietary purine clearance by engineered gut bacteria.

Uric acid, the end product of purine degradation, causes hyperuricemia and gout, afflicting hundreds of millions of people. The debilitating effects of gout are exacerbated by dietary purine intake, and thus a potential therapeutic strategy is to enhance purine degradation in the gut microbiome. Aerobic purine degradation involves oxidative dearomatization of uric acid catalyzed by the O2-dependent uricase. The enzymes involved in purine degradation in strictly anaerobic bacteria remain unknown. Here we report the identification and characterization of these enzymes, which include four hydrolases belonging to different enzyme families, and a prenyl-flavin mononucleotide-dependent decarboxylase. Introduction of the first two hydrolases to Escherichia coli Nissle 1917 enabled its anaerobic growth on xanthine as the sole nitrogen source. Oral supplementation of these engineered probiotics ameliorated hyperuricemia in a Drosophila melanogaster model, including the formation of renal uric acid stones and a shortened lifespan, providing a route toward the development of purinolytic probiotics.

Dietary factors potentially impacting thiaminase I-mediated thiamine deficiency.

Fish population declines from thiamine (vitamin B1) deficiency have been widespread in ecologically and economically valuable organisms, ranging from the Great Lakes to the Baltic Sea and, most recently, the California coast. Thiamine deficiencies in predatory fishes are often attributed to a diet of prey fishes with high levels of thiamine-degrading (e.g., thiaminase) enzymes, such as alewives, rainbow smelt, and anchovies. Since their discovery, thiaminase I enzymes have been recognized for breaking down thiamine into its pyrimidine and thiazole moieties using various nucleophilic co-substrates to afford cleavage, but these studies have not thoroughly considered other factors that could modify enzyme activity. We found the thiaminase I enzyme from Clostridium botulinum efficiently degrades thiamine in the presence of pyridoxine (vitamin B6) as a co-substrate but has relatively limited activity in the presence of nicotinic acid (vitamin B3). Using fluorescence measurements, thiamine degradation in an over-the-counter complete multivitamin formulation was inhibited, and a B-complex formulation required co-substrate supplementation for maximal thiamine depletion. These studies prompted the evaluation of specific constituents contributing to thiaminase I inhibition by both chromatography and fluorescence assays: Cu2+ potently and irreversibly inhibited thiamine degradation; ascorbic acid was a strong but reversible inhibitor; Fe2+, Mn2+ and Fe3+ modulated thiamine degradation to a lesser degree. The enhancement by pyridoxine and inhibition by Cu2+ extended to thiaminase-mediated degradation from Burkholderia thailandensis, Paenibacillus thiaminolyticus, and Paenibacillus apiarius in tryptic soy broth supernatants. These co-substrate limitations and the common presence of inhibitory dietary factors complement recent studies reporting that the intended function of thiaminase enzymes is to recycle thiamine breakdown products for thiamine synthesis, not thiamine degradation.

Reconnoitring the antioxidant and antibacterial potential of different fruits after tannin acyl hydrolase mediated biotransformation.

Condensed and hydrolyzable tannins are secondary metabolites present in almost every plant part. Tannase enzyme acts on hydrolyzable tannins to produce gallic acid and tannase-mediated end-products with immense therapeutic potential. Seven different fruits with significant presence of hydrolyzable tannin content were selected to check for phenol, tannin, and hydrolyzable tannin contents. Prunus domestica had the maximum phenol content, that is, 85.4 ± 0.207, followed by Syzygium cumini, Fragaria ananassa, Rubus fruticosus, and Psidium guajava. Plum showed the maximum number of hydrolyzable tannins. Fruit extracts were subjected to tannase hydrolysis and their antimicrobial and antioxidant activities were determined. There was a significant increase in the antioxidant abilities of the fruits with Punica granatum extract, displaying the highest decline of 132 units of IC50 followed by F. ananassa hydrolyzable extract, showing a decrease from 224.75 to 119.98 µg/mL. The extracts also depicted a significant increase in antibacterial activity after hydrolysis against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus with Rubus idaeus aqueous extract observed to be most effective against E. coli. The increase in antioxidant and antibacterial activity can be attributed to the production of tannase-mediated products formed after the biotransformation of hydrolyzable tannins present in the aqueous extracts.

