Exploring the variability of the intestinal flora of patients with hepatic blastocysticercosis and searching for members of the intestinal microflora that may play a role in the disease process by means of macro-genome sequencing technology. A case-control study was used to include fecal samples from patients with hepatic vesicular schistosomiasis admitted to Qinghai Provincial People′s Hospital between October 2023 and January 2024 and individuals attending health checkups. The experimental group (AE group) consisted of 10 patients with liver vesicular schistosomiasis and the control group (NC group) consisted of 9 individuals attending health checkups. Macrogenomic sequencing was performed on these two groups of samples using the Illumina Novaseq 6000 sequencing platform, using fastp (v0.20.1) to remove junctions, and bbmap (v38.93-0) to remove the hosted sequences, followed by sequence splicing using MEGAHIT (v1.2.9), and then using prodigal (v2.6.3) to The spliced scaffold was subjected to ORF prediction and translated into amino acid sequences, followed by the construction of a non-redundant gene set using MMSeqs2 (v13.45111), and finally compared with the non-redundant gene set using salmon (v1.8.0). Species were annotated by the non-redundant database, species abundance was calculated in each sample, and the two sets were tested using Wilcoxon rank sum test. Finally, the differences in intestinal flora between the two groups were statistically analyzed using linear discriminant analysis, and the correlation between the differential intestinal flora and clinical indicators was analyzed using redundancy analysis (RDA). The results showed that the effective data volume of each sample was distributed from 10.41 to 12.46 G. The number of ORFs in the de-redundantly constructed gene catalogue (non-redundant gene set) was 4 951 408, and the annotation rate of the non-redundant genes was 97.97% when compared with the NR database. The ages of the study subjects in the two groups were (44.78±4.58) years in the NC group and (42.90±10.44) years in the AE group, and the difference was not statistically significant ( t=0.530, P=0.476). The two groups were matched for body mass index (BMI) ( t=2.368, P=0.142), gender ( χ2=0.200, P=0.655), and dietary habits. There was no statistically significant difference in alpha diversity in the AE group (ACE index, t=0.942; chao1 index, t=0.947; shannon index, t=0.813, the simpson′s index, t=0.613, P>0.05), while beta diversity analysis showed significant differences in the overall structure of the two communities (Stress=0.054 5). A total of 120 species were annotated at the phylum level, of which two differed. While 1 736 species were annotated at the genus level, 69 were different, and 309 were different at the species level. The AE group ranked the top 6 in terms of abundance of Anaplasma, Escherichiaceae, Clostridium, Alternaria, Ruminalia, and Treponema spp. at the genus level; whereas, Segatella, Prevotella, E. faecalis, Rossella, and beneficial rod-shaped bacteria were more abundant in the NC group. There were differences in the abundance and diversity of intestinal flora between the two groups, and the structure of community composition was significantly different. Statistical results by linear discriminant analysis (LDA) showed that LDA scores >2 in the NC group included beneficial bacillus spp. and E. faecalis spp. in young infants, etc. LDA scores >2 in the AE group at the mid-species level included Clostridium polterococcus, unknown microorganisms in the genus Clostridium intestinalis, Hathaway′s Henkett′s bacillus, and Clostridium oryzae in the genus Clostridium refractory to culture and small Clostridium spp. in the AE group. Clostridium intestinalis. The RDA results showed a negative correlation between beneficial rod genera and liver function indices, and a positive correlation between Clostridium intestinalis genera and liver function indices. In conclusion, patients with hepatic blastomycosis have altered intestinal flora abundance and diversity, with significant structural changes in community composition and differences in several genera, including Mycobacterium anisopliae and Clostridium intestinalis, and imbalances in the intestinal flora may affect hepatic function by influencing intestinal metabolites and may have an impact on the development of hepatic blastomycosis, a finding that warrants further in-depth study.

