Hepatic stellate cells (HSCs) are the primary fibrogenic cells in the liver, and their activation plays a crucial role in the development and progression of hepatic fibrosis. Here, we report that retinoid X receptor-alpha (RXRα), a unique member of the nuclear receptor superfamily, is a key modulator of HSC activation and liver fibrosis. RXRα exerts its effects by modulating calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ)-mediated activation of AMP-activated protein kinase-alpha (AMPKα). In addition, we demonstrate that K-80003, which binds RXRα by a unique mechanism, effectively suppresses HSC activation, proliferation, and migration, thereby inhibiting liver fibrosis in the CCl₄ and amylin liver NASH (AMLN) diet animal models. The effect is mediated by AMPKα activation, promoting mitophagy in HSCs. Mechanistically, K-80003 activates AMPKα by inducing RXRα to form condensates with CaMKKβ and AMPKα via a two-phase process. The formation of RXRα condensates is driven by its N-terminal intrinsic disorder region and requires phosphorylation by CaMKKβ. Our results reveal a crucial role of RXRα in liver fibrosis regulation through modulating mitochondrial activities in HSCs. Furthermore, they suggest that K-80003 and related RXRα modulators hold promise as therapeutic agents for fibrosis-related diseases.

ObjectiveBody fluid stains left at crime scenes are frequently trace amounts, while the identification of body fluids through real time fluorogenic quantitative technique often necessitates the repeated detection within the limited sample, as multiple miRNA markers are the basis for the identification. Based on the goal of both the throughput and efficiency improvement of miRNA analysis in trace samples, a duplex real time fluorogenic quantitative PCR assay system was designed to accurately quantify two miRNAs simultaneously, and the system should be further verified by actual sample for the body fluid identification. MethodsThe duplex real time fluorogenic quantitative PCR system of miR-451a to miR-21-5p was established with specially designed primers and probes, and the concentrations of the primers and probes were both optimized. The specificity, sensitivity and reproducibility of the system were validated, while its capability for body fluid identification was assessed using the miR-451a to miR-21-5p ratio. ResultsThe optimized assay system exhibited excellent specificity and repeatability, with coefficients of variation consistently below 8% for both intra- and inter-batch variability. The amplification efficiency of miR-451a and miR-21-5p reached 71.77% and 74.81%, respectively, with high and relatively consistent results. By utilizing this duplex real time fluorogenic quantitative PCR assay system, a total of 58 body fluid samples were analyzed, exhibiting a discrimination rate of 100% between blood and non-blood samples, as well as between peripheral blood and menstrual blood samples. Moreover, the results, obtained from single real time fluorogenic quantitative PCR assay system and duplex real time fluorogenic quantitative PCR assay system, showed no statistically significant difference with randomly selected blood samples (n=20). Compared to previous single real time fluorogenic quantitative PCR assay system, the sensitivity of duplex real time fluorogenic quantitative PCR assay system exhibited remarkable improvement. A minimum input of only 0.1 ng total RNA was sufficient for accurate detection of peripheral blood and menstrual blood samples, while saliva, semen, and vaginal secretion required only 1 ng total RNA for precise identification purposes. Additionally, the duplex real time fluorogenic quantitative PCR assay system successfully differentiated between different types of body fluids in simulated samples under natural outdoor conditions. ConclusionThe duplex real time fluorogenic quantitative PCR assay system effectively reduced both the time and material costs by half compared to the single system, especially suitable for the examination of body fluid stains left at crime scenes, solving the contradiction between the trace amount and the multiple sample volumes demand of repeated real time fluorogenic quantitative PCR. The duplex real time fluorogenic quantitative PCR assay successfully distinguished blood and other body fluid, as well as peripheral blood and menstrual blood samples, which maintains an equivalent capability for body fluid identification with half sample, time and reagent consumption. This system provides an efficient tool for identifying suspicious body fluids, as well as a foundation for more multiplexed real time fluorogenic quantitative PCR assay system research.

