Combined analysis of the effects of hypoxia and oxidative stress on DNA methylation and the transcriptome in HTR-8/SVneo trophoblast cells.

The alterations in DNA methylation and transcriptome in trophoblast cells under conditions of low oxygen and oxidative stress have major implications for pregnancy-related disorders. However, the exact mechanism is still not fully understood. In this study, we established models of hypoxia (H group) and oxidative stress (HR group) using HTR-8/SVneo trophoblast cells and performed combined analysis of genome-wide DNA methylation changes using reduced representation bisulphite sequencing and transcriptome expression changes using RNA sequencing. Our findings revealed that the H group exhibited a higher number of differentially methylated genes and differentially expressed genes than the HR group. In the H group, only 0.90% of all differentially expressed genes displayed simultaneous changes in DNA methylation and transcriptome expression. After the threshold was expanded, this number increased to 6.29% in the HR group. Notably, both the H group and HR group exhibited concurrent alterations in DNA methylation and transcriptome expression within Axon guidance and MAPK signalling pathway. Among the top 25 differentially methylated KEGG pathways in the promoter region, 11 pathways were commonly enriched in H group and HR group, accounting for 44.00%. Among the top 25 KEGG pathways in transcriptome with significant differences between the H group and HR group, 10 pathways were consistent, accounting for 40.00%. By integrating our previous data on DNA methylation from preeclamptic placental tissues, we identified that the ANKRD37 and PFKFB3 genes may contribute to the pathogenesis of preeclampsia through DNA methylation-mediated transcriptome expression under hypoxic conditions.

Molecular characterization and expression analyses of five genes involved in the MyD88-dependent pathway of yellow catfish (Pelteobagrus fulvidraco) responding to challenge of Aeromonas hydrophila.

MyD88-dependent pathway mediated by Toll-like receptor is one of the vital ways activating immune responses. In order to identify the role of MyD88-dependent signaling pathway in yellow catfish, the Pf_MyD88, Pf_IRAK4, Pf_IRAK1, Pf_TRAF6 and Pf_NFκB1 (p105) (Pf: abbreviation of Pelteobagrus fulvidraco) were cloned and characterized respectively. The Pf_MyD88, Pf_IRAK4, Pf_IRAK1 and Pf_TRAF6 were all highly conserved among species and showed the highest homology to that of Pangasianodon hypophthalmus. Pf_NFκB1 showed the highest homology to that of Ictalurus punetaus. All of the five genes showed similar expression patterns in various tissues, with the highest expression level in the liver. These genes also showed similar expression levels in different embryonic development stages, except Pf_IRAK4. The higher expression level was detected from fertilized eggs to 1 day post hatching (dph), lower expression from 3 dph to 30 dph. After stimulation of inactivated Aeromonas hydrophila, the mRNA expressions of Pf_MyD88, Pf_IRAK4, Pf_IRAK1, Pf_TRAF6 and Pf_NFκB1 were significantly increased at 24 h in the liver, spleen, head kidney and trunk kidney, suggesting that all the five genes were involved in the innate immune response of yellow catfish. These results showed that MyD88-dependent signaling pathway plays important roles for disease defensing in the innate immune response. Meanwhile, inactivated A. hydrophila can cause strong innate immune response, which provides theoretical bases for the application of inactivated vaccines in defense against bacterial diseases of teleost.

Detection of drug safety signal of drug-induced neutropenia and agranulocytosis in all-aged patients using electronic medical records.

