Steroidogenic Effects of Salinity Change on the Hypothalamus-Pituitary-Gonad (HPG) Axis of Male Chinese Sea Bass (Lateolabrax maculatus).

As lower vertebrates, teleost species could be affected by dynamic aquatic environments and may respond to environmental changes through the hypothalamus-pituitary-gonad (HPG) axis to ensure their normal growth and sexual development. Chinese sea bass (Lateolabrax maculatus), euryhaline marine teleosts, have an extraordinary ability to deal with a wide range of salinity changes, whereas the salinity decrease during their sex-maturation season may interfere with the HPG axis and affect their steroid hormone metabolism, resulting in abnormal reproductive functioning. To this end, in this study, 40 HPG axis genes in the L. maculatus genome were systematically characterized and their copy numbers, phylogenies, gene structures, and expression patterns were investigated, revealing the conservation of the HPG axis among teleost lineages. In addition, freshwater acclimation was carried out with maturing male L. maculatus, and their serum cortisol and 11-ketotestosterone (11-KT) levels were both increased significantly after the salinity change, while their testes were found to be partially degraded. After salinity reduction, the expression of genes involved in cortisol and 11-KT synthesis (cyp17a, hsd3b1, cyp21a, cyp11c, hsd11b2, and hsd17b3) showed generally upregulated expression in the head kidneys and testes, respectively. Moreover, cyp11c and hsd11b2 were involved in the synthesis and metabolism of both cortisol and 11-KT, and after salinity change their putative interaction may contribute to steroid hormone homeostasis. Our results proved the effects of salinity change on the HPG axis and steroidogenic pathway in L. maculatus and revealed the gene interactions involved in the regulation of steroid hormone levels. The coordinated interaction of steroidogenic genes provides comprehensive insights into steroidogenic pathway regulation, as well as sexual development, in teleost species.

Characterization of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification.

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

Anti-oxidative and anti-hyperglycemic properties of Agastache foeniculum essential oil and oily fraction in hyperglycemia-stimulated and lipopolysaccharide-stimulated macrophage cells: In vitro and in silico studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Hyperglycemia (HG) and lipopolysaccharide (LPS) often promote superoxide accumulation, which may increase oxidative stress. Reducing superoxide production in hyperglycemia and the inflammatory condition is an emerging way to reduce protein and lipid oxidation and diabetes complication. AIM OF STUDY: To examine the effect of Agastache foeniculum essential oil (AFEO) and oil fraction (AFoil) on HG- and LPS-stimulated oxidative stress, the pathogenicity of AFEO and AFoil on oxidative stress was assessed. METHODS: The stimulatory effects of AFEO and AFoil on the activity and expression of NADH oxide (NOX), catalase (CAT), superoxide dismutase (SOD), and the expression of nuclear respiratory factor 2 (NRF2) and nuclear factor-kappa B (NF-kB) in the stimulated macrophage cell line, J774.A1, was studied. The interaction patterns of AFEO and AFoil components with NOX, SOD, CAT, NRF2, and NF-kB proteins were also deduced using molecular docking. RESULTS: Estragole was the main ingredient in AFEO (97%). Linolenic acid (32.10%), estragole (16.22%), palmitic acid (12.62%), linoleic acid (12.04%), and oleic acid (8.73%) were the major chemical components of the AFoil. NOX activation was stimulated in macrophage cells by HG and LPS. At 20 µg/mL, AFEO and AFoil decreased NOX activity while increased SOD and CAT activities in stimulated macrophages. AFoil with estragole and omega-3 fatty acids was better than AFEO with estragole in anti-hyperglycemic and anti-oxidative activity. According to molecular docking research, estragole, linoleic acid, and linolenic acid bind to different hydrophobic pockets of NOX, SOD, CAT, NFR2, and NF-kB using hydrogen bonds, van der Waals bonds, pi-alkyl, and pi-anion interactions, with different binding energies. CONCLUSION: AFEO and AFoil showed antioxidant and anti-diabetic activity. The mechanisms in lowering oxidative stress markers depended on down-regulating superoxide-producing enzymes and up-regulating superoxide-removing enzymes at gene and protein levels. The AFoil emulsion can be used to reduce the detrimental impacts of hyperglycemia and oxidative stress.

