This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

Objective: To investigate the association between gestational diabetes mellitus (GDM) and preterm birth subtypes. Methods: Based on the cohort of pregnant women in Anqing Prefectural Hospital, the pregnant women who received prenatal screening in the first or second trimesters were recruited into baseline cohorts; and followed up for them was conducted until delivery, and the information about their pregnancy status and outcomes were obtained through electronic medical record system and questionnaire surveys. The log-binomial regression model was used to explore the association between GDM and preterm birth [iatrogenic preterm birth, spontaneous preterm birth (preterm premature rupture of membranes and preterm labor)]. For multiple confounding factors, the propensity score correction model was used to compute the adjusted association. Results: Among the 2 031 pregnant women with a singleton delivery, the incidence of GDM and preterm birth were 10.0% (204 cases) and 4.4% (90 cases) respectively. The proportions of iatrogenic preterm birth and spontaneous preterm birth in the GDM group (n=204) were 1.5% and 5.9% respectively, while the proportions in non-GDM group (n=1 827) were 0.9% and 3.2% respectively, and the difference in the proportion of spontaneous preterm birth between the two groups was significant (P=0.048). Subtypes of spontaneous preterm were further analyzed, and the results showed that the proportions of preterm premature rupture of membranes and preterm labor in the GDM group were 4.9% and 1.0% respectively, while the proportions in the non-GDM group were 2.1% and 1.1% respectively. It showed that the risk of preterm premature rupture of membranes in GDM pregnant women was 2.34 times (aRR=2.34, 95%CI: 1.16-4.69) higher than that in non-GDM pregnant women. Conclusions: Our results showed that GDM might increase the risk of preterm premature rupture of membranes. No significant increase in the proportion of preterm labor in pregnant women with GDM was found.
Infant, Newborn ; Female ; Pregnancy ; Humans ; Premature Birth ; Diabetes, Gestational ; Obstetric Labor, Premature ; Hospitals ; Iatrogenic Disease

The ribose and phosphorus contents in Haemophilus influenzae type b (Hib) capsular polysaccharide (CPS) are two important chemical indexes for the development and quality control of Hib conjugate vaccine. A quantitative ¹H- and ³¹P-NMR method using a single internal standard was developed for simultaneous determination of ribose and phosphorus contents in Hib CPS. Hexamethylphosphoramide (HMPA) was successfully utilized as an internal standard in quantitative ¹H-NMR method for ribose content determination. The ribose and phosphorus contents were found to be affected by the concentration of polysaccharide solution. Thus, 15-20 mg·L^-1 was the optimal concentration range of Hib CPS in D₂O solution for determination of ribose and phosphorus contents by this method. The ribose and phosphorus contents obtained by the quantitative NMR were consistent with those obtained by traditional chemical methods. In conclusion, this quantitative ¹H- and ³¹P-NMR method using a single internal standard shows good specificity, accuracy and precision, providing a valuable approach for the quality control of Hib glycoconjugate vaccines.
Haemophilus Vaccines ; Haemophilus influenzae type b ; Phosphorus ; Polysaccharides, Bacterial ; Ribose

Humans ; COVID-19 ; Consensus ; SARS-CoV-2 ; China

OBJECTIVES: This study aimed to explore the effects of silencing isoprenylcysteine carboxyl methyltransfe-rase (Icmt) through small interfering RNA (siRNA) interference on the proliferation and apoptosis of tongue squamous cell carcinoma (TSCC). METHODS: Three siRNA were designed and constructed for the Icmt gene sequence and were then transfected into TSCC cells CAL-27 and SCC-4 to silence Icmt expression. The tested cells were divided as follows: RNA interference groups Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The transfection efficiency of siRNA was detected by the fluorescent group Cy3-labeled siRNA, and the expression of Icmt mRNA was screened by quantitive real-time polymerase chain reaction (qRT-PCR) selected the experimental group for subsequent experiments. The expression of Icmt, RhoA, Cyclin D1, p21, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) were analyzed by Western blot. The proliferation abilities of TSCC cells were determined by cell counting kit-8 assay. The change in apoptosis was detected by AnnexinV-APC/propidium staining (PI) assay. Cell-cycle analysis was conducted by flow cytometry. RESULTS: The expression of Icmt mRNA and protein in TSCC cells significantly decreased after Icmt-siRNA transfection ( CONCLUSIONS Silencing Icmt can effectively downregulate its expression in TSCC cells, reduce the RhoA membrane targeting localization and cell proliferation, and induce apoptosis. Thus, Icmt may be a potential gene therapy target for TSCC.
Apoptosis ; Carcinoma, Squamous Cell ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Protein Methyltransferases ; RNA, Small Interfering ; Tongue ; Tongue Neoplasms

