Single-cell RNA transcriptomics reveals Du-Zhong-Wan promotes osteoporotic fracture healing via YAP/ß-catenin/VEGF axis in BMSCs.

BACKGROUND: Our previous study demonstrated that Du-Zhong-Wan (DZW) promoted osteoporotic fracture (OPF) healing by enhancing osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and angiogenesis of endothelial cells (ECs). However, the heterogeneity of BMSCs and ECs, as well as the specific molecular mechanism underlying these effects, still require further evaluation. PURPOSE: The primary objective of this study was to elucidate the heterogeneity of BMSCs and ECs, as well as the cellular-level mechanism of DZW against OPF through single-cell RNA sequencing. METHODS: In this study, we presented a single-cell atlas of mouse femoral callus, comparing samples with and without DZW treatment, utilizing single-cell RNA sequencing. Variable genes were identified using the FindVariableGenes (FVG) and principal component analysis (PCA) analysis. Additionally, uniform manifold approximation and projection (U-MAP) was employed to reduce and visualize the distinct subclusters. The CellPhoneDB2 method was employed to analyze intercellular communication and quantify the interaction between ligands and receptors within distinct cell clusters. The osteogenic differentiation capacity of BMSCs was assessed by micro-CT, alkaline phosphatase (ALP), and alizarin red S (ARS) assay. The scratch wound assay and tube formation assay were utilized to assess the angiogenic capabilities of ECs in vitro. Additionally, western blot and immunofluorescence experiments were utilized to elucidate the related protein expression. RESULTS: Consistent with our previous studies, DZW obviously promoted osteoporotic fracture healing. Moreover, this study discovered 14 cell clusters at the femoral fracture callus, where the BMSCs most actively interacted with ECs, through single-cell sequencing. Notably, DZW significantly elevated the proportion of Lepr+ BMSCs and Podxl+ ECs subgroup, which were respectively considered essential cells for osteoblastogenesis and angiogenesis of arteriolar vessels. The increased proportion of Podxl+ ECs was partially attributed to vascular endothelial growth factor (VEGF), secreted by BMSCs, which were able to be reversed by YAP pharmacological inhibitor verteporfin. Furthermore, the western blot assay revealed elevated expression levels of YAP/ß-catenin, VEGF, RUNX2, and OCN in BMSCs treated with DZW, which were counteracted by verteporfin. CONCLUSION: The data above indicates that DZW elevates the proportion of LEPR+ BMSCs and Podxl+ ECs, therefore contributing for the osteogenic ability of BMSCs and BMSCs-mediated angiogenesis via activation of the YAP/ß-catenin/VEGF axis, which provides novel potential targets and mechanism for DZW in treating OPF in sub-clusters and molecular level.

De Novo Biosynthesis of a Bioactive Meroterpene Bakuchiol in Yeast.

Bakuchiol (BAK), a specialized meroterpene, is known for its valuable biological properties and has recently gained prominence in cosmetology for its retinol-like functionality. However, low abundance in natural sources leads to environmentally unfriendly and unsustainable practices associated with crop-based manufacturing and chemical synthesis. Here, we identified a prenyltransferase (PT) from Psoralea corylifolia that catalyzes the reverse geranylation of a nonaromatic carbon in para-coumaric acid (p-CA), coupled with a decarboxylation step to form BAK. Given that the biosynthesis pathway of BAK is well elucidated, we engineered Saccharomyces cerevisiae to produce BAK, starting from glucose. To enhance the titer of BAK, we employed a multifaceted approach that included increasing the supply of precursors, balancing the fluxes in the two parallel biosynthetic pathways, engineering of prenyltransferase, and fusing enzymes. Consequently, the engineered yeast strains showed a marked improvement of 117.3-fold in BAK production, reaching a titer of 9.28 mg/L from glucose. Our work provides a viable approach for the sustainable microbial production of complex natural meroterpenes.

Single-cell sequencing reveals that specnuezhenide protects against osteoporosis via activation of METTL3 in LEPR+ BMSCs.

