Background: Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype–phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes. Methods: In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results: Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants. Conclusions We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.

Objective : To explore the mechanism of hippocampal corticotropin-releasing hormone ( CRH) receptor type 1 ( CRHR1 ) in chronic stress-induced learning and memory impairment in early aged mice． Methods: C57BL /6J mice aged 12 －14 months were divided into two groups according to gender，and then divided into wild type (WT) group and hippocampal CRHR1 conditional gene knockout (KN) group according to genotype．Mice in each group were randomly divided into control group and stress group，and the stress group was subjected to chronic unpredictable stress ( CUS ) for 30 days． Genotyping of mice was performed using polymerase chain reaction ( PCR) ，agarose gel electrophoresis and real-time fluorescence quantitative PCR (RT-qPCR) ．The new object rec- ognition experiment and Morris Water maze measured learning and memory ability．Golgi-Cox staining was used to observe damage to hippocampal neuronal dendrites． The protein expressions of target protein of rapamycin (mTOR) ，p-mTOR (Ser2448) ，ribosomal protein S6 kinase ( p70S6K) and p-p70S6K ( Thr389 / Thr412 ) were detected by Western blot．Serum levels of corticotropin releasing hormone ( CRH) were measured by ELISA. Results : Compared to mice without chronic stress，the cognitive coefficient of WT stress groups decreased after chron- ic stress，and the difference was statistically significant (P ＜0. 05) ，while there was no significant difference in cognitive coefficient of KN stress groups before and after chronic stress．Compared with the WT stress group，the escape latency of the WT control group was shortened (P＜0. 05) ，and the number of crossing the platform and tar- get quadrant increased (P ＜0. 01) ，and there was no significant difference in the KN groups above． Compared with the WT control group，the WT stress group had a significant reduction in the neuronal complexity in the hipp- ocampal CA1，CA3 and DG regions (P ＜0. 05) and significant reductions in the expression of p-mTOR and p- p70S6K in the hippocampus (P＜0. 05) ．There was no significant difference in the expression of p-mTOR between the KN stress group and the KN control group (P＞0. 05) ，except that the expression of p-mTOR in the hippocam- pus of the female group decreased (P＜0. 05) ．In addition，the serum level of CRH in the stress group was higher than that in the control group (P＜0. 01) ． Conclusion Hippocampal CRHR1 regulates learning and memory im- pairment and neuronal dendrite damage in early aged mice induced by chronic stress．The mechanism may be that high levels of CRH induced by chronic stress cannot bind to CRHR1 receptor，thereby enhancing the expression of down-regulated mTOR / p70S6K signaling pathway.

Objective To explore the effects of mechanical ventilation and mechanical ventilation com-bined with phrenic nerve electrical stimulation on the morphology of the phrenic nerve-diaphragm neuromuscular junction in rats.Methods The rats were divided into a control group(CON group,n=6);an 18-hour mechanical ventilation group(MV group,n=6);an 18-hour ventilation combined with sham electrical stimulation group(S-MS group,n=6);and an 18-hour ventilation combined with electrical stimulation group(MS group,n=6),in which the diaphragm contraction force was detected through the biological signal acquisition system,the stimula-tion frequency-contraction curve was fitted,and the muscle fatigue index was calculated.The muscle fiber cross-sectional area(CSA)of the diaphragm specimen was observed through HE staining;the endplate membrane area of the neuromuscular junction(NMJ)was observed through immunohistochemistry(IHC)staining of the diaphragm combined with laser confocal technology.Results As compared with the CON group,the contractility of isolated diaphragm muscles in the MV group and S-MS group decreased;the fatigue resistance of isolated diaphragm,as compared with the CON group(0.686 8±0.049 42),decreased in the MV group(0.360 3±0.066 89)and the S-MS group(0.367 9±0.034 94),with a greater decline in the MS group(0.536 2±0.054 38);as compared with the CON group[674.3(523.9,863.6)],diaphragm CSA was reduced in the MV group[374.5(293.3,522.7)]and S-MS group[392.8(309.5,542.6)],with a relatively lower reduction in the MS group[592.7(425.1,820.0)];as compared with the CON group[393.7(293.5,471.3)],the diaphragm endplate membrane area decreased in the MV group[289.7(227.0,354.2)]and S-MS group[243.1(190.8,331.7)],with a lower degree of reduction in the MS group[331.0(262.8,413.7).Conclusions Mechanical ventilation causes diaphragm dysfunction in rats and decreases the endplate membrane area of the diaphragm NMJ.Bilateral phrenic nerve electrical stimulation for 10 min/h can improve diaphragm dysfunction and changes in the endplate membrane area of the diaphragm NMJ caused by mechanical ventilation.

