Objective To investigate the mechanism by which peripubertal exposure to di-2-ethylhexyl phthalate(DEHP)induces reproductive injury in adult male mice through impairment of Sertoli cell function and the blood-testis barrier.Methods A total of 180 male C57BL/6J mice(3 weeks old)were randomly divided into corn oil group(control group),and 5,25,125,250 and 500 mg/kg DEHP exposure groups.All groups received daily oral gavage for 5 consecutive weeks.Ten mice from each group were sacrificed for first material collection when they were 9 weeks old.The remaining F0 generation mice were mated with unexposed adult female mice at a male-to-female ratio of 1∶2 at 14 weeks of age for a period of 2 weeks.The second material collection was completed at 16 weeks of age,with 20 mice taken from each group.A computer-assisted analysis system was used to detect sperm density and motility in 9-week-old and 16-week-old male mice.The average birth body weight of F1 generation pups,survival rate of pups at 4 d after birth per litter,number of pups per litter,and male-to-female sex ratio within the litter were recorded.ELISA was used to detect hormone levels in the serum.Sperm abnormalities were observed using eosin staining.HE staining was used to observe pathological changes in testicular tissue.Immunofluorescence assay was utilized to detect the protein expression of tight junction protein 1(zonula occludens-1,ZO-1)and gap junction protein 43(connexin43,Cx43)related to the blood-testis barrier in testicular tissue,as well as the protein expression of apoptosis-related molecule Cleaved-Caspase3.Immunohistochemistry was used to detect the expression of Sertoli cell marker SOX9 protein in testicular tissue.TUNEL was used to detect apoptosis in testicular tissue.Comparative toxicogenomics database(CTD)was used to screen for mono-2-ethylhexyl phthalate(MEHP)induced reproductive damage-associated genes and combined with GO/KEGG and Reactome enrichment analysis to predict possible signaling pathways.An in vitro exposure model was established by treating TM4 cells with MEHP at concentrations of 0,50,100,200 and 400 μmol/L,respectively.CCK-8 assay,flow cytometry and Western blotting were applied to detect the cell viability,apoptosis,and protein expression of apoptosis-related proteins B-cell lymphoma 2(Bcl-2)and Bcl-2-associated X protein(Bax),and blood-testis barrier-related proteins ZO-1 and Cx43.Results Compared with the control group,the pregnancy rate was decreased from 80.0%to 52.5%,and the total number of pups was decreased from 212 to 125 in the female mice mating with the male from the 500 mg/kg group.Compared with the control group,the sperm density and motility were significantly reduced,and the total rate of sperm abnormalities was obviously increased in the 9-week-old DEHP-exposed mice(P<0.05),and even in the 16-week-old mice,the sperm motility was still in a dose-dependent downward trend(P<0.05).In the 25,125,250 and 500 mg/kg DEHP exposure groups,the thickness of the seminiferous epithelium was obviously decreased,with more cellular vacuolization and notable epithelial atrophy and degeneration.Cell apoptosis in the testicular tissue was enhanced,while the expression of ZO-1 and Cx43 were decreased in the 250 and 500 mg/kg exposure groups.The levels of androgen-binding protein,gonadotropin-releasing hormone,and testosterone in the serum were decreased in a dose-dependent manner(P<0.05),while those of luteinizing hormone and follicle-stimulating hormone showed a dose-dependent increase(P<0.05).Bioinformatics analysis showed that 153 genes related to male system diseases were screened out by CTD for MEHP.GO enrichment,KEGG pathway,and Reactome enrichment analyses displayed that the apoptosis signaling pathway was significantly enriched.CCK-8 assay showed that TM4 cell viability was decreased in a dose-dependent manner after MEHP treatment when compared with the control group(P<0.05).Western blotting indicated that MEHP treatment reduced the expression levels of Bcl-2,ZO-1 and Cx43(P<0.05),and enhanced that of Bax in comparison with the control group(P<0.05).Conclusion Peripubertal DEHP exposure may lead to reduced sperm quality and reproductive damage in adult male mice by disrupting the blood-testis barrier and activating Sertoli cell apoptosis.

