RING E3 ubiquitin (UB) ligases rely on signature RING domains for mediating UB transfer to substrate proteins. The large number of RING E3s and their weak association with substrates pose a significant challenge in identifying the substrates of individual E3s, thereby hindering the elucidation of their biological functions. Here, we utilized phage display to engineer an "orthogonal UB transfer" (OUT) cascade with RING E3 RNF38, enabling the exclusive transfer of an engineered UB (xUB) to its substrates in the cell. The OUT screen revealed RNF38 substrates regulating nucleocytoplasmic transport (Ran, RanGAP1, and KPNA2), protein translation (HuR and Rack1), and endosomal sorting (VPS35). Furthermore, RNF38-catalyzed ubiquitination was found to induce the degradation of the substrate proteins and negatively affect the translocation of transcription factors E2F1 and phosphorylated STAT3 (p-STAT3) into the nucleus. Phage selection of the RNF38 RING library also revealed hotspot residues for E2 interaction, which may guide the engineering of orthogonal E2-E3 pairs with other RING E3s. Overall, our work discovered new roles of RNF38 in regulating nuclear transport and established an anchoring point for expanding OUT cascades within the large family of RING E3s for revealing their UB transfer targets and cellular functions.

Background Targeted drug delivery systems achieve precise drug enrichment at lesion sites through mechanisms such as passive targeting, active targeting, and stimulus-responsive release, offering novel pathways to enhance therapeutic efficacy while reducing systemic toxicity. With advancements in technologies like antibody-drug conjugates and carrier-based encapsulation, these systems have demonstrated remarkable success in treating multiple diseases.Discussion In the field of cardiovascular diseases (CVD), the large patient population and persistently high mortality rates, coupled with limitations of conventional drugs and surgeries, such as narrow therapeutic windows, insufficient targeting precision, and high invasiveness, make the development of novel targeted delivery strategies crucial for overcoming current therapeutic bottlenecks. Proteins and small molecule drugs are regarded as ideal candidates for targeted delivery due to their high specificity, favorable stability, and tissue penetration capabilities, while delivery carriers such as exosomes and liposomes play a central role in protecting drug activity and guiding targeted accumulation. Furthermore, the appropriate selection of administration routes significantly influences the efficacy of targeted delivery and clinical outcomes. At present, there is still a lack of systematic summaries on the application of targeted drug delivery systems composed of proteins and small molecule drugs in cardiovascular diseases.Conclusions This review focuses on targeted drug delivery systems for CVD, to systematically examine the functional properties of carriers designed for proteins and small molecule drugs, as well as the efficacy of different administration routes. It also offers a forward-looking perspective on future trends, aiming to provide theoretical and practical insights for precision medicine.

Rapid triage and effective treatment of patients with acute chest pain are critical. Enhancing the clinical competency of emergency resident physicians in managing these patients has emerged as a key challenge in contemporary clinical education. Emergency department resident physicians were divided into two groups in 2022–2023. After receiving either traditional lecture-based training or standardized patient (SP) training, we assessed whether there was an improvement in the physicians’ clinical competency in managing chest pain patients, and whether the time-to-treatment was reduced. Following SP training, resident physicians showed statistically significant improvements in physical exam (p = 0.036), management and treatment (p = 0.002), and total scores (p = 0.016). However, no significant differences were observed in history-talking (p = 0.941), differential diagnosis (p = 0.336), laboratory test (p = 0.523) and diagnosis (p = 0.131). Furthermore, the time required for residents to complete point-of-care testing (POCT) cardiac troponin I (cTnI), establish final diagnosis, and activate the catheterization laboratory was significantly reduced. Following SP training, resident physicians improved their abilities in physical examination, management and treatment of patients with acute chest pain, and less time was spend compared to the physicians without SP training. The use of SP and the generalization of this training technique is recommended, particularly in resident physicians in emergency department where more timely treatment of patients is required.

