Objective: To explore the correlation between the composition of bone marrow lymphocyte subsets and the clinical attributes observed in de novo AML patients, as well as their influence on prognosis. Methods: A detailed study was carried out on a cohort of 191 de novo acute myeloid leukemia patients who were admitted to our medical center between October 2022 and September 2024. In addition, a group of 24 patients with iron deficiency anemia individuals was carefully chosen as the control cohort. The proportions of lymphocyte subsets within the bone marrow of de novo AML patients were analyzed. Furthermore, an in-depth analysis was performed to investigate the association between the expression levels of these subsets in de novo AML patients and their clinical attributes, as well as their prognostic implications. Results: The proportion of CD19 and CD56 lymphocytes within the bone marrow of de novo AML patients significantly diminished compared to the control cohort (8.5% vs 13.2% P<0.05, and 15.5% vs 18.0%, P<0.05). Conversely, no significant discrepancies were observed in the CD3 , CD3 CD4 , and CD3 CD8 lymphocyte percentages between the AML patients and control group (71.7% vs 72.1%, 32.5% vs 33.7% and 32.8% vs 35.7%, P>0.05). When analyzing the relationships between lymphocyte subsets within the bone marrow of de novo patients and their respective clinical characteristics, patients aged 60 years and above exhibited diminished percentages of CD3 CD8 lymphocytes in the bone marrow compared to their younger counterparts (31.6% vs 34.1%, P<0.05), while the CD56 lymphocyte subsets demonstrated an increased prevalence (17.2% vs 14.4%, P<0.05). Furthermore, patients with leukocytosis (WBC≥100×10 /L) presented lower levels of CD3 and CD3 CD4 lymphocytes in the bone marrow compared with those without it (65.3% vs 72.9% P<0.05, and 28.9% vs 33.2%, P<0.05), respectively. The AML1-ETO fusion gene-positive cohort exhibited a higher prevalence of CD3 CD8 lymphocytes in the bone marrow than in the negative group (38.2% vs 32.3%, P<0.05), whereas the FLT3-ITD mutation-positive group presented a decreased prevalence of CD56 lymphocytes compared with the negative group (12.4% vs 16.8%, P<0.05). In addition, the NPM1 mutation-positive group demonstrated lower levels of CD3 CD8 lymphocytes in the bone marrow than in the negative group (29.1% vs 33.3%, P<0.05). Variables such as tumor protein p53(TP53) mutation positive, the absence of hematopoietic stem cell transplantation, and CD3 CD4 lymphocyte proportions below 25% were identified as independent adverse prognostic indicators for AML patients (P<0.05). Conclusion: The pathogenesis of AML is closely associated with an imbalance in bone marrow lymphocyte subsets. The FLT3-ITD mutation potentially contributes to the dysregulation of CD56 lymphocyte subset expression. The AML1-ETO fusion gene and NPM1 mutation are implicated in the abnormal expression of CD3 CD8 lymphocytes within the bone marrow. Moreover, the percentage of CD3 CD4 lymphocytes in the bone marrow serves as a prognostic factor for de novo AML patients.

Objective:To analyze the survival outcomes and prognostic factors of patients with ovarian carcino-sarcoma(OCS)based on SEER database.Methods:The data of 1285 OCS patients from 2000 to 2018 in SEER database were retrospectively analyzed.Univariate and multivariate Cox proportional hazards models were used to evaluate the prognostic factors associated with overall survival(OS)and cancer specific survival(CSS).Kap-lan-Meier survival curve was drawn to evaluate the survival analysis of patients' prognosis after clinical treatment.Results:①The study cohort included a total of 1285 OCS patients,The mean age of these patients was 66.21±11.71 years.Most patients had already experienced regional(22.80％)or distant(72.22％)metastasis at the time of diagnosis.②Multivariate Cox regression revealed,SEER stage of regional or distant metastasis,no surger-y,no chemotherapy,and no lymphadenectomy were independent risk factors for both patient OS and CSS(HR＞1,P＜0.05).Age ≥67 years was an independent risk factor for OS(HR＞1,P＜0.05).Age ≥ 83 years was an in-dependent risk factors for CSS(HR＞1,P＜0.05).③Kaplan-Meier survival analysis showed that among surgical patients with adjacent tissue invasion or distant metastasis had significantly better overall survival rate after lymph node dissection than those without(P＜0.001);We didn't see the significantly different effects of lymphadenecto-my on patients with localized disease(P=0.266).Among all patients who underwent surgery,the overall survival rate of all patients who received adjuvant chemotherapy after surgery was significantly better than that of those who did not(P＜0.001).Conclusions:Prognosis of OCS patients is associated with age,SEER comprehensive stage,surgery status,chemotherapy status,lymphadenectomy status.Patients with OCS who underwent cytore-ductive surgery and adjuvant chemotherapy had a better prognosis.However,it is questionable whether lymph-adenectomy is necessary in OCS patients with very early stage.

