Objective To study the microscopic structure and morphological characteristics of Zebrafish eyeball and retina at different developmental stages, and to lay a foundation for visual research model. Methods Select eight groups of zebrafish at different ages, with six fish in each group, 48 fish in total. Optical microscopy and transmission electron microscopy were used to observe the eyeball structure of Zebrafish at different developmental stages, and the thickness of retinal each layer was measured to analyze the temporal and spatial development pattern. The morphological characteristics of various cells in the retina and the way of nerve connection were observed from the microscopic and ultrastructural aspects, especially the structural differences between rod cells and cone cells. Results The retina of Zebrafish can be divided into ten layers including retinal pigment epithelial layer, rod cells and cone cells layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, inner limiting membrane. Rod cells had a smaller nucleus and a higher electron density than cone cells. Photoreceptor terminals were neatly arranged in the outer plexiform layer, forming neural connections with horizontal cells and bipolar cells, and several synaptic ribbons are clearly visible within them. In Zebrafish retina, ganglion cell layer and inner plexiform layer are the earliest developed. With the growth and development of Zebrafish, the thickness of rod cells and cone cells layer and retinal pigment epithelial layer gradually increases, and the retinal structure was basically developed in about 10 weeks. Conclusion The retinal structure of Zebrafish is typical, with obvious stratification and highly differentiated nerve cells. There are abundant neural connections in the outer plexiform layer. The ocular development characteristics of Zebrafish are similar to those of most mammals.

This study was aimed at creating an engineered strain of Bacillus subtilis for efficient expression of biologically active type 2 toxin B(TcdB2)derived from a highly virulent strain of Clostridium difficile.The TcdB2 gene was cloned from ST1/RT027 strain genome DNA,incorporated into the PHT01 vector,and then transformed into B.subtilis strain WB800N for prokaryotic expression.Cell toxicity assays revealed that the recombinant TcdB2 exhibited cytotoxic effects in various cells.The engineered B.subtilis strain effectively expressed biologically active TcdB2,thus providing a basis for further exploration of the pathogenic mechanisms of highly virulent strains of C.difficile and establishing a foundation for potential vaccine can-didate targets.

Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.