Myocardial fibrosis is a serious complication in sepsis and leads to cardiac dysfunction. The carboxy terminus of Hsc70-interacting protein (CHIP), a U-box E3 ligase, defends against sepsis-caused cardiac injury. Here, we explored a novel therapeutic effect of α1-adrenoceptor (α1-AR) blockage on lipopolysaccharide (LPS)-induced myocardial fibrosis and clarified that its molecular mechanism was related to the restoration of CHIP expression. The results showed that LPS increased the release of norepinephrine (NE) in the myocardium and promoted myocardial fibrosis. NE promoted the cardiac fibroblasts (CFs) differentiation characterized by increased α-SMA and collagen I/III. Blockage of α1-AR by prazosin apparently alleviated LPS-induced cardiac fibrosis and NE-caused CFs differentiation. Prazosin decreased phosphorylation of protein kinase C (PKC), p38 and Smad2/3, and reduced nuclear c-Jun level, as well as increased CHIP expression in the NE-stimulated CFs and the myocardium in LPS-treated mice. In vitro and in vivo data suggested that the overexpression of CHIP restrained α-SMA and collagen I/III production, and downregulated TGF-β receptor 1 (TGF-BR1) expression and Smad2/3 phosphorylation induced by NE or LPS respectively. Conversely, the knockdown of CHIP weakened the effect of prazosin. Furthermore, we innovatively revealed that α1A-AR is the dominant α1-AR subtype in CFs, and its specific antagonist, silodosin, eliminated NE-mediated CFs differentiation and LPS-induced myocardial fibrosis, which was consistent with the action of prazosin. These findings demonstrate the protective effect of α1-AR blockage against LPS-mediated myocardial fibrosis, which is achieved by directly inhibiting the PKC-p38-Smad2/3 signaling pathway and promoting TGF-BR1 downregulation through restoring CHIP expression.

BACKGROUND:Bipolar disorder (BD) is a severe mental disorder with high, approximately 70% heritability. Here, to identify novel risk genes of BD, we conducted whole-exome sequencing and pathway enrichment analyses across one multi-affected, southern Chinese Han pedigree. METHODS:Whole-exome sequencing (WES) was performed on five patients with BD and three unaffected members in one multi-affected pedigree. The analyses focused on variants that (i) were shared by affected members but were not present in the unaffected members, and (ii) were rare and damaging. Bioinformatic analyses, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, were used for functional annotation and pathway analysis. RESULTS:We identified a rare and potentially damaging single nucleotide variations (SNVs) in NTN1, MYH10, and RILP that were shared by affected family members but were absent in unaffected members. Their functions were predicted to be associated with actin binding, substrate-dependent cell migration, actin cytoskeleton, and nucleotide excision repair. CONCLUSION:Our findings suggest that NTN1, MYH10, and RILP may represent novel candidate risk genes for BD, although further validation in larger cohorts is needed.

Previous studies demonstrated that dexmedetomidine (Dex) posttreatment aggravated myocardial dysfunction and reduced survival in septic mice. Yet, whether Dex elicits similar effects in septic patients as defined by Sepsis-3 remains unknown. This study sought to assess the effects of Dex-based sedation on mortality and cardiac dysfunction in septic patients defined by Sepsis-3 and to further reveal the mechanisms in septic rats. In the retrospective cohort study, patients were categorised into sepsis with Dex, other sedatives (propofol or midazolam) or without sedatives, mortality at 28 days were compared, and patients with measurements of cardiovascular biomarkers and echocardiography were used to examine the effect of Dex on cardiac dysfunction. Septic rats and Langendorff-perfused isolated rat hearts were used, cardiac function, mortality and pro-inflammatory mediators were analyzed. The all-cause mortality of septic patients receiving Dex reached to 35.2 % on Day 28, significantly higher than that of patients with other sedatives (16.1 %), while no difference with group of no sedatives (27.3 %). Patients in Dex group showed lower left ventricular EF and lateral mitral annular early diastolic peak velocities, but higher interventricular septum diastolic dimension compared to those with other sedatives. The plasma levels of H-FABP, NT-proBNP and HMGB1 in Dex and other sedative groups showed no difference, while both were significantly lower than the group of no sedative. Notably, Dex posttreatment deteriorated cardiac dysfunction, increasing mortality in septic rats with enhanced systemic and myocardial proinflammatory mediators, including TNF-α, IL-1β, IL-6 and VCAM-1. Mechanistical study by Langendorff-perfusion revealed that Dex directly acted on the heart, aggravating LPS-induced myocardial inflammation and dysfunction. These results suggest that Dex increases mortality and deteriorates myocardial dysfunction compared with other sedatives in septic patients defined by Sepsis 3.0, maybe partly through promoting proinflammatory response via directly acting on the heart.

