OBJECTIVE: To explore the application of targeted mRNA sequencing in fusion gene diagnosis of hematologic diseases. METHODS: Bone marrow or peripheral blood samples of 105 patients with abnormally elevated eosinophil proportions and 291 acute leukemia patients from January 2015 to June 2023 in the First Affiliated Hospital of Soochow University were analyzed and gene structural variants were detected by targeted mRNA sequencing. RESULTS: Among 105 patients with abnormally elevated eosinophil proportions, 6 cases were detected with gene structural variants, among which fusion gene testing results in 5 cases could serve as diagnostic indicators for myeloid neoplasms with eosinophilia. In addition, a IL3∷ETV6 fusion gene was detected in one patient with chronic eosinophilic leukemia, not otherwise specified. Among 119 patients with acute myeloid leukemia (AML), 38 cases were detected structural variants by targeted mRNA sequencing, accounting for 31.9%, which was significantly higher than 20.2% (24/119) detected by multiple quantitative PCR (P < 0.05). We also found one patient with AML had both NUP98∷PRRX2 and KCTD5∷JAK2 fusion genes. A total of 104 patients were detected structural variants by targeted mRNA sequencing in 172 cases with acute B-lymphoblastic leukemia who were tested negative by multiple quantitative PCR, with a detection rate of 60.5% (102/172). CONCLUSION Targeted mRNA sequencing can effectively detect fusion gene and has potential clinical application value in diagnosis and classificatation in hematologic diseases.
Humans ; Hematologic Diseases/diagnosis* ; RNA, Messenger/genetics* ; Oncogene Proteins, Fusion/genetics* ; Sequence Analysis, RNA ; Leukemia, Myeloid, Acute/diagnosis*

Objective:To evaluate the clinical efficacy of individualized thrombolysis-assisted comprehensive intervention for deep vein thrombosis (DVT) in the lower limbs.Methods:This study included 32 patients with acute lower limb DVT diagnosed by angiography who received treatment at the Jianhu Clinical Medical College of Yangzhou University from March 2012 to November 2021. These patients first received implantation of an inferior vena cava filter. Then they were divided into a control group and an observation group based on treatment methods. The control group received thrombolytic catheterization and a routine infusion of urokinase. In the observation group, balloon dilation was performed first, and a large lumen catheter was used to draw blood clots. Subsequently, urokinase at a dose based on fibrinogen measurement was injected through a thrombolytic catheter. Swelling reduction, venous patency, and complications of the affected limbs were monitored.Results:In the control group, the difference in thigh circumference before treatment was (4.65 ± 1.06) cm, and after treatment, it was (2.76 ± 1.25) cm. In the observation group, the difference in thigh circumference before treatment was (4.73 ± 1.03) cm, and it was (1.40 ± 0.83) cm after treatment. In the control group, the difference in calf circumference before treatment was (2.24 ± 0.90) cm, and it was (1.56 ± 0.86) cm after treatment. In the observation group, the difference in calf circumference before treatment was (2.40 ± 0.83) cm, and it was (0.80 ± 0.73) cm after treatment. After treatment, the differences in thigh circumference and calf circumference between the healthy and affected sides were statistically significant ( t = 3.58, 2.67, both P < 0.05). After treatment, there was a significant difference in venous patency between the control and observation groups (34.02% [33/97] vs. 68.18% [60/88], t = 3.44, P < 0.05). After 12 months of follow-up, the Villalta scale score, which was used to evaluate post-thrombotic syndrome, was (9.23 ± 4.07) points in the control group, which was significantly different from (5.73 ± 3.39) points in the observation group ( t = 2.62, P < 0.05). Conclusion:Individualized thrombolysis-assisted comprehensive intervention is highly effective in the treatment of DVT in the lower limbs and results in few complications.

ObjectiveTo explore the correlation between skeletal muscle mass and metabolic syndrome （MS） disease risk among middle-aged and elderly community residents in Urumqi， and to provide a theoretical basis for understanding the relationship between skeletal muscle mass and MS among middle-aged and elderly community residents in China. MethodsA total of 1 438 community residents ≥ 50 years old were selected as the research subjects from July 2018 to January 2019 in Urumqi. They were selected from a multi-ethnic natural population cohort in Xinjiang. Data were collected through questionnaires， physical examination， bioelectrical impedance analysis （BIA）， laboratory tests， etc. Skeletal muscle mass was evaluated using the limb skeletal muscle mass index （SMI） corrected for body weight； MS was defined as it at least includes three of the following： abdominal obesity， hypertension， hyperglycemia， high triglycerides and low high-density lipoprotein cholesterol. SMI was divided into four quantile arrays of Q₁‒Q₄. Trend χ² test was applied to explore whether there was a correlation between SMI changes and MS. A multivariate logistic regression model was used to analyze whether there is a difference in the risk of MS between the higher SMI group （Q₂， Q₃， Q₄） and the reference group Q₁. ResultA total of 560 MS patients were detected in this study， with a prevalence rate of 38.94%. Among them， the prevalence rate of MS was 39.16% in males and 38.80% in females. The increase in male SMI grading level is not correlated with the prevalence of MS （trend P>0.05）； After adjusting for confounding factors （model 4）， the increase in SMI was still not related to the prevalence of MS （P_trend=0.995）. There was no statistical difference in the risk of MS between the lowest quartile group Q₁ and the highest quartile group Q₄ （OR=1.01， 95%CI： 0.69‒1.78）. The prevalence of MS in women gradually decreased with the increase of SMI grading level （P_trend<0.001）； After adjusting for confounding factors （model 4）， there was still a correlation between the increase of SMI and the prevalence of MS （P_trend=0.005）. With the lowest quartile of SMI Q₁ as the reference group， the risk of MS in Q₂ （OR=0.63， 95%CI： 0.40‒1.00）， Q₃ （OR=0.56， 95%CI： 0.34‒0.94）， Q₄ （OR=0.42， 95%CI： 0.23‒0.76） decreased. ConclusionAn increase in skeletal muscle mass may be beneficial for preventing MS， especially among middle-aged and elderly female residents. Considering the intensification of aging in China and the close relationship between MS and related comorbidities， managing skeletal muscle mass may contribute to potential MS prevention.

