Enhancing therapeutic efficacy in luminal androgen receptor triple-negative breast cancer: exploring chidamide and enzalutamide as a promising combination strategy.

Extensive exploration of the molecular subtypes of triple-negative breast cancer (TNBC) is critical for advancing precision medicine. Notably, the luminal androgen receptor (LAR) subtype has attracted attention for targeted treatment combining androgen receptor antagonists and CDK4/6 inhibitors. Unfortunately, this strategy has proven to be of limited efficacy, highlighting the need for further optimization. Using our center's comprehensive multiomics dataset (n = 465), we identified novel therapeutic targets and evaluated their efficacy through multiple models, including in vitro LAR cell lines, in vivo cell-derived allograft models and ex vivo patient-derived organoids. Moreover, we conducted flow cytometry and RNA-seq analysis to unveil potential mechanisms underlying the regulation of tumor progression by these therapeutic strategies. LAR breast cancer cells exhibited sensitivity to chidamide and enzalutamide individually, with a drug combination assay revealing their synergistic effect. Crucially, this synergistic effect was verified through in vivo allograft models and patient-derived organoids. Furthermore, transcriptomic analysis demonstrated that the combination therapeutic strategy could inhibit tumor progression by regulating metabolism and autophagy. This study confirmed that the combination of histone deacetylase (HDAC) inhibitors and androgen receptor (AR) antagonists possessed greater therapeutic efficacy than monotherapy in LAR TNBC. This finding significantly bolsters the theoretical basis for the clinical translation of this combination therapy and provides an innovative strategy for the targeted treatment of LAR TNBC.

Discovery of novel coumarin-based derivatives as inhibitors of tubulin polymerization targeting the colchicine binding site with potent anti-gastric cancer activities.

In this work, a series of novel coumarin-based derivatives were designed and synthesized as tubulin polymerization inhibitors targeting the colchicine binding site, and their antiproliferative activities against MGC-803, HCT-116 and KYSE30 cells were evaluated. Among them, the compound I-3 (MY-1442) bearing a 6-methoxy-1,2,3,4-tetrahydroquinoline group exhibited most potent inhibitory activities on MGC-803 (IC50 = 0.034 µM), HCT-116 (IC50 = 0.081 µM) and KYSE30 cells (IC50 = 0.19 µM). Further mechanism studies demonstrated that compound I-3 (MY-1442) could directly bind to the colchicine binding site of ß-tubulin to inhibit tubulin polymerization and microtubules at the cellular level. The results of molecular docking indicated there were well binding interactions between compound I-3 (MY-1442) and the colchicine binding site of ß-tubulin. Compound I-3 (MY-1442) also exhibited effective anti-proliferation, pro-apoptosis, and anti-migration abilities against gastric cancer cells MGC-803. Additionally, compound I-3 (MY-1442) could regulate the expression of cell cycle- and apoptosis-related proteins. Importantly, compound I-3 (MY-1442) could significantly inhibit tumor growth in the MGC-803 xenograft tumor model with a TGI rate of 65.5 % at 30 mg/kg/day. Taken together, this work suggested that the coumarin skeleton exhibited great potential to be a key pharmacophore of tubulin polymerization inhibitors for the discovery of anticancer agents.

MUC15 is an independent prognostic factor that promotes metastases of MYCN non-amplified neuroblastoma.

Background: Neuroblastoma (NB) is a cancer that arises from neural-crest-derived sympathoadrenal lineage. Less is known about the pathogenesis and molecular characteristics of MYCN non-amplified (MYCN-NA) NB. Methods: We constructed a signature model targeting mucin family according to RNA sequencing data from GSE49710 dataset, and validated the prognostic performance. We also analyzed the gene expression matrix using DESeq2 R packages to screen the most differential mucin in high-risk NB samples. We further assessed its prognostic value, particularly in MYCN-NA NB samples. Moreover, we performed functional experiments to evaluate the impact of MUC15 overexpression on the migration of MYCN-NA NB cell lines. Results: The 8-mucin signature model showed good prognostic performance in the GSE49710 dataset. Among the mucin genes, MUC15 was significantly upregulated in the high-risk NB cohort and was associated with poor prognosis, especially in MYCN-NA NB samples. Furthermore, MUC15 overexpression and exogenous MUC15 protein enhanced the migration of MYCN-NA NB cell lines. Mechanistically, MUC15 promoted the phosphorylation of focal adhesion kinase (FAK) by inhibiting the expression of MYCT1, a target of c-Myc. Conclusions: Our findings suggested a potential network in controlling NB cell metastasis. Targeting MUC15 in MYCN-NA NB patients could be a promising therapeutic strategy.

