Orelabrutinib for the treatment of relapsed or refractory marginal zone lymphoma: A phase 2, multicenter, open-label study.

Marginal zone lymphoma (MZL) is an indolent type of non-Hodgkin lymphoma that develops through pathological B cell receptor signaling. Orelabrutinib, a new-generation oral small molecule Bruton's tyrosine kinase inhibitor, was evaluated in relapsed/refractory (r/r) MZL patients. Previously treated r/r MZL patients received orelabrutinib 150 mg once daily in a phase 2, multicenter, single-arm study conducted in China. The primary endpoint was overall response rate (ORR) assessed by an Independent Review Committee (IRC) based on the Lugano 2014 classification. Other efficacy, safety, and pharmacokinetic profiles were evaluated as secondary outcome measures. A total of 111 patients were enrolled, of which 90 patients had MZL confirmed by central pathology review, who were mainly with extra-nodal MZL of mucosa-associated lymphoid tissue (MALT, 46.7%) and nodal MZL (35.6%). The majority had late-stage disease, with stage IV accounting for 75.6%. After a median follow-up duration of 24.3 months, the IRC-assessed ORR was 58.9% (95% confidence interval [CI], 48.0-69.2), with rates of complete response and partial response being 11.1% and 47.8%, respectively. The IRC-assessed median duration of response was 34.3 months, and the IRC-assessed median progression-free survival (PFS) was not reached with a 12-month PFS rate of 82.8% (95% CI, 72.6-89.5). The rate of overall survival at 12 months was 91.0% (95% CI, 82.8-95.4). Common all-grade treatment-related adverse events (TRAEs) included anemia (27.9%), neutrophil count decrease (23.4%), white blood cell count decrease (18.0%), platelet count decrease (17.1%), blood present in urine (16.2%), rash (14.4%), and upper respiratory tract infection (10.8%). Thirty-four patients (30.6%) experienced grade 3 or higher TRAEs. Serious TRAEs occurred in 18 patients (16.2%), of which pneumonia (5.4%) was the most common. Seven patients (6.3%) discontinued orelabrutinib due to TRAEs. Orelabrutinib demonstrated high response rates with durable disease remission and was well tolerated in Chinese patients with r/r MZL.

A Novel Inhibitor of the Obesity-Related Protein FTO.

Fe(II) and α-ketoglutarate-dependent fat mass and obesity associated protein (FTO)-dependent demethylation of m6A is important for regulation of mRNA splicing and adipogenesis. Developing FTO-specific inhibitors can help probe the biology of FTO and unravel novel therapeutic targets for treatment of obesity or obesity-associated diseases. In the present paper, we have identified that 4-chloro-6-(6'-chloro-7'-hydroxy-2',4',4'-trimethyl-chroman-2'-yl)benzene-1,3-diol (CHTB) is an inhibitor of FTO. The crystal structure of CHTB complexed with human FTO reveals that the novel small molecule binds to FTO in a specific manner. The identification of the novel small molecule offers opportunities for further development of more selective and potent FTO inhibitors.

Changes in expression levels of immune cells and inflammatory cytokines in pre-eclampsia patients before and after delivery.

OBJECTIVE: By analyze the expression levels of immune cells and cytokines secreted by preeclampsia patients before and after delivery. Furthermore, to determine the inflammatory and immunological mechanism responsible for preeclampsia, to provide better future prevention and treatment. METHODS: Twenty-one preeclampsia-diagnosed pregnant women were involved in this study as an observation group. Twenty pregnant women with no history of PE, age-matched, were added to the control group. Thirty NP women, age-matched were included as the reference. The levels of cytokines secreted by T, B, NK and Treg immune cells and Th1, Th2 and Th17 cells were detected before and after delivery. RESULTS: The number of CD3 + cells in women without preeclampsia before delivery was not statistically different from that in PE patients, but the number of CD4 + and CD8 + cells was lower than that in women without preeclampsia. After delivery, CD4 + cells were larger than those in pregnant women without preeclampsia, while CD8 + cells were smaller than those in pregnant women without preeclampsia. The level of cytokines secreted by Th1 in pregnant women without preeclampsia before delivery was higher than that in PE patients. The levels of cytokines produced by Th1, Th2 and Th17 cells before and after delivery were higher in pregnant women without preeclampsia than in NP women, while Th2 and Th17 were in PE group. CONCLUSION: PE patients may reduce CD4 + cells and CD8 + cells, and down-regulate the level of cytokines (especially TNF-ß) secreted by Th1 cells to reduce the maternal rejection of embryos, thereby reducing the risk of premature delivery, which is a protective adaptive compensation mechanism in favor of embryos.