Temporomandibular joint (TMJ) diseases are common clinical conditions. The number of patients with TMJ diseases is large, and the etiology, epidemiology, disease spectrum, and treatment of the disease remain controversial and unknown. To understand and master the current situation of the occurrence, development and prevention of TMJ diseases, as well as to identify the patterns in etiology, incidence, drug sensitivity, and prognosis is crucial for alleviating patients′suffering.This will facilitate in-depth medical research, effective disease prevention measures, and the formulation of corresponding health policies. Cohort construction and research has an irreplaceable role in precise disease prevention and significant improvement in diagnosis and treatment levels. Large-scale cohort studies are needed to explore the relationship between potential risk factors and outcomes of TMJ diseases, and to observe disease prognoses through long-term follw-ups. The consensus aims to establish a standard conceptual frame work for a cohort study on patients with TMJ disease while providing ideas for cohort data standards to this condition. TMJ disease cohort data consists of both common data standards applicable to all specific disease cohorts as well as disease-specific data standards. Common data were available for each specific disease cohort. By integrating different cohort research resources, standard problems or study variables can be unified. Long-term follow-up can be performed using consistent definitions and criteria across different projects for better core data collection. It is hoped that this consensus will be facilitate the development cohort studies of TMJ diseases.

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 investigate the effects of zalcitabine(ddC),a mitochondrial DNA polymerase γ(POLG)inhibitor,on the migration,invasion,and to preliminarily explore mitochondrial biogenesis of human tri-ple-negative breast cancer MDA-MB-231 cells.Methods The effect of ddC on cell viability was detected using the MTT assay.The migration and invasion abilities of the cells were evaluated using the cell scratch and Transwell in-vasion assays.Cell apoptosis was determined using flow cytometry and a V-FITC/PI cell apoptosis detection kit.The protein expression of POLG,NADH dehydrogenase subunit Ⅰ(NADH1),NADH dehydrogenase subunit Ⅱ(NADH2),ATP synthase subunit 6(ATPase6),cytochrome c oxidase subunit Ⅰ(COX-1)and cytochrome c ox-idase subunit Ⅲ(COX-3)were determined using Western blot.The POLG mRNA level and mtDNA copy number were determined using qPCR.The mitochondrial content and ATP levels were determined using MitoTracker Green fluorescent probe staining and an ATP determination kit.MDA-MB-231 cells were transfected with pcDNA3.1-EG-FP-POLG plasmids to overexpress POLG.The inhibitory effects of ddC on cell migration and invasion were detected in POLG-overexpressed MDA-MB-231 cells.Results POLG expression was higher in MDA-MB-231 cells than in normal mammary epithelial cells(MCF-10A)(P＜0.01).ddC inhibited cell viability in a dose-dependent man-ner.ddC inhibited the migration(P＜0.01)and invasion(P＜0.01)of MDA-MB-231 cells;however,it dis-played no significant inhibitory effects on cell viability in normal mammary epithelial cells(MCF-10A)at the same concentration.ddC downregulated the protein(P＜0.01)and mRNA(P＜0.01)levels of POLG,reduced mtD-NA copy number(P＜0.01)and downregulated mtDNA-coded NADH1,NADH2,ATPase6,COX-1 and COX-3 protein expression(P＜0.01)in MDA-MB-231 cells.Furthermore ddC inhibited mitochondrial content(P＜0.01)and ATP(P＜0.01)levels in MDA-MB-231 cells.POLG overexpression increased the migration(P＜0.05)and invasion(P＜0.05)abilities of MDA-MB-231 cells,while ddC did not significantly inhibit the migra-tion and invasion abilities of MDA-MB-231 cells overexpressing POLG.Conclusion ddC downregulates POLG ex-pression in MDA-MB-231 cells and inhibits mitochondrial biogenesis and ATP levels,thereby inhibiting the migra-tion and invasion of MDA-MB-231 cells.