Objective: To explore the correlation between the composition of bone marrow lymphocyte subsets and the clinical attributes observed in de novo AML patients, as well as their influence on prognosis. Methods: A detailed study was carried out on a cohort of 191 de novo acute myeloid leukemia patients who were admitted to our medical center between October 2022 and September 2024. In addition, a group of 24 patients with iron deficiency anemia individuals was carefully chosen as the control cohort. The proportions of lymphocyte subsets within the bone marrow of de novo AML patients were analyzed. Furthermore, an in-depth analysis was performed to investigate the association between the expression levels of these subsets in de novo AML patients and their clinical attributes, as well as their prognostic implications. Results: The proportion of CD19 and CD56 lymphocytes within the bone marrow of de novo AML patients significantly diminished compared to the control cohort (8.5% vs 13.2% P<0.05, and 15.5% vs 18.0%, P<0.05). Conversely, no significant discrepancies were observed in the CD3 , CD3 CD4 , and CD3 CD8 lymphocyte percentages between the AML patients and control group (71.7% vs 72.1%, 32.5% vs 33.7% and 32.8% vs 35.7%, P>0.05). When analyzing the relationships between lymphocyte subsets within the bone marrow of de novo patients and their respective clinical characteristics, patients aged 60 years and above exhibited diminished percentages of CD3 CD8 lymphocytes in the bone marrow compared to their younger counterparts (31.6% vs 34.1%, P<0.05), while the CD56 lymphocyte subsets demonstrated an increased prevalence (17.2% vs 14.4%, P<0.05). Furthermore, patients with leukocytosis (WBC≥100×10 /L) presented lower levels of CD3 and CD3 CD4 lymphocytes in the bone marrow compared with those without it (65.3% vs 72.9% P<0.05, and 28.9% vs 33.2%, P<0.05), respectively. The AML1-ETO fusion gene-positive cohort exhibited a higher prevalence of CD3 CD8 lymphocytes in the bone marrow than in the negative group (38.2% vs 32.3%, P<0.05), whereas the FLT3-ITD mutation-positive group presented a decreased prevalence of CD56 lymphocytes compared with the negative group (12.4% vs 16.8%, P<0.05). In addition, the NPM1 mutation-positive group demonstrated lower levels of CD3 CD8 lymphocytes in the bone marrow than in the negative group (29.1% vs 33.3%, P<0.05). Variables such as tumor protein p53(TP53) mutation positive, the absence of hematopoietic stem cell transplantation, and CD3 CD4 lymphocyte proportions below 25% were identified as independent adverse prognostic indicators for AML patients (P<0.05). Conclusion: The pathogenesis of AML is closely associated with an imbalance in bone marrow lymphocyte subsets. The FLT3-ITD mutation potentially contributes to the dysregulation of CD56 lymphocyte subset expression. The AML1-ETO fusion gene and NPM1 mutation are implicated in the abnormal expression of CD3 CD8 lymphocytes within the bone marrow. Moreover, the percentage of CD3 CD4 lymphocytes in the bone marrow serves as a prognostic factor for de novo AML patients.