Efficient Production of 2'-Fucosyllactose from l-Fucose via Self-Assembling Multienzyme Complexes in Engineered Escherichia coli.

2'-Fucosyllactose (2'-FL) has been widely used as a nutritional additive in infant formula due to its multifarious nutraceutical and pharmaceutical functions in neonate health. As such, it is essential to develop an efficient and extensive microbial fermentation platform to cater to the needs of the 2'-FL market. In this study, a spatial synthetic biology strategy was employed to promote 2'-FL biosynthesis in recombinant Escherichia coli. First, the salvage pathway for 2'-FL production from l-fucose and lactose was constructed by introducing a bifunctional enzyme l-fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) derived from Bacteroides fragilis and an α-1,2-fucosyltransferase (FutC) derived from Helicobacter pylori into engineered E. coli BL21(DE3). Next, the endogenous genes involved in the degradation and shunting of the substrate and key intermediate were inactivated to improve the availability of precursors for 2'-FL biosynthesis. Moreover, to further improve the yield and titer of 2'-FL, a short peptide pair (RIAD-RIDD) was used to form self-assembling multienzyme complexes in vivo. The spatial localization of peptides and stoichiometry of enzyme assemblies were subsequently optimized to further improve 2'-FL production. Finally, cofactor regeneration was also considered to alleviate the potential cofactor deficiency and redox flux imbalance in the biocatalysis process. Fed-batch fermentation of the final WLS20 strain accumulated 30.5 g/L extracellular 2'-FL with the yield and productivity of 0.661 mol/mol fucose and 0.48 g/L/h, respectively. This research has demonstrated that the application of spatial synthetic biology and metabolic engineering strategies can dramatically enlarge the titer and yield of 2'-FL biosynthesis in engineered E. coli.

Cytotoxic effect of xyloglucan and oxovanadium (IV/V) xyloglucan complex in HepG2 cells.

It is well known that the chemical structure of polysaccharides is important to their final biological effect. In this study we investigated the cytotoxic effect of xyloglucan from Copaifera langsdorffii seeds (XGC) and its complex with oxovanadium (XGC:VO) on hepatocellular carcinoma cells (HepG2). After 72 h of incubation, XGC and XGC:VO (200 µg/mL) reduced cell viability in ~20% and ~40%, respectively. At same conditions, only XGC:VO increased in ~20% the LDH enzyme release. In permeabilized cells, incubated with XGC and XGC:VO (200 µg/mL) for 72 h, NADH oxidase activity was reduced by ~45% with XGC and XGC:VO. The succinate oxidase activity was reduced by ~35% with XGC and ~65% with XGC:VO, evidencing that polysaccharide complexation with vanadium could intensify its effects on the respiratory chain. According to this result, the mitochondrial membrane potential was also reduced by ~9% for XGC and ~30% for XGC:VO, when compared to the control group. Interestingly, ATP levels were more elevated for XGC:VO in respect to XGC, probably due the enhance in glycolytic flux evidenced by increased levels of lactate. These results show that the xyloglucan complexation with oxovanadium (IV/V) potentiates the cytotoxic effect of the native polysaccharide, possibly by impairment of oxidative phosphorylation.

Progesterone dose during synchronization treatment alters luteinizing hormone receptor and steroidogenic enzyme mRNA abundances in granulosa cells of Nellore heifers.

