Clinical characteristics and predictive value of lower CD4+T cell level in patients with moderate and severe COVID-19: a multicenter retrospective study.

BACKGROUND: In December 2019, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, Hubei, China. Moreover, it has become a global pandemic. This is of great value in describing the clinical symptoms of COVID-19 patients in detail and looking for markers which are significant to predict the prognosis of COVID-19 patients. METHODS: In this multicenter, retrospective study, 476 patients with COVID-19 were enrolled from a consecutive series. After screening, a total of 395 patients were included in this study. All-cause death was the primary endpoint. All patients were followed up from admission till discharge or death. RESULTS: The main symptoms observed in the study included fever on admission, cough, fatigue, and shortness of breath. The most common comorbidities were hypertension and diabetes mellitus. Patients with lower CD4+T cell level were older and more often male compared to those with higher CD4+T cell level. Reduced CD8+T cell level was an indicator of the severity of COVID-19. Both decreased CD4+T [HR:13.659; 95%CI: 3.235-57.671] and CD8+T [HR: 10.883; 95%CI: 3.277-36.145] cell levels were associated with in-hospital death in COVID-19 patients, but only the decrease of CD4+T cell level was an independent predictor of in-hospital death in COVID-19 patients. CONCLUSIONS: Reductions in lymphocytes and lymphocyte subsets were common in COVID-19 patients, especially in severe cases of COVID-19. It was the CD8+T cell level, not the CD4+T cell level, that reflected the severity of the patient's disease. Only reduced CD4+T cell level was independently associated with increased in-hospital death in COVID-19 patients. TRIAL REGISTRATION: Prognostic Factors of Patients With COVID-19, NCT04292964 . Registered 03 March 2020. Retrospectively registered.

Formation of heat-induced cottonseed congossypin(7S) fibrils at pH 2.0.

BACKGROUND: Heat-induced protein aggregation is important for the texture of various food products. Many types of food proteins have been found to assemble into fibrillar structures under certain conditions. We studied fibril formation of cottonseed 7S storage protein upon heating (for 0-720 min) at 90°C and pH 2.0, investigated the conversion rate, and determined the extent of thermal aggregation. RESULTS: Thioflavin-T fluorescence and Congo-red analysis indicated the formation of amyloid-like fibrils upon heating. Centrifugal filtration indicated that the conversion was very low (<10%) until congossypin concentration up to 2 mg mL(-1), and the conversion increases with increasing heating time, but levels off after longer heating times. Dynamic light scattering and atomic force microscopy showed that the extent of thermal aggregation at pH 2.0, or contour length of the worm-like and fine-stranded aggregates, progressively increased with increasing heating time. Furthermore, reducing electrophoresis analyses indicated that progressive polypeptide hydrolysis occurred upon heating. Experiments indicate that congossypin can form heat-induced amyloid-like aggregates and the conversion of congossypin monomers into fibrils increased with heating time and protein concentration. CONCLUSION: The results would be of vital importance for the utilisation of cottonseed proteins to produce thermally induced fibrillar gels with excellent properties.

Application of neoadjuvant chemoradiotherapy and neoadjuvant chemotherapy in curative surgery for esophageal cancer: A meta-analysis.

BACKGROUND: The effectiveness of neoadjuvant therapy in esophageal cancer (EC) treatment is still a subject of debate. AIM: To compare the clinical efficacy and toxic side effects between neoadjuvant chemoradiotherapy (nCRT) and neoadjuvant chemotherapy (nCT) for locally advanced EC (LAEC). METHODS: A comprehensive search was conducted using multiple databases, including PubMed, EMBASE, MEDLINE, Science Direct, The Cochrane Library, China National Knowledge Infrastructure, Wanfang Database, Chinese Science and Technology Journal Database, and Chinese Biomedical Literature Database Article. Studies up to December 2022 comparing nCRT and nCT in patients with EC were selected. RESULTS: The analysis revealed significant differences between nCRT and nCT in terms of disease-free survival. The results indicated that nCRT provided better outcomes in terms of the 3-year overall survival rate (OSR) [odds ratio (OR) = 0.95], complete response rate (OR = 3.15), and R0 clearance rate (CR) (OR = 2.25). However, nCT demonstrated a better 5-year OSR (OR = 1.02) than nCRT. Moreover, when compared to nCRT, nCT showed reduced risks of cardiac complications (OR = 1.15) and pulmonary complications (OR = 1.30). CONCLUSION: Overall, both nCRT and nCT were effective in terms of survival outcomes for LAEC. However, nCT exhibited better performance in terms of postoperative complications.

