Objective:To investigate the clinical characteristics and prognostic significance of germline mutations in patients with myelodysplastic neoplasms (MDS) .Methods:Clinical data from 407 patients with MDS [male, 252; female, 155; median age, 64 (range, 19-85) years] diagnosed at the First Affiliated Hospital of Zhejiang University School of Medicine were retrospectively analyzed. The clinical features and prognostic effects of germline mutations were evaluated.Results:The prevalence of germline mutations in patients with MDS was 5.9% (24/407), peaking at 20.0% in the group aged 21-30 years. The spectrum of germline mutations comprised DDX41 (9 cases, 2.2%), TP53 (3 cases, 0.7%), and single cases of RUNX1, TET2, MPL, CBL, ATRX, CEBPA, ETV6, IDH1, KDM5C, SBDS, GNAS, and CTC1. Patients with germline mutations exhibited significantly lower peripheral WBC counts than those without (1.87×10 9/L vs 2.50×10 9/L, P=0.018), but showed comparable median overall survival (21.3 months vs 21.1 months, P=0.97). Patients with DDX41 germline mutations, compared with those with other germline mutations, had a significantly older median age (65 vs 54 years, P=0.010), lower WBC counts (1.51×10 9/L vs 2.31×10 9/L, P=0.040), increased mean corpuscular volume (111.80 fl vs 97.25 fl, P=0.003), and a higher prevalence of normal karyotypes (100.0% vs 53.3%, P=0.022). The most frequently co-occurring somatic mutations in DDX41 germline mutation carriers were ASXL1, TET2, and RUNX1. Conclusion:In this study, the detection rate of germline mutations in MDS patients was 5.9% (24/407), peaking at 20% in the group aged 21-30 years. DDX41 and TP53 were the most prevalent germline mutations. DDX41 mutation carriers displayed distinct clinical characteristics; however, germline mutations overall showed no significant prognostic effect.

BACKGROUND:Chinese herbal prescription with head acupuncture exercise therapy has significant clinical efficacy in the treatment of limb spasticity after ischemic stroke;however,the exact mechanism is not clear.OBJECTIVE:To explore the possible mechanism of the combination of Chinese herbal prescription with head acupuncture exercise therapy in improving limb spasticity after stroke.METHODS:Seventy Sprague-Dawley rats were divided into blank,model,exercise therapy,head-acupuncture,Chinese herbal prescription,Chinese herbal prescription with head acupuncture exercise therapy,and sham-operation groups using the random number method,with 10 rats in each group.Except for the blank group and the sham-operation group,a rat model of cerebral ischemia/reperfusion injury was made by using the middle cerebral artery cable embolism method in the rest of the groups.Interventions in each group started on the 3rd day after modeling.In the Chinese herbal prescription group,rats were given 1.5 g/kg Yi'nao Fujian herbal prescription by gavage;in the head acupuncture group,acupuncture was applied to Baihui and 2 mm on the left and right sides of Baihui,and was left in place for 30 minutes;in the exercise therapy group,rats were exercised on a treadmill for 30 minutes;in the Chinese herbal prescription with head acupuncture exercise therapy group,1.5 g/kg Yi'nao Fujian herbal prescription was given by gavage followed by acupuncture treatment(identical to that in the head acupuncture group,and then the animals were exercised for 30 minutes on the treadmill while the acupuncture needles remained in place.Rats in the model and sham-operation groups were subjected to grasping under the same environmental conditions and placed on a rat immobilizer for 30 minutes,and the blank group did not have any intervention.The frequency of intervention in each group was once a day for 7 consecutive days.The neurological deficit score and muscle tone were evaluated in each group before and after treatment.Pathological changes in rat brain tissue were observed.Protein and mRNA expressions of glutamic acid decarboxylase 67,succinate semialdehyde dehydrogenase,and γ-aminobutyric acid transaminase in the cerebral cortex of the rats were detected.RESULTS AND CONCLUSION:(1)Compared with the model group,the Chinese herbal prescription with head acupuncture exercise therapy group had lower neurological deficit scores and modified Ashworth spasticity scale(MAS)muscle tone scores(P＜0.01),decreased expression of succinate semialdehyde dehydrogenase andγ-aminobutyric acid transaminase at protein and mRNA levels,and increased expression of expression of glutamic acid decarboxylase 67 at protein and mRNA levels(P＜0.01);the neuronal cell structure was relatively intact,with only some irregular cavities visible and reduced nuclear consolidation.(2)Compared with the Chinese herbal prescription with head acupuncture exercise therapy group,the neurological deficit scores and MAS muscle tone scores were higher in the exercise therapy group(P＜0.05),and the MAS muscle tone scores were higher in the head-acupuncture group and the Chinese herbal prescription group(P＜0.05,P＜0.01),while the expressions of glutamic acid decarboxylase 67 protein and succinate semialdehyde dehydrogenase andγ-aminobutyric acid transaminase mRNAs were increased in all the other intervention groups(P＜0.05,P＜0.01).Neuronal cells in the Chinese herbal prescription with head acupuncture exercise therapy group were more orderly arranged and clearly outlined,with less nuclear consolidation and shape irregularity compared with the other intervention groups.(3)The neurological deficit score and MAS muscle tone score in the Chinese herbal prescription with head acupuncture exercise therapy group were significantly lower after treatment(P＜0.01).To conclude,Yi'nao Fujian herbal prescription combined with head-acupuncture exercise therapy can improve post-stroke spasticity by affecting the expression of γ-aminobutyric acid transaminase in rats,which is more effective than Chinese herbal prescription alone,exercise therapy alone,and head-acupuncture alone.

