Objective:To investigate the effect of DEAD-box helicase (DDX) 21 on myocardial ischemia-reperfusion (I/R) injury and its potential mechanisms.Methods:In vivo, adult male Bama pigs and C57BL/6J mice were used to establish a myocardial I/R injury model by ligating the left anterior descending coronary artery, with sham-operated groups set as controls. The expression of DDX21 in myocardium after I/R injury was assessed by quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence staining. Following the establishment of the myocardial I/R injury model in mice, AAV9 vectors with cardiac-specific expression were injected in situ into the peri-infarct region (The I/R+DDX21 group, I/R+negative control (NC) group, I/R+sh-NC group and I/R+sh-DDX21 group were injected with AAV9:cTnT-DDX21, AAV9:cTnT-NC, AAV9:cTnT-sh-NC and AAV9:cTnT-sh-DDX21, respectively). Additionally, the I/R+A-485 group received intraperitoneal injections of the cAMP response element-binding protein (CREB) binding protein inhibitor A-485, while the I/R+PBS group was injected with an equivalent volume of phosphate-buffered saline (PBS) as the control. Echocardiography was performed on postoperative days 1 and 28 to evaluate cardiac function (left ventricular ejection fraction and fractional shortening). At 28 days post-surgery, mice were euthanized and heart tissues were harvested for histological sectioning. Myocardial fibrosis was evaluated using Masson′s trichrome staining. In vitro, primary cardiomyocytes were isolated from neonatal day 1 C57BL/6J mice using enzymatic digestion method. Cardiomyocytes were transfected with plasmids or small interfering RNA (siRNA). The cardiomyocytes transfected with DDX21-siRNA were assigned to the siDDX21 group, those transfected with the DDX21 plasmid were assigned to the DDX21 group, and those transfected with the corresponding empty plasmid or siRNA were assigned to the NC group. Additionally, cardiomyocytes were treated with A-485 (A-485 group) or PBS (PBS group). An oxygen-glucose deprivation/reoxygenation (OGD/R) model was used to simulate cellular injury. Transcriptome sequencing was performed to identify downstream mechanisms of DDX21. Differential gene expression analysis was conducted using software such as DESeq2, and alternative splicing events in the mRNA transcriptome were analyzed using rMATS software. Mitochondrial superoxide, mitochondrial membrane potential, ATP content, and mitochondrial respiratory chain complex enzyme activity in cardiomyocytes were detected using immunofluorescence staining and commercial assay kits. The oxidative phosphorylation level of the cells was assessed by the Seahorse extracellular flux analyzer. Acetylated DDX21 levels were measured using co-immunoprecipitation and Western blot assays.Results:The expression levels of DDX21 in myocardium from the Bama pigs and mice in the I/R injury model were significantly higher than those in the sham group (all P<0.001). Echocardiographic results showed that at 28 days post-surgery, compared to the I/R+NC group, the I/R+DDX21 group exhibited higher left ventricular ejection fraction and fractional shortening, while the I/R+sh-DDX21 group showed lower values; Masson staining results demonstrated that, compared to the I/R+NC group, the myocardial fibrosis area in the I/R+DDX21 group was significantly reduced, whereas it was significantly increased in the I/R+sh-DDX21 group (all P<0.001). Transcriptomic sequencing results suggested that DDX21 may influence myocardial injury by regulating mitochondrial metabolic activity. In vitro, compared to the OGD/R+NC group, the OGD/R+DDX21 group exhibited lower mitochondrial superoxide levels, higher polymer/monomer ratio, maximal oxygen consumption, reserve capacity, and ATP content. In contrast, the OGD/R+siDDX21 group showed the opposite results, with reduced activity of mitochondrial respiratory chain complex V (all P<0.05). Mechanistically, rMATS software and other analyses indicated that knockdown of DDX21 affected the alternative 3′ splicing sites of ATP5J precursor mRNA, inhibiting the splicing of certain exonic sequences. Overexpression of DDX21 upregulated both mRNA and protein levels of ATP5J. Co-immunoprecipitation experiments showed that, compared to the PBS group, acetylated DDX21 levels were reduced in the A-485 group. Further in vivo experiments showed that, compared to the I/R+PBS group, the I/R+A-485 group exhibited higher left ventricular ejection fraction and fractional shortening, and a lower proportion of left ventricular fibrosis (all P<0.001). Conclusions:DDX21 improves cardiomyocyte energy metabolism and alleviates I/R injury by regulating the alternative splicing of ATP5J. A-485 holds potential as a novel small molecule candidate for the treatment of myocardial injury.