Intraoperative Sufentanil Consumption and the Risk of Postoperative Nausea and/or Vomiting: A Retrospective Observational Study.

INTRODUCTION: Post-operative nausea and/or vomiting (PONV) is a common post-operative adverse reaction and has been associated with post-operative sufentanil injection. The assessment of the relationship between intraoperative opioid consumption and PONV has been understudied. This study examined the relationship between intraoperative sufentanil administration and PONV. METHODS: This was a single-center retrospective observational study. Patients who underwent video-assisted thoracoscopic surgery under general anesthesia with the preoperative thoracic paravertebral block between January 2017 and June 2020 at the Peking University People's Hospital were recruited for this study. Patients were grouped into two groups according to whether or not PONV occurred on postoperative day 1 (POD1). The factors associated with PONV were analyzed using logistic regression. RESULTS: A total of 2733 patients, 1510 males and 1223 females, were included in this study. Among them, 143 patients developed PONV, a 5.2% (143/2733) PONV incidence. Logistic regression analysis showed that female, nonsmoking, sufentanil patient-controlled intravenous analgesia (PCIA), POD1 opioids consumption, and a time-weighted average of intraoperative sufentanil (twSuf) were associated with PONV. All patients were further divided into four subgroups based on intraoperative twSuf. Logistic regression analysis revealed that twSuf higher than 0.21 µg kg-1 h-1 was an independent risk factor for PONV. CONCLUSIONS: Intraoperative sufentanil injection with a twSuf higher than 0.21 µg kg-1 h-1 increased the risk of PONV in patients undergoing thoracoscopic surgery under general anesthesia after a preoperative thoracic paravertebral block.

Transcutaneous electrical acupoint stimulation in adult patients receiving gastrectomy/colorectal resection: A randomized controlled trial.

BACKGROUND: Acupuncture promotes the recovery of gastrointestinal function and provides analgesia after major abdominal surgery. The effects of transcutaneous electrical acupoint stimulation (TEAS) remain unclear. AIM: To explore the potential effects of TEAS on the recovery of gastrointestinal function after gastrectomy and colorectal resection. METHODS: Patients scheduled for gastrectomy or colorectal resection were randomized at a 2:3:3:2 ratio to receive: (1) TEAS at maximum tolerable current for 30 min immediately prior to anesthesia induction and for the entire duration of surgery, plus two 30-min daily sessions for 3 consecutive days after surgery (perioperative TEAS group); (2) Preoperative and intraoperative TEAS only; (3) Preoperative and postoperative TEAS only; or (4) Sham stimulation. The primary outcome was the time from the end of surgery to the first bowel sound. RESULTS: In total, 441 patients were randomized; 405 patients (58.4 ± 10.2 years of age; 247 males) received the planned surgery. The time to the first bowel sounds did not differ among the four groups (P = 0.90; log-rank test). On postoperative day 1, the rest pain scores differed significantly among the four groups (P = 0.04; Kruskal-Wallis test). Post hoc comparison using the Bonferroni test showed lower pain scores in the perioperative TEAS group (1.4 ± 1.2) than in the sham stimulation group (1.7 ± 1.1; P = 0.04). Surgical complications did not differ among the four groups. CONCLUSION: TEAS provided analgesic effects in adult patients undergoing major abdominal surgery, and it can be added to clinical practice as a means of accelerating postoperative rehabilitation of these patients.

A review of progress in o-aminobenzamide-based HDAC inhibitors with dual targeting capabilities for cancer therapy.

