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*

OBJECTIVES: To investigate the application value of thromboelastography (TEG) in assessing coagulation function in children with severe hemophilia A (HA) after emicizumab (EMI) therapy. METHODS: A retrospective analysis was performed on the activated partial thromboplastin time (APTT) and TEG testing results of 17 children with severe HA before and after EMI treatment at Shenzhen Children's Hospital from January 2023 to July 2024. Correlation analysis was conducted between coagulation factor VIII (FVIII) equivalent activity and reaction time (R value) measured by TEG. RESULTS: After EMI treatment, the mean bleeding rate for children with severe HA was 1.6 events per year, with 15 children (88%) without spontaneous bleeding or joint bleeding. The children with severe HA showed a significant reduction in APTT after EMI treatment (P<0.05), with a significantly shorter APTT than the normal control group (P<0.05). There was no correlation between APTT and FVIII equivalent activity after treatment (P>0.05). After EMI treatment, TEG parameters, including R value, kinetic time, alpha angle (α), maximum amplitude, clot strength, and coagulation index, shifted from a hypocoagulable state before treatment to a nearly normal state after treatment (P<0.05). The R value demonstrated a strong negative correlation with FVIII equivalent activity (r=-0.758, P<0.05). CONCLUSIONS The bleeding condition of children with severe HA can be effectively controlled after EMI treatment. Routine APTT testing cannot reflect true coagulation function, whereas TEG testing is clinically valuable in assessing the coagulation function of children with severe HA undergoing EMI treatment.
Humans ; Thrombelastography ; Hemophilia A/physiopathology* ; Male ; Child ; Antibodies, Bispecific/therapeutic use* ; Antibodies, Monoclonal, Humanized/therapeutic use* ; Blood Coagulation/drug effects* ; Child, Preschool ; Retrospective Studies ; Female ; Partial Thromboplastin Time ; Adolescent ; Infant

Endo-beta-N-acetylglucosaminidase (ENGase) is widely distributed in various organisms. The first reported ENGase activity was detected in Diplococcus pneumoniae in 1971. The protein (Endo D) was purified and its peptide sequence was determined in 1974. Three ENGases (Endo F1-F3) were discovered in Flavobacterium meningosepticum from 1982 to 1993. After that, the activity was detected from different species of bacteria, yeast, fungal, plant, mice, human, etc. Multiple ENGases were detected in some species, such as Arabidopsis thaliana and Trichoderma atroviride. The first preliminary crystallographic analysis of ENGase was conducted in 1994. But to date, only a few ENGases structures have been obtained, and the structure of human ENGase is still missing. The currently identified ENGases were distributed in the GH18 or GH85 families in Carbohydrate-Active enZyme (CAZy) database. GH18 ENGase only has hydrolytic activity, but GH85 ENGase has both hydrolytic and transglycosylation activity. Although ENGases of the two families have similar (β/α)8-TIM barrel structures, the active sites are slightly different. ENGase is an effective tool for glycan detection andglycan editing. Biochemically, ENGase can specifically hydrolyze β‑1,4 glycosidic bond between the twoN-acetylglucosamines (GlcNAc) on core pentasaccharide presented on glycopeptides and/or glycoproteins. Different ENGases may have different substrate specificity. The hydrolysis products are oligosaccharide chains and a GlcNAc or glycopeptides or glycoproteins with a GlcNAc. Conditionally, it can use the two products to produce a new glycopeptides or glycoprotein. Although ENGase is a common presentation in cell, its biological function remains unclear. Accumulated evidences demonstrated that ENGase is a none essential gene for living and a key regulator for differentiation. No ENGase gene was detected in the genomes of Saccharomyces cerevisiae and three other yeast species. Its expression was extremely low in lung. As glycoproteins are not produced by prokaryotic cells, a role for nutrition and/or microbial-host interaction was predicted for bacterium produced enzymes. In the embryonic lethality phenotype of the Ngly1-deficient mice can be partially rescued by Engase knockout, suggesting down regulation of Engase might be a solution for stress induced adaptation. Potential impacts of ENGase regulation on health and disease were presented. Rabeprazole, a drug used for stomach pain as a proton inhibitor, was identified as an inhibitor for ENGase. ENGases have been applied in vitro to produce antibodies with a designated glycan. The two step reactions were achieved by a pair of ENGase dominated for hydrolysis of substrate glycoprotein and synthesis of new glycoprotein with a free glycan of designed structure, respectively. In addition, ENGase was also been used in cell surface glycan editing. New application scenarios and new detection methods for glycobiological engineering are quickly opened up by the two functions of ENGase, especially in antibody remodeling and antibody drug conjugates. The discovery, distribution, structure property, enzymatic characteristics and recent researches in topical model organisms of ENGase were reviewed in this paper. Possible biological functions and mechanisms of ENGase, including differentiation, digestion of glycoproteins for nutrition and stress responding were hypothesised. In addition, the role of ENGase in glycan editing and synthetic biology was discussed. We hope this paper may provide insights for ENGase research and lay a solid foundation for applied and translational glycomics.