Objective:To investigate the effect of DEAD-box helicase (DDX) 21 on myocardial ischemia-reperfusion (I/R) injury and its potential mechanisms.Methods:In vivo, adult male Bama pigs and C57BL/6J mice were used to establish a myocardial I/R injury model by ligating the left anterior descending coronary artery, with sham-operated groups set as controls. The expression of DDX21 in myocardium after I/R injury was assessed by quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence staining. Following the establishment of the myocardial I/R injury model in mice, AAV9 vectors with cardiac-specific expression were injected in situ into the peri-infarct region (The I/R+DDX21 group, I/R+negative control (NC) group, I/R+sh-NC group and I/R+sh-DDX21 group were injected with AAV9:cTnT-DDX21, AAV9:cTnT-NC, AAV9:cTnT-sh-NC and AAV9:cTnT-sh-DDX21, respectively). Additionally, the I/R+A-485 group received intraperitoneal injections of the cAMP response element-binding protein (CREB) binding protein inhibitor A-485, while the I/R+PBS group was injected with an equivalent volume of phosphate-buffered saline (PBS) as the control. Echocardiography was performed on postoperative days 1 and 28 to evaluate cardiac function (left ventricular ejection fraction and fractional shortening). At 28 days post-surgery, mice were euthanized and heart tissues were harvested for histological sectioning. Myocardial fibrosis was evaluated using Masson′s trichrome staining. In vitro, primary cardiomyocytes were isolated from neonatal day 1 C57BL/6J mice using enzymatic digestion method. Cardiomyocytes were transfected with plasmids or small interfering RNA (siRNA). The cardiomyocytes transfected with DDX21-siRNA were assigned to the siDDX21 group, those transfected with the DDX21 plasmid were assigned to the DDX21 group, and those transfected with the corresponding empty plasmid or siRNA were assigned to the NC group. Additionally, cardiomyocytes were treated with A-485 (A-485 group) or PBS (PBS group). An oxygen-glucose deprivation/reoxygenation (OGD/R) model was used to simulate cellular injury. Transcriptome sequencing was performed to identify downstream mechanisms of DDX21. Differential gene expression analysis was conducted using software such as DESeq2, and alternative splicing events in the mRNA transcriptome were analyzed using rMATS software. Mitochondrial superoxide, mitochondrial membrane potential, ATP content, and mitochondrial respiratory chain complex enzyme activity in cardiomyocytes were detected using immunofluorescence staining and commercial assay kits. The oxidative phosphorylation level of the cells was assessed by the Seahorse extracellular flux analyzer. Acetylated DDX21 levels were measured using co-immunoprecipitation and Western blot assays.Results:The expression levels of DDX21 in myocardium from the Bama pigs and mice in the I/R injury model were significantly higher than those in the sham group (all P<0.001). Echocardiographic results showed that at 28 days post-surgery, compared to the I/R+NC group, the I/R+DDX21 group exhibited higher left ventricular ejection fraction and fractional shortening, while the I/R+sh-DDX21 group showed lower values; Masson staining results demonstrated that, compared to the I/R+NC group, the myocardial fibrosis area in the I/R+DDX21 group was significantly reduced, whereas it was significantly increased in the I/R+sh-DDX21 group (all P<0.001). Transcriptomic sequencing results suggested that DDX21 may influence myocardial injury by regulating mitochondrial metabolic activity. In vitro, compared to the OGD/R+NC group, the OGD/R+DDX21 group exhibited lower mitochondrial superoxide levels, higher polymer/monomer ratio, maximal oxygen consumption, reserve capacity, and ATP content. In contrast, the OGD/R+siDDX21 group showed the opposite results, with reduced activity of mitochondrial respiratory chain complex V (all P<0.05). Mechanistically, rMATS software and other analyses indicated that knockdown of DDX21 affected the alternative 3′ splicing sites of ATP5J precursor mRNA, inhibiting the splicing of certain exonic sequences. Overexpression of DDX21 upregulated both mRNA and protein levels of ATP5J. Co-immunoprecipitation experiments showed that, compared to the PBS group, acetylated DDX21 levels were reduced in the A-485 group. Further in vivo experiments showed that, compared to the I/R+PBS group, the I/R+A-485 group exhibited higher left ventricular ejection fraction and fractional shortening, and a lower proportion of left ventricular fibrosis (all P<0.001). Conclusions:DDX21 improves cardiomyocyte energy metabolism and alleviates I/R injury by regulating the alternative splicing of ATP5J. A-485 holds potential as a novel small molecule candidate for the treatment of myocardial injury.