PURPOSE: We explored the adverse drug reaction signals of drug-induced neutropenia (DIN) and drug-induced agranulocytosis (DIA) in hospitalized patients and evaluated the novelty of these correlations. METHOD: A two-step method was established to identify the relationship between drugs and DIN or DIA using 5-year electronic medical records (EMRs) obtained from 242 000 patients at Qilu Hospital of Shandong University. First, the drugs suspected to induce DIN or DIA were selected. The associations between suspected drugs and DIN or DIA were evaluated by a retrospective cohort study using unconditional logistic regression analysis and multiple linear regression model. RESULTS: Twelve suspected drugs (vancomycin, meropenem, voriconazole, acyclovir, ganciclovir, fluconazole, oseltamivir, linezolid, compound borax solution, palonosetron, polyene phosphatidylcholine, and sulfamethoxazole) were associated with DIN, and six suspected drugs (vancomycin, voriconazole, acyclovir, ganciclovir, fluconazole, and oseltamivir) were associated with DIA. The multivariate linear regression model revealed that nine drugs (vancomycin, meropenem, voriconazole, ganciclovir, fluconazole, oseltamivir, compound borax solution, palonosetron, and polyene phosphatidylcholine) and four drugs (vancomycin, voriconazole, ganciclovir, and fluconazole) were found to be associated with DIN and DIA, respectively. While logistic regression analysis revealed that palonosetron and ganciclovir were associated with DIN and DIA, respectively. CONCLUSION: Palonosetron and ganciclovir were found to be correlated with drug-induced granulocytopenia. The results of this study provide an early warning of drug safety signals for drug-induced granulocytopenia, facilitating a quick and appropriate response for clinicians.

Successful Kidney-Sparing Systemic Therapy for a High-Risk Ureteral Carcinoma Case.

Upper tract urothelial carcinoma (UTUC) refers to the malignancies located from renal calices toward the end of the ureter and could be classified as renal pelvis carcinoma and ureteral carcinoma. For high-risk UTUC cases with a normal contralateral kidney, radical nephroureterectomy is the standard treatment. As for low-risk UTUC cases or solitary kidney cases, kidney-sparing therapy may be a better choice. Besides, to prevent postoperative recurrence, systemic therapy should be applied, though the investigation is still ongoing. In this case report, we reported a rare case diagnosed with high-risk ureteral carcinoma, but he underwent kidney-sparing therapy due to chronic kidney disease. Recurrence has occurred after segmental ureterectomy. But through the utilization of ablation, bladder instillation, and tislelizumab, endoscopy and CT were normal in the follow-up (the patient refused to take washings from the upper urinary tract) for more than a year. In all, the utilization of ureteroscopic retrograde tumor ablation, BCG bladder instillation, and tislelizumab injection to treat high-risk ureteral carcinoma for kidney-sparing therapy have filled in the gap in this field, which should be promoted to help more patients in similar situations.

Identification of microRNA transcriptome throughout the lifespan of yak (Bos grunniens) corpus luteum.

The corpus luteum (CL) is a temporary organ that plays a critical role for female fertility by maintaining the estrous cycle. MicroRNA (miRNA) is a class of non-coding RNAs involved in various biological processes. However, there exists limited knowledge of the role of miRNA in yak CL. In this study, we used high-throughput sequencing to study the transcriptome dynamics of miRNA in yak early (eCL), middle (mCL) and late-stage CL (lCL). A total of 6,730 miRNAs were identified, including 5,766 known and 964 novels miRNAs. Three miRNAs, including bta-miR-126-3p, bta-miR-143 and bta-miR-148a, exhibited the highest expressions in yak CLs of all the three stages. Most of the miRNAs were 20-24 nt in length and the peak was at 22 nt. Besides, most miRNAs with different lengths displayed significant uracil preference at the 5'-end. Furthermore, 1,067, 280 and 112 differentially expressed (DE) miRNAs were found in eCL vs. mCL, mCL vs. lCL, and eCL vs. lCL, respectively. Most of the DE miRNAs were down-regulated in the eCL vs. mCL and eCL vs. lCL groups, and up-regulated in the mCL vs. lCL group. A total of 18,904 target genes were identified, with 18,843 annotated. Pathway enrichment analysis of the DE miRNAs target genes illustrated that the most enriched cellular process in each group included pathways in cancer, PI3K-Akt pathway, endocytosis, and focal adhesion. A total of 20 putative target genes in 47 DE miRNAs were identified to be closely associated with the formation, function or regression of CL. Three DE miRNAs, including bta-miR-11972, novel-miR-619 and novel-miR-153, were proved to directly bind to the 3'-UTR of their predicated target mRNAs, including CDK4, HSD17B1 and MAP1LC3C, respectively. Both of these DE miRNAs and their target mRNAs exhibited dynamic expression profiles across the lifespan of yak CL. This study presents a general basis for understanding of the regulation of miRNA on yak CL and also provides a novel genetic resource for future analysis of the gene network during the estrous cycle in the yak.