[Pilose antler polypeptide CNT14 promoted proliferation and migration of human oral mucosa fibroblast cells].

PURPOSE: To explore the effect of pilose antler polypeptides CNT14 on proliferation and migration of human oral mucosa fibroblast (hOMF) cells and the related molecular mechanism. METHODS: The biosafety of pilose antler polypeptides CNT14 on hOMF cells was verified by live-dead cell staining kit.CCK-8 assay was used to detect the effect of pilose antler polypeptides CNT14 on hOMF cell proliferation. The effect of pilose antler polypeptides CNT14 on hOMF cell migration was detected by scratch test. Western blot was used to detect the expression of α-SMA, TGF-ß1, Smad2 and p-Smad2 proteins in hOMF cells stimulated by pilose antler polypeptides CNT14. The effect of Smad2 inhibitors on fibroblast activation induced by pilose antler polypeptides CNT14 was evaluated.The model of keratinized gingival defect was established in New Zealand white rabbits, and the regenerated gingival tissue was stained with H-E. The expression levels of α-SMA, TGF-ß1, Smad2 and p-Smad2 proteins in the gingival tissues of regenerated New Zealand white rabbits were detected by immunohistochemistry, and the ability of pilose antler polypeptides CNT14 to promote regeneration of oral gingival tissues was verified. Statistical analysis was performed with SPSS 20.0 software package. RESULTS: The survival rate of hOMF cells was above 95% after treated with pilose antler polypeptides CNT14. After stimulation of hOMF cells with pilose antler polypeptides CNT14, the proliferation and migration rates of hOMF cells were increased compared with the control group (P＜0.05). The expression of α-SMA, TGF-ß1, Smad2 and p-Smad2 proteins in hOMF cells stimulated by pilose antler peptide CNT14 was increased, and the difference was statistically significant(P＜0.05). The expression of α-SMA in fibroblasts induced by Smad2 inhibitor was decreased. In animal experiments, H-E staining showed that the inflammatory response of oral mucosal wounds of New Zealand white rabbits treated with CNT14 was less than that of the control group. Immunohistochemical staining results showed that the expressions of α-SMA, TGF-ß1, Smad2 and p-Smad2 in the regenerated gingival tissues of New Zealand white rabbits treated with CNT14 were significantly increased compared with those in the control group on the 9th and 11th days within the gingival wounds(P＜0.05). CONCLUSIONS: Pilose antler polypeptides CNT14 has good biosafety and can promote the proliferation and migration of human oral mucosa fibroblast cells, and the expression levels of α-SMA, TGF-ß1, Smad2 and p-Smad2 were increased, promoting the regeneration of gingival tissues.

Enhancement on antioxidant, anti-hyperglycemic and antibacterial activities of blackberry anthocyanins by processes optimization involving extraction and purification.

This research aimed to recover anthocyanin-rich extracts from blackberry (Rubus spp. Hull cultivar) by optimizing the processing conditions, and to characterize anthocyanin individuals and determine influences of optimization on enhancement of antioxidant and anti-hyperglycemic activities of anthocyanins as natural supplements. The ethanol concentration of 69.87%, HCl dosage of 0.53%, solid-to-liquid ratio of 1:19.06 at 47.68°C for 17.04 h were optimal to obtain the highest extraction yield of anthocyanins at 0.72 mg/g. By using AB-8 macroporous resins, the anthocyanin concentration of 3.0 mg/mL, ethanol concentration of 90%, and elution rate of 2.0 mL/min were selected to boost the anthocyanin purity up to be 60.11%. Moreover, the purified anthocyanin extracts from blackberry contained nine main pigments which could be divided into three aglycone-based forms, and cyanidin-3-O-glucoside was the most abundant among them. Due to the successive processes of extraction and purification, the blackberry purified anthocyanin extracts (BA-PAE) showed much higher bioactive capacities than the blackberry crude anthocyanin extracts (BA-CAE) and blackberry fruit slurry extracts (BA-FSE), e.g., DPPH and ABTS radical scavenging activities (EC50 = 0.08 and 0.04, 0.32 and 0.24, and 1.31 and 0.41 mg/mL), oxygen radical absorbance capacity (1.60, 0.59, and 0.15 mmol TEAC/g), cytoprotective effects against oxidative stress in PC12 cells (1.69-, 1.58-, and 1.50-fold cell viability compared to oxidative group), α-amylase and α-glucosidase inhibitory activities (IC50 = 0.10 and 0.06, 0.56 and 0.32, and 3.98 and 2.16 mg/mL), and antibacterial activity (93.23, 40.85, and 80.42% reduced biofilm).