Objective:To investigated the safety and efficacy of donor-derived CD19+ or sequential CD19+ CD22+ chimeric antigen receptor T-cell (CAR-T) therapy in patients with B-cell acute lymphoblastic leukemia (B-ALL) after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:The data of 22 patients with B-ALL who relapsed after allo-HSCT and who underwent donor-derived CAR-T therapy at the Zhujiang Hospital of Southern Medical University and the 920th Hospital of Joint Logistics Support Force of the People’s Liberation Army of China from September 2015 to December 2022 were retrospectively analyzed. The primary endpoint was overall survival (OS), and the secondary endpoints were event-free survival (EFS), complete remission (CR) rate, and Grade 3-4 adverse events.Results:A total of 81.82% ( n=18) of the 22 patients achieved minimal residual disease-negative CR after CAR-T infusion. The median follow-up time was 1037 (95% CI 546–1509) days, and the median OS and EFS were 287 (95% CI 132-441) days and 212 (95% CI 120-303) days, respectively. The 6-month OS and EFS rates were 67.90% (95% CI 48.30%-84.50%) and 58.70% (95% CI 37.92%-79.48%), respectively, and the 1-year OS and EFS rates were 41.10% (95% CI 19.15%-63.05%) and 34.30% (95% CI 13.92%-54.68%), respectively. Grade 1-2 cytokine release syndrome occurred in 36.36% ( n=8) of the patients, and grade 3-4 occurred in 13.64% of the patients ( n=3). Grade 2 and 4 graft-versus-host disease occurred in two patients. Conclusion:Donor-derived CAR-T therapy is safe and effective in patients with relapsed B-ALL after allo-HSCT.

Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75％(27/36);boys accounted for 64％(23/36).Among the 36 neonates,31(86％)had disease onset within 3 days after birth,and 19(53％)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61％)had left cerebral infarction and 13(36％)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75％)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72％)and seizures in 10 neonates(34％).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33％,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44％(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.

The effect of Tujia medicine Berberidis Radix on endogenous metabolites in the serum and feces of mice with ulcerative colitis(UC) induced by dextran sulfate sodium(DSS) was analyzed by metabolomics technology to explore the metabolic pathway and underlying mechanism of Berberidis Radix in the intervention of UC. The UC model was induced in mice by DSS. Body weight, disease activity index(DAI), and colon length were recorded. The levels of tumor necrosis factor-α(TNF-α) and interleukin-10(IL-10) in colon tissues were determined by ELISA. The levels of endogenous metabolites in the serum and feces were detected by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS). Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were employed to characterize and screen differential metabolites. The potential metabolic pathways were analyzed by MetaboAnalyst 5.0. The results showed that Berberidis Radix could significantly improve the symptoms of UC mice and increase the level of the anti-inflammatory factor IL-10. A total of 56 and 43 differential metabolites were identified in the serum and feces, respectively, belonging to lipids, amino acids, fatty acids, etc. After the intervention by Berberidis Radix, the metabolic disorder gradually recovered. The involved metabolic pathways included biosynthesis of phenylalanine, tyrosine, and tryptophan, linoleic acid metabolism, phenylalanine metabolism, and glycerophospholipid metabolism. Berberidis Radix can alleviate the symptoms of mice with DSS-induced UC, and the mechanism may be closely related to the re-gulation of lipid metabolism, amino acid metabolism, and energy metabolism.
Mice ; Animals ; Colitis, Ulcerative/drug therapy* ; Interleukin-10 ; Metabolomics/methods* ; Chromatography, High Pressure Liquid

OBJECTIVE: To investigate the anti-angiogenic activity of Kunxian Capsule (KX) extract and explore the underlying molecular mechanism using zebrafish. METHODS: The KX extract was prepared with 5.0 g in 100 mL of 40% methanol followed by ultrasonication and freeze drying. Freeze dried KX extract of 10.00 mg was used as test stock solution. Triptolide and icariin, the key bioactive compounds of KX were analyzed using ultra-high performance liquid chromatography. The transgenic zebrafish Tg(flk1:GFP) embryos were dechorionated at 20-h post fertilization (hpf) and treated with PTK 787, and 3.5, 7, 14 and 21 µg/mL of KX extract, respectively. After 24-h post exposure (hpe), mortality and malformation (%), intersegmental vessels (ISV) formation, and mRNA expression level of angiogenic pathway genes including phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), extracellular signal-regulated kinases (ERKs), mitogen-activated protein kinase (MAPK), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2) were determined. Further, the embryos at 72 hpf were treated with KX extract to observe the development of sub-intestinal vein (SIV) after 24 hpe. RESULTS: The chromatographic analysis of test stock solution of KX extract showed that triptolide and icariin was found as 0.089 mg/g and 48.74 mg/g, respectively, which met the requirements of the national drug standards. In zebrafish larvae experiment, KX extract significantly inhibited the ISV (P<0.01) and SIV formation (P<0.05). Besides, the mRNA expression analysis showed that KX extract could significantly suppress the expressions of PI3K and AKT, thereby inhibiting the mRNA levels of ERKs and MAPK. Moreover, the downstream signaling cascade affected the expression of VEGF and its receptors (VEGFR and VEGFR-2). FGF-2, a strong angiogenic factor, was also down-regulated by KX treatment in zebrafish larvae. CONCLUSION KX extract exhibited anti-angiogenic effects in zebrafish embryos by regulating PI3K/AKT-MAPK-VEGF pathway and showed promising potential for RA treatment.
Animals ; Fibroblast Growth Factor 2 ; Human Umbilical Vein Endothelial Cells ; Mitogen-Activated Protein Kinases/metabolism* ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Zebrafish

