BACKGROUND: The addition of growth factiors is commonly applied to improve the osteogenic differentiation of stem cells. However, for human pluripotent stem cells (hPSCs), their complex differentiation processes result in the unknown effect at different stages. In this study, we focused on the widely used bone forming peptide-1 (BFP-1) and investigated the effect and mechanisms of its addition on the osteogenic induction of hPSCs as a function of the supplementation period. METHODS: Monolayer-cultured hPSCs were cultured in osteogenic induction medium for 28 days, and the effect of BFP-1 peptide addition at varying weeks was examined. After differentiation for varying days (0, 7, 14, 21 and 28), the differentiation efficiency was determined by RT–PCR, flow cytometry, immunofluorescence, and alizarin red staining assays. Moreover, the expression of marker genes related to germ layers and epithelial-mesenchymal transition (EMT) was investigated at day 7. RESULTS: Peptide treatment during the first week promoted the generation of mesoderm cells and mesenchymal-like cells from hiPSCs. Then, the upregulated expression of osteogenesis marker genes/proteins was detected in both hESCs and hiPSCs during subsequent inductions with BFP-1 peptide treatment. Fortunately, further experimental design confirmed that treating the BFP-1 peptide during 7–21 days showed even better performance for hESCs but was ineffective for hiPSCs. CONCLUSION The differentiation efficiency of cells could be improved by determining the optimal treatment period.Our study has great value in maximizing the differentiation of hPSCs by adding osteogenesis peptides based on the revealed mechanisms and promoting the application of hPSCs in bone tissue regeneration.

Nonalcoholic fatty liver disease (NAFLD), especially nonalcoholic steatohepatitis (NASH), is a common hepatic manifestation of metabolic syndrome. However, there are no effective therapy to treat this devastating disease. Accumulating evidence suggests that the generation of elastin-derived peptides (EDPs) and the inhibition of adiponectin receptors (AdipoR)1/2 plays essential roles in hepatic lipid metabolism and liver fibrosis. We recently reported that the AdipoR1/2 dual agonist JT003 significantly degraded the extracellular matrix (ECM) and ameliorated liver fibrosis. However, the degradation of the ECM lead to the generation of EDPs, which could further alter liver homeostasis negatively. Thus, in this study, we successfully combined AdipoR1/2 agonist JT003 with V14, which acted as an inhibitor of EDPs-EBP interaction to overcome the defect of ECM degradation. We found that combination of JT003 and V14 possessed excellent synergistic benefits on ameliorating NASH and liver fibrosis than either alone since they compensate the shortage of each other. These effects are induced by the enhancement of the mitochondrial antioxidant capacity, mitophagy, and mitochondrial biogenesis via AMPK pathway. Furthermore, specific suppression of AMPK could block the effects of the combination of JT003 and V14 on reduced oxidative stress, increased mitophagy and mitochondrial biogenesis. These positive results suggested that this administration of combination of AdipoR1/2 dual agonist and inhibitor of EDPs-EBP interaction can be recommended alternatively for an effective and promising therapeutic strategy for the treatment of NAFLD and NASH related fibrosis.