Objective: Sialidosis type 2 has variants that are both catalytically inactive (severe), while sialidosis type 1 has at least one catalytically active (mild) variant. This study aimed to discuss the structural changes associated with these variants in a newly reported family carrying N-acetyl-α-neuraminidase-1 (NEU1) variants and explore the clinical characteristics of different combinations of variants in sialidosis type 1. Methods: First, whole-exome sequencing and detailed clinical examinations were performed on the family. Second, structural analyses, including assessments of energy, flexibility and polar contacts, were conducted for several NEU1 variants, and a sialidase activity assay was performed. Third, previous NEU1 variants were systematically reviewed, and the clinical characteristics of patients in the severe-mild and mild-mild groups with sialidosis type 1 were analyzed. Results: We report a novel family with sialidosis type 1 and the compound heterozygous variants S182G and V143E. The newly identified V143E variant was predicted to be a mild variant through structural analysis and was confirmed by a sialidase activity assay. Cherry-red spots were more prevalent in the severe-mild group, and ataxia was more common in the mild-mild group. Impaired cognition was found only in the severe-mild group. Moreover, patients with cherry-red spots and abnormal electroencephalographies and visual evoked potentials had a relatively early age of onset, whereas patients with myoclonus had a late onset. Conclusion Changes in flexibility and local polar contacts may be indicators of NEU1 pathogenicity. Sialidosis type 1 can be divided into two subgroups according to the variant combinations, and patients with these two subtypes have different clinical characteristics.

Objective:To analyze the clinical characteristics and medication options of patients with elderly-onset epilepsy and influencing factors for medication efficacy.Methods:A total of 213 patients with elderly-onset epilepsy (age of onset≥65 years) were selected from Epilepsy Outpatient, Department of Neurology, First Medical Center of Chinese PLA General Hospital from February 1999 to March 2023. General data, imaging findings and follow-up results of these patients were collected. Seizure frequencies and types, medication types, and medication efficacy were analyzed retrospectively. According to medication efficacy, these patients were divided into effective anti-seizure medications (ASMs) group and ineffective ASMs group (effective ASMs was defined as having no seizures or seizure reduction>50% at 6 months after medication, and ineffective ASMs as having seizure reduction≤50% or seizure increase. Univariate and multivariate Logistic regression analyses were used to identify the influencing factor for ASMs efficacy.Results:In these 213 patients with elderly-onset epilepsy, 143 (67.1%) were males and 70 (32.9%) were females. Onset age was 70.0 (67.0, 74.5) years, with duration of 12 (4, 32) months. Time from first onset to treatment was 2.0 (1.0, 10.5) months, with that<2 months enjoying the largest proportion ( n=101). MRI/CT in 102 patients indicated potential epileptogenic abnormal structures, such as post-stroke gliosis/encephalomalacia ( n=67) and post-traumatic gliosis/encephalomalacia ( n=13). MRI/CT in 78 patients indicated non-epileptogenic abnormal structures, such as ischemic changes of small and medium vessels ( n=51) and brain atrophy ( n=15). Structural change was the most common cause ( n=160). Sixty-nine patients (32.4%) did not take medicine and 144 (67.6%) took medicine at the visiting; sodium valproate was mostly used ( n=74), followed by levetiracetam ( n=35) and carbamazepine ( n=24). Five patients had sodium valproate combined with levetiracetam, and 4 patients had sodium valproate combined with carbamazepine. Multivariate Logistic regression analysis showed that disease duration and medication combination were independent influencing factors for ASMs efficacy. Conclusion:Structural change is the main cause for elderly-onset epilepsy; medication efficacy is worse in patients with longer disease course and medication combination therapy.

Opioid receptors μOR,δOR,κOR and NOPR are all G protein-coupled receptors(GPCRs),which mainly function through G protein and β-ar-restin.Recent studies have found that G protein mediates analgesia,while β-arrestin reduces anal-gesia and is related to the side effects of opioids.Oliceridine is the first biased μOR agonist approved for commerce.It mainly exerts analgesic effect by activating G protein.It has rapid onset of action and reliable analgesic effect.Due to its low activity on β-arrestin,the incidence of side effects is low,comparing to the classic opioid morphine.Oliceri-dine can be safely used in patients with liver or kid-ney insufficiency and its metabolite is inactive.This article summarizes the current progress of pharma-cological research and clinical application of oliceri-dine,aiming to provide reference for the clinical practice of oliceridine.

