The aim of this investigation was to determine the optimal storage medium for testicular hypothermic transportation and identify the ideal concentration for the application of the protective agent 5-aminolevulinic acid (5-ALA). Furthermore, this study aimed to explore the underlying mechanism of the protective effects of 5-ALA. First, we collected and stored mouse testicular fragments in different media, including Hank's balanced salt solution (HBSS; n = 5), Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; n = 5), and alpha-minimum essential medium (αMEM; n = 5). Storage of testicular tissue in HBSS preserved the integrity of testicular morphology better than that in the DMEM/F12 group ( P < 0.05) and the αMEM group ( P < 0.01). Testicular fragments were subsequently placed in HBSS with various concentrations of 5-ALA (0 [control], 1 mmol l -1 , 2 mmol l -1 , and 5 mmol l -1 ) to determine the most effective concentration of 5-ALA. The 2 mmol l -1 5-ALA group ( n = 3) presented the highest positive rate of spermatogonial stem cells compared with those in the control, 1 mmol l -1 , and 5 mmol l -1 5-ALA groups. Finally, the tissue fragments were preserved in HBSS with control ( n = 3) and 2 mmol l -1 5-ALA ( n = 3) under low-temperature conditions. A comparative analysis was performed against fresh testes ( n = 3) to elucidate the underlying mechanism of 5-ALA. Gene set enrichment analysis (GSEA) for WikiPathways revealed that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was downregulated in the 2 mmol l -1 5-ALA group compared with that in the control group (normalized enrichment score [NES] = -1.57, false discovery rate [FDR] = 0.229, and P = 0.019). In conclusion, these data suggest that using 2 mmol l -1 5-ALA in HBSS effectively protected the viability of spermatogonial stem cells upon hypothermic transportation.
Male ; Animals ; Testis/cytology* ; Aminolevulinic Acid/pharmacology* ; Mice ; Organ Preservation/methods* ; Organ Preservation Solutions/pharmacology* ; Cryopreservation/methods*

OBJECTIVE: To investigate the whole-genome differential methylation profile of patients with high-altitude polycythemia (HAPC). METHODS: In this study, a total of 20 adult male patients with HAPC were included, including 10 Tibetan and 10 Han patients. The control group consisted of 20 healthy adult males, including 10 Tibetan and 10 Han patients. Peripheral blood was collected from each group for DNA extraction and quality inspection, and DNA libraries were constructed. The differential methylation regions (DMRs) between groups were detected using reduced representation bisulfite sequencing, with enriched regions compared to those of the control group. The differential enrichment regions were selected, and the intersection of the enriched regions was associated with genes. The methylation enrichment regions that differed significantly between groups were filtered based on the number of enriched samples in the enriched regions between the groups. GO, KEGG functional, and pathway analysis were performed on the differentially associated gene sets to reveal significant differences between the patients and control groups at the functional and pathway levels. RESULTS: In comparison with the control group, 17 152 sites with more than 25% difference and 15 558 sites with less than -25% difference were identified in Tibetan patients. The top 5 genes with the largest methylation differences between the two groups were MCCC2, RP3-399L15.3, ZNF621, RP11-394A14.2 and SLC39A10. The top significantly different pathways annotated in the differentially expressed genes pathway was serotonergic synapse. In comparison with the control group, 2 687 CpG sites with a greater than 25% difference and 2 602 CpG sites with a less than -25% difference were identified in Han patients. The top 5 genes with the largest methylation differences between the two groups were NAA25, CORO2B, PDC, ZNF853, and MLLT10. The top significantly different pathways annotated in the differentially expressed genes pathway were glutamatergic synapse, retrograde endocannabinoid signaling, Rap1 signaling pathway and cholinergic synapse. In comparison with the control group, 3 895 CpG sites with a greater than 25% difference and 3 969 CpG sites with a less than -25% difference were identified in HAPC patients. The maximum methylation difference between the two groups could reach 78.1%, while the minimum was -42.6%. The top 5 genes with the largest methylation differences between the two groups were MCCC2, ARSJ, CTNNA3, SLC39A10, and SWAP70. The top significantly different pathways annotated in the differentially expressed genes pathway was signaling pathways regulating pluripotency of stem cells. CONCLUSION The occurrence of HAPC may be related to abnormal changes in DNA methylation, and methylation sites may be helpful for the early diagnosis of HAPC.
Humans ; DNA Methylation ; Altitude ; Polycythemia/genetics* ; Male ; Adult ; CpG Islands