Histone deacetylases, as a new class of anticancer targets, could maintain homeostasis by catalyzing histone deacetylation and play important roles in regulating the expression of target genes. Due to the fact that simultaneous intervention with dual tumor related targets could improve treatment effects, researches on innovative design of dual-target drugs are underway. HDAC is known as a "sensitizer" for the synergistic effects with other anticancer-target drugs because of its flexible structure design. The synergistic effects of HDAC inhibitor and other target inhibitors usually show enhanced inhibitory effects on tumor cells, and also provide new strategies to overcome multidrug resistance. Many research groups have reported that simultaneously inhibiting HDAC and other targets, such as tubulin, EGFR, could enhance the therapeutic effects. The o-aminobenzamide group is often used as a ZBG group in the design of HDAC inhibitors with potent antitumor effects. Given the prolonged inhibitory effects and reduced toxic side effects of HDAC inhibitors using o-aminobenzamide as the ZBG group, the o-aminobenzamide group is expected to become a more promising alternative to hydroxamic acid. In fact, o-aminobenzamide-based dual inhibitors of HDAC with different chemical structures have been extensively prepared and reported with synergistic and enhanced anti-tumor effects. In this work, we first time reviewed the rational design, molecular docking, inhibitory activities and potential application of o-aminobenzamide-based HDAC inhibitors with dual targeting capabilities in cancer therapy, which might provide a reference for developing new and more effective anticancer drugs.

Tubulin degradation: Principles, agents, and applications.

The microtubule system plays an important role in the mitosis and growth of eukaryotic cells, and it is considered as an appealing and highly successful molecular target for cancer treatment. In fact, microtubule targeting agents, such as paclitaxel and vinblastine, have been approved by FDA for tumor therapy, which have achieved significant therapeutic effects and sales performance. At present, microtubule targeting agents mainly include microtubule-destabilizing agents, microtubule-stabilizing agents, and a few tubulin degradation agents. Although there are few reports about tubulin degradation agents at present, tubulin degradation agents show great potential in overcoming multidrug resistance and reducing neurotoxicity. In addition, some natural drugs could specifically degrade tubulin in tumor cells, but have no effect in normal cells, thus showing a good biosafety profile. Therefore, tubulin degradation agents might exhibit a better application. Currently, some small molecules have been designed to promote tubulin degradation with potent antiproliferative activities, showing the potential for cancer treatment. In this work, we reviewed the reports on tubulin degradation, and focused on the degradation mechanism and important functional groups of chemically synthesized compounds, hoping to provide help for the degradation design of tubulin.

Discovery of novel tranylcypromine-based derivatives as LSD1 inhibitors for gastric cancer treatment.

As an important epigenetic regulator, histone lysine specific demethylase 1 (LSD1) has become an attractive target for the discovery of anticancer agents. In this work, a series of tranylcypromine-based derivatives were designed and synthesized. Among them, compound 12u exhibited the most potent inhibitory potency on LSD1 (IC50 = 25.3 nM), and also displayed good antiproliferative effects on MGC-803, KYSE450 and HCT-116 cells with IC50 values of 14.3, 22.8 and 16.3 µM, respectively. Further studies revealed that compound 12u could directly act on LSD1 and inhibit LSD1 in MGC-803 cells, thereby significantly increasing the expression levels of mono-/bi-methylation of H3K4 and H3K9. In addition, compound 12u could induce apoptosis and differentiation, inhibit migration and cell stemness in MGC-803 cells. All these findings suggested that compound 12u was an active tranylcypromine-based derivative as a LSD1 inhibitor that inhibited gastric cancer.

Analysis of the Influencing Factors of Seeking Intention on COVID-19 Risk Information: A Cross-Sectional Study.

Background: Information seeking, as an important part of the prevention and control of infectious diseases, can lead to positive outcomes by reducing uncertainty and alleviating panic. However, most previous studies have limited their analysis to individual-level psychosocial factors, and little is known about how social-level factors influence individuals' information-seeking intentions. Methods: The cross-sectional survey was conducted from July 30, 2020 to August 15, 2020 in China. We used a convenience sampling strategy to recruit participants from among the Internet users. The structural equation model was used to identify the incentives associated with coronavirus disease 2019 (COVID-19) risk information-seeking intention. Results: In this study, the responses of 871 Internet users who reflected a response rate of 85% were analyzed. Information-seeking intention was found to be directed by informational subjective norms (ISNs), perceived information need, risk knowledge, the sense of community (SOC), and negative affective responses, and ISNs were found to be the strongest driving factor. Individuals with a stronger SOC, which was associated with greater pressure and expectations, show negative affective responses. COVID-19 risk knowledge can affect the information-seeking intention of Internet users not only directly but also indirectly through their perceived information need. In addition, more risk knowledge was associated with a lower perceived risk likelihood. Conclusion: When formulating risk communication strategies, governments and health institutions should take targeted measures to improve the public's SOC and knowledge. This will provide an opportunity to explore the role of individual cognition and environmental risk information in public health.