Objective: To analyze the clinical epidemiological characteristics including composition of pathogens , clinical characteristics, and disease prognosis acute bacterial meningitis (ABM) in Chinese children. Methods: A retrospective analysis was performed on the clinical and laboratory data of 1 610 children <15 years of age with ABM in 33 tertiary hospitals in China from January 2019 to December 2020. Patients were divided into different groups according to age,<28 days group, 28 days to <3 months group, 3 months to <1 year group, 1-<5 years of age group, 5-<15 years of age group; etiology confirmed group and clinically diagnosed group according to etiology diagnosis. Non-numeric variables were analyzed with the Chi-square test or Fisher's exact test, while non-normal distrituction numeric variables were compared with nonparametric test. Results: Among 1 610 children with ABM, 955 were male and 650 were female (5 cases were not provided with gender information), and the age of onset was 1.5 (0.5, 5.5) months. There were 588 cases age from <28 days, 462 cases age from 28 days to <3 months, 302 cases age from 3 months to <1 year of age group, 156 cases in the 1-<5 years of age and 101 cases in the 5-<15 years of age. The detection rates were 38.8% (95/245) and 31.5% (70/222) of Escherichia coli and 27.8% (68/245) and 35.1% (78/222) of Streptococcus agalactiae in infants younger than 28 days of age and 28 days to 3 months of age; the detection rates of Streptococcus pneumonia, Escherichia coli, and Streptococcus agalactiae were 34.3% (61/178), 14.0% (25/178) and 13.5% (24/178) in the 3 months of age to <1 year of age group; the dominant pathogens were Streptococcus pneumoniae and the detection rate were 67.9% (74/109) and 44.4% (16/36) in the 1-<5 years of age and 5-<15 years of age . There were 9.7% (19/195) strains of Escherichia coli producing ultra-broad-spectrum β-lactamases. The positive rates of cerebrospinal fluid (CSF) culture and blood culture were 32.2% (515/1 598) and 25.0% (400/1 598), while 38.2% (126/330)and 25.3% (21/83) in CSF metagenomics next generation sequencing and Streptococcus pneumoniae antigen detection. There were 4.3% (32/790) cases of which CSF white blood cell counts were normal in etiology confirmed group. Among 1 610 children with ABM, main intracranial imaging complications were subdural effusion and (or) empyema in 349 cases (21.7%), hydrocephalus in 233 cases (14.5%), brain abscess in 178 cases (11.1%), and other cerebrovascular diseases, including encephalomalacia, cerebral infarction, and encephalatrophy, in 174 cases (10.8%). Among the 166 cases (10.3%) with unfavorable outcome, 32 cases (2.0%) died among whom 24 cases died before 1 year of age, and 37 cases (2.3%) had recurrence among whom 25 cases had recurrence within 3 weeks. The incidences of subdural effusion and (or) empyema, brain abscess and ependymitis in the etiology confirmed group were significantly higher than those in the clinically diagnosed group (26.2% (207/790) vs. 17.3% (142/820), 13.0% (103/790) vs. 9.1% (75/820), 4.6% (36/790) vs. 2.7% (22/820), χ²=18.71, 6.20, 4.07, all P<0.05), but there was no significant difference in the unfavorable outcomes, mortility, and recurrence between these 2 groups (all P>0.05). Conclusions: The onset age of ABM in children is usually within 1 year of age, especially <3 months. The common pathogens in infants <3 months of age are Escherichia coli and Streptococcus agalactiae, and the dominant pathogen in infant ≥3 months is Streptococcus pneumoniae. Subdural effusion and (or) empyema and hydrocephalus are common complications. ABM should not be excluded even if CSF white blood cell counts is within normal range. Standardized bacteriological examination should be paid more attention to increase the pathogenic detection rate. Non-culture CSF detection methods may facilitate the pathogenic diagnosis.
Adolescent ; Brain Abscess ; Child ; Child, Preschool ; Escherichia coli ; Female ; Humans ; Hydrocephalus ; Infant ; Infant, Newborn ; Male ; Meningitis, Bacterial/epidemiology* ; Retrospective Studies ; Streptococcus agalactiae ; Streptococcus pneumoniae ; Subdural Effusion ; beta-Lactamases