Ammonium sulfate supplementation enhances erythromycin biosynthesis by augmenting intracellular metabolism and precursor supply in Saccharopolyspora erythraea.

In this study, the cellular metabolic mechanisms regarding ammonium sulfate supplementation on erythromycin production were investigated by employing targeted metabolomics and metabolic flux analysis. The results suggested that the addition of ammonium sulfate stimulates erythromycin biosynthesis. Targeted metabolomics analysis uncovered that the addition of ammonium sulfate during the late stage of fermentation resulted in an augmented intracellular amino acid metabolism pool, guaranteeing an ample supply of precursors for organic acids and coenzyme A-related compounds. Therefore, adequate precursors facilitated cellular maintenance and erythromycin biosynthesis. Subsequently, an optimal supplementation rate of 0.02 g/L/h was determined. The results exhibited that erythromycin titer (1311.1 µg/mL) and specific production rate (0.008 mmol/gDCW/h) were 101.3% and 41.0% higher than those of the process without ammonium sulfate supplementation, respectively. Moreover, the erythromycin A component proportion increased from 83.2% to 99.5%. Metabolic flux analysis revealed increased metabolic fluxes with the supplementation of three ammonium sulfate rates.

Promoted photocarrier separation by dipole engineering in two-dimensional perovskite/C2N van der Waals heterostructures.

Due to the aggravation of environmental pollution and the energy crisis, it is urgent to develop and design environment-friendly and efficient photocatalysts for water splitting. van der Waals heterostructures composed of different two-dimensional materials offer an easily accessible way to combine properties of individual materials for applications. Herein, a novel Cs3Bi2I9/C2N heterostructure is proposed through first-principles calculations. The structural, electronic, and optical properties, as well as the charge transfer mechanism at the interface of Cs3Bi2I9/C2N are systematically investigated. Due to the difference between the work functions of Cs3Bi2I9 and C2N monolayers, when they are constructed into heterostructures, redistribution of charge occurs in the whole structure, and some of the charge transfer occurs at the interface due to the formation of an internal electric field. The band structure of Cs3Bi2I9/C2N has type-II band alignment, and the band edge position as well as the band-gap value of the heterostructure are suitable for visible light water splitting. The in-plane biaxial strain, interfacial spacing, and external electric field can effectively modulate the electronic structure and photocatalytic performance of the heterostructure. Under certain conditions, the heterostructure can be changed from type-II to type-I band alignment, accompanied by the transition from an indirect band-gap semiconductor to a direct band-gap semiconductor. Moreover, the intrinsic anion defect (I vacancy) at different positions, as donor defects, can introduce defect levels near the conduction band edge, which affects the transition of photogenerated carriers in these systems. Our findings provide a theoretical design for strategies to improve the performance of two-dimensional perovskites/C2N in photocatalytic and optoelectronic applications.

miR-26a-5p protects against drug-induced liver injury via targeting bid.

BACKGROUNDS: miR-26a-5p is a short noncoding RNA that is abnormally expressed in drug-induced liver injury (DILI), but its pathophysiologic role in the mechanism of disease in DILI is still vague. METHODS: The expression of miR-26a-5p, viability of hepatic stellate cells (HSCs) proliferation, and apoptosis were explored via real-time PCR, CCK-8 assay, Tunel fluorescence, and flow cytometry. The expression of Bid was detected via Western blot assays, real-time PCR, and immunofluorescence. The apoptosis-associated proteins were determined through Western blot. The interaction between miR-26a-5p and Bid was measured via Dual luciferase reporter assay. RESULTS: miR-26a-5p expression was greatly decreased in HSCs and serum treated with azithromycin, simvastatin and diclofenac sodium, respectively. Hepatocyte viability was largely suppressed while hepatocyte apoptosis was markedly increased in DILI. Correspondingly, the apoptosis-associated proteins including Bid, caspase-8 and cytochrome C in HSCs were significantly upregulated when treated with either of these drugs. Moreover, miR-26a-5p interacted with Bid, and hepatocyte proliferation and apoptosis influenced by miR-26a-5p mimics were obviously reversed when co-treated with overexpressed Bid plasmids. CONCLUSIONS: miR-26a-5p played a protective role against DILI via targeting Bid.

