The Sec1-Munc18 protein VPS33B forms a uniquely bidirectional complex with VPS16B.

Loss-of-function variants of vacuolar protein sorting proteins VPS33B and VPS16B (VIPAS39) are causative for arthrogryposis, renal dysfunction, and cholestasis syndrome, where early lethality of patients indicates that VPS33B and VPS16B play essential cellular roles. VPS33B is a member of the Sec1-Munc18 protein family and thought to facilitate vesicular fusion via interaction with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, like its paralog VPS33A in the homotypic fusion and vacuole sorting complex. VPS33B and VPS16B are known to associate, but little is known about the composition, structure, or function of the VPS33B-VPS16B complex. We show here that human VPS33B-VPS16B is a high molecular weight complex, which we expressed in yeast to perform structural, composition, and stability analysis. Circular dichroism data indicate VPS33B-VPS16B has a well-folded α-helical secondary structure, and size-exclusion chromatography-multiangle light scattering revealed a molecular weight of ∼315 kDa. Quantitative immunoblotting indicated a VPS33B:VPS16B ratio of 2:3. Expression of arthrogryposis, renal dysfunction, and cholestasis syndrome-causing VPS33B missense variants showed L30P disrupts complex formation but not S243F or H344D. Truncated VPS16B (amino acids 143 to 316) was sufficient to form a complex with VPS33B. Small-angle X-ray scattering and negative-staining EM revealed a two-lobed shape for VPS33B-VPS16B. Avidin tagging indicated that each lobe contains a VPS33B molecule, and they are oriented in opposite directions. We propose a structure for VPS33B-VPS16B that allows the VPS33B at each end to interact with separate SNARE bundles and/or SNAREpins, plus associated membrane components. These observations reveal the only known potentially bidirectional Sec1-Munc18 protein complex.

Spatial-temporal trends in leprosy burden and its associations with socioeconomic and physical geographic factors: results from the Global Burden of Disease Study 2019.

OBJECTIVES: The purpose of our study was to assess the multiscalar changes in leprosy burden and its associated risk factors over the last three decades. STUDY DESIGN: We conducted an in-depth examination of leprosy's spatial-temporal trends at multiple geographical scale (global, regional, and national), utilizing information from Global Burden of Disease, Injuries, and Risk Factors Study (GBD 2019). METHODS: Incidence and the estimated annual percentage change (EAPC) in age-standardized incidence rate (ASIR) of leprosy were determined, with countries categorized based on leprosy incidence changes. We examined socioeconomic and physical geography influences on leprosy incidence via Spearman correlation analysis, using ternary phase diagrams to reveal the synergetic effects on leprosy occurrence. RESULTS: Globally, incident cases of leprosy decreased by 27.86% from 1990 to 2019, with a reduction in ASIR (EAPC = -2.53), yet trends were not homogeneous across regions. ASIR and EAPC correlated positively with sociodemographic index (SDI), and an ASIR growth appeared in high SDI region (EAPC = 3.07). Leprosy burden was chiefly distributed in Tropical Latin America, Oceania, Central Sub-Saharan Africa, and South Asia. Negative correlations were detected between the incidence of leprosy and factors of SDI, GDP per capita, urban population to total population, and precipitation, whereas the number of refugee population, temperature, and elevation showed opposite positive results. CONCLUSIONS: Despite a global decline in leprosy over the past three decades, the disparities of disease occurrence at regional and national scales still persisted. Socioeconomic and physical geographic factors posed an obvious influence on the transmission risk of leprosy. The persistence and regional fluctuations of leprosy incidence necessitate the ongoing dynamic and multilayered control strategies worldwide in combating this ancient disease.

[How to think about the clinical classifications of acquired aplastic anemia].

Paying attention to the diagnosis and classification of acquired aplastic anemia (AA) is the basis for improving the efficacy and the guarantee for the correct exploration of the pathological mechanism, which is of great clinical and academic significance. At present, AA classification is still based on clinical characteristics, which is a historical product of academic development.It is beneficial to guide symptomatic treatment and for the onset of curative treatment. However, the clinical classification of AA cannot replace the pathological mechanism classification to guide the treatment of the root cause. The classification of the pathological mechanism of AA determines the choice of treatment strategy, and can provide a basis for the study of etiology and prevention, and is also the future research direction. Paying attention to the classification of the pathological mechanism of AA is the basis for improving the efficacy and the guarantee for the correct exploration of the pathological mechanism. Modern medicine has entered the era of "molecular targets" and "precision", and how to treat clinical classification based on clinical characteristics is an important issue faced by clinicians. When many different mechanisms of bone marrow failure isolated from AA patients can be accurately identified, that is, when the clinically diagnosed AA has been truly purified into a disease with a clear pathological mechanism, the clinical classification of AA can help to choose the root cause strategy. This article mainly focuses on how to view the clinical classification of AA for the reference of colleagues.

[Analysis of risk factors for venous thromboembolism in patients with polycythemia vera and establishment of a prediction model].