Design, Synthesis and Biological Evaluation of [1,2,4]Triazolo[1,5-a]pyrimidine Indole Derivatives against Gastric Cancer Cells MGC-803 via the Suppression of ERK Signaling Pathway.

[1,2,4]Triazolo[1,5-a]pyrimidine and indole skeletons are widely used to design anticancer agents. Therefore, in this work, a series of [1,2,4]triazolo[1,5-a]pyrimidine indole derivatives were designed and synthesized by the molecular hybridization strategy. The antiproliferative activities of the target compounds H1-H18 against three human cancer cell lines, MGC-803, HCT-116 and MCF-7, were tested. Among them, compound H12 exhibited the most active antiproliferative activities against MGC-803, HCT-116 and MCF-7 cells, with IC50 values of 9.47, 9.58 and 13.1 µM, respectively, which were more potent than that of the positive drug 5-Fu. In addition, compound H12 could dose-dependently inhibit the growth and colony formation of MGC-803 cells. Compound H12 exhibited significant inhibitory effects on the ERK signaling pathway, resulting in the decreased phosphorylation levels of ERK1/2, c-Raf, MEK1/2 and AKT. Furthermore, compound 12 induced cell apoptosis and G2/M phase arrest, and regulated cell cycle-related and apoptosis-related proteins in MGC-803 cells. Taken together, we report here that [1,2,4]triazolo[1,5-a]pyrimidine indole derivatives, used as anticancer agents via the suppression of ERK signaling pathway and the most active compound, H12, might be a valuable hit compound for the development of anticancer agents.

Discovery of N-benzylarylamide derivatives as novel tubulin polymerization inhibitors capable of activating the Hippo pathway.

Novel N-benzylarylamide saderivatives were designed and synthesized, and their antiproliferative activities were explored. Some of 51 target compounds exhibited potent inhibitory activities against MGC-803, HCT-116 and KYSE450 cells with IC50 values in two-digit nanomolar. Compound I-33 (MY-875) displayed the most potent antiproliferative activities against MGC-803, HCT-116 and KYSE450 cells (IC50 = 0.027, 0.055 and 0.067 µM, respectively) and possessed IC50 values ranging from 0.025 to 0.094 µM against other 11 cancer cell lines. Further mechanism studies indicated that compound I-33 (MY-875) inhibited tubulin polymerization (IC50 = 0.92 µM) by targeting the colchicine bingding site of tubulin. Compound I-33 (MY-875) disrupted the construction of the microtubule networks and affected the mitosis in MGC-803 and SGC-7901 cells. In addition, although it acted as a colchicine binding site inhibitor, compound I-33 (MY-875) also activated the Hippo pathway to promote the phosphorylation status of MST and LATS, resulting in the YAP degradation in MGC-803 and SGC-7901 cells. Due to the degradation of YAP, the expression levels of TAZ and Axl decreased. Because of the dual actions on colchicine binding site and Hippo pathway, compound I-33 (MY-875) dose-dependently inhibited cell colony formatting ability, arrested cells at the G2/M phase and induced cells apoptosis in MGC-803 and SGC-7901 cells. Moreover, compound I-33 (MY-875) could regulate the levels of cell cycle and apoptosis regulatory proteins in MGC-803 and SGC-7901 cells. Furthermore, molecular docking analysis suggested that the hydrogen bond and hydrophobic interactions made compound I-33 (MY-875) well bind into the colchicine binding site of tubulin. Collectively, compound I-33 (MY-875) is a novel anti-gastric cancer agent and deserves to be further investigated for cancer therapy by targeting the colchicine binding site of tubulin and activating the Hippo pathway.

Responses of earthworm Metaphire vulgaris gut microbiota to arsenic and nanoplastics contamination.

The growing contamination of arsenic and plastics has severely effects on the soil fauna health, including shifts of gut microbiota community. A few studies have focused on effects of microplastics and metal(loid) in soil and fauna gut microbiome. However, the environmental effect of nanoplastics and arsenic on the earthworm gut microbiota, especially on arsenic biotransformation in the gut, remain largely unknown. Here, a microcosm study was performed to explore the effects of nanoplastics and arsenic on the microbiota characteristics in earthworm Metaphire vulgaris gut using Illumina high throughput sequencing, and to investigate changes in the gut microbiota-mediated arsenic biotransformation genes (ABGs) by using high-throughput quantitative PCR. Our results demonstrated that the concentration of arsenic in the earthworm body tissues after exposure to arsenic and nanoplastics was significantly lower from that with arsenic alone exposure. Moreover, the clearly different bacterial community was observed in the soil compared with the earthworm gut, which was dominated by Proteobacteria, Actinobacteria, and Firmicutes at phylum level. Arsenic exposure significantly disturbed bacterial community structure in the earthworm gut, but exposure to nanoplastics did not induce gut microbiota changes. More interestingly, nanoplastics can relieve adverse effect of arsenic on the gut microbiota possibly by adsorbing arsenic. In addition, a total of 16 ABGs were detected, and predominant genes involved in arsenic reduction and transport process were observed in the earthworm guts. In short, this study provides a new picture of the effects of nanoplastics and arsenic on the gut microbiota and arsenic biotransformation in soil fauna gut.