Objective:To establish a conditional knockout mouse model of polycystic kidney disease 1 ( Pkd1) gene based on CRISPR/Cas9 and Cre-loxP gene editing technology, and to provide an animal model for in-depth research on the role of Pkd1 gene in the development of polycystic kidney disease. Methods:In-Fusion technology was used to construct a targeting vector. Corresponding gRNAs, Cas9 mRNAs, and donor vectors carrying the loxP site were prepared based on the Pkd1 gene, and injected into the fertilized eggs of C57BL/6N mice. The fertilized eggs were transferred to the fallopian tubes of female mice with pseudopregnancy. After the newborn mice were identified by PCR and sequencing analysis, Pkd1 flox/flox F0 generation positive mice were selected. The F0 generation positive mice were bred with wild-type mice, and F1 generation heterozygous mice with Pkd1 flox/+ genotype were selected for offspring. F2 generation homozygous mice with Pkd1 flox/flox genotype were obtained through internal expansion, and then hybridized with Cre positive Ggt1/ Cre mice. F3 generation mice with Pkd1 flox/+Ggt1 Cre genotype were obtained. F4 generation mice with Pkd1 flox/flox Ggt1 Cre genotype were obtained by self crossing or backcrossing with F2 generation Pkd1 flox/flox, namely kidney-specific Pkd1 gene knockout mice ( Ggt1-cKO mice). PCR method was used to identify the genotype of mice, and then the mice were divided into wild-type control (WT) group ( n=6), Pkd1 homozygous control (PKD) group ( n=6), and Ggt1-cKO knockout validation (CKO) group ( n=6) according to the gene identification results. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of Pkd1 mRNA in the kidneys and other organs of mice in each group. HE staining was used to detect the pathological changes in renal tissues of mice in each group. The automatic biochemical detector was used to detect the blood urea nitrogen and serum creatinine levels of mice, and the kidney coefficient was calculated. Results:The PCR detection results showed that the genotype of offspring mice in CKO group was consistent with Pkd1floxflox Ggt1 Cre. Pkd1 gene was only specifically expressed in the kidney, but not in other tissues. The RT-qPCR results showed that the relative expression of Pkd1 mRNA in the renal medulla of CKO group was significantly lower than that of WT and PKD groups. The kidney volume of the CKO group had increased by about twice compared to the WT group. Under the microscope, it could be observed that there were multiple vacuoles of varying sizes and shapes in the kidneys of the CKO group, and there was a significant increase in the interstitial space of the medullary tissue. The kidney coefficient, blood urea nitrogen, and serum creatinine in the CKO group were significantly higher than those in the WT and PKD groups (all P<0.05). Conclusion:Based on CRISPR/Cas9 and Cre-loxP gene editing technology, Pkd1 gene kidney conditional knockout mice can be successfully constructed, providing an animal model for further studying the action mechanism of Pkd1 gene in polycystic kidney disease.

Objective: To study the effect of stem cell factor (SCF) on the angiogenic ability of cocultured dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs). Methods : This study has been reviewed and approved by the Ethics Committee. The experiment was split into the HUVECs, SCF+HUVECs, DPSCs+HUVECs, and SCF+DPSCs+HUVECs groups. A mixture of SCF and culture medium was used to prepare a mixed culture medium with an SCF concentration of 100 ng/mL. In vitro coculture of DPSCs and HUVECs was performed at a 1∶5 ratio. CCK-8 proliferation assay was used to observe the proliferative capacity of cells in each group on days 1, 3, 5, and 7. Wound healing and Transwell migration assays were used to detect the effect of SCF on cell migration under either direct or indirect coculture conditions, respectively. In vitro angiogenesis experiments were performed to detect the angiogenic capacity of the cells in each group. The vascular endothelial growth factor A (VEGFA) concentration in the cell culture supernatant was detected using ELISAs, and the protein expression levels of CD31, CD34, and VEGFA were detected using Western blot analysis. Results : Wound healing and Transwell migration experiments showed that SCF significantly promoted the migration of cocultured DPSCs and HUVECs (P<0.05). The in vitro angiogenesis experiment showed that the number of branches and the total length of branches of tubular structures in the SCF+DPSCs+HUVECs group were significantly greater than those of the other groups (P<0.05), and the expression levels of the vascular-related proteins CD31, CD34, and VEGFA in this group were greater (P<0.01). Conclusion SCF can enhance the migration and in vitro angiogenesis of cocultured DPSCs and HUVECs.