Objective: Compare and analyze the results of the domestic Lanyi AH600 glycated hemoglobin analyzer and other different detection systems to understand the comparability of the detection results of different detectors, and establish the best cut point of Lanyi AH600 determination of haemoglobin A1c (HbA1c) in the diagnosis of diabetes. Methods: Multi center cohort study was adopted. The clinical laboratory departments of 18 medical institutions independently collected test samples from their respective hospitals from March to April 2022, and independently completed comparative analysis of the evaluated instrument (Lanyi AH600) and the reference instrument HbA1c. The reference instruments include four different brands of glycosylated hemoglobin meters, including Arkray, Bio-Rad, DOSOH, and Huizhong. Scatter plot was used to calculate the correlation between the results of different detection systems, and the regression equation was calculated. The consistency analysis between the results of different detection systems was evaluated by Bland Altman method. Consistency judgment principles: (1) When the 95% limits of agreement (95% LoA) of the measurement difference was within 0.4% HbA1c and the measurement score was≥80 points, the comparison consistency was good; (2) When the measurement difference of 95% LoA exceeded 0.4% HbA1c, and the measurement score was≥80 points, the comparison consistency was relatively good; (3) The measurement score was less than 80 points, the comparison consistency was poor. The difference between the results of different detection systems was tested by paired sample T test or Wilcoxon paired sign rank sum test; The best cut-off point of diabetes was analyzed by receiver operating characteristic curve (ROC). Results: The correlation coefficient R² of results between Lanyi AH600 and the reference instrument in 16 hospitals is≥0.99; The Bland Altman consistency analysis showed that the difference of 95% LoA in Nanjing Maternity and Child Health Care Hospital in Jiangsu Province (reference instrument: Arkray HA8180) was -0.486%-0.325%, and the measurement score was 94.6 points (473/500); The difference of 95% LoA in the Tibetan Traditional Medical Hospital of TAR (reference instrument: Bio-Rad Variant II) was -0.727%-0.612%, and the measurement score was 89.8 points; The difference of 95% LoA in the People's Hospital of Chongqing Liang Jiang New Area (reference instrument: Huizhong MQ-2000PT) was -0.231%-0.461%, and the measurement score was 96.6 points; The difference of 95% LoA in the Taihe Hospital of traditional Chinese Medicine in Anhui Province (reference instrument: Huizhong MQ-2000PT) was -0.469%-0.479%, and the measurement score was 91.9 points. The other 14 hospitals, Lanyi AH600, were compared with 4 reference instrument brands, the difference of 95% LoA was less than 0.4% HbA1c, and the scores were all greater than 95 points. The results of paired sample T test or Wilcoxon paired sign rank sum test showed that there was no statistically significant difference between Lanyi AH600 and the reference instrument Arkray HA8180 (Z=1.665,P=0.096), with no statistical difference. The mean difference between the measured values of the two instruments was 0.004%. The comparison data of Lanyi AH600 and the reference instrument of all other institutions had significant differences (all P<0.001), however, it was necessary to consider whether it was within the clinical acceptable range in combination with the results of the Bland-Altman consistency analysis. The ROC curve of HbA1c detected by Lanyi AH600 in 985 patients with diabetes and 3 423 patients with non-diabetes was analyzed, the area under curve (AUC) was 0.877, the standard error was 0.007, and the 95% confidence interval 95%CI was (0.864, 0.891), which was statistically significant (P<0.001). The maximum value of Youden index was 0.634, and the corresponding HbA1c cut point was 6.235%. The sensitivity and specificity of diabetes diagnosis were 76.2% and 87.2%, respectively. Conclusion: Among the hospitals and instruments currently included in this study, among these four hospitals included Nanjing Maternity and Child Health Care Hospital in Jiangsu Province (reference instrument: Arkray HA8180), Tibetan Traditional Medical Hospital of TAR (reference instrument: Bio-Rad Variant Ⅱ), the People's Hospital of Chongqing Liang Jiang New Area (reference instrument: Huizhong MQ-2000PT), and the Taihe Hospital of traditional Chinese Medicine in Anhui Province (reference instrument: Huizhong MQ-2000PT), the comparison between Lanyi AH600 and the reference instruments showed relatively good consistency, while the other 14 hospitals involved four different brands of reference instruments: Arkray, Bio-Rad, DOSOH, and Huizhong, Lanyi AH600 had good consistency with its comparison. The best cut point of the domestic Lanyi AH600 for detecting HbA1c in the diagnosis of diabetes is 6.235%.
Pregnancy ; Child ; Humans ; Female ; Glycated Hemoglobin ; Cohort Studies ; Diabetes Mellitus/diagnosis* ; Sensitivity and Specificity ; ROC Curve

