ObjectiveTo investigate the potential mechanism of Danggui Buxuetang （DBT） in the treatment of vascular dementia （VAD）. MethodsSixty male SD rats were randomly assigned to the sham-operated group， model group， DBT low-， medium-， and high-dose groups， and the donepezil group. Except for the sham-operated group， rats in all other groups underwent bilateral common carotid artery ligation. After successful modeling， DBT was administered at doses of 9.2， 18.4， 36.8 g·kg^-1 for the low-， medium-， and high-dose groups， respectively， while the donepezil group received 3 mg·kg^-1 donepezil solution by gavage once daily. After 4 consecutive weeks of drug treatment， rats underwent the Morris water maze test， novel object recognition test， Nissl staining to observe hippocampal neurons， and immunofluorescence staining to detect the expression of neuronal nuclear protein （NeuN） in the hippocampus. Western blot was used to assess the expression of PTEN-induced kinase 1 （PINK1）， Parkin， microtubule-associated protein 1 light chain 3Ⅱ （LC3Ⅱ）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax）. Transmission electron microscopy was used to observe hippocampal neuronal ultrastructure. Real-time PCR was used to detect the expression of NADPH oxidase subunits p22^phox and p47^phox in hippocampal tissues. The levels of malondialdehyde （MDA）， glutathione （GSH）， superoxide dismutase （SOD）， and total antioxidant capacity were measured to evaluate oxidative stress levels. ResultsIn the Morris water maze test， escape latency changed significantly over time in all groups except the model group. Compared with the sham-operated group， the model group showed significantly prolonged escape latency （P<0.01）. Compared with the model group， rats in the DBT groups and the donepezil group exhibited significantly shorter escape latency （P<0.05， P<0.01）. The number of crossings over the original platform was significantly reduced in the model group compared with the sham-operated group （P<0.01）， whereas rats in the DBT and donepezil groups showed significantly increased platform crossings compared with the model group （P<0.05， P<0.01）. Compared with the sham-operated group， exploration time of new objects was significantly reduced in the model group （P<0.01）. Compared with the model group， exploration time of new objects increased significantly in the medium- and high-dose DBT groups and the donepezil group （P<0.05， P<0.01）， while no significant change was observed in the low-dose DBT group. Compared with the high-dose DBT group， rats in the donepezil group had significantly prolonged escape latency and reduced platform crossings and new-object exploration time （P<0.05）. Nissl staining showed decreased density of healthy neurons in the CA1 and CA3 regions of the hippocampus in the model group， with loss of Nissl bodies and nuclear atrophy or disappearance. In the high-dose DBT group， neuronal density in CA1 and CA3 increased， with neurons arranged closely and displaying normal morphology. Immunofluorescence showed that compared with the sham-operated group， the hippocampal NeuN⁺ cell count in the VAD model group was significantly decreased（P<0.01）， compared with the VAD model group， the hippocampal NeuN⁺ cell count in the high-dose DBT group was significantly increased（P<0.01）. Compared with the sham-operated group， the expression of PINK1， Parkin， LC3Ⅱ， and Bax proteins was significantly increased（P<0.01）， while the expression of Bcl-2 was significantly decreased in the VAD model group（P<0.01）. Compared with the VAD model group， the high-dose DBT group showed significantly decreased expression of PINK1， Parkin， LC3Ⅱ， and Bax proteins（P<0.01）and significantly upregulated Bcl-2 expression（P<0.01）. The medium-dose DBT group exhibited significantly reduced expression of Parkin， LC3Ⅱ， and Bax proteins（P<0.05，P<0.01） and significantly increased Bcl-2 expression（P<0.01）， while no statistically significant differences were observed in the low-dose DBT group. Transmission electron microscopy showed mitochondrial pyknosis， thickened cristae， increased electron density， and the presence of mitochondrial autophagy in the model group. In contrast， hippocampal neurons in the high-dose DBT group contained abundant mitochondria with intact morphology， clear cristae， and uniform matrix. Compared with the sham-operated group， total antioxidant capacity， SOD activity， and GSH levels were significantly decreased， while MDA levels were significantly increased in the model group （P<0.01）. Compared with the model group， total antioxidant capacity and antioxidant levels （SOD， GSH） increased significantly， and MDA decreased significantly in the medium- and high-dose DBT groups （P<0.01）， while no significant changes were observed in the low-dose DBT group. Compared with the sham-operated group， mRNA expression of p22^phox and p47^phox was significantly increased in the model group （P<0.01）. Compared with the model group， expression of p22^phox and p47^phox was significantly decreased in the DBT groups （P<0.05， P<0.01）. ConclusionDBT may exert neuroprotective effects by regulating PINK1/Parkin-mediated mitochondrial autophagy， thereby improving learning and memory abilities and treating VAD.