Objective To find potential biomarkers and analyzing metabolic pathways of the treatment by honey-processed Hedysari Radix,the cecal contents of rats with spleen-Qi deficiency were used as samples for analysis.Methods Sixty male SD rats were randomly divided into blank,model,experimental and control groups.The rats in other groups except the control group were carried out by using the three-factor compound modeling method of bitter-cold diarrhea,excessive exertion and hunger and satiety disorders.Experimental group was given 12.60 g·kg-1 honey-processed Hedysari Radix;control group was given 0.63 g·kg-1 lactobacillus bifidum triplex tabletsa;control and model groups received with equal volume of distilled water for a total of 15 days.Measure body weight,anal temperature,immune organ index of rats.Ultra-pressure liquid chromatography-quadrupole-exactive-mass spectrometry technology was used to measure the levels of endogenous metabolites in cecum contents.Orthogonal partial least squares discriminant analysis and database"Kyoto Encyclopedia of Genes and Genomes"were used to identify potential differential metabolites and possible metabolic pathways.Results After the intervention,the average body weight of the experimental,control,model and blank groups was(216.87±7.85),(210.96±9.03),(159.47±5.18)and(293.51±22.98)g;anal temperature was(36.14±0.48),(35.40±0.64),(34.50±0.78)and(36.61±0.34)℃;the thymus indexes were(1.19±0.20),(1.24±0.25),(0.47±0.15)and(1.31±0.21)mg·g-1;the spleen indexes were(1.95±0.33),(2.18±0.28),(1.61±0.27)and(2.29±0.24)mg·g-1.Compared with the model group,the above indexes of the experimental group and the control group were significantly increased(all P＜0.01).A total of 14 potential biomarkers of Honey-processed Hedysari Radix in treating spleen-Qi deficiency syndrome were screened out in this study,which mainly involved amino acid metabolism such as tryptophan and glutamate,riboflavin metabolism and adenosine 5'-monophosphate-activated protein kinase metabolism.Conclusion Honey-processed Hedysari Radix can further protect the intestinal mucosal barrier and reduce the intestinal inflammatory response by improving the metabolic level of cecum contents in rats with spleen-Qi deficiency in cecum contents,thus exerting the effect of strengthening the spleen and tonifying the Qi.

Objective To investigate the risk factors for bronchopulmonary dysplasia(BPD)in twin preterm infants with a gestational age of<34 weeks,and to provide a basis for early identification of BPD in twin preterm infants in clinical practice.Methods A retrospective analysis was performed for the twin preterm infants with a gestational age of<34 weeks who were admitted to 22 hospitals nationwide from January 2018 to December 2020.According to their conditions,they were divided into group A(both twins had BPD),group B(only one twin had BPD),and group C(neither twin had BPD).The risk factors for BPD in twin preterm infants were analyzed.Further analysis was conducted on group B to investigate the postnatal risk factors for BPD within twins.Results A total of 904 pairs of twins with a gestational age of<34 weeks were included in this study.The multivariate logistic regression analysis showed that compared with group C,birth weight discordance of>25%between the twins was an independent risk factor for BPD in one of the twins(OR=3.370,95%CI:1.500-7.568,P<0.05),and high gestational age at birth was a protective factor against BPD(P<0.05).The conditional logistic regression analysis of group B showed that small-for-gestational-age(SGA)birth was an independent risk factor for BPD in individual twins(OR=5.017,95%CI:1.040-24.190,P<0.05).Conclusions The development of BPD in twin preterm infants is associated with gestational age,birth weight discordance between the twins,and SGA birth.