Objective To explore the role and mechanism of RNA demethylase fat mass and obesity-associated protein(FTO)in the ferroptosis in testicular interstitial cells induced by di(2-ethylhexyl)phthalate(DEHP).Methods Forty 3-week-old C57BL/6 male mice were randomly divided into a control group(corn oil)and 3 dosed DEHP treatment groups(5,250 and 500 mg/kg),and received an intragastric infusion of corresponding agents for 35 d,respectively.After mouse testicular interstitial TM3 cells was treated with 0,100,200 and 400 μmol/L mono-2-ethylhexyl phthalate(MEHP)for 24 h,corresponding plasmids were transfected to construct Fto overexpressing TM3 cells.Serum testosterone level was detected by ELISA,expression of testicular proteins was detected with immunohistochemical assay,and contents of Fe2+,malondialdehyde(MDA)and lipid peroxides in the testicle were detected by colorimetry.Methylated RNA immunoprecipitation,RT-PCR,and Western blotting were used to detect the level of N6-methyladenosine(m6A)modification.Results In the mice exposed to 250 and 500 mg/kg DEHP,the serum testosterone level was significantly reduced(P＜0.01),contents of Fe2+,MAD and lipid peroxides in testicular tissue were obviously increased(P＜0.01),and protein levels of RNA demethylase FTO,and ferroptosis related molecules ferritin heavy chain 1(FTH1)and glutathione peroxidase 4(GPX4)were significantly down-regulated(P＜0.05),while those of transferrin receptor(TFRC),ferroportin(FPN),cyclooxygenase-2(COX-2),and acyl-CoA synthetase long-chain family member 4(ACSL4)were notably up-regulated(P＜0.05).MEHP treatment for 24 h resulted in remarkably decreased cell viability in the TM3 cells,increased production of intracellular reactive oxygen species(ROS),reduced mitochondrial membrane potential(MMP)(P＜0.01),down-regulated mRNA and protein levels of Fto(P＜0.01),and the changes in other ferroptosis related proteins were consistent with the trend in testicular tissue,indicating ferroptosis in testicular interstitial cells.Intervention with ferroptosis inhibitor Fer-1 or overexpression of Fto significantly inhibited MEHP-induced toxicity and ferroptosis in TM3 cells(P＜0.05),and overexpression of Fto reduced the m6A modification of Gpx4 and Fth1 mRNA(P＜0.05).Conclusion Abnormal m6A modification of Gpx4 and Fth1 caused by inhibiting FTO expression may be the mechanism of ferroptosis in testicular interstitial cells induced by DEHP.

Background: Ophiopogon japonicus (L.f) Ker-Gawl. growing in Zhejiang is recognized as the Dao-di medicinal herb for the production of Ophiopogonis Radix. Borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside, a prominent pharmacologically active compound, serves as a marker distinguishing O. japonicus in Zhejiang from those in other geographical areas. It is synthesized from borneol through glycosylation, with terpene synthase (TPS) being the critical enzyme catalyzing the conversion of terpene precursors into borneol. Objective: The aim of the study was to define key genes involved in biosynthesis of borneol in O. japonicus. Methods: The candidate terpene synthase genes were identified from the root and leaf transcriptome data of O. japonicus in Zhejiang and the functions of these enzymes were identified using engineered Escherichia coli. Results: This study developed a rapid expression system for monoterpene and sesquiterpene synthases based on engineered E. coli. Seven terpene synthase genes (OjTPS1 to OjTPS7) were identified in different terpene synthase subfamilies, including 2 from TPS-a, 4 from TPS-b, and 1 from TPS-g. Biochemical analysis using an engineered system E. coli demonstrated that all the 7 terpene synthases produced monoterpenes, and OjTPS3, OjTPS5, and OjTPS6 also yielded sesquiterpenes. Conclusions: These 7 terpene synthases produced 13 monoterpenes and 8 sesquiterpenes. Notably, OjTPS1 produced borneol establishing the groundwork for elucidating the biosynthetic pathways of borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside and other volatile oil components.

Objective To investigate the effects of intrathecal administration of Maresin 1 on pain behavior and glial cells activation in spinal dorsal horn of mice with bone cancer pain(BCP).Methods Experiment Ⅰ,twenty-eight clean male C57BL/6 mice,aged 4-6 weeks,weighing 18-22 g,were ran-domly divided into two groups:sham 1 group(group S1)and BCP 1 group(group B1),14 mice in each group.BCP model was established in group B1 by injecting PBS 20 μl containing 2 × 105 lewis lung carcino-ma cells into the intramedullary space.Group S,received PBS 20 μl without tumor cells.Six mice in each group were randomly selected to determine the mechanical withdrawal threshold(MWT)and thermal with-drawal latency(TWL)1 day before implantation,3,7,14,21 days after implantation.Four mice were randomly sacrificed on days 7,14 and 21 in group S,and group B1.The expressions of spinal glial fibrillary acidic protein(GFAP)and ionized calcium-binding adapter molecule 1(Iba1)were detected by Western blot.Experiment Ⅱ,thirty-six clean male C57BL/6 mice were randomly divided into three groups:sham 2 group(group S2),BCP 2 group(group B2),and Maresin 1 group(group M),12 mice in each group.BCP models were established in groups B2 and M while group S2 received PBS 20 μl without tumor cells.Maresin 1 50 ng/5 μl were intrathecally injected from days 14 to 16 after tumor cells implantation.Six mice in each group were randomly selected to determine the MWT and TWL on days 14 to 18 after implantation.Mice were sacrificed on day 18 after pain behavior tests.The expressions of spinal GFAP and Iba1 were de-tected by Western blot and immunofluorescence staining.The levels of spinal IL-1 β,IL-6,and TNF-α were detected by ELISA.Results Experiment Ⅰ:compared with group S1,the MWT and TWL were significantly lower on days 7,10,14,and 21 after implantation in group B,(P＜0.05),the expressions of spinal GFAP and Iba1 were upregulated on days 7,14,and 21 after implantation in group B1(P＜0.05).Experiment Ⅱ:compared with group S2,the levels of spinal GFAP,Iba1,IL-1β,IL-6,and TNF-α were increased on day 18 after implantation in group B2(P＜0.05).Compared with group B2,the MWT and TWL were significantly higher on days 15-18 after implantation while levels of spinal GFAP,Iba1,IL-1β,IL-6,and TNF-α on day 18 were decreased in group M(P＜0.05).Conclusion Intrathecal administra-tion of Maresin 1 attenuates BCP maybe by inhibiting the glial cells activation and neuroinflammation in the spinal cord.