Objective: To explore the effects of expanded frontal-parietal pedicled flap in reconstructing cervical scar contracture deformity in children after burns. Methods: A retrospective observational study was conducted. From January 2015 to December 2020, 18 male children with cervical scar contracture deformity after burns who met the inclusion criteria were admitted to Zhengzhou First People's Hospital, aged 4 to 12 years, including 10 cases with degree Ⅱ cervical scar contracture deformity and 8 cases with degree Ⅲ scar contracture deformity, and were all reconstructed with expanded frontal-parietal pedicled flap. The surgery was performed in 3 stages. In the first stage, a cylindrical skin and soft tissue expander (hereinafter referred to as expander) with rated capacity of 300 to 500 mL was placed in the frontal-parietal region. The expansion time was 4 to 6 months with the total normal saline injection volume being 2.1 to 3.0 times of the rated capacity of expander. In the second stage, expander removal, scar excision, contracture release, and flap transfer were performed, with the flap areas of 18 cm×9 cm to 23 cm×13 cm and the secondary wound areas of 16 cm×8 cm to 21 cm×11 cm after scar excision and contracture release. After 3 to 4 weeks, in the third stage, the flap pedicle was cut off and restored. The rated volume of placed expander, total normal saline injection volume, type of vascular pedicle of flap, survival of flap and reconstruction of scar after the second stage surgery were recorded. The neck range of motion and cervico-mental angle were measured before surgery and one-year after surgery. The appearance of neck, occurrence of common complications in the donor and recipient sites of children, and satisfaction of children's families for treatment effects were followed up. Data were statistically analyzed with paired sample t test. Results: All the patients successfully completed the three stages of operation. The rated volume of implanted expander was 300 mL in 6 children, 400 mL in 9 children, and 500 mL in 3 children, with the volume of normal saline injection being 630 to 1 500 mL. The type of vascular pedicle of flap was double pedicle in 13 cases and was single pedicle in 5 cases. All the flaps in 17 children survived well, and the secondary wounds after neck scar excision and contracture release were all reconstructed in one procedure. In one case, the distal blood supply of the single pedicled flap was poor after the second stage surgery, with necrosis of about 2.5 cm in length. The distal necrotic tissue was removed on 10 days after the operation, and the wound was completely closed after the flap was repositioned. In the follow-up of 6 months to 3 years post operation, the cervical scar contracture deformity in 18 children was corrected without recurrence. The flap was not bloated, the texture was soft, and the appearances of chin and neck were good. The range of motion of cervical pre-buckling, extension, left flexion, and right flexion, and cervico-mental angle in one year after operation were improved compared with those before operation (with t values of 43.10, 22.64, 27.96, 20.59, and 88.42, respectively, P<0.01). The incision in the frontal donor site was located in the hairline, the scar was slight and concealed. No complication such as cranial depression was observed in expander placement site, and the children's families were satisfied with the result of reconstruction. Conclusions: Application of expanded frontal-parietal pedicled flap in reconstructing the cervical scar contracture deformity in children after burns can obviously improve the appearance and function of neck, with unlikely recurrence of postoperative scar contractures, thus it is an ideal method of reconstruction.
Burns/surgery* ; Child ; Cicatrix/surgery* ; Contracture/surgery* ; Humans ; Male ; Perforator Flap ; Reconstructive Surgical Procedures/methods* ; Saline Solution ; Skin Transplantation ; Treatment Outcome

Objective: To understand immune escape mutation, drug resistance mutation, and genome evolution information of HBV genome sequence in China. Methods: The whole genome sequence information of HBV in China submitted in GenBank from 1998 to 2021 was selected as the object for analysis. MAFFT method was used for cluster analysis. Analysis of immune escape and drug-resistant mutations was performed using the online tool Gen2pheno. The BEAST 1.10.4 was used for analysis the time evolution of HBV sequences. Results: A total of 5 426 sequences were included in the dataset and distributed in 19 provinces of China. Type C accounted for the highest proportion (59.1%, 3 211/5 426), followed by type B (33.7%, 1 833/5 426). Immune escape mutations were found in 764 sequences (14.1%, 764/5 426). At least one reverse transcriptase region mutation occurred in 98.1% of the sequences. The evolutionary roots of most HBV sequences in China date from around 1801 AD. Conclusion: HBV-resistant mutation rate is high in China. HBV genomes evolve slowly.
China/epidemiology* ; DNA, Viral/genetics* ; Drug Resistance, Viral/genetics* ; Genome, Viral ; Genotype ; Hepatitis B virus/genetics* ; Humans ; Mutation

We used metabolomics technology to identify and understand the biomarkers and therapeutic mechanisms of umbilical compress therapy based on Xiaozhang Tie (XT) to provide scientific evidence for its clinical application. A total of 10 patients with cirrhotic ascites and gastrointestinal motility disorders who were hospitalized in the Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from November 2017 to June 2018 were divided into a placebo group (4 cases) or an XT group (5 cases), and 10 healthy volunteers were included as controls. This clinical trial was approved according to the Ethics Committee of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (2017-528-11-01). The patients in the XT group were given umbilical compress therapy with Xiaozhang Tie, and patients in the placebo group were administered a plaster patch in which the drug content was less than 5%, receiving one patch per day for three consecutive days. Non-targeted metabolomics technology and UPLC-Q/Orbitrap-MS/MS analysis technology were utilized to investigate the fluctuations in endogenous metabolic profiles in the patient's urine prior to and after administration of XT. By analyzing and comparing the urine metabolic profiles of patients with cirrhotic ascites to those of healthy volunteers, a total of 31 biomarkers were identified, 14 of which were significantly decreased by the intervention with Xiaozhang Tie (P <0.05). Pathway enrichment analysis revealed that phenylalanine metabolism and tryptophan metabolism are key pathways affected by XT treatment. The results suggest that XT can alleviate cirrhotic ascites by modulating abnormalities in amino acid metabolism.

