Nuclear isolation is crucial for studying gene expression and regulatory mechanisms in eukaryotic cells. This study aimed to improve nuclear isolation and compare the yield, purity, and efficiency of several methods. Human umbilical vein endothelial cells were used to evaluate four different techniques: sucrose centrifugation, a simplified method, homogenization, and the NE-PER kit. For sucrose centrifugation, cells were scraped in Tween buffer, washed with sucrose buffer, and homogenized in a Dounce homogenizer. The pellet was washed with glycerol buffer to isolate the nuclei. In the simplified method, cells were scraped in scraping buffer, washed with sucrose buffer, and the pellet was washed with glycerol buffer to isolate the nuclei. For homogenization, cells were washed with phosphate buffered saline, followed by two washes in extract buffer and lysed with 10 strokes in a Kontes Dounce homogenizer. The NE-PER kit was used according to the manufacturer's protocol. Nuclei isolated by each method were tested by immunoblotting, co-immunoprecipitation (Co-IP), and chromatin immunoprecipitation (Ch-IP) assays. The simplified method produced nuclei with fewer organelles and less cytoplasm than those isolated by homogenization or the NE-PER kit. It was similarly effective as sucrose centrifugation but faster. Co-IP and Ch-IP assays confirmed that the simplified method enriched target proteins and DNA fragments. Overall, the simplified method provides a highly pure nuclear sample optimal for downstream applications requiring purified nuclei.

重症患者病情复杂、变化迅速，需快速判断与处理病情变化。诊疗过程中的医疗差错是加剧患者死亡的重要原因，合理、规范、精准的重症诊疗尤为重要。提升诊疗质量需要有具体着力点，因此从“认知、认识、流程”3个位点进行阐述：对重症正确认知即为对疾病本质及重症化发展规律的清晰掌握；对病情的认识就是通过对临床信息及指标的正确解读来准确掌握病情状态；规范可行的诊疗流程有助于引导医生按照更佳的路径收集、分析信息，形成更合理的判断与治疗行为，由此提高“单兵”诊疗质量，提升团队协作效率。

Turning to critical illness is a common stage of various diseases and injuries before death. Patients usually have complex health conditions, while the treatment process involves a wide range of content, along with high requirements for doctor's professionalism and multi-specialty teamwork, as well as a great demand for time-sensitive treatments. However, this is not matched with critical care professionals and the current state of medical care in China. Telemedicine, which shortens the distance of medical professionals and the gap of disease diagnosis and treatments in various regions through electronic information, can effectively solve the current problem. Therefore, there is an urgent need to develop a standardized, high-quality visualization telemedicine round system .Therefore, experts have been organized to search domestic and foreign literature on telemedicine round for critically ill patients and to form this consensus based on clinical experiences so as to further improve the level of critical care treatments in regions.

Mesenchymal stromal cells (MSCs) and stem cells are distinct types of cells, but they are practically undistinguishable by currently commonly-used identification markers. A single-cell transcriptomic analysis was used to solve this problem. There are eight critical genes involved in self-renewal and differentiation, SOX2, NANOG, POU5F1, SFRP2, DPPA4, SALL4, ZFP42 and MYCN expressed in ESCs, iPSCs and adult stem cells (ASCs), but not in MSCs. There are five functional genes of MSCs, TMEM119, FBLN5, KCNK2, CLDN11 and DKK1, which are not expressed in stem cells. Trajectory analysis displayed clear developmental cliffs from ESCs/iPSCs to ASCs and to MSCs. Adipose-derived MSCs, relative to other types of MSCs, exhibit a more consistent and broader spectrum of gene expression for regulatory and excrete function. This study identifies distinction markers between MSCs and stem cells, providing an alternative approach for quality control of MSCs in their propagation and further mechanistic insights into their action.

