Search

Clavulanic acid (CA) is usually used together with other β‐lactam antibiotics as combination drugs to inhibit bacterial β‐lactamases, which is mainly produced from the fermentation of microorganism such as Streptomyces clavuligerus. Recently, it is still a challenge for downstream processing of low concentration and unstable CA from fermentation broth with high solid content, high viscosity, and small cell size. In this study, an integrated process was developed for simultaneous solid–liquid separation and primary purification of CA from real fermentation broth of S. clavuligerus using salting‐out extraction system (SOES). First, different SOESs were investigated, and a suitable SOES composed of ethanol/phosphate was chosen and further optimized using the pretreated fermentation broth. Then, the optimal system composed of 20% ethanol/15% K2HPO4 and 10% KH2PO4 w/w was used to direct separation of CA from untreated fermentation broth. The result showed that the partition coefficient (K) and recovery yield (Y) of CA from untreated fermentation broth were 29.13 and 96.8%, respectively. Simultaneously, the removal rates of the cells and proteins were 99.8% and 63.3%, respectively. Compared with the traditional method of membrane filtration or liquid–liquid extraction system, this developed SOES showed the advantages of simple operation, shorter operation time, lower process cost and higher recovery yield of CA. These results demonstrated that the developed SOES could be used as an attractive alternative for the downstream processing of CA from real fermentation broth.

Type 2 diabetes mellitus (T2DM) is a global metabolic disease with potentially life- threatening complications. Liver metabolism plays a vital role in the occurrence and development of diabetes mellitus. It has been reported that the Chinese medicinal

The ComPA two-component signal transduction system (TCS) is essential in Bacillus spp. However, the molecular mechanism of the histidine kinase ComP remains unclear. Here, we predicted the structure of ComP from Bacillus amyloliquefaciens Q-426 (BaComP) using an artificial intelligence approach, analyzed the structural characteristics based on the molecular docking results and compared homologous proteins, and then investigated the biochemical properties of BaComP. We obtained a truncated ComPS protein with high purity and correct folding in solution based on the predicted structures. The expression and purification of BaComP proteins suggested that the subdomains in the cytoplasmic region influenced the expression and stability of the recombinant proteins. ComPS is a bifunctional enzyme that exhibits the activity of both histidine kinase and phosphotransferase. We found that His571 played an obligatory role in the autophosphorylation of BaComP based on the analysis of the structures and mutagenesis studies. The molecular docking results suggested that the HATPase_c domain contained an ATP-binding pocket, and the ATP molecule was coordinated by eight conserved residues from the N, G1, and G2 boxes. Our study provides novel insight into the histidine kinase BaComP and its homologous proteins.

Prediabetes is a prevalent metabolic disorder with multiple complications, including nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the combinatorial effect of baicalein, a dietary flavonoid abundant in multiple edible plants, and acarbose on prediabetes-associated NAFLD. Baicalein and its metabolites inhibited de novo lipogenesis (DNL), thereby decreasing lipid accumulation and hepatokine secretion in oleic acid-induced hepatocytes. Carbohydrate restriction, which mimicked the effect of acarbose, led to comparable results. The combinatorial effect of baicalein and acarbose was further verified in prediabetic mice with NAFLD. Through the 16-week intervention, baicalein and acarbose inhibited DNL and improved glucose tolerance, oxidative stress, liver histology, and hepatokine secretion, thereby ameliorating insulin resistance and NAFLD. Our study demonstrated that baicalein enhanced the effect of acarbose on improving NAFLD and explored the underlying multitarget mechanism, laying a theoretical foundation for the development of flavonoid dietary supplements for the simultaneous improvement of NAFLD and prediabetes.