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannan-binding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP. Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays. Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05). Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

Objective:To understand the prevalence of major chronic diseases of diabetes, cardiovascular disease and malignant tumor in people living with HIV in Taizhou.Methods:The data were collected from China Information System for Disease Control and Prevention and Taizhou Chronic Disease Information Management System. A total of 5 126 people living HIV under follow-up in Taizhou from 1998 to 2022 were included in the analysis. Software SAS 9.4 was used for χ 2 test, trend analysis and logistic regression analysis. Results:In the 5 126 people living with HIV, the reported prevalence rates of diabetes,cardiovascular disease and malignant tumor were 10.28% (527/5 126),3.98% (204/5 126) and 6.01% (308/5 126), respectively. 37.00% (195/527) and 48.58% (256/527), 40.20% (82/204) and 48.53% (99/204), 37.66% (116/308) and 48.38% (149/308) were diagnosed as diabetes, cardiovascular disease and malignant tumor before and after confirmation of HIV infection. From 2013 to 2022, the proportion of HIV infected people diagnosed with diabetes, cardiovascular disease and malignant tumor after confirmation increased (trend χ2=79.98, P<0.001; trend χ2=17.44, P<0.001; trend χ2=32.06, P<0.001). Based on the analysis on the factors for complicated chronic diseases in people living with HIV, it was found that women under 60 years old (a OR=0.66, 95% CI: 0.50-0.86) and those with access to antiviral treatment for >5 years before 2016 (a OR=0.54,95% CI:0.37-0.78) were less likely to develop complicated chronic diseases, and those under 60 years old with initial CD4 +T lymphocytes counts <200 cells/μl (a OR=1.32, 95% CI: 1.02-1.70), those aged 40-49 and 50-59 years (a OR=2.88, 95% CI:2.20-3.79; a OR=5.43, 95% CI: 4.10-7.21) as well as those without a record of treatment medication use after 2016 (a OR=1.95,95% CI:1.20-3.16) were more likely to develop complicated chronic diseases. The probability of developing complicated chronic diseases might increase with age in people living with HIV. Conclusions:From 1998 to 2022, there was a certain proportion of complicated chronic diseases among HIV infected individuals in Taizhou, and the proportion of diagnosed cases increased after HIV infection was confirmed. It is necessary to conduct early chronic disease screening, behavior intervention and standardized management in people living with HIV.

Objective To explore the biomarkers and potential mechanisms of chronic restraint stress-induced myocardial injury in hyperlipidemia ApoE-/-mice.Methods The hyperlipidemia combined with the chronic stress model was established by restraining the ApoE-/-mice.Proteomics and bioinformatics techniques were used to describe the characteristic molecular changes and related regulatory mechanisms of chronic stress-induced myocardial injury in hyperlipidemia mice and to explore potential diagnostic biomarkers.Results Proteomic analysis showed that there were 43 significantly up-regulated and 58 sig-nificantly down-regulated differentially expressed proteins in hyperlipidemia combined with the restraint stress group compared with the hyperlipidemia group.Among them,GBP2,TAOK3,TFR1 and UCP1 were biomarkers with great diagnostic potential.KEGG pathway enrichment analysis indicated that fer-roptosis was a significant pathway that accelerated the myocardial injury in hyperlipidemia combined with restraint stress-induced model.The mmu_circ_0001567/miR-7a/Tfr-1 and mmu_circ_0001042/miR-7a/Tfr-1 might be important circRNA-miRNA-mRNA regulatory networks related to ferroptosis in this model.Conclusion Chronic restraint stress may aggravate myocardial injury in hyperlipidemia mice via ferrop-tosis.Four potential biomarkers are selected for myocardial injury diagnosis,providing a new direction for sudden cardiac death(SCD)caused by hyperlipidemia combined with the restraint stress.

Objective:To establish an in vivo infection model of West Nile virus (WNV) pseudovirus and evaluate the neutralizing activity of antibody WNV-XH1.Methods:A stable cell line that can package the WNV pseudovirus was established in the early stage to prepare the pseudovirus supernatant. The supernatant was concentrated and infected BHK21 cells to detect the titer of the pseudovirus. After intraperitoneal injection of the pseudovirus into C57BL/J mice, bioluminescence imaging was performed to observe the infection status of the pseudovirus in the mice. After simultaneous infection, blood was collected and ELISA was used to detect NS1 levels in mouse serum. The in vivo functional activity of antibody WNV-XH1 was evaluated using the established mouse infection model.Results:Fluorescence was detected in C57BL/J mice infected with WNV pseudovirus, and the NS1 levels in the peripheral blood serum of mice infected with pseudovirus were significantly higher than those of non infected mice (1.453±0.09vs0.305±0.018). After intravenous administration of WNV-XH1 antibody before the attack, the fluorescence signal in the mice decreased and the serum NS1 level decreased (0.384±0.015).Conclusions:A successful in vivo infection model of WNV pseudovirus was established, and it was confirmed that the antibody WNV-XH1 had a protective effect against WNV pseudovirus infection in vivo.