The objective was to investigate effects of progesterone (P4) dose on abundance of luteinizing hormone receptor (LHCGR), aromatase (CYP19A1), 3ß-hydroxysteroid dehydrogenase (HSD3B1), and other steroidogenic mRNA transcripts in granulosa cells from dominant follicles. Nellore heifers were assigned to one of six groups: new, first-use controlled internal drug release device (CIDR1) inserted for 5 days (Large-P4-dose-D5; n = 7) or 6 days (Large-P4-dose-D6; n = 8), prostaglandin (PG)F2α administered on D0 and 1 previously-used CIDR (CIDR3) inserted for 5 days (Small- P4-dose-D5; n = 8) or 6 days (Small-P4-dose-D6; n = 8), CIDR1 inserted on D0 and removed plus PGF2α on D5 (Large-P4-dose-proestrus (PE); n = 7), and CIDR3 and PGF2α on D0 and 1, CIDR3 removed plus PGF2α on D5 (Small-P4-dose-PE; n = 7). Duration of P4 treatment (D5 compared to D6) affected abundances of CYP19A1 mRNA transcripts, with there being greater abundances on D6 than D5 (P ≤ 0.05). Heifers treated with the large dose of P4 had a smaller dominant follicle, less serum and intra-follicular estradiol (E2) concentrations (P ≤ 0.05) and lesser LHCGR, CYP19A1, and HSD3B1 transcript abundances (P ≤ 0.05). Heifers treated to induce PE had a larger follicle diameter (P = 0.09), greater intra-follicular E2 concentrations and larger abundances of CYP19A1 mRNA transcript (P ≤ 0.05) than heifers of the D6 group. Overall, treatment with larger doses of P4 resulted in lesser abundances of LHCGR, HSD3B1, and CYP19A1 mRNA transcripts; thus, potentially leading to development of smaller dominant follicles and lesser E2 concentrations.

Renoprotective effects of enzyme-hydrolyzed polysaccharides from Auricularia polytricha on adenine-induced chronic kidney diseases in mice.

The present work was aimed to investigate the protective effects of enzymatic-hydrolyzed Auricularia polytricha polysaccharides (EnAPS) on renal functions. The characterizations were analyzed by physicochemical methods, and the renoprotections were processed in adenine-induced chronic kidney diseases (CKD) models of mice. Animal experiments exhibited that EnAPS showed superior renal-protections contributing to its antioxidant effects of increasing the enzyme activities and decreasing the lipid contents, and anti-inflammatory effects of reducing proinflammatory cytokines than A. polytricha polysaccharides (APS). Besides, the anti-apoptosis effects of EnAPS was proved by down-regulating Bax and Caspase-3 expressions and up-regulating Bcl-2 expressions by molecular biotechnology, and the anti-fibrosis effects was confirmed by histopathological observations of staining. The characterizations indicated that lower molecular weights possibly contributed to the superior renoprotective effects. These results suggested that enzymatic hydrolysis had potential effects in enhancing the bioactivities, and the polysaccharides could be used in the development of functional foods supplement against CKD.

Initial in vivo testing of TPO-receptor agonist eltrombopag in osteosarcoma patient-derived xenograft models by the pediatric preclinical testing consortium.

Eltrombopag is a small molecule, thrombopoietin receptor agonist approved for the treatment of patients with aplastic anemia and chronic immune thrombocytopenia. It is also a polyvalent cation chelator and inhibits leukemia cell proliferation via reduction of intracellular iron. The in vivo efficacy of eltrombopag was tested against a panel of six Pediatric Preclinical Testing Consortium osteosarcoma xenografts at doses of 5 mg/kg/day (moderate dose) and 50 mg/kg/day (high dose). Eltrombopag, at moderate doses, failed to significantly improve event-free survival (EFS) in 6/6 models. At high doses, eltrombopag significantly prolonged EFS in 2/2 models, though the effect size was small. All models tested demonstrated progressive disease. While eltrombopag did not meaningfully inhibit osteosarcoma growth, it also did not stimulate tumor growth, suggesting it may be safely investigated as a supportive care agent to enhance platelet recovery post chemotherapy.

Protocatechualdehyde protects oxygen-glucose deprivation/reoxygenation-induced myocardial injury via inhibiting PERK/ATF6α/IRE1α pathway.

