Construction of an economical xylose-utilizing Saccharomyces cerevisiae and its ethanol fermentation.

Traditional industrial Saccharomyces cerevisiae could not metabolize xylose due to the lack of a specific enzyme system for the reaction from xylose to xylulose. This study aims to metabolically remould industrial S. cerevisiae for the purpose of utilizing both glucose and xylose with high efficiency. Heterologous gene xylA from Piromyces and homologous genes related to xylose utilization were selected to construct expression cassettes and integrated into genome. The engineered strain was domesticated with industrial material under optimizing conditions subsequently to further improve xylose utilization rates. The resulting S. cerevisiae strain ABX0928-0630 exhibits a rapid growth rate and possesses near 100% xylose utilization efficiency to produce ethanol with industrial material. Pilot-scale fermentation indicated the predominant feature of ABX0928-0630 for industrial application, with ethanol yield of 0.48 g/g sugars after 48 hours and volumetric xylose consumption rate of 0.87 g/l/h during the first 24 hours. Transcriptome analysis during the modification and domestication process revealed a significant increase in the expression level of pathways associated with sugar metabolism and sugar sensing. Meanwhile, genes related to glycerol lipid metabolism exhibited a pattern of initial increase followed by a subsequent decrease, providing a valuable reference for the construction of efficient xylose-fermenting strains.

Effect of low-molecular-weight heparins on anti-Xa peak levels and adverse reactions in Chinese patients with recurrent spontaneous abortion: a single-center, observational study.

OBJECTIVE: To compare three commonly used low-molecular-weight heparins (LWMHs) in the treatment of recurrent spontaneous abortion (RSA) by evaluating the anti-Xa peak levels and adverse reactions. METHODS: In this single-center, observational study, we enrolled 310 patients with RSA in whom anti-Xa levels were measured during pregnancy. Patients were divided into three groups according to the LMWH they used: the nadroparin group, enoxaparin group and dalteparin group. We compared the peak anti-Xa levels and the coagulation status of each group, and analyzed the incidence of adverse reactions, including local allergy, liver and renal dysfunction, and the impact on platelet. RESULTS: Patients in the enoxaparin group had a higher anti-Xa peak level than those in the nadroparin group (0.80 ± 0.22 IU/ml vs. 0.61 ± 0.24 IU/ml; P <  0.0001), although most patients in the three groups reached the target concentration of anti-Xa. Furthermore, patients in the enoxaparin group had a more stable anti-Xa levels during pregnancy. In addition, patients in the nadroparin group had a higher rate of local allergy than those in the enoxaparin group (60.5% vs. 42.5%; P = 0.004) and those in the dalteparin group (60.5% vs. 33.3%; P = 0.002). Further examination by the type of local allergy indicated a dramatic difference in pruritus and induration between the nadroparin group and the other two groups. No difference was found in the incidence of liver and renal dysfunction and thrombocytopenia. CONCLUSION: Compared with nadroparin and daltepatin, enoxaparin showed a better performance regarding anti-Xa levels and the incidence of adverse reactions in the treatment of RSA.

Decidua-derived granulocyte macrophage colony-stimulating factor induces polymorphonuclear myeloid-derived suppressor cells from circulating CD15+ neutrophils.