Phages as a potential alternative antibiotic for multidrug-resistant bacterial infections are receiving great attention worldwide. However, the traditional separation and purification of phage are cumbersome, time-consuming, costly and inefficient. In this study, phage phiAB9 for multidrug-resistant Acinetobacter baumannii was separated and purified by a simple and cost-saving one-step salting-out extraction (SOE). Several kinds of salts and organic solvents without effect on phage survival were chosen to form the SOE systems, and the composition of SOE systems were optimized according to the phage recovery rate and impurity removal rate. After one-step SOE by an optimal system composed of 18% (w/w) ammonium citrate and 40% (w/w) ethyl acetate, the recovery rate of phage in the middle phase could reach to 90.82%, and most proteins (99.57%), cells (97.98%) and endotoxin (84.08%) were well removed, with a concentration factor of 210 and the purification factors of phage to proteins, cells and endotoxin were 303.64, 133.55 and 5.36, respectively. Comparing with two-step SOE and traditional aqueous two-phase extraction, one-step SOE may be an available method for the separation and purification of phages.

1-Deoxynojirimycin (1-DNJ) is the major effective component of mulberry leaves, exhibiting inhibitory activity against α-glucosidase. However, due to the low content of 1-DNJ in mulberry products, its level cannot meet the lowest dose to exhibit its activity. In this study, a combination of dietary 5,6,7-trihydroxy-flavonoid aglycones with 1-DNJ showed synergistic inhibitory activity against maltase of mice α-glucosidase and recombinant C- and N-termini of maltase-glucoamylase (MGAM) and baicalein with 1-DNJ exhibited the strongest synergistic effect. The synergistic effect of the combination was also confirmed by the maltose tolerance test in vivo. Enzyme kinetics, molecular docking, fluorescence spectrum, and circular dichroism spectrometry studies indicated that the major mechanism of the synergism is that baicalein was a positive allosteric inhibitor and bound to the noncompetitive site of MGAM, causing an increase of the binding affinity of 1-DNJ to MGAM. Our results might provide a theoretical basis for the design of dietary supplements containing mulberry products.

Trichinosis is a worldwide zoonotic disease closely related to cultural and dietary habits caused by a nematode Trichinella spp. The drugs for its prevention and treatment are still not thoroughly defined. Wortmannilactone F was used to value the therapeutic effects on the worm reduction rates, change of the intestinal mucosa, and the host's body's immune activity in this experiment. BALB/c mice were orally fed with 200 infective Trichinella spiralis larvae. Then, T. spiralis-infected mice were treated with wortmannilactone F (50, 100, and 200 mg/kg). The number and morphological analysis of adult worm, the expression of Factor associated suicide (Fas), and the level of SIgA in the mice were investigated. Wortmannilactone F showed dose-dependent anthelmintic effects by causing mortality of worms, obvious damaging effects on mature T. spiralis' surface and their digestive systems, decreasing the expression of mice's intestinal mucosa's Fas protein, and reducing intestinal mucosa's level of SIgA secretions. Wortmannilactone F is expected to be a potential therapeutic drug for trichinellosis treatment.

Interspecific interaction mediated crosstalk was regarded as an important feature among the microorganisms under natural conditions and also the key mechanism for the biosynthesis of newly induced compounds in the co-culture. However, there is still a lack of direct experimental evidence for this mechanism. In this study, the interaction between A. sydowii and B. subtilis in the co-culture was firstly verified by the morphological observation. Subsequently, through the strategy combining substrate feeding, stable isotope labeling and gene expression analysis, the crosstalk in the co-culture for the production of five benzoic acid derivatives (N1-N4 and N7) were demonstrated: the secondary metabolites 10-deoxygerfelin of A. sydowii acted as an inducer to induce B. subtilis to produce benzoic acid, which was further converted into 3-OH-benzoic acid by A. sydowii. Subsequently, A. sydowii used 3-OH-benzoic acid as the substrate to synthesize the new compound N2, and then N1, N3, N4 and N7 were biosynthesized upon the up-regulation of hydrolase, hydroxylase, and acyltransferase during co-culture. The plate zone analysis suggested that the biosynthesis of the newly induced compounds N1-N4 was mainly attributed to A. sydowii, and both A. sydowii and B. subtilis were indispensable for the biosynthesis of N7. The current study provides an important basis for a better understanding of the metabolites transfer and the biosynthetic pathways of the newly induced compounds in the co-culture, providing a new insight into the interaction-mediated crosstalk among microorganisms.

