African swine fever virus pB318L, a trans-geranylgeranyl-diphosphate synthase, negatively regulates cGAS-STING and IFNAR-JAK-STAT signaling pathways.

African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by the ASF virus (ASFV). ASFV has evolved multiple strategies to escape host antiviral immune responses. Here, we reported that ASFV pB318L, a trans-geranylgeranyl-diphosphate synthase, reduced the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs). Mechanically, pB318L not only interacted with STING to reduce the translocation of STING from the endoplasmic reticulum to the Golgi apparatus but also interacted with IFN receptors to reduce the interaction of IFNAR1/TYK2 and IFNAR2/JAK1. Of note, ASFV with interruption of B318L gene (ASFV-intB318L) infected PAMs produces more IFN-I and ISGs than that in PAMs infected with its parental ASFV HLJ/18 at the late stage of infection. Consistently, the pathogenicity of ASFV-intB318L is attenuated in piglets compared with its parental virus. Taken together, our data reveal that B318L gene may partially affect ASFV pathogenicity by reducing the production of IFN-I and ISGs. This study provides a clue to design antiviral agents or live attenuated vaccines to prevent and control ASF.

African swine fever virus pH240R enhances viral replication via inhibition of the type I IFN signaling pathway.

African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by ASF virus (ASFV) infection. At present, there are still no safe and effective drugs and vaccines to prevent ASF. Mining the important proteins encoded by ASFV that affect the virulence and replication of ASFV is the key to developing effective vaccines and drugs. In this study, ASFV pH240R, a capsid protein of ASFV, was found to inhibit the type I interferon (IFN) signaling pathway. Mechanistically, pH240R interacted with IFNAR1 and IFNAR2 to disrupt the interaction of IFNAR1-TYK2 and IFNAR2-JAK1. Additionally, pH240R inhibited the phosphorylation of IFNAR1, TYK2, and JAK1 induced by IFN-α, resulting in the suppression of the nuclear import of STAT1 and STAT2 and the expression of IFN-stimulated genes (ISGs). Consistent with these results, H240R-deficient ASFV (ASFV-∆H240R) infection induced more ISGs in porcine alveolar macrophages compared with its parental ASFV HLJ/18. We also found that pH240R enhanced viral replication via inhibition of ISGs expression. Taken together, our results clarify that pH240R enhances ASFV replication by inhibiting the JAK-STAT signaling pathway, which highlights the possibility of pH240R as a potential drug target.IMPORTANCEThe innate immune response is the host's first line of defense against pathogen infection, which has been reported to affect the replication and virulence of African swine fever virus (ASFV) isolates. Identification of ASFV-encoded proteins that affect the virulence and replication of ASFV is the key step in developing more effective vaccines and drugs. In this study, we found that pH240R interacted with IFNAR1 and IFNAR2 by disrupting the interaction of IFNAR1-TYK2 and IFNAR2-JAK1, resulting in the suppression of the expression of interferon (IFN)-stimulated genes (ISGs). Consistent with these results, H240R-deficient ASFV (ASFV-∆H240R) infection induces more ISGs' expression compared with its parental ASFV HLJ/18. We also found that pH240R enhanced viral replication via inhibition of ISGs' expression. Taken together, our findings showed that pH240R enhances ASFV replication by inhibiting the IFN-JAK-STAT axis, which highlights the possibility of pH240R as a potential drug target.

African Swine Fever Virus HLJ/18 CD2v Suppresses Type I IFN Production and IFN-Stimulated Genes Expression through Negatively Regulating cGMP-AMP Synthase-STING and IFN Signaling Pathways.

