Spliceosome component Usp39 contributes to hepatic lipid homeostasis through the regulation of autophagy.

Regulation of alternative splicing (AS) enables a single transcript to yield multiple isoforms that increase transcriptome and proteome diversity. Here, we report that spliceosome component Usp39 plays a role in the regulation of hepatocyte lipid homeostasis. We demonstrate that Usp39 expression is downregulated in hepatic tissues of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) subjects. Hepatocyte-specific Usp39 deletion in mice leads to increased lipid accumulation, spontaneous steatosis and impaired autophagy. Combined analysis of RNA immunoprecipitation (RIP-seq) and bulk RNA sequencing (RNA-seq) data reveals that Usp39 regulates AS of several autophagy-related genes. In particular, deletion of Usp39 results in alternative 5' splice site selection of exon 6 in Heat shock transcription factor 1 (Hsf1) and consequently its reduced expression. Importantly, overexpression of Hsf1 could attenuate lipid accumulation caused by Usp39 deficiency. Taken together, our findings indicate that Usp39-mediated AS is required for sustaining autophagy and lipid homeostasis in the liver.

Bud31-mediated alternative splicing is required for spermatogonial stem cell self-renewal and differentiation.

Alternative splicing (AS) is tightly regulated during cell differentiation and development. AS events are prevalent in the testis, but the splicing regulation in spermatogenesis remains unclear. Here we report that the spliceosome component Bud31 plays a crucial role during spermatogenesis in mice. Germ cell-specific knockout of Bud31 led to loss of spermatogonia and to male infertility. We further demonstrate that Bud31 is required for both spermatogonial stem cell pool maintenance and the initiation of spermatogenesis. SMART-seq revealed that deletion of Bud31 in germ cells causes widespread exon-skipping and intron retention. Particularly, we identified Cdk2 as one of the direct splicing targets of Bud31, knockout of Bud31 resulted in retention of the first intron of Cdk2, which led to a decrease in Cdk2 expression. Our findings suggest that Bud31-mediated AS within spermatogonial stem cells regulates the self-renewal and differentiation of male germ cells in mammals.

RAD51 is essential for spermatogenesis and male fertility in mice.

The recombinase RAD51 catalyzes the DNA strand exchange reaction in homologous recombination (HR) during both mitosis and meiosis. However, the physiological role of RAD51 during spermatogenesis remains unclear since RAD51 null mutation is embryonic lethal in mice. In this study, we generated a conditional knockout mouse model to study the role of RAD51 in spermatogenesis. Conditional disruption of RAD51 in germ cells by Vasa-Cre led to spermatogonial loss and Sertoli cell-only syndrome. Furthermore, tamoxifen-inducible RAD51 knockout by UBC-CreERT2 confirmed that RAD51 deletion led to early spermatogenic cells loss and apoptosis. Notably, inducible knockout of RAD51 in adult mice caused defects in meiosis, with accumulated meiotic double-strand breaks (DSBs), reduced numbers of pachytene spermatocytes and less crossover formation. Our study revealed an essential role for Rad51 in the maintenance of spermatogonia as well as meiotic progression in mice.

Investigation of the status quo of massive blood transfusion in China and a synopsis of the proposed guidelines for massive blood transfusion.

The aim of this study was to provide an overview of massive transfusion in Chinese hospitals, identify the important indications for massive transfusion and corrective therapies based on clinical evidence and supporting experimental studies, and propose guidelines for the management of massive transfusion. This multiregion, multicenter retrospective study involved a Massive Blood Transfusion Coordination Group composed of 50 clinical experts specializing in blood transfusion, cardiac surgery, anesthesiology, obstetrics, general surgery, and medical statistics from 20 tertiary general hospitals across 5 regions in China. Data were collected for all patients who received ≥10 U red blood cell transfusion within 24 hours in the participating hospitals from January 1 2009 to December 31 2010, including patient demographics, pre-, peri-, and post-operative clinical characteristics, laboratory test results before, during, and after transfusion, and patient mortality at post-transfusion and discharge. We also designed an in vitro hemodilution model to investigate the changes of blood coagulation indices during massive transfusion and the correction of coagulopathy through supplement blood components under different hemodilutions. The experimental data in combination with the clinical evidence were used to determine the optimal proportion and timing for blood component supplementation during massive transfusion. Based on the findings from the present study, together with an extensive review of domestic and international transfusion-related literature and consensus feedback from the 50 experts, we drafted the guidelines on massive blood transfusion that will help Chinese hospitals to develop standardized protocols for massive blood transfusion.