Endoplasmic reticulum (ER) stress has been considered as a promising strategy in developing novel therapeutic agents for cardiovascular diseases through inhibiting cardiomyocyte apoptosis. Protocatechualdehyde (PCA) is a natural phenolic compound from medicinal plant Salvia miltiorrhiza with cardiomyocyte protection. However, the potential mechanism of PCA on cardiovascular ischemic injury is largely unexplored. Here, we found that PCA exerted markedly anti-apoptotic effect in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced H9c2 cells (Rat embryonic ventricular H9c2 cardiomyocytes), which was detected by 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH), Hoechst 33258 and acridine orange/ethidium bromide (AO/EB) assays. PCA also obviously protected cardiomyocytes in myocardial fibrosis model of mice, which was determined by hematoxylin-eosin (HE) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining. Transcriptomics coupled with bioinformatics analysis revealed a complex pharmacological signaling network especially for PCA-mediated ER stress on cardiomyocytes. Further mechanism study suggested that PCA suppressed ER stress via inhibiting protein kinase R-like ER kinase (PERK), inositol-requiring enzyme1α (IRE1α), and transcription factor 6α (ATF6α) signaling pathway through Western blot, DIOC6 and ER-Tracker Red staining, leading to a protective effect against ER stress-mediated cardiomyocyte apoptosis. Taken together, our observations suggest that PCA is a major component from Salvia miltiorrhiza against cardiovascular ischemic injury by suppressing ER stress-associated PERK, IRE1α and ATF6α signaling pathways.

Roles of the EnvZ/OmpR Two-Component System and Porins in Iron Acquisition in Escherichia coli.

Escherichia coli secretes high-affinity Fe3+ chelators to solubilize and transport chelated Fe3+ via specific outer membrane receptors. In microaerobic and anaerobic growth environments, where the reduced Fe2+ form is predominant, ferrous transport systems fulfill the bacterial need for iron. Expression of genes coding for iron metabolism is controlled by Fur, which when bound to Fe2+ acts as a repressor. Work carried out here shows that the constitutively activated EnvZ/OmpR two-component system, which normally controls expression of the ompC and ompF porin genes, dramatically increases the intracellular pool of accessible iron, as determined by whole-cell electron paramagnetic resonance spectroscopy, by inducing the OmpC/FeoB-mediated ferrous transport pathway. Elevated levels of intracellular iron in turn activated Fur, which inhibited the ferric transport pathway but not the ferrous transport pathway. The data show that the positive effect of constitutively activated EnvZ/OmpR on feoB expression is sufficient to overcome the negative effect of activated Fur on feoB In a tonB mutant, which lacks functional ferric transport systems, deletion of ompR severely impairs growth on rich medium not supplemented with iron, while the simultaneous deletion of ompC and ompF is not viable. These data, together with the observation of derepression of the Fur regulon in an OmpC mutant, show that the porins play an important role in iron homeostasis. The work presented here also resolves a long-standing paradoxical observation of the effect of certain mutant envZ alleles on iron regulon.IMPORTANCE The work presented here solved a long-standing paradox of the negative effects of certain missense alleles of envZ, which codes for kinase of the EnvZ/OmpR two-component system, on the expression of ferric uptake genes. The data revealed that the constitutive envZ alleles activate the Feo- and OmpC-mediated ferrous uptake pathway to flood the cytoplasm with accessible ferrous iron. This activates the ferric uptake regulator, Fur, which inhibits ferric uptake system but cannot inhibit the feo operon due to the positive effect of activated EnvZ/OmpR. The data also revealed the importance of porins in iron homeostasis.

Enhancement of Production of D-Glucosamine in Escherichia coli by Blocking Three Pathways Involved in the Consumption of GlcN and GlcNAc.

D-Glucosamine is a commonly used dietary supplement that promotes cartilage health in humans. Metabolic flux analysis showed that D-glucosamine production could be increased by blocking three pathways involved in the consumption of glucosamine-6-phosphate and acetylglucosamine-6-phosphate. By homologous single-exchange, two key genes (nanE and murQ) of Escherichia coli BL21 were knocked out, respectively. The D-glucosamine yields of the engineered strains E. coli BL21ΔmurQ and E. coli BL21ΔnanE represented increases by factors of 2.14 and 1.79, respectively. Meanwhile, for bifunctional gene glmU, we only knocked out its glucosamine-1-phosphate acetyltransferase domain by 3D structural analysis to keep the engineered strain E. coli BL21glmU-Δgpa survival, which resulted in an increase in the production of D-glucosamine by a factor of 2.16. Moreover, for further increasing D-glucosamine production, two genes encoding rate-limiting enzymes, named glmS and gna1, were coexpressed by an RBS sequence in those engineered strains. The total concentrations of D-glucosamine in E. coli BL21 glmU-Δgpa', E. coli BL21ΔmurQ', and E. coli BL21ΔnanE' were 2.65 g/L, 1.73 g/L, and 1.38 g/L, which represented increases by factors of 8.83, 5.76, and 3.3, respectively.