Proteomic Analysis Reveals a Critical Role of the Glycosyl Hydrolase 17 Protein in Panax ginseng Leaves under Salt Stress.

Ginseng, an important crop in East Asia, exhibits multiple medicinal and nutritional benefits because of the presence of ginsenosides. On the other hand, the ginseng yield is severely affected by abiotic stressors, particularly salinity, which reduces yield and quality. Therefore, efforts are needed to improve the ginseng yield during salinity stress, but salinity stress-induced changes in ginseng are poorly understood, particularly at the proteome-wide level. In this study, we report the comparative proteome profiles of ginseng leaves at four different time points (mock, 24, 72, and 96 h) using a label-free quantitative proteome approach. Of the 2484 proteins identified, 468 were salt-responsive. In particular, glycosyl hydrolase 17 (PgGH17), catalase-peroxidase 2, voltage-gated potassium channel subunit beta-2, fructose-1,6-bisphosphatase class 1, and chlorophyll a-b binding protein accumulated in ginseng leaves in response to salt stress. The heterologous expression of PgGH17 in Arabidopsis thaliana improved the salt tolerance of transgenic lines without compromising plant growth. Overall, this study uncovers the salt-induced changes in ginseng leaves at the proteome level and highlights the critical role of PgGH17 in salt stress tolerance in ginseng.

γ-Glutamyl-transpeptidase CsGGT2 functions as light-activated theanine hydrolase in tea plant (Camellia sinensis L.).

Theanine is an important secondary metabolite endowing tea with umami taste and health effects. It is essential to explore the metabolic pathway and regulatory mechanism of theanine to improve tea quality. Here, we demonstrated that the expression patterns of CsGGT2 (γ-glutamyl-transpeptidase), participated in theanine synthesis in vitro in our previous research, are significantly different in the aboveground and underground tissues of tea plants and regulated by light. Light up-regulated the expression of CsHY5, directly binding to the promoter of CsGGT2 and acting as an activator of CsGGT2, with a negative correlation with theanine accumulation. The enzyme activity assays and transient expression in Nicotiana benthamiana showed that CsGGT2, acting as bifunctional protein, synthesize and degrade theanine in vitro and in planta. The results of enzyme kinetics, Surface plasmon resonance (SPR) assays and targeted gene-silencing assays showed that CsGGT2 had a higher substrate affinity of theanine than that of ethylamine, and performed a higher theanine degradation catalytic efficiency. Therefore, light mediates the degradation of theanine in different tissues by regulating the expression of the theanine hydrolase CsGGT2 in tea plants, and these results provide new insights into the degradation of theanine mediated by light in tea plants.

Valorization of leftover green tea residues through conversion to bioactive peptides using probiotics-aided anaerobic digestion.

Bioactive peptides (BPs) are protein fragments that benefit human health. To assess whether leftover green tea residues (GTRs) can serve as a resource for new BPs, we performed in silico proteolysis of GTRs using the BIOPEP database, revealing a wide range of BPs embedded in GTRs. Comparative genomics and the percentage of conserved protein analyses enabled us to select a few probiotic strains for GTR hydrolysis. The selected probiotics digested GTRs anaerobically to yield GTR-derived peptide fractions. To examine whether green tea (GT) peptide fractions could be potential mediators of host-microbe interactions, we comprehensively screened agonistic and antagonistic activities of 168 human G protein-coupled receptors (GPCRs). NanoLC-MS/MS analysis and thin-layer chromatography allowed the identification of peptide sequences and the composition of glycan moieties in the GTRs. Remarkably, GT peptide fractions produced by Lactiplantibacillus plantarum APsulloc 331261, a strain isolated from GT, showed a potent-binding activity for P2RY6, a GPCR involved in intestinal homeostasis. Therefore, this study suggests the potential use of probiotics-aided GTR hydrolysates as postbiotic BPs, providing a biological process for recycling GTRs from agro-waste into renewable resources as health-promoting BPs.