Objective: To investigate the risk factors of venous thrombosis in patients with polycythemia vera (PV) and establish a prediction model for venous thrombosis. Methods: PV patients with JAK2V617F gene mutation positive in the Second Hospital of Tianjin Medical University from September 2017 to November 2023 were retrospectively included. The patients were divided into groups according to whether they had venous thrombosis. After matching age and gender factors with propensity scores, 102 patients were included in the venous thrombosis group [46 males, 56 females, with a median age M (Q1, Q3) of 52 (44, 60) years] and 204 cases were included in the group without venous thrombosis [92 males, 112 females, with a median age of 52 (44, 59) years]. The clinical and laboratory characteristics, disease progression and incidence of gene mutation were compared between the two groups. The follow-up cohort ended on November 20, 2023, with a median follow-up [M (Q1, Q3)] of 11 (1, 53) years. Multivariate Cox risk model was used to analyze the influencing factors of venous thrombosis in PV patients, and establish a scoring system for the venous thrombosis risk factor prediction model of PV patients. Receiver operating characteristic (ROC) curve was used to evaluate the predictive efficiency of the model. Results: Hemoglobin concentration, the ratio of hematopoietic volume≥55%, neutrophil to lymphocyte ratio≥5, hypertension, subcostal spleen≥5 cm and secondary myelofibrosis in venous thrombosis group were higher than those in non-venous thrombosis group (all P<0.05). In addition, the proportion of history of thromboembolism, V617F gene mutation load (V617F%)≥50%, diabetes mellitus, ASXL1 mutation and secondary reticular silver staining≥3 in the venous thrombosis group were higher than those in the non-venous thrombosis group (all P<0.05). The proportion of PV patients with 3 or more gene mutations was 44.1% (45/102) in venous thrombosis group, which was higher than that of PV patients without venous thrombosis 29.9% (61/204) (P=0.014). The proportion of ASXL1 gene mutation in venous thrombosis group was 17.6% (18/102), which was higher than the 4.9% (10/204) in non-venous thrombosis group (P<0.001). Multivariate Cox risk model analysis showed that previous thromboembolism history (HR=2.031, 95%CI: 1.297-3.179, P=0.002), V617F%≥50% (HR=2.141, 95%CI: 1.370-3.347, P=0.001), ASXL1 mutation (HR=4.632, 95%CI: 1.497-14.336, P=0.008), spleen subcostal≥5 cm (HR=1.771, 95%CI: 1.047-2.996, P=0.033) are the risk factors of venous thrombosis in PV patients. According to HR values, a score system for predicting risk of venous thrombosis in PV patients was established: previous history of thromboembolism, V617F%≥50% and spleen subcostoal≥5 cm were assigned 1 point respectively, and ASXL1 mutation was assigned 2 points. Low risk group: score 0, medium risk group: score 1-2, high risk group: score≥3. The ROC curve analysis of the model for predicting venous thrombosis in PV patients showed that the area under the curve (AUC) was 0.807 (95%CI: 0.755-0.860), with the sensitivity of 88.2% and the specificity of 59.8% when the Youden index was 0.48. Conclusions: Previous thromboembolism history, V617F%≥50%, ASXL1 mutation, spleen subcostoal≥5 cm are risk factors of venous thrombosis in PV patients. The established prediction model has good prediction efficiency.

Pathogenic infection characteristics and risk factors for bovine respiratory disease complex based on the detection of lung pathogens in dead cattle in Northeast China.