BACKGROUND: Osteoporosis (OP) has garnered significant attention due to its substantial morbidity and mortality rates, imposing considerable health burdens on societies worldwide. However, the molecular mechanisms underlying osteoporosis pathogenesis remain largely elusive, and the available therapeutic interventions are limited. Therefore, there is an urgent need for innovative strategies in the treatment of osteoporosis. PURPOSE: The primary objective of this study was to elucidate the molecular mechanisms underlying osteoporosis pathogenesis using single-cell RNA sequencing (scRNA-seq), thereby proposing novel therapeutic agents. METHODS: The mice osteoporosis model was established through bilateral ovariectomy. Micro-computed tomography (µCT) and hematoxylin and eosin (H&E) staining were employed to assess the pathogenesis of osteoporosis. scRNA-seq was utilized to identify and analyze distinct molecular mechanisms and sub-clusters. Gradient dilution analysis was used to obtain specific sub-clusters, which were further validated by immunofluorescence staining and flow cytometry analysis. Molecular docking and cellular thermal shift assay (CETSA) were applied for screening potential agents in the TCMSPs database. Alkaline phosphatase (ALP) activity and alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Osteogenic organoids analysis was employed to assess the proliferation and sphere-forming ability of BMSCs. Quantitative real-time PCR (qRT-PCR) and western blot analysis were conducted to investigate signaling pathways. Wound healing assay and tube formation analysis were employed to evaluate the angiogenesis of endothelial cells. RESULTS: The scRNA-seq analysis revealed the crucial role of LEPR+ BMSCs in the pathogenesis of osteoporosis, which was confirmed by immunofluorescence staining of the epiphysis. Subsequently, the LEPR+ BMSCs were obtained by gradient dilution analysis and identified by immunofluorescence staining and flow cytometry. Accordingly, specnuezhenide (Spe) was screened and identified as a potential compound targeting METTL3 from the TCMSPs database. Spe promoted bone formation as evidenced by µ-CT, and H&E analysis. Additionally, Spe enhanced the osteogenic capacity of LEPR+ BMSCs through ALP and ARS assay. Notably, METTL3 pharmacological inhibitors S-Adenosylhomocysteine (SAH) attenuated the aforementioned osteo-protective effects of Spe. Particularly, Spe enhanced the LEPR+ BMSCs-dependent angiogenesis through the secretion of SLIT3, which was abolished by SAH in LEPR+ BMSCs. CONCLUSION: Collectively, these findings suggest that Spe could enhance the osteogenic potential of LEPR+ BMSCs and promote LEPR+ BMSCs-dependent angiogenesis by activating METTL3 in LEPR+ BMSCs, indicating its potential as an ideal therapeutic agent for clinical treatment of osteoporosis.

ITRAQ-based proteomics analysis of human ectopic endometrial stromal cells treated by Maqian essential oil.

BACKGROUND: Endometriosis is a common and complex syndrome characterized by the presence of endometrial-like tissue outside the uterus. Chinese medicine has been recently found to show good efficacy in treating endometriosis. Our previous results revealed that Maqian fruit essential oil (MQEO) could inhibit the proliferation and induce apoptosis of ectopic endometrial stromal cells (EESCs), but the mechanisms remain unclear. In this study, we aim to explore the molecular mechanism of MQEO's specific effects in EESCs. METHODS: We conducted a quantitative proteomics analysis by iTRAQ on EESCs treated with MQEO or DMSO. Then deep analysis was performed based on differentially expressed proteins, including Gene Ontology enrichment analysis, pathway enrichment analysis and protein interaction analysis. Candidate protein targets were subsequently verified by western blotting. RESULTS: Among 6575 identified proteins, 435 proteins exhibited altered expression levels in MQEO-treated EESCs. Of these proteins, most were distributed in signal transduction as well as immune system and the most significantly altered pathway was complement and coagulation cascades. Moreover, two differentially expressed proteins (Heme oxygenase 1 and Acyl-CoA 6-desaturase) were verified and they can be potential biomarkers for endometriosis treatment. CONCLUSIONS: Our proteomic analysis revealed distinct protein expression patterns induced by MQEO treatment in EESCs, highlighting the potential of MQEO for endometriosis treatment and biomarker discovery.