ObjectiveTo compare the contents and relative molecular weight distributions of proteins and polypeptides in Naoxuekang dropping pills， Huoxue Tongmai capsules and Maixuekang capsules of Hirudo single medicinal preparations， to evaluate the in vitro anticoagulant， antiplatelet and fibrinolytic activities of the three preparations， and to investigate the effects of temperature， pH and digestive enzymes on the anticoagulant activities of the three preparations. MethodsThe contents of soluble proteins and polypeptides in the three preparations were determined by bicinchoninic acid assay（BCA） and Bradford method， and the relative molecular weight distributions of the three preparations were determined by electrophoresis combined with gel chromatography. The antithrombin activity of the three preparations was evaluated by fibrinogen-thrombin time（Fibg-TT） method， and their anticoagulant activities were further assessed by the elongations of activated partial thromboplastin time（APTT）， prothrombin time（PT） and thrombin time（TT）. The antiplatelet aggregation activities of the three preparations were measured by turbidimetry and the fibrinolytic activities were measured by fibrin plate method. Relative TT was used as index to investigate the effects of temperature， pH and digestive enzyme buffer on anticoagulant activities of the three preparations. ResultsAt the lowest single dosage， the contents of proteins and polypeptides were in the order of Maixuekang capsules>Huoxue Tongmai capsules>Naoxuekang dropping pills. Both Huoxue Tongmai capsules and Maixuekang capsules had 11 electrophoretic bands between 4.0 kDa and 90 kDa， the bands of Maixuekang capsules were more clear in the range of >25 kDa， and there was 1 obvious band at 14 kDa for the two capsules. Huoxue Tongmai capsules had one specific band at 9.0 kDa and Maixuekang capsules had one specific band at 48.0 kDa. Naoxuekang dropping pills only had 2 electrophoretic bands at 6.5 kDa and 8.5 kDa， primarily containing peptides below 2 kDa， most of which were oligopeptides. The anticoagulant activity concentrations of the three preparations exhibited a certain dose-dependent effect. At the lowest single dosage， The anticoagulant activity concentrations were ranked as Naoxuekang dropping pills>Huoxue Tongmai capsules>Maixuekang capsules. The prolongation effect of the three preparations on coagulation time was dose-dependent. At the same concentration， the prolongation effect of Naoxuekang dropping pills and Huoxue Tongmai capsules was APTT prolongation rate>TT prolongation rate>PT prolongation rate， whereas for Maixuekang capsules， the sequence was TT prolongation rate>APTT prolongation rate>PT lengthening rate. At the single minimum dosage， the order of APTT prolongation rate was Maixuekang capsules>Huoxue Tongmai capsules≈Naoxuekang dropping pills， the order of PT prolongation rate was Naoxuekang dropping pills≈Maixuekang capsules>Huoxue Tongmai capsules， and the order of TT prolongation rate was Maixuekang capsules>Huoxue Tongmai capsules>Naoxuekang dropping pills. The three preparations showed dose-dependent effects on platelet aggregation induced by adenosine diphosphate（ADP） and arachidonic acid（AA）， and the effect induced by ADP was stronger than that induced by AA. The anti-platelet aggregation effect of Naoxuekang dropping pills was significantly stronger than that of Maixuekang capsules（P<0.01）， whereas Huoxue Tongmai capsules had the effect of promoting platelet aggregation. None of the three preparations had the ability to dissolve fibrin. The anticoagulant activity of Naoxuekang dropping pills was least affected by heating， while the activities of the two capsules decreased significantly within 5 min above 80 ℃， and continued to decrease within 2 h. Compared with pure water， the anticoagulant activities of the three preparations could be increased by 1-3 times under strong acidity（pH 1-3）. In the pepsin buffer， the anticoagulant activity of Naoxuekang dropping pills could be increased by 1-3 times， while the anticoagulant activities of Huoxue Tongmai capsules and Maxuekang capsules were significantly decreased， the lowest levels were about 60% and 20%， respectively. In trypsin buffer， the anticoagulant activities of Naoxuekang dropping pills， Huoxue Tongmai capsules and Maixuekang capsules decreased significantly， and the lowest levels decreased to about 41%， 41% and 35%， respectively. ConclusionThe contents of proteins and polypeptides and relative molecular weights of the preparations derived from lyophilized fresh Hirudo powder， dried Hirudo powder and reflux extract of Hirudo decrease sequentially， and the anticoagulant activity decrease gradually， but the anticoagulant pathway is different. And the anti-platelet aggregation activity of the reflux extract is significantly enhanced. The heat resistance and gastrointestinal stability of the three preparations increase successively， and the first two are suitable for enteric-soluble preparations， while the latter is suitable for routine oral administration. The above results can provide data reference for the rationality of different preparation methods， active substances， pharmacodynamics and mechanism of Hirudo preparations.