Mechanisms for Induction of Microbial Extracellular Proteases in Response to Exterior Proteins.

Proteins are a main organic nitrogen source for microorganisms. Many heterotrophic microorganisms secrete extracellular proteases (ex-proteases) to efficiently decompose proteins into oligopeptides and amino acids when exterior proteins are required for growth. These ex-proteases not only play important roles in microbial nutrient acquisition or host infection but also contribute greatly to the global recycling of carbon and nitrogen. Moreover, may microbial ex-proteases have important applications in industrial, medical, and biotechnological areas. Therefore, uncovering the mechanisms by which microorganisms initiate the expression of ex-protease genes in response to exterior proteins is of great significance. In this review, the progress made in understanding the induction mechanisms of microbial ex-proteases in response to exterior proteins is summarized, with a focus on the inducer molecules, membrane sensors, and downstream pathways. Problems to be solved for better understanding of the induction mechanisms of microbial ex-proteases are also discussed.

The Putative Methyltransferase TlLAE1 Is Involved in the Regulation of Peptaibols Production in the Biocontrol Fungus Trichoderma longibrachiatum SMF2.

The biocontrol fungus Trichoderma longibrachiatum SMF2 secretes a large quantity of peptaibols that have been shown to have a range of biological activities and therefore great application values. However, the mechanism of the regulatory expression of peptaibols is still unclear. The putative methyltransferase LaeA/LAE1 is a global regulator involved in the biosynthesis of some secondary metabolites in filamentous fungi. In this study, we demonstrated that the ortholog of LaeA/LAE1 in the biocontrol fungus T. longibrachiatum SMF2, TlLAE1, plays an important role in the regulation of peptaibols production. Deletion of Tllae1 resulted in a slight negative impact on mycelial growth, and a significant defect in conidial production. Deletion of Tllae1 also compromised the production of peptaibols to a large degree. Further analyses indicated that this defect occurred at the transcriptional level of the two synthetases-encoding genes, tlx1 and tlx2, which are responsible for peptaibols production. By contrast, constitutive expression of Tllae1 in T. longibrachiatum SMF2 led to 2-fold increased peptaibols production, suggesting that this is a strategy to improve peptaibols production in Trichoderma fungi. These results demonstrate the important role of LAE1 in the regulation of peptaibols production in T. longibrachiatum SMF2.

Traditional Chinese medicine (Liang-Xue-Di-Huang Decoction) for hemorrhoid hemorrhage: Study Protocol Clinical Trial (SPIRIT Compliant).

BACKGROUND: Hemorrhoidal disease (HD) is one of the commonest proctologic condition in the general population. Medical therapy for HD has not been formally confirmed due to the inconsistent of results. Liang-Xue-Di-Huang Decoction, a kind of ancient Chinese classical prescription, has been used to treat HD from the 19th century in China. However, clinical research of Liang-Xue-Di-Huang Decoction in the treatment of HD was lack. We designed this study to evaluate the efficacy and safety of Liang-Xue-Di-Huang Decoction in the treatment of HD. METHODS/DESIGN: A randomized, controlled, double blind, double-mimetic agent, and multicenter trial to evaluate the efficacy and safety of Liang-Xue-Di-Huang Decoction is proposed. HD patients (stage I, II, III) will be randomly assigned into experimental group or control group. HD patients will receive a 7-day treatments and a 7-day follow-up. The primary outcome measure is the Hemorrhoid Bleeding Score in 7 and 14 days. The Secondary outcome measures are Goligher prolapse score and quality-of-life score in 7 and 14 days. DISCUSSION: This study will provide objective evidences to evaluate the efficacy and safety of Liang-Xue-Di-Huang Decoction in treatment of HD.