Chinese Ecliptae herba (Eclipta prostrata (L.) L.) extract and its component wedelolactone enhances osteoblastogenesis of bone marrow mesenchymal stem cells via targeting METTL3-mediated m6A RNA methylation.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese Ecliptae herba (Eclipta prostrata (L.) L.) is an ethnomedicinal herb, which is used mainly to nourish kidney and thus strengthen bones according to traditional Chinese medicine theory. Pharmacological studies have supported the ethnomedicine use, showing that Ecliptae herba extract has an anti-osteoporotic effect in vivo and promoted osteoblast proliferation and activity in vitro. However, the molecular mechanism of Ecliptae herba on osteoblast differentiation from bone marrow mesenchymal stem cells (BMSC), the progenitors of osteoblasts, is still unclear. AIM OF THE STUDY: N6-methyladenosine (m6A) mRNA epigenetic modification may play a key role in promoting osteoblastic differentiation, and thus treating osteoporosis. This study sought to assess the mechanism through which Eclipate herba and its component wedelolactone influence m6A modification during the process of osteoblastogenesis from BMSC. MATERIAL AND METHODS: The alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were applied to determine osteoblastogenesis from BMSC. Western blot and quantitative real-time PCR were performed. RNA sequencing analysis was used to determine the characteristics of m6A methylation. Stable knocking down of METTL3 using lentiviral-based shRNA was performed. RESULTS: Upon 9 d treatment of BMSC with ethyl acetate extract of Ecliptae herba (MHL), ALP activity and ossification level increased in comparison with osteogenic medium (OS)-treated control. The expression of methyltransferase METTL3 and METTL14 was significantly increased, but WTAP expression had no change in response to MHL treatment. Knocking down of METTL3 resulted in a decrease in MHL-induced ALP activity, ossification level as well as mRNA expression of Osterix and Osteocalcin, two bone formation-related markers. The level of m6A increased when BMSC was treated with MHL for 9 d. RNA sequencing analysis indicated that MHL treatment altered mRNA m6A modification of genes associated with osteoblastogenesis. By kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, HIF-1α, PI3K/Akt, and Hippo signaling pathways were enriched and associated with m6A modification. The expression of m6A-modified genes including HIF-1α, VEGF-A, and RASSF1, was upregulated by MHL, but the upregulation was reversed after METTL3 knockdown. Additionally, the enhanced expression of METTL3 was also observed after treatment with wedelolactone, a component from MHL. CONCLUSIONS: These results suggested a previously uncharacterized mechanism of MHL and wedelolactone on osteoblastogenesis, by which METTL3-mediated m6A methylation is involved and thus contributes to the enhancement of osteoblastogenesis.

Shared Decision-Making in Hemophilic Arthropathy Rehabilitation: A Qualitative Study.

Purpose: To probe into the needs and barriers underlying patients' participation in shared decision-making related to rehabilitation nursing for hemophilic arthropathy. Patients and Methods: The phenomenological research approach was adopted to conduct a series of semi-structured, in-depth interviews with 15 patients with hemophilic arthropathy undergoing rehabilitative treatments, 10 caregivers, and 7 healthcare providers from a hemophilia treatment center in Shanxi province, China. Colaizzi's seven-step method of data analysis was applied to organize, analyze, and extract the themes from the interview materials. Results: Three main themes emerged from the analysis: the status quo of the healthcare system (insufficient decision support systems and mismatch between healthcare providers' and patients' resources), circumstances of provider-patient interactions (lack of information exchange and unbalanced power structure between healthcare providers and patients), and patient-related factors influencing participation in decision-making (lack of self-efficacy, personal characteristics, family and social decision support, and attitude toward participation in decision-making). Conclusion: Participation in rehabilitation decision-making among patients with hemophilic arthropathy is affected by multiple barriers. Healthcare professionals should improve their understanding of shared decision-making, offer patients active guidance on participating in the decision-making process, prioritize their affective needs, and formulate professional and effective solutions to support shared decision-making as early as possible.

Predictive value of left ventricular dyssynchrony for short-term outcomes in three-vessel disease patients undergoing coronary artery bypass grafting with preserved or mildly reduced left ventricular ejection fraction.

Background and objective: Coronary artery bypass grafting (CABG) is the reference standard intervention in coronary artery disease (CAD) patients with three-vessel disease (3VD). We aimed to evaluate the predictive value of left ventricular (LV) dyssynchrony for short-term adverse outcomes in patients with 3VD undergoing CABG with preserved or mildly reduced LV ejection fraction (LVEF). Materials and methods: This study involved ninety-five 3VD patients with preserved or mildly reduced LVEF undergoing scheduled on-pump CABG. The pre-operative diameters and volumes of LV and LVEF were obtained by two-dimensional echocardiography. LV dyssynchrony parameters were acquired by real-time three-dimensional echocardiography (RT-3DE) and analyzed by HeartModel quantification software. And the perfusion index of LV was obtained by contrast echocardiography. The clinical endpoints of short-term adverse outcomes comprised 30-day mortality and/or composite outcomes of postoperative complications. Univariate and multivariate logistic regression analyses were used to identify risk factors for the occurrence of post-CABG short-term adverse outcomes. Results: Short-term adverse outcomes occurred in 12 (12.6%) patients. These patients had higher LV dyssynchrony parameters obtained through RT-3DE. The standard deviation (SD) of the time to minimum systolic volume (Tmsv) corrected by heart rate over 16 segments (Tmsv16-SD%) [odds ratio (OR), 1.362; 95% confidence interval (CI) (1.090-1.702); P = 0.006], one of the LV dyssynchrony parameters, was independently associated with short-term adverse outcomes. Patients with poor synchronization tended to spend more time in the intensive care unit (ICU) and hospital after surgery. Conclusion: Pre-operative LV dyssynchrony parameter Tmsv16-SD% obtained through RT-3DE could be a useful additional predictor of postoperative short-term adverse outcomes in 3VD patients with preserved or mildly reduced LVEF undergoing CABG.