Bovine respiratory disease complex (BRDC) involves multiple pathogens, shows diverse lung lesions, and is a major concern in calves. Pathogens from 160 lung samples of dead cattle from 81 cattle farms in Northeast China from 2016 to 2021 were collected to characterize the molecular epidemiology and risk factors of BRDC and to assess the major pathogens involved in bovine suppurative or caseous necrotizing pneumonia. The BRDC was diagnosed by autopsy, pathogen isolation, PCR, or reverse transcription-PCR detection, and gene sequencing. More than 18 species of pathogens, including 491 strains of respiratory pathogens, were detected. The positivity rate of bacteria in the 160 lung samples was 31.77%, including Trueperella pyogenes (9.37%), Pasteurella multocida (8.35%), Histophilus somni (4.48%), Mannheimia haemolytica (2.44%), and other bacteria (7.13%). The positivity rate of Mycoplasma spp. was 38.9%, including M. bovis (7.74%), M. dispar (11.61%), M. bovirhinis (7.94%), M. alkalescens (6.11%), M. arginini (0.81%), and undetermined species (4.68%). Six species of viruses were detected with a positivity rate of 29.33%, including bovine herpesvirus-1 (BoHV-1; 13.25%), bovine respiratory syncytial virus (BRSV; 5.50%), bovine viral diarrhea virus (BVDV; 4.89%), bovine parainfluenza virus type-3 (BPIV-3; 4.28%), bovine parainfluenza virus type-5 (1.22%), and bovine coronavirus (2.24%). Mixed infections among bacteria (73.75%), viruses (50%), and M. bovis (23.75%) were the major features of BRDC in these cattle herds. The risk analysis for multi-pathogen co-infection indicated that BoHV-1 and H. somni; BVDV and M. bovis, P. multocida, T. pyogenes, or Mann. haemolytica; BPIV-3 and M. bovis; BRSV and M. bovis, P. multocida, or T. pyogenes; P. multocida and T. pyogenes; and M. bovis and T. pyogenes or H. somni showed co-infection trends. A survey on molecular epidemiology indicated that the occurrence rate of currently prevalent pathogens in BRDC was 46.15% (6/13) for BoHV-1.2b and 53.85% (7/13) for BoHV-1.2c, 53.3% (8/15) for BVDV-1b and 46.7% (7/15) for BVDV-1d, 29.41% (5/17) for BPIV-3a and 70.59% (12/17) for BPIV-3c, 100% (2/2) for BRSV gene subgroup IX, 91.67% (33/36) for P. multocida serotype A, and 8.33% (3/36) for P. multocida serotype D. Our research discovered new subgenotypes for BoHV-1.2c, BRSV gene subgroup IX, and P. multocida serotype D in China's cattle herds. In the BRDC cases, bovine suppurative or caseous necrotizing pneumonia was highly related to BVDV [odds ratio (OR) = 4.18; 95% confidence interval (95% CI): 1.6-10.7], M. bovis (OR = 2.35; 95% CI: 1.1-4.9), H. somni (OR = 8.2; 95% CI: 2.6-25.5), and T. pyogenes (OR = 13.92; 95% CI: 5.8-33.3). The risk factor analysis found that dairy calves <3 mo and beef calves >3 mo (OR = 5.39; 95% CI: 2.7-10.7) were more susceptible to BRDC. Beef cattle were more susceptible to bovine suppurative or caseous necrotizing pneumonia than dairy cattle (OR = 2.32; 95% CI: 1.2-4.4). These epidemiological data and the new pathogen subgenotypes will be helpful in formulating strategies of control and prevention, developing new vaccines, improving clinical differential diagnosis by necropsy, predicting the most likely pathogen, and justifying antimicrobial use.

[Research status and challenges of advanced myeloproliferative neoplasms].

Classical myeloproliferative neoplasms (MPN), also known as Ph-MPN, includes polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Secondary myelofibrosis (sMF) and secondary acute myeloid leukemia (sAML) are important disease progressions of MPN. After MPN disease progression, hematopoietic stem cells undergo new clonal evolution, leading to drug resistance, poor treatment effect and poor survival of patients. In recent years, the exploration of the mechanism of disease progression and the precise diagnosis and treatment of MPN have attracted much attention. This article summarizes the research status of MPN disease progression, including the pathogenesis, risk stratification, and precision treatment, in order to provide reference for exploring new diagnosis and treatment methods of MPN disease progression.

[The expression and correlation analysis of TOX and inhibitory receptors on peripheral blood CD8+T cells in patients with aplastic anemia].

Objective: To investigate the expression levels of thymocyte selection related high mobility group proteins (TOX) and different inhibitory receptors in peripheral blood CD8+T cells of patients with aplastic anemia (AA), and to conduct correlation analysis. Methods: From September 2019 to November 2020, 27 AA patients in the Department of Hematology, General Hospital of Tianjin Medical University were retrospectively selected, including 21 males and 6 females, with a median age [M (Q1, Q3)] of 48 (30, 72) years. Thirty-three healthy controls, included 17 males and 16 females, with a median age of 46 (27, 69) years. The expression levels of TOX, programmed cell death receptor-1 (PD-1), T-cell immunoglobulin and mucin domain 3 (TIM-3), cytotoxic T-lymphocyte associated antigen-4 (CTLA-4), T-cell immune receptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT), perforin and granzyme B in peripheral blood CD8+T cells from AA patients and healthy controls were detected by flow cytometry. The correlation between TOX expression levels and different inhibitory receptors was analyzed using Pearson correlation analysis. Results: The expression levels of TOX, PD-1, TIM-3, CTLA-4, TIGIT, perforin, and granzyme B in peripheral blood CD8+T cells of AA patients were 47.33%(41.47%, 56.61%), (30.61±12.37)%, (39.94±10.84)%, (6.21±3.40)%, (51.45±20.21)%, (71.32±22.46)%, and (52.39±23.99)%, respectively, which were higher than those of healthy controls 27.32%(21.64%, 46.96%), (21.29±10.01)%, (21.11±3.00)%, (1.31±0.34)% (30.80±13.40)%, (46.72±22.53)%, (21.75±16.43)% (all P<0.05). The expression level of TOX in CD8+T cells was positively correlated with the expression levels of PD-1, TIM-3, CTLA-4, TIGIT, perforin, and granzyme B (r=0.49, 0.65, 0.70, 0.54, 0.58, 0.48, all P<0.05). Conclusion: The expression levels of TOX and different inhibitory receptors on peripheral blood CD8+T cells in AA patients are higher than those in the healthy control group, and the expression levels of TOX and different inhibitory receptors are positively correlated.

[Abnormal expression of CXCR5+CD8+ T cells and CXCL13 in severe aplastic anemia patients and their correlation with hematological parameters].