BACKGROUND:Chinese herbal prescription with head acupuncture exercise therapy has significant clinical efficacy in the treatment of limb spasticity after ischemic stroke;however,the exact mechanism is not clear.OBJECTIVE:To explore the possible mechanism of the combination of Chinese herbal prescription with head acupuncture exercise therapy in improving limb spasticity after stroke.METHODS:Seventy Sprague-Dawley rats were divided into blank,model,exercise therapy,head-acupuncture,Chinese herbal prescription,Chinese herbal prescription with head acupuncture exercise therapy,and sham-operation groups using the random number method,with 10 rats in each group.Except for the blank group and the sham-operation group,a rat model of cerebral ischemia/reperfusion injury was made by using the middle cerebral artery cable embolism method in the rest of the groups.Interventions in each group started on the 3rd day after modeling.In the Chinese herbal prescription group,rats were given 1.5 g/kg Yi'nao Fujian herbal prescription by gavage;in the head acupuncture group,acupuncture was applied to Baihui and 2 mm on the left and right sides of Baihui,and was left in place for 30 minutes;in the exercise therapy group,rats were exercised on a treadmill for 30 minutes;in the Chinese herbal prescription with head acupuncture exercise therapy group,1.5 g/kg Yi'nao Fujian herbal prescription was given by gavage followed by acupuncture treatment(identical to that in the head acupuncture group,and then the animals were exercised for 30 minutes on the treadmill while the acupuncture needles remained in place.Rats in the model and sham-operation groups were subjected to grasping under the same environmental conditions and placed on a rat immobilizer for 30 minutes,and the blank group did not have any intervention.The frequency of intervention in each group was once a day for 7 consecutive days.The neurological deficit score and muscle tone were evaluated in each group before and after treatment.Pathological changes in rat brain tissue were observed.Protein and mRNA expressions of glutamic acid decarboxylase 67,succinate semialdehyde dehydrogenase,and γ-aminobutyric acid transaminase in the cerebral cortex of the rats were detected.RESULTS AND CONCLUSION:(1)Compared with the model group,the Chinese herbal prescription with head acupuncture exercise therapy group had lower neurological deficit scores and modified Ashworth spasticity scale(MAS)muscle tone scores(P＜0.01),decreased expression of succinate semialdehyde dehydrogenase andγ-aminobutyric acid transaminase at protein and mRNA levels,and increased expression of expression of glutamic acid decarboxylase 67 at protein and mRNA levels(P＜0.01);the neuronal cell structure was relatively intact,with only some irregular cavities visible and reduced nuclear consolidation.(2)Compared with the Chinese herbal prescription with head acupuncture exercise therapy group,the neurological deficit scores and MAS muscle tone scores were higher in the exercise therapy group(P＜0.05),and the MAS muscle tone scores were higher in the head-acupuncture group and the Chinese herbal prescription group(P＜0.05,P＜0.01),while the expressions of glutamic acid decarboxylase 67 protein and succinate semialdehyde dehydrogenase andγ-aminobutyric acid transaminase mRNAs were increased in all the other intervention groups(P＜0.05,P＜0.01).Neuronal cells in the Chinese herbal prescription with head acupuncture exercise therapy group were more orderly arranged and clearly outlined,with less nuclear consolidation and shape irregularity compared with the other intervention groups.(3)The neurological deficit score and MAS muscle tone score in the Chinese herbal prescription with head acupuncture exercise therapy group were significantly lower after treatment(P＜0.01).To conclude,Yi'nao Fujian herbal prescription combined with head-acupuncture exercise therapy can improve post-stroke spasticity by affecting the expression of γ-aminobutyric acid transaminase in rats,which is more effective than Chinese herbal prescription alone,exercise therapy alone,and head-acupuncture alone.