African swine fever is a fatal infectious disease caused by African swine fever virus (ASFV). The high mortality caused by this infectious disease is a significant challenge to the swine industry worldwide. ASFV virulence is related to its ability to antagonize IFN response, yet the mechanism of antagonism is not understood. Recently, a less virulent recombinant virus has emerged that has a EP402R gene deletion within the parental ASFV HLJ/18 (ASFV-ΔEP402R) strain. EP402R gene encodes CD2v. Hence we hypothesized that ASFV uses CD2v protein to evade type I IFN-mediated innate immune response. We found that ASFV-ΔEP402R infection induced higher type I IFN response and increased the expression of IFN-stimulated genes in porcine alveolar macrophages when compared with parental ASFV HLJ/18. Consistent with these results, CD2v overexpression inhibited type I IFN production and IFN-stimulated gene expression. Mechanistically, CD2v, by interacting with the transmembrane domain of stimulator of IFN genes (STING), prevented the transport of STING to the Golgi apparatus, and thereby inhibited the cGMP-AMP synthase-STING signaling pathway. Furthermore, ASFV CD2v disrupted IFNAR1-TYK2 and IFNAR2-JAK1 interactions, and thereby inhibited JAK-STAT activation by IFN-α. In vivo, specific pathogen-free pigs infected with the mutant ASFV-ΔEP402R strain survived better than animals infected with the parental ASFV HLJ/18 strain. Consistent with this finding, IFN-ß protein levels in the peripheral blood of ASFV-ΔEP402R-challenged pigs were significantly higher than in the blood of ASFV HLJ/18-challenged pigs. Taken together, our findings suggest a molecular mechanism in which CD2v inhibits cGMP-AMP synthase-STING and IFN signaling pathways to evade the innate immune response rendering ASFV infection fatal in pigs.

Correlation between immunotherapy biomarker PD-L1 expression and genetic alteration in patients with non-small cell lung cancer.

Programmed death-ligand 1 (PD-L1) has been widely used in immunotherapy evaluation of patients with non-small cell lung cancer (NSCLC). However, the effect is not particularly ideal, and the association between PD-L1 and genetic alterations requires more exploration. Here, we performed targeted next-generation sequencing and PD-L1 immunohistochemistry (IHC) testing for PD-L1 expression on both tumor cells (TCs) and tumor-infiltrating immune cells (ICs) in 1549 patients. Our studies showed that surgical method of resection was positively correlated with IC+, and a low tumor mutation burden (TMB) was negatively correlated with TC+. Furthermore, we found that EGFR was mutually exclusive with both ALK and STK11. In addition, the features between PD-L1 expression status and genomic alterations were characterized. These results suggest that clinical characteristics and molecular phenotypes are associated with PD-L1 expression signatures, which may provide novel insights for improving the efficiency of immune checkpoint inhibitors (ICIs) in immunotherapy.

High production of valencene in Saccharomyces cerevisiae through metabolic engineering.

BACKGROUND: The biological synthesis of high value compounds in industry through metabolically engineered microorganism factories has received increasing attention in recent years. Valencene is a high value ingredient in the flavor and fragrance industry, but the low concentration in nature and high cost of extraction limits its application. Saccharomyces cerevisiae, generally recognized as safe, is one of the most commonly used gene expression hosts. Construction of S. cerevisiae cell factory to achieve high production of valencene will be attractive. RESULTS: Valencene was successfully biosynthesized after introducing valencene synthase into S. cerevisiae BJ5464. A significant increase in valencene yield was observed after down-regulation or knock-out of squalene synthesis and other inhibiting factors (such as erg9, rox1) in mevalonate (MVA) pathway using a recyclable CRISPR/Cas9 system constructed in this study through the introduction of Cre/loxP. To increase the supplement of the precursor farnesyl pyrophosphate (FPP), all the genes of FPP upstream in MVA pathway were overexpressed in yeast genome. Furthermore, valencene expression cassettes containing different promoters and terminators were compared, and PHXT7-VS-TTPI1 was found to have excellent performance in valencene production. Finally, after fed-batch fermentation in 3 L bioreactor, valencene production titer reached 539.3 mg/L with about 160-fold improvement compared to the initial titer, which is the highest reported valencene yield. CONCLUSIONS: This study achieved high production of valencene in S. cerevisiae through metabolic engineering and optimization of expression cassette, providing good example of microbial overproduction of valuable chemical products. The construction of recyclable plasmid was useful for multiple gene editing as well.