Investigation of the current situation of massive blood transfusion in different surgical departments: a large multicenter study in China.

OBJECTIVE: This study aims to learn about the current situation of surgical massive blood transfusion of different surgical departments in China's Tertiary hospitals, which could provide the basis for the formulation of guidelines on massive blood transfusion. METHOD: A multicenter retrospective research on the application status of blood constituents during massive blood transfusion was conducted and a comparative analyses of survival and length of hospitalization in patients from different departments (trauma, cardiac surgery, obstetric conditions, or other common surgeries), were performed. RESULT: In China, during massive blood transfusion the ratio of the dosage of fresh frozen plasma to the dosage of red blood cell suspension reached 1:1-2, while the dosage of platelet and cryoprecipitate appeared to be very small. The risk of in-hospital death were associated with the primary disease in patients receiving massive blood transfusion (Log-Rank P = 0.000), cardiac surgery and trauma patients who received massive blood transfusion have a higher risk of death rate. CONCLUSIONS: Patients undergoing massive blood transfusion among different surgical departments have a certain difference in use of blood transfusion, mortality rate and the time of death. Our findings suggested that we should set up an independent transfusion program in cardiac surgery and trauma patients of massive blood transfusion.

Hemostatic function of packed red blood cells: an in-vitro study.

Clinical observations suggest that red blood cells (RBCs) participate directly in hemostasis. We designed an in-vitro system aimed at evaluating the hemostatic function of RBCs. Blood samples were collected from 20 healthy volunteers and packed RBCs (PRBCs) were supplied by the Shaanxi Province Blood Center. We investigated the effect of RBCs and hemoglobin concentration on the hemostatic function in vitro by thromboelastography. The activation of platelets was evaluated by detecting their active markers through flow cytometry. PRBCs ameliorated the coagulation disorders induced by dilution of the blood in vitro. However, addition of hemoglobin did not increase the blood coagulation, as the level of hemoglobin was negatively correlated to the clot index. Furthermore, washing PRBCs to remove contaminating residual clotting factors and platelets excluded that the coagulation effect of the PRBCs transfusion was because of the RBCs itself. Platelet activity in PRBCs exposed to storage greater than 3 weeks was not significantly reduced consistent with it being a possible contributor. Therefore, we postulate that the suspected coagulation effects ascribed to the PRBCs at transfusion may simply be because of residual clotting factors and active platelets incompletely removed in the preparation of PRBCs rather than because of the red cell membrane or its contents.

Balanced ratio of plasma to packed red blood cells improves outcomes in massive transfusion: A large multicenter study.

Resuscitation with the early administration of plasma can improve the survival of patients undergoing surgery or trauma patients who require massive transfusion. To ascertain the optimal ratio of fresh frozen plasma (FFP) to packed red blood cells (pRBCs) in massive transfusions, the records of 1,048 patients who received a massive transfusion at 20 hospitals were retrospectively reviewed. The patients were stratified into three groups according to the ratio of FFP to pRBCs. These were the low (<1:2.3), middle (1:2.3-0.75) and high (≥1:0.75) ratio groups. For 24-h treatment, the middle FFP:pRBC ratio led to a lower mortality rate (9.31%) compared with that in the low (11.83%) and high (11.44%) ratio groups (P=0.477). For 72-h treatment, the middle FFP:pRBC ratio also lead to the lowest mortality rate (7.25%), which was significantly lower than the ratios in the low (10.39%) and high (13.65%) ratio groups (P=0.007). The length of hospital stay, ICU stay, and FFP:pRBC ratio in 72 h were found to be significant associated with mortality. The optimal ratio of FFP to pRBCs of 1:2.3-0.75 in 72 h can improve the survival of patients undergoing massive transfusions.

Association between frequency of blood tests and mortality rate in patients undergoing massive blood transfusion: a multicenter study in five regions of China.