Objective:To investigate the clinical characteristics and prognostic significance of germline mutations in patients with myelodysplastic neoplasms (MDS) .Methods:Clinical data from 407 patients with MDS [male, 252; female, 155; median age, 64 (range, 19-85) years] diagnosed at the First Affiliated Hospital of Zhejiang University School of Medicine were retrospectively analyzed. The clinical features and prognostic effects of germline mutations were evaluated.Results:The prevalence of germline mutations in patients with MDS was 5.9% (24/407), peaking at 20.0% in the group aged 21-30 years. The spectrum of germline mutations comprised DDX41 (9 cases, 2.2%), TP53 (3 cases, 0.7%), and single cases of RUNX1, TET2, MPL, CBL, ATRX, CEBPA, ETV6, IDH1, KDM5C, SBDS, GNAS, and CTC1. Patients with germline mutations exhibited significantly lower peripheral WBC counts than those without (1.87×10 9/L vs 2.50×10 9/L, P=0.018), but showed comparable median overall survival (21.3 months vs 21.1 months, P=0.97). Patients with DDX41 germline mutations, compared with those with other germline mutations, had a significantly older median age (65 vs 54 years, P=0.010), lower WBC counts (1.51×10 9/L vs 2.31×10 9/L, P=0.040), increased mean corpuscular volume (111.80 fl vs 97.25 fl, P=0.003), and a higher prevalence of normal karyotypes (100.0% vs 53.3%, P=0.022). The most frequently co-occurring somatic mutations in DDX41 germline mutation carriers were ASXL1, TET2, and RUNX1. Conclusion:In this study, the detection rate of germline mutations in MDS patients was 5.9% (24/407), peaking at 20% in the group aged 21-30 years. DDX41 and TP53 were the most prevalent germline mutations. DDX41 mutation carriers displayed distinct clinical characteristics; however, germline mutations overall showed no significant prognostic effect.

Objective:To evaluate the efficacy and safety of hypomethylating agents (HMA) in patients with myelodysplastic syndromes (MDS) .Methods:A total of 409 MDS patients from 45 hospitals in Zhejiang province who received at least four consecutive cycles of HMA monotherapy as initial therapy were enrolled to evaluate the efficacy and safety of HMA. Mann-Whitney U or Chi-square tests were used to compare the differences in the clinical data. Logistic regression and Cox regression were used to analyze the factors affecting efficacy and survival. Kaplan-Meier was used for survival analysis. Results:Patients received HMA treatment for a median of 6 cycles (range, 4-25 cycles) . The complete remission (CR) rate was 33.98% and the overall response rate (ORR) was 77.02%. Multivariate analysis revealed that complex karyotype ( P=0.02, OR=0.39, 95% CI 0.18-0.84) was an independent favorable factor for CR rate. TP53 mutation ( P=0.02, OR=0.22, 95% CI 0.06-0.77) was a predictive factor for a higher ORR. The median OS for the HMA-treated patients was 25.67 (95% CI 21.14-30.19) months. HMA response ( P=0.036, HR=0.47, 95% CI 0.23-0.95) was an independent favorable prognostic factor, whereas complex karyotype ( P=0.024, HR=2.14, 95% CI 1.10-4.15) , leukemia transformation ( P<0.001, HR=2.839, 95% CI 1.64-4.92) , and TP53 mutation ( P=0.012, HR=2.19, 95% CI 1.19-4.07) were independent adverse prognostic factors. There was no significant difference in efficacy and survival between the reduced and standard doses of HMA. The CR rate and ORR of MDS patients treated with decitabine and azacitidine were not significantly different. The median OS of patients treated with decitabine was longer compared with that of patients treated with azacitidine (29.53 months vs 20.17 months, P=0.007) . The incidence of bone marrow suppression and pneumonia in the decitabine group was higher compared with that in the azacitidine group. Conclusion:Continuous and regular use of appropriate doses of hypomethylating agents may benefit MDS patients to the greatest extent if it is tolerated.