The DNA damage response (DDR) is susceptible to occurrence during the reprogram-ming process induced by Yamanaka factors, which constrains the generation and ap-plication of induced pluripotent stem cells (iPSCs). The present study demonstrates that the knockdown (KD) of MBD3, a member of the nucleosome remodeling and deacetylase (NuRD) complex, reduced DDR during the early stages of bovine somatic cell re-programming, and enhanced pluripotency and in vitro differentiation ability of bovine iPSCs. MBD3 KD reduced the generation of reactive oxygen species (ROS) by upregulated of HIF1A expression, which mitigate DNA damage and apoptosis induced by oxidative stress in bovine iPSCs. Conversely, the overexpression of MBD3 increased DNA damage throughout the reprogramming process. In conclusion, our results demonstrate that the knockdown of MBD3 during reprogramming can reduce DNA damage and promote the generation of bovine iPSCs with enhanced pluripotency and in vitro differentiation ability.

Geranylgeranylacetone (GGA), an isoprenoid compound widely utilized as an antiulcer agent in Asia, confers protection against ischemia, anoxia, and oxidative stress by rapidly enhancing the expression of HSP70. Nevertheless, the impact of GGA on sepsis-associated intestinal injury remains unexplored. Thus, this study is crafted to elucidate the protective efficacy and underlying mechanisms of GGA against septic intestinal damage. Our findings revealed that GGA significantly extended the survival duration of septic mice, and mitigated lipopolysaccharide (LPS)-induced alterations in intestinal permeability and tissue damage. Furthermore, GGA effectively suppressed LPS-induced cytokine release, attenuated levels of reactive oxygen species (ROS) and malondialdehyde, and bolstered antioxidant-related parameters within the intestinal tissue of LPS-stimulated mice. Mechanistically, GGA significantly increased HSP70 expression and promoted E3 ubiquitin ligase CHIP to play the role in ubiquitination and degradation of karyopherin-α2 (KPNA2), resulting in inhibition of nuclear translocation of NF-κB and reduced NOX1, NOX2 and NOX4 expression. The inhibitory action of GGA on cytokine release and ROS generation was abolished by CHIP knockdown in IEC-6 cells treated with LPS. Simultaneously, the downregulation of CHIP reversed the suppressive role of GGA in the LPS-induced NF-κB activation and the expression of NOX1, NOX2 and NOX4 in IEC-6 cells. The effects of GGA on mitigating intestinal damage, inflammation and oxidative stress caused by LPS were eliminated in CHIP knockout mice. Our results demonstrate that the protective effect of GGA against LPS-caused intestinal injury of mice is dependent on CHIP activation, which promotes KPNA2 degradation and restrains translocation of NF-κB into nucleus, leading to suppressing LPS-induced inflammatory response and oxidative stress.

Introduction Due to policy changes in the context of COVID-19 pandemic, online teaching has become the main form of class in many Chinese universities. Flipped classroom has been widely used in other disciplines, but there is a dearth of evidence available about the use in online teaching of emergency medicine. This study aimed to develop a flipped classroom for online emergency medicine teaching and evaluate its effectiveness by comparing it with traditional lecture-based online teaching.Methods A total of 62 clinical medical undergraduates from Jinan University participated in this study from September to December in 2022. An online flipped classroom approach was developed (FC group, n = 31). Traditional lecture-based online teaching was applied as a contrast (LBT group, n = 31). The undergraduates completed examinations and questionnaires at the end of the course. A course experience questionnaire and course examination score were used to evaluate the effectiveness of the flipped classroom approach.Results Regarding the five dimensions of the course experience questionnaire, the scores for good teaching (3.47 +/- 0.50 vs. 2.34 +/- 0.48, p < .001), appropriate assessment (3.31 +/- 0.68 vs. 2.95 +/- 0.71, p = .043) and generic skills (3.16 +/- 0.60 vs. 2.72 +/- 0.39, p < .001) were higher for the FC group than for the LBT group. There was no significant difference between the two groups in clear goals and standards, and appropriate workload. The undergraduates in the FC group showed significantly higher overall satisfaction than those in the LBT group (3.52 +/- 0.1.03 vs. 2.87 +/- 0.92, p = .012). The examination scores (77.936 +/- 11.573 vs. 70.484 +/- 7.434, p < .001), especially the scores for questions related to case analysis (33.032 +/- 5.363 vs. 26.968 +/- 7.657, p < .001), were significantly higher in the FC group than in the LBT group.Conclusions The flipped classroom for online teaching was efficient in improving undergraduates' emergency medical academic performance and promoting the development of clinical case analysis ability. These findings provide an alternative flipped classroom approach for online teaching of emergency medicine.