Objective: To analyze the expression of C-X-C chemokine receptor 5 (CXCR5)+CD8+ T cells and plasma C-X-C motif chemokine 13 (CXCL13) in severe aplastic anemia (SAA) patients and their correlations with hematological parameters. Methods: The clinical data of 35 SAA patients in the Hematology Department of Tianjin Medical University General Hospital from January 2018 to September 2021 were retrospectively analyzed. The patients were divided into two groups according to whether they had received the medication: untreated SAA group and recovery SAA group. In untreated group, there were 18 patients who had not received any medication, with 9 males and 9 females, and aged 51 (18-76) years. In recovery SAA group, there were 17 patients who were separated from component blood transfusion after the immunosuppressive treatment with anti-thymocyte globulin (ATG) combined with cyclosporine A (CsA), with 7 males and 10 females, and aged 46 (16-70) years. Meanwhile, 20 healthy controls were also selected, including 8 males and 12 females, and aged 45(15-72) years. Peripheral blood and bone marrow samples were collected from SAA patients, while peripheral blood samples were obtained from healthy controls. Flow cytometry was used to detect the percentage of CXCR5+CD8+ T cells in peripheral blood and bone marrow samples. The concentration of plasma CXCL13 was measured by enzyme-linked immunosorbent assay (ELISA). The correlations between the percentage of CXCR5+CD8+ T cells and the concentration of CXCL13, as well as the correlations between these two parameters and the hematological parameters were analyzed by Spearman correlation analysis. Results: The proportion of CXCR5+CD8+ T cells in the bone marrow of untreated SAA group was (4.9±2.9)%, which was higher than that of recovery SAA group (2.7±1.5)%, with a statistically significant difference (t=2.34, P=0.027). The proportion of CXCR5+CD8+ T cells in peripheral blood of untreated SAA group, recovery SAA group and healthy control group was (8.4±4.2)%, (3.8±2.3)% and (2.6±2.0)% respectively. The proportion of CXCR5+CD8+ T cells in peripheral blood of untreated SAA group was higher than that of recovery SAA group and healthy control group (both P<0.05). The plasma CXCL13 concentration in untreated SAA group was (97.2±46.8) ng/L, which was significantly higher than that in recovery SAA group [(54.9±20.9) ng/L] and healthy control group [(47.6±17.3) ng/L] (both P<0.05). The proportion of CXCR5+CD8+ T cells in peripheral blood of SAA patients was positively correlated with the concentration of plasma CXCL13 (r=0.545, P<0.001). The proportion of peripheral blood CXCR5+CD8+ T cells in SAA patients was negatively correlated with white blood cell count, platelets count, percentage of neutrophils, absolute neutrophils count, percentage of reticulocytes, absolute reticulocytes count, bone marrow myeloid cells, bone marrow erythroid cells and megakaryocytes count (r=-0.556, -0.392, -0.617, -0.615, -0.395, -0.543, -0.432, -0.484 and -0.523, all P<0.05). The proportion of peripheral blood CXCR5+CD8+ T cells was positively correlated with the percentage of peripheral blood lymphocytes and bone marrow lymphoid cells (r=0.593 and 0.556, both P<0.05). Meanwhile, the concentration of plasma CXCL13 in SAA patients was negatively correlated with white blood cell count, absolute neutrophils count, percentage of reticulocytes, absolute reticulocytes count and bone marrow myeloid cells (r=-0.447, -0.446, -0.498, -0.407 and -0.456, all P<0.05), but positively correlated with bone marrow lymphoid cells (r=0.384, P<0.05). Conclusions: The proportion of CXCR5+CD8+ T cells and the concentration of plasma CXCL13 increases in SAA patients. The proportion of CXCR5+CD8+ T cells in peripheral blood is positively correlated with the concentration of CXCL13. Moreover, the proportion of CXCR5+CD8+ T cells and the concentration of CXCL13 are correlated with many hematological parameters, which may play a critical role in the immune pathogenesis of SAA.

[Clinical and laboratory features of SF3B1-mutated myeloproliferative neoplasms].