Cannabinoid receptor type 2( CB2 R),a member of the G protein-coupled receptor( GPCR) superfamily,has a variety of biological activities,such as regulating pain response,resisting inflammation and fibrosis,and mediating bone metabolism. Some CB2 R regulators exhibit a good regulatory effect on bone metabolism. Cannabinoids in Cannabis sativa can cause psychoactive effects despite various pharmacological actions they exerted by targeting CB2 R. Therefore,it is of great significance to discover CB2 R regulators in non-Cannabis plants for finding new lead compounds without psychoactive effects and elucidating the action mechanism of plant drugs. The present study clarifies the discovery,structure,and physiological functions of CB2 R,especially its regulatory effects on bone metabolism,summarized CB2 R regulators extracted from non-Cannabis plants,and systematically analyzes the regulatory effects of CB2 R regulators on bone metabolism in animals,osteoblasts,and osteoclasts,to provide a scientific basis for the discovery of new CB2 R regulators and the development of anti-osteoporotic drugs.
Animals ; Cannabinoids/pharmacology* ; Cannabis ; Osteoblasts ; Osteoclasts ; Receptors, Cannabinoid

Objective: To analyze the long-term outcome of unoperated Ebstein's anomaly (EA) patients aged over 18 years, and to evaluate the related factor of outcomes. Methods: The data of 48 unoperated EA patients from March 2004 to December 2008 in the First Hospital of Tsinghua University, were analyzed. The clinical data of the patients were collected, and patients received regular echocardiography, ECG and chest X-ray examinations. Septal leaflet attachment ratio (SLAr) was calculated based on transthoracic echocardiography imagines. The patients were divided into 3 groups according to SLAr: SLAr<0.45 (n=18), 0.45≤SLAr≤0.60 (n=21) and SLAr>0.60 (n=9). Chest X-ray was used for measurement of cardiothoracic ratio (CTR). Kaplan Meier survival curve was used to calculate the long-term survival rate. Cox proportional hazards regression model was used to analyze the influencing factors of death. Results: There were 19 males, and the mean age at diagnosis was (21.3±11.1) years. Forty-two patients (87.5%) were complicated with arrhythmia, including W-P-W syndrome (n=4), supraventricular tachycardia (n=16), right bundle branch block (n=37), and atrial fibrillation (n=2). The mean duration of follow-up was (148.8±16.8) months, the follow-up rate was 100% with no loss-to-follow up. Nine cases (18.8%) died during follow-up: 6 cases (12.5%) died of cardiac origin, including 3 cases of heart failure, 1 case of arrhythmia, and 2 cases of sudden death; 1 case died of accident; 2 cases died from unknown causes. During the follow-up period, the survival rates were 17/18, 19/21 (90.5%) and 3/9 in the SLAr<0.45, 0.45≤SLAr≤0.60 and SLAr>0.60 group, respectively. According to Kaplan-Meier survival curve, the 5-year survival rates among the three groups were 100%, 100% and 78%, respectively. The 10-year survival rates among the three groups were 94%, 95% and 44%, respectively. Decreased activity tolerance and heart failure were found in 7 patients (6 patients in SLAr>0.60 group and 1 patient in 0.45≤SLAr≤0.60 group). Two patients had cerebrovascular embolism. There were 3 cases with tachyarrhythmia lasting more than 24 hours. Cox regression analysis showed that the risk of death was higher in patients with SLAr>0.60 than in patients with SLAr<0.45 (HR=12.375, 95%CI 1.692-22.146, P=0.015); the risk of death in patients with CTR≥0.65 was 1.306 times higher than that in patients with CTR<0.65 (HR=1.306, 95%CI 0.417-12.754, P=0.038). Conclusions: EA patients often combines with arrhythmia. For unoperated EA patients, SLAr>0.60 and CTR≥0.65 are risk factors of death. EA patients with arrhythmia should be actively treated with drugs or radiofrequency ablation.