Alcohol-related liver disease is accompanied by hepatic zinc deficiency. We synthesized a zinc-glutathione (ZnGSH) complex as a zinc delivery system to test the hypothesis that effective zinc supplementation can recover the liver from alcohol-related injury. Mice were fed an ethanol-containing liquid diet for 6 months to develop alcohol-related liver injury. ZnGSH, Zn, GSH, or Zn+GSH was independently administered to mice for one month after the 6-month alcohol feeding. All of the treatments elevated hepatic Zn levels. ZnGSH increased total GSH levels and the GSH-to-GSSG ratio, similar to that caused by GSH or Zn+GSH. Importantly, only ZnGSH, no other treatments, significantly recovered alcohol-related liver injury, as judged by reductions in hepatic steatosis, fat deposition, and inflammation. ZnGSH increased metallothionein (MT) mRNA and protein levels in the liver, an effect was not observed by other treatments. The therapeutic effect of ZnGSH on alcohol-related liver injury was diminished in an MT-II-knockout (MT-KO) mouse model of alcohol-related liver injury, in which ZnGSH did not increase the total hepatic GSH or zinc levels. Thus, ZnGSH recovers the liver from alcohol-related injury depending on the production of MT, a distinguished effect not caused by treatment with GSH or zinc, or their simple combination.

Long-term alcohol overconsumption impairs intestinal and hepatic structure and function, along with dysregulation of zinc homeostasis. We previously found that zinc-glutathione (Zn-GSH) complex effectively suppressed alcohol-induced liver injury in mice. This study was undertaken to test the hypothesis that Zn-GSH suppresses alcohol-induced liver injury by modulating intestinal zinc transporters. Mice were subjected to long-term ethanol feeding, as per the NIAAA model, with groups receiving either an ethanol diet alone or an ethanol diet supplemented with Zn-GSH. Treatment groups were carefully monitored for alcohol consumption and subjected to a final binge drinking exposure. The results showed that Zn-GSH increased the survival rate and decreased the recovery time from binge drinking-induced drunkenness. Histopathological analyses demonstrated a reduction in liver steatosis and the preservation of intestinal integrity by Zn-GSH. It was observed that Zn-GSH prevented the reduction of Zn and GSH levels while increasing alcohol dehydrogenase and aldehyde dehydrogenase in both liver and intestine. Importantly, the expression and protein abundance of zinc transporters ZnT-1, ZIP-1, ZIP-4, ZIP-6, and ZIP-14, all of which are critically involved in intestinal zinc transport and homeostasis, were significantly increased or preserved by Zn-GSH in response to alcohol exposure. This study thus highlights the critical role of Zn-GSH in maintaining intestinal zinc homeostasis by modulating zinc transporters, thereby preventing alcohol-induced intestinal and hepatic injury.

Abstract Background Body Mass Index (BMI) has been recognized as a prognosticator for heart failure (HF) in that higher BMI relates to lower mortality. However, limited data are available whether body composition underneath BMI would further differentiate patients with a better or worse outcome, especially in those with lower or normal BMI. Purpose To investigate the characteristics of body fat and body muscle in HF patient groups with different BMI, and their impact on prognosis. Methods Between July 2021 and July 2022, hospitalized HF patients who were suitable for the examination underwent body composition analysis by using bioelectrical analyses (BIA) in the stable state after decongestion therapy and before discharge. Percentage of body fat (PBF) and percentage of body muscle (PBM) were calculated by dividing the mass of body fat, or body muscle, respectively. Patients were divided into underweight (<18.5kg/m2), normal weight (20-24.9 kg/m2), overweight/obese (≥25kg/m2) based on BMI, who were followed for 180 days for all-cause death. Results 127 consecutive patients with obtained data were finally enrolled (60±15 years, 70% males), including 20 (15.75%) underweight, 75 (59.06%) normal weight, and 32 (25.19%) overweight/obese. As shown in Table 1, As shown in Table 1, the body fat mass and muscle mass varied with BMI, though they showed a different rate of change. Compared with patients with normal weight, the fat and muscle mass in those overweight/obese was 81.8% and 12.4% higher; whereas it was 39.2%, and 23.1% lower in those underweight, respectively. As results, the overweight/obesity group had a higher PBF, but a lower percentage of PBM. Consequently, the overweight/obesity group had a higher PBF, a lower PBM and a decreased muscle mass/fat mass ratio (MFR); conversely, the underweight group had a lower PBF, a higher PBM and an increased MFR. During 180-day follow-up, 17 (13.4%) died for all cause. By Cox regression analysis, MFR independently predict all-cause death (HR, 1.273, 95% CI: 1.056-1.534, p=0.011) after adjusted for age, BMI, ejection fraction and NT-proBNP. Of note, in patients with normal weight, Receiver Operating Characteristic (ROC) curve analysis suggested the capability of MFR (AUC=0.780, p=0.003) as well as its superiority over BMI in association with all-cause mortality (Figure 1). Conclusions The change of body fat or muscle mass with BMI displayed a different rate in HF patients, so that the MFR was higher in underweight but lower in overweight/obese when compared to normal weight group. Apart from BMI, MFR emerged as a predictor of mortality, in particular in patients with normal weight.