STUDY QUESTION: Do decidua-derived factors stimulate the conversion of circulating neutrophils to polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in early human pregnancy? SUMMARY ANSWER: Circulating neutrophils can acquire PMN-MDSC-like phenotypes and function via phosphorylated signal transducer and activator of transcription 5/programmed death ligand 2 (pSTAT5/PD-L2) signalling after stimulation with decidua-derived granulocyte macrophage colony-stimulating factor (GM-CSF). WHAT IS KNOWN ALREADY: PMN-MDSCs are an important immunoregulatory cell type in early pregnancy. Neutrophils are of high heterogeneity and plasticity and can polarize to immunosuppressive PMN-MDSCs upon stimulation. STUDY DESIGN, SIZE, DURATION: For analysis of myeloid-derived suppressor cell (MDSC) subset proportions, 12 endometrium tissues and 12 peripheral blood samples were collected from non-pregnant women, and 40 decidua tissues and 16 peripheral blood samples were obtained from women with normal early pregnancy undergoing elective surgical pregnancy termination for nonmedical reasons with gestation age of 6-10 weeks. Twenty-nine decidua tissues were collected for isolation of CD15+ PMN-MDSCs. Twenty endometrium tissues and 30 decidua tissues were collected for cytokine analysis, immunohistochemistry or neutrophil stimulation. Peripheral blood samples were obtained from 36 healthy donors for isolation of CD3+ T cells and CD15+ neutrophils. PARTICIPANTS/MATERIALS, SETTING, METHODS: The proportion of MDSC subsets in the decidua and peripheral blood of normal early pregnancy, endometrium and peripheral blood of non-pregnant women was analysed by flow cytometry. The phenotypes and function of decidual PMN-MDSCs and circulating neutrophils were compared by flow cytometry. Circulating neutrophils were stimulated with decidual explant supernatant (DES) and the phenotypes were measured by flow cytometry and immunofluorescence. The suppressive capacity of decidual PMN-MDSCs and DES-conditioned neutrophils was analysed by flow cytometry with or without anti-programmed cell death-1 (PD-1) antibody. Cytokines from DES and endometrial explant supernatant (EES) were detected by a Luminex assay. GM-CSF expression was determined by ELISA and immunohistochemistry. Neutrophils were stimulated with DES, EES, DES with anti-GM-CSF antibody or EES with GM-CSF. CD11b, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), PD-L2 and pSTAT5 expression were measured by flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: The frequency of PMN-MDSCs was significantly increased in the decidua of early pregnancy compared with peripheral blood of non-pregnant women, the endometrium of non-pregnant women or peripheral blood during early pregnancy. Decidual PMN-MDSCs suppressed T-cell proliferation and cytokine production. Phenotypes of decidual PMN-MDSCs were similar to mature activated neutrophils. DES-induced CD11b, LOX-1, PD-L2 expression and STAT5 phosphorylation in neutrophils. The PD-L2 expression in neutrophils was dependent on STAT5 phosphorylation. Both decidual PMN-MDSCs and DES-conditioned neutrophils suppressed T-cell proliferation via PD-1 signalling. GM-CSF was up-regulated in the decidua and induced CD11b, LOX-1 and PD-L2 expression on neutrophils. DES significantly induced CD11b, LOX-1, PD-L2 expression and STAT5 phosphorylation. Anti-GM-CSF antibody remarkably blocked such stimulation in neutrophils. EES did not induce CD11b, LOX-1, PD-L2 expression or STAT5 phosphorylation, while GM-CSF treatment sufficiently stimulated CD11b, LOX-1, PD-L2 expression and STAT5 phosphorylation in neutrophils. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The study was based on in vitro experiments and we were not able to evaluate neutrophils differentiation to PMN-MDSCs in other sites before entering the maternal-foetal interface due to the limited availability of human samples. This needs to be explored using murine models. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study demonstrating that decidual PMN-MDSCs are a group of immunoregulatory cells with mature status, and that neutrophils can be induced to a PMN-MDSC-like phenotype with decidua-derived GM-CSF via pSTAT5/PD-L2 signalling. This study indicates that GM-CSF can facilitate immune tolerance of early pregnancy through regulating PMN-MDSCs and further provides a potential role of GM-CSF in prevention and treatment for pregnancy complications. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (81671481) and National Natural Science Foundation of China (81871179). All authors have no competing interests to declare.

Intelligent microbial cell factory with genetic pH shooting (GPS) for cell self-responsive base/acid regulation.

BACKGROUND: In industrial fermentation, pH fluctuation resulted from microbial metabolism influences the strain performance and the final production. The common way to control pH is adding acid or alkali after probe detection, which is not a fine-tuned method and often leads to increased costs and complex downstream processing. Here, we constructed an intelligent pH-sensing and controlling genetic circuits called "Genetic pH Shooting (GPS)" to realize microbial self-regulation of pH. RESULTS: In order to achieve the self-regulation of pH, GPS circuits consisting of pH-sensing promoters and acid-/alkali-producing genes were designed and constructed. Designed pH-sensing promoters in the GPS can respond to high or low pHs and generate acidic or alkaline substances, achieving endogenously self-responsive pH adjustments. Base shooting circuit (BSC) and acid shooting circuit (ASC) were constructed and enabled better cell growth under alkaline or acidic conditions, respectively. Furthermore, the genetic circuits including GPS, BSC and ASC were applied to lycopene production with a higher yield without an artificial pH regulation compared with the control under pH values ranging from 5.0 to 9.0. In scale-up fermentations, the lycopene titer in the engineered strain harboring GPS was increased by 137.3% and ammonia usage decreased by 35.6%. CONCLUSIONS: The pH self-regulation achieved through the GPS circuits is helpful to construct intelligent microbial cell factories and reduce the production costs, which would be much useful in industrial applications.