Rhizosphere bacteria play a vital role in plant nutrition absorption,growth and disease resistance. In this study,high-throughput sequencing technology was used to analyze the rhizosphere bacterial communities of Salvia miltiorrhiza and S. miltiorrhiza f. alba. Moreover,the function of dominant rhizosphere bacterial communities was analyzed. We found that Sphingobacteriales,Sphingomonadales and Nitrosomonadaceae were both dominant and specific bacteria in the rhizosphere of S. miltiorrhiza. The main functions of dominant rhizosphere bacteria communities in both species include promoting transformation of soil nutrients,improving plant immunity and ability of stress tolerance. This study was the first to compare rhizobacterial communities structure and function of S. miltiorrhiza and S. miltiorrhiza f. alba,which provided a new theoretical reference for studing the rhizosphere mechanism of healthy S. miltiorrhiza planting in the future.
Bacteria ; classification ; High-Throughput Nucleotide Sequencing ; Rhizosphere ; Salvia miltiorrhiza ; microbiology ; Soil Microbiology

To ensure the consistency of quality in recombinant protein production, the cell bank for biologics should be derived from a single clone. A number of techniques have been used for cloning and assurance from the cellular pool after transfection with a target gene. Here, using CHO cell as an example, we summarize the knowledge and understanding of monoclonality of production cell bank from both industries and regulatory authorities, and propose general considerations on the requirements of monoclonality for clinical trial application and new drug application based on current techniques. Furthermore, we suggest quality control strategies and assessment methods for those cell banks from non-single clones.

Sequence analysis of DNA, mRNA and protein is an essential component of biologics or bioprocess development. Analysis of sequences at the DNA, mRNA, and protein levels after the transfer of the gene of interest into a host cell is an important part of quality control. This article reviews the application of new technologies such as next-generation sequencing and LC-MS/MS in biological drug development such as monoclonal antibodies. These techniques have different requirements in term of cost, handling time and expertise. Selecting an appropriate technique with a sound rationale at different stages of drug development will add to the success rate of research and development, and ensure product quality, thus ensuring the clinical efficacy and safety.

Along with the increase of clinical application, the safety of traditional Chinese medicine gained more and more attentions. In particular, the safety evaluation of Chinese medical injections has become a mandatory task should be completed by pharmaceutical companies under the supervision of China Food and Drug Administration(CFDA). Due to the weak foundation of previous studies, the safety issues of Chinese medical injections have not been fully understood, and lack of scientific and rational risk management programs. Clinical safety centralized monitoring(CSCM) is an important method for post-market safety evaluation of Chinese medicine. Due to the lack of appropriate norms and procedures, the quality of similar research is uneven, and the results vary. Combined with practical experience with experts' suggestions, we developed this expert consensus on the design and implementation of CSCM from three stages (design, implementation and report) with 20 technical points, which will provide technical support for future CSCM studies.

Objective To evaluated the safety and feasibility of excimer laser atherectomy (ELA) combined with drug-eluting balloon angioplasty in treating chronic ischemia of lower limbs.Methods ELA combined with paclitaxel-eluting balloon angioplasty was adopted to treat chronic ischemia of lower limbs caused by arteriosclerosis occlusive disease of lower extremity in three patients.All three patients had arteriosclerosis occlusive disease of superficial femoral artery;in two of them the disease was primary occlusive lesion and in another patient the disease was in-stent re-occlusion lesion after sten implantation.Results After the treatment,the blood flow in the diseased arteries was unobstructed,the blood supply of the lower limbs was obviously improved.No procedure-related complications occurred.Two weeks after the treatment,no recurrence of ischemic symptoms was observed,the blood flow in superficial femoral artery kept unobstructed.The patients recovered smoothly.Conclusion For the treatment of chronic ischemia of lower limbs,which are caused by the primary arteriosclerosis occlusive disease of lower extremity or by the in-stent re-occlusion lesion after sten implantation,ELA combined with paclitaxel-eluting balloon angioplasty is clinically safe and feasible,although its long-term effect needs to be clarified with more studies.