Objective:To investigate the correlation between serum albumin, urea nitrogen and Fazekas scores and cognitive function scores in patients with mild and medium ischemic stroke.Methods:Clinical data of 160 patients with acute ischemic stroke with the National Institutes of Health Stroke Scale (NIHSS)≤7 scores admitted to the Department of Neurology of the First Affiliated Hospital of Hainan Medical College from June 2021 to April 2023 were selected for a cross-sectional study. According to the Montreal Cognitive Assessment (MoCA) score, they were divided into normal cognitive group (28 cases) (MoCA≥26 scores), mild to moderate cognitive impairment group (74 cases) (MoCA 15-<26 scores), and severe cognitive impairment group (58 cases) (MoCA<15 scores). Demographic characteristics, serological indicators and imaging data of patients were collected, and the correlation between serum albumin, urea nitrogen and Fazekas scores and the total score of MoCA and the scores of each cognitive domain was analyzed. One-way ANOVA was used for comparison between the normal distribution and homogeneous variance data sets, LSD analysis was used for pairwise comparison, Kruskal-Wallis H test was used between the skew distribution or heterogeneous variance data sets. Bonferroni correction analysis was used for pairwise comparison. Chi-square test or Fisher exact probability method was used after the comparison between the count data sets. Spearman Spearman correlation analysis was performed on serum albumin, urea nitrogen and Fazekas scores with MoCA scores and cognitive domain scores. Multivariate ordered Logistic regression was used to analyze the independent risk factors of cognitive function in acute stage of mild and medium ischemic stroke patients. Results:The incidence of cognitive impairment in patients with acute mild and medium ischemic stroke was 82.50% (132/160). Comparison of age ((56.71±7.35), (60.32±10.20), (66.40±11.88) years old), sex (male/female: (23/5, 58/16, 33/25)), the proportion of education level above high school (25.0%(7/28), 16.2%(12/74), 6.9%(4/58)), hemoglobin ((149.26±14.91), (144.85±16.85), (137.63±17.22) g/L), albumin (39.5 (37.0, 41.2), 38.6(35.6, 40.8), 37.4 (34.5, 39.8) g/L), urea nitrogen (5.30 (4.00, 6.60), 4.81 (4.00, 6.32), 5.86 (4.55, 6.97) mmol/L), Hamilton Anxiety Scale (HAMA) score (5.0 (2.0, 10.0), 7.5 (5.0, 11.0), 10.0 (6.0, 14.3) scores),Hamilton Depression Scale (HAMA) score (5.5 (3.0, 12.5), 7.0 (4.0, 11.0), 9.5 (5.0, 14.0) scores), and Fazekas score (2.00 (1.25, 3.00), 2.00 (1.00, 4.00), 3.00 (2.00, 5.00) scores) among cognitive normal group, mild to moderate cognitive impairment group, and severe cognitive impairment group of patients, the difference were statistically significant (the statistical values were F=9.68, χ 2=9.29, χ 2=30.77, F=5.31, H=7.06, H=6.71, H=12.37, H=8.91, and H=10.96, respectively;the P values were <0.001, 0.010, <0.001, 0.006, 0.029, 0.035, 0.002, 0.012, and 0.004, respectively ). The total score of MoCA was negatively correlated with Fazekas score and serum urea nitrogen, but positively correlated with serum albumin ( r s values were -0.250, -0.168, and 0.212, respectively; P values were 0.001, 0.036, and 0.009, respectively). Serum albumin was positively correlated with scores in visual space and execution, naming, attention and orientation, serum urea nitrogen was negatively correlated with scores in language and orientation, and Fazekas score was negatively correlated with scores in visual space and execution, orientation, attention and language ( r s values were 0.291, 0.196, 0.191, 0.209, -0.205, -0.180, -0.248, -0.193, -0.188, and -0.183, respectively; P values were <0.001, 0.017, 0.020, 0.011, 0.012, 0.027, 0.002, 0.016, 0.020, and 0.023, respectively). Multiple Logistic regression analysis showed that low albumin ( OR=0.884, 95% CI: 0.813-0.963, P=0.005) and high urea nitrogen ( OR=1.195, 95% CI: 1.003-1.425, P=0.047) and high Fazekas scores ( OR=1.401, 95% CI: 1.132-1.733, P=0.002) were independent risk factors for cognitive function, while high education level was a protective factor ( OR=0.062, 95% CI: 0.019-0.202, P<0.001). Conclusion:The incidence of acute cognitive impairment is high in patients with mild and medium ischemic stroke. Higher education level is a protective factor for cognitive function. Low albumin, high urea nitrogen and high Fazekas score are independent risk factors for cognitive function.

Deficiencies in the clearance of peripheral amyloid β (Aβ) play a crucial role in the progression of Alzheimer's disease (AD). Previous studies have shown that the ability of blood monocytes to phagocytose Aβ is decreased in AD. However, the exact mechanism of Aβ clearance dysfunction in AD monocytes remains unclear. In the present study, we found that blood monocytes in AD mice exhibited decreases in energy metabolism, which was accompanied by cellular senescence, a senescence-associated secretory phenotype, and dysfunctional phagocytosis of Aβ. Improving energy metabolism rejuvenated monocytes and enhanced their ability to phagocytose Aβ in vivo and in vitro. Moreover, enhancing blood monocyte Aβ phagocytosis by improving energy metabolism alleviated brain Aβ deposition and neuroinflammation and eventually improved cognitive function in AD mice. This study reveals a new mechanism of impaired Aβ phagocytosis in monocytes and provides evidence that restoring their energy metabolism may be a novel therapeutic strategy for AD.
Animals ; Mice ; Alzheimer Disease ; Amyloid beta-Peptides ; Monocytes ; Cognition ; Energy Metabolism ; Phagocytosis