Objective:To investigate the role of ubiquitin-specific protease 21 (USP21) in enterovirus group A type 71 (EV-A71) infection.Methods:Peripheral blood mononuclear cells (PBMC) were obtained from a cohort of 24 children infected with EV-A71 and 24 healthy children. Expression of USP21 was determined by real-time fluorescence quantitative PCR (qPCR). Additionally, the impact of USP21 overexpression or knockout on EV-A71 replication was evaluated using a combination of qPCR and western blot (WB) analysis. Furthermore, WB was employed to measure the levels of EV-A71 structural protein VP1, phosphorylated interferon regulatory factor 3 (IRF3) and other key molecules in the RIG-I-like receptor (RLR) signaling pathway. Co-immunoprecipitation (Co-IP) was utilized to investigate the effects of USP21 on the ubiquitin levels of EV-A71 nonstructural protein 2A protease (2A pro). Results:In comparison to healthy children, the expression of USP21 mRNA in PBMC of children infected with EV-A71 was notably elevated. The overexpression of USP21 significantly enhanced the cytopathic effects induced by EV-A71, upregulated levels of VP1 mRNA and protein, and facilitated EV-A71 replication, leading to a decrease in cell activity with increasing levels of USP21 transfection. Following the knockout of the USP21 gene, the VP1 mRNA levels were significantly declined in comparison to the control group. Furthermore, the overexpression of USP21 was found to have no impact on the transcriptional activity of EV-A71 2A pro. However, it was observed to enhance the expression of 2A pro protein, reduce the ubiquitination of 2A pro, suppress the protein levels of mitochondrial antiviral signaling protein (MAVS) and melanoma differentiation-associated gene 5 (MDA5), as well as decrease the phosphorylation of IRF3. Additionally, the induction of IFN-β mRNA by EV-A71 infection was downregulated. Conclusions:USP21 has been shown to enhance the replication of EV-A71 through the downregulation of 2A pro ubiquitination, suppression of MAVS and MDA5 protein expression, and inhibition of the interferon signaling pathway.

Objective:To analyze the survival outcomes and prognostic factors of patients with ovarian carcino-sarcoma(OCS)based on SEER database.Methods:The data of 1285 OCS patients from 2000 to 2018 in SEER database were retrospectively analyzed.Univariate and multivariate Cox proportional hazards models were used to evaluate the prognostic factors associated with overall survival(OS)and cancer specific survival(CSS).Kap-lan-Meier survival curve was drawn to evaluate the survival analysis of patients' prognosis after clinical treatment.Results:①The study cohort included a total of 1285 OCS patients,The mean age of these patients was 66.21±11.71 years.Most patients had already experienced regional(22.80％)or distant(72.22％)metastasis at the time of diagnosis.②Multivariate Cox regression revealed,SEER stage of regional or distant metastasis,no surger-y,no chemotherapy,and no lymphadenectomy were independent risk factors for both patient OS and CSS(HR＞1,P＜0.05).Age ≥67 years was an independent risk factor for OS(HR＞1,P＜0.05).Age ≥ 83 years was an in-dependent risk factors for CSS(HR＞1,P＜0.05).③Kaplan-Meier survival analysis showed that among surgical patients with adjacent tissue invasion or distant metastasis had significantly better overall survival rate after lymph node dissection than those without(P＜0.001);We didn't see the significantly different effects of lymphadenecto-my on patients with localized disease(P=0.266).Among all patients who underwent surgery,the overall survival rate of all patients who received adjuvant chemotherapy after surgery was significantly better than that of those who did not(P＜0.001).Conclusions:Prognosis of OCS patients is associated with age,SEER comprehensive stage,surgery status,chemotherapy status,lymphadenectomy status.Patients with OCS who underwent cytore-ductive surgery and adjuvant chemotherapy had a better prognosis.However,it is questionable whether lymph-adenectomy is necessary in OCS patients with very early stage.