In Vitro Inhibitory Effects of Maqian Essential Oil against Ectopic Endometrial Stromal Cells and LPS-Induced Endometrial Epithelial Cells.

Previous studies revealed that MQEO (Maqian fruits essential oil), which is extracted from the fruit of Maqian (Zanthoxylum myriacanthum var. Pubescens), had a good anti-inflammatory effect, but the effect on endometriosis in vitro remains unknown. In the present study, the inhibitory effects of MQEO against the EESCs (ectopic endometrial stromal cells) were investigated. Cells were treated with a concentration gradient (from 0.025 % to 0.15 %) of MQEO for 24 h and cell viability was detected by CCK-8. In addition, apoptotic rates were investigated using flow cytometry. The effect of MQEO on cell migration was determined by wound-healing and transwell assay. The expression of apoptosis-associated and cell adhesion-related proteins was assessed by western blotting. The transcriptional levels of IL-1, IL-6 and TNF-α were determined by Real-time qPCR. RNA-seq was used to identify the DEGs (differentially expressed genes) in MQEO-pretreated EESCs. We found that the MQEO condition dosage-dependently reduced the cell viability of EESCs. Based on flow cytometry results, the number of apoptotic cells increased significantly with dosage. The wound-healing and transwell results showed that MQEO group exhibited a significantly decreased cell motility and migration ability in comparison with the normal group. Western blotting results showed that MQEO down-regulated the expression of Bcl-2, ICAM-1 (intercellular adhesion molecule 1) and CD44, but up-regulated the cleaved caspase-3 expression in EESCs. What's more, MQEO also inhibited the LPS-induced inflammation in human EECs (endometrial epithelial cells). RNA-seq revealed that 221 DEGs were up-regulated genes and 284 DEGs were down-regulated in MQEO-pretreated EESCs. Our data uncovered the beneficial effects of MQEO in endometriosis and provided new insights into the mechanism of the effect of MQEO on EESCs, suggesting MQEO could be a promising new therapeutic agent for endometriosis.

Diagnosis of left anterior descending branch-right ventricular fistula with giant coronary artery aneurysm by contrast echocardiography: A case report.

BACKGROUND: Coronary fistulae are communications between a coronary artery and a heart chamber or vessel. The final diagnosis is usually made by coronary angiography or computed tomographic (CT) angiography. Here we report a case by employing contrast echocardiography in diagnosis of a giant coronary aneurysm with right ventricle (RV) fistula. CASE PRESENTATION: The patient, a 29-year-old woman, referred to our institution with a complaint of palpitation occasionally. Transthoracic echocardiogram showed a spherical, echogenic structure in the apex of RV. Proximal to the aneurysm, the left anterior descending branch (LAD) remained enlarged (8-9 mm) and showed a fistulous communication with the echogenic structure. A contrast echocardiography was performed, and 4-5 cardiac cycle after the left ventricle was enhanced, the echogenic structure started to become more prominent and several fistulae were seen between RV and the echogenic structure. Computed tomography (CT) angiography and coronary angiography confirmed the dilation (9 mm in diameter) of the LAD with an aneurysm at the distal segment of the LAD, with a small amount of iodinated contrast agent flowing into the subsequent region of the RV, thereby characterizing a LAD-to-RV fistula. CONCLUSION: The final diagnosis of fistula is usually made by coronary angiography or CT angiography. However, contrast echocardiography is also a well-established method for the demonstration of intracardiac shunting. In this case, the contrast echocardiography clearly revealed one of the fistulae between the aneurysm and RV.

Protective effect of Du-Zhong-Wan against osteoporotic fracture by targeting the osteoblastogenesis and angiogenesis couple factor SLIT3.