Ectopic bronchogenic cyst in the retroperitoneal region: a case report and literature review of adult patients.

BACKGROUND: Bronchogenic cyst is congenital aberration of bronchopulmonary malformation, which is rarely encountered in the abdomen and retroperitoneum. We present a case report and literature review of retroperitoneal bronchogenic cyst. CASE PRESENTATION: A 53-year-old female presented to outpatient clinic for a routine checkup of lumbar intervertebral disc herniation. She received a contrast computed tomography scan of the abdomen which revealed a retroperitoneal cystic lesion below the left crura of diaphragm. Afterward, the patient underwent a laparoscopic excision of the cystic lesion and was discharged uneventfully at postoperative day 4. Histopathological findings confirmed the diagnosis of retroperitoneal bronchogenic cyst. Our literature review identified 55 adult cases in recent two decades. The average age at diagnosis was 43.2 (range 17-69) years. 44 (80%) cases had a retroperitoneal cyst on the left side, and 52 (94.5%) cases underwent curative excision through open or laparoscopic surgery. In the available follow up of cases, there was no recurrence after surgery. CONCLUSIONS: Bronchogenic cyst is rare in the retroperitoneal region. It should be considered as one of the differential diagnoses of a retroperitoneal neoplasm.

A concealed inguinal presentation of a gastrointestinal stromal tumor (GIST): a case report and literature review.

BACKGROUND: Gastrointestinal stromal tumor (GIST) can arise anyplace along the gastrointestinal (GI) tract. The uncommon tumor location in groin area is rarely reported. CASE PRESENTATION: We herein reported a metastasized case presented as GI hemorrhage complicated with indirect hernia, and underwent tumor cytoreduction, herniorrhaphy and chemotherapy for jejunal GIST. The case was described consecutively based on the process of surgical management, with a good follow-up result. A literature review by searching similar case reports from two national medical databases was performed to summarize clinical features of such unusual presentation of GIST, which included hernia characteristics, short- and long-term outcomes of this disease. It showed GIST presenting as groin hernia was rarely reported and all available 11 cases suggested a primary tumor and required both tumor resection and hernia repair. The long-term results indicated 64.3% overall survival at 5 years after the incidental diagnosis. CONCLUSIONS: Inguinal hernia is an extremely rare presentation of GIST, with limited case reports available in the literature. A radical involving tumor resection plus hernia repair is an optimal surgical approach for such uncommon condition. An adjuvant medication mounting on mutated KIT gene should be strictly followed for high risk cases.

CT-based radiomics scores predict response to neoadjuvant chemotherapy and survival in patients with gastric cancer.

BACKGROUND: Neoadjuvant chemotherapy is a promising treatment option for potential resectable gastric cancer, but patients' responses vary. We aimed to develop and validate a radiomics score (rad_score) to predict treatment response to neoadjuvant chemotherapy and to investigate its efficacy in survival stratification. METHODS: A total of 106 patients with neoadjuvant chemotherapy before gastrectomy were included (training cohort: n = 74; validation cohort: n = 32). Radiomics features were extracted from the pre-treatment portal venous-phase CT. After feature reduction, a rad_score was established by Randomised Tree algorithm. A rad_clinical_score was constructed by integrating the rad_score with clinical variables, so was a clinical score by clinical variables only. The three scores were validated regarding their discrimination and clinical usefulness. The patients were stratified into two groups according to the score thresholds (updated with post-operative clinical variables), and their survivals were compared. RESULTS: In the validation cohort, the rad_score demonstrated a good predicting performance in treatment response to the neoadjuvant chemotherapy (AUC [95% CI] =0.82 [0.67, 0.98]), which was better than the clinical score (based on pre-operative clinical variables) without significant difference (0.62 [0.42, 0.83], P = 0.09). The rad_clinical_score could not further improve the performance of the rad_score (0.70 [0.51, 0.88], P = 0.16). Based on the thresholds of these scores, the high-score groups all achieved better survivals than the low-score groups in the whole cohort (all P < 0.001). CONCLUSION: The rad_score that we developed was effective in predicting treatment response to neoadjuvant chemotherapy and in stratifying patients with gastric cancer into different survival groups. Our proposed strategy is useful for individualised treatment planning.