Background: Current replacement procedures for stenosis or occluded arteries using prosthetic grafts have serious limitations in clinical applications, particularly, endothelialization of the luminal surface is a long-standing unresolved problem. Method: We produced a cell-based hybrid vascular graft using a bioink engulfing adipose-derived mesenchymal stromal cells (ADSCs) and a 3D bioprinting process lining the ADSCs on the luminal surface of GORE-Tex grafts. The hybrid graft was implanted as an interposition conduit to replace a 3-cm-long segment of the infrarenal abdominal aorta in Rhesus monkeys. Results: Complete endothelium layer and smooth muscle layer were fully developed within 21 days post-implantation, along with normalized collagen deposition and crosslinking in the regenerated vasculature in all monkeys. The regenerated blood vessels showed normal functionality for the longest observation of more than 1650 days. The same procedure was also conducted in miniature pigs for the interposition replacement of a 10-cm-long right iliac artery and showed the same long-term effective and safe outcome. Conclusion: This cell-based vascular graft is ready to undergo clinical trials for human patients.

Atherosclerosis has traditionally been considered as a disorder characterized by the accumulation of cholesterol and thrombotic materials within the arterial wall. However, it is now understood to be a complex inflammatory disease involving multiple factors. Central to the pathogenesis of atherosclerosis are the interactions among monocytes, macrophages, and neutrophils, which play pivotal roles in the initiation, progression, and destabilization of atherosclerotic lesions. Recent advances in our understanding of atherosclerosis pathogenesis, coupled with results obtained from experimental interventions, lead us to propose the hypothesis that atherosclerosis may be reversible. This paper outlines the evolution of this hypothesis and presents corroborating evidence that supports the potential for atherosclerosis regression through the restoration of vascular copper homeostasis. We posit that these insights may pave the way for innovative therapeutic approaches aimed at the reversal of atherosclerosis.

随着重症超声应用日益广泛，合理规范的培训越来越重要。现有重症超声培训多以短期培训为主，培训内容多、时间短，学员的重症医学基础与学习能力差异大，培训后对所学掌握不一，不论是超声图像获取抑或临床解读及应用。因此提出基于CPVAP（clinical analysis，protocol，view，approach and practice-workflow）理念的体系化培训方案，将培训内容拆解为基础理论和技能入门培训、技能标准化练习、技能强化练习、基于视觉积累的判读和解读训练、基于标准案例的诊疗决策训练五个模块，匹配与之适应的基地培训新模式，以基地为承载，以重症超声三维立体虚拟演示系统为依托，以阶梯化培训课程体系为核心，以学员、培训师与基地培训保障人员为主要要素，以培训机制为运行保障，探索未来重症超声培训的发展方向。