Objective:To report the blood group antigen and antibody specificity identification methods for a patient with high-frequency antibodies, and the process of finding and providing compatible blood for the patient.Methods:A patient sent from the Blood Transfusion Department of Shanxi Provincial People′s Hospital to Taiyuan Blood Center in November 2022 was selected for the study. Classical serological methods were used to determine the patient′s blood type, screen for unexpected antibodies, identify antibodies, and perform crossmatching. High-frequency antibody identification was carried out using red blood cells treated with various enzymes. Blood group genotyping was conducted using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) and Sanger sequencing. Multiple strategies were employed to address the patient′s blood source problem. The study was approved by the Medical Ethics Committee of Taiyuan Blood Center [Ethics No. 2024 Ethics Review No.(2)].Results:①The patient′s blood type was B, RhD positive. Initial screening of the patient′s serum with multiple screening cells and antibody identification cells in saline medium was negative, but positive in antiglobulin medium. The patient′s serum showed varying reaction intensities with red blood cells treated with different enzymes. ②MALDI-TOF mass spectrometry and Sanger sequencing revealed a homozygous nonsense variant c. 376C>T (p.Gln126Ter) in the ABCG2 gene, resulting in the Jr(a-) phenotype. During family donor selection, the patient′s son was found to have a heterozygous variant c. 376C>T (p.Gln126Ter), and another heterozygous variant c. 421C>A (p.Gln141Lys), which predicted a Jr(a+ w) phenotype. ③Crossmatch tests confirmed the compatibility of blood from the patient′s son, which was used to address the urgent blood requirement. Later, rare blood from a Jr(a-) donor from the Guangzhou Blood Center was used for the patient′s ongoing treatment, saving the patient′s life. Conclusion:Combining classic serological testing with blood group gene typing techniques successfully identified the rare Jr(a-) blood type and high-frequency anti-Jra antibodies. Enzyme-treated red blood cell identification methods confirmed the presence of anti-Jra antibodies. By searching within the family and seeking help from other blood centers, compatible blood was found. This approach may provide insights for resolving similar complex blood matching problems in the future.

Objective:To establish a rapid and effective melanin-bleaching method to standardize and improve the molecular pathological analyses of melanoma.Methods:Fifteen cases of melanoma with melanin content exceeding 50% collected at the Precision Pathology Diagnosis Center, Weifang People′s Hospital, Weifang, China between 2023 and 2024 were included in the study. These tissue samples were subject to melanin-bleaching treatments using H 2O 2, potassium permanganate (KMnO 4), Tris-HCl or PBS method. The bleaching effects in each group were evaluated using hematoxylin and eosin (HE) staining. Thereafter, the total amount, purity, and integrity of DNA extracted from bleached tissues were analyzed using UV spectrophotometry and Qsep1 Bio-fragment analyzer. Finally, in order to compare the efficiency of DNA amplification, C-KIT and PDGFRA were examined using Sanger sequencing, and BRAF was detected using real-time quantitative PCR. Results:Among the 15 cases, there were seven males and eight females whose median age was 68 (range, 63 to 71) years. Bleached tissue in Tris-HCl group had the highest HE score, which was significantly higher than those in the groups of PBS, H 2O 2, KMnO 4, and control ( F=113.3, P<0.05). The total DNA amount in the control and Tris-HCl groups was significantly higher than those in the groups of PBS, H 2O 2, and KMnO 4, respectively ( F=275, P<0.05). Meanwhile, the mean A260/ A280 values of DNA obtained from bleached tissue in Tris-HCl and KMnO 4 were better than that of control, and the mean A260/ A230 values of Tris-HCl and KMnO 4 were higher than that of control, PBS, and H 2O 2. Meanwhile, the proportion of >5 000 bp,>3 000 bp, and >1 000 bp DNA fragments in Tris-HCl was all significantly higher than those in other groups ( F=147.9, F=127.9 and F=61.9, respectively, P<0.05). C-KIT and PDGFRA genes were both successfully amplified based on DNA obtained from bleached tissue in Tris-HCl group, and the sequencing peaks were clear and free of noticeable noises. Real-time quantitative PCR results showed that Tris-HCl method had lower cycle threshold values than other methods in detecting BRAF gene ( F=30.36, P<0.05). Conclusions:Tris-HCl method is a fast and effective melanin-bleaching method for analyzing melanoma, which could remove melanin effectively, confirm high quality of DNA and good integrity of DNA, and improve the amplification efficiency of sequencing for melanoma related genes. This new method may help pathologists with reliable molecular typing and identifying therapeutic targets for melanoma. It may greatly improve clinical management of melanoma patients.