Objective:To explore the effect of "Internet +" blood glucose management model in patients with gestational diabetes mellitus (GDM) .Methods:From January 2020 to March 2021, convenience sampling was used to select 95 GDM patients who were treated in the Outpatient Department of Lu'an People's Hospital of Anhui Province as the research object. According to the random number table method, the patients were divided into the control group ( n=47) and the intervention group ( n=48) . The control group received routine nursing, and the intervention group was given the "Internet +" blood glucose management model on the basis of routine nursing. The scores of the Diabetes Management Self-Efficacy Scale (DMSES) , GDM Self-management Behavior Scale and blood glucose control, including fasting blood glucose, 2-hour postprandial blood glucose (2hPG) , and glycosylated hemoglobin (HbA1c) , were compared before and after management, and the pregnancy outcomes were calculated. Results:After management, the DMSES and GDM self-management behavior scores of the intervention group were higher than those of the control group, and the 2 hPG, fasting blood glucose and HbA1c were lower than those of the control group, and the differences were statistically significant ( P<0.05) . The incidences of cesarean section, abnormal amniotic fluid volume and neonatal hypoglycemia in the intervention group were lower than those in the control group, and the differences were statistically significant ( P<0.05) . Conclusions:The "Internet +" blood glucose management model can improve the self-efficacy of GDM patients, strengthen self-management behaviors, effectively control blood glucose, and improve maternal and infant pregnancy outcomes.

Objective:To explore the effect of "Internet +" blood glucose management model in patients with gestational diabetes mellitus (GDM) .Methods:From January 2020 to March 2021, convenience sampling was used to select 95 GDM patients who were treated in the Outpatient Department of Lu'an People's Hospital of Anhui Province as the research object. According to the random number table method, the patients were divided into the control group ( n=47) and the intervention group ( n=48) . The control group received routine nursing, and the intervention group was given the "Internet +" blood glucose management model on the basis of routine nursing. The scores of the Diabetes Management Self-Efficacy Scale (DMSES) , GDM Self-management Behavior Scale and blood glucose control, including fasting blood glucose, 2-hour postprandial blood glucose (2hPG) , and glycosylated hemoglobin (HbA1c) , were compared before and after management, and the pregnancy outcomes were calculated. Results:After management, the DMSES and GDM self-management behavior scores of the intervention group were higher than those of the control group, and the 2 hPG, fasting blood glucose and HbA1c were lower than those of the control group, and the differences were statistically significant ( P<0.05) . The incidences of cesarean section, abnormal amniotic fluid volume and neonatal hypoglycemia in the intervention group were lower than those in the control group, and the differences were statistically significant ( P<0.05) . Conclusions:The "Internet +" blood glucose management model can improve the self-efficacy of GDM patients, strengthen self-management behaviors, effectively control blood glucose, and improve maternal and infant pregnancy outcomes.

Objective To observe the effect of low-dose remifentanil combined with propofol on the blood pressure(BP),heart rate(HR)and neonatal Apgar score in parturients undergoing cesarean section. Methods Forty-two parturients undergoing cesarean section were placed in two groups:low-dose remifentanil combined with propofol general anesthesia group(group GA,n=20)and neuraxial anesthesia group(group RA, n=22).BP and HR at the basis line(T0),pre-incision(T1),instant incision(T2),1 min(T3),3 min(T4),5 min(T5)after incision,cutting the umbilical cord(T6)were recorded,and neonatal Apgar scores at 1 min and 5 min after birth were also recorded. Results In group GA,the systolic blood pressure(SBP)and the diastolic blood pressure(DBP)remained unchanged at any time(P > 0.05). Compared with T0,HRs at T1~ T4were de-creased significantly(P<0.05).In group RA,compared with T0,the levels of SBP,DBP,HR at T1~T4were de-creased significantly(P<0.05).The levels of SBP and DBP at T1~T4in group GA were significantly higher than those in group RA(P<0.05),but there was no significant difference in HR between the two groups(P>0.05). The number of cases of ephedrine in group RA was significantly larger than that in the group GA(P<0.05).Neo-natal Apgar scores at 1 min and 5 min after birth were similar between the two groups(P > 0.05). Conclusion The application of low-doseremifentanil and propofol can be safely used in cesarean section,for the stability of par-turient BP and HR and no risk of neonatal respiratory depression.