In order to provide Chinese clinicians with guidelines for the management of massive blood loss, we investigated the correlation between the frequency of blood tests and the mortality rate in patients undergoing massive blood transfusion (MBT). The aim of this study is to provide Chinese clinicians with guidelines for the management of massive blood loss. We retrospectively reviewed the medical records of patients who underwent massive blood transfusion (MBT) from 20 tertiary hospitals in 5 regions of China. The frequency of blood tests performed within 24 or 72 hours was compared between patients infused with < 10 and ≥ 10 U of red blood cells (RBC). The correlation between the frequency of blood tests and the mortality rate was determined. A high frequency of blood tests was associated with a low mortality rate in MBT cases. The frequency of all blood tests performed within 24 hours was negatively correlated with the mortality rate in patients infused with ≥ 10 U of RBC, while the frequency of blood coagulation tests performed within 72 hours was negatively correlated with the mortality rate in both patients infused with ≥ 10 and < 10 U of RBC. In conclusions: Measuring the blood indices frequently within the first 24 hours of MBT links to lower mortality rate. Coagulation indices in MBT patients should be closely monitored in the long term to help improve survival.

Coagulation defects associated with massive blood transfusion: A large multicenter study.

The variations in the coagulation indices of patients receiving massive blood transfusion were investigated across 20 large­scale general hospitals in China. The data of 1,601 surgical inpatients receiving massive transfusion were retrospectively collected and the trends in the platelet counts and coagulation indices prior to and at 16 different time points during packed red blood cell (pRBC; after 2­40 units of pRBC) transfusion were evaluated by linear regression analysis. Temporal variations in the means of prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (APTT) and fibrinogen (FIB) concentration were also assessed and the theoretical estimates and actual measurements of the platelet count were compared. The results demonstrated that the platelet count decreased linearly with an increase in the number of pRBC units transfused (Y=150.460­3.041X; R2 linear=0.775). Following transfusion of 18 units of pRBC (0.3 units of pRBC transfused per kilogram of body weight), the average platelet count decreased to 71x10(9)/l (<75x10(9)/l). Furthermore, variations in the means of PT, INR, APTT and FIB did not demonstrate any pronounced trends and actual platelet counts were markedly higher than the theoretical estimates. In conclusion, no variations in the means of traditional coagulation indices were identified, however, the platelet count demonstrated a significant linear decrease with an increase in the number of pRBC units transfused. Furthermore, actual platelet counts were higher than theoretical estimates, indicating the requirement for close monitoring of actual platelet counts during massive pRBC transfusion.

The plasma and platelet are important in reducing the mortality in surgical massive blood transfusion: a large multicenter study in China.

OBJECTIVE: The aim of this study was to learn the current situation of surgical massive transfusion of death and survival groups in China, which could provide the basis for the formulation of guidelines on massive transfusion. METHODS: A multicenter retrospective research for the application status of blood constituents during massive blood transfusion was conducted, the differences of fresh frozen plasma and platelet application between death group and survival group were compared, and the transfusion volume and the distribution of other blood constituents were analyses at different periods of time when red blood cells are infused between death group and survival group. RESULTS: The patients with fresh frozen plasma compare the patients with red blood cell was 1:1-2 during massive transfusion, while the dosage of platelet and cryocepitate were transfused very small. Results showed that the average amount of platelet and plasma in death group was significantly lower than those in survival group. CONCLUSION: During massive transfusion, clinicians in 20 Chinese hospitals paid more attention to the infusion of fresh frozen plasma while making the infusion of red blood cells. However, they paid little attention to the supplement of platelet and cryocepitate. The average quantity of plasma and platelet in survival group were also higher than those in death group.

Correlation between red blood cell transfusion volume and mortality in patients with massive blood transfusion: A large multicenter retrospective study.