The BioH carboxylesterase which is a typical α/β-hydrolase enzyme involved in biotin synthetic pathway in most bacteria. BioH acts as a gatekeeper and blocks the further elongation of its substrate. In the pathogen Klebsiella pneumoniae, BioH plays a critical role in the biosynthesis of biotin. To better understand the molecular function of BioH, we determined the crystal structure of BioH from K. pneumoniae at 2.26 A resolution using X-ray crystallography. The structure of KpBioH consists of an α-β-α sandwich domain and a cap domain. B-factor analysis revealed that the α-β-α sandwich domain is a rigid structure, while the loops in the cap domain shows the structural flexibility. The active site of KpBioH contains the catalytic triad (Ser82-Asp207-His235) on the interface of the α-β-α sandwich domain, which is surrounded by the cap domain. Size exclusion chromatography shows that KpBioH prefers the monomeric state in solution, whereas two-fold symmetric dimeric formation of KpBioH was observed in the asymmetric unit, the conserved Cys31-based disulfide bonds can maintain the irreversible dimeric formation of KpBioH. Our study provides important structural insight for understanding the molecular mechanisms of KpBioH and its homologous proteins.

Fusobacterium nucleatum is associated with the incidence and development of multiple diseases, such as periodontitis and colorectal cancer (CRC). Till now, studies have proved only a few proteins to be associated with such pathogenic diseases. The two-component system is one of the most prevalent forms of bacterial signal transduction related to intestinal diseases. Here we report a novel, recombinant, two-component, response-regulator protein ArlR from the genome of F. nucleatum strain ATCC 25586. We optimized the expression and purification conditions of ArlR; in addition, we characterized the interaction of this response regulator protein to the corresponding histidine kinase and DNA sequence. The full-length ArlR was successfully expressed in six of the E. coli host strains. However, optimum expression conditions of ArlR were present only in E. coli strain BL21 Condon plus (DE3) RIL that was later induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) for 8 h at 25 °C. The SDS-PAGE analysis revealed the molecular weight of the recombinant protein as 27.3 kDa and approximately 90% purity after gel filtration chromatography. ArlR was biologically active after its purification. Therefore, it accepted the corresponding phosphorylated histidine-kinase phosphate group and bound to the analogous DNA sequence. The binding constant between ArlR and the corresponding histidine kinase is 1.28 µM, while the binding constant between ArlR and the bound DNA sequence is 37.5 µM. Altogether, these results illustrate an effective expression and purification method for the novel two-component system protein ArlR.

MalE is a maltose/maltodextrin-binding protein (MBP) that plays a critical role in most bacterial maltose/maltodextrin-transport systems. Previously reported wild-type MBPs are monomers comprising an N-terminal domain (NTD) and a C-terminal domain (CTD), and maltose-like molecules are recognized between the NTD and CTD and transported to the cell system. Because MBP does not undergo artificial dimerization, it is widely used as a tag for protein expression and purification. Here, the crystal structure of a domain-swapped dimeric MalE from Salmonella enterica (named SeMalE) in complex with maltopentaose is reported for the first time, and its structure is distinct from typical monomeric MalE family members. In the domain-swapped dimer, SeMalE comprises two subdomains: the NTD and CTD. The NTD and CTD of one molecule of SeMalE interact with the CTD and NTD of the partner molecule, respectively. The domain-swapped dimeric conformation was stabilized by interactions between the NTDs, CTDs and linkers from two SeMalE molecules. Additionally, a maltopentaose molecule was found to be located at the interface between the NTD and CTD of different SeMalE molecules. These results provide new insights that will improve the understanding of maltodextrin-binding MalE proteins.