Sesquiterpenoids Produced by Combining Two Sesquiterpene Cyclases with Promiscuous Myxobacterial CYP260B1.

Sesquiterpenes represent a class of important terpenoids with high structural diversity and a wide range of applications. The cyclized core skeletons are generated by sesquiterpene cyclases, and the structural diversity is further increased by a series of modification steps. Cytochromes P450 (P450s) are a class of monooxygenases and one of the main contributors to the structural diversity of natural products. Some of these P450s show a broad substrate range and might be promising candidates for the implementation of cascade reactions. In this study, a combinatorial biosynthesis approach was utilized by the combination of a promiscuous myxobacterial P450 (CYP260B1) with two sesquiterpene cyclases (FgJ01056, FgJ09920) of filamentous fungi. Two oxygenated products, culmorin and culmorone, and a new compound, koraidiol, were successfully generated and characterized. This approach suggests the potential use of noncognate P450s to produce novel oxygenated terpenoids, or to generate a novel biosynthetic route for known terpenoids by a combinatorial biosynthesis strategy.

Predictive ability of prognostic nutritional index in surgically resected gastrointestinal stromal tumors: a propensity score matching analysis.

BACKGROUND: Recent findings have shown that inflammation indices are associated with prognosis in various malignancies. However, the usefulness of inflammation indices including platelet-to-lymphocyte ratio, neutrophil-to-lymphocyte ratio and prognostic nutritional index for gastrointestinal stromal tumors (GISTs) remains controversial. METHODS: We retrospectively reviewed 340 primary localized GIST patients who had received surgical resection between 2005 and 2015 to investigate the effect of platelet-to-lymphocyte ratio, neutrophil-to-lymphocyte ratio and prognostic nutritional index on prognosis. 206 patients were selected by propensity score matching to control selection biases. RESULTS: Kaplan-Meier analysis and the log rank test demonstrated that high prognostic nutritional index (≥43.9) was significantly correlated with better recurrence-free survival (RFS) (P<0.001). Among the three inflammatory indices, only preoperative high prognostic nutritional index was an independent prognostic factor for survival [hazard ratio (HR) 0.509; 95% confidence interval (CI) 0.266-0.872; P = 0.031] in multivariate analysis. After propensity score matching, elevated prognostic nutritional index was still a predictor for RFS (HR = 0.498; 95% CI 0.253-0.971; P = 0.042) in the multivariate analyses. In addition, prognostic nutritional index was a significant prognostic factor for GISTs within the National Institutes of Health (NIH) high and very low/low-risk categories. Incorporation prognostic nutritional index into the NIH risk criteria improved the prognostic stratification (c-index, 0.725 vs. 0.686, p = 0.039). CONCLUSIONS: High prognostic nutritional index is a predictor of improved survival for surgically resected GISTs and incorporation prognostic nutritional index into NIH risk criteria improves the predictive accuracy.

Deperitoneum biological mesh repair for abdominal wall hernia: a novel wound healing promotion idea.

PURPOSE: Nowadays, biological matrix has become more widely applied than synthetic mesh for the surgical management of ventral hernia. Conventionally, such biodegradable matrix is commonly placed in an intraperitoneal or extraperitoneal position to reinforce the abdominal wall during surgery. Herein, we introduce our novel idea to deliver such biological material. MATERIAL AND METHODS: After contrast-enhanced CT-scan via lateral decubitus confirmed the position of ventral hernias, 11 patients underwent deperitoneum biological mesh repair by open or laparoscopic approach. During surgery, biological material was placed in preperitoneal position with elimination of matrix-covered peritoneum meanwhile. No bridge repair was allowed for this technique. Postoperative complications were prospectively documented. RESULTS: Laparoscopic and open repair were performed in six and five patients, respectively. The mean operative time was 115 min, with no significant difference between the two procedures. All patients had quick recovery and returned to their normal life, with median five days (range, 3-12 days) of hospital stay after surgery. Although wound dehiscence and chronic pain occurred in three (27.3%) patients, no additional surgery was required. No recurrence case was observed within the one-year follow-up period. CONCLUSION: This novel approach could be safely performed in ventral hernia patients. Early evaluation of this surgical technique demonstrates quick recovery and minimal complications.