Inpatient case characteristics of SGLT2 inhibitor-associated diabetic ketoacidosis: a retrospective study.

OBJECTIVES: Diabetic ketoacidosis (DKA) is a serious complication in patients treated with sodium-glucose co-transporter 2 inhibitors (SGLT2i). The aim of this study was to investigate the relationship between SGLT2i and the risk of DKA, and to identify high-risk groups and characteristics that should be emphasised. METHODS: A retrospective case series study was conducted to collect medical records of inpatients diagnosed with DKA and using SGLT2i before the onset of the disease from September 2022 to September 2023 in a tertiary hospital in Shanghai. Cases that met the inclusion criteria were retrieved through the electronic medical record system. Information was collected to compare the risk of DKA in patients with different characteristics. RESULTS: A total of 21 patients (12 men and 9 women) met the criteria for SGLT2i-associated DKA. The mean diabetes duration was 10.4 years, with 47.6% (10/21) of patients diagnosed with euglycaemic DKA. The drug treatment regimen most commonly used was the combination of SGLT2i and metformin, representing 52.4% (11/21) of cases. The most common clinical symptoms were nausea, vomiting, abdominal pain and malaise. Common predisposing factors were acute infections, acute pancreatitis (predominantly hyperlipidaemic type), dietary inappropriateness, acute cardiovascular and cerebrovascular events and surgery. 71.4% of patients (15/21) had multiple risk factors. CONCLUSION: The use of SGLT2i in diabetic patients is associated with an increased risk of DKA, particularly in the presence of predisposing factors such as infection. Furthermore, long diabetes duration, decreased pancreatic ß-cell function and the combined use of metformin may also contribute to the risk of DKA in patients treated with SGLT2i. The findings of this study provide valuable insights for better identification and management of DKA risks associated with SGLT2i in clinical practice.

[Stable integration sites in Saccharomyces cerevisiae: identification and application in the biosynthesis of valencene].

Valencene, a high-value sesquiterpene with a citrus aroma, is widely employed in the food and cosmetic fields and the industrial synthesis of nootkatone. In this study, 16 genomic loci in the intergenic regions (IGRs) of Saccharomyces cerevisiae were identified. A Ypet expression cassette was successfully integrated into various genomic loci by CRISPR-Cas9, with an impressive integration success rate of 87.50% and exhibiting expression variations of up to 1.91-fold depending on the insertion site. The study demonstrates that the positional effect exhibits relative stability in gene expression, and is essentially unaffected by changes in promoters and reporter genes. Furthermore, a high-expression element combination, PTDH3-TPRC1, was selected. The iterative integration of the valencene synthase gene VSm from Callitropsis nootkatensis at the selected loci increased the valencene yield to 254.67 mg/L. Overexpression of key genes tHMG1-ERG20 with multiple copies increased the valencene yield by 93.49%. The engineered strain L-13 achieved the valencene yield of 9 530.18 mg/L by two-stage fed-batch fermentation in a 3 L fermenter. This yield represents a nearly 100-fold increase compared with that of the starting strain, highlighting the significant potential of the screened genomic loci in optimizing valencene production.

Intermediate product control in cascade reaction for one-pot production of ε-caprolactone by Escherichia coli.

ε-Caprolactone is an important non-toxic compound for polymer synthesis like polycaprolactone which has been widely used in drug delivery and degradable plastics. To meet the demand for a green economy, a bi-enzymatic cascade, consisting of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO), was designed and introduced into Escherichia coli to synthesize ε-caprolactone from cyclohexanol with a self-sufficient NADPH-cofactor regeneration system. To further improve the catalytic efficiency, a carbonyl group-dependent colorimetric method using inexpensive 2,4-dinitrophenylhydrazine (DNPH) was developed for assay of cyclohexanone, an intermediate production of cascade reaction. It can be used to screen mutant strains with high catalytic efficiency from high-throughput library by detecting the absorbance value in microtiter plates (MTP) instead of gas chromatography (GC) analysis. Moreover, an RBS combinatorial library was constructed for balancing the expression of ADH and CHMO from two independent transcriptional units. After the high-throughput screening based on intermediate product control, an optimal variant with higher substrate tolerance and long-term stability was obtained from RBS combinatorial library. Through a fed-batch process, ε-caprolactone production reached 148.2 mM after 70 h of reaction under the optimized conditions, which was the highest yield achieved to date.