1-Deoxynojirimycin (DNJ), the major alkaloid in Morus alba L., is the main effective constituent in "Mulberry twig Alkaloids Tablets" launched in China in 2020. Prediabetes, characterized by insulin resistance, is regarded as the key period for reversing Type 2 diabetes mellitus (T2DM) through lifestyle intervention and glucose-lowering drugs. Besides the excellent activity as an α-glucosidase inhibitor, DNJ also improves insulin sensitivity in T2DM murine models, yet the mechanism is still unclear. Besides, the pharmaceutical effect of DNJ on prediabetes is also undocumented.The aim of this study was to investigate the pharmaceutical effect of DNJ on high-fat and streptozotocin (STZ)-induced prediabetes mice, and to elucidate the mechanism of insulin resistance ameliorated by DNJ.Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed to detect blood glucose level and insulin sensitivity in mice. The levels of circulating lipopolysaccharide (LPS), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in the plasma of mice were measured by limulus reagent and enzyme-linked immunosorbent assay (ELISA), respectively. Next-generation sequencing (NGS) and intestinal microbiota sequencing were used to screen the alterations in the transcriptome of liver tissues and to assess the differences in intestinal flora composition, respectively. Expression of cytokine signaling pathway inhibitor 3 (SOCS3), insulin receptor substrate (IRS1), p-IRS1 (Tyr896), occludin, and toll like receptor 4 (TLR4)/NF-κB signaling pathway were confirmed by western blotting.Our study revealed that DNJ decreased the blood glucose level and improve insulin sensitivity in prediabetic mice. DNJ significantly reduced the relative risk of T2DM in prediabetic mice by approximately 83.7%. Mechanistically, DNJ treatment suppressed the circulating levels of LPS, IL-6, and TNF-α in plasma and decreased the inflammatory infiltration in liver and colon tissues. DNJ-treatment increased the abundance of Akkermansia, Bifidobacterium, and Lactobacillus, and decreased the abundance of Enterococcaceae and Lachnospiraceae. Moreover, DNJ suppressed the expression of SOCS3 and the activity of TLR4/NF-κB signaling pathway, meanwhile improving the expression of occludin and the ratio of p-IRS1 (Tyr896)/IRS1.DNJ effectively ameliorates glucose and lipid metabolism in prediabetic mice, and decreased the relative risk of progression into T2DM from prediabetes. The suppressed immune responses play essential roles in the improvement of insulin resistance by DNJ treatment. In conclusion, DNJ from Morus alba L. is a promising alternative agent in T2DM prevention.

Combination of dietary flavonoid-baicalein and acarbose reduces the risk that prediabetes will develop into type 2 diabetes mellitus; however, the mechanism underlying this effect has not been clarified. In this study, the in vitro culture conditions of intestinal microorganisms from prediabetic mice were optimized to increase over 30% similarity between in vitro cultured and fecal samples. Baicalein and acarbose alone and in combination, and their corresponding starch hydrolysate were assayed by the in vitro model. The results indicated that the combination of baicalein with acarbose decreased gas production by reducing the residual starch ratio in starch hydrolysate and decreasing the dosage of acarbose, and that reducing the relative abundance of gut bacteria correlated with gas production is the main mechanism. This study provided a theoretical foundation for the development of flavonoid dietary supplements to enhance the efficacy of oral hypoglycemic agents with fewer side effects and higher efficacy.

Prediabetes is a prevalent metabolic disorder with multiple complications, including nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the combinatorial effect of baicalein, a dietary flavonoid abundant in multiple edible plants, and acarbose on prediabetes-associated NAFLD. Baicalein and its metabolites inhibited

Separation and purification of secondary metabolites from wild microorganisms remains a challenge due to the analogues existing in the fermentation broth. In this study, a three-liquid-phase salting-out extraction system (TLP-SOES) coupled with column chromatography was developed to purify wortmannilactone F and its dehydrated derivative wortmannilactone L. The TLP-SOES composed of 25.5% petroleum ether/20.0% ethanol/12.9% ammonium sulfate (w/w) at pH 5.6 and room temperature was found to separate wortmannilactone F in the middle (ethanol-rich) phase and wortmannilactone L in the top (petroleum ether-rich) phase with the recovery rates of 88.9% and 83.3%, respectively. The two compounds were further purified by ODS column and semi-preparative HPLC. The total recovery rates of the two compounds were over 2-fold higher than that of the conventional method, which included ethyl acetate/hexane extraction and silica gel column chromatography. Additionally, the calculated logP (ClogP) value of wortmannilactones showed a good linear correlation with their natural logarithm of partition coefficient according to the tie line length (TLL), and that the critical ClogP value might be useful for the selection of TLL or the composition of the extraction system. In summary, our study provided an effective approach for the separation and purification of analogues of natural products.