This study aims to investigate the role of claudin-5 (Cldn5) in cardiac structural integrity. Proteomic analysis was performed to screen the protein profiles in enlarged left atrium from atrial fibrillation (AF) patients. Cldn5 shRNA adeno-associated virus (AAV) or siRNA was injected into the mouse left ventricle or added into HL1 cells respectively to knockdown Cldn5 in cardiomyocytes to observe whether the change of Cldn5 influences cardiac morphology and function, and affects those protein expressions stem from the proteomic analysis. Mitochondrial density and membrane potential were also measured by Mitotracker staining and JC-1 staining under the confocal microscope in HL1 cells. Cldn5 was reduced in cardiomyocytes from the left atrial appendage of AF patients compared to non-AF donors. Proteomic analysis showed 83 proteins were less abundant and 102 proteins were more abundant in AF patients. KEGG pathway analysis showed less abundant CACNA2D2, CACNB2, MYL2 and MAP6 were highly associated with dilated cardiomyopathy. Cldn5 shRNA AAV injection caused severe cardiac atrophy, dilation and myocardial dysfunction in mice. The decreases in mitochondrial numbers and mitochondrial membrane potentials in HL1 cells were observed after Cldn5 knockdown. We demonstrated for the first time the mechanism of Cldn5 downregulation-induced myocyte atrophy and myocardial dysfunction might be associated with the downregulation of CACNA2D2, CACNB2, MYL2 and MAP6, and mitochondrial dysfunction in cardiomyocytes.

ABSTRACT β3-adrenergic receptor (β3-AR) has been proposed as a new therapy for several myocardial diseases. However, the effect of β3-AR activation on sepsis-induced myocardial apoptosis is unclear. Here, we investigated the effect of β3-AR activation on the cardiomyocyte apoptosis and cardiac dysfunction in cecal ligation and puncture (CLP)-operated rats and lipopolysaccharide (LPS)-treated cardiomyocytes. We found that β3-AR existed both in adult rat ventricular myocytes (ARVMs) and H9c2 cells. The expression of β3-AR was upregulated in LPS-treated ARVMs and the heart of CLP rats. Pretreatment with β3-AR agonist, BRL37344, inhibited LPS-induced cardiomyocyte apoptosis and caspase-3, -8 and -9 activation in ARVMs. BRL37344 also reduced apoptosis and increased the protein levels of PI3K, p-AktSer473 and p-eNOSSer1177 in LPS-treated H9c2 cells. Inhibition of PI3K using LY294002 abolished the inhibitory effect of BRL37344 on LPS-induced caspase-3, -8, and -9 activation in H9c2 cells. Furthermore, administration of β3-AR antagonist, SR59230A (5 mg/kg), significantly decreased the maximum rate of left ventricular pressure rise (+dP/dt) in CLP-induced septic rats. SR59230A not only increased myocardial apoptosis, reduced p-AktSer473 and Bcl-2 contents, but also increased mitochondrial Bax, cytoplasm cytochrome c, cleaved caspase-9 and cleaved caspase-3 levels of the myocardium in septic rats. These results suggest that endogenous β3-AR activation alleviates sepsis-induced cardiomyocyte apoptosis via PI3K/Akt signaling pathway and maintains intrinsic myocardial systolic function in sepsis.