Three New Monoterpenoid Indole Alkaloids from Ervatamia pandacaqui.

Three new monoterpenoid indole alkaloids, ervapandine A (1), (3R)-hydroxyibogaine (3), 12-hydroxyakuammicine N(4)-oxide (6), along with four known ones, were isolated from the twigs and leaves of Ervatamia pandacaqui. The structures of the new alkaloids were elucidated by spectroscopic methods and sugar hydrolysis experiment. All of the compounds were evaluated for their cytotoxicity against three human cell lines. Compound 7 showed moderate cytotoxic activity.

[Studies on alkaloids from Ervatamia pandacaqui].

To inverstigate the alkaloids from the twigs and leaves of Ervatamia pandacaqui, eleven known alkaloids were isolated by silica gel, Sephadex LH-20, and ODS column chromatography, as well as RP-HPLC. Their structures were elucidated by UV, IR, MS, and NMR spectral data as coronaridine (1), 3-oxocoronaridine (2), 19S-heyneanine (3), 19R-heyneanine (4), voacangine (5), 3-oxovoacangine (6), voacristine (7), 19-epi-voacristine (8), iso-voacangine (9), coronaridine 7-hydroxyindolenine (10), and voacangine 7-hydroxyindolenine (11). Compounds 1-11 were isolated from E. pandacaqui for the first time.

The biological principles and advanced applications of DSB repair in CRISPR-mediated yeast genome editing.

To improve the performance of yeast cell factories for industrial production, extensive CRISPR-mediated genome editing systems have been applied by artificially creating double-strand breaks (DSBs) to introduce mutations with the assistance of intracellular DSB repair. Diverse strategies of DSB repair are required to meet various demands, including precise editing or random editing with customized gRNAs or a gRNA library. Although most yeasts remodeling techniques have shown rewarding performance in laboratory verification, industrial yeast strain manipulation relies only on very limited strategies. Here, we comprehensively reviewed the molecular mechanisms underlying recent industrial applications to provide new insights into DSB cleavage and repair pathways in both Saccharomyces cerevisiae and other unconventional yeast species. The discussion of DSB repair covers the most frequently used homologous recombination (HR) and nonhomologous end joining (NHEJ) strategies to the less well-studied illegitimate recombination (IR) pathways, such as single-strand annealing (SSA) and microhomology-mediated end joining (MMEJ). Various CRISPR-based genome editing tools and corresponding gene editing efficiencies are described. Finally, we summarize recently developed CRISPR-based strategies that use optimized DSB repair for genome-scale editing, providing a direction for further development of yeast genome editing.

Antiphosphatidylserine/prothrombin antibodies (aPS/PT) and risk of obstetric anti-phospholipid syndrome.

OBJECTIVE: Assessment of the prevalence of anti-phosphatidylserine-prothrombin antibodies (aPS/PT) with OAPS and SN-OAPS in Chinese patients. METHODS: This retrospective study proceeded at Ren Ji Hospital, Shanghai, China, from January 2019 to January 2020. Two hundred eleven OAPS, 68 SN-OAPS, 81 disease controls, and 30 healthy donors were enrolled. IgM and IgG aPS/PT, IgM/IgG - aCL, and IgM/ IgG/ anti-ß2GPI antibodies were tested by ELISA while LAC was tested by clotting assays. All the patients were followed up and tested at least twice over 12 weeks apart. RESULTS: Thirty-three OAPS (15.64%) and 31 SN-OAPS (45.59%) were positive for aPS/PT. aPS/PT IgM showed a high Youden index (.813), which classified OAPS and SN-OAPS patients from healthy controls and other autoimmune diseases. aPS/PT showed a stronger relationship with LAC. Of the 25 OAPS women positive for IgM aPS/PT, 19 (79%) LAC were positive, while of the eight women positive for IgG aPS/PT, 100% were found LAC positive. CONCLUSION: aPS/PT antibody showed an efficient diagnostic value for Chinese patients with OAPS and SN-OAPS, which could be a potential risk predictor for obstetric complications.

Low-dose aspirin treatment improves endometrial receptivity in the midluteal phase in unexplained recurrent implantation failure.