Baicalein is a dietary flavonoid that exhibits a variety of intestinal pharmacological activities with limited oral bioavailability. In the present study, the intestinal pharmacokinetics of baicalein was determined by HPLC-ESI-MS/MS and HPLC-PDA analysis. After oral administration, the intestinal concentration of baicalein was peaked at 1 h, and the amount of baicalein in the intestine corresponded to 87%, 45%, and 20% of the administered dose at 1 h, 8 h, and 12 h, respectively. Glucuronidation, sulfation, and methylation metabolites were the major metabolites of baicalein in the small intestine, including duodenum, jejunum, and ileum. Dehydroxylation, sulfation, and methylation metabolites were the major metabolites in the large intestine, including cecum and colon. In vitro, baicalein was transformed into its glucuronidation and methylation metabolites in hepatic and intestinal S9 fraction. The glucuronidation metabolites was hydrolyzed to baicalein by gut microbiota, but the dehydroxylation and methylation metabolites were stable during gut microbiota incubation. In conclusion, the intestinal concentration of baicalein can support its intestinal pharmacological activities, and the present study may provide useful information for understanding the bioactivities of flavonoids with limited systemic bioavailability.

Pu-erh tea is produced from the leaves of large-leaf tea species (Camellia sinensis var. assamica) in the Yunnan province of China and divided into ripened pu-erh tea (RIPT, with pile-fermentation) and raw pu-erh tea (RAPT) according to processing methods. RIPT extract showed more potent anti-diabetic effects on two-hour postprandial blood glucose (2h-PBG) and fasting blood glucose (FBG) than RAPT extract. UHPLC-Q-TOF/MS and UHPLC-PDA analyses found that 17 newly formed components and the increased components after fermentation, such as quinic acid, gallic acid, caffeine, puerin I and so on, might be the main contributors to the enhanced activities of RIPT. In addition, the probiotic role of RIPT to some beneficial gut bacteria, such as lactobacillus, Prevotellaceae NK3B31 group, Alloprevotella and Prevotella, was observed in our study. These results might provide a clue to anti-diabetic mechanism and active components of pu-erh tea, and use as a functional beverage worth to be further studied.

Background The co-culture strategy which mimics natural ecology by constructing an artificial microbial community is a useful tool to activate the biosynthetic gene clusters to generate new metabolites. However, the conventional method to study the co-culture is to isolate and purify compounds separated by HPLC, which is inefficient and time-consuming. Furthermore, the overall changes in the metabolite profile cannot be well characterized. Results A new approach which integrates computational programs, MS-DIAL, MS-FINDER and web-based tools including GNPS and MetaboAnalyst, was developed to analyze and identify the metabolites of the co-culture of Aspergillus sydowii and Bacillus subtilis. A total of 25 newly biosynthesized metabolites were detected only in co-culture. The structures of the newly synthesized metabolites were elucidated, four of which were identified as novel compounds by the new approach. The accuracy of the new approach was confirmed by purification and NMR data analysis of 7 newly biosynthesized metabolites. The bioassay of newly synthesized metabolites showed that four of the compounds exhibited different degrees of PTP1b inhibitory activity, and compound N2 had the strongest inhibition activity with an IC50 value of 7.967 μM. Conclusions Co-culture led to global changes of the metabolite profile and is an effective way to induce the biosynthesis of novel natural products. The new approach in this study is one of the effective and relatively accurate methods to characterize the changes of metabolite profiles and to identify novel compounds in co-culture systems.

The SaeRS two-component system in Staphylococcus aureus controls the expression of a series of virulence factors, such as hemolysins, proteases, and coagulase. The response regulator, SaeR, belongs to the OmpR family with an N-terminal regulatory domain and a C-terminal DNA binding domain. To improve the production and stability of the recombinant protein SaeR, l -arginine (L-Arg) was added to the purification buffers. L-Arg enhanced the solubility and stability of the recombinant protein SaeR. The thermal denaturation temperature of SaeR in 10 mM L-Arg buffer was significantly increased compared to the buffer without L-Arg. Microscale Thermophoresis (MST) analysis results showed that the SaeR protein could bind to the P1 promoter under both phosphorylated and non-phosphorylated status in buffer containing 10 mM L-Arg. These results illustrate an effective method to purify SaeR and other proteins.