This study aimed to explore the correlation between red blood cell (RBC) transfusion volume and patient mortality in massive blood transfusion. A multicenter retrospective study was carried out on 1,601 surgical inpatients who received massive blood transfusion in 20 large comprehensive hospitals in China. According to RBC transfusion volume and duration, the patients were divided into groups as follows: 0-4, 5-9, 10-14, 15-19, 20-24, 25-29, 30-39 and ≥40 units within 24 or 72 h. Mortality in patients with different RBC transfusion volumes was analyzed. It was found that patient mortality increased with the increase in the volume of RBC transfusion when the total RBC transfusion volume was ≥10 units within 24 or 72 h. Survival analysis revealed significant differences in mortality according to the RBC transfusion volume (χ2=72.857, P<0.001). Logistic regression analysis revealed that RBC transfusion volume is an independent risk factor [odds ratio (OR) = 0.52; confidence interval (CI): 0.43-0.64; P<0.01] for the mortality of patients undergoing a massive blood transfusion. When RBCs were transfused at a volume of 5-9 units within 24 and 72 h, the mortality rate was the lowest, at 3.7 and 2.3% respectively. It is concluded that during massive blood transfusion in surgical inpatients, there is a correlation between RBC transfusion volume within 24 or 72 h and the mortality of the patients. Patient mortality increases with the increase in the volume of RBC transfusion. RBC transfusion volume, the length of stay at hospital and intensive care unit stay constitute the independent risk factors for patient mortality.

Changes in platelet function following cold storage of RBC suspensions.

OBJECTIVE: To provide a basis for the cold-storage of human platelets as a way to assess changes in platelet function. METHODS: Red blood cell suspensions (11 U and 50 U) were randomly selected at different storage times (3-28 days) and evidence of platelet activation (CD62P) and thromboelastography (TEG) reaction times were investigated. RESULTS: After 21 days of storage at 4°C, a large number of activated platelets (PAC1+62P+, PAC1-62P+) within the red blood cell suspension (RBCs) retained their function and had TEG-maximum amplitude (TEG-MA) indices in the normal range. CONCLUSION: We report that platelets in RBC suspensions retain high activity when stored at 4°C for 21 days. The results provide important information for studies that involve storing platelets under cold conditions.

Correction of blood coagulation dysfunction and anemia by supplementation of red blood cell suspension, fresh frozen plasma, and apheresis platelet: results of in vitro hemodilution experiments.

PURPOSE: This study aimed to determine the optimal composition and timing for the administration of blood supplements during in vivo blood transfusion with red blood cells suspension (pRBC), fresh frozen plasma (FFP), and apheresis platelet (PLT) administered for the correction of anemia and coagulation dysfunction caused by in vitro hemodilution. MATERIALS AND METHODS: We collected blood samples from 24 healthy volunteers and prepared various dilutions of whole blood with normal saline: 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9. The diluted blood samples were then supplemented with blood components at various proportions and then analyzed to determine the values of the routine blood indices, coagulation indices, and thromboelastogram measures. RESULTS: At hemodilutions of 40%, 50%, and 60%, the hemoglobin, coagulation indices, and platelet number and function reached critical levels, necessitating supplementation with pRBC, FFP, and PLT, respectively. When hemodilution was 90%, the supplementation required was approximately 1:1.3:0.9 of pRBC/FFP/PLT. CONCLUSION: The use of pRBC, FFP, and PLT in appropriate proportions can correct the blood coagulation dysfunction and anemia caused by in vitro hemodilution, and these proportions can be used as guidelines for in vivo massive transfusion.

Application status of blood constituents during massive blood transfusion in some regions of China.

OBJECTIVE: This study aims to learn about the current situation of surgical massive blood transfusion in China's Class III general hospitals, which could provide the basis for the formulation of guidelines on massive blood transfusion. METHODS: A multicenter retrospective research on the application status of blood constituents during massive blood transfusion was conducted and a comparative analysis on the distribution of the population infused with other blood constituents and the transfusion volume at different periods of time when red blood cells are infused in different units within 24 hours as well as on the blood applied for both the death group and survival group was made in this study. RESULTS: In China, during massive blood transfusion the ratio of the dosage of fresh frozen plasma to the dosage of red blood cell suspension reached 1:1-2, while the dosage of platelet and cryocepitate appeared to be very small. CONCLUSION: During massive blood transfusion, clinicians in 20 Chinese hospitals paid more attention to the infusion of fresh frozen plasma while making the infusion of red blood cells. However, they paid little attention to the supplement of platelet and cryocepitate.

[Construction for detecting of human papillomaviruses types by using a multiplexed Luminex assay].