Expression of Th1/2/17 Cytokines in CML with or without Pulmonary Bacterial and Fungal Coinfection.

Background: Tyrosine kinase inhibitors (TKIs) are the standard therapy for patients with chronic myeloid leukemia (CML). While their use greatly increases patient survival rates and can lead to normal life expectancy, bacterial infections in the lungs continue to play a significant role in determining patient outcomes. Methods: In this study, the medical records of 272 CML and 53 healthy adults were analyzed. Information on age, sex, body temperature, procalcitonin (PCT), C-reactive protein (CRP), and cytokine levels were collected from patients. Since the data belonged to a nonstate distribution, we used the Mann-Whitney U test to examine differences between groups. Cut-off values were analyzed by receiver operating characteristic (ROC) curves. Results: No significant differences in the Th1/2/17 levels were observed in relation to TKI treatment. Further analysis showed that the levels of the interleukins IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-22, IL-12p70, IL-17A, IL-17F, and IL-1ß, interferon (IFN-γ), and tumor necrosis factors (TNF α and ß) were higher in patients with pulmonary bacterial infections compared with uninfected patients. IL-6, IL-8, and IL-10 levels in CML patients with bacterial and fungal coinfection were higher than those in patients without infection. The areas under the ROC curves (AUCs) were found to be 0.73 for IL-5, 0.84 for IL-6, 0.82 for IL-8, 0,71 for IL-10, and 0.84 for TNF-α. AUC values were higher for patients with pulmonary bacterial infection, especially IL-6 (AUC = 0.84, cut-off = 13.78 pg/ml) and IL-8 (AUC = 0.82, cut-off = 14.35 pg/ml), which were significantly better than those for CRP (AUC = 0.80, cut-off = 6.18 mg/l), PCT (AUC = 0.71, cut-off = 0.25 ng/ml), and body temperature (AUC = 0.68, cut-off = 36.8°C). In addition, according to the cut-off values, we found that 83.33% of patients with pulmonary bacterial infections had IL-6 ≥ 13.78 pg/ml, while when IL-6, IL-8, and IL-10 levels simultaneously exceeded the cut-off values, the probability of pulmonary bacterial infection was 93.55%. Conclusions: TKI treatment did not appear to affect cytokine expression in CML patients. However, CML patients with pulmonary bacterial infection had significantly higher levels of Th1/2/17 cytokines. In particular, abnormally elevated IL-6, IL-8, and IL-10 levels were associated with a pulmonary bacterial infection in patients with CML.

Orelabrutinib for the treatment of relapsed or refractory MCL: a phase 1/2, open-label, multicenter, single-arm study.

Relapsed or refractory (r/r) mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a poor prognosis. Bruton tyrosine kinase (BTK) is a mediator of B-cell receptor signaling and is associated with the development of B-cell lymphomas. Patients with r/r MCL were enrolled in this phase 1/2 study and treated with orelabrutinib, a novel, highly selective BTK inhibitor. The median number of prior regimens was 2 (range, 1-4). The median age was 62 years (range, 37-73 years). Eligible patients received oral orelabrutinib 150 mg once daily (n = 86) or 100 mg twice daily (n = 20) until disease progression or unacceptable toxicity. A dose of 150 mg once daily was chosen as the preferred recommended phase 2 dose. After a median follow-up duration of 23.8 months, the overall response rate was 81.1%, with 27.4% achieving a complete response and 53.8% achieving a partial response. The median duration of response and progression-free survival were 22.9 and 22.0 months, respectively. The median overall survival (OS) was not reached, and the rate of OS at 24 months was 74.3%. Adverse events (AEs) occurring in >20% of patients were thrombocytopenia (34.0%), upper respiratory tract infection (27.4%), and neutropenia (24.5%). Grade ≥3 AEs were infrequent and most commonly included thrombocytopenia (13.2%), neutropenia (8.5%), and anemia (7.5%). Three patients discontinued treatment because of treatment-related adverse events (TRAEs), but no fatal TRAEs were reported. Orelabrutinib showed substantial efficacy and was well tolerated in patients with r/r MCL. This trial was registered at www.clinicaltrials.gov as #NCT03494179.