This study aims to obtain two whole-genome sequences of enzootic nasal tumor virus of goats(ENTV-2)from Anhui Province and analyzed the genetic diversity of ENTV-2 gene.Nasal secretion samples and blood samples of six goats with enzootic nasal adenocarcinoma(ENA)were collected from a goat farm in Anhui Province.The total RNA was extracted by the TRIzol method.The DNA interference was removed by the two-step reverse transcription.The ENTV-2 was detec-ted by PCR.Then,two positive samples were selected and five pairs of primers were used to ampli-fy the whole-genome sequences of ENTV-2.After sequencing and splicing,two sequences were up-loaded to the database for comparative analysis with the sequences in the NCBI database.Finally,the genetic evolution tree was constructed.ENTV-2 was detected in the nasal secretion samples,but not in the blood of the six ENA goats.The ENTV-2 genes were approximately 7 400 bp in length,named ENTV-2AH1(DDBJ accession no.:LC762616)and ENTV-2AH2(DDBJ accession no.:LC762617),respectively.Two sequences showed 99.2％and 99.1％homology with the Fujian strain(ENTV-2FJ)and Guangxi strain(ENTV-2-DA0),respectively.They were in the same evo-lutionary branch.In this study,two whole-genome sequences of ENTV-2 were obtained in Anhui for the first time,which can help to further study the genetic diversity of ENTV-2 in China.

Objective:To investigate the causal association between immune cell and different types of sepsis by using Mendelian randomization (MR) method, and to find the immune cell phenotypes causally associated with sepsis.Methods:Summary data for various circulating immune cell phenotypes were obtained from the GWAS catalog (GCST90001391-GCST90002121). Sepsis data were sourced from the UK Biobank database. Single nucleotide polymorphisms (SNP) were used as instrumental variables. The correlation threshold of P < 5×10 -6 was used to identify the strongly correlated instrumental variables, and the code was used to remove the linkage disequilibrium and the instrumental variables with F-value < 10. Inverse variance weighting (IVW) was used as the main research method to evaluate the stability and reliability of the results, including Cochran's Q test, MR-Egger regression and Leave one out. Reverse MR analysis was performed based on the immunophenotypic results of the removal of horizontal pleiotropy, and the immune cell phenotype with one-way causal association was obtained. Odds ratio ( OR) and 95% confidence interval (95% CI) were used to represent the effect value of the results. Results:CD16 on CD14 -CD16 + monocyte had horizontal pleiotropy in sepsis ( OR = 0.965?4, 95% CI was 0.933?5-0.998?3, P = 0.039?6). There were five immunophenotypes that had reverse causal associations with the types associated with sepsis. After excluding immune cell phenotypes with horizontal pleiotropy and reverse causation, a total of 42 immune cell phenotypes with sepsis, 36 immune cell phenotypes with sepsis (28-day death in critical care), 32 immune cell phenotypes with sepsis (critical care), 44 immune cell phenotypes with sepsis (28-day death), and 30 immune cell phenotypes had potential causal associations with sepsis (under 75 years old). After false discovery rate (FDR) correction, the correlations between BAFF-R on IgD - CD38br and sepsis (28-day death) were negative and strong ( OR = 0.737?8, 95% CI was 0.635?9-0.856?0, P = 6.05×10 -5, PFDR = 0.044?2). Conclusion:A variety of immune cell phenotypes may have a protective effect on sepsis, especially BAFF-R on IgD - CD38br expression is negatively correlated with sepsis (28-day death), which provides a new idea for immune modulation therapy in sepsis.

Objective:To establish and optimize a detection method of dibutyryl cyclic adenosine phosphate related substances.Methods:On the basis of the JP,the experimental conditions were optimized to obtain the optimal detection conditions.The column was CAPCEILPAK C18;mobile phase was pH 6.0 phosphate buffer solution-meth-anol with gradient elution;detection wavelength was 258 mm.The method validation was carried out.Results:In the system applicability solution,the separation degree of the main peak and adjacent peaks in each destruction solution was greater than 1.5;the solution was stable within 48 h.The content of each impurity showed a good line-ar relationship with the peak area in the range of 50％-150％of the limit concentration,R2＞0.999 4.In the spike experiment,the average recovery rate of N6-butyryl 3',5-cyclophosphatin sodium was 99.4％-106.6％.The average recoveries of 2'-0 butyryl 3,5-cycloadenosine monophosphate sodium were 88.8％-103.6％,and the average recoveries of cyclophosphaminate sodium were 96.9％-102.5％,which met the requirements.Three bat-ches of samples were tested,and the above impurities were detected.Conclusion:This method has high sensitivi-ty,good separation effect,accuracy and durability,and can be used for the detection of bucladesine sodium,provi-ding a basis for its quality control.