Green Production of a High-value Mosquito Insecticide of Nootkatone from Seaweed Hydrolysates.

Nootkatone is a type of valuable sesquiterpene that is widely used in food, cosmetics, fragrance, and other fields. The industry is faced with a major challenge due to the high expenses associated with plant-extracted nootkatone. We have developed a fermentation process for valencene production using seaweed hydrolysate as a carbon source via engineered Saccharomyces cerevisiae. Reduced-pressure distillation purified valencene was used as a substrate, and a yeast strain carrying HPO/AtCPR1 and ADH genes was constructed for whole-cell catalysis. After biotransformation at 25 °C for 3 h, a high yield of 73% for nootkatone production was obtained. Further, simple rotary evaporation was used to obtain nootkatone with a high purity of 97.4%. Mosquito-repellent testing showed that 1% nootkatone has a mosquito-repellent effect lasting up to 6 h, which is comparable to the 20% N,N-diethyl-meta-toluamide (DEET) effect. This study provided practical experience for developing third-generation biomass resources, generating new ideas for green manufacturing of valuable chemical products, and serving as a reference for creating efficient and eco-friendly mosquito repellents.

Analysis of clinical characteristics of centrally mediated abdominal pain syndrome, exploration of diagnostic markers and its relationship with the efficacy of duloxetine treatment.

INTRODUCTION: Centrally mediated abdominal pain syndrome (CAPS) is characterized by severe abdominal pain. Diagnosis of CAPS is still an exclusionary diagnosis, there remain no effective diagnostic biomarkers so far. Duloxetine is the major pharmacotherapy of CAPS, while some CAPS patients do not respond to duloxetine treatment. However, there is a lack of molecular markers to predict the efficacy of duloxetine. In our pilot study, we have found differential expression profiles of serum miRNAs between CAPS patients and healthy controls. Our study aims to explore the clinical characteristics, specific miRNAs in serum as diagnostic biomarkers of CAPS and predictive biomarkers of the efficacy of duloxetine. METHODS/DESIGN: In this prospective cohort study, we plan to enroll 430 participants including 215 CAPS patients and 215 healthy controls. The CAPS group takes duloxetine 30 mg per day as an initial dose. Patients will have 24-week medication period and follow up at week 0, 4, 12, 24 and 36. Blood samples will be obtained from patients at every visits and health controls at the initial visit and a series of questionnaires will be completed by the participants. The primary end points are: The differential expression of miRNAs between CAPS groups and healthy control groups at baseline. The changes in abdominal pain scores before and after duloxetine treatment in patients with CAPS and their relationship with the changes in miRNAs. The secondary end point is the changes in scores of depression, anxiety, sleep quality and quality of life before and after duloxetine treatment in patients with CAPS and their relationship with changes in miRNAs. DISCUSSION: Findings of study will provide the reliable basis for diagnosis and the predictor of duloxetine efficacy of CAPS. Importantly, findings grant patients a chance to benefit from treatment.

Systematic Optimization of HPO-CPR to Boost (+)-Nootkatone Synthesis in Engineered Saccharomyces cerevisiae.

As an important and expensive natural sesquiterpene compound in grapefruit, the interest in (+)-nootkatone is stimulated by its strong grapefruit-like odor and physiological activities, which induce efforts for its microbial production. However, the low catalytic efficiency of the cytochrome P450-P450 reductase (HPO-CPR) system is the main challenge. We developed a high-throughput screening (HTS) method using the principle of the color reaction between carbonyl compounds and 2,4-dinitrophenylhydrazine (DNPH), which could rapidly screen the activity of candidate HPO mutants. After optimizing the pairing of HPO and CPR and through semirational design, the optimal mutant HPO_M18 with catalytic performance 2.54 times that of the initial was obtained. An encouraging (+)-nootkatone titer of 2.39 g/L was achieved through two-stage fed-batch fermentation after metabolic engineering and endoplasmic reticulum engineering, representing the highest titer reported to date. Our findings lay the foundation for the development of an economically viable bioprocess for (+)-nootkatone.