Cytokines help suggest aplastic anemia with pulmonary bacterial or co-fungal infection.

Although aplastic anemia (AA) does not come under the category of blood malignant diseases, the infection that frequently occurs in this bone marrow failure can make it worse. Pulmonary infection is the most prevalent but limiting clinical diagnosis. To find biomarkers predicting bacterial or bacterial-combined fungal infections in the lungs, we reviewed 287 AA medical records including 151 without any infection, 87 with pure pulmonary bacterial infection, and 49 with bacterial and fungal infection were reviewed. There were substantial changes in IL-17F, IL-17A, IFN-γ, IL-6, IL-8, and IL-10 levels between the non-infected and lung bacterial infection groups (P < 0.05). Further, a significant variation in IL-17A, TNF-ß, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-22, and IL-12p70, between the uninfected group and the pulmonary bacterial and fungal infection group (P < 0.05) was observed. The results further revealed significant differences in TNF-ß, IL-12p70, IL-6, IL-8, and IL-10 between the pulmonary bacterial infection group and the fungal infection group (P < 0.05). Moreover, by calculating ROC and cut-off values, we determined that IL-6 (AUC = 0.98, Cut-off = 14.28 pg/ml, P = 0.0000) had a significant advantage than other cytokines, body temperature (AUC = 0.61, P = 0.0050), PCT (AUC = 0.57, P = 0.0592), and CRP (AUC = 0.60, P = 0.0147) in the detection of lungs bacterial infections. In addition, IL-6 (AUC = 1.00, Cut-off = 51.50 pg/ml, P = 0.000) and IL-8 (AUC = 0.87, Cut-off = 60.53 pg/ml, P = 0.0000) showed stronger advantages than other cytokines, body temperature (AUC = 0.60, P = 0.0324), PCT (AUC = 0.72, Cut-off = 0.63 ng/ml, P = 0.0000) and CRP (AUC = 0.79, Cut-off = 5.79 mg/l, P = 0.0000) in distinguishing bacteria from fungi. This may suggest that IL-8 may play a role in differentiating co-infected bacteria and fungi. Such advantages are repeated in severe aplastic anemia (SAA) and very severe aplastic anemia (VSAA).In conclusion, aberrant IL-6 elevations in AA patients may predict the likelihood of bacterial lung infection. The concurrent increase of IL-6 and IL-8, on the other hand, should signal bacterial and fungal infections in patients.These findings may help to suggest bacterial or fungal co-infection in patients with AA (Focus on VSAA and SAA).

Increased Cytokine Levels Assist in the Diagnosis of Respiratory Bacterial Infections or Concurrent Bacteremia in Patients With Non-Hodgkin's Lymphoma.