Objective:To explore the regulatory mechanisms underlying the weak expression of ABO blood group antigens due to variants in the non-coding regions of the ABO gene. Methods:From June 2014 to October 2023, a total of 29 samples from the Taiyuan Blood Center and local hospitals, which were serologically identified as having weak ABO antigen expression without detectable coding region mutations, were selected for this study. Full-length ABO gene sequencing was performed using third-generation long-read sequencing technology (Pacific Biosciences) to obtain complete haplotype sequences of the ABO gene. Variants in the non-coding regions were compared and identified to infer their regulatory effects on weak antigen expression. The procedures followed in this study were in accordance with the ethical standards of the World Medical Association′s Declaration of Helsinki (2013 revision). The Medical Ethics Committee of Taiyuan Blood Center has granted an exemption from ethical review. Results:18 bp deletions in the -35 to -18 region of the promoter were identified in 7 samples. Variants in intron 1 (+ 5.8 kb) were detected in 7 samples, including ABO* A (28+ 5792_5793delCT (1 case) and ABO* B (28+ 5793T>C) located in the GATA binding region; ABO* B (28+ 5808C>T) (1 case) in the E-box region; and ABO* B (28+ 5875C>T) (4 cases) in the RUNX1 binding region. Nucleotide variants at splice sites were detected in 2 samples, namely ABO* B (C.98+ 1G>A) and ABO* B (C.204-2A>C). Conclusion:Variants in the non-coding regulatory sequences of the ABO gene are a significant factor contributing to weak ABO antigen expression. In clinical ABO sequencing, it is essential to screen not only the conventional coding regions but also the flanking sequences, introns, and splice sites of the ABO gene to facilitate precise blood transfusion.

Parkinson’s disease （PD） is a common chronic neurodegenerative disease with movement disorders as the main clinical manifestation. The nuclear factor-erythroid 2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1） signaling pathway is closely associated with the occurrence and progression of PD. TCM flavonoid monomers （luteolin， rutin， etc.）， alkaloids （camptothecin， sinomenine， and alkaloids extracted from Uncaria rhynchophylla）， terpenes （tanshinone ⅡA， carvacrol， paeoniflorin）， phenols （ellagic acid， rosmarinic acid）， saponins （ginsenoside RK1）， and traditional Chinese medicine compounds （Wuzi yanzong pill and PD formula-2） can resist oxidative stress damage， inhibit inflammatory responses and abnormal aggregation of α-synuclein， and regulate neurotrophic factors by activating the Nrf2/HO-1 signaling pathway， thereby alleviating dopaminergic neuronal damage.

Objective: To identify the phenotypes, antibodies and explore the molecular mechanisms of a patient who carries antibodies to RBC high-frequency antigens and his family members. Methods: The antibody identification test was performed for the proband by serological methods, and targeted NGS was subsequently used to detect mutations that occurred in blood group genes. Blood samples were collected from the proband and his family members. Sanger sequencing was used to verify the mutation of the XK gene. The expression of Kell blood group antigens was detected by serological methods and flow cytometry. K cells were used to detect the antibody specificity of the proband. The morphology of red blood cells was detected by the scanning electron microscopy. The serum creatine kinase levels of the proband and his family members were analyzed by colorimetric methods. Results: The results of the antibody identification test suggested that the proband might have antibodies to high-frequency antigens. NGS results suggested a homozygous mutation (c. 107G>A) in exon 1 of the XK gene in the proband, resulting in a truncated XK protein. The Sanger sequencing results of the proband were consistent with the NGS results, and the mutation was not found in other family members. The expression of Kell blood group antigens of the proband was not found by serological methods and flow cytometry. The results of the antibody specificity test showed that the proband had anti-Km antibodies. Spike-like changes were identified on red blood cells, and serum creatine kinase level was elevated in the proband. Conclusion: In this study, the McLeod phenotype caused by homozygous mutation (c. 107G>A) of XK gene was identified in Chinese individuals for the first time by the phenotype and molecular mechanism studies. The results of genotyping and phenotyping suggested that the McLeod phenotype caused by the mutation was compatible with the phenotypes of McLeod and K .