Changes in Soil Organic C Fractions and C Pool Stability Are Mediated by C-Degrading Enzymes in Litter Decomposition of Robinia pseudoacacia Plantations.

Litter decomposition is the main source of soil organic carbon (SOC) pool, regarding as an important part of terrestrial ecosystem C dynamics. The turnover of SOC is mainly regulated by extracellular enzymes secreted by microorganisms. However, the response mechanism of soil C-degrading enzymes and SOC in litter decomposition remains unclear. To clarify how SOC fraction dynamics respond to C-degrading enzymes in litter decomposition, we used field experiments to collect leaf litter and SOC fractions from the underlying layer in Robinia pseudoacacia plantations on the Loess Plateau. Our results showed that SOC, easily oxidizable organic C, dissolved organic C, and microbial biomass C increased significantly during the decomposition process. Litter decomposition significantly decreased soil hydrolase activity, but slightly increased oxidase activity. Correlation analysis results showed that SOC fractions were significantly positively correlated with the litter mass, lignin, soil moisture, and oxidase activity, but significantly negatively correlated with cellulose content and soil pH. Partial least squares path models revealed that soil C-degrading enzymes can directly or indirectly affect the changes of soil C fractions. The most direct factors affecting the SOC fractions of topsoil during litter decomposition were litter lignin and cellulose degradation, soil pH, and C-degrading enzymes. Furthermore, regression analysis showed that the decrease of SOC stability in litter decomposition was closely related to the decrease of soil hydrolase to oxidase ratio. These results highlighted that litter degradation-induced changes in C-degrading enzyme activity significantly affected SOC fractions. Furthermore, the distribution of soil hydrolases and oxidases affected the stability of SOC during litter decomposition. These findings provided a theoretical framework for a more comprehensive understanding of C turnover and stabilization mechanisms between plant and soil.

Metal Ions and Chemical Modification Reagents Inhibit the Enzymatic Activity of Lecithin-Dependent Hemolysin from Vibrio parahaemolyticus.

Lecithin-dependent thermolabile hemolysin (LDH) is a virulence factor excreted by Vibrio parahaemolyticus, a marine bacterium that causes important losses in shrimp farming. In this study, the function of LDH was investigated through its inhibition by metal ions (Mg2+, Ca2+, Mn2+, Co2+, Ni2+ and Cu2+) and chemical modification reagents: ß-mercaptoethanol (ßME), phenylmethylsulfonyl fluoride (PMSF) and diethyl pyrocarbonate (DEPC). LDH was expressed in the Escherichia coli strain BL-21, purified under denaturing conditions, and the enzymatic activity was evaluated. Cu2+, Ni2+, Co2+ and Ca2+ at 1 mmol/L inhibited the LDH esterase activity by 20−95%, while Mg2+ and Mn2+ slightly increased its activity. Additionally, PMSF and DEPC at 1 mmol/L inhibited the enzymatic activity by 40% and 80%, respectively. Dose-response analysis showed that DEPC was the best-evaluated inhibitor (IC50 = 0.082 mmol/L), followed by Cu2+ > Co2+ > Ni2+ and PMSF (IC50 = 0.146−1.5 mmol/L). Multiple sequence alignment of LDH of V. parahaemolyticus against other Vibrio species showed that LDH has well-conserved GDSL and SGNH motifs, characteristic of the hydrolase/esterase superfamily. Additionally, the homology model showed that the conserved catalytic triad His-Ser-Asp was in the LDH active site. Our results showed that the enzymatic activity of LDH from V. parahaemolyticus was modulated by metal ions and chemical modification, which could be related to the interaction with catalytic amino acid residues such as Ser153 and/or His 393.

