Technique optimization of intravenous or intraperitoneal injection of Evans blue combined with retinal preparations in mice / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To optimize the technique of intravenous injection of Evans blue and retinal preparations in mice, improving the accuracy and repeatability of staining experiment of retinal preparations.METHODS: C57BL/6 male mice were intravenous injected with 10g/L(1%)Evans Blue 0.3mL and circulated in vivo for 10 or 20min, and the eyes were removed after sacrificed and fixed in 4% paraformaldehyde for 20, 40 or 60min. When failure of intravenous injection, the experiment was remediated by intraperitoneal injection of 1% Evans Blue 0.3mL, circulated in vivo for 3h and fixed for 60min to observe morphology, distribution and leakage of the retinal vessels. Besides, we compared the morphology, distribution and leakage of the retinal vessels after intravenous injection with those after intraperitoneal injection to determine the optimal conditions for in vivo circulation time and retinal preparations.RESULTS: After intravenous injection, compared to the retinal vascular condition under 20min in vivo circulation time of Evans blue and 20 or 40min of fixation, with 10min of in vivo Evans blue circulation and 60min of fixation, the morphology of retinal vascular was more intact with less retinal vascular leakage, and the vascular branches are clear. When intravenous injection failed, remediated results from intraperitoneal injection showed that the morphology and distribution of retinal vessels were intact. There was no significant difference in morphology, distribution and leakage of the retinal vessels after 3h of intraperitoneal Evans blue circulation compared to 10min intravenous Evans blue circulation.CONCLUSION: This experiment optimizes the protocol, improves the accuracy and reproducibility of retinal preparations, and provides a reference for the study of related retinal vascular diseases.

Role of NLRP3 Inflammasome in Bone and Joint Diseases and Traditional Chinese Medicine Intervention： A Review / 中国实验方剂学杂志

Nucleotide binding oligomeric dome-like receptor protein 3 （NLRP3） inflammasome is an intracellular sensing protein complex， and it is an important player in the innate immune system， capable of sensing foreign pathogens and endogenous danger signals. After tissue injury， the activation of the NLRP3 inflammasome induces the release of the pro-inflammatory cytokines interleukin （IL）-1β and IL-18， while promoting gasdermin D-mediated pyroptosis. Existing studies have shown that NLRP3 inflammasome plays a key role in the occurrence and development of common bone and joint diseases such as osteoporosis， osteoarthritis， rheumatoid arthritis， and gouty arthritis by inducing inflammatory cascade reaction and accelerating bone resorption and cartilage destruction. Therefore， blocking the NLRP3 inflammasome signaling pathway may be an effective strategy to treat or prevent bone and joint diseases. Currently， researchers have developed and tested several drugs that selectively target the NLRP3 inflammasome in animal and clinical studies， but the progress has been poor due to obvious side effects and high prices. Traditional Chinese medicine （TCM） has been widely recognized in the treatment of bone and joint diseases due to its unique advantages of multi-target， multi-pathway， multi-mechanism synergism， low price， and low side effects. With the deepening of research， the targeted intervention of NLRP3 inflammasome by TCM in the treatment of bone and joint diseases has attracted wide attention. In this paper， the mechanism of NLRP3 inflammasome in osteoarthritis was summarized by analyzing relevant literature in China and abroad in recent years， and the progress of targeted intervention of NLRP3 inflammasome by TCM in the treatment of bone and joint diseases was systematically reviewed， so as to provide new ideas and theoretical basis for the treatment of bone and joint diseases.

Clinical Characteristics and Prognostic Factors of Primary FL Patients with Grade 3 or Large B Cell Transformation / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical characteristics and prognostic factors of primary follicular lymphoma (FL) patients with grade 3 or large B cell transformation, so as to provide more reference for the subsequent clinical diagnosis and treatment.@*METHODS@#Forty-seven primary FL patients with grade 3 or large B cell transformation from March 2010 to March 2018 were selected, the clinical characteristics and survival of patients were analyzed. Cox regression model were used to evaluate the related prognostic factors.@*RESULTS@#The cumulative progression-free survival rate and cumulative overall survival rate of 47 patients in 3-year follow-up reached to 55.32% (26/47) and 80.85% (38/47) respectively. There were significant differences in cumulative progression-free survival rate and cumulative overall survival rate among different subgroups of IPI, FLIPI-1 and FLIPI-2 in 3-year follow-up (P3 cm lymph node-involved site number≥3, extranodal lesion site number≥2, IPI score=2-3, FLIPI-1 score and FLIPI-2 score≥3 were the risk factors for progression-free survival (P<0.05); LDH≥240 U/ml, IPI score=2-3 and FLIPI-2 score≥3 were risk factors for overall survival (P<0.05). Cox regression model multivariate analysis showed that IPI score=2-3 was the independent risk factor for progression-free survival and overall survival (P<0.05). FLIPI-2 score≥3 was the independent risk factor for overall survival (P<0.05).@*CONCLUSION@#Primary FL patients with grade 3 or large B cell transformation by using the existing treatment regimen might be possibly curable, and the current treatment strategies and IPI score can be used to predict the clinical prognosis of patients.