Objective: To explore the clinical and laboratory characteristics of SF3B1 gene mutations in myeloproliferative neoplasms (MPN) patients. Methods: The clinical data of 273 MPN patients who were diagnosed MPN and treated in the Second Hospital of Tianjin Medical University from November 2017 to March 2023 were retrospectively analyzed. There were 133 males and 140 females, with a median age M(Q1,Q3)of 56(46, 67) years. The molecular biology and cytogenetic characteristics were detected by second-generation sequencing (NGS) and R+G banding techniques, and the clinical and laboratory characteristics of patients with SF3B1 gene mutation were analyzed. Results: SF3B1 gene mutations were found in 13 patients (4.8%, 13/273).The types of SF3B1 mutations included missense (92.3%, 12/13) and nonsense mutations (7.7%, 1/13).Compared to the non-mutant cohort, patients in SF3B1 mutant cohort had older ages [68(51, 76) vs 56(45, 66)years,P=0.025], higher proportion of splenomegaly [46.2%(6/13) vs 15.8%(41/259),P=0.014]and secondary tumor [23.1%(3/13)vs 3.8%(10/260), P=0.018]with higher proportion of bone marrow blast [0.5%(0, 1.5%) vs 0(0, 0.5%),P=0.002] and lower hemoglobin[(104±36) vs (137±40) g/L,P=0.004] and hematocrit [31%(22%, 40%) vs 41%(35%, 52%),P=0.003]. All of the 10 patients in the SF3B1 mutant cohort whose ring sideroblast (RS) could be evaluated showed no RS formation. The overall survival, thrombosis-free survival and leukemia free survival of MPN patients in SF3B1 mutant cohort were 4.0 (2.0, 6.0), 2.0 (0.5, 4.5) and 4.0 (2.0, 6.0) years, respectively, while patients in the non-mutant cohort were 6.0 (3.0, 10.0), 5.0 (1.0, 8.0), 6.0 (3.0, 10.0) years, respectively, there were no statistical significance between two groups (Z=3.69, 1.66, 2.05, all P>0.05).The secondary tumor free survival of SF3B1 mutant cohort patients was 4.0 (2.0, 6.0) years, which was lower than that of non-mutant cohort patients [5.5 (3.0, 10.0) years, Z=18.18, P<0.001). Conclusions: MPN patients with SF3B1 gene mutations are older, more prone to splenomegaly and secondary tumors. They also have a higher proportion of bone marrow blast, lower hemoglobin and hematocrit, and show no RS formation.

[Efficacy and safety of pegylated interferon alpha-2b for patients with myeloproliferative neoplasm].

Objective: To evaluate the efficacy and safety of pegylated interferon alpha-2b (PEG-IFN-α2b) in the treatment of myeloproliferative neoplasm (MPN). Methods: Thirty-four MPN patients receiving PEG-IFN-α2b treatment in the Second Hospital of Tianjin Medical University from August 2019 to October 2022 were prospectively included. Among the patients, 9 were male and 25 were female, and the median age [M (Q1, Q3)] was 57 (19, 78) years. Patients' clinical characteristics were collected and the follow-up was performed. As of January 30, 2023, the follow-up period [M(Q1, Q3)] was 24 (16, 33) months. The efficacy, safety and changes in immune cell and cytokine levels after 12 and 24 months of treatment were analyzed. Results: During the follow-up period, 4 patients dropped out, and the efficacy was evaluable in 30 patients. Following 12 and 24 months of treatment, the complete hematologic response (CHR) rates were 57.1% (16/28) and 75.0% (18/24), respectively. The complete molecular response (CMR)+partial molecular response (PMR) rates were 27.3% (6/22) and 55.0% (11/20), respectively. The bone marrow histopathological overall response rates (ORR) were 34.6% (9/26) and 47.6% (10/21), respectively. At 12 and 24 months of treatment, the proportions of CD8+HLA-DR+T cells, effector T cell subpopulations, CD56bright natural killer (NK) cells, and plasmacytoid dendritic cells (pDC) were higher than the pre-treatment levels, while the proportion of CD56dim NK cells was lower than the pre-treatment level (all P<0.05). The levels of motif chemokine ligand 10 (CXCL10), tumor necrosis factor (TNF)-α and TNF-ß in bone marrow all increased from those prior to treatment, while the levels of vascular endothelial growth factor (VEGF) and interleukin (IL-4) decreased from those prior to treatment (all P<0.05). Among hematological adverse reactions, white blood cells decrease [47% (16/34)] was observed with high incidence. Among non-hematological adverse reactions, asthenia [44.1% (15/34)] and transaminases increase [32.3% (11/34)] were observed with high incidences. Conclusions: PEG-IFN-α2b has high hematologic, molecular, and bone marrow histopathological response rates in the treatment of MPN. It can reduce malignant clone loads and regulate the immune microenvironment and is safe and well tolerated overall.

[Analysis of risk factors for thromboembolism in patients with JAK2V617F gene mutation positive myeloproliferative neoplasms].

Objective: To analyze the risk factors of thrombosis in patients with JAK2V617F mutation positive myeloproliferative neoplasms (MPN). Methods: A total of 223 MPN patients with JAK2V617F mutation in the Second Hospital of Tianjin Medical University from September 2017 to May 2023 were retrospectively enrolled, including 111 males and 112 females, aged [M(Q1,Q3)] 57(21,66) years. According to the presence or absence of thromboembolism during follow-up, the patients were divided into thrombosis group (n=102) and non-thrombosis group (n=121). The clinical characteristics, laboratory characteristics, cytogenetics and other disease progression and survival of the two groups of patients were analyzed. As of March 31, 2023, the follow-up period [M (Q1, Q3)] was 6 (3, 10) years. The influencing factors of thrombosis in JAK2V617F positive MPN patients were analyzed by using the Cox risk model. Results: Among 223 JAK2V617F positive MPN patients, 144 were polycythemia vera (PV), 51 were essential thrombocythemia (ET) and 28 were primary myelofibrosis (PMF). The mutation rates of ASXL1 and BCORL1 genes in the thrombosis group were 19.6% (20/102) and 6.9% (7/102), respectively, which were higher than those in the non-thrombosis group [9.1% (11/121) and 0.8% (1/121)] (both P<0.05). The proportion of monocytes, C-reactive protein (CRP), interleukin-1ß (IL)-1ß, IL-8 and tumor necrosis factor-ß (TNF-ß) increased in the thrombosis group were higher than those in the non-thrombosis group (all P<0.05). Multivariate analysis showed that age≥60 years (HR=2.132, 95%CI: 1.376-3.303, P=0.001), history of thrombosis (HR=3.636, 95%CI: 2.121-6.202, P<0.001), ASXL1 mutation positive (HR=2.245, 95%CI: 1.093-3.231, P=0.022) and elevated TNF-ß (HR=2.009, 95%CI: 1.113-3.624, P=0.021) were risk factors for thrombosis in JAK2V617F positive MPN patients. Conclusions: In addition to age, history of thrombosis and positive ASXL1 mutation, elevated TNF-ß is also an influencing factor of thrombosis in JAK2V617F positive MPN patients. Intervention of inflammation may have a certain effect on the prevention and treatment of thrombosis.