ETHNOPHARMACOLOGICAL RELEVANCE: Du-Zhong-Wan (DZW) is a traditional Chinese medicine (TCM) composed of Eucommia ulmoides Oliv. and Dipsacus asper Wall. ex C.B. Clarke in the ratio 1:1. Based on the TCM theory, DZW nourishes the kidney to strengthen the bones. The literature research revealed that DZW possesses anti-fatigue, anti-depressant, and anti-osteoporotic properties. However, the action and mechanism of DZW on osteoporotic fracture remains slightly unclear. AIM OF THE STUDY: To evaluate the pharmacological effect of DZW on ovariectomized mice with an open femoral fracture and reveal the underlying mechanism. MATERIALS AND METHODS: We conducted ovariectomy for 5 weeks, followed by unilateral open transverse femoral fracture for another 3 weeks in C57BL/6 mice; during this process, DZW was administrated. The femur bone and vertebra tissues were collected and analyzed by micro-computed tomography, histomorphometry, mechanical strength testing, immunohistochemistry staining, and qRT-PCR analyses. In addition, alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were performed to determine the extent of osteoblastogenesis from bone marrow mesenchymal stem cells (BMSCs). Western blotting was performed to examine the protein expression. RESULTS: DZW treatment significantly improved the bone histomorphometric parameters in mice undergoing ovariectomy when combined with the femoral fracture, including an increase in the bone volume, trabecular number, and bone formation rate and a decrease in the bone erosion area. Simultaneously, DZW treatment histologically promoted fractured callus formation. Mechanical strength testing revealed significantly higher stiffness and an ultimate load after treatment with DZW. The angiogenesis of H-type vessels was enhanced by DZW, as evidenced by increased levels of CD31 and endomucin (EMCN), the H-type vessel endothelium markers, at the fractured endosteum and metaphysis regions. Relative to the osteoporotic fracture mice, the DZW treatment group showed an increased proangiogenic factor SLIT3 level. The increased level of SLIT3 was also recorded during the process of DZW-stimulated osteoblastogenesis from BMSCs. CONCLUSIONS: For the first time, we demonstrated that DZW promoted osteoporotic fracture healing by enhancing osteoblastogenesis and angiogenesis of the H-type vessels. This enhanced combination of osteoblastogenesis and angiogenesis was possibly related to the production of proangiogenic factor SLIT3 induced by DZW.

Redirection of acyl donor metabolic flux for lipopeptide A40926B0 biosynthesis.

The metabolic flux of fatty acyl-CoAs determines lipopeptide biosynthesis efficiency, because acyl donor competition often occurs from polyketide biosynthesis and homologous pathways. We used A40926B0 as a model to investigate this mechanism. The lipopeptide A40926B0 with a fatty acyl group is the active precursor of dalbavancin, which is considered as a new lipoglycopeptide antibiotic. The biosynthetic pathway of fatty acyl-CoAs in the A40926B0 producer Nonomuraea gerenzanensis L70 was efficiently engineered using endogenous replicon CRISPR (erCRISPR). A polyketide pathway and straight-chain fatty acid biosynthesis were identified as major competitors in the malonyl-CoA pool. Therefore, we modified both pathways to concentrate acyl donors for the production of the desired compound. Combined with multiple engineering approaches, including blockage of an acetylation side reaction, overexpression of acetyl-CoA carboxylase, duplication of the dbv gene cluster and optimization of the fermentation parameters, the final strain produced 702.4 mg l-1 of A40926B0, a 2.66-fold increase, and the ratio was increased from 36.2% to 81.5%. Additionally, an efficient erCRISPR-Cas9 editing system based on an endogenous replicon was specifically developed for L70, which increased conjugation efficiency by 660% and gene-editing efficiency was up to 90%. Our strategy of redirecting acyl donor metabolic flux can be widely adopted for the metabolic engineering of lipopeptide biosynthesis.

Prevalence of the methylenetetrahydrofolate reductase 677C>T polymorphism in the pregnant women of Yunnan Province, China.

Mutations in the methylenetetrahydrofolate reductase (MTHFR) gene can result in a reduced ability to utilize folic acid. The MTHFR 677C>T polymorphism in particular has been linked to both birth defects and pregnancy-associated diseases. This study aimed to evaluate the prevalence of the MTHFR 677C>T mutation among pregnant women in Yunnan Province so as to collect baseline data that may be utilized to guide folic acid supplementation efforts and to support related disease prevention programs. We retrospectively reviewed 3387 pregnant women from Yunnan Province. The MTHFR 677C>T polymorphism was identified using polymerase chain reaction (PCR) and DNA sequencing. In total, 1350 (39.9%) subjects were homozygous for the C allele (CC), 1540 (45.4%) subjects were heterozygous (CT), and 497 (14.7%) subjects were homozygous for the T allele (TT). The MTHFR 677C>T polymorphism was found to be present within the studied population, with ∼60% of these patients being either heterozygous or homozygous for the mutant allele and with an overall T allele frequency of 0.37. The frequency of the T allele was significantly higher among pregnant women with complications relative to women with healthy pregnancies, particularly among women <30 years old. As such, the maternal MTHFR 677C>T polymorphism may be a genetic risk factor associated with pregnancy complications and may help identify pregnant women at a high risk of such complications.