Non-Hodgkin's lymphoma (NHL) is a form of tumor that originates in the lymphoid tissues. Bacterial infections are very common in NHL patients. Because most of the patients do not experience apparent symptoms during the initial stage of infection, it is difficult to detect the underlying condition before it progresses to a more critical level. The activation of the cytokines is a hallmark of inflammation. Due to the advantages of short detection time and high sensitivity of cytokines, many studies have focused on relationship between cytokines and infection. However, few studies have been conducted on NHL patients with infection. Therefore, we reviewed the cytokine profiles of 229 newly diagnosed NHL patients and 40 healthy adults to predict respiratory bacterial infection and bacteremia. Our findings revealed that IL-6(41.67 vs 9.50 pg/mL), IL-8(15.55 vs 6.61 pg/mL), IL-10(8.02 vs 4.52 pg/mL),TNF-ß(3.82 vs 2.96 pg/mL), IFN- γ(4.76 vs 2.96 pg/mL), body temperature(37.6 vs 36.5°C), CRP(20.80 vs 4.37 mg/L), and PCT(0.10 vs 0.04 ng/mL) levels were considerably greater in NHL cases with respiratory bacterial infections relative to NHL cases without infection (P<0.05). Furthermore, IL-6(145.00 vs 41.67 pg/mL), IL-8(34.60 vs 15.55 pg/mL),temperature(38.4 vs 37.6°C), PCT(0.79 vs 0.10 ng/mL), and CRP(93.70 vs 20.80 mg/L) levels in respiratory infectious NHL patients with more severe bacteremia were considerably elevated than in patients with respiratory bacterial infections only (P<0.05). Remarkably, increased levels of IL-6 and IL-8 are effective in determining whether or not pulmonary bacterial infectious NHL patients have bacteremia. Temperature, PCT, and CRP all have lower sensitivity and specificity than IL-6. IL-6 ≥18.79pg/mL indicates the presence of pulmonary bacterial infection in newly diagnosed NHL patients, and IL-6 ≥102.6pg/mL may suggest pulmonary bacterial infection with bacteremia. In short, this study shows that cytokines can be advantageous in the diagnosis and differentiation of pulmonary bacterial infection and bacteremia in newly diagnosed NHL patients and may also guide for the use of clinical antibiotics.

Evaluation of orelabrutinib monotherapy in patients with relapsed or refractory Waldenström's macroglobulinemia in a single-arm, multicenter, open-label, phase 2 study.

Background: Orelabrutinib is a novel, small molecule, selective irreversible Bruton tyrosine kinase inhibitor. The purpose of this study was to evaluate the efficacy and safety of orelabrutinib in patients with relapsed or refractory Waldenström's macroglobulinemia (R/R WM). Methods: This is a prospective, multicenter study of orelabrutinib in patients with WM who had at least one prior line of treatment. Orelabrutinib was administered orally at a daily dose of 150 mg until disease progression or unacceptable toxicity. The primary endpoint was major response rate (MRR) assessed by the Independent Review Committee (IRC) according to IWWM-6. This study is registered with ClinicalTrials.gov, NCT04440059. This trial was also registered on Center for Drug Evaluation (www.chinadrugtrials.org.cn) in March 2019, with a number of CTR2019036. Findings: Between August 2019 and December 2020, 66 R/R WM patients were assessed for eligibility. Forty-seven eligible patients were evaluated for efficacy at a median follow-up of 16.4 months (interquartile range: 12.5, 19.5). As assessed by IRC, the MRR was 80.9%, and the overall response rate was 89.4%. The median time to at least a minor response was 1.9 months. The PFS rates was 89.4% at 12 months. For patients with MYD88L265P /CXCR4NEG, MYD88L265P /CXCR4 S338X, and MYD88NEG /CXCR4NEG mutations, the MRRs were 84.6%, 100%, and 25.0%. Most adverse events were Grades 1 or 2 (91.0%). The common grade 3 or higher adverse events occurring were neutropenia (10.6%), thrombocytopenia (6.4%), and pneumonia (4.3%). Serious adverse events (SAE) occurred in 10 patients (21.3%). One treatment-related death was reported (hepatitis B reactivation). Interpretation: Orelabrutinib has shown good efficacy and manageable safety profiles in patients with R/R WM. Funding: InnoCare Pharma.

Cytokine Expression of Lung Bacterial Infection in Newly Diagnosed Adult Hematological Malignancies.