Different effects of long-term and short-term repeated restraints on the hematopoietic stem cells in mice / 生理学报

Humans with chronic psychological stress are prone to develop multiple disorders of body function including impairment of immune system. Chronic psychological stress has been reported to have negative effects on body immune system. However, the underlying mechanisms have not been clearly demonstrated. All immune cells are derived from hematopoietic stem cells (HSC) in the bone marrow, including myeloid cells which comprise the innate immunity as a pivotal component. In this study, to explore the effects of chronic psychological stress on HSC and myeloid cells, different repeated restraint sessions were applied, including long-term mild restraint in which mice were individually subjected to a 2 h restraint session twice daily (morning and afternoon/between 9:00 and 17:00) for 4 weeks, and short-term vigorous restraint in which mice were individually subjected to a 16 h restraint session (from 17:00 to 9:00 next day) for 5 days. At the end of restraint, mice were sacrificed and the total cell numbers in the bone marrow and peripheral blood were measured by cell counting. The proportions and absolute numbers of HSC (LinCD117Sca1CD150CD48) and myeloid cells (CD11bLy6C) were detected by fluorescence activated cell sorting (FACS) analysis. Proliferation of HSC was measured by BrdU incorporation assay. The results indicated that the absolute number of HSC was increased upon long-term mild restraint, but was decreased upon short-term vigorous restraint with impaired proliferation. Both long-term mild restraint and short-term vigorous restraint led to the accumulation of CD11bLy6C cells in the bone marrow as well as in the peripheral blood, as indicated by the absolute cell numbers. Taken together, long-term chronic stress led to increased ratio and absolute number of HSC in mice, while short-term stress had opposite effects, which suggests that stress-induced accumulation of CD11bLy6C myeloid cells might not result from increased number of HSC.

Effect of MiRNA-145 on Apoptosis of Leukemia HuT 78 Cells and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect and mechanism of miRNA-145 on leukemic cell apoptosis.@*METHODS@#After transfection of miRNA-145 mimic and negative control mimic in leukemia cells by Lipofectamine 2000 liposome, the MTT assay was used to detect the effect of miRNA-145 on cell proliferation. Flow cytometry was used to detect the effect of miRNA-145 on cell cycle and apoptosis. Western blotting assay was used to detect the expression levels of BCL-2, CDK6, Cyclin D1, BAX, PI3K p-PI3K, p-AKT and AKT.@*RESULTS@#The relative level of microRNA in HuT 78 cells transfected with miRNA-145 was 2.3±02, which was significantly higher than that in blank control group and miRNA-NC group (P＜0.05). MTT assay showed that the proliferation level of HuT 78 cells transfected with miRNA-145 mimic was significantly lower than that of blank control and miRNA-NC group (P＜0.05). Flow cytometry showed that the cells at G/G, S and G2/M phase of HuT 78 cells were significantly decreased after transfection with miRNA-145 mimic (P＜0.05). Annexin V/PI double staining assay showed that the apoptosis rate of HuT 78 cells was 17.6%±3.4%，which was significantly higher than that in blank control group and miRNA-NC group (P＜0.05). Western blot showed that the expression levels of BCL-2, CDK6 and Cyclin D1 in HuT 78 cells were significantly lower than those in blank control and miRNA-NC group (P＜0.05), and BAX expression in HuT 78 cells was significantly higher than that in blank control and miRNA-NC group (P＜0.05). Western blot showed that expression of PI3K, p-PI3K, AKT and p-AKT in HuT 78 cells transfected with miRNA-145 mimic were significantly lower than that in blank control and miRNA-NC group (P＜0.05).@*CONCLUSION@#Upregulation of miRNA-145 may inhibit the proliferation of leukemia cells and promote the apoptosis, which may be related with the inhibition of PI3K/AKT signaling pathway.

Clinical Efficiency and Safety of the First-line CHOP Regimen Containing PLD Applied to Treat Aged Patients with Advanced DLBCL / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the clinical efficiency and safety of CHOP regimen containing pegylated liposomal doxorubicin (PLD) for the aged patients with advanced diffuse large B-cell lymphoma (DLBCL).</p><p><b>METHODS</b>Fifty aged patients with advanced DLBCL treated in our hospital from February 2010 to February 2014 were selected and divided into two groups. Out of 50 cases, 25 cases received standard CHOP regimen (sCHOP group), other 25 cases received CHOP regimen containing PLD at dose of 30 mg/m2 (PLD+CHOP). These patients were followed up for 18 months, and the total effective rate, the survival rate and the adverse reaction rate were compared between these two groups.</p><p><b>RESULTS</b>After receiving different treatments, the survival rate of patients on 6, 12 and 18 months in PLD+CHOP group was 88.0%, 80.0% and 76.0%, respectively, and the survival rate of 18 month was significantly higher than that in the sCHOP group (P<0.05); The total effective rate in the PLD+CHOP group was statistically higher than that in the sCHOP group (P<0.05); and all the incidences of non-hematological toxicity, peripheral sensory neuropathy, lung infection, gastrointestinal reaction and hepatotoxicity were not statistically different between two groups (P>0.05), while the incidence of cardiac toxicity including acute myocardial infarction, congestive heart failure, atrioventricular block (AV block) and paroxysmal atrial tachycardia significantly decreased in the PLD+CHOP group (P<0.05).</p><p><b>CONCLUSION</b>The efficiency of CHOP regimen containing PLD for the aged patients with advanced DLBCL has been confirmed to be significant, and its cardiac toxicity is low, thus being worth to be popularized and applied for the treatment of advanced diffuse large B-cell lymphoma.</p>

Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study.</p><p><b>METHODS</b>A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography.</p><p><b>RESULTS</b>Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)₃ adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10⁶ CFU of Y. pestis virulent strain 141.</p><p><b>CONCLUSION</b>The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.</p>

Barbiturate infusion for intractable intracranial hypertension and its effect on brain tissue oxygen / 中国医师杂志

Objective To examine the barbiturate infusion for intractable intracranial hypertension and its effect on brain tissue oxygen ( PbtO2 ). Method 60 patients with intractable intracranial hypertension were divided into tow groups, experimental group and control group. PbtO2 and intracranial pressure (ICP) were continuous monitored. Follow-up was more than 3 months and the prognosis was compared between two groups. Results When pentobarbital administration began, the mean PbtO2 [ 72 h: (26. 7 ±6. 7)mmHg] at 24h, 48h and 72h in experimental group were significantly higher than that of control group [ 72 h: ( 21. 1 ± 7. 2) mmHg ] ( P < 0. 05 ). The mean ICP [ 5 d: (2. 48 ± 1.11 ) kPa ] in the third and fifth day of experimental group were lower than that of control group [ 5 d: (3. 12 ± 1.09 ) kPa ]. Prognosis of pentobarbital group was better than control group( P < 0. 05 ). Conclusions Pentobarbital can effectively reduce intracranial pressure in patients with refractory intracranial hypertension and improve brain tissue oxygen. PbtO2 is an ideal monitoring marker, and it can predict prognosis to a certain extent.

High-level production of neutral endoglucanase 1 in Pichia pastoris / 生物工程学报

The gene (eg1) encoding for novel endoglucanase 1 was cloned previously from Chinese straw mushroom Volvariella volvacea. EG1 has high thermal stability and optimal pH at neutral and shows great potential in textile and paper industry applications. To improve the expression level of EG1 in Pichia pastoris, the increasing copy number of clone, and its high cell density fermentation in 3.2L fermenter for its high-level expression were investigated in this work. By electro-transformation of pPICZalphaB-egl into GS115EG11 integrated with single copy of eg1 gene, A resistant transformant with 3.8 times higher level expression than GS115EG11 was screened from YPDSZ plate containing 2000 microg/mL of Zeocin. The effect of initial cell density, pH and methanol on its expression and biomass accumulation was evaluated in shaking culture. Optimal EG1 production was observed when initial cell density OD600 was 5.0. EG1 production and biomass accumulation did not seem to vary when cells were induced at different pH values. Both of EG1 and cell density were found to increase with higher methanol concentrations, reaching 62.48 IU/mL and 31.7 (OD600) respectively after 120 h induction with 2.0% (V/V) methanol compared to 30.24 IU/mL and 17.79 (OD60) with 0.25% methanol induction. EG1 expression was further increased by 6.4 times higher than shaking culture after 95.5 hours induction with methanol in fed-batch fermentation, so totally 34 times higher than that for GS115EG11 was achieved by screening of high Zeocin resistant clone and high cell density fermentation. The production of EG1 with 543.36IU/mL CMC activity and 8.80mg/mL protein expression was obtained in Pichia pastoris.

Effect of Indapamide on the Rehabilitation of Cardiac Function in Patients with Primary Hypertension Disease / 中国康复理论与实践

@#ObjectiveTo observe the effect of Indapamide on the rehabilitation of cardiac function in patients with primary hypertension disease.Methods56 patients with primary hypertension disease were randomly divided into the Indapamide group and Hydrochlorothiazide group with 28 cases in each group, and treated with Indapamide and Hydrochlorothiazide respectively assisted with conventional non-drug treatment such as dietetic and weight control. The cardiac functions of patients in two groups were examined with echocardiography before and two months after treatment.ResultsAfter treatment, the left ventricular ejection fraction (LVEF) raised and relative wall thickness (RWT) declined in patients of two groups, but the effect of the Indapamide group was better than that of the Hydrochlorothiazide group ( P<0.05).ConclusionThe effect of Indapamide on cardiac function and structure is better than Hydrochlorothiazide.