[Review and perspective of clinical research involving chest tightness variant asthma in China].

Chest tightness variant asthma (CTVA) was first reported and named by Chinese scholars in 2013. It is a new clinical type of asthma characterized by chest tightness as the only or primary symptom, without typical asthma manifestations such as recurrent wheezing and shortness of breath, and without wheezing sounds heard during lung auscultation. The overall epidemiological data on CTVA is currently unavailable. Its pathogenesis is similar to that of typical asthma, involving eosinophilic airway inflammation. Due to the lack of typical clinical manifestations, insufficient knowledge of this disease in some clinicians and some other reasons, CTVA is susceptible to misdiagnosis or missed diagnosis. Currently, the diagnostic criteria for CTVA are: chest tightness as the only or primary symptom, without typical asthma symptoms and signs such as wheezing and shortness of breath, and with any one of the objective indicators of variable airflow limitation. Effective anti-asthma treatment is required, and other diseases that cause chest tightness, such as cardiovascular, digestive, nervous, muscular, and mental diseases should be excluded. CTVA treatment follows that of typical asthma, but the specific treatment duration is uncertain and may require long-term management. Traditional Chinese medicine has shown some therapeutic effects on CTVA. Most CTVA patients have a good prognosis after active anti-asthma treatment. This paper analyzes and summarizes the research of CTVA in China from 2013 and provides new perspectives for further exploration of CTVA.

[Developmental effects of TCIPP and TnBP on zebrafish (Danio rerio) embryos].

Objective: To investigate the toxicity of tris (2-chloropropyl) phosphate (TCIPP) and tributyl phosphate (TnBP) on the growth and development of zebrafish embryos, as well as to explore the underlying mechanisms at the transcriptional level. Methods: With zebrafish as a model, two hpf zebrafish embryos were exposed to TCIPP and TnBP (0.1, 1, 10, 100, 500, and 1 000 µmol/L) using the semi-static method, and their rates of lethality and hatchability were determined. The transcriptome changes of 120 hpf juvenile zebrafish exposed to environmentally relevant concentrations of 0.1 and 1 µmol/L were measured. Results: The 50% lethal concentrations (LC50) of TCIPP and TnBP for zebrafish embryos were 155.30 and 27.62 µmol/L (96 hpf), 156.5 and 26.05 µmol/L (120 hpf), respectively. The 72 hpf hatching rates of TCIPP (100 µmol/L) and TnBP (10 µmol/L) were (23.33±7.72)% and (91.67±2.97)%, which were significantly decreased compared with the control group (P<0.05). Transcriptome analysis showed that TnBP had more differential genes (DEGs) than TCIPP, with a dose-response relationship. These DEGs were enriched in 32 pathways in total, including those involved in oxidative stress, energy metabolism, lipid metabolism, and nuclear receptor-related pathways, using the IPA pathway analysis. Among them, three enriched pathways overlapped between TCIPP and TnBP, including TR/RXR activation and CAR/RXR activation. Additionally, DEGs were also mapped onto pathways of LXR/RXR activation and oxidative stress for TnBP exposure only. Conclusion: Both TCIPP and TnBP have growth and developmental toxicities in zebrafish embryos, with distinct biomolecular mechanisms, and TnBP has a stronger effect than TCIPP.

[Occupational protection effect of two protective devices for manual cleaning and oiling of dental handpieces on operators].