Growth, carcass characteristics, and meat quality of broilers fed a low-energy diet supplemented with a multienzyme preparation.

The effect of a low-ME diet with a multienzyme (Kemzyme Plus, Kemin, Des Moines, IA) blend on performance, meat quality, and carcass traits was evaluated in Hubbard broiler chicks. A total of 120 Hubbard broiler chicks were allocated to the following 4 experimental groups and every group was separated into 6 replicates, with 5 birds per replicate: control (3,180 kcal/kg of ME), control + 0.50 g/kg diet of enzyme (Cont-Enz), low-ME diet (3,080 kcal/kg), and low-ME + 0.50 g/kg diet of enzyme (low-ME-Enz). The trail lasted for 16 D (32 to 48 D of age). No significant differences in growth parameters or carcass traits were observed among treatments. However, liver weight increased with the low-ME-Enz diet (P = 0.038). The low-ME diet recorded the highest weight for the bursa (P = 0.043) and thymus (P = 0.019). Dietary treatments had significant impacts on the length of duodenum, ileum, and cecum, as well as the weight of duodenum. The length of duodenum, ileum, and cecum increased with enzyme supplementation. The myofibril fragmentation index was lower with the Cont-Enz, low-ME, and low-ME-Enz diets than with the control diet (P = 0.043). The shear force increased with the low-ME-Enz diet (P = 0.022) than the control diet. Dietary treatments influenced breast meat yellowness (P = 0.019), whereas the low-ME diet had the lowest yellowness at the slaughtering age. The dietary treatments affected the breast meat pH (P = 0.001), with the control diet having the highest pH value after 24 hours. Thus, there was no effect of low-ME or enzyme supplementation to the control or low-ME diet on growth performance or carcass yield. However, feeding a low-ME diet or Cont-Enz preparation influenced organ and small intestine weights and meat characteristics.

Cytosolic Acetyl-CoA Generated by ATP-Citrate Lyase Is Essential for Acetylation of Cell Wall Polysaccharides.

Plant cell wall polysaccharides, including xylan, glucomannan, xyloglucan and pectin, are often acetylated. Although a number of acetyltransferases responsible for the acetylation of some of these polysaccharides have been biochemically characterized, little is known about the source of acetyl donors and how acetyl donors are translocated into the Golgi, where these polysaccharides are synthesized. In this report, we investigated roles of ATP-citrate lyase (ACL) that generates cytosolic acetyl-CoA in cell wall polysaccharide acetylation and effects of simultaneous mutations of four Reduced Wall Acetylation (RWA) genes on acetyl-CoA transport into the Golgi in Arabidopsis thaliana. Expression analyses of genes involved in the generation of acetyl-CoA in different subcellular compartments showed that the expression of several ACL genes responsible for cytosolic acetyl-CoA synthesis was elevated in interfascicular fiber cells and induced by secondary wall-associated transcriptional activators. Simultaneous downregulation of the expression of ACL genes was demonstrated to result in a substantial decrease in the degree of xylan acetylation and a severe alteration in secondary wall structure in xylem vessels. In addition, the degree of acetylation of other cell wall polysaccharides, including glucomannan, xyloglucan and pectin, was also reduced. Moreover, Golgi-enriched membrane vesicles isolated from the rwa1/2/3/4 quadruple mutant were found to exhibit a drastic reduction in acetyl-CoA transport activity compared with the wild type. These findings indicate that cytosolic acetyl-CoA generated by ACL is essential for cell wall polysaccharide acetylation and RWAs are required for its transport from the cytosol into the Golgi.