Sepsis is a severe inflammatory disorder that can lead to life-threatening multiple organ injury. Lipopolysaccharide (LPS)-induced inflammation is the leading cause of multiple organ failure in sepsis. This study aimed to explore the effect of a novel agent, 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole (YL-109), on LPS-induced multiple organ injury and the molecular mechanisms underlying these processes. The results showed that YL-109 protected against LPS-induced high mortality, cardiac dysfunction, pulmonary and intestinal injury through inhibiting the proinflammatory response, NLRP3 expression and pyroptosis-associated indicators in mouse tissues. YL-109 suppressed LPS-initiated cytokine release, pyroptosis and pyroptosis-related protein expression in HL-1, IEC-6 and MLE-12 cells, which was consistent with the results of the in vivo experiments. Mechanistically, YL-109 reduces phosphorylated ERK (extracellular signal-regulated kinase) levels and NF-κB activation, which are achieved through upregulating CHIP (carboxy terminus of Hsc70-interacting protein) expression, thereby inhibiting c-Jun and c-Fos activation as well as NLRP3 expression. As an E3 ligase, CHIP overexpression obviously promoted the degradation of phosphorylated ERK and inhibited the expression of NF-κB-mediated NLRP3 in cells stimulated with LPS. The protective effects of YL-109 against cardiac, pulmonary and intestinal damage, inflammation and pyroptosis caused by LPS were eliminated in CHIP knockout mice. Our results not only reveal the protective effect and molecular mechanism of YL-109 against LPS-mediated organs damage but also provide additional insights into the effect of CHIP on negatively regulating pyroptosis and inflammatory pathways.

Cardiomyopathy is particularly common in septic patients. Our previous studies have shown that activation of the alpha 1 adrenergic receptor (α1-AR) on cardiomyocytes inhibits sepsis-induced myocardial dysfunction. However, the role of cardiac endothelial α1-AR in septic cardiomyopathy has not been determined. Here, we identified α1-AR expression in mouse and human endothelial cells and showed that activation of α1-AR with phenylephrine (PE) improved cardiac function and survival by preventing cardiac endothelial injury in septic mice. Mechanistically, activating α1-AR with PE decreased the expression of ICAM-1, VCAM-1, iNOS, E-selectin, and p-p38MAPK, while promoting PKC and ERK1/2 phosphorylation in LPS-treated endothelial cells. These effects were abolished by a PKC inhibitor or α1-AR antagonist. PE also reduced p65 nuclear translocation, but this suppression is not blocked by PKC inhibition. Treatment with U0126 (a specific ERK1/2 inhibitor) reversed the effects of PE on p38MAPK phosphorylation. Our results demonstrate that cardiac endothelial α1-AR activation prevents sepsis-induced myocardial dysfunction in mice by inhibiting the endothelial injury via PKC-ERK/p38MAPK signaling pathway and a PKC-independent inhibition of p65 nuclear translocation. These findings offer a new perspective for septic patients with cardiac dysfunction by inhibiting cardiac endothelial cell injury through α1-AR activation.

Doxorubicin (DOX)-related cardiotoxicity has been recognized as a serious complication of cancer chemotherapy. Effective targeted strategies for myocardial protection in addition to DOX treatment are urgently needed. The purpose of this paper was to determine the therapeutic effect of berberine (Ber) on DOX-triggered cardiomyopathy and explore the underlying mechanism. Our data showed that Ber markedly prevented cardiac diastolic dysfunction and fibrosis, reduced cardiac malondialdehyde (MDA) level and increased antioxidant superoxide dismutase (SOD) activity in DOX-treated rats. Moreover, Ber effectively rescued the DOX-induced production of reactive oxygen species (ROS) and MDA, mitochondrial morphological damage and membrane potential loss in neonatal rat cardiac myocytes and fibroblasts. This effect was mediated by increases in the nuclear accumulation of nuclear erythroid factor 2-related factor 2 (Nrf2) and levels of heme oxygenase-1 (HO-1) and mitochondrial transcription factor A (TFAM). We also found that Ber suppressed the differentiation of cardiac fibroblasts (CFs) into myofibroblasts, as indicated by decreased expression of α-smooth muscle actin (α-SMA), collagen I and collagen III in DOX-treated CFs. Pretreatment with Ber inhibited ROS and MDA production and increased SOD activity and the mitochondrial membrane potential in DOX-challenged CFs. Further investigation indicated that the Nrf2 inhibitor trigonelline reversed the protective effect of Ber on both cardiomyocytes and CFs after DOX stimulation. Taken together, these findings demonstrated that Ber effectively alleviated DOX-induced oxidative stress and mitochondrial damage by activating the Nrf2-mediated pathway, thereby leading to the prevention of myocardial injury and fibrosis. The current study suggests that Ber is a potential therapeutic agent for DOX-induced cardiotoxicity that exerts its effects by activating Nrf2.