Genome mining in Trichoderma viride J1-030: discovery and identification of novel sesquiterpene synthase and its products.

Sesquiterpene synthases in Trichoderma viride have been seldom studied, despite the efficiency of filamentous fungi for terpenoid production. Using the farnesyl diphosphate-overexpressing Saccharomyces cerevisiae platform to produce diverse terpenoids, we herein identified an unknown sesquiterpene synthase from T. viride by genome mining and determined the structure of its corresponding products. One new 5/6 bicyclic sesquiterpene and its esterified derivative were characterised by GC-MS and 1D and 2D NMR spectroscopy. To the best of our knowledge, this is the first well-identified sesquiterpene synthase from T. viride to date.

Long Non-Coding RNA RP11-789C1.1 Suppresses Epithelial to Mesenchymal Transition in Gastric Cancer Through the RP11-789C1.1/MiR-5003/E-Cadherin Axis.

BACKGROUND/AIMS: Gastric cancer (GC) is a common malignancy with a global incidence that ranks fourth among all tumor types. Epithelial-to-mesenchymal transition (EMT) is a tumor biological process with a role in GC cell metastasis. Long non-coding RNAs (lncRNAs) and microRNAs possess important regulatory functions at the cellular level and in diverse pathophysiological processes. This study was conducted to investigate whether lncRNA RP11-789C1.1 regulates EMT in GC by mediating the miR-5003/E-cadherin pathway. METHODS: RP11-789C1.1 and miR-5003 expression was detected in GC specimens and cell lines by quantitative real-time PCR. Western blotting and immunohistochemistry were performed to detect EMT markers in GC. Cell Counting Kit 8 assays were carried out to explore cell proliferation. Wound healing and Transwell assays were conducted to determine the migration and invasion of GC cells. To clarify the correlation between RP11-789C1.1, miR-5003, and E-cadherin, dual-luciferase reporter assays were applied. RESULTS: LncRNA RP11-789C1.1 was significantly down-regulated in GC patients and cell lines, along with the concomitant up-regulation of miR-5003. Silencing RP11-789C1.1 and over-expressing miR-5003 significantly promoted the tumor behavior of GC cells. Dual-luciferase reporter assays confirmed that miR-5003 was the target of both RP11-789C1.1 and E-cadherin. Furthermore, at both the mRNA and protein level, silencing RP11-789C1.1 remarkably reduced the expression of E-cadherin and promoted EMT, which were reversed by knocking down miR-5003. CONCLUSIONS: LncRNA RP11-789C1.1 inhibited EMT in GC through the RP11-789C1.1/miR-5003/E-cadherin axis, which could be a promising therapeutic target for GC.

The efficiency of electronic list-based multidisciplinary team meetings in management of gastrointestinal malignancy: a single-center experience in Southern China.