Aim To investigate the effect of DNA methyltransferase 3A (DNMT3A) on the proliferation and migration of cardiac fibroblasts (CFs) in C57 mice under high glucose environment. Methods The hearts of C57 mice were taken from 1 to 3 days. After cutting and digesting, CFs were extracted by differential adherance centrifugattion and observed under microscope. After cell attachment, the cells were cultured under low glucose (5.5 mmol • L

An HPLC pre-column derivatization detection method was established to detect and analyze the formaldehyde and acetaldehyde in polysorbate 80 and polysorbate 20 from different manufacturers.The effects of aldehyde and acetaldehyde on the aggregation of adalimumab under different conditions were monitored.Based on the control of genotoxic impurities and the influence on the stability of monoclonal antibody preparations, the control limits of the two chemicals were preliminarily obtained.2, 4-dinitrophenylhydrazine (2, 4-DNPH) was applied as the derivatization reagent in HPLC pre-column derivatization; acetonitrile and water were used as mobile phase to perform a gradient elution on a C8 (4.6 mm × 150 mm, 5 μm) chromatographic column.The detection wavelength was 360 nm, and the external standard method was used for quantification.Verification results showed that the method was suitable for the quantitative analysis of trace formaldehyde and acetaldehyde in polysorbate 80 and polysorbate 20 . The detection and analysis of formaldehyde or acetaldehyde in different batches of polysorbate 80 and polysorbate 20 from different manufacturers showed that the content of formaldehyde and acetaldehyde were quite different. The content of formaldehyde and acetaldehyde in polysorbate 80 were significantly higher than those of polysorbate 20. After monitoring the changes of adalimumab aggregates treated by formaldehyde and acetaldehyde by size exclusion chromatography (SEC), it was found that the effect of formaldehyde on adalimumab aggregation was significantly higher than that of acetaldehyde.According to the requirements of ICH M7 (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, M7: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk), the impurity limits of formaldehyde and acetaldehyde in polysorbate 80 and polysorbate 20 for monoclonal antibody preparations were calculated from the perspective of risk assessment.Combined with the influence on the aggregation stability of monoclonal antibodies, the preliminary limis for acetaldehyde and acetaldehyde were recommended to be ≤ 7 μg/g and ≤ 765 μg/g, respectively.

The processing of Chinese medicine is a unique and dialectical treatment of traditional Chinese medicine in clinic.The processing theory of "leading vinegar-processing Chinese medicine into liver" is one of the traditional processing theories of Chinese medicine.The vinegar-processing Chinese medicine under the guidance of the processing theory typically reflects the characteristics of "reducing toxicity and enhancing efficacy" of the processing of Chinese medicine.This paper traced the origin and discussed the connotation of the traditional theory of "leading vinegar-processing Chinese medicine into liver".Combined with the research status of "lea-ding vinegar-processing Chinese medicine into liver", this paper explored the mechanism of "leading vinegar-processing Chinese medicine into liver" from the aspects of material basis, medicine effect, and traditional Chinese medicine(TCM) meridian, and analyzed the existing problems in the current research.This paper reviewed the modern study on reducing toxicity and enhancing efficacy of vinegar-processing Chinese medicine, and deeply explored the scientific connotation of the traditional processing theory of "leading vinegar-processing Chinese medicine into liver".At the same time, the research trend and idea of the effect mechanism of "leading vinegar-processing Chinese medicine into liver" based on the Quality markers(Q-Marker) of TCM, biological targets, and clinical prescriptions were put forward, providing references for the further study on "leading vinegar-processing Chinese medicine into liver".This paper also provided a scientific basis for the rational selection of processed products in TCM clinical practice.
Acetic Acid ; Biomarkers ; Drugs, Chinese Herbal ; Liver ; Medicine, Chinese Traditional ; Meridians