The production of small-diameter artificial vascular grafts continues to encounter numerous challenges, with concerns regarding the degradation rate and endothelialization being particularly critical. In this study, porous PCL scaffolds were prepared, and PCL vascular grafts were fabricated by 3D bioprinting of collagen materials containing adipose-derived mesenchymal stem cells (ADSCs) on the internal wall of the porous PCL scaffold. The PCL vascular grafts were then implanted in the abdominal aorta of Rhesus monkeys for up to 640 days to analyze the degradation of the scaffolds and regeneration of the aorta. Changes in surface morphology, mechanical properties, crystallization property, and molecular weight of porous PCL revealed a similar degradation process of PCL in PBS at pH 7.4 containing Thermomyces lanuginosus lipase and in situ in the abdominal aorta of rhesus monkeys. The contrast of in vitro and in vivo degradation provided valuable reference data for predicting in vivo degradation based on in vitro enzymatic degradation of PCL for further optimization of PCL vascular graft fabrication. Histological analysis through hematoxylin and eosin (HE) staining and fluorescence immunostaining demonstrated that the PCL vascular grafts successfully induced vascular regeneration in the abdominal aorta over the 640-day period. These findings provided valuable insights into the regeneration processes of the implanted vascular grafts. Overall, this study highlights the significant potential of PCL vascular grafts for the regeneration of small-diameter blood vessels.

Background: Copper (Cu), by inhibiting the factor inhibiting HIF-1 (FIH-1), promotes the transcriptional activity of hypoxia-inducible factor-1 (HIF-1).Objective: The present study was undertaken to understand the molecular mechanism by which Cu inhibits FIH-1.Methods: Human umbilical vein endothelial cells (HUVECs) were treated with dimethyloxalylglycine (DMOG) resulting in HIF-1 alpha accumulation and the FIH-1 protein complexes were pulled down for candidate protein analysis. The metal binding sites were predicted by both MetalDetector V2.0 and Metal Ion-Binding Site Prediction Server, and then the actual ability to bind to Cu in vitro was tested by both Copper-Immobilized metal affinity chromatography (Cu-IMAC) and Isothermal Titration Calorimetry (ITC). Subsequently, subcellular localization was monitored by immunocytochemistry, GFP-fusion protein expression plasmid and Western blotting in the nuclear extract. The interaction of candidate protein with HIF-1 alpha and FIH-1 was validated by CoImmunoprecipitation (Co-IP). Finally, the effect of candidate protein on the FIH-1 structure and HIF-1 alpha transcriptional activity was analyzed by the InterEvDock3 web server and real-time quantitative RT-PCR.Results: ATP-binding cassette E1 (ABCE1) was present in the FIH-1 complexes and identified as a leading Cu binding protein as indicated by a number of possible Cu binding sites. The ability of ABCE1 to bind Cu was demonstrated in vitro. ABCE1 entered the nucleus along with FIH-1 under hypoxic conditions. Protein interaction analysis revealed that ABCE1 prevented FIH-1 to bind iron ions, inhibiting FIH-1 enzymatic activity. ABCE1 silencing suppressed the expression of Cu-dependent HIF-1 target gene BNIP3, not that of Cu-independent IGF-2.Conclusion: The results demonstrate that ABCE1, as a Cu-binding protein, enters the nucleus under hypoxic conditions and inhibits FIH-1degradation of HIF-1 alpha, thus promoting HIF-1 transactivation of angiogenic gene expression.

Zinc depletion is associated with alcohol-associated liver injury. We tested the hypothesis that increasing zinc availability along with alcohol consumption prevents alcohol-associated liver injury. Zinc-glutathione (ZnGSH) was synthesized and directly added to Chinese Baijiu. Mice were administered a single gastric dose of 6 g/kg ethanol in Chinese Baijiu with or without ZnGSH. ZnGSH in Chinese Baijiu did not change the likeness of the drinkers but significantly reduced the recovery time from drunkenness along with elimination of high-dose mortality. ZnGSH in Chinese Baijiu decreased serum AST and ALT, suppressed steatosis and necrosis, and increased zinc and GSH concentrations in the liver. It also increased alcohol dehydrogenase and aldehyde dehydrogenase in the liver, stomach, and intestine and reduced acetaldehyde in the liver. Thus, ZnGSH in Chinese Baijiu prevents alcohol-associated liver injury by increasing alcohol metabolism timely with alcohol consumption, providing an alternative approach to the management of alcohol-associated drinking.