Objective:To investigate the in vitro bacteriostatic effect of ethyl alcohol extract of Moutan Cortex (EAEMC) on Candida tropicalis and its effect on virulence factors, including aspartic protease, hemolysin, phospholipase, esterase, lipase activities and biofilm. Methods:EAEMC powder was obtained by ultrasonic extraction, decompression concentration and lyophilization; the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EAEMC on 21 clinical strains and one standard strain of Candida tropicalis were determined by microdilution. Five extracellular enzyme activities of Candida tropicalis and the effect of EAEMC on them were detected by the plate assay, and the results were analyzed by ANOVA. The biofilm model of Candida tropicalis was constructed in vitro, and the inhibition rate of EAEMC on Candida tropicalis biofilm was evaluated using the thiazolyl blue (MTT) method. Results:The MIC of EAEMC against Candida tropicalis BNCC335988 was 12.5 g/L and the MBC value was 25 g/L, while for the clinical strains, the MIC was 12.5-25 g/L and the MBC was 25-50 g/L. Aspartic protease, esterase and hemolytic activities of Candida tropicalis were positive, but phospholipase and lipase showed negative activities. At a concentration of 1/2 MIC of EAEMC, the aspartic protease and hemolytic activities of Candida tropicalis were completely inhibited the aspartic protease and hemolytic activities of Candida tropicalis were completely inhibited and the esterase activity was completely inhibited at a concentration of MIC of EAEMC. The inhibition of Candida tropicalis BNCC335988 biofilm by EAEMC reached more than 70% at a concentration of 2MIC, more than 80% at a concentration of 4MIC, and more than 90% at a concentration of 8MIC. Conclusion:EAEMC can achieve bacteriostatic effects by reducing the aspartic protease, esterase and hemolysin activities of Candida tropicalis, as well as inhibiting biofilm formation.

Exploring the variability of the intestinal flora of patients with hepatic blastocysticercosis and searching for members of the intestinal microflora that may play a role in the disease process by means of macro-genome sequencing technology. A case-control study was used to include fecal samples from patients with hepatic vesicular schistosomiasis admitted to Qinghai Provincial People′s Hospital between October 2023 and January 2024 and individuals attending health checkups. The experimental group (AE group) consisted of 10 patients with liver vesicular schistosomiasis and the control group (NC group) consisted of 9 individuals attending health checkups. Macrogenomic sequencing was performed on these two groups of samples using the Illumina Novaseq 6000 sequencing platform, using fastp (v0.20.1) to remove junctions, and bbmap (v38.93-0) to remove the hosted sequences, followed by sequence splicing using MEGAHIT (v1.2.9), and then using prodigal (v2.6.3) to The spliced scaffold was subjected to ORF prediction and translated into amino acid sequences, followed by the construction of a non-redundant gene set using MMSeqs2 (v13.45111), and finally compared with the non-redundant gene set using salmon (v1.8.0). Species were annotated by the non-redundant database, species abundance was calculated in each sample, and the two sets were tested using Wilcoxon rank sum test. Finally, the differences in intestinal flora between the two groups were statistically analyzed using linear discriminant analysis, and the correlation between the differential intestinal flora and clinical indicators was analyzed using redundancy analysis (RDA). The results showed that the effective data volume of each sample was distributed from 10.41 to 12.46 G. The number of ORFs in the de-redundantly constructed gene catalogue (non-redundant gene set) was 4 951 408, and the annotation rate of the non-redundant genes was 97.97% when compared with the NR database. The ages of the study subjects in the two groups were (44.78±4.58) years in the NC group and (42.90±10.44) years in the AE group, and the difference was not statistically significant ( t=0.530, P=0.476). The two groups were matched for body mass index (BMI) ( t=2.368, P=0.142), gender ( χ2=0.200, P=0.655), and dietary habits. There was no statistically significant difference in alpha diversity in the AE group (ACE index, t=0.942; chao1 index, t=0.947; shannon index, t=0.813, the simpson′s index, t=0.613, P>0.05), while beta diversity analysis showed significant differences in the overall structure of the two communities (Stress=0.054 5). A total of 120 species were annotated at the phylum level, of which two differed. While 1 736 species were annotated at the genus level, 69 were different, and 309 were different at the species level. The AE group ranked the top 6 in terms of abundance of Anaplasma, Escherichiaceae, Clostridium, Alternaria, Ruminalia, and Treponema spp. at the genus level; whereas, Segatella, Prevotella, E. faecalis, Rossella, and beneficial rod-shaped bacteria were more abundant in the NC group. There were differences in the abundance and diversity of intestinal flora between the two groups, and the structure of community composition was significantly different. Statistical results by linear discriminant analysis (LDA) showed that LDA scores >2 in the NC group included beneficial bacillus spp. and E. faecalis spp. in young infants, etc. LDA scores >2 in the AE group at the mid-species level included Clostridium polterococcus, unknown microorganisms in the genus Clostridium intestinalis, Hathaway′s Henkett′s bacillus, and Clostridium oryzae in the genus Clostridium refractory to culture and small Clostridium spp. in the AE group. Clostridium intestinalis. The RDA results showed a negative correlation between beneficial rod genera and liver function indices, and a positive correlation between Clostridium intestinalis genera and liver function indices. In conclusion, patients with hepatic blastomycosis have altered intestinal flora abundance and diversity, with significant structural changes in community composition and differences in several genera, including Mycobacterium anisopliae and Clostridium intestinalis, and imbalances in the intestinal flora may affect hepatic function by influencing intestinal metabolites and may have an impact on the development of hepatic blastomycosis, a finding that warrants further in-depth study.