Objective To investigate whether advanced glycation end products (AGEs) can induce the expression of Ros,JC-1 and its apoptosis-related proteins in glomerular mesangial cells under high glucose environment,induce apoptosis and injury of glomerular mesangial cells.Methods Rat glomerular mesangial cell line HBZY-1 was cultured in vitro.The cells were cultured with different concentrations of AGEs for 0,12,24 and 48 hours respectively.MTT assay was used to observe the cell proliferation ability.After the optimal time and concentration of AGEs were selected,the caspase enzyme inhibitor Z-VAD-fmk and reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) were cultured and the apoptosis rate was detected by cell death detection apoptosis ELISA plus and Annexin V-FITC/PI kit.JC-1 staining was used to detect the changes of mitochondrial membrane potential (MMP).Cell ROX deep red flow cytometry was used to detect the total ROS level.The expression of anti-apoptotic protein Bcl-2,pro-apoptotic protein BAX,caspase-9,caspase-3 and poly ADP-ribose polymerase (PARP)-activated fragments was detected by Western blotting.Results AGEs could decrease the activity of glomerular mesangial cells in a time and concentration-dependent manner,and induce cell death.The percentage of apoptotic cells in glomerular mesangial cells was significantly increased after treatment with 250 mg/L AGEs for 24 h (P ＜ 0.01),and Z-VAD-fmk could significantly alleviate AGEs-induced glomerular mesangial cell apoptosis (P ＜ 0.01).Compared with the control group,AGEs increased the level of intracellular reactive oxygen species and decreased MMP in a time-dependent manner,and the two time points that AGEs significantly caused the change were 1 h and 2 h (all P ＜ 0.01).AGEs also reduced the expression of antiapoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bax,cleaved caspase-9,cleaved caspase-3 and cleaved PARP (all P ＜ 0.01).Compared with AGEs group,NAC could significantly stabilize MMP (P ＜ 0.01),increase Bcl-2 expression (P ＜ 0.01),and decrease the expression of BAX,cleaved caspase-9,cleaved caspase-3 and cleaved PARP (all P ＜ 0.01).Conclusion AGEs induce mitochondrial pathway apoptosis in glomerular mesangial cells by increasing intracellular ROS level and destroying MMP.

[ ABSTRACT] AIM: To study the protective effect of ischemia preconditioning ( IPC ) on ischemia/reperfusion ( IR)-damaged myocardium in young and old rats .METHODS: Male Wistar rats aged at 3 months ( young ) and 20 months ( old) were used to establish myocardial IPC model and IR model with the method of Langendorff heart perfusion . The rats were divided into young ischemia/reperfusion ( YIR) group, young ischemic preconditioning ( YPC) group, old ischemia/reperfusion ( OIR) group and old ischemic preconditioning ( OPC) group.Transmission electron microscopy was used to observe the ultrastructural changes of myocardial tissue and myocardial mitochondria .The myocardial infarction area was determined by TTC staining .The lactate dehydrogenase ( LDH) content in coronary effluent fluid and the levels of su-peroxide dismutase ( SOD) and malondialdehyde ( MDA) in myocardial tissues were detected by the method of colorimetry . The levels of nitrated and carbonylated proteins in myocardial tissue were measured by ELISA .The myocardial cell apopto-sis was analyzed by TUNEL assay .The mitochondrial respiratory function and mitochondrial permeability transition pore o-pening induced by calcium load were evaluated by oxygen electrode method .RESULTS: Compared with YIR group , the myocardial infarction area in YPC group was obviously smaller , SOD activity in myocardial tissues increased , LDH activity in coronary effluent fluid and the content of MDA decreased , and the levels of nitrated and carbonylated proteins in the car-diac tissues reduced .In YPC group, the mitochondrial membrane structure appeared intact , cristae of the mitochondria showed close arrangement , and the matrix was compressed under the electron microscope .Myocardial mitochondrial respir-atory control rate , state Ⅲoxygen consumption and the P/O ratio in YIR group all significantly increased , proton leak de-creased, mitochondrial swelling induced by calcium distinctly reduced , and myocardial apoptosis rate declined .No signifi-cant difference of the above indexes between OIR group and OPC group was observed .Compared with YPC group , myocar-dial ultrastructural damage increased clearly , cardiac oxidative stress increased , mitochondrial respiratory function de-clined, and cell apoptosis and necrosis increased in OPC group .CONCLUSION:Ischemic preconditioning has protective effect against myocardial IR injury in young rat hearts , while old rat hearts were less sensitive to ischemic preconditioning , leading to bluntness of cardioprotection with IPC in aging hearts .This may be related to mitochondrial injury and severe cel-lular apoptosis caused by increase of cardiac oxidative stress levels in the aging ischemic preconditioning heart .