Nitrophenols suppress steroidogenesis in prehierarchical chicken ovarian follicles by targeting STAR, HSD3B1, and CYP19A1 and downregulating LH and estrogen receptor expression.

To assess the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the chicken ovary, white (WF, 1-4 mm) and yellowish (YF, 4-8 mm) prehierarchical follicles were incubated in a medium supplemented with PNP or PNMC (10-8-10-4 M), ovine LH (oLH; 10 ng/mL), and combinations of oLH with PNP or PNMC (10-6 M). Testosterone (T) and estradiol (E2) concentrations in media and mRNA expression for steroidogenic proteins (STAR, HSD3B1, and CYP19A1), and LH receptors (LHR), estrogen receptor α (ESR1) and ß (ESR2) in follicles were determined by RIA and real-time qPCR, respectively. PNP and PNMC decreased T and E2 secretion by the WF and YF, and oLH-stimulated T secretion from these follicles. PNP decreased basal STAR and HSD3B1 mRNA levels both in the WF and YF, and CYP19A1 mRNAs in the WF. PNP reduced oLH-affected mRNA expression of these genes in the YF. PNMC inhibited basal STAR, HSD3B1, and CYP19A1 mRNA expression in the WF, but not in the YF. PNMC reduced oLH-stimulated STAR and CYP19A1 expression in the YF and WF, respectively. PNP decreased basal mRNA expression of LHR, ESR1, and ESR2 in the WF, but it increased ESR1 and ESR2 mRNA levels in the YF. PNMC reduced both basal and oLH-affected LHR, ESR1, and ESR2 mRNA expression in the WF; however, it did not influence expression of these genes in the YF. We suggest that nitrophenols by influencing sex steroid synthesis and transcription of LH and estrogen receptors in prehierarchical ovarian follicles may impair their development and selection to the preovulatory hierarchy.

[Lipoic acid: physiological role and prospects for clinical application].

α-Lipoic acid (also known as thioctic acid) is a natural vitamin-like compound. Lipoic acid contains asymmetrical carbon, which causes the presence of two possible optical isomers (enantiomers): R-lipoic acid (levogyrate isomer) and S-lipoic acid (rightspinning isomer). Lipoic acid functions as a cofactor for several important mitochondrial multienzyme complexes, enhances the uptake of glucose by the cells, and modulates the activity of various signaling molecules and transcription factors. It was shown that α-lipoic acid and its derivative, dihydrolipoic acid, have a direct antioxidant effect due to the neutralization of reactive oxygen species that are destructive to DNA, proteins and lipids of cells. Dihydrolipoic acid enhances the antioxidant properties of ascorbic acid, glutathione and ubiquinone. Available evidence suggests that supplementation with lipoic acid reduces the symptoms of peripheral diabetic neuropathy. Results from randomized controlled trials show that high doses of lipoic acid can improve the glycemic profile of subjects with metabolic disorders. Lipoic acid can be used to control body weight in people with obesity. R-Lipoic acid is synthesized in the human body and is contained in foods in a form covalently associated with lysine (lipoyllysine). Its dose in dietary supplements significantly exceeds the amount in the diet. Most dietary supplements contain a racemic mixture of R- and S-lipoic acid.

Structural insights into Escherichia coli phosphopantothenoylcysteine synthetase by native ion mobility-mass spectrometry.

CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility-mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery.

Dual and selective inhibitors of pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Leishmania chagasi.

Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this disease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other protozoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized. Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 diaminopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1 Ki = 1.50-2.30 µM and LcDHFR Ki = 0.28-3.00 µM) with poor selectivity index. On the other hand, compound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki = 0.47 µM, selectivity index = 20).

Atmospheric carbon monoxide oxidation is a widespread mechanism supporting microbial survival.