Objective:To investigate the in vitro bacteriostatic effect of ethyl alcohol extract of Moutan Cortex (EAEMC) on Candida tropicalis and its effect on virulence factors, including aspartic protease, hemolysin, phospholipase, esterase, lipase activities and biofilm. Methods:EAEMC powder was obtained by ultrasonic extraction, decompression concentration and lyophilization; the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EAEMC on 21 clinical strains and one standard strain of Candida tropicalis were determined by microdilution. Five extracellular enzyme activities of Candida tropicalis and the effect of EAEMC on them were detected by the plate assay, and the results were analyzed by ANOVA. The biofilm model of Candida tropicalis was constructed in vitro, and the inhibition rate of EAEMC on Candida tropicalis biofilm was evaluated using the thiazolyl blue (MTT) method. Results:The MIC of EAEMC against Candida tropicalis BNCC335988 was 12.5 g/L and the MBC value was 25 g/L, while for the clinical strains, the MIC was 12.5-25 g/L and the MBC was 25-50 g/L. Aspartic protease, esterase and hemolytic activities of Candida tropicalis were positive, but phospholipase and lipase showed negative activities. At a concentration of 1/2 MIC of EAEMC, the aspartic protease and hemolytic activities of Candida tropicalis were completely inhibited the aspartic protease and hemolytic activities of Candida tropicalis were completely inhibited and the esterase activity was completely inhibited at a concentration of MIC of EAEMC. The inhibition of Candida tropicalis BNCC335988 biofilm by EAEMC reached more than 70% at a concentration of 2MIC, more than 80% at a concentration of 4MIC, and more than 90% at a concentration of 8MIC. Conclusion:EAEMC can achieve bacteriostatic effects by reducing the aspartic protease, esterase and hemolysin activities of Candida tropicalis, as well as inhibiting biofilm formation.

Objective In this study,we analyzed the transcriptome sequences of Kupffer cells exposed to simulated microgravity for 3 d and conducted biological experiments to determine how microgravity initiates apoptosis in Kupffer cells. Methods Rotary cell culture system was used to construct a simulated microgravity model.GO and KEGG analyses were conducted using the DAVID database.GSEA was performed using the R language.The STRING database was used to conduct PPI analysis.qPCR was used to measure the IL1B,TNFA,CASP3,CASP9,and BCL2L11 mRNA expressions.Western Blotting was performed to detect the level of proteins CASP3 and CASP 9.Flow cytometry was used to detect apoptosis and mitochondrial membrane cells.Transmission electron microscopy was used to detect changes in the ultrastructure of Kupffer cells. Results Transcriptome Sequencing indicated that simulated microgravity affected apoptosis and the inflammatory state of Kupffer cells.Simulated microgravity improved the CASP3,CASP9,and BCL2L11 expressions in Kupffer cells.Annexin-V/PI and JC-1 assays showed that simulated microgravity promoted apoptosis in Kupffer cells.Simulated microgravity causes M1 polarization in Kupffer cells. Conclusion Our study found that simulated microgravity facilitated the apoptosis of Kupffer cells through the mitochondrial pathway and activated Kupffer cells into M1 polarization,which can secrete TNFA to promote apoptosis.