Objective: To explore the occupational protective effect of different protective devices on the operators during manual cleaning and oiling of dental handpieces, and to provide a basis for the selection of appropriate protective methods. Methods: From November 2020 to December 2021, 20 high-speed dental handpieces of the same brand were selected and randomly divided into disposable protective bag group and small aerosol safety cabinet group by drawing lots, with 10 in each group. After recording the model, they were distributed to the clinical fixed consulting room for use, and were collected by specially-assigned personnel every day for manual cleaning under the protection of the two devices. By measuring the number of airborne colonies, the concentrations of particulate matter and the satisfaction of operators, the occupational protection effect of the two protective devices on operators was evaluated. Results: Under the protection of the two devices, the average number of airborne colonies after operation was less than 1 CFU/ml. When no protective device was used, the number concentration of particulate matter produced during operation was (21595.70±8164.26) pieces/cm(3). The number concentrations of particles produced by disposable protective bag group [ (6800.24±515.05) pieces/cm(3)] and small aerosol safety cabinet group [ (5797.15±790.50) pieces/cm(3)] were significantly lower than those without any protective device (P<0.001). The number concentration of particle matter of small aerosol safety cabinet group was significantly lower than that of disposable protective bag group (P<0.001). In the satisfaction evaluation of operators, small aerosol safety cabinet group [ (3.53±0.82) points] was significantly better than disposable protective bag group [ (2.23±1.10) points] (P<0.001) . Conclusion: The use of small aerosol safety cabinet during manual cleaning and oiling of dental handpieces has good protective effect, superior safety performance and strong clinical applicability, and has advantages in occupational protection of clinical operators.

Overall survival in the OlympiA phase III trial of adjuvant olaparib in patients with germline pathogenic variants in BRCA1/2 and high-risk, early breast cancer.

BACKGROUND: The randomized, double-blind OlympiA trial compared 1 year of the oral poly(adenosine diphosphate-ribose) polymerase inhibitor, olaparib, to matching placebo as adjuvant therapy for patients with pathogenic or likely pathogenic variants in germline BRCA1 or BRCA2 (gBRCA1/2pv) and high-risk, human epidermal growth factor receptor 2-negative, early breast cancer (EBC). The first pre-specified interim analysis (IA) previously demonstrated statistically significant improvement in invasive disease-free survival (IDFS) and distant disease-free survival (DDFS). The olaparib group had fewer deaths than the placebo group, but the difference did not reach statistical significance for overall survival (OS). We now report the pre-specified second IA of OS with updates of IDFS, DDFS, and safety. PATIENTS AND METHODS: One thousand eight hundred and thirty-six patients were randomly assigned to olaparib or placebo following (neo)adjuvant chemotherapy, surgery, and radiation therapy if indicated. Endocrine therapy was given concurrently with study medication for hormone receptor-positive cancers. Statistical significance for OS at this IA required P < 0.015. RESULTS: With a median follow-up of 3.5 years, the second IA of OS demonstrated significant improvement in the olaparib group relative to the placebo group [hazard ratio 0.68; 98.5% confidence interval (CI) 0.47-0.97; P = 0.009]. Four-year OS was 89.8% in the olaparib group and 86.4% in the placebo group (Δ 3.4%, 95% CI -0.1% to 6.8%). Four-year IDFS for the olaparib group versus placebo group was 82.7% versus 75.4% (Δ 7.3%, 95% CI 3.0% to 11.5%) and 4-year DDFS was 86.5% versus 79.1% (Δ 7.4%, 95% CI 3.6% to 11.3%), respectively. Subset analyses for OS, IDFS, and DDFS demonstrated benefit across major subgroups. No new safety signals were identified including no new cases of acute myeloid leukemia or myelodysplastic syndrome. CONCLUSION: With 3.5 years of median follow-up, OlympiA demonstrates statistically significant improvement in OS with adjuvant olaparib compared with placebo for gBRCA1/2pv-associated EBC and maintained improvements in the previously reported, statistically significant endpoints of IDFS and DDFS with no new safety signals.

Docosahexaenoic Acid Reverses Epithelial-Mesenchymal Transition and Drug Resistance by Impairing the PI3K/AKT/ Nrf2/GPX4 Signalling Pathway in Docetaxel-Resistant PC3 Prostate Cancer Cells.

Drug resistance is a serious problem in cancer therapy. Growing evidence has shown that docosahexaenoic acid has anti-inflammatory and chemopreventive abilities. Studies have shown that autophagy inhibition and ferroptosis are promising therapeutic strategies for overcoming multidrug resistance. This study was aimed to examine whether docosahexaenoic acid (DHA) could reverse docetaxel resistance in prostate cancer cells. Cell survival was examined by MTT and colony formation. Protein expression was determined by Western blot. Reactive oxygen species (ROS) production was measured by flow cytometry. DHA displayed anti-cancer effects on proliferation, colony formation, migration, apoptosis, autophagy and epithelial mesenchymal transition. Glutathione-S-transferase π is an enzyme that plays an important role in drug resistance. DHA inhibited GSTπ protein expression and induced cytoprotective autophagy by regulating the PI3K/AKT signalling pathway in PC3R cells. DHA combined with PI3K inhibitor (LY294002) enhanced apoptosis by alleviating the expression of LC3B, (pro-) caspase- 3 and (uncleaved) PARP. DHA induced ferroptosis by attenuating the expression of glutathione peroxidase 4 (GPX4) and nuclear erythroid 2-related factor 2 (Nrf2). DHA-treated PC3R cells produced ROS. The ROS and cytotoxicity were reversed by treatment with ferrostatin-1. DHA combined with docetaxel inhibited EMT by regulating the expression of E-cadhein and N-cadherin. In summary, DHA reversed drug resistance and induced cytoprotective autophagy and ferroptosis by regulating the PI3K/AKT/Nrf2/GPX4 signalling pathway in PC3R cells. We propose that DHA could be developed as a chemosensitizer and that the PI3K/AKT /Nrf2/GPX4 signalling pathway might be a promising therapeutic target for overcoming cancer drug resistance.