Mesenchymal stromal/stem cells (MSCs) are a promising therapeutic agent for various diseases, including sepsis. However, translating MSC therapy to clinical applications remains challenging due to variations in the properties of MSCs under different preparation conditions. In this study, the gene expression profiles of human adipose-derived mesenchymal stromal/stem cells (ADSCs) under different culture conditions were compared in relation to their therapeutic efficacy for sepsis. Results showed that ADSCs cultured in media supplemented with human platelet lysates (hPL) (hPL-ADSCs) exhibited a smaller cell size and higher proliferative capacity, whereas ADSCs cultured in media supplemented with fetal bovine serum (FBS) (FBS-ADSCs) showed a broader and flatter shape. Both hPL-ADSCs and FBS-ADSCs exhibited a protective effect in a mouse model of sepsis; however, hPL-ADSCs displayed a better potency for immunosuppressive function, as evidenced by a better improvement of survival rate and further reduction of tissue injury and infectious biomarkers (alanine transaminase and procalcitonin). Furthermore, hPL-ADSCs caused a more anti-inflammatory transcriptomic shift, whereas FBS-ADSCs led to more depression of proinflammatory transcriptomic response. This study thus demonstrates that both hPL-ADSCs and FBS-ADSCs are effective for antiseptic therapy via different mechanisms of inflammatory manipulation, although hPL-ADSCs may imply a better preference.

After myocardial infarction (MI) occurs, progressive pathological cardiac remodeling results in heart dysfunction and even heart failure during the following months or years. The present study explored the molecular mechanisms underlying the late phase of MI at the global transcript level. A rhesus monkey model of myocardial ischemia induced by left anterior descending (LAD) artery ligation was established, and the heart tissue was collected eight weeks after ligation for transcriptome analysis by DNA microarray technology. Differentially expressed genes in the core infarcted area and remote infarcted area of the ischemic heart were detected with significance analysis of microarray (SAM), and related pathways were detected by Gene Ontology (GO)/pathway analysis. We found that compared to the sham condition, prolonged ischemia increased the levels of 941 transcripts, decreased the levels of 380 transcripts in the core infarcted area, and decreased the levels of 8 transcripts in the remote area in monkey heart tissue. Loss of coordination between the expression of genes, including natriuretic peptide A ( NPPA), NPPB, and corin (Corin, serine peptidase), may aggravate cardiac remodeling. Furthermore, imbalance in the enriched significantly changed pathways, including fibrosis-related pathways, cardioprotective pathways, and the cardiac systolic pathway, likely also plays a key role in regulating the development of heart remodeling.

Cell encapsulation has proven to be promising in stem cell therapy. However, there are issues needed to be addressed, including unsatisfied yield, unmet clinically friendly formulation, and unacceptable viability of stem cells after cryopreservation and thawing. We developed a novel biosynsphere technology to encapsulate stem cells in clinically-ready biomaterials with controlled microsphere size. We demonstrated that biosynspheres ensure the bioviability and functionality of adipose-derived stromal cells (ADSCs) encapsulated, as delineated by a series of testing procedures. We further demonstrated that biosynspheres protect ADSCs from the hardness of clinically handling such as cryopreservation, thawing, high-speed centrifugation and syringe/nozzle injection. In a swine full skin defect model, we showed that biosynspheres were integrated to the destined tissues and promoted the repair of injured tissues with an accelerating healing process, less scar tissue formation and normalized deposition of collagen type I and type III, the ratio similar to that found in normal skin. These findings underscore the potential of biosynsphere as an improved biofabrication technology for tissue regeneration in clinical setting.