OBJECTIVES: To provide reference basis for reducing the mortality for children under 5 years old and promote the healthy development, the mortality for children under 5 years old and the main causes for death in Liuyang City from 2013 to 2020 are analyzed. METHODS: The data of 725 cases of death for children under 5 years old in Liuyang City from 2013 to 2020 were collected.The causes and difference of death among the children were analyzed retrospectively by descriptive statistic methods. RESULTS: There were a total of 144 516 live births in Liuyang City from 2013 to 2020. The mortality for children under 5 years old was 5.01‰, for infants was 3.39‰, and for newborns was 1.63‰. The male child mortality was 5.28‰, and the female child mortality rate was 4.72‰, with significant difference (P>0.05). The mortality for children under 5 years old was seasonal fluctuation, without significant difference among seasons (P>0.05). For the past 5 years, the top 3 causes for death among children under 5 years old were preterm birth and low birth weight, congenital heart disease, and pneumonia. Before death, 341 cases (47.04%) were treated in provincial hospitals, 198 cases (27.31%) in county-level hospitals, 56 cases (7.72%) in village-level hospitals, and 130 cases (17.93%) were not treated. CONCLUSIONS The mortality for children under 5 years old in Liuyang City is gradually reduced in the past 5 years. The main causes for death are premature birth and low birth weight, congenital heart disease and pneumonia. We should develop healthy education, improve the rate of prenatal diagnosis, promote the construction of obstetrics and paediatrics, and fundamentally reduce the mortality for children under 5 years old.
Cause of Death ; Child ; Child Mortality ; Child, Preschool ; Female ; Heart Defects, Congenital ; Humans ; Infant ; Infant Mortality ; Infant, Newborn ; Male ; Pneumonia/epidemiology* ; Pregnancy ; Premature Birth ; Retrospective Studies

ObjectiveTo obtain HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect by transfecting HSC-T6 cells with CRISPR/Cas9 lentiviral vector, and to provide a good method for further functional research and new strategies for the clinical treatment of liver fibrosis. MethodsThe COX-2 gene-specific sgRNAs (COX-2-sgRNA-1, COX-2-sgRNA-2, COX-2-sgRNA-3) were designed, synthesized, and connected to the GV371 vector, and the recombinant plasmid and the packaging plasmid were transfected into 293T cells to form lentivirus particles; the fluorescence method was used to measure virus titer. The most appropriate amount of the virus was calculated based on MOI. Lenti-Cas9-puro was transfected into HSC-T6 cells, and HSC-T6-Cas9 cells were screened out by puromycin; Lenti-COX-2-sgRNA-EGFP was transfected into HSC-T6-Cas9 cells to obtain HSC-T6-COX-2-/- cells. Cruiser enzyme digestion and Western blot were used to verify gene knockout at the gene and protein levels. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsSequencing verified that the COX-2-sgRNA expression vector was constructed successfully. Recombinant expression plasmids and packaging plasmids were transfected into 293T cells to form lentivirus particles, and the fluorescence method showed a virus titer of ＞1×108. HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect were successfully constructed. The HSC-T6-Cas9 group had significantly higher relative mRNA expression of LV-Cas9-Puro than the CON group (541.93±105.76 vs 1.00±0.02, t=12.995, P＜0.01). Cruiser enzyme digestion and Western blot showed that the CRISPR/Cas9 lentivirus expression vectors played a role in the target, among which COX-2-sgRNA-2 knockout had the most significant effect, and this group had a significant reduction in the protein expression level of COX-2 compared with the CON group and the NC group (both P＜0.05), suggesting that COX-2-sgRNA was active. ConclusionA CRISPR/Cas9 lentivirus vector is successfully constructed for COX-2 target gene, and HSC-T6-COX-2-/- cells with stable COX-2 gene knockout are obtained.

Objective:To analyze the malignant probability of thyroid nodules with the diagnosis of atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS) determined by fine-needle aspiration (FNA) and to explore the value of the combined application of BRAFV600E gene detection for the diagnosis of benign and malignant thyroid nodules. Methods:A total of 114 patients including 20 males and 94 females, aged 16-76 years old with thyroid nodules underwent FNA examination and surgical treatment in the Affiliated Cancer Hospital of Zhengzhou University from October 2018 to November 2019 were retrospectively analyzed. Postoperative histopathological results were used as the gold standard for the diagnosis of malignant thyroid nodules. The malignant rate of thyroid nodules with the diagnosis of AUS/FLUS was evaluated. Differential diagnostic efficacy of preoperative FNA combined with BRAFV600E gene detection for papillary thyroid carcinoma (PTC) was analyzed by McNemer test and diagnostic test evaluation method. Results:The mutation rate of BRAFV600E gene was 84.76% (89/105) in PTC. PTC accounted for 57.14% (12/21) of the patients with the diagnoses of AUS/FLUS determined by FNA. The specificity, sensitivity, positive predictive value and negative predictive value of BRAFV600E mutation examination for the diagnosis of malignant thyroid nodules determined preoperatively as AUS/FLUS were 9/9, 5/12, 5/5 and 9/16, respectively. BRAFV600E mutation examination could improve the detection rate of PTC in patients with AUS/FLUS ( OR=0.438, 95% CI=0.251-0.763, P=0.016). Conclusion:FNA combined with BRAFV600E mutation examination can significantly improve the detection rate of malignant thyroid nodules diagnosed preoperatively as AUS/FLUS.