Catabolite control protein E (CcpE) is a LysR-type transcriptional regulator that positively regulates the transcription of the first two enzymes of the TCA cycle, namely, citZ and citB, by sensing accumulated intracellular citrate. CcpE comprises an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD) and senses citrate with conserved arginine residues in the RD. Although the crystal structure of the apo SaCcpE-RD has been reported, the citrate-responsive and DNA-binding mechanisms by which CcpE regulates TCA activity remain unclear. Here, we report the crystal structure of the apo and citrate-bound SaCcpE-RDs. The SaCcpE-RD exhibits conformational changes between the two subdomains via hinge motion of the central β4 and β10 strands. The citrate molecule is located in a positively charged cavity between the two subdomains and interacts with the highly conserved Ser98, Leu100, Arg145, and Arg256 residues. Compared with that of the apo SaCcpE-RD, the distance between the two subdomains of the citrate-bound SaCcpE-RD is more than ∼3 Å due to the binding of the citrate molecule, and this form exhibits a closed structure. The SaCcpE-RD exhibits various citrate-binding-independent conformational changes at the contacting interface. The SaCcpE-RD prefers the dimeric state in solution, whereas the SaCcpE-FL prefers the tetrameric state. Our results provide insight into the molecular function of SaCcpE.

The nicotinamidase/pyrazinamidase PncA is a member of a large family of hydrolase enzymes that catalyze the deamination of nicotinamide to nicotinic acid. PncA also functions as a pyrazinamidase in a wide variety of eubacteria and is an essential coenzyme in many cellular redox reactions in living systems. We report the crystal structure of substrate-free PncA from Bacillus subtilis (BsPncA) at 2.0 A resolution to improve our understanding of the PncA family. The structure of BsPncA consists of an α/β domain and a subdomain. The subdomain of BsPncA has a different conformation than that of PncA enzymes from other organisms. The B-factor analysis revealed a rigid structure of the α/β domain, while the subdomain is highly flexible. Both dimers and tetramers were observed in BsPncA protein crystals, but only dimers were observed in solution. Our results provide useful information that will further enhance our understanding of the molecular functions of PncA family members.

Baicalein, a dietary flavonoid, is the major constituent of Oroxylum indicum and Scutellaria baicalensis which are consumed as teas or dietary supplements in Asia, European, and the United States. The compound exhibits a variety of bioactivities despite its limited bioavailability. In the present work, 4-week treatment of baicalein significantly decreased the levels of blood glucose and circulating lipopolysaccharide (LPS) and improved insulin resistance, inflammation, and lipid profile in diabetic rats induced by STZ and high-fat-high-sugar-diet. These anti-diabetic effects were attributed to the increased SCFAs content and the increased thickness of gut mucus layer, and were associated with the modulation of gut microbiota. Among the key phylotypes, Bacteroides and Bacteroidales S24-7 had the highest relative abundance in the rats receiving high-dose baicalein and showed positive correlation with the phenotypes related to the improvement of type 2 diabetes (T2DM). Our study supports the use of baicalein as a dietary supplement and a potential prebiotic for its ability to modify gut microbiota and to improve T2DM-related biochemical abnormalities.

Biotransformation of wortmannilactone F (3) using the marine-derived fungus DL1103 generated wortmannilactone M (1), a novel analog of wortmannilactone, which was a reduction product of 3 at the C-3 carbonyl group. The in vitro inhibitory activities of 10 wortmannilactones, including 1, against electron transport enzymes indicated that all the wortmannilactones were selective inhibitors of NADH-fumarate reductase and NADH-rhodoquinone reductase. The structure-activity relationship analysis showed that the relative configuration of C1" and C5", the positions of double bonds, the oxygen atoms in the dihydropyran moiety, and the keto-carbonyl group in the oxabicyclo-[2.2.1]-heptane moiety were important to the inhibitory activity of wortmannilactones. In vivo studies indicated that 3 significantly decreased the number and size of adult worms in Trichinella spiralis-infected mice in a dose-dependent manner. Notable changes in the cuticle and microvilli of T. spiralis were also observed. Our data provided useful information in the research and development of polyketides with dihydropyran and oxabicyclo [2.2.1] heptane moieties as antihelminthics.