Adult patients with hematological malignancies are frequently accompanied by bacterial infections in the lungs when they are first diagnosed. Sputum culture, procalcitonin (PCT), C-reactive protein (CRP), body temperature, and other routinely used assays are not always reliable. Cytokines are frequently abnormally produced in adult hematological malignancies associated with a lung infection, it is uncertain if cytokines can predict lung bacterial infections in individuals with hematological malignancies. Therefore, we reviewed 541 adult patients newly diagnosed with hematological malignancies, of which 254 patients had lung bacterial infections and 287 patients had no other clearly diagnosed infections. To explore the predictive value of cytokines for pulmonary bacterial infection in adult patients with hematological malignancies. Our results show that IL-4, IL-6, IL-8, IL-10, IL-12P70, IL-1ß, IL-2, IFN-γ, TNF-α, TNF-ß and IL-17A are in the lungs The expression level of bacterially infected individuals was higher than that of patients without any infections (P<0.05). Furthermore, we found that 88.89% (200/225) of patients with IL-6 ≥34.12 pg/ml had a bacterial infection in their lungs. With the level of IL-8 ≥16.35 pg/ml, 71.67% (210/293) of patients were infected. While 66.10% (193/292) of patients had lung bacterial infections with the level of IL-10 ≥5.62 pg/ml. When IL-6, IL-8, and IL-10 were both greater than or equal to their Cutoff-value, 98.52% (133/135) of patients had lung bacterial infection. Significantly better than PCT ≥0.11 ng/ml [63.83% (150/235)], body temperature ≥38.5°C [71.24% (62/87)], CRP ≥9.3 mg/L [53.59% (112/209)] the proportion of lung infection. In general. IL-6, IL-8 and IL-10 are abnormally elevated in patients with lung bacterial infections in adult hematological malignancies. Then, the abnormal increase of IL-6, IL-8 and IL-10 should pay close attention to the possible lung bacterial infection in patients.

Analysis of cytokine risk factors in the early death of patients with secondary phagocytic lymphocytic histiocytosis.

Secondary hemophagocytic lymphohistiocytosis (sHLH) is an excessive inflammatory response syndrome caused by immune abnormalities. Up to date, the risk factors for cytokines causing early death in sHLH patients have not been elucidated. Our study reviewed the cytokine expression levels in peripheral blood of 50 sHLH patients. Through Cox proportional hazard model analysis, we found that IL-17F ≥2.835 pg/mL (HR = 5.922, 95% CI = 1.793-19.558, P = 0.004) was an independent death risk factor in sHLH patients, and it was also 30 days (Cutoff-value = 2.890 pg/mL, HR = 16.568, 95% CI = 1.917-143.195, P = 0.011), 60 days (Cutoff-value = 2.890 pg/mL, HR = 7.559, 95% CI = 1.449-39.423, P = 0.016), 90 day death risk factor (Cutoff-value = 2.835 pg/mL, HR = 7.649, 95% CI = 1.965-29.778, P = 0.003); IL-10 ≥16.730 pg/mL (HR = 4.821, 95% CI = 1.151-20.116, P = 0.031) is not only a death risk factor within 90 days, but also within 10 days (Cutoff-value = 944.350 pg/mL, HR = 13.321, 95% CI = 1.123-158.03, P = 0.027); and IL-5 ≥2.495 pg/mL (HR = 15.687, 95% CI = 1.377-178.645, P = 0.04) was also a death risk factor within 10 days. Besides, IL-17F, IL-10, IL-5, and the previously reported common risk factors Age, platelets, activated partial thromboplastin time, triglyceride, and lactate dehydrogenase were analyzed together. It was found that the patient age ≥56 years-old is was an important risk factor for death within 30 days, IL-17 ≥2.89 pg/mL and IL-10 ≥16.73 pg/mL are important risk factors for patient death. In summary, our data indicate that age, IL-10 and IL-17F are important risk factors for early death in sHLH patients.

Traditional Chinese medicine compound, Bu Sheng Hui Yang Fang, promotes the proliferation of lymphocytes in the immunosuppressed mice potentially by upregulating IL-4 signaling.

The immune system plays a pivotal role in defending against infection and cancer immunosurveillance during the onset and procession of malignant disease. Cancer patients are frequently immunocompromised and subject to refractory infection and relapse of leukemia, due to the cytotoxic agents and immunosuppressive glucocorticoids in the chemotherapy regimens. Bu Shen Hui Yang Fang (BSHY), a traditional Chinese compound, was widely used in China to enhance the immune system of leukemia patients combined with chemotherapy and effectively lowered their risk of infection, with specific mechanism unknown yet. Thus, we investigated the effects of BSHY on the immune system using immunosuppressive mouse models. By analyzing the immune system of immunosuppressed BALB/C mice induced by hydrocortisone, we found an increase of CD4+ and CD8+ lymphocytes in the spleens of mice after BSHY treatment. Furthermore, we found the enhanced immune system in BSHY treated group was due to increased proliferation and decreased apoptosis of lymphocytes. Cytokine array analysis revealed that interleukin 4 (IL-4) was reduced in the plasma of immunosuppressed mice but returned to a normal level after BSHY treatment. Moreover, we found IL-4 was an adverse prognostic factor in acute myeloid leukemia patients and part of them could be elevated by BSHY. Mechanistically, we found BSHY enhances the proliferation of lymphocytes in a Stat6-dependent manner. In summary, our current study demonstrates that BSHY enhances the proliferation of lymphocytes in the immunosuppressed mice via upregulating IL-4 signaling.