Previous studies have shown that cardiomyocytes in the subendocardial region of myocardium survive from ischemic insult. This study was undertaken to explore possible mechanisms for the survival of these cardiomyocytes, focusing on changes in endothelial cells (ECs) and blood supply. C57/B6 mice were subjected to permanent ligation of left anterior descending (LAD) coronary artery to induce myocardial ischemia (MI). The hearts were harvested at 1, 4, and 7 days post MI and examined for histological changes. It was found that the survival of cardiomyocytes was associated with a preservation of ECs in the subendocardial region, as revealed by EC-specific tdTomato expression transgenic mice ( Tie2 tdTomato ). However, the EC selective proteins, PECAM1 and VEGFR2, were significantly depressed in these ECs. Consequently, the ratio of PECAM1/tdTomato was significantly decreased, indicating a transformation from PECAM1 + ECs to PECAM1 − ECs. Furthermore, EC junction protein, VE-cadherin, was not only depressed but also disassociated from PECAM1 in the same region. These changes led to an increase in EC permeability, as evidenced by increased blood infiltration in the subendocardial region. Thus, the increase in the permeability of ECs due to their transformation in the subendocardial region allows blood infiltration, creating a unique microenvironment and ensuring the survival of cardiomyocytes under ischemic conditions.

Abstract Background Mesenchymal stem cells (MSCs) therapy for sepsis has been extensively studied in the past decade; however, the treatment regimen and mechanism of action of MSCs remain elusive. Here, we attempted to understand the efficacy and mechanism of action of MSCs on rescuing mice with sepsis. Methods A mouse model of sepsis was produced by cecal ligation and puncture (CLP). Allogeneic adipose-derived MSCs (ADSCs) were administered by intravenous infusion at 6 h after CLP, and dose-related effects of ADSCs on these mice were determined by survival rate, histopathological changes, biochemical and coagulation parameters, bacterial load, and plasma levels of endotoxin and inflammatory cytokines. The tissue distribution of intravenously infused ADSCs in septic mice was investigated by pre-labeling ADSCs with the lipophilic membrane dye PKH26. RNA sequencing analysis was performed to assess the transcriptional changes in peripheral blood mononuclear cells (PBMCs) and the liver. Results A significant therapeutic effect of ADSCs at a dose of 2 × 107 cells/kg in septic mice was evidenced by a remarkable reduction in mortality (35.89% vs. 8.89% survival rate), blood bacterial burden, systemic inflammation, and multiple organ damage. In contrast, ADSCs at a lower dose (1 × 107 cells/kg) failed to achieve any beneficial outcomes, while ADSCs at a higher dose (4 × 107 cells/kg) caused more early death within 24 h after CLP, retaining a steady survival rate of 21.42% thereafter. PKH26-labeled ADSCs were predominantly localized in the lungs of septic mice after intravenous infusion, with only a smaller proportion of PKH26-positive signals appearing in the liver and spleen. RNA sequencing analysis identified that insufficient phagocytic activity of PBMCs in addition to a hyperactivation of the hepatic immune response was responsible for the ineffectiveness of low-dose ADSCs therapy, and acute death caused by high-dose ADSCs infusion was associated with impaired coagulation signaling in PBMCs and exacerbated hepatic hypoxic injury. Conclusions Our findings demonstrate a dose-specific effect of ADSCs on the treatment of sepsis due to dose-related interactions between exogenous stem cells and the host’s microenvironment. Therefore, a precise dosing regimen is a prerequisite for ADSCs therapy for sepsis.

Here, we present a protocol to identify the pro-embolic sub-population of human adipose-derived multipotent stromal cells (ADSCs) and predict fatal embolism risks from ADSC infusion. We describe steps for the collection, processing, and classification of ADSC single-cell RNA-seq data. We then detail the development of a mathematical model for predicting ADSC embolic risk. This protocol allows for the development of prediction models to enhance the assessment of cell quality and advance the clinical applications of stem cells. For complete details on the use and execution of this protocol, please refer to Yan et al. (2022).1.