Objective:To investigate the role of ubiquitin-specific protease 21 (USP21) in enterovirus group A type 71 (EV-A71) infection.Methods:Peripheral blood mononuclear cells (PBMC) were obtained from a cohort of 24 children infected with EV-A71 and 24 healthy children. Expression of USP21 was determined by real-time fluorescence quantitative PCR (qPCR). Additionally, the impact of USP21 overexpression or knockout on EV-A71 replication was evaluated using a combination of qPCR and western blot (WB) analysis. Furthermore, WB was employed to measure the levels of EV-A71 structural protein VP1, phosphorylated interferon regulatory factor 3 (IRF3) and other key molecules in the RIG-I-like receptor (RLR) signaling pathway. Co-immunoprecipitation (Co-IP) was utilized to investigate the effects of USP21 on the ubiquitin levels of EV-A71 nonstructural protein 2A protease (2A pro). Results:In comparison to healthy children, the expression of USP21 mRNA in PBMC of children infected with EV-A71 was notably elevated. The overexpression of USP21 significantly enhanced the cytopathic effects induced by EV-A71, upregulated levels of VP1 mRNA and protein, and facilitated EV-A71 replication, leading to a decrease in cell activity with increasing levels of USP21 transfection. Following the knockout of the USP21 gene, the VP1 mRNA levels were significantly declined in comparison to the control group. Furthermore, the overexpression of USP21 was found to have no impact on the transcriptional activity of EV-A71 2A pro. However, it was observed to enhance the expression of 2A pro protein, reduce the ubiquitination of 2A pro, suppress the protein levels of mitochondrial antiviral signaling protein (MAVS) and melanoma differentiation-associated gene 5 (MDA5), as well as decrease the phosphorylation of IRF3. Additionally, the induction of IFN-β mRNA by EV-A71 infection was downregulated. Conclusions:USP21 has been shown to enhance the replication of EV-A71 through the downregulation of 2A pro ubiquitination, suppression of MAVS and MDA5 protein expression, and inhibition of the interferon signaling pathway.

Objective:To investigate the expression of DARS2 and its clinical significance in colorectal cancer.Methods:In this study, bioinformatics tools, especially gene expression profile interactive analysis 2 (GEPIA2), were used to conduct an in-depth analysis of DARS2 expression in colorectal cancer tissues. Immunohistochemical staining was carried out in 108 colorectal cancer specimens and 30 normal colorectal tissues obtained from the First Affiliated Hospital of Nanchang University, Nanchang, China. Colorectal cancer cell lines (HCT116 and SW480) were transfected with small interfering RNA (siRNA) and DARS2 overexpression plasmid to examine the effects of DARS2 knockdown and overexpression on cell function. To assess the effects on cell function, CCK8 and transwell migration assays were used to assess proliferation and cell motility, respectively. Additionally, protein immunoblotting was employed to scrutinize the expression of proteins associated with the epithelial-mesenchymal transition of colorectal cancer cells.Results:DARS2 exhibited a pronounced upregulation in expression within colorectal cancer tissues compared to their normal epithelial counterparts. Furthermore, DARS2 expression was higher in colorectal cancer of stage Ⅲ-Ⅳ than those of stage Ⅰ-Ⅱ, exhibiting a significant correlation with N staging, M staging, and pathological staging ( P<0.05). Kaplan-Meier analyses showed a decreased overall survival rate in colorectal cancer with DARS2 expression compared to those without DARS2 expression ( P<0.05). In the siRNA transfection group, there was a significant reduction in cell proliferation and migration ( P<0.01 and P<0.05, respectively). Conversely, the transfection of DARS2 overexpression plasmids substantially increased both cell proliferation and migration ( P<0.05). Additionally, immunoblotting revealed that DARS2 knockdown led to an upregulation of E-cadherin expression and a downregulation of N-cadherin and vimentin expression. In contrast, DARS2 overexpression resulted in increased N-cadherin and vimentin expression, coupled with reduction in E-cadherin expression. Conclusions:There is a strong association between DARS2 expression and colorectal cancer progression. Silencing DARS2 inhibits cell proliferation and migration, exerting a discernible influence on the epithelial-mesenchymal transition process.