Next-generation sequencing: a follow-up of 36,913 singleton pregnancies with noninvasive prenatal testing in central China.

PURPOSE: To evaluate the noninvasive prenatal testing (NIPT) results of 36,913 cases in Jiangxi province of central China and explore its application value in prenatal screening and diagnosis. METHODS: This retrospective analysis included 36,913 singleton pregnant women who underwent NIPT because of moderate-/high-risk pregnancy or voluntary requirements between January 2017 and December 2019 in our hospital. Chromosomal abnormalities such as trisomies 21, 18, and 13 (T21, T18, T13) and sex chromosome aneuploidies (SCAs) were judged by standard Z-score analysis. Positive NIPT results were confirmed by amniocentesis and karyotyping. Pregnancy outcomes were followed up via telephone interview. RESULTS: A total of 1.01% (371/36,913) positive cases were detected by NIPT, comprising 137, 46, 31, and 157 cases of T21, T18, T13, and SCAs, respectively. A total of 116 of T21, 27 of T18, 13 of T13, and 51 of SCAs were confirmed to be true positive; all normal cases that had been followed up were verified to be true negative. The NIPT sensitivity in T21, T18, T13, and SCAs was 100.00% individually, whereas the specificity was 99.94% (36,488/36,509), 99.95% (36,579/36,598), 99.95% (36,594/36,612), and 99.72% (36,472/36,574), respectively. Furthermore, the negative predictive values of T21, T18, T13, and SCAs were all 100%, while the positive predictive values were 84.67%, 58.70%, 41.94%, and 33.33%, respectively. CONCLUSION: NIPT is highly sensitive and has a low false positive rate in testing clinically significant fetal aneuploidies of general reproductive women. However, this technique cannot substitute for amniocentesis and karyotyping, and detailed genetic counseling is also essential for the high-risk group of NIPT.

[Effects of hypothermia on the repolarization duration of ventricular myocytes in rats and its mechanism].

Objective: To observe the effects of hypothermia on the repolarization duration and the expression of Kir2.1 protein of ventricular myocytes in isolated rat heart and explore the role of Kir2.1 protein.Methods: Eighteen healthy adult male Sprague-Dawley rats were randomly divided into three groups (n=6 per group): Control group (C group), 35℃ group (H1 group), 32℃ group (H2 group). Langendorff isolated heart models were established. After 15 min 37℃ K-H fluid banlanced perfusion, C group continued to perfuse the K-H solution at 37℃ for 30 minutes, H1 group continued to perfuse the K-H solution at 35℃ for 30 minutes, H2 group continued to perfuse the K-H solution at 32℃ for 30 minutes. At 15 min of balanced perfusion (T1), and 30 min of continuous perfusion (T2), the heart rate,and the MAP in the three layers of the left ventricular anterior wall were recorded, the action potential duration at 50% repolarization (MAPD50), the action potential duration at 90% repolarization (MAPD90) and transmural dispersion of repolarization(TDR) were calculated. At the same time, the occurrence of arrhythmia was recorded. The expression of Kir2.1 protein was measured by Western blot. The average optical density (AOD) and the distribution of Kir2.1 protein were measured by immunohistochemistry in the ventricular tissue measured by electrophysiology. Results: Compared with T0, the heart rate was decreased, MAPD50 and MAPD90 were prolonged significantly (P＜0.05), and TDR was increased significantly (P＜0.05) in H1 group, H2 group at T1. Compared with C group, the HR was decreased, the MAPD90 was prolonged significantly (P＜0.05), TDR was increased significantly (P＜0.05),the expression and the AOD of Kir2.1 protein were decreased significantly (P＜0.05) in H1group, H2group at T1. Compared with H1 group, the heart rate of H2 group was decreased significantly (P＜0.05), MAPD50 and MAPD90 were prolonged significantly (P＜0.05), and TDR was increased significantly (P＜0.05) at T1. The distribution of Kir2.1 protein in group C was normal, while the distribution of Kir2.1 in H1 group and H2 group was disordered. Conclusion: Hypothermia prolonged the ventricular duration of repolarization and increased the dispersion of repolarization. The mechanism is related to the down-regulation the expression of Kir2.1 protein and the disorder of the distribution of Kir2.1 protein.