Probing the Synergistic Ratio of P450/CPR To Improve (+)-Nootkatone Production in Saccharomyces cerevisiae.

(+)-Nootkatone is an expensive sesquiterpene substance found in grapefruit peels and the heartwood of yellow cedar. It can be used as a food additive, perfume, and insect repellent; therefore, its highly efficient production is greatly needed. However, the low catalytic efficiency of the membrane-anchored cytochrome P450/P450 reductase system (HPO/AtCPR) is the main challenge and limits the production of (+)-nootkatone. We developed an effective high-throughput screening system based on cell wall destruction to probe the optimal ratio of HPO/AtCPR, which achieved a twofold elevation in (+)-valencene oxidation in Saccharomyces cerevisiae. An engineered strain PK2RI-AtC/Hm6A was constructed to realize de novo (+)-nootkatone production by a series of metabolic engineering strategies. In biphasic fed-batch fermentation, maximum titers of 3.73 and 1.02 g/L for (+)-valencene and (+)-nootkatone, respectively, were achieved. The dramatically improved performance of the constructed S. cerevisiae provides an excellent approach for economical production of (+)-nootkatone from glucose.

Effects of genetic and nongenetic factors on hyperuricemia in Chinese patients with coronary artery disease.

Aim: The relationship between hyperuricemia and polymorphisms of transporter genes in coronary artery disease (CAD) patients in China remains unclear. Materials & methods: A total of 258 hyperuricemia patients with CAD and 242 control patients with CAD were recruited in this case-control study. Twenty-four SNPs in genes of ABCG2, PDZK1, URAT1, OAT4, GLUT9, ABCC4, NPT1 and NPT4 were genotyped using direct sequencing in all subjects. Results: The mutation of ABCG2 rs2231142 locus increases the risk of hyperuricemia, and there is a gene dose effect in the influence of mutant heterozygotes and homozygotes. rs3825017 in URAT1 and rs62293298 in GLUT9 were also confirmed to be associated with hyperuricemia. Conclusion: Age, weight, creatinine clearance rate, diuretics and SNPs on ABCG2, URAT1 and GLUT9 were all risk factors of hyperuricemia.

Patient expectations and awareness of information regarding adverse reactions in drug labelling in China.

Background The misunderstanding of adverse drug reaction labelling information is not conducive to the rational use of drugs. There has been no research on how doctors can effectively transmit information on adverse drug reactions to patients in China. Objective To assess how well patients understand the adverse reactions presented in the labelling of drugs and how much information they want from their doctor regarding the adverse reactions. Setting The study was conducted in secondary medical institutions, tertiary medical institutions and community healthcare centres in Shanghai. Method A cross-sectional self-administered survey was conducted from November 2018 to March 2019. Mixed methods involving paper questionnaires and online surveys (scan a QR code by the WeChat app) were used. Main outcome measure Participants' demand for adverse reaction information. Results A total of 295 people completed the questionnaires, of which 31.8% of people thought that the greater the number of adverse reactions listed on the label of a drug, the more insecure they felt about that drug. At the same time, 30.13% of people thought that if the adverse reactions listed on a label were undefined, then the drug was safe for use (for example, some Chinese patent medicines). Most of the respondents (45.4%) thought that it was better to give a brief description of possible adverse reactions and to answer patients' questions in detail only if necessary. Conclusions Most patients wanted doctors to give them a brief introduction to serious and common adverse reactions when they prescribed drugs, and only a small percentage of people wanted to obtain all the information about adverse reactions. It was found that many people misunderstood the contents of the adverse reactions provided on the labels and equated the number of adverse reactions with drug safety.

Tuning a bi-enzymatic cascade reaction in Escherichia coli to facilitate NADPH regeneration for ε-caprolactone production.

ε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.