The Possible Anti-Inflammatory Effect of Dehydrocostus Lactone on DSS-Induced Colitis in Mice.

BACKGROUND: Dehydrocostus lactone (DL), one of the main active constituents in Aucklandia lappa Decne. (Muxiang), reported to have anti-inflammatory, antiulcer, and immunomodulatory properties. However, the effect of DL on ulcerative colitis (UC) has not been reported. To analyze the anti-inflammatory potential role of DL in UC, we provide a mechanism for the pharmacological action of DL. METHODS: The experimental model of UC was induced by using oral administration of 2% dextran sulfate sodium (DSS) with drinking water in BALB/c mice. Mesalazine (Mes, 0.52 g/kg/d), DL-high doses (DL-H, 20 mg/kg/d), DL-middle doses (DL-M, 15 mg/kg/d), DL-low doses (DL-L, 10 mg/kg/d) were gavaged once a day from day 4 to day 17. Disease activity index (DAI) was calculated daily. On day 18, mice were rapidly dissected and the colorectal tissues were used to detect the levels of UC-related inflammatory cytokines (TNF-α, IL-1ß, MCP-1, MPO, SOD, IL-6, IL-17, and IL-23), IL-6/STAT3 inflammatory signaling pathway (iNOS, COX2, IL-6, GP130, L-17, and IL-23), and colorectal mucosal barrier-related regulatory factors (MUC2, XBP1s, and α, IL-1. RESULTS: DL reduced the colorectal inflammation histological assessment, decreased UC-related inflammatory cytokines (TNF-α, IL-1ß, MCP-1, MPO, SOD, IL-6, IL-17, and IL-23), IL-6/STAT3 inflammatory signaling pathway (iNOS, COX2, IL-6, GP130, L-17, and IL-23), and colorectal mucosal barrier-related regulatory factors (MUC2, XBP1s, and α, IL-1. CONCLUSIONS: DL possessed the potential of anti-inflammatory effect to treated colitis. The protective mechanism of DL may involve in reducing inflammation and improving colorectal barrier function via downregulating the IL-6/STAT3 signaling.

Characterization of a highly stable α-galactosidase from thermophilic Rasamsonia emersonii heterologously expressed in a modified Pichia pastoris expression system.

BACKGROUND: Structurally stable α-galactosidases are of great interest for various biotechnological applications. More thermophilic α-galactosidases with high activity and structural stability have therefore to be mined and characterized. On the other hand, few studies have been performed to prominently enhance the AOX1 promoter activity in the commonly used Pichia pastoris system, in which production of some heterologous proteins are insufficient for further study. RESULTS: ReGal2 encoding a thermoactive α-galactosidase was identified from the thermophilic (hemi)cellulolytic fungus Rasamsonia emersonii. Significantly increased production of ReGal2 was achieved when ReGal2 was expressed in an engineered Pastoris pichia expression system with a modified AOX1 promoter and simultaneous fortified expression of Mxr1 that is involved in transcriptionally activating AOX1. Purified ReGal2 exists as an oligomer and has remarkable thermo-activity and thermo-tolerance, exhibiting maximum activity of 935 U/mg towards pNPGal at 80 °C and retaining full activity after incubation at 70 °C for 60 h. ReGal2 is insensitive to treatments by many metal ions and exhibits superior tolerance to protein denaturants. Moreover, ReGal2 efficiently hydrolyzed stachyose and raffinose in soybeans at 70 °C in 3 h and 24 h, respectively. CONCLUSION: A modified P. pichia expression system with significantly enhanced AOX1 promoter activity has been established, in which ReGal2 production is markedly elevated to facilitate downstream purification and characterization. Purified ReGal2 exhibited prominent features in thermostability, catalytic activity, and resistance to protein denaturants. ReGal2 thus holds great potential in relevant biotechnological applications.

Enhancing peptaibols production in the biocontrol fungus Trichoderma longibrachiatum SMF2 by elimination of a putative glucose sensor.