Migrant adolescents' behavioral problems compared to host adolescents and adolescents in their region of origin: a longitudinal study.

BACKGROUND: Since the 1990s, families from the ecologically hostile mountainous southern areas of Ningxia Province, China, have been migrating to the northern areas of the province. This study compared the prevalence of behavioral problems among migrant adolescents to those among host adolescents (adolescents from the northern areas) and adolescents in the region of origin (adolescents from the southern areas), to determine whether ecological migration is related to adolescent behavioral problems, and possible changes in such problems over time. METHODS: We used the Children and Adolescents Ecological Migration Survey on Mental Health, administered to 4805 students aged 12-16 years and their parents between 2012 and 2014 (W1), of whom 1753 students and their parents completed the follow-up between 2014 and 2017 (W2). Parents answered questions related to adolescent behavioral problems, main source of family income, parents' desire to reverse migrate, improved standard of living, and parents' educational attainment, while children completed the Eysenck Personality Questionnaire and a classroom environment questionnaire. RESULTS: The prevalence of behavioral problems among the migrant adolescents (28.04%) was significantly higher than among host adolescents (21.59%) or adolescents in the region of origin (24.37%; p < 0.001) at W1. After adjusting for gender and age, parents' work outside the home was the main source of family income (OR = 1.42, 95% CI = 1.13-1.78), and adolescents' learning burden (OR = 1.04, 95% CI = 1.01-1.06) in school negatively influenced behavioral problems. Strong student-teacher relationships (OR = 0.97,95% CI = 0.94-0.99) and parents who had no intention to move back to the original residence (OR = 0.70, 95% CI = 0.52-0.94) exerted a protective effect at W1; at W2, a protective effect was associated with improved living conditions (OR = 0.39-0.55, 95% CI = 0.25-0.84). The extent of behavioral problems among migrant adolescents significantly decreased after two years. CONCLUSION: Ecological migration will increase children's behavioral problems in the early stage, with various factors influencing the extent of these problems.

A novel delins (c.773_819+47delinsAA) mutation of the PCCA gene associated with neonatal-onset propionic acidemia: a case report.

BACKGROUND: Propionic acidemia (PA)(OMIM#606054) is an inborn error of branched-chain amino acid metabolism, caused by defects in the propionyl-CoA carboxylase (PCC) enzyme which encoded by the PCCA and PCCB genes. CASE PRESENTATION: Here we report a Chinese neonate diagnosed with suspected PA based on the clinical symptoms, gas chromatography-mass spectrometry (GC/MS), and brain imaging tests. Targeted next-generation sequencing (NGS) was performed on the proband. We detected only one heterozygous recurrent nonsense variant (c.937C > T, p.Arg313Ter) in the PCCA gene. When we manually checked the binary alignment map (BAM) diagram of PCCA gene, we found a heterozygous deletion chr13:100915039-100915132delinsAA (c.773_819 + 47delinsAA) (GRCh37.p13) inside the exon 10 in the PCCA gene. The results were validated by Sanger sequencing and qPCR method in the family: the variant (c.937C > T, p.Arg313Ter) was in the maternal allele, and the delins was in the paternal allele. When the mother was pregnant again, prenatal diagnosis was carried out through amniocentesis at 18 weeks gestation, the fetus carried neither of the two mutations. After birth, newborn screening was undertaken, the result was negative. CONCLUSIONS: We identified a recurrent c.937C > T and a novel c.773_819 + 47delinsAA mutations in the PCCA gene, which may be the genetic cause of the phenotype of this patient. Our findings expanded the spectrum of causative genotype-phenotype of the PCCA gene. For the cases, the NGS results revealed only a heterozygous mutation in autosomal recessive disease when the gene is associated with phenotypes, it is necessary to manually check the BAM diagram to improve the detection rate. Targeted NGS is an effective technique to detect the various genetic lesions responsible for the PA in one step. Genetic testing is essential for genetic counselling and prenatal diagnosis in the family to avoid birth defects.

Myocardial contrast echocardiography in the diagnosis of postoperative takotsubo myocardiopathy: case report and literature review.