Humans ; Abdomen ; Dantrolene ; East Asian People ; Malignant Hyperthermia

Objective:To systematically evaluate the disease experience of patients with gestational hypertension, provide targeted care support for patients with gestational hypertension and promote their physical and mental health.Methods:This study is a Meta-synthesis. Qualitative studies on the experience of hypertensive patients during pregnancy were searched through PubMed, Web of Science, Cochrane Library, Embase, China National Knowledge Infrastructure, Wanfang Database, VIP and China Biology Medicine disc. The retrieval time limit was from the establishment of the databases to May 8, 2022. The quality of the studies were evaluated using the quality evaluation criteria for qualitative research of the Australian Joanna Briggs Institute Evidence-Based Health Care Center, and the results were integrated by the pooled integration method.Results:A total of 13 studies were included, 55 complete research results were extracted, 9 new categories were summarized and 3 integrated results were synthesized. Integration result 1 was the physical, psychological and behavioral changes that occurred in patients with gestational hypertension after diagnosis of the disease. Integration result 2 was that patients with gestational hypertension had coping deficits and hoped to receive support from multiple sources. Integration result 3 was the current and future challenges for patients with gestational hypertension.Conclusions:Gestational hypertension will bring many impacts to the patients, which needs support from many aspects. There are many challenges for disease management in the future. Medical staff should fully understand the experience of patients with gestational hypertension, give guidance and support to the most authentic feelings and experiences of the patients and encourage them to actively deal with the disease. At the same time, an effective and comprehensive support system should be built to help them better manage the disease.

Objective:To systematically integrate qualitative research on the decision-making experience of fertility preservation in cancer patients of childbearing age, so as to provide evidence-based basis for improving patients' fertility preservation decision-making ability and formulating targeted intervention plans.Methods:Qualitative research on the decision-making experience of fertility preservation in cancer patients of childbearing age was systematically searched on PubMed, Web of Science, Cochrane Library, Embase, China National Knowledge Infrastructure (CNKI) , Wanfang Database, VIP and China Biology Medicine disc. The search period was from database establishment to December 2, 2022. The quality of literature was evaluated using the quality evaluation criteria for qualitative research of the Australia Joanna Briggs Institute Evidence-Based Health Care Center. The results were integrated using the Meta-synthesis method.Results:A total of 11 articles were included, 28 results were extracted, summarized into 6 categories, and formed 3 integrated results. The integration results included the challenges and opportunities faced by fertility preservation decision-making, fertility preservation decision-making was influenced by individual, family and social factors, and lack of sufficient decision-making support.Conclusions:Cancer patients of childbearing age lack decision-making support for fertility preservation and face decision-making difficulties. Medical and nursing staff should comprehensively evaluate the fertility needs of cancer patients of childbearing age, pay attention to their cognition and experience, provide professional fertility counseling, and help patients make high-quality decision-making.

Currently, there is still no effective curative treatment for the development of late-stage liver fibrosis. Here, we have illustrated that TB001, a dual glucagon-like peptide-1 receptor/glucagon receptor (GLP-1R/GCGR) agonist with higher affinity towards GCGR, could retard the progression of liver fibrosis in various rodent models, with remarkable potency, selectivity, extended half-life and low toxicity. Four types of liver fibrosis animal models which were induced by CCl₄, α-naphthyl-isothiocyanate (ANIT), bile duct ligation (BDL) and Schistosoma japonicum were used in our study. We found that TB001 treatment dose-dependently significantly attenuated liver injury and collagen accumulation in these animal models. In addition to decreased levels of extracellular matrix (ECM) accumulation during hepatic injury, activation of hepatic stellate cells was also inhibited via suppression of TGF-β expression as well as downstream Smad signaling pathways particularly in CCl₄-and S. japonicum-induced liver fibrosis. Moreover, TB001 attenuated liver fibrosis through blocking downstream activation of pro-inflammatory nuclear factor kappa B/NF-kappa-B inhibitor alpha (NFκB/IKBα) pathways as well as c-Jun N-terminal kinase (JNK)-dependent induction of hepatocyte apoptosis. Furthermore, GLP-1R and/or GCGR knock-down results represented GCGR played an important role in ameliorating CCl₄-induced hepatic fibrosis. Therefore, TB001 can be used as a promising therapeutic candidate for the treatment of multiple causes of hepatic fibrosis demonstrated by our extensive pre-clinical evaluation of TB001.