Objective To evaluate the diagnostic performance of Xpert MTB/RIF for the diagnosis of tuberculous meningitis (TBM).Methods This was a prospective, single center clinical trial.A total of 116 consecutive patients with suspected meningitis who were admitted to Xijing Hospital from October 2013 to June 2015 were recruited.Mycobacterium tuberculosis ( MTB) and rifampicin ( RIF) resistance mutations in 1 ml cerebrospinal fluid ( CSF) were detected with Xpert MTB/RIF and the remaining sample was tested by Ziehl-Neelsen staining , MGIT960 liquid culture and other laboratory tests .And the enrolled patients were grouped according to the 2010 South African expert consensus .The diagnostic performance of Xpert MTB/RIF was evaluated by comparing against clinical score >5 points and MGIT960 liquid culture as reference standards respectively .The comparison was performed using a χ2 test or Fisher′s exact test for categorical variables and a nonparametric rank sum test for continuous variables .Results Among the enrolled 116 subjects, 23 subjects were diagnosed as definite-TBM by MGIT960 liquid culture, 16 subjects were classified as probable TBM , 27 subjects were classified as possible TBM , and 50 subjects were classified as non-TBM.When clinical score >5 points was used as a reference standard , the sensitivity of Xpert MTB/RIF (39.4%) was comparable with that of MGIT960 liquid culture (34.8%) (χ2 =0.292, P=0.589), and significantly better than that of Ziehl-Neelsen staining (9.9%) (χ2 =16.500, 12.771, P<0.001). No significant differences were found among the specificities of Xpert MTB /RIF, MGIT960 liquid culture and Ziehl-Neelsen staining ( 98.0%, 100.0% vs 98.0%, χ2 =1.014, P=0.602 ) .When tested against MGIT960 liquid culture as a reference standard , the sensitivity of Xpert MTB/RIF was 91.3%. Conclusions Xpert MTB/RIF is a rapid and specific method to detect MTB and RIF resistance in CSF .It exhibits a good rule in value for the diagnosis of TBM and a comparable sensitivity with MGIT 960 liquid culture, thus it can be used as the initial method for the diagnosis of tuberculous meningitis .

Objective To investigate the influence of the different second stage of labor duration on the maternal and neo‐natal outcomes .Methods Data were retrospectively analyzed from 201 nulliparous women delivering a singleton infant in cephalic presentation without labor analgesia hospitalized in our hospital between January 2012 and May 2015 ,whose second stage pro‐gressed beyond 2 hours .They were divided into observation group and control group according to different second stage of labor du‐ration ,and cases with a second stage of labor duration of three hours were in the observation group (163 cases) ,and those within two hours were in the control group (38 cases) .Results Whether the limit of the second stage of labor duration of 2 hours ,or 3 hours ,the risk of postpartum hemorrhage ,postpartum fever ,urinary retention and neonatal asphyxia ,admission to the neonatology department in two groups were not significantly different(P>0 .05) .However ,there was significance difference in the rate of soft birth canal laceration(72 cases in the observation group) ,poor wound healing (0 case in the observation group and 3 cases in the control group ,P<0 .05) .Conclusion a second stage of labor duration of 3 hours could reduce the rate of cesarean section ,and with no increase in severe adverse maternal and neonatal outcomes .

We describe an interactive viewer for the All-Species Living Tree (LVTree). The viewer incorporates treeing and lineage information from the ARB-SILVA website. It allows collapsing the tree branches at different taxonomic ranks and expanding the collapsed branches as well, keeping the overall topology of the tree unchanged. It also enables the user to observe the consequence of trial lineage modifications by re-collapsing the tree. The system reports taxon statistics at all ranks automatically after each collapsing and re-collapsing. These features greatly facilitate the compar-ison of the 16S rRNA sequence phylogeny with prokaryotic taxonomy in a taxon by taxon manner. In view of the fact that the present prokaryotic systematics is largely based on 16S rRNA sequence analysis, the current viewer may help reveal discrepancies between phylogeny and taxonomy. As an application, we show that in the latest release of LVTree, based on 11,939 rRNA sequences, as few as 24 lineage modifications are enough to bring all but two phyla (Proteobacteria and Firmicutes) to monophyletic clusters.