OBJECTIVES: The effect of isoprenylcysteine carboxymethyltransferase (ICMT) silencing on the migration and invasion of tongue squamous cell carcinoma was investigated by constructing the small interfering RNA (siRNA) of ICMT. METHODS: Through liposomal transfection, siRNA was transfected into human tongue squamous cell carcinoma CAL-27 and SCC-4 cells (ICMT-siRNA group) with a negative control group (transfected with NC-siRNA) and a blank control group (transfected with a transfection reagent but not with siRNA). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of ICMT and RhoA in each group of cells after transfection and to measure the silencing efficiency. Western blot was applied to examine the expression levels of ICMT, total RhoA, membrane RhoA, ROCK1, matrix metalloproteinase (MMP)-2, and MMP-9 proteins in each group. The migration and invasion abilities were evaluated via wound healing and Transwell motility assays. RESULTS: After CAL-27 and SCC-4 cells were transfected with ICMT-siRNA, the expression levels of ICMT genes and proteins decreased significantly in the experimental group compared with those in the negative and blank control groups ( CONCLUSIONS The migration and invasion abilities of CAL-27 and SCC-4 cells were reduced significantly after the transfection of ICMT-siRNA, and the involved mechanism might be related to the RhoA-ROCK signaling pathway.
Carcinoma, Squamous Cell ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Neoplasm Invasiveness ; Protein Methyltransferases ; RNA, Small Interfering ; Tongue ; Tongue Neoplasms ; Transfection ; rho-Associated Kinases

The species within the family Cunninghamellaceae are widely distributed and produce important metabolites. Morphological studies along with a molecular phylogeny based on the internal transcribed spacer (ITS) and large subunit (LSU) of ribosomal DNA revealed two new species in this family from soils in China, that is, Absidia ovalispora sp. nov. andCunninghamella globospora sp. nov. The former is phylogenetically closely related to Absidia koreana, but morphologically differs in sporangiospores, sporangia, sporangiophores, columellae, collars, and rhizoids. The latter is phylogenetically closely related to Cunninghamella intermedia, but morphologically differs in sporangiola and colonies. They were described and illustrated.

The species within the family Cunninghamellaceae are widely distributed and produce important metabolites. Morphological studies along with a molecular phylogeny based on the internal transcribed spacer (ITS) and large subunit (LSU) of ribosomal DNA revealed two new species in this family from soils in China, that is, Absidia ovalispora sp. nov. andCunninghamella globospora sp. nov. The former is phylogenetically closely related to Absidia koreana, but morphologically differs in sporangiospores, sporangia, sporangiophores, columellae, collars, and rhizoids. The latter is phylogenetically closely related to Cunninghamella intermedia, but morphologically differs in sporangiola and colonies. They were described and illustrated.

OBJECTIVE: This study aimed to explore the influence of Rce1 on invasion and migration of tongue squamous cell carcinoma cells by silencing the Rce1 gene with RNA interference. METHODS: The tongue squamous cell carcinoma Cal-27 and SCC-4 cells were cultured in vitro. The small interfering RNA (siRNA) of the Rce1 gene was designed, and the Rcel gene expression was silenced vialiposome transfection. According to the siRNA transfected by liposome, the experimental group was divided into three groups, namely, Rce1-siRNA-1, Rce1-siRNA-2, and Rce1-siRNA-3 groups. Negative control group was transfected by siCON, and the blank control group was untransfected by siRNA. The Rce1, RhoA, and K-Ras gene expression levels in each group were analyzed by real-time quantitative polymerase chain reaction. The Rce1, RhoA, K-Ras, MMP-2, and MMP-9 protein expression levels were analyzed by Western blot. The invasiveness of tongue cancer cell Cal-27 and SCC-4 were determined by Transwell invasion assay, and cell migration assay was performed by cell scratch assay. RESULTS: Real-time quantitative polymerase chain reaction and Western blot results showed that compared with the negative and blank control groups, the Rce1 gene and protein expression levels in three experimental groups decreased (P<0.05). The RhoA, K-Ras gene and protein expression levels were insignificantly different among groups (P>0.05). Meanwhile, the MMP-2 and MMP-9 expression levels decreased (P<0.05). Transwell invasion assay results showed that the total number of cells in the PET film of the experimental groups was significantly decreased compared with the control group (P<0.05). The cell scratch test showed that the cell closure time of the scratch in the interference group was significantly longer than those in the control and blank groups (P<0.05). CONCLUSIONS Silencing Rce1 in vitro can effectively downregulate its expression in tongue squamous cell carcinoma cells Cal-27 and SCC-4 and reduce the migration and invasion abilities of these cells.
Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Endopeptidases ; metabolism ; Humans ; Neoplasm Invasiveness ; RNA Interference ; RNA, Small Interfering ; Tongue Neoplasms ; metabolism ; therapy ; Transfection