The current study was designed to explore the brain protection mechanism of Xinglou Chengqi Decoction (XCD) based on gut microbiota analysis and network pharmacology. A transient middle cerebral artery occlusion (MCAO) model of mice was established, followed by behavioral evaluation, TTC and TUNEL staining. Additionally, to investigate the effects of gut microbiota on neurological function after stroke, C57BL/6 mice were treated with anti-biotic cocktails 14 days prior to ischemic stroke (IS) to deplete the gut microbiota. High-throughput 16S rDNA gene sequencing, metabonomics technique, and flow multifactor technology were used to analyze bacterial communities, SCFAs and inflammatory cytokines respectively. Finally, as a supplement, network pharmacology and molecular docking were applied to fully explore the multicomponent-multitarget-multichannel mechanism of XCD in treating IS, implicated in ADME screening, target identification, network analysis, functional annotation, and pathway enrichment analysis. We found that XCD effectively improved neurological function, relieved cerebral infarction and decreased the neuronal apoptosis. Moreover, XCD promoted the release of anti-inflammatory factor like IL-10, while down-regulating pro-inflammatory factors such as TNF-α, IL-17A, and IL-22. Furthermore, XCD significantly increased the levels of short chain fatty acids (SCFAs), especially butyric acid. The mechanism might be related to the regulation of SCFAs-producing bacteria like Verrucomicrobia and Akkermansia, and bacteria that regulate inflammation like Paraprevotella, Roseburia, Streptophyta and Enterococcu. Finally, in the network pharmacological analysis, 51 active compounds in XCD and 44 intersection targets of IS and XCD were selected. As a validation, components in XCD docked well with key targets. It was obviously that biological processes were mainly involved in the regulation of apoptotic process, inflammatory response, response to fatty acid, and regulation of establishment of endothelial barrier in GO enrichment. XCD can improve neurological function in experimental stroke mice, partly due to the regulation of gut microbiota. Besises, XCD has the characteristic of "multi-component, multi-target and multi-channel" in the treatment of IS revealed by network pharmacology and molecular docking.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Gastrointestinal Microbiome ; Mice ; Mice, Inbred C57BL ; Molecular Docking Simulation ; Network Pharmacology ; Stroke/drug therapy*