OBJECTIVE: To report the blood group antigen and antibody specificity identification methods for a patient with high-frequency antibodies, and the process of finding and providing compatible blood for the patient. METHODS: A patient sent from the Blood Transfusion Department of Shanxi Provincial People's Hospital to Blood Transfusion Technology Research Laboratory of Taiyuan Blood Center in November 2022 was selected for the study. Classical serological methods were used to determine the patient's blood type, screen for unexpected antibodies, identify antibodies, and perform crossmatching. High-frequency antibody identification was carried out using red blood cells treated with various enzymes. Blood group genotyping was conducted using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) and Sanger sequencing. Multiple strategies were employed to address the patient's blood source problem. The study was approved by the Medical Ethics Committee of Taiyuan Blood Center [Ethics No. 2024 Ethics Review No.(2)]. RESULTS: The patient's blood type was B, RhD positive. Initial screening of the patient's serum with multiple screening cells and antibody identification cells in saline medium was negative, but positive in antiglobulin medium. The patient's serum showed varying reaction intensities with red blood cells treated with different enzymes. MALDI-TOF mass spectrometry and Sanger sequencing revealed a homozygous nonsense variant c.376C>T (p.Gln126Ter) in the ABCG2 gene, resulting in the Jr(a-) phenotype. During family donor selection, the patient's son was found to have a heterozygous variant c.376C>T (p.Gln126Ter), and another heterozygous variant c.421C>A (p.Gln141Lys), which predicted a Jr(a+w) phenotype. Crossmatch tests confirmed the compatibility of blood from the patient's son, which was used to address the urgent blood requirement. Later, rare blood from a Jr(a-) donor from the Guangzhou Blood Center was used for the patient's ongoing treatment, saving the patient's life. CONCLUSION Combining classic serological testing with blood group gene typing techniques successfully identified the rare Jr(a-) blood type and high-frequency anti-Jra antibodies. Enzyme-treated red blood cell identification methods confirmed the presence of anti-Jra antibodies. By searching within the family and seeking help from other blood centers, compatible blood was found. This approach may provide insights for resolving similar complex blood matching problems in the future.
Humans ; Blood Grouping and Crossmatching/methods* ; Blood Group Antigens/immunology* ; Blood Transfusion ; Male ; Isoantibodies/blood* ; Female ; Genotype

OBJECTIVE: To explore the regulatory mechanisms underlying the weak expression of ABO blood group antigens due to variants in the non-coding regions of the ABO gene. METHODS: From June 2014 to October 2023, a total of 29 samples from the Taiyuan Blood Center and local hospitals, which were serologically identified as having weak ABO antigen expression without detectable coding region mutations, were selected for this study. Full-length ABO gene sequencing was performed using third-generation long-read sequencing technology (Pacific Biosciences) to obtain complete haplotype sequences of the ABO gene. Variants in the non-coding regions were compared and identified to infer their regulatory effects on weak antigen expression. The procedures followed in this study were in accordance with the ethical standards of the World Medical Association's Declaration of Helsinki (2013 revision). The Medical Ethics Committee of Taiyuan Blood Center has granted an exemption from ethical review. RESULTS: 18 bp deletions in the -35 to -18 region of the promoter were identified in 7 samples. Variants in intron 1 (+5.8 kb) were detected in 7 samples, including ABO*A (28+5792_5793delCT (1 case) and ABO*B (28+5793T>C) located in the GATA binding region; ABO*B (28+5808C>T) (1 case) in the E-box region; and ABO*B (28+5875C>T) (4 cases) in the RUNX1 binding region. Nucleotide variants at splice sites were detected in 2 samples, namely ABO*B (C.98+1G>A) and ABO*B (C.204-2A>C). CONCLUSION Variants in the non-coding regulatory sequences of the ABO gene are a significant factor contributing to weak ABO antigen expression. In clinical ABO sequencing, it is essential to screen not only the conventional coding regions but also the flanking sequences, introns, and splice sites of the ABO gene to facilitate precise blood transfusion.
ABO Blood-Group System/genetics* ; Humans ; Alleles ; Promoter Regions, Genetic ; Haplotypes ; Introns