Sepsis is a dysregulated systemic inflammatory response caused by infection that leads to multiple organ injury and high mortality without effective treatment. Corilagin, a natural polyphenol extracted from traditional Chinese herbs, exhibits strong anti-inflammatory properties. However, the role for Corilagin in lipopolysaccharide (LPS)-induced sepsis and the molecular mechanisms underlying this process have not been completely explored. Here we determine the effect of Corilagin on LPS-treated mice and use a screening approach integrating surface plasmon resonance with liquid chromatography-tandem mass spectrometry (SPR-LC-MS/MS) to further explore the therapeutic target of Corilagin. We discovered that Corilagin significantly prolonged the survival time of septic mice, attenuated the multi-organ injury and the expression of pyroptosis-related proteins in tissues of LPS-treated mice. In vitro studies revealed that Corilagin inhibited pyroptosis and NLRP3 inflammasome activation in LPS-treated macrophages followed with ATP stimulation, as reflected by decreased levels of GSDMD-NT and activated caspase-1, and reduced ASC specks formation. Mechanistically, Corilagin alleviated the formation of ASC specks and blocked the interaction of ASC and pro-caspase1 by competitively binding with the caspase recruitment domain (CARD) of ASC. Additionally, Corilagin interrupted the TLR4-MyD88 interaction through targeting TIR domain of MyD88, leading to the inhibition of NF-κB activation and NLRP3 production. In addition, Corilagin downregulated genes associated with several inflammatory responses and inflammasome-related signaling pathways in LPS-stimulated macrophages. Overall, our results indicate that the inhibitory effect of Corilagin on pyroptosis through targeting TIR domain of MyD88 and binding the CARD domain of ASC in macrophages plays an essential role in protection against LPS-induced sepsis.

ABSTRACT:Pulmonary fibrosis is an important factor affecting the prognosis of severe septic patients with acute lung injury. The objective of this study was to explore the effect of norepinephrine (NE) and α 2 -adrenoreceptor (AR) on sepsis-associated pulmonary fibrosis and the mechanism underlying these effects. We found pulmonary fibrotic changes, and increased NE production and α 2A -AR expression in the pulmonary tissue of mice subjected to cecal ligation and puncture surgery. Reserpine and yohimbine alleviated pulmonary fibrosis in mice with sepsis by exhausting NE derived from the lung's adrenergic nerve and blocking α 2 -AR, respectively. There was no significant difference in the expression of the three α 1 -AR subtypes. The effect of NE on promoting pulmonary fibroblast differentiation in vitro was suppressed by yohimbine. Both the protein and mRNA expression levels of α 2A -AR were increased in pulmonary fibroblasts treated with LPS. Clonidine, a selective α 2 -AR agonist, enhanced LPS-induced differentiation in pulmonary fibroblasts, as indicated by the increase in α-smooth muscle actin and collagen I/III, which was mitigated by inhibiting PKC and p38. Further in vivo results indicated that yohimbine alleviated pulmonary fibrosis and inhibited the phosphorylation of PKC, p38, and Smad2/3 in lung tissue of mice exposed to LPS for 4 weeks. Clonidine showed the opposite effect to yohimbine, which aggravated LPS-induced pulmonary fibrosis. These findings demonstrated that the sepsis-induced increase in NE promoted fibroblast differentiation via activating α 2 -AR. Blockage of α 2 -AR effectively ameliorated sepsis-associated pulmonary fibrosis by abolishing NE-induced lung fibroblast differentiation and inhibiting the PKC-p38-Smad2/3 pathway.