[Level of Microparticles in Patients with Essential Thrombocythemia and Its Relation with JAK2V617F Mutation].

OBJECTIVE: To detect the levels of microparticles (MP) in plasma of patients with esseutial thrombo-cythermia(ET) and analyze the relationship between the JAK2V617F mutant and MP in ET patients. METHODS: The numerical values of MPs were analysed by using flow cytometry. Venous blood of 56 ET patients and 28 healthy persons was collected in the morning and anticoagulated with sodium citrate (1∶9). The RMP, PMP, TF+MP and EMP were detected by FCM using phycoerythrin (PE)-conjugated monoclonal antibodies to CD235a for red blood cells, CD61 for platelets, CD142 for tissue factor (TF) and CD62E for endothelial cells, respectively. Forward scatter was set in scale using fluorescent microspheres of 0.8 µm. Standard fluorescent microbeads (0-0.8 µm) in diameter were used to set the microparticles gate. Genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen 26 JAK2V617F mutations. RESULTS: The detection results showed that the MP levels in ET group were higher than those in normal control group: RMP (157.2±304.9/µl vs 21.3±18.4/µl), PMP (1378.9±2454/µl vs 113.8±97.1/µl), TF+MP (123±354.6/µl vs 21±15.7/µl) and EMP (1434.7±2601.9/µl vs 291.7±297.7/µl) (P<0.05). In 8 patients with thrombus, the levels of these four kinds of MP were higher than those of controls without thrombus: RMP (258.2±327.7/µl vs 55.6±63.8/µl), PMP (1853±1874.7/µl vs 444.7±712/µl), TF+MP (120.4±112.5/µl vs 60.3±128.3/µl) and EMP (1637.1±1563.5/µl vs 629.4±1000.2/µl) (P<0.05). The levels of those 4 kinds of MP in 17 patients with splenomegaly were significantly higher than those in 11 patients without splenomegaly: RMP (306.8±491.1/µl vs 59.3±51/µl), PMP (2944.8±3767.6/µl vs 334.8±420.2/µl), TF+MP (162.9±166.8/µl vs 31.0±28.7/µl) and EMP (3031.8±4048.8/µl vs 701.5±729.2/µl) (P<0.05). The level of other MP showed no difference between MPN patients with JAK2V617F mutation and without JAK2V617F mutation (P>0.05), except TF+MP (154.7±516.3/µl vs 100.5±126.6/µl) (P<0.05). CONCLUSION: The numerical values of MP detected are more in ET patients than those in healthy controls. The number of MP is higher in patients with thrombus than that without thrombus, so do in patients with splenomegaly and without splenomegaly. Patients with JAK2V617F mutation show higher number of TF+MP than that without JAK2V617F mutation. But the other three kinds of MP show no this difference.

Extracts from Glycine max (soybean) induce elastin synthesis and inhibit elastase activity.

Elastic fibres are essential extracellular matrix components of the skin, contributing to its resilience and elasticity. In the course of skin ageing, elastin synthesis is reduced, and elastase activity is accelerated, resulting in skin sagging and reduced skin elasticity. Our studies show that non-denatured Glycine max (soybean) extracts induced elastin promoter activity, inhibited elastase activity and protected elastic fibres from degradation by exogenous elastases in vitro. Mouse and swine skins topically treated with soybean extracts showed enhanced elastic fibre network and increased desmosine content. Elastin expression was also augmented in human skin transplanted onto SCID mice in response to soy treatment. These data suggest that non-denatured soybean extracts may be used as skin care agents to reduce the signs of skin ageing.