Objective:To observe the genetic variation of SH2B3 in patients with myeloid neoplasms.Methods:The results of targeted DNA sequencing associated with myeloid neoplasms in the Department of Hematology,Xuanwu Hospital,Capital Medical University from November 2017 to November 2022 were retrospectively analyzed,and the patients with SH2B3 gene mutations were identified.The demographic and clinical data of these patients were collected,and characteristics of SH2B3 gene mutation,co-mutated genes and their correlations with diseases were analyzed.Results:The sequencing results were obtained from 1 005 patients,in which 19 patients were detected with SH2B3 gene mutation,including 18 missense mutations(94.74％),1 nonsense mutation(5.26％),and 10 patients with co-mutated genes(52.63％).Variant allele frequency(VAF)ranged from 0.03 to 0.66.The highest frequency mutation was p.Ile568Thr(5/19,26.32％),with an average VAF of 0.49,involving 1 case of MDS/MPN-RS(with SF3B1 mutation),1 case of MDS-U(with SF3B1 mutation),1 case of aplastic anemia with PNH clone(with PIGA and KMT2A mutations),2 cases of MDS-MLD(1 case with SETBP1 mutation).The other mutations included p.Ala567Thr in 2 cases(10.53％),p.Arg566Trp,p.Glu533Lys,p.Met437Arg,p.Arg425Cys,p.Glu314Lys,p.Arg308*,p.Gln294Glu,p.Arg282Gln,p.Arg175Gln,p.Gly86Cys,p.His55Asn and p.Gln54Pro in 1 case each.Conclusion:A wide distribution of genetic mutation sites and low recurrence of SH2B3 is observed in myeloid neoplasms,among of them,p.Ile568Thr mutation is detected with a higher incidence and often coexists with characteristic mutations of other diseases.

Lipids,including fats(triglycerides)and lipoids(phospholipids and sterols),not only serve as an energy source for the body but also play a pivotal role throughout the reproductive process,particularly in the establishment and maintenance of early pregnancy.This encompasses the regulate of early embryonic development and uterine tolerance,and the facilitation of embryo implantation.Given the diversity of lipids,this review focuses on extensively studied lipid mediators such as polyunsaturated fatty acids,endocannabinoids,prostaglandins,lysophosphatidic acid,sphingolipids and steroid hormones.It systematically elaborates on the regulatory effects of fatty acid,phospholipid,and cholesterol metabolism on the formation of endometrial receptivity and embryo implantation,as well as the potential underlying mechanisms.The review aims to provide new insights and feasible intervention approaches for predicting and improving the outcomes of natural pregnancy and/or assisted reproductive technology.

AIM To investigate the role of Tripterygium wilfordii polyglycosides as an inhibitor of pulmonary fibrosis progression in rats.METHODS In contrast to the six rats randomly selected into the blank group,another 34 rats were induced into a rat model of pulmonary fibrosis by bleomycin sulfate ( 5 mg/mL) injection using tracheal puncture followed by 7 days later random assignment of the survival thirty rats into the model group,the prednisone acetate group (1.17 mg/kg) and the low,medium and high dose tripterygium wilfordii polyglycosides groups (1,3,6 mg/kg) for 21 days corresponding dosing of the drugs.The general characteristics of rats were observed during administration.After the administration,the rats had their pulmonary morphology observed;their lung coefficients of each group compared;their structural changes of lung tissue observed using HE staining;their collagen deposition observed by Masson staining;and their hyperplastic conditions assessed by the criteria for judging pulmonary fibrous hyperplasia;their serum HYP,TNF-α,IL-1β and IL-6 levels detected by ELISA;their pulmonary expressions of miR-146a,NF-κB and Col-I mRNA detected by RT-qPCR method;and their pulmonary protein expressions of p-P65,IRAK1,TRAF6 and α-SMA detected by Western blot.RESULTS Compared with the blank group,the model group displayed increased levels of serum TNF-α,IL-6,IL-1β and HYP ( P<0.01);decreased pulmonary miR-146a mRNA expression ( P<0.01);and increased expressions of NF-κB,Col-I mRNA and p-P65,IRAK1,TRAF6 and α-SMA protein ( P<0.01 ).Compared with the model group,the groups intervened with either prednisone acetate or Tripterygium wilfordii polyglycoside shared differently improved alveolar structure and inflammatory cell infiltration;reduced fibrosis;increased pulmonary miR-146a mRNA expression ( P<0.01);increased levels of TRAF6-α,IL-6,IL-1β and HYP in serum,and expressions of NF-κB and Col-I mRNA and expression of p-P65,IRAK1,TRAF6 and α-SMA protein of the lung tissue ( P<0.05,P<0.01) as well.CONCLUSION Tripterygium wilfordii polyglycosides can delay the progression of pulmonary fibrosis in rats,and its mechanism may be related to the regulation via miR-146/NF-κB signaling pathway.