Objective:To investigate the role of speckle-type POZ(pox virus and zinc finger protein) protein (SPOP) in enterovirus 71 (EV71) infection.Methods:Immunoprecipitation analysis was employed to examine the impact of SPOP on the ubiquitin level of EV71 non-structural protein 2A protease (2A pro), while the phosphorylation level of IFR3 protein was assessed through Western blot. Cells were either overexpressed or knockdown of SPOP, followed by infection with EV71. RT-qPCR was utilized to analyze the transcription level of IFN-β, and the transcription level and protein level of EV71 structural protein VP1 were determined using RT-qPCR and Western blot, respectively. Results:The inhibition of EV71 infection in RD cells was observed following transfection with HA-SPOP. Additionally, it was found that the ubiquitin level of EV71-2A pro increased in a gradient-dependent manner. Subsequent transfection with shSPOP plasmid for endogenous SPOP knockdown resulted in a dose-dependent decrease in the levels of melanoma differentiation-associated gene 5 (MDA5), mitochondrial antiviral signaling (MAVS), and p-IRF3. Conversely, transfection with HA-SPOP plasmid led to a dose-dependent increase in the levels of MDA5, MAVS, and p-IRF3. The expression of SPOP, whether high or low, had an impact on the expression of IFN-β in cells. Additionally, the levels of VP1 mRNA or protein were found to be inhibited or increased. Conclusions:SPOP plays a role in increasing the ubiquitination level of EV71-2A pro, which in turn promotes the phosphorylation level of IRF3 and secretion of IFN-β. This effect is achieved by inhibiting the cleavage of 2A pro against key molecules MAVS and MDA5 in the RLR signaling pathway, ultimately leading to the inhibition of EV71 replication.

Objective To establish an artificial intelligence (AI)-assisted platform for detection of parasite eggs, and to evaluate its detection efficiency and accuracy, so as to provide technical supports for elimination of parasitic diseases. Methods A total of 1 003 slides of Enterobius vermicularis, horkworm, Trichuris trichiura, Clonorchis sinensis, Taenia, Ascaris lumbricoides, Schistosoma japonicum, Paragonimus westermani and Fasciolopsis buski eggs were collected, and converted into digital images with an automatated scanning microscope to create a dataset. Based on the Object Detection platform on the Baidu Easy DL model, an AI-assisted platform for detection of parasite eggs was created through procedures of uploading, labeling, training, evaluation and optimization. Then, 70% of the datasets were randomly selected for model training, and the precision, recall and average accuracy were calculated to evaluate the effectiveness of platform for recognition of parasite eggs. In addition, the platform was deployed on the computer and smart phone terminals for use. Results An AI-assisted platform for detection of parasite eggs was successfully created. If the platform was deployed using the public cloud application programming interface (API), the average accuracy, precision and recall of the platform were 93.42%, 92.55% and 89.32% for recognition of parasite eggs. If the platform was deployed using the offline software development kit (SDK), the average accuracy, precision and recall of the platform were 92.97%, 94.78% and 87.63% for recognition of parasite eggs. In addition, the precision of the platform was 97.00% and 96.23% for identification of Taenia and C. sinensis eggs, respectively. Conclusions The AI-assisted platform for detection of parasite eggs has been successfully created, which is high in the accuracy for recognition of parasite eggs and convenient in use. This platform may provide a powerful technical support for parasitic disease diagnosis.