BACKGROUND: Takotsubo cardiomyopathy (TCM) is a brief ventricular dysfunction that usually occurs after emotional or physical stress. Here, we report a patient who underwent cardiac surgery and then developed TCM during the postoperative period. CASE PRESENTATION: A 51-year-old woman was admitted to our hospital complaining of chest tightness, palpitations and dyspnoea after activity. An echocardiogram performed by our hospital showed rheumatic heart disease (severe mitral stenosis and regurgitation) with normal cardiac function and wall motion. After mitral valve replacement, this patient developed heart failure with low blood pressure and tachycardia. Urgent bedside echocardiography demonstrated akinesis in the middle and apical segments of the left ventricle and a depressed ejection fraction (EF) of 36%. Myocardial contrast echocardiography (MCE) showed similar enhancement intensity in the basal, middle and apical segments. Quantitative analysis showed approximately equivalent maximum intensity in these regions. The diagnosis was considered TCM instead of myocardial infarction. Then, an intra-aortic balloon pump was inserted to maintain effective circulation and reduce the postcardiac load. Given ventilation therapy, postoperative anticoagulation therapy and anti-infection treatment, the patient recovered quickly. In the follow-up examination, the patient remained asymptomatic and showed normalization of ventricular wall motion in the apical segment. CONCLUSION: This report presents a case of TCM in which MCE was used to demonstrate intact microvascular perfusion despite apical akinesis. This report might support the use of MCE as a substitute for invasive coronary angiography.

Wedelolactone inhibits osteoclastogenesis but enhances osteoblastogenesis through altering different semaphorins production.

Our previous study showed that wedelolactone, isolated from Ecliptae herba, enhanced osteoblastogenesis but inhibited osteoclastogenesis through Sema3A signaling pathway. This study aims to investigate the role of other semaphorins in wedelolactone-enhanced osteoblastogenesis and -inhibited osteoclastogenesis. Wedelolactone inhibited RANKL-induced Sema4D and Sema7A production, but had no effect on RANKL-reduced Sema6D expression in osteoclastic RAW264.7 cells. In mouse bone marrow mesenchymal stem cells (BMSC), wedelolactone reversed osteogenic medium(OS)-reduced Sema7A expression and OS-enhanced Sema3E mRNA expression, but no effect on OS-reduced Sema3B mRNA expression. Addition of Sema4D antibody promoted wedelolactone-reduced TRAP activity and bone resorption pit formation. Wedelolactone combined with Sema4D antibody inhibited the formation of Sema4D-Plexin B1 complex. In co-culture of BMSC with RAW264.7 cells, Sema7A antibody, similar with Sema 3A antibody, reversed wedelolactone-enhanced ALP activity and mineralization level, but promoted wedelolactone-inhibited TRAP activity. However, Sema3E and Sema3B antibodies had no effect. Further, wedelolactone enhanced the binding of Sema7A with PlexinC1 and Beta1, but addition of Sema7A antibody partially blocked this binding. Our data demonstrated that wedelolactone inhibited Sema4D production and Sema4D-PlexinB1 complex formation in RAW264.7 cells, thereafter inhibiting osteoclastogenesis. At the same time, wedelolactone enhanced osteoblastogenesis through promoting Sema7A production and Sema7A-PlexinC1-Beta1 complex formation in BMSC.

Wedelolactone Enhances Osteoblastogenesis through ERK- and JNK-mediated BMP2 Expression and Smad/1/5/8 Phosphorylation.

Our previous study showed that wedelolactone, a compound isolated from Ecliptae herba, has the potential to enhance osteoblastogenesis. However, the molecular mechanisms by which wedelolactone promoted osteoblastogenesis from bone marrow mesenchymal stem cells (BMSCs) remain largely unknown. In this study, treatment with wedelolactone (2 µg/mL) for 3, 6, and 9 days resulted in an increase in phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal protein kinase (JNK), and p38. Phosphorylation of mitogen-activated protein kinases (MAPKs), ERK and JNK started to increase on day 3 of treatment, and p38 phosphorylation was increased by day 6 of treatment. Expression of bone morphogenetic protein (BMP2) mRNA and phosphorylation of Smad1/5/8 was enhanced after treatment of cells with wedelolactone for 6 and 9 days. The addition of the JNK inhibitor SP600125, ERK inhibitor PD98059, and p38 inhibitor SB203580 suppressed wedelolactone-induced alkaline-phosphatase activity, bone mineralization, and osteoblastogenesis-related marker genes including Runx2, Bglap, and Sp7. Increased expression of BMP2 mRNA and Smad1/5/8 phosphorylation was blocked by SP600125 and PD98059, but not by SB203580. These results suggested that wedelolactone enhanced osteoblastogenesis through induction of JNK- and ERK-mediated BMP2 expression and Smad1/5/8 phosphorylation.