Objective:Resorption can occur after lumbar disc herniation, and Thymic stromal lymphopoietin (TSLP) is considered to be a key factor mediating reabsorption. Studies have found that under the action of inflammatory factors like TNF-α, IL-6, etc,the expression of TSLP in intervertebral disc tissue was increased, and then mononuclear macrophage chemokine-1 (MCP-1) was induced to induce infiltration of macrophage to promote reabsorption. To determine the mechanism, we design the experiment to explore the mechanism of IL-6-mediated JAK/STAT pathway and TGF-β/Smad pathway to promote the reabsorption of intervertebral disc by regulating the expression of TSLP.Methods:Rat bone marrow mesenchymal stem cells (MSC) and rat nucleus pulposus cells (NPC) were cultured in vitro, treating the cells withrat IL-6 recombinant protein, STAT3inhibitor and Smad2/3 inhibitorrespectively, and use real-time quantitative PCR (qRT-PCR) technology to detect the expression of JAK1, STAT3, Smad2, TSLP mRNA under different conditions; Western blot to detect the expression of TSLP, Smad2 and phosphate STAT3 protein; using enzyme-linked immunosorbent assay (Elisa) to detect the expression of TGF-β in two rat cells under the treatment of IL-6.Results:After stimulation ofIL-6 (10 ng/ml or 100 ng/ml) the expression of JAK1in MSC (10 ng/ml: 5.13±1.21; 100 ng/ml: 5.23±0.35; control group: 0.97±0.03), STAT3 (10 ng/ml: 6.50±0.38; 100 ng/ml: 6.74±0.61; control group: 0.87±0.19) was significantly increased, and TSLP also showed high expression in MSC (10 ng/ml: 4.26±0.38; 100 ng/ml: 5.05±0.46; control group: 1.04±0.04).The expression of STAT3 (10 ng/ml: 2.91±0.08; control group: 1.12±0.11), TSLP (10 ng/ml: 7.32±0.37; control group: 1.03±0.03) in NPC also increased. After stimulation of IL-6, the expression of Smad2 increasing in MSC was observed (10 ng/ml: 15.92±0.62; 100 ng/ml: 20.28±0.58; control group: 0.96±0.08), and increased expression of Smad2 in NPC (10 ng/ml: 5.01±0.17; control group: 0.96±0.03). The expression of TSLP in MSC decreased after adding STAT3 inhibitor (BP group, BP group: 0.17±0.01; control group: 0.90±0.09), the expression of TSLP also decreased in NPC (BP group: 0.42±0.11; control group: 0.90±0.11). After adding Smad2/3 inhibitor (SB group), the expression of TSLP in MSC decreased (SB group: 0.33±0.01; control group: 1.02±0.02), and the expression of TSLP in NPC also decreased (SB group: 0.40±0.04; control group: 0.99±0.01).Conclusion:IL-6 up-regulates TSLP expression via the JAK1/STAT3 signaling pathway and promotes prominent intervertebral disc reabsorption. At the same time, IL-6 can activate the TGF-β/Smad2/3 pathway and up-regulate the expression of TSLP, which play a synergistic role in the reabsorption process.

Objective The aim of current study is to determine the effect and mechanism of thymic stromal lymphopoietin on apoptosis of mouse nucleus pulposus cells by investigating the apoptotic activity and variation of intracellular phosphorylated protein kinase B (p-Akt),X-linkedinhibitor of apoptosis protein (XIAP),cysteinyl aspartate specific proteinase-3 (caspase-3),with the treatment of thymic stromal lymphopoietin.Methods Mouse lumbar nucleus pulposus cells were cultured and identified under a fluorescence microscope.Second or third passage cells maintained in monolayers were used for the following experiments.The groups were divided randomly into normal group,TNF-α treated group,TSLP treated group,TSLP+LY94002 treated group and TSLP+Embelin treated group.As a control,normal group was treated with PBS.TNF-α treated group was treated with 500 ng/ml TNF-αt as a positive control.TSLP treated group was treated with 10 ng/ml rhTSLP.TSLP+LY94002 treated group and TSLP+ Embelin treated group were treated with 10 ng/ml TSLP with the pretreatment of different pathway inhibitors for 30 ain in different corresponding experiments,for which 10 μ mol LY294002 or 50 LY294002 responding experimentsreatment of different pathway inhibitors formouse nucleus pulposus cells was detected by FACS.The expression levels of the intracellular p-Akt,XIAP,caspase-3 were investigated by Western blot analysis.Results As the culture cell type Ⅱ collagen staining was positive observed by fluorescence microscopy,we confirmed that the cuhured cells were nucleus pulposus cells.In comparison with negative control,the levels of p-Akt,XIAP in TSLP treated group were elevated (t=9.510,P=0.001;t=8.851,P=0.001).Thecaspase-3 activity were slightly enhanced and the rate of cells apoptosis was no significance.Compared with TSLP treated group,downregulated level of pAkt and XIAPand upregulatedcaspase-3 activity in TSLP+LY294002 treated group were observed (t=8.798,P=0.001;t=7.032,P=0.002;t=5.908,P=0.004).Upregulated caspase-3 activity were also observed in TSLP+ Embelin treated group (t=7.990,P=0.001).Furthermore,significant increased apoptotic cell rate was observed in TSLP+LY294002 or TSLP+Embelin treated groups (t=21.268,P=0.001;t=21.279,P=0.001).Conclusion TSLP may have a potential anti-apoptotic effect on mouse NP cells via upregulating XIAP in PI3K/Akt signaling pathway to restrain the activation of caspase-3.