ObjectiveTo investigate the effect and significance of cyclooxygenase-2 (COX-2) inhibitors on the expression of the Acsl gene family in the ileum of rats with nonalcoholic fatty liver disease (NAFLD). MethodsA total of 45 Sprague-Dawley rats were randomly divided into normal control group (15 rats given normal diet), NAFLD model group (15 rats given high-fat diet), and nimesulide group (15 rats given high-fat diet and nimesulide). All rats were sacrificed after 12 weeks of feeding, and then blood samples were collected from the inferior vena cava to measure total cholesterol (TC) and triglyceride (TG). HE staining and oil red O staining were performed for the liver to evaluate the degree of hepatic steatosis in each group, and quantitative real-time PCR was used to measure the mRNA expression of the Acsl family genes in the ileum. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the normal control group, the NAFLD model group had significant increases in serum TC and TG and marked hepatic steatosis (all P＜0.05); compared with the NAFLD model group, the nimesulide group had significant reductions in serum TC and TG and degree of hepatic steatosis (all P＜0.05). Compared with the normal control group, the NAFLD model group had a significant increase in the expression of COX-2 in the ileum (P＜0.05), and compared with the NAFLD model group, the nimesulide group had a significant reduction in the expression of COX-2 in the ileum (P＜005). Compared with the normal control group, the NAFLD model group had significant increases in the mRNA expression of Acsl3 and Acsl5 in the ileum (both P＜0.05), and compared with the NAFLD model group, the nimesulide group had significant reductions in the mRNA expression of Acsl3 and Acsl5 (both P＜0.05). ConclusionThe COX-2 inhibitor nimesulide can regulate the expression of the Acsl gene family in the ileum of rats with NAFLD, suggesting that COX-2 inhibitors may inhibit the progression of NAFLD through the Acsl gene.

Objective: To investigate the histological and biomechanical changes of rabbit ear cartilage induced by long pulse width 1064 nm Nd: YAGlaser. Methods: Seven New Zealand rabbits weighing 2.0-2.5 kg, male, 4-6 months old were selected. For a self-control, the right ear was irradiated by laser and the left ear was the normal control group. Each rabbit ear was divided into 3 cm × 1 cm, three 2 cm × 1 cm and 1 cm× 1 cm experimental area. After the experimental area of the laser irradiation group was irradiated by long pulse width 1064 nm Nd: YAG laser, Three pieces of 2 cm × 1 cm cartilage in each ear of the experimental rabbits were immediately cut and stained with HE, Sirius red and Masson to observe the histological changes of chondrocytes. The 3 cm × 1 cm and 1 cm× 1 cm cartilage of each rabbit ear was cut for biomechanical examination, and the changes of biomechanical properties such as tension, fracture, elastic modulus and compression of rabbit ear cartilage were observed immediately after the operation. Results: Histological observation showed that the chondrocytes became small, the matrix was stained deeply, the refraction was obvious/evident and the collagen was relatively increased. After being irradiated by long pulse width 1064 nm Nd: YAG laser, the peak tensile stress, tensile elastic modulus, tensile fracture load, compressive stress peak, compressive elastic modulus and compressive failure load of rabbit ear cartilage in laser irradiation group were (5.22 ±0.80) MPa, (42.40 ±9.78) MPa, (22.86 ±4.85) N, (16.04 ±5.57) MPa, (28.71 ±13.97) MPa, (1 211.63 ±427.86) N. All of them were smaller than those of the control group[(6.07±0.64) MPa, (48.44±6.30) MPa, (26.94±4.19) N, (25.12±9.10) MPa, (45.30±19.24) MPa, (1 962.83±896.71) N], and the difference was statistically significant (P<0.05). Conclusions After the operation of long pulse width 1064 nm Nd: YAG laser, the stress of rabbit ear cartilage resisting elastic deformation is reduced, the shape is easier to change, and the remodeling effect is more stable.