BACKGROUND: The multidisciplinary team (MDT) discussion has earned increasing popularity for the delivery of cancer care. However, MDT meeting (MDTM) is time and resource intensive, and some efforts to optimize discussion processes are required. This study aims to investigate the efficiency of electronic list-based MDTM in treatment of gastrointestinal (GI) malignancy. METHODS: Between January 2015 and December 2016, patients with GI cancers were retrospectively reviewed. Patients permitting an MDTM with our novel technique (eMDT group) were compared with those undergoing a traditional discussion (cMDT group). The efficiency of MDT working, including time cost per meeting or case and overall number of reviewed cases, was checked, with accuracy of clinical staging and other outcomes explored meanwhile. RESULTS: Three thousand six hundred seventy-four patients were included, with 2156 (58.7%) and 1518 (41.3%) cases for eMDT and cMDT groups, respectively. Comparisons in age (P = 0.529), gender (P = 0.844), cancer type (P = 0.218), treatment plan (P = 0.737), and pathological stage (P = 0.098) were not significant between groups. However, the average time cost in both each meeting (149.4 vs. 205.1 min; P < 0.001) and each case (3.1 vs. 6.2 min; P < 0.001) was markedly reduced. Besides, this novel technique was associated with improved accuracy of clinical staging (P = 0.070) and reduced hospital stay (P < 0.001) compared with the traditional approach, with similar incidence of complications observed (P = 0.243). CONCLUSIONS: The MDT working based on an intelligent checklist could save considerable time while not affecting treatment of GI malignancies. The improved efficiency also earns an increased capacity of hospital admission and in-patient care.

A Clade II-D Fungal Chimeric Diterpene Synthase from Colletotrichum gloeosporioides Produces Dolasta-1(15),8-diene.

Based on a terpenoid overproduction platform in yeast for genome mining, a chimeric diterpene synthase from the endophytic fungus Colletotrichum gloeosporioides ES026 was characterized as the (5R,12R,14S)-dolasta-1(15),8-diene synthase. The absolute configuration was independently verified through the use of enantioselectively deuterated terpene precursors, which unequivocally established the predicted C1-III-IV cyclization mode for this first characterized clade II-D enzyme. Extensive isotopic labeling experiments and isolation of the intermediate (1R)-δ-araneosene supported the proposed cyclization mechanism.

Releasing the potential power of terpene synthases by a robust precursor supply platform.

Terpenoids represent the largest family of natural products. Their structural diversity is largely due to variable skeletons generated by terpene synthases. However, terpene skeletons found in nature are much more than those generated from known terpene synthases. Most promiscuous terpene synthases (i.e. those that can generate more than one product) have not been comprehensively characterised. Here, we first demonstrated that the promiscuous terpene synthases can produce more variable terpenoids in vivo by converting precursor polyisoprenoid diphosphates of different lengths (C10, C15, C20, C25). To release the synthetic potential of these enzymes, we integrated the engineered MVA pathway, combinatorial biosynthesis, and point mutagenesis to depict the comprehensive product profiles. In total, eight new terpenoids were characterised by NMR and three new skeletons were revealed. This work highlights the key role of metabolic engineering for natural product discovery.

Expression analysis of nine Toll-like receptors in yellow catfish (Pelteobagrus fulvidraco) responding to Aeromonas hydrophila challenge.

Toll-like receptors (TLRs) are important components of pattern recognition receptors (PRRs), which play significant roles in innate immunity to defense against pathogen invasion. Many TLRs have been found in teleosts, but there are no reports about cloning and expression of TLR genes in yellow catfish (Pelteobagrus fulvidraco). In this study, we analyzed the sequence characters and the relative mRNA expression levels of nine TLRs (TLR1, TLR2, TLR3, TLR4-1, TLR5, TLR7, TLR8-2, TLR9 and TLR22) in different tissues of yellow catfish. The results showed that all nine TLR genes are highly expressed in head kidney, trunk kidney, spleen and liver, all of which are related to host immunity. Subsequently we used Aeromonas hydrophila as a stimulating agent to detect the expression profiles of these nine TLRs in the liver, spleen, trunk kidney and head kidney of yellow catfish at different time points after injection with killed Aeromonas hydrophila. All nine TLRs responded to A. hydrophila challenge with tissue-specific patterns in different immune tissues. The kinetics of up- or down-regulation of these nine TLRs exhibited a similar trend, rising to an elevated level at first and then falling to the basal level, but the peak value differed at different time points in different tissues. The expression levels of the TLR3, TLR4-1, TLR9 and TLR22 genes were significantly up-regulated after bacterial challenge in the liver, spleen, head kidney and trunk kidney. The relatively high expression of TLR genes in the immune tissues in response to the A. hydrophila challenge indicated that TLRs may play important roles in the innate immune response against gram-negative bacteria in yellow catfish.