Objective:To analyze the electroencephalogram features of visual working memory in the delay stage based on microstate analysis.Methods:In this cross-sectional study, ten healthy male subjects aged 19-29 years old from Tianjin Medical University were selected from January to July 2015. The 32-channel electroencephalogram experimental data were recorded from these subjects during the visual working memory eye-open state for 10 min (resting-state) and the delayed matching-to-sample (DMS) task (task-state) conditions, respectively. After data pre-processing, a short-time Fourier transform was applied for time-frequency analysis to extract the characteristic time periods and frequency bands, and electroencephalogram microstates of the resting and task states of visual working memory were computed separately, using an improved k-means clustering algorithm. The Mann-Whitney U test was used to analyze the statistical differences between resting-state and task-state microstate parameters, including global field power (GFP), global explained variance (GEV), time proportion, mean spatial correlation, frequency of occurrence and duration. Results:The time-frequency distribution of the Fz channel in the responsible brain region in DMS task visual working memory showed that the energy was mainly concentrated in the theta band (4-8 Hz) during the delay stage (4.039-7.039 s). The bipolar locations of microstates A, B, C, and D were prefrontal and occipital, central frontal and occipital, right occipital and left frontal, and left occipital and right frontal, respectively. In microstates A and C, the mean spatial correlation of the task-state was both higher than that of the resting state. The comparison results for microstate A were (0.632 5±0.105 8 vs 0.624 5±0.461 4, P<0.05), and the results for microstate C were (0.589 4±0.233 4 vs 0.561 3±0.066 8, P<0.01). The parameters of the task-state microstate D were lower than those of the resting-state, including GFP (1.125±0.061 vs 1.277±0.741, P<0.05), GEV (0.077±0.061 vs 0.101±0.057, P<0.05), and time proportion (0.191±0.165 vs 0.224±0.094, P<0.05) and mean spatial correlation (0.561 2±0.211 6 vs 0.612 1±0.315 2, P<0.01). Conclusions:The critical period of visual working memory is the delay stage and the critical frequency band is the theta band. Microstate D is the characteristic feature of visual working memory in the delay stage of the theta band, and microstates A and C can also serve as its potential features.

ObjectiveCardiovascular diseases pose a major public health challenge in China. The burden of cardiovascular disease associated with high low-density lipoprotein cholesterol （LDL-C） has increased steadily nationwide. A comprehensive analysis of secular trends in cardiovascular disease burden and its determinants is crucial for developing targeted interventions and evidence-based health policies. MethodsBased on data from the Global Burden of Disease Study 2021， we analyzed the trends in deaths， disability-adjusted life years （DALYs）， age-standardized mortality rates （ASMR）， and age-standardized DALY rates （ASDR） of cardiovascular diseases attributable to high LDL-C in China from 1990 to 2021 using Joinpoint regression analysis. An age-period-cohort model was applied to assess the contributions of age， period， and cohort effects to changes in the cardiovascular disease burden attributable to high LDL-C. Projections of the high LDL-C-attributable cardiovascular disease burden in China from 2022 to 2030 were generated using a Bayesian age-period-cohort model. ResultsBetween 1990 and 2021， China saw a substantial rise in both deaths and disability-adjusted life years （DALYs） from cardiovascular disease linked to high LDL-C. Joinpoint regression revealed key turning points in this trend： an overall increase continued until 2004， after which the burden began to fall starting in 2011. Throughout this period， age-standardized mortality and DALY rates were consistently higher in males than in females. Age-period-cohort analysis further indicated that mortality and DALY rates due to high LDL-C increased almost exponentially with age， while period and cohort risks generally decreased over time. Projections suggest a continued decline in age-standardized mortality rates from LDL-C-related cardiovascular disease for both Chinese men and women by 2030. ConclusionRapid population growth and accelerated ageing in China emerge as primary drivers of the escalating cardiovascular diseases burden linked to elevated LDL-C. The burden of cardiovascular diseases is higher in men compared to women. By 2030， the burden of cardiovascular disease caused by high LDL-C in China will remain severe. These findings underscore the critical need for gender-specific screening protocols， age-tailored interventions， and personalized management frameworks to mitigate this public health challenge.