Membrane fusion proteins (MFPs) play an essential role in the action of the drug efflux pumps and protein secretion systems in bacteria. The sporulation delaying protein (SDP) efflux pump YknWXYZ has been identified in diverse Bacillus species. The MFP YknX requires the ATP-binding cassette (ABC) transporter YknYZ and the Yip1 family protein YknW to form a functional complex. To date, the crystal structure, molecular function and mechanism of action of YknX remain unknown. In this study, to characterize the structural and biochemical roles of YknX in the functional assembly of YknWXYZ from B. amyloliquefaciens, we successfully obtained crystals of the YknX protein that diffracted X-rays to a resolution of 4.4 Å. We calculated an experimentally phased map using single-wavelength anomalous diffraction (SAD), revealing that YknX forms a hexameric assembly similar to that of MacA from Gram-negative bacteria. The hexameric assembly of YknX exhibited a funnel-like structure with a central channel and a conical mouth. Functional studies in vitro suggest that YknX can bind directly to peptidoglycan. Our study provides an improved understanding of the assembly of the YknWXYZ efflux pump and the role of YknX in the complex.

In this paper, the inhibition of α-amylase and α-glucosidase by nine pentacyclic triterpenes was determined. For α-amylase inhibitory activity, the IC50 values of ursolic acid, corosolic acid, and oleanolic acid were 22.6±2.4μM, 31.2±3.4μM, and 94.1±6.7μM, respectively. For α-glucosidase inhibition, the IC50 values of ursolic acid, corosolic acid, betulinic acid, and oleanolic acid were 12.1±1.0μM, 17.2±0.9μM, 14.9±1.9μM, and 35.6±2.6μM, respectively. The combination of corosolic acid and oleanolic acid with acarbose showed synergistic inhibition against α-amylase. The combination of the tested triterpenes with acarbose mainly exhibited additive inhibition against α-glucosidase. Kinetic studies revealed that corosolic acid and oleanolic acid showed non-competitive inhibition and acarbose showed mixed-type inhibition against α-amylase. The results provide valuable implications for the triterpenes (ursolic acid, corosolic acid, and oleanolic acid) alone or in combination with acarbose as a therapeutic agent for the treatment of diabetes mellitus.

Baicalein, a major component in Scutellaria baicalensis and Oroxylum indicum, is used as dietary supplement in Asia. In this study, we characterized the insulin-sensitizing activity of baicalein and found that baicalein improved insulin resistance by reducing the expression of SOCS3, increasing the phosphorylation of IRS1 and Akt1, reducing the phosphorylation of GSK3β and increasing hepatic glycogen level. We further evaluated the preventive effect of baicalein combined with acarbose in mice, aiming to enhance the therapeutic effect and reduce the side-effects of acarbose. The combination reduced the relative risk of progression from prediabetes to diabetes by 83.3% and the dose of acarbose by 80%. The underlying mechanisms include reducing postprandial hyperglycemia, improving insulin resistance, improving lipid metabolism, and reducing oxidative stress. Our results suggest that the consumption of 195–780 mg/d baicalein might improve the efficacy of acarbose on diabetes prevention and reduce the side-effects of acarbose in long-term acarbose users.

The inhibition of porcine pancreatic α-amylase and mammalian α-glucosidase by 16 individual flavonoids was determined. The IC50 values for baicalein, (+)-catechin, quercetin, and luteolin were 74.1 ± 5.6, 175.1 ± 9.1, 281.2 ± 19.2, and 339.4 ± 16.3 μM, respectively, against α-glucosidase. The IC50 values for apigenin and baicalein were 146.8 ± 7.1 and 446.4 ± 23.9 μM, respectively, against α-amylase. The combination of baicalein, quercetin, or luteolin with acarbose showed synergistic inhibition, and the combination of (+)-catechin with acarbose showed antagonistic inhibition of α-glucosidase. The combination of baicalein or apigenin with acarbose showed additive inhibition of α-amylase at lower concentrations and antagonistic inhibition at a higher concentration. Kinetic studies of α-glucosidase activity revealed that baicalein alone, acarbose alone, and the combination showed noncompetitive, competitive, and mixed-type inhibition, respectively. Molecular modeling revealed that baicalein had higher affin...