Objective: To investigate the correlation between CLOCK and BMAL1 genes and MEN2 medullary thyroid carcinoma (MTC). Methods: Thirteen cases with MEN2 MTC and thirteen cases with non-MEN2 MTC were selected who were treated in the Yantai Yuhuangding Hospital between January 2013 and September 2021. Clinical indicators such as blood calcitonin level, tumor diameter and metastatic lymph node of patients were collected. The expression differences of CLOCK and BMAL1 between MEN2 MTC and para-carcinoma tissue as well as between MEN2 MTC and non-MEN2 MTC were detected by immunohistochemistry and qPCR. The correlation between lymph node metastasis and CLOCK or BMAL1 expression was analyzed. Protein-protein interaction (PPI) network analysis combined with qPCR and correlation analysis was used to explore the expression regulation relationship between RET and circadian clock genes. The rhythm disorder of MEN2 cells was verified by lipopolysaccharide cell stimulation experiment after dexamethasone rhythm synchronization. Results: MEN2 MTC exhibited typical RET gene mutation. The mean blood calcitonin level, the tumor diameter and the number of metastatic lymph nodes of patients with MEN2 MTC were higher than those of patients with non-MEN2 MTC (t value was 2.76, 2.53, 2.26, all P<0.05). Immunohistochemical results showed that the expression levels of CLOCK and BMAL1 in MEN2 MTC were higher than those in non-MEN2 MTC, while negatively expressed in para-cancerous thyroid follicle. qPCR displayed that the expression of CLOCK gene in cancer tissues was higher than that in non-MEN2 MTC and para-cancerous tissues (t value was 2.68 and 2.86, all P<0.05); the expression of BMAL1 gene in MEN2 MTC was higher than that in non-MEN2 MTC and para-cancerous tissues (t value was 2.21 and 2.35, all P<0.05). Correlation analysis showed that the expression levels of CLOCK and BMAL1 genes were positively correlated with the number of lymph node metastases in patients with MEN2 MTC (r=0.65, P<0.001; r=0.52, P=0.005). PPI network analysis indicated that the expression of CLOCK gene was positively correlated with the abnormal expression of RET gene (r=0.96, P<0.001). With lipopolysaccharide to stimulate cultured cells in vitro after dexamethasone rhythm synchronization, the expressions of CLOCK and BMAL1 in MEN2 MTC cells (0.47±0.22 and 2.60±1.48) at 12 hours of synchronization were significantly lower than those in para-cancerous tissues (1.70±1.62 and 8.23±2.52), the difference was statistically significant(t=5.04, P=0.007; t=3.34, P=0.029). Conclusion: CLOCK and BMAL1 are correlated with the occurrence and development of MEN2 MTC, and may be potential targets for the development of new therapeutic strategies for MEN2 MTC.
ARNTL Transcription Factors/genetics* ; CLOCK Proteins/genetics* ; Calcitonin ; Carcinoma, Neuroendocrine/genetics* ; Dexamethasone ; Humans ; Lipopolysaccharides ; Lymphatic Metastasis ; Multiple Endocrine Neoplasia Type 2a/genetics* ; Thyroid Neoplasms/surgery*

Antibodies, Monoclonal, Humanized/adverse effects* ; Humans ; Immune Checkpoint Inhibitors/adverse effects* ; Myocarditis/drug therapy*

ObjectiveTo analyze the difference in esophageal microbiome between the patients with esophageal squamous cell carcinoma （ESCC group） and the healthy controls （HC group）， and to screen out the characteristic bacteria in tumor tissues of patients with esophageal squamous cell carcinoma. MethodsEsophageal tissues were collected from 105 patients with esophageal squamous cell carcinoma and 54 healthy controls. The total DNA of the samples was extracted， and the 16S rDNA V4 region was amplified by PCR， followed by high-throughput sequencing. The data were subjected to OTU clustering and diversity analysis to screen the characteristic bacteria in the ESCC group. Finally， the screening results were to be verified by Q-PCR amplification. ResultsDiversity analysis showed that there were differences in diversity and composition between the ESCC group and the HC group. Species analysis showed that the main components of microbiome in the ESCC group were Fusobacterium， Gemella， Neisseria， Porphyromonas and Streptococcus. While in the HC group the main components of microbiome were Fusobacterium， Actinobacillus， Streptococcus and Prevotella. The LEfSe analysis showed that the expression of porphyrin and Fusobacteria in the ESCC group increased and the difference is statistically significant （P<0.05）. The results of Q-PCR showed that the presence of Porphyromonas gingivalis and Fusobacterium nucleatum in the ESCC group significantly increased. ConclusionsEsophageal cancer has its characteristic flora composition. Porphyromonas gingivalis and Fusobacterium nucleatum in esophageal squamous cell carcinoma tumor tissue are the characteristic bacteria.