Trichoderma spp. are main producers of peptide antibiotics known as peptaibols. While peptaibols have been shown to possess a range of biological activities, molecular understanding of the regulation of their production is largely unclear, which hampers the production improvement through genetic engineering. Here, we demonstrated that the orthologue of glucose sensors in the outstanding biocontrol fungus Trichoderma longibrachiatum SMF2, TlSTP1, participates in the regulation of peptaibols production. Deletion of Tlstp1 markedly impaired hyphal growth and conidiation, but significantly increased peptaibols yield by 5-fold for Trichokonins A and 2.6-fold for Trichokonins B. Quantitative real-time polymerase chain reaction analyses showed that the increased peptaibols production occurs at the transcriptional levels of the two nonribosomal peptide synthetase encoding genes, tlx1 and tlx2. Transcriptome analyses of the wild type and the Tlstp1 mutant strains indicated that TlSTP1 exerts a regulatory effect on a set of genes that are involved in a number of metabolic and cellular processes, including synthesis of several other secondary metabolites. These results suggest an important role of TlSTP1 in the regulation of vegetative growth and peptaibols production in T. longibrachiatum SMF2 and provide insights into construction of peptaibol-hyperproducing strains through genetic engineering.

Morphological and molecular identification of Habronema spp. (Nematoda: Habronematidae) from donkeys in Xinjiang, China, and notes on the taxonomical status of Habronema majus (Creplin, 1849) and H. microstoma (Schneider, 1866).

Habronematid nematodes were collected from the stomachs of donkeys, Equus asinus L., in the Tarim Basin, Xinjiang, China. After examination by light and scanning electron microscopy, Habronema muscae (Carter, 1861) and H. majus (Creplin, 1849) were identified. The morphology of our specimens representing H. muscae (Carter, 1861) agreed well with previous redescriptions in the shape of the lateral lips, origin of the lateral alae, ratio of left and right spicules, and number and arrangement of caudal papillae. However, H. majus (Creplin, 1849) differs from H. microstoma (Schneider, 1866) in the arrangement of the caudal papillae in the male. Moreover, molecular analysis also showed interspecific differences of 26.2-28.2% in ITS2 and 8.6-8.9% in cox1 between H. majus and H. microstoma, a divergence much higher than the known intraspecific variation of Habronema spp. (6.6-8.7% in ITS2; 0.2-2.2% in cox1). The results indicate that both H. microstoma (Schneider, 1866) and H. majus (Creplin, 1849) are valid species.

Structural insights into the multispecific recognition of dipeptides of deep-sea gram-negative bacterium Pseudoalteromonas sp. strain SM9913.

UNLABELLED: Peptide uptake is important for nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria absorb peptides is still not fully understood. DppA is the periplasmic dipeptide binding protein of dipeptide permease (Dpp; an important peptide transporter in bacteria) and exclusively controls the substrate specificity of Dpp. Here, the substrate binding specificity of deep-sea Pseudoalteromonas sp. strain SM9913 DppA (PsDppA) was analyzed for 25 different dipeptides with various properties by using isothermal titration calorimetry measurements. PsDppA showed binding affinities for 8 dipeptides. To explain the multispecific substrate recognition mechanism of PsDppA, we solved the crystal structures of unliganded PsDppA and of PsDppA in complex with 4 different types of dipeptides (Ala-Phe, Met-Leu, Gly-Glu, and Val-Thr). PsDppA alternates between an "open" and a "closed" form during substrate binding. Structural analyses of the 4 PsDppA-substrate complexes combined with mutational assays indicate that PsDppA binds to different substrates through a precise mechanism: dipeptides are bound mainly by the interactions between their backbones and PsDppA, in particular by anchoring their N and C termini through ion-pair interactions; hydrophobic interactions are important in binding hydrophobic dipeptides; and Lys457 is necessary for the binding of dipeptides with a C-terminal glutamic acid or glutamine. Additionally, sequence alignment suggests that the substrate recognition mechanism of PsDppA may be common in Gram-negative bacteria. All together, our results provide structural insights into the multispecific substrate recognition mechanism of marine Gram-negative bacterial DppA, which provides a better understanding of the mechanisms of marine bacterial peptide uptake. IMPORTANCE: Peptide uptake plays a significant role in nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria recognize and absorb peptides is still unclear. This study analyzed the substrate binding specificity of deep-sea Pseudoalteromonas sp. strain SM9913 DppA (PsDppA; the dipeptide-binding protein of dipeptide permease) and solved the crystal structures of unliganded PsDppA and PsDppA in complex with 4 different types of dipeptides. The multispecific recognition mechanism of PsDppA for dipeptides is explained based on structural and mutational analyses. We also find that the substrate-binding mechanism of PsDppA may be common in Gram-negative bacteria. This study sheds light on marine Gram-negative bacterial peptide uptake and marine nitrogen cycling.