Objective:To compare the prognostic values of different classification by using transpulmonary pressure gradient (TPG), diastolic pressure gradient (DPG) and pulmonary vascular resistance (PVR) in patients with pulmonary hypertension due to left heart disease (PH-LHD), and investigated hemodynamic and clinical factors associated with mortality in patients with PH-LHD.Methods:This was a single-center prospective cohort study. In-hospital patients diagnosed with PH-LHD via right heart catheterization at the Department of Cardiology, the First Affiliated Hospital of Nanjing Medical University, from September 2013 to December 2019 were enrolled. Patients were divided according to TPG (cutoff value 12 mmHg; 1 mmHg=0.133 kPa), DPG (cutoff value 7 mmHg), PVR (cutoff value 3 Wood Units), and the combination of TPG and PVR. Baseline characteristic was recorded. All patients were followed up until the occurrence of endpoint event, defined as all-cause death that occurred during the follow-up period, or until April 18, 2022. Receiver operating characteristic curves were used to compare the predictive value of 3 classification methods for all-cause death in PH-LHD patients. The optimal cutoff values were calculated using Jorden index. Survival analysis was performed using Kaplan-Meier analysis, and log-rank test was used to compare the predictive efficacy of classification methods based on optimal cutoff values or guidance-recommended thresholds for the survival of PH-LHD patients. Variables showing statistical significance in the univariate analysis were incorporated into multivariate Cox regression model to analyze the independent risk factors for all-cause mortality.Results:A total of 243 patients were enrolled, aged (54.9±12.7) years old, including 169 (69.5%) males. During a median follow-up of 57 months, there were 101 (41.6%) deaths occurred. Grouping results were as follows: (1) TPG: TPG≤12 mmHg group 115 patients, TPG>12 mmHg group 128 patients; (2) DPG: DPG<7 mmHg group 193 patients, DPG≥7 mmHg group 50 patients; (3) PVR: PVR≤3 Wood Units group 108 patients, PVR>3 Wood Units group 135 patients; (4) TPG and PVR: TPG≤12 mmHg and PVR≤3 Wood Units group 89 patients, TPG>12 mmHg and PVR>3 Wood Units group 109 patients. PVR ( AUC=0. 698,95% CI:0.631-0.766) had better predictive value for all-cause mortality than TPG ( AUC=0.596, 95% CI: 0.523-0.669) and DPG ( AUC=0.526, 95% CI: 0.452-0.601) (all P<0.05). The optimal cutoff values for TPG, DPG, and PVR were13.9 mmHg, 2.8 mmHg, and 3.8 Wood Units, respectively. Kaplan-Meier analysis based on the optimal cutoff values or guidance-recommended thresholds showed that PVR and TPG were the predictors of survival ( P<0.05), while DPG did not showed significance ( P>0.05). Multivariate Cox regression analysis showed that age, PVR and log 2N-terminal pro-B-type natriuretic peptide were independent risk factors for all-cause mortality in PH-LHD patients (all P<0.05). Conclusion:Classification according to PVR was most valuable in predicting all-cause death in PH-LHD patients, while TPG showed moderate predictive ability and DPG had no predictive value.

Acute pancreatitis (AP) is a devastating disease characterized by an inflammatory disorder of the pancreas. P-selectin glycoprotein ligand-1 (PSGL-1) plays a crucial role in the initial steps of the adhesive at process to inflammatory sites, blockade of PSGL-1 might confer potent anti-inflammatory effects. In this study, we generated two non-human primate derived monoclonal antibodies capable of efficiently targeting human PSGL-1, RH001-6 and RH001-22, which were screened from immunized rhesus macaques. We found that RH001-6, can effectively block the binding of P-selectin to PSGL-1, and abolish the adhesion of leukocytes to endothelial cells in vitro. In vivo, we verified that RH001-6 relieved inflammatory responses and pancreatic injury in both caerulein and l-arginine induced AP models. We also evaluated the safety profile after RH001-6 treatment in mice, and verified that RH001-6 did not cause any significant pathological damages in vivo. Taken together, we developed a novel non-human primate derived PSGL-1 blocking antibody with high-specificity, named RH001-6, which can interrupt the binding of PSGL-1 and P-selectin and attenuate inflammatory responses during AP. Therefore, RH001-6 is highly potential to be further developed into therapeutics against acute inflammatory diseases, such as AP.

Depression has become a serious global public health problem due to its high incidence and great harm, and has aroused the attention of the society. Ketamine, a commonly used intravenous anesthetic and analgesic in clinical practice, has been found to have unique advantages in antidepressant therapy in recent years. With the development of research, the enantiomeric isomers of ketamine, (S)-ketamine and (R)ketamine have entered the research field of antidepressant therapy. In this paper, we reviewed the progress of research on the antidepressant effects and mechanisms of ketamine, (S )-ketamine and (R)-ketamine, and provide a brief overview of the antidepressant effects of metabolites of ketamine, thereby deepening the understanding of the readers in the field of antidepressant effects of ketamine.