Objective:To analyze the urine of normal healthy left-behind children under 1 year old and left-behind children with zinc deficiency under 1 year old in Zunyi area using hydrogen nuclear magnetic resonance ( 1HNMR), thus providing a new biomarker for the early diagnosis of zinc deficiency. Methods:From January to August 2018, a total of 40 normal healthy left-behind children under 1 year old in Zunyi area(healthy control group)[22 males and 18 females, average age of (7.78±3.62) months, average height of (65.01±2.67) cm and average body mass of (7.15±1.59) kg] and 40 age-matched left-behind children with zinc deficiency in the same region(zinc deficiency group)[19 males and 21 females, average age of (7.89±3.57) months, average height of (64.25±2.95) cm and average body mass of (7.02±1.68) kg] were included for a cross-sectional study by stratified sampling.The urine 1HNMR spectra of children in the 2 groups were measured, and the age, height, body mass and serum zinc content of children in the 2 groups were compared.The metabolites of the 2 groups were compared by metabono-mics technology combined with multivariate statistical analysis, and the differential metabolites of children with zinc deficiency were screened out. Results:There were no significant differences in age, height and body mass between the 2 groups (all P>0.05). The serum zinc level of healthy control group was significantly higher than that of zinc deficiency group [(54.3±3.06) mmol/L vs.(39.2±3.77) mmol/L, t=22.65, P<0.05]. Urine 1HNMR spectrogram results showed that compared with healthy controls, 4-hydroxyphenylpyruvic acid, phenyl acetyl glycine, and hippuric acid salt water were significantly lower in zinc deficiency group ( r=-0.620, -0.689, and -0.721, respectively, all | r|>0.602, all P<0.05). Conclusions:Zinc deficiency in left-behind children under 1 year old in Zunyi area is mainly manifested by decreased metabolites of 4-hydroxyphenylpyruvic acid, phenylacetyl glycine and horse-urate, suggesting metabolic disorder of intestinal flora.Differentially expressed metabolites have a potential application value in the early diagnosis of zinc deficiency.

Objective:To investigate the distribution of pathogen species isolated from cerebrospinal fluid culture (CSF) in children and analyze the antibiotic-resistance of the main isolates in vitro, which provides reference for interpreting the pathogens and choosing antibiotics in empiric therapy for pediatric patients. Methods:The results of cerebrospinal fluid culture were collected by checking laboratory information system of the Children′s Hospital of Zhejiang University and the clinical characteristics of these children were analyzed retrospectively by checking electronic medical record system.Results:A total of 1 312 isolates were detected, including 1 294 isolates of bacteria and 18 isolates of fungi. A total of 497 (37.9%) isolates were pathogenic microorganisms, of which 288 (57.9%) isolates were gram-positive, 200 (40.3%) isolates were gram-negative, and 9 (1.8%) isolates were fungi. The top 5 pathogens were Escherichia coli (102 isolates, 20.5%), Streptococcus pneumoniae (64 isolates, 12.9%), Streptococcus agalactiae (52 isolates, 10.5%), Enterococcus faecium (33 isolates, 6.6%) and Staphylococcus aureus (28 isolates, 5.6%). Most of the Streptococcus pneumoniae strains were isolated from children more than 1 year old (76.6%, 49/64), while the other top 4 bacteria were mainly isolated from infants less than 1 year old, with the rate of 95.1%(97/102) for Escherichia coli, 98.1%(51/52) for Streptococcus agalactiae, 81.8%(27/33) for Enterococcus faecium and 71.4% (20/28) for Staphylococcus aureus. A total of 815 (62.1%) isolates were considered to be contaminated pathogens according to the analysis on clinical manifestations and other laboratory findings in CSF, and coagulase-negative Staphylococcus (680 isolates), Micrococcus (50 isolates), Corynebacterium (28 isolates) and Enterococcus faecium (23 isolates), which accounted for 41.1% (23/56) of the total detected Enterococcus faecium, were the top 4 contaminated bacteria. During the study period, the isolation rate of the pathogenic microorganisms increased year by year (χ2=34.84, P<0.001), while the isolation rate of the contaminated pathogens, which detected mainly in summer and autumn, decreased year by year (χ2=13.26, P<0.001). Conclusions:The predominant bacteria causing pediatric purulent meningitis were Escherichia coli, Streptococcus pneumoniae, Streptococcus agalactiae, Enterococcus faecium and Staphylococcus aureus. Coagulase-negative Staphylococcus, Micrococcus, Corynebacterium and Enterococcus faecium were common contaminated bacteria in CSF culture, therefore clinicians should interpret the results of CSF culture cautiously according to the bacterial species and clinical manifestations.