Citrullination is linked to reduced Ca2+ sensitivity in hearts of a murine model of rheumatoid arthritis.

AIMS: Cardiac contractile dysfunction is prevalent in rheumatoid arthritis (RA), with an increased risk for heart failure. A hallmark of RA has increased levels of peptidyl arginine deaminases (PAD) that convert arginine to citrulline leading to ubiquitous citrullination, including in the heart. We aimed to investigate whether PAD-dependent citrullination in the heart was linked to contractile function in a mouse model of RA during the acute inflammatory phase. METHODS: We used hearts from the collagen-induced arthritis (CIA) mice, with overt arthritis, and control mice to analyze cardiomyocyte Ca2+ handling and fractional shortening, the force-Ca2+ relationship in isolated myofibrils, the levels of PAD, protein post-translational modifications, and Ca2+ handling protein. Then, we used an in vitro model to investigate the role of TNF-α in the PAD-mediated citrullination of proteins in cardiomyocytes. RESULTS: Cardiomyocytes from CIA mice displayed larger Ca2+ transients than controls, whereas cell shortening was similar in the two groups. Myofibrils from CIA hearts required higher [Ca2+ ] to reach 50% of maximum shortening, ie Ca2+ sensitivity was lower. This was associated with increased PAD2 expression and α-actin citrullination. TNF-α increased PAD-mediated citrullination which was blocked by pre-treatment with the PAD inhibitor 2-chloroacetamide. CONCLUSION: Using a mouse RA model we found evidence of impaired cardiac contractile function linked to reduced Ca2+ sensitivity, increased expression of PAD2, and citrullination of α-actin, which was triggered by TNF-α. This provides molecular and physiological evidence for acquired cardiomyopathy and a potential mechanism for RA-associated heart failure.

The potential role of betaine in enhancement of microbial-assisted phytoremediation of benzophenone-3 contaminated soil.

Benzophenone-3 (BP-3) is an emerging environmental pollutant used in personal care products, helping to reduce the risk of ultraviolet radiation to human skin. The BP-3 removal potential from soil by tobacco (Nicotiana tabacum) assisted with Methylophilus sp. FP-6 was explored in our previous study. However, the reduced BP-3 remediation efficiency by FP-6 in soil and the inhibited plant growth by BP-3 limited the application of this phytoremediation strategy. The aim of the present study was to reveal the potential roles of betaine, as the methyl donor of methylotrophic bacteria and plant regulator, in improving the strain FP-6-assisted phytoremediation capacity of BP-3 contaminated soil. The results revealed that strain FP-6 could use betaine as a co-metabolism substrate to enhance the BP-3 degradation activity. About 97.32% BP-3 in soil was effectively removed in the phytoremediation system using tobacco in combination with FP-6 and betaine for 40 d while the concentration of BP-3 in tobacco significantly reduced. Moreover, the biomass and photosynthetic efficiency of plants were remarkably improved through the combined treatment of betaine and strain FP-6. Simultaneously, inoculation of FP-6 in the presence of betaine stimulated the change of local microbial community structure, which might correlate with the production of a series of hydrolases and reductases involved in soil carbon, nitrogen and phosphorus cycling processes. Meantime, some of the dominant bacteria could secrete various multiple enzymes involved in degrading organic pollutants, such as laccase, to accelerate the demethylation and hydroxylation of BP-3. Overall, the results from this study proposed that the co-metabolic role of betaine could be utilized to strengthen microbial-assisted phytoremediation process by increasing the degradation ability of methylotrophic bacteria and enhancing plant tolerance to BP-3. The present results provide novel insights and perspectives for broadening the engineering application scope of microbial-assisted phytoremediation of organic pollutants without sacrificing economic crop safety.