Carbon monoxide (CO) is a ubiquitous atmospheric trace gas produced by natural and anthropogenic sources. Some aerobic bacteria can oxidize atmospheric CO and, collectively, they account for the net loss of ~250 teragrams of CO from the atmosphere each year. However, the physiological role, genetic basis, and ecological distribution of this process remain incompletely resolved. In this work, we addressed these knowledge gaps through culture-based and culture-independent work. We confirmed through shotgun proteomic and transcriptional analysis that the genetically tractable aerobic soil actinobacterium Mycobacterium smegmatis upregulates expression of a form I molydenum-copper carbon monoxide dehydrogenase by 50-fold when exhausted for organic carbon substrates. Whole-cell biochemical assays in wild-type and mutant backgrounds confirmed that this organism aerobically respires CO, including at sub-atmospheric concentrations, using the enzyme. Contrary to current paradigms on CO oxidation, the enzyme did not support chemolithoautotrophic growth and was dispensable for CO detoxification. However, it significantly enhanced long-term survival, suggesting that atmospheric CO serves a supplemental energy source during organic carbon starvation. Phylogenetic analysis indicated that atmospheric CO oxidation is widespread and an ancestral trait of CO dehydrogenases. Homologous enzymes are encoded by 685 sequenced species of bacteria and archaea, including from seven dominant soil phyla, and we confirmed genes encoding this enzyme are abundant and expressed in terrestrial and marine environments. On this basis, we propose a new survival-centric model for the evolution of aerobic CO oxidation and conclude that, like atmospheric H2, atmospheric CO is a major energy source supporting persistence of aerobic heterotrophic bacteria in deprived or changeable environments.

Compound K producing from the enzymatic conversion of gypenoside by naringinase.

Compound K is a type of protopanaxadiol-type ginsenosides (PPDs) that has strong bioactivities due to fewer glycosyls. However, compound K is not present in raw and unprocessed ginseng. Some PPDs have the same structure with gypenosides, and could be obtained from Gynostemma pentaphyllum. The enzymolysis of PPD-type gypenosides of G. pentaphyllum by naringinase has been reported for the first time in this research. In addition, isolation and identification of enzymolysis end product, and the optimization of enzymolysis parameters were investigated. The results showed that compound K was produced from the enzymolysis of PPD-type gypenosides by naringinase, and could be isolated and purificated by HP-20 macroporous resin and C-18 column chromatography. The optimum enzymolysis conditions determined by the response surface methodology (RSM) are pH 4.1, 50 °C, and 71 h, with a yield of 65.44 ±â€¯4.52% for compound K. These results demonstrated that enzymolysis could be a promising method for producing compound K from the biotransformation of PPD-type gypenosides of G. pentaphyllum.

Effects of multi-enzyme on production performance, egg quality, nutrient digestibility, and excreta noxious gas emission of early phase Hy-line brown hens.

The purpose of this study was to investigate the effects of dietary supplementation of non-starch polysaccharide multi-enzyme (NME) in early laying phase of hens on production performance, egg quality, nutrient digestibility, and excreta noxious gas emission. In total, 432 Hy-line brown laying hens at 18 wk of age were used in a 10-wk feeding trail. Hens were randomly assigned to 1 of 3 treatments with 24 replication and 6 hens per replication (1 hen per cage). Dietary treatments were corn-soybean meal-DDGS-based diets supplemented with 0 (based diet, CON), 0.05% (NME1), and 0.1% (NME2) of NME. No significant (P > 0.05) response to increasing NME supplementation was observed for damaged egg rate shown throughout the experiment. Significant (P < 0.05) linear increase was observed for egg production at week 4, 6, and 8; moreover, egg production at week 8 also showed quadratic (P = 0.0344) increase. No significant effects were found on yolk color, eggshell strength, and eggshell thickness during the experiment (P > 0.05) with the increase in NME supplemental levels. Albumin height, haugh unit, and egg color values were linearly (P < 0.05) increased at week 2 and 6 following the increasing NME supplementation, respectively. Additionally, quadratic (P = 0.0013) effect was observed on egg weight at week 6 with the increasing level of NME. Moreover, apparent total tract digestibility of nitrogen and excreta ammonia emission was linearly (P < 0.05) affected increasing NME supplementation. In summary, inclusion of NME containing xylanase, ß-glucanase, galactosidase, and galactomannanase activities in corn-soybean meal-DDGS-based diets increased nitrogen digestibility, decreased excreta ammonia emission, and had no negative effects on production performance and egg quality parameters.