Tuberculosis(TB)is still a serious threat to global public health,causing millions of people infected ev-ery year.Mycobacterium tuberculosis(MTB)is the main pathogen causing TB.In recent years,MTB extracellular vesicles(MEVs)as important carriers for MTB-secreted Mycobacterium antigens have attracted the attention of re-searchers.MEVs enable MTB to secrete phospholipid,nucleic acid,lipopolysaccharide,and periplasmic component in a centralized protective mode,and interact with the host.Some progress has been made on the study of partial contents of MEVs,but the understanding of their biological mechanisms,functions,and roles in the immune re-sponses during MTB infection is still at its early stages.This article reviews the current progress in the biogenesis of MEVs,their roles in MTB infection and immune responses regulation,and discusses their applications in vaccine development and diagnostic techniques.

Tuberculosis(TB)is still a serious threat to global public health,causing millions of people infected ev-ery year.Mycobacterium tuberculosis(MTB)is the main pathogen causing TB.In recent years,MTB extracellular vesicles(MEVs)as important carriers for MTB-secreted Mycobacterium antigens have attracted the attention of re-searchers.MEVs enable MTB to secrete phospholipid,nucleic acid,lipopolysaccharide,and periplasmic component in a centralized protective mode,and interact with the host.Some progress has been made on the study of partial contents of MEVs,but the understanding of their biological mechanisms,functions,and roles in the immune re-sponses during MTB infection is still at its early stages.This article reviews the current progress in the biogenesis of MEVs,their roles in MTB infection and immune responses regulation,and discusses their applications in vaccine development and diagnostic techniques.

AIM To investigate the role of Tripterygium wilfordii polyglycosides as an inhibitor of pulmonary fibrosis progression in rats.METHODS In contrast to the six rats randomly selected into the blank group,another 34 rats were induced into a rat model of pulmonary fibrosis by bleomycin sulfate ( 5 mg/mL) injection using tracheal puncture followed by 7 days later random assignment of the survival thirty rats into the model group,the prednisone acetate group (1.17 mg/kg) and the low,medium and high dose tripterygium wilfordii polyglycosides groups (1,3,6 mg/kg) for 21 days corresponding dosing of the drugs.The general characteristics of rats were observed during administration.After the administration,the rats had their pulmonary morphology observed;their lung coefficients of each group compared;their structural changes of lung tissue observed using HE staining;their collagen deposition observed by Masson staining;and their hyperplastic conditions assessed by the criteria for judging pulmonary fibrous hyperplasia;their serum HYP,TNF-α,IL-1β and IL-6 levels detected by ELISA;their pulmonary expressions of miR-146a,NF-κB and Col-I mRNA detected by RT-qPCR method;and their pulmonary protein expressions of p-P65,IRAK1,TRAF6 and α-SMA detected by Western blot.RESULTS Compared with the blank group,the model group displayed increased levels of serum TNF-α,IL-6,IL-1β and HYP ( P<0.01);decreased pulmonary miR-146a mRNA expression ( P<0.01);and increased expressions of NF-κB,Col-I mRNA and p-P65,IRAK1,TRAF6 and α-SMA protein ( P<0.01 ).Compared with the model group,the groups intervened with either prednisone acetate or Tripterygium wilfordii polyglycoside shared differently improved alveolar structure and inflammatory cell infiltration;reduced fibrosis;increased pulmonary miR-146a mRNA expression ( P<0.01);increased levels of TRAF6-α,IL-6,IL-1β and HYP in serum,and expressions of NF-κB and Col-I mRNA and expression of p-P65,IRAK1,TRAF6 and α-SMA protein of the lung tissue ( P<0.05,P<0.01) as well.CONCLUSION Tripterygium wilfordii polyglycosides can delay the progression of pulmonary fibrosis in rats,and its mechanism may be related to the regulation via miR-146/NF-κB signaling pathway.