PP4C facilitates lung cancer proliferation and inhibits apoptosis via activating MAPK/ERK pathway.

PURPOSE: Protein phosphatase 4 catalytic subunit (PP4C) has been shown to play crucial regulatory roles in biological process and is frequently upregulated in cancer such as breast and colorectal carcinoma. However, the function and potential molecular mechanism of PP4C in lung cancer remains unclear. METHODS: Bioinformatic analysis was used to detect the expression level and prognosis of patients. Western blot, quantitative real-time PCR (qRT-PCR), CCK8, 5-Ethynyl-2'-deoxyuridine (Edu) proliferation assay and flow cytometric were used to explore the function in lung cancer cells. RESULTS: In this study, we found that PP4C was upregulated in lung cancer tissues as compared with that in normal lung tissues. Furthermore, patients with high expression level of PP4C were correlated with a poor prognosis in lung cancer patients. In vitro, CCK8, Edu proliferation assays and flow cytometry analysis showed that PP4C could promote lung cancer cell growth and inhibit apoptosis. Mechanistic investigations revealed that PP4C may interact with PP4R1 and promote ERK activation. Additionally, PP4C depletion resulted in lower tumor growth in vivo. CONCLUSIONS: Taken together, these data showed the oncogenic of PP4C in NSCLC tumorigenesis and provide a new insight of PP4C in the progression of NSCLC.

A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs.

African swine fever (ASF) is a devastating infectious disease in swine that is severely threatening the global pig industry. An efficacious vaccine is urgently required. Here, we used the Chinese ASFV HLJ/18 as a backbone and generated a series of gene-deleted viruses. The virulence, immunogenicity, safety, and protective efficacy evaluation in specific-pathogen-free pigs, commercial pigs, and pregnant sows indicated that one virus, namely HLJ/18-7GD, which has seven genes deleted, is fully attenuated in pigs, cannot convert to the virulent strain, and provides complete protection of pigs against lethal ASFV challenge. Our study shows that HLJ/-18-7GD is a safe and effective vaccine against ASFV, and as such is expected to play an important role in controlling the spread of ASFV.

On a maximally permissive deadlock prevention policy for automated manufacturing systems by using resource-oriented Petri nets.

It is theoretically and practically significant to synthesize a maximally permissive (optimal) controller to prevent deadlocks in an automated manufacturing system (AMS). With an AMS being modeled with Petri nets, by the existing methods, integer linear programming (ILP) problems are usually formulated and solved to obtain optimal policies by forbidding illegal markings at the same time no legal marking is excluded. Without an efficient technique for solving an ILP, such a method is usually computationally prohibitive. A resource-oriented Petri net (ROPN) is employed to model a class of AMS for resolving the deadlock control problem with maximal permissiveness in this paper. Efficient methods are developed to figure out the key structures in an ROPN model for deadlock prevention. Based on the structural properties of ROPN models, this work explores several types of illegal markings that can be prohibited optimally by structural analysis. For these markings, a deadlock prevention policy can be derived in an algebraic way without solving a notorious ILP problem. For the other markings, linear programming (LP), instead of ILP, approaches are developed to forbid them optimally. Thus, a maximally permissive controller can be developed while the computational cost is reduced greatly. The proposed methods are verified by typical examples in the literature.

Clinical pharmacy service practice in a Chinese tertiary hospital.

Clinical pharmacy service is focused on the rationality and safety of medication therapy. Clinical pharmacists play an important role in designing therapeutic regimen, preventing medication errors, reducing the incidence of adverse drug reaction, and saving medical costs. Although clinical pharmacy service in China is in its early stage, its development is rapid. In this manuscript, the working model of clinical pharmacists in a Chinese tertiary hospital is introduced, including ward rounds, consultation, stewardship of antimicrobial therapy, drug adverse reaction monitoring, therapeutic drug monitoring, clinical pharmacokinetics and pharmacogenetics, and training system. With the efforts of clinical pharmacists, there will be a significant increase in the optimization of medication therapy and a notable reduction in preventable adverse drug events as well as health-care cost in China.