OBJECTIVES: Cardiac hypertrophy and fibrosis are major pathological manifestations observed in left ventricular remodeling induced by angiotensin II (AngII). Low-intensity pulsed ultrasound (LIPUS) has been reported to ameliorate cardiac dysfunction and myocardial fibrosis in myocardial infarction (MI) through mechano-transduction and its downstream pathways. In this study, we aimed to investigate whether LIPUS could exert a protective effect by ameliorating AngII-induced cardiac hypertrophy and fibrosis and if so, to further elucidate the underlying molecular mechanisms. METHODS: We used AngII to mimic animal and cell culture models of cardiac hypertrophy and fibrosis. LIPUS irradiation was applied in vivo for 20 min every 2 d from one week before mini-pump implantation to four weeks after mini-pump implantation, and in vitro for 20 min on each of two occasions 6 h apart. Cardiac hypertrophy and fibrosis levels were then evaluated by echocardiographic, histopathological, and molecular biological methods. RESULTS: Our results showed that LIPUS could ameliorate left ventricular remodeling in vivo and cardiac fibrosis in vitro by reducing AngII-induced release of inflammatory cytokines, but the protective effects on cardiac hypertrophy were limited in vitro. Given that LIPUS increased the expression of caveolin-1 in response to mechanical stimulation, we inhibited caveolin-1 activity with pyrazolopyrimidine 2 (pp2) in vivo and in vitro. LIPUS-induced downregulation of inflammation was reversed and the anti-fibrotic effects of LIPUS were absent. CONCLUSIONS These results indicated that LIPUS could ameliorate AngII-induced cardiac fibrosis by alleviating inflammation via a caveolin-1-dependent pathway, providing new insights for the development of novel therapeutic apparatus in clinical practice.

OBJECTIVES: To evaluate the value of thrombospond in Type I domain-containing 7A (THSD7A) and M-type phospholipase A2 receptor (PLA2R) in primary membranous nephropathy (PMN) and to explore the relationship between their antibody levels and prognosis. METHODS: Renal tissues in 128 patients with membranous nephropathy in the Second Xiangya Hospital of Central South University were collected from February 2015 to August 2017, including 108 patients with primary membranous nephropathy (PMN group) and 20 patients with secondary membranous nephropathy (SMN) (SMN group). Indirect immunofluorescence method was used to detect the expression of PLA2R antigen in kidney tissues,and the glomerular expression of THSD7A antigen was examined by immunohistochemistry and indirect immunofluorescence. The serum levels of anti-PLA2R antibodies and THSD7A antibodies were also detected by ELISA. According to the results of PMN examination,the patients were also divided into a PLA2R-related membranous nephropathy group and a THSD7A-related membranous nephropathy group. RESULTS: The positive rate of PLA2R in the renal tissues in the PMN group was higher than that in the SMN group (78% in the PMN group, 35% in the SMN group, <0.01),while the positive rate of anti-PLA2R antibody in the PMN group was also higher than that in the SMN group (50% in the PMN group, 25% in the SMN group, <0.05).The serum level of anti-PLA2R antibody was positively correlated with 24 h urine protein (=0.254, <0.05) and negatively correlated with serum albumin (=-0.236, <0.05). The expression of THSD7A was positive in glomeruli in 7 cases of the PMN group (6%) by immuno-histochemistry, and which was positive in 1case of the SMN group (5%).The serum levels of anti-THSD7A antibody in the PMN group were higher than those in the SMN group [(0.49±0.26) pg/mL in the PMN group,(0.34±0.27) pg/mL in the SMN group, <0.05]. There was no difference in the clinical characteristics between the PLA2R-related membranous nephropathy group and the THSD7A-related membranous nephropathy group. CONCLUSIONS PLA2R and THSD7A are the target antigen of PMN, and the associated autoantibodies are helpful for the differential diagnosis of PMN. The anti-PLA2R antibody levels can reflect the severity of the disease and evaluate the effect of treatment. The incidence of THSD7A membranous nephropathy is low, and monitoring the serum anti-THSD7A antibody levels can assess patients' condition and predict disease outcome.
Autoantibodies ; Glomerulonephritis, Membranous ; Humans ; Immunohistochemistry ; Receptors, Phospholipase A2 ; Thrombospondins