Objective: This cross-sectional investigation aimed to determine the incidence, clinical characteristics, prognosis, and related risk factors of olfactory and gustatory dysfunctions related to infection with the SARS-CoV-2 Omicron strain in mainland China. Methods: Data of patients with SARS-CoV-2 from December 28, 2022, to February 21, 2023, were collected through online and offline questionnaires from 45 tertiary hospitals and one center for disease control and prevention in mainland China. The questionnaire included demographic information, previous health history, smoking and alcohol drinking, SARS-CoV-2 vaccination, olfactory and gustatory function before and after infection, other symptoms after infection, as well as the duration and improvement of olfactory and gustatory dysfunction. The self-reported olfactory and gustatory functions of patients were evaluated using the Olfactory VAS scale and Gustatory VAS scale. Results: A total of 35 566 valid questionnaires were obtained, revealing a high incidence of olfactory and taste dysfunctions related to infection with the SARS-CoV-2 Omicron strain (67.75%). Females(χ2=367.013, P<0.001) and young people(χ2=120.210, P<0.001) were more likely to develop these dysfunctions. Gender(OR=1.564, 95%CI: 1.487-1.645), SARS-CoV-2 vaccination status (OR=1.334, 95%CI: 1.164-1.530), oral health status (OR=0.881, 95%CI: 0.839-0.926), smoking history (OR=1.152, 95%CI=1.080-1.229), and drinking history (OR=0.854, 95%CI: 0.785-0.928) were correlated with the occurrence of olfactory and taste dysfunctions related to SARS-CoV-2(above P<0.001). 44.62% (4 391/9 840) of the patients who had not recovered their sense of smell and taste also suffered from nasal congestion, runny nose, and 32.62% (3 210/9 840) suffered from dry mouth and sore throat. The improvement of olfactory and taste functions was correlated with the persistence of accompanying symptoms(χ2=10.873, P=0.001). The average score of olfactory and taste VAS scale was 8.41 and 8.51 respectively before SARS-CoV-2 infection, but decreased to3.69 and 4.29 respectively after SARS-CoV-2 infection, and recovered to 5.83and 6.55 respectively at the time of the survey. The median duration of olfactory and gustatory dysfunctions was 15 days and 12 days, respectively, with 0.5% (121/24 096) of patients experiencing these dysfunctions for more than 28 days. The overall self-reported improvement rate of smell and taste dysfunctions was 59.16% (14 256/24 096). Gender(OR=0.893, 95%CI: 0.839-0.951), SARS-CoV-2 vaccination status (OR=1.334, 95%CI: 1.164-1.530), history of head and facial trauma(OR=1.180, 95%CI: 1.036-1.344, P=0.013), nose (OR=1.104, 95%CI: 1.042-1.171, P=0.001) and oral (OR=1.162, 95%CI: 1.096-1.233) health status, smoking history(OR=0.765, 95%CI: 0.709-0.825), and the persistence of accompanying symptoms (OR=0.359, 95%CI: 0.332-0.388) were correlated with the recovery of olfactory and taste dysfunctions related to SARS-CoV-2 (above P<0.001 except for the indicated values). Conclusion: The incidence of olfactory and taste dysfunctions related to infection with the SARS-CoV-2 Omicron strain is high in mainland China, with females and young people more likely to develop these dysfunctions. Active and effective intervention measures may be required for cases that persist for a long time. The recovery of olfactory and taste functions is influenced by several factors, including gender, SARS-CoV-2 vaccination status, history of head and facial trauma, nasal and oral health status, smoking history, and persistence of accompanying symptoms.
Female ; Humans ; Adolescent ; SARS-CoV-2 ; Smell ; COVID-19/complications* ; Cross-Sectional Studies ; COVID-19 Vaccines ; Incidence ; Olfaction Disorders/etiology* ; Taste Disorders/etiology* ; Prognosis

Objective To improve the fixation method of the transmission electron microscope for better morphological preservation of mitochondria and lipid droplets in mouse brown adipose tissue. Methods The fixation method for mouse brown adipose tissue was optimized, mainly including an increased concentration of paraformaldehyde from 2% to 4% in the pre-fixative, employment of transcardial perfusion followed by immersion fixation in pre-fixation, and using imidazole-buffered osmium tetroxide as the post-fixative. The ultrastructures of brown adipocytes prepared by the improved method were observed and compared with those of a known standard protocol (3 mice in each group). The improved method was further validated in the quantitative analysis of mitochondrial cristae density and lipid droplets. Results The mitochondrial cristae and membrane structure of other organelles of brown adipocytes were better preserved using the optimized method compared with those of the standard method. Lipid droplets were presented as round structures with high electron density instead of vacuolated appearances. Using this method, we observed that the density of mitochondrial cristae and the content of lipid droplets increased in brown adipocytes after cold adaptation. Conclusion The optimized method can better preserve the ultrastructure of organelles in brown adipocytes, especially mitochondria and lipid droplets, and ma)' be applicable for studying the ultrastructures remodeling of brown adipose tissue under different physiological or pathological conditions.