[Clinical Efficacy of Autologous Transplantation of Hematopoietic Stem Cells Cryopreserved by Ladder-style Freezing from Low Temperature Refrigerator to Liquid Nitrogen].

OBJECTIVE: To investigate the efficacy of hematopoietic stem cells cryopreserved by ladder-style freezing from low temperature refrigerator to liquid nitrogen in treatment of hematological malignancies, and to analyze the survival condition of patients after hematopoietic stem cell transplantation. METHODS: The coyoprotectant formed by 3% hydroxyethyl starch, 4% albumin and 5% dimethyl sulfoxide (DMSO) was need for cryopreservation of hematopoietic stem cells,which were first placed in -800C low temperature refrigerator and then were stored in -1960C liquid nitrogen tank. 98 cases of hemafologic malignancies (io cases of ALL, 24 cases of AML, L-cases of MM and 53 case of malignant lymphoma) were selected from January 2002 to December 2016, and recived transplantatin auto-hematopoiehc stem cells cryopresorved by above-mentined method. The overall survival rate (OS), progression-free survival (PFS) were analyzed statistically. RESULTS: One case failed in implantation due to intracranial hemorrhage and the other 97 cases all succeeded in hematopoietic reconstitution. The average time needed for neutrophil count ≥0.5×109/L was 9.24±1.89 d, and the average time needed for blood platelet ≥20×109/L without platelet transfusion for 3 days was 11.04±1.84 d. The median survival time was 47.6 months (1-80 months). The 3 and 5 year OS rates were (97.2±1.9) %, (84.2±4.6) % and (77.8±5.6) %, respectively. 3- and 5-year PFS of patients were (74.4±5.1)% and (61.2±6.2)%. CONCLUSION: ladder-style freezing from low temperature refrigerator to liquid nitrogen can reach the same clinical transplantation effect with traditional programmed cooling freezing method in autologous hematopoietic stem cells transplantation. moreover the incidence of complications after transpeantatim does not show increase.

[Impact of JAK2V617F Mutation Burden on Clinial Presentation and Survival in ET Patients].

OBJECTIVE: To better define the effect of JAK2V617F mutant allele burden on clinical presentation of patients with essential thrombo cythamia (ET), especially thrombosis. METHODS: Two ml of heparin anti-coagulated bone marrow was collected from 229 ET cases, who were diagnosed and treated in the First People's Hospital of Yunnan Province during 2013.10 to 2016.12. and then the mononuclear cells were separated by Red Blood Cell Lysis Buffer, genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen the JAK2V617F mutation, then the JAK2V617F mutation burden was detected by real-time polymerase chain reaction (RT-PCR) in 120 patients with JAK2V617F mutation. Meanwhile, these samples were sequenced in order to verify the accuracy of the PCR screewing. RESULTS: ET patients with thrombotic events had significantly higher JAK2V617F allele burden than those without thrombosis (23.2% vs 14.2%) ( P<0.05). Meanwhile, ET patients showed increased JAK2V617F allele burden in the group with higher leukocytosis (WBC > 10×109/L) (P<0.001) and hemoglobin (> 150 g/L) (P<0.05). JAK2V617F mutation burden in 17 patients with splenomegaly was higher than that in 45 patients without splenomegaly (28.1% vs 11.8%) (P<0.05). but the JAK2V617F mutation burden was regatively correlated with platelet count (P<0.05). On the other hand, no correlation was found between JAK2V617F mutation burden and sex (P > 0.05). Univariate analysis showed that the JAK2V617F allele burden did not affect survival. Multivariable analysis showed that prognostic variable including WBC counts, hemoglobin level, age, sex, and splenomegaly not affected survival, (P > 0.05). CONCLUSION: The clinical presentations of ET patients, such as WBC counts, hemoglobin level and splenomegaly, are influenced by the JAK2V617F mutation burden. ET patients with thrombotic events has significantly higher JAK2V617F allele burden than those in ET palients without thrombosis.JAK2V617F mutation burden has no relations with sex and age..