[Exclusive diagnosis of aplastic anemia].

As an independent disease, aplastic anemia (AA) has been recognized for more than a century. When AA is diagnosed, other non-AA bone marrow failures should be excluded. It is termed as exclusive diagnosis of AA. The exclusive diagnosis of AA is helplessly based on that there is no parameter by which AA can be sensitively and specifically diagnosed now. So further searching for the meaningful diagnostic parameters of AA should be carried on to establish a direct diagnostic protocol of this disease and make it possible to differentiate it clearly from other bone marrow failure disease such as congenenital bone marrow failure, hypoplastic myelodysplastic syndromes, AA-paroxysmal nocturnal hemoglobinuria syndromes, large granules lymphocyte leukemia, clonal cytopenia of undetermined significance, immunorelated pancytopenia, acute hemopoietic arresting and idiopathic cytopenia of undetermined significance. The new markers and technologies being helpful for distinguishing AA from other bone marrow failures should be used in diagnosing AA. Correct understanding and application of exclusive diagnosis is not only related to the correctness of diagnosis and treatment of excluded diseases, but also to the quality of AA diagnosis, treatment and research.

[Effect and prognosis of new type of re-dilated stent in the treatment of pulmonary artery bifurcation stenosis].

the early and med-term follow-up results and technical points of new re-dilated stent in the treatment of pulmonary artery bifurcation opening stenosis, and explore its feasibility and advantages. From March 2019 to October 2020, 10 children [5 males, mean age (7±3) years], mean weight 18.75(13.35,23.05) kg with pulmonary artery bifurcation opening stenosis were treated with new re-dilated stents in the Central China Fuwai Hospital. Including 5 cases of tetralogy of Fallot, 4 cases of pulmonary atresia, 1 case of anomalous origin of coronary artery, all children were given new re-dilated stent implantation. Echocardiography, chest X-ray and electrocardiogram were performed 1 day, 3, 6, 12 months after intervention. Pulmonary artery CTA was performed after 6 or 12 months to evaluate the results, including restenosis, malposition and rupture. A total of 16 stents were implanted in 10 children, 5 cases had simultaneous stenosis of bifurcation openings of pulmonary arteries, and 1 stent was implanted in each of the left and right pulmonary artery openings. The pressure of right ventricular and gradient was significantly decreased immediately after intervention, from preoperative (38-80) mmHg(1 mmHg=0.133 kPa) to postoperative (0-22) mmHg, only one patient's pressure gradient is over 20 mmHg (22 mmHg), and all cases discharged successfully. Stent restenosis, malposition, fracture and other abnormalities were not observed in follow-up. Stents implantation for patients with pulmonary artery bifurcation opening stenosis is very more difficult. A good strategy can ensure that the intervention is safe and effective. It not only avoids the risk of repeated surgery, but also achieves good med-term follow-up results.

[Serotyping methods of Streptococcus pneumonia].

More than 100 serotypes of Streptococcus pneumonia have been identified, which has been one bottleneck problem for pneumococcal disease diagnosis, surveillance, development of pneumococcal vaccine and effectiveness evaluation of pneumococcal vaccines. Three categories of approaches for pneumococcal serotyping will be discussed including phenotyping based on anti-serum, biochemical typing based on pneumococcal capsular characteristics and genotyping based on pneumococcal capsular locus sequences. We reviewed the development and applications of different serotyping of pneumococcus to provide guidance for pneumococcal disease prevention and control.

[Research progress on immunogenicity evaluation of serogroup B meningococcal vaccine].

Epidemic cerebrospinal meningitis (meningococcal meningitis) is an acute respiratory infectious disease with high mortality and serious sequelae. Meningococcal vaccine is an effective measure to prevent and control meningococcal meningitis. At present, group B meningococcal meningitis has become the main prevalent serum group in the world, including China. Meningococcal ACYW and other vaccines are mainly composed of capsular polysaccharides, while the main component of group B meningococcal vaccine is protein, including outer membrane vesicles (OMV) and recombinant protein vaccine. The methods for evaluating the immunogenicity of group B meningococcal vaccine include hSBA and alternative methods such as meningococcal antigen typing system (MATS), flow cytometric meningococcal antigen surface expression assay (MEASURE), genetic meningococcal antigen typing system (gMATS) and bexsero antigen sequence type (BAST). The evaluation of vaccine immunogenicity is the basis of vaccine development and clinical trial research, However, at present, there is no group B meningococcal vaccine in China. Therefore, in this paper, the research progress of immunogenicity evaluation of group B meningococcal vaccine has been reviewed, in order to provide technical guidance for the research and development of group B meningococcal vaccine, immunogenicity evaluation and clinical trial research in China.