OBJECTIVE: To compare endometrial receptivity in patients with and without unexplained recurrent implantation failure (URIF) and the benefits of low-dose aspirin treatment in women with URIF. METHODS: A retrospective study was conducted at Ren Ji Hospital, Shanghai, from January 2014 to January 2017. Endometrial thickness, pulsatility index (PI), resistive index (RI), and systolic-to-diastolic ratio (S/D) values of endometrial and uterine perfusion were recorded and compared between women with and without URIF. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive accuracy of the risk of URIF. Ultrasonography examination was repeated after 2 months of treatment with low-dose aspirin. RESULTS: PI, RI, and S/D values for endometrial blood flow were significantly higher in URIF patients than the control group (P < 0.001). The predictive indexes were 0.833, 0.857, and 0.839, respectively. Differences between the groups for endometrial thickness and impedance of uterine perfusion were not significant (P > 0.05). After low-dose aspirin treatment, endometrial and uterine arterial blood flow resistance in URIF patients was significantly lower than before treatment (P < 0.05). CONCLUSION: URIF patients had inappropriate endometrial blood flow. Doppler parameters are promising for predicting women at high risk of URIF. Low-dose aspirin treatment can improve endometrial receptivity.

Low-dose aspirin improves blood perfusion of endometrium of unexplained recurrent biochemical pregnancy loss.

OBJECTIVE: To assess the differences in Doppler parameters of endometrial receptivity in unexplained recurrent biochemical pregnancy loss (URBPL) and the therapeutic effect of low-dose aspirin (LDA). METHODS: A retrospective study was conducted at Ren Ji Hospital, Shanghai, PR China, from January 2017 to January 2019. Doppler parameters of endometrium and uterus were recorded as the evaluation of the endometrial receptivity. Receiver operating characteristic (ROC) curve was managed to predict the risk of URBPL. Ultrasonography tests were repeated after 2 months of treatment with LDA. RESULTS: Biochemical pregnancies did not correlate with maternal age. The resistance of endometrial perfusion (pulsatility index, resistive index, and systolic-to-diastolic ratio) was significantly higher in URBPLs (P < 0.001) and had predictive values (0.739, 0.779, and 0.760, respectively). Endometrial thickness and impedance to uterine blood flow showed no statistically significant difference (P > 0.05). After treatment with LDA, patients with URBPL improved the blood perfusion of endometrium significantly (P < 0.01). CONCLUSION: There was no relation between BPL and maternal age. Patients with URBPL had inappropriate endometrial blood velocity. Doppler indices are capable of predicting the risk of URBPL. LDA exerts the therapeutic effect on improving blood perfusion of endometrium in URBPL.

Ovarian teratoma related anti-N-methyl-D-aspartate receptor encephalitis: A case series and review of the literature.

BACKGROUND: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a rare but important complication of ovarian teratoma. Between July 2012 and December 2019, six patients with ovarian teratoma-associated anti-NMDAR encephalitis were enrolled in our hospital and their clinical characteristics, treatment, and follow-up were reviewed. We also conducted a systematic literature review of ovarian teratoma related anti-NMDAR encephalitis reports between January 2014 and December 2019. AIM: To better understand anti-NMDAR encephalitis through literature review and patients enrolled in our hospital. METHODS: The six patients enrolled in the study were those diagnosed with anti-NMDAR encephalitis. Their history, clinical manifestations, and medications were recorded and optimum treatment provided in addition to maintaining a record of the follow-ups. In addition, we also extensively surveyed the literature and provide summarized data from 155 published cases of anti-NMDAR encephalitis from 130 case reports. PubMed and Scopus were the sources of these publications and the time period covered was 6 years ranging from January 2014 through December 2019. RESULTS: The six patients enrolled for this study presented with typical symptoms resulting in a diagnosis of ovarian teratoma induced anti-NMDAR encephalitis. Appropriate interventions led to a positive outcome in all the patients, with five of six patients reporting full recovery and the sixth patient recovering with a few deficits. No death was recorded. The literature survey comprising of 155 patients cases across 130 case reports of anti-NMDAR encephalitis clearly indicated an upward trend in the reports/diagnosis in China, particularly in the surveyed time from 2014 through 2019. The majority of patients (150/155) underwent surgical intervention resulting in positive outcome. No treatment intervention was mentioned for one case while the four patients who were not surgically operated succumbed to the disease. CONCLUSION: Suspected anti-NMDAR encephalitis should be quickly evaluated for anti-NMDAR antibodies since early diagnosis is important. In case of a tumor, its earliest and complete removal is recommended. Finally, early use of corticosteroids and IgG-depleting strategies (intravenous immunoglobulin or plasma exchange) may improve outcome.

Intracellular Lipid Accumulation Drives the Differentiation of Decidual Polymorphonuclear Myeloid-Derived Suppressor Cells via Arachidonic Acid Metabolism.