Background: Dexmedetomidine (DEX) administered before or at 30 min after sepsis induction was reported to alleviate septic cardiomyopathy in experimental models. However, sepsis is a life-threatening organ dysfunction due to infection-induced dysregulated host response, whether DEX treatment in the presence of organ dysfunction affects septic cardiomyopathy is unknown. This study investigated the effect of DEX posttreatment on septic cardiomyopathy.Methods: Male wild-type and alpha 2A-adrenergic receptor (AR) knockout mice were exposed to lipopolysaccharide (LPS) or cecal ligation puncture (CLP), and cultured cardiac endothelial cells were used. Mouse survival, myocardial function, inflammatory response and related signaling pathways were determined.Results: DEX treatment at 6, 9 h after LPS challenge significantly reduced survival rate of LPS-challenged mice, especially at 9 h. DEX administered at 9 h after LPS injection or CLP significantly reduced survival in LPS or CLP-induced sepsis in wild-type mice, but not in alpha 2A-AR knockout mice. LPS treatment for 20 h decreased the left ventricle + dp/dt, increased myocardial interleukin (IL)-1 beta and IL-6 concentrations as well as cardiac endothelial tumor necrosis factor (TNF)-alpha, vascular cell adhesion molecule-1 (VCAM-1) and ICAM-1 expression, which were enhanced by DEX treated at 9 h after LPS injection in wild-type mice, but not in alpha 2A-AR knockout mice. Furthermore, DEX posttreatment increased p38 phosphorylation, c-Fos nuclear translocation and VCAM-1 expression in LPS-treated cardiac endothelial cells, which were eliminated by alpha 2A-AR knockout or PKC inhibitor.Conclusions: DEX posttreatment aggravates LPS-induced cardiac inflammation and myocardial dysfunction, at least in part, via activating cardiac endothelial alpha 2A-AR-mediated PKC signal pathway.

Yeast is an essential model organism for studying protein ubiquitination pathways; however, identifying the direct substrates of E3 in the cell presents a challenge. Here, we present a protocol for using the orthogonal ubiquitin transfer (OUT) cascade to profile the substrate specificity of yeast E3 Rsp5. We describe steps for OUT profiling, proteomics analysis, in vitro and in cell ubiquitination, and stability assay. The protocol can be adapted for identifying and verifying the ubiquitination targets of other E3s in yeast. For complete details on the use and execution of this protocol, please refer to Wang et al.1

Background: Fecal microbiota transplantation (FMT) based on the positive ion mode of metabonomics has a good therapeutic benefit for slow transit constipation (STC) patients. However, a piece of comprehensive metabolomics information is yet to be established. The aim of the study was to explore the efficacy and mechanism of FMT in the treatment of STC under metabonomics. Methods: Eight STC patients meeting the set inclusion and exclusion criteria were enrolled and treated with FMT (three times). The Patient Assessment of Constipation-Symptoms (PAC-SYM), weekly total defecation times, and defecation frequency scores of these STC patients were compared before and after treatment. Feces and serum of STC patients before and after treatment were analyzed using 16SrDNA and metabolomics. Results: After FMT treatment, the PAC-SYM score of constipated patients decreased [(5.00 ± 2.94) vs (5.20 ± 2.87)], while the number of complete defecations per week increased [(2.00 ± 1.79) vs (1.69 ± 1.80)]. The score of defecation frequency decreased [(0.83 ± 1.03) vs (0.86 ± 0.95)]. The metabolites in the feces and serum of patients receiving FMT changed significantly ( P < 0.05). The results from 16SrDNA analysis showed that the α and β diversity of the fecal microbiome changed significantly ( P < 0.05) after transplantation, and the contents of genera Lactobacillus , Bacillus , Succiniclasticum , Cellvibrio , and Escherichia increased in FMT treated patients. Conclusion: FMT may treat STC by increasing the beneficial intestinal flora and metabolites in the anion mode of metabolomics.

Cardiomyopathy is a common complication and significantly increases the risk of death in septic patients. Our previous study demonstrated that post-treatment with dexmedetomidine (DEX) aggravates septic cardiomyopathy. However, the mechanisms for the side effect of DEX post-treatment on septic cardiomyopathy are not well-defined. Here we employed a cecal ligation and puncture (CLP) model and α2A-adrenoceptor deficient (Adra2a-/-) mice to observe the effects of DEX post-treatment on myocardial metabolic disturbances in sepsis. CLP mice displayed significant cardiac dysfunction, altered mitochondrial dynamics, reduced cardiac lipid and glucose uptake, impaired fatty acid and glucose oxidation, enhanced glycolysis and decreased ATP production in the myocardium, almost all of which were dramatically enhanced by DEX post-treatment in septic mice. In Adra2a-/- mice, DEX post-treatment did not affect cardiac dysfunction and metabolic disruptions in CLP-induced sepsis. Additionally, Adra2a-/- mice exhibited impaired cardiac function, damaged myocardial mitochondrial structures, and disturbed fatty acid metabolism and glucose oxidation. In sum, DEX post-treatment exacerbates metabolic disturbances in septic cardiomyopathy in a α2A-adrenoceptor dependent manner.