AIM: To develop multiplex human papillomavirus genotyping, a quick and sensitive high-throughput procedure for the identification of multiple HPV genotypes by using a multiplexed Luminex array. METHODS: Standard plasmids containing HPV genomic DNA were developed a standard multiplexed suspension array for detection 13 HPV types on Luminex analysis platform. the primer and probes were selected, HPV type-specific oligonucleotide probes were coupled to fluorescence-labeled polystyrene beads. RESULTS: The one or two of mixed HPV standard plasmids were identified HPV type by using 13 HPV genetypes multiplexed suspension array, different HPV types can be correctly detected simultaneously. The positives of HPV clinical samples were detected by using this method, and the genetypes of samples by detecting were the same results with using BLAST in NCBI. CONCLUSION: The proposed method allowed a high through-out, special, simple, rapid and economical identification of HPV DNA genotypes, It is expected to be an extremely useful tool in the identification of HPV genetypes for a lot of clinical samples.

Evaluation of specific antibodies for early diagnosis and management of syphilis.

For many years, tests for cardiolipin antibodies Tp15, Tp17, Tp45, Tp47, and other specific Treponema pallidum antibodies, have been used to diagnose syphilis, but the timing and rates of antibody detection differ in primary and secondary syphilis. Our objective was to determine the value of the rapid plasma reagin (RPR) test, T. pallidum passive particle agglutination (TPPA) assay and Western blotting (WB) in the diagnosis of early syphilis, by studying 67 patients (20 with primary and 47 with secondary syphilis) over a recent 5-year period. All patients were tested before and 21/67 were tested after treatment. Western blotting showed that while there was a difference within the study group in terms of the number of bands detected, all patients with primary syphilis demonstrated the 47-kDa antibody, but RPR and TPPA were negative in some patients. Eighteen to 24 months after treatment, 21/67 patients were tested by WB and TPPA; antibodies to T. pallidum became undetectable within 24 months after treatment in 29% of patients. By RPR, 29% patients had negative titers within 6 months of treatment, and 86% returned to normal within 24 months. We conclude that detection of specific 47-kDa T. pallidum antibodies can be used to diagnose primary syphilis. By RPR, antibodies disappear in 6-24 months after treatment in many patients, suggesting that a change in titer may be an indicator of treatment success.

Effects of single herbal drugs on adhesion and migration of melanocytes.

OBJECTIVE: To find herbs with effects on adhesion and migration of melanocytes in vitro. MATERIALS AND METHODS: Ethanol extracts from 14 herbs were tested. Normal human melanocytes were obtained from neonatal foreskin, and the 48-well culture dish covered with fibronectin was used for the melanocyte adhesion assay. Motility was assessed by using the micropore filter method. RESULTS: The extracts of Danshen (Radix Salviae Miltiorrhizae), Tusizi (Semen Cuscutae) and Honghua (Flos Carthami) could enhance melanocyte adhesion to fibronectin, while Cijili (Fructus Tribuli) and Huangqi (Radix Astragali) promote melanocyte migration in vitro. Buguzhi (Fructus Psoraleae), Nü Zhen Zi (Fructus Ligustri Lucidi) and Baizhi (Radix Angelicae Dahuricae) could promote both adhesion and migration of melanocytes. CONCLUSION: The above herbs may play a role through promoting adhesion and/or migration of melanocytes in the treatment of vitiligo.

Epidemiologic study of the predisposing factors in erythema toxicum neonatorum.

BACKGROUND: Erythema toxicum neonatorum (ETN) is a very common disease, but its predisposing factors are still unknown. OBJECTIVE: To determine the predisposing factors of ETN. METHODS: Seven hundred and eighty-three neonates born in the same hospital during the same period were investigated, and the factors predisposing to ETN were evaluated in a case-control study. RESULTS: (1) The incidence of ETN is about 43.68%, and it is significantly higher in males than in females (p < 0.001). (2) Term birth (p < 0.05), first-pregnancy birth (p < 0.001), the birth season (summer and autumn, p < 0.005), being fed with milk powder substitute or a mixed diet (p < 0.001) and vaginal delivery (p < 0.001) are the predisposing factors of ETN. (3) The severity of ETN in neonates born by vaginal delivery is significantly correlated with the total length of labor (p < 0.001). CONCLUSION: Our findings suggest that environmental factors play an important role in the onset of ETN.