The complete mitochondrial genome of black-footed ferret, Mustela nigripes (Mustela, Mustelinae).

In this study, the complete mitochondrial genome sequence of black-footed ferret, Mustela nigripes, is determined for the first time. This mitogenome is 16,556 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). The overall base composition is A (32.9%), C (26.1%), G (13.8%), and T (27.2%), so the percentage of A and T (60.1%) is higher than that of G and C. Most of the genes are encoded on H-strand, except for the ND6 subunit gene and six tRNA genes. The complete mitochondrial genome sequence reported here would be useful for further phylogenetic analysis and conservation genetic studies in M. nigripes.

Complete mitochondrial genome of the Algerian honeybee, Apis mellifera intermissa (Hymenoptera: Apidae).

In this study, the complete mitochondrial genome sequence of Algerian honeybee, Apis mellifera intermissa, is analyzed for the first time. The results show that this genome is 16,336 bp in length, and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). The overall base composition is A (43.2%), C (9.8%), G (5.6%), and T (41.4%), so the percentage of A and T (84.6%) is considerably higher than that of G and C. All the genes are encoded on H-strand, except for four subunit genes (ND1, ND4, ND4L, and ND5), two rRNA genes (12S and 16S rRNA), and eight tRNA genes. The complete mitochondrial genome sequence reported here would be useful for further phylogenetic analysis and conservation genetic studies in A. m. intermissa.

The Demethylase Activity of FTO (Fat Mass and Obesity Associated Protein) Is Required for Preadipocyte Differentiation.

FTO (fat mass and obesity associated gene) was genetically identified to be associated with body mass index (BMI), presumably through functional regulation of energy homeostasis. However, the cellular and molecular mechanisms by which FTO functions remain largely unknown. Using 3T3-L1 preadipocyte as a model to study the role of FTO in adipogenesis, we demonstrated that FTO is functionally required for 3T3-L1 differentiation. FTO knock-down with siRNA inhibited preadipocyte differentiation, whereas ectopic over-expression of FTO enhanced the process. The demethylase activity of FTO is required for differentiation. Level of N6-methyladenosine (m6A) is decreased in cells over-expressing FTO. In contrast, overexpression of R96Q, a FTO missense mutant lack of demethylase activity, had no effect on cellular m6A level and impeded differentiation. Treatment with Rosiglitazone, a PPARγ agonist, could overcome the differentiation inhibition imposed by R96Q mutant, suggesting the effect of FTO is mediated through PPARγ.

Identification of A Novel Small-Molecule Binding Site of the Fat Mass and Obesity Associated Protein (FTO).

N-(5-Chloro-2,4-dihydroxyphenyl)-1-phenylcyclobutanecarboxamide (N-CDPCB, 1a) is found to be an inhibitor of the fat mass and obesity associated protein (FTO). The crystal structure of human FTO with 1a reveals a novel binding site for the FTO inhibitor and defines the molecular basis for recognition by FTO of the inhibitor. The identification of the new binding site offers new opportunities for further development of selective and potent inhibitors of FTO, which is expected to provide information concerning novel therapeutic targets for treatment of obesity or obesity-associated diseases.

SNPstream UHT: ultra-high throughput SNP genotyping for pharmacogenomics and drug discovery.

Single nucleotide polymorphism (SNP) genotyping is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discovery. To date, genome-wide scans and studies involving thousands of SNPs and samples have been hampered by the lack of a system that can perform genotyping with cost-effective throughput, accuracy, and reliability. To address this need, Orrhid has developed an automated, ultra-high throughput system, SNPstream UHT, which uses multiplexed PCR in conjunction with our next generation SNP-IT tag array single base extension genotyping technology The system employs oligonucleotide microarrays manufactured in a 384-well format on a novel glass-bottomed plate. Multiplexed PCR and genotyping are performed in homogeneous reactions, and assay results are read by direct two-color fluorescence on the SNPstream UHTArray Imager. The systems flexibility enables large projects involving thousands of SNPs and thousands of samples as well as small projects that have hundreds of SNPs and hundreds of samples to be done cost effectively. We have successfully demonstrated this system in greater than 1,000,000 genotyping assays with >96% of samples giving genotypes with >99% accuracy