The combined effect of Oroxylum indicum seed extracts (OISE) or major flavonoids from OISE and acarbose on reducing postprandial blood glucose (PBG) levels was investigated in vitro and in vivo. In vitro, the IC50 values of OISE and baicalein against α-glucosidase were 43.4±0.731μgmL-1 and 25.9±0.412μgmL-1 respectively. A combination of acarbose with OISE or baicalein synergistically inhibited rat intestinal α-glucosidase. The combination index (CI) values for acarbose with OISE ranged from 0.33 to 0.75, suggesting a synergistic but not additive effect. OISE was determined to be a non-competitive inhibitor of maltose-hydrolyzing activity. In vivo, OISE were administered to normoglycemic and diabetic mice, either alone or in combination with acarbose. At doses between 50 and 200mgkg-1, OISE enhanced the efficacy of acarbose by up to 5-fold. These results demonstrated that OISE enhances the efficacy of acarbose in vivo, and that the combination of OISE and acarbose displayed a synergistic effect in vitro. Therefore, OISE can be used to design dietary supplements to treat diabetes.

Many bioactive components such as poly-unsaturated fatty acids (e.g. EPA and DHA), phospholipids and astaxanthin are known in Antarctic krill ( Euphausia superba ) oil. The krill DHA and EPA are generally considered to be similar to natural ones. However, two chemical compounds which were separated from Antarctic krill oil and identified as EPA and DHA by HRESIMS and NMR acted much more effective inhibitive activities on growth of several cell lines (U937, K562, SMMC-7721, PC-3, MDA-MB-231, HL60 and MCF-7) than those from sturgeon liver and commercial fish oil. Taking MCF-7 as an example, the IC 50 values of Antarctic krill EPA and DHA were 14.01 and 19.94μM，while the IC 50 values of sturgeon liver and commercial fish EPA and DHA were 81.45, 73.13, 82.11 and 75.31μM, respectively. Raman spectra revealed that the Antarctic krill EPA and DHA have E-configuration structures, which were different from those in commercial fish oil. Additionally, the Antarctic krill EPA and DHA had no effects on human normal liver cell line HL7702. These results indicated that the Antarctic krill E-EPA and E-DHA had a great prospect in cancer therapy.

Aim To screen a potential PTP1b inhibitor from the microbial origin-based compound library and to investigate the potential anti-diabetic effects of the inhibitor in vivo and determine its primary anti-diabetic mechanism in vitro and in silico. Methods PTP1b inhibitory activity was measured using recombination protein as the enzyme and p-NPP as the substrate. The binding of the inhibitor to PTP1b was analysed by docking in silico and confirmed by ITC experiments. The intracellular signalling pathway was detected by Western blot analysis in HepG2 cells. The anti-diabetic effects were evaluated using a diabetic mice model in vivo. Results Among 545 microbial origin-based pure compounds tested, trivaric acid, a tridepside, was selected as a PTP1B inhibitor exhibiting strong inhibitory activity with an IC50 of 173 nM. Docking and ITC studies showed that trivaric acid was able to spontaneously bind to PTP1b and may inhibit PTP1b by blocking the catalytic domain of the phosphatase. Trivaric acid also enhanced the ability of insulin to stimulate the IR/IRS/Akt/GLUT2 pathway and increase the glucose consumption in HepG2 cells. In diabetic mice, trivaric acid that had been encapsulated into Eudrgit L100-5.5 showed significant anti-diabetic effects, improving insulin resistance, leptin resistance and lipid profile and weight control at doses of 5 mg/kg and 50 mg/kg. Significance Trivaric acid is a potential lead compound in the search for anti-diabetic agents targeting PTP1b.

Three new polyketides, wortmannilactones I1-I3, were purified from Talaromyces wortmannii using the One Strain Many Compounds (OSMAC) strategy. The polyketides’ structures were established using IR, 1D and 2D NMR, and HRESIMS analyses and the absolute configurations were identified by comparison of experimental and calculated ECDs. These polyketides exhibit selective inhibitory activity against NADH-fumarate reductase.