Decidual polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are essential to immune tolerance during pregnancy. A reduction in the number of these cells is associated with unexplained recurrent pregnancy loss (URPL). In our previous study, we reported that PMN-MDSCs are a group of mature neutrophils that are activated by the decidua microenvironment. In the present study, we show that the decidua microenvironment induces substantial lipid accumulation in neutrophils during their differentiation to PMN-MDSCs. Lower levels of lipid accumulation are detected in PMN-MDSCs from URPL patients, and the amount of lipid in the PMN-MDSCs is positively correlated with the proportion of PMN-MDSCs. Next, we demonstrate that decidua-derived IL6 with the presence of arachidonic acid upregulates fatty acid-binding protein 5 (FABP5) via the phosphorylation of signal transducer and activator of transcription 3 (STAT3). Fy -60ABP5 then continuously stimulates intracellular lipid accumulation. Increased intracellular lipid accumulation mediates arachidonic acid metabolism, a pathway that is significantly activated by the induction of the decidua microenvironment, to stimulate the synthesis of prostaglandin E2 (PGE2) and finally induce the differentiation of PMN-MDSCs. To summarize, decidua-derived IL6 facilitates the differentiation of PMN-MDSCs from neutrophils via the pSTAT3/FABP5/PGE2 pathway. Defects in the process may result in impaired differentiation and dysfunction of PMN-MDSCs in URPL. These findings enhance our understanding of the physiological mechanisms of immune tolerance in pregnancy and provide therapeutic options for URPL.

Management of heparin resistance due to antithrombin deficiency in a Chinese pregnant woman: a case report.

Untreated individuals with antithrombin (AT) deficiency are at higher risk of thrombosis and adverse pregnancy outcomes. The present recommendations are mostly empirical for treating patients with AT deficiency during pregnancy because of the absence of guidelines. We report a rare case of heparin resistance due to AT deficiency in a pregnant 32-year-old Chinese woman. We also reviewed the English medical literature for AT deficiency and its association with thromboembolism and treatment. This patient suffered two early miscarriages because of thrombosis due to AT deficiency. The patient was administered the combination of adequate low molecular weight heparin with fresh frozen plasma and warfarin because of her heparin resistance. She delivered a healthy female newborn without any adverse effects of the anticoagulation therapy. Our findings suggest that the combination of adequate low molecular weight heparin with fresh frozen plasma and warfarin is effective for preventing thrombus during pregnancy.

Long-term follow-up of 46 cases of primary fallopian tube carcinoma: a single institute study.

BACKGROUND: Primary fallopian tube carcinoma (PFTC) is a rare malignancy. In recent years the incidence of PFTC has been rising. This study retrospectively analyzed 46 cases of PFTC to identify prognostic factors that may impact the survival of patients with PFTC and explored the clinical characteristics. METHODS: The clinical data of patients who had undergone surgery and adjuvant chemotherapy in Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University from 1995 to 2015 were retrospectively analyzed. We analyzed clinical data. Cox proportional hazards model was used for univariate and multivariate survival analysis. RESULTS: The level of CA125 increased in almost all patients with advanced-stage (stage III-IV) carcinoma and about half the patients with early stage (stage I-II) carcinoma. On ultrasound examination, 41 cases had pelvic mass, and five cases had intrauterine space-occupying lesion. Nine patients underwent curettage (19.6%). By the International Federation of Gynecology and Obstetricians (FIGO) staging system, the distribution of patients was 18 at stage I, 16 at stage II, 10 at stage III, and 2 at stage IV. The mainstay of treatment was surgical cytoreduction and platinum-based chemotherapy. Four patients had residual tumors diameter ≤1 cm (R1), 10 had residual tumors diameter >1 cm, and 32 had no macroscopic residual tumor (R0). Forty patients received postoperative intravenous (IV) chemotherapy. The five-year overall survival (OS) was 94.7% in stage I, 80.0% in stage II, 44.4% in stage III, and 0% in stage IV. Univariate and multivariate analysis revealed that residual tumor was independent prognostic variable for OS. Univariate and multivariate analysis revealed that ascites tumor cells and residual tumor were independent prognostic variables for progression free survival (PFS). CONCLUSIONS: Any postmenopausal women with vaginal bleeding, vaginal discharge, or lower abdominal pain should be alert to PFTC. Complete tumor markers and imaging examination should be conducted as soon as possible to improve the early diagnosis rate of the disease. Regardless of whether the operation is a comprehensive staging operation or cytoreductive surgery (CRS), achieving satisfactory R0 can improve OS and PFS. It is important the ascitic fluid is tested for tumor markers in order to predict PFS.