TGFbeta/BMP type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.

[Evaluation of shensongyangxin capsules in the treatment of paroxysmal atrial fibrillation: a randomized, double-blind and controlled multicenter trial].

OBJECTIVE: To evaluate the efficacy and safety of Chinese medicinal shensongyangxin capsules in the treatment of paroxysmal atrial fibrillation. METHODS: From August 2007 to July 2008, Beijing Chaoyang Hospital conducted a multicenter study, select the eleven hospital's outpatient subjects, aged 18 to 75 years old, male or female, paroxysmal atrial fibrillation (at least one electrocardiogram diagnosis) seizure frequency ≥ 2 times/month, according to the ratio 1:1:1, subjects were randomly divided into three groups: a. shensongyangxin group, taking shensongyangxin capsule 4 + propafenone analogues 150 mg, 3 times a day; b. propafenone group, taking propafenone tablets 150 mg + 4 shensongyangxin analogues, 3 times a day; shensongyangxin capsule + propafenone group, taking shensongyangxin capsule 4 + propafenone 150 mg, 3 times a day. The treatment course is 8 weeks, with 3 times of follow-up. RESULTS: Total of 349 cases of paroxysmal atrial fibrillation, which 117 cases in shensongyangxin group, 115 cases in propafenone group; 117 cases in shensongyangxin + propafenone group. The baseline data analysis showed that there were no significantly difference (P > 0.05) among the three groups of atrial fibrillation seizure frequency, vital signs, general condition, medical history, 24-hour ambulatory ECG, 12-lead normal electrocardiogram, cardiac ultrasound and symptoms. The comparison before and after (8 weeks) treatment showed that the frequency (from 6 times/m to 2 times/m in each group, P < 0.01), number of cases [from 46 (43.3%) to 22 (20.8%), 43 (43.4%) to 25 (25.3%), and 40 (40.6%) to 31 (29.2%), respectively P < 0.01] and duration time of attack of atrial fibrillation (from 4 h to 0.5 h, 4 h to 0.5 h, and 4.25 h to 0.5 h, respectively P < 0.01) all decreased in three groups. No significant difference among the three groups comparing the overall effect (62.3%, 58.6%, and 58.5%, respectively, P > 0.05), while the efficacy of TCM symptoms in shensongyangxin group (80.2%) was better than that of propafenone group (67.7%) (P < 0.05). Safety evaluation showed that adverse reaction rate was 1.8% in shensongyangxin group, and 8.2% and 5.4% in propafenone group and shensongyangxin + propafenone group. CONCLUSION: Shensongyangxin capsules and propafenone have comparable efficacies in the treatment of PAF. The efficacy of TCM symptoms is better than propafenone. Shensongyangxin capsules have an excellent profile of safety.

Peripheral artery disease and clinical outcomes in patients with atrial fibrillation: A systematic review and meta-analysis.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac rhythm disturbance and leads to morbidity and mortality. Peripheral artery disease (PAD) is associated with atherosclerotic risk factors and always classified as a vascular disease and deemed to be a bad complication of AF. In patients with AF, the risk and prognostic value of PAD have not been estimated comprehensively. HYPOTHESIS: PAD is associated with all-cause mortality, cardiovascular (CV) mortality, and other outcomes in patients with AF. METHODS: We searched PubMed, Embase, and Cochrane Library databases for prospective studies published before April 2021 that provided outcomes data on PAD in confirmed patients with AF. Heterogeneity was estimated using the I2 statistic. The fixed-effects model was used for low to moderate heterogeneity studies, and the random-effects model was used for high heterogeneity studies. RESULTS: Eight prospective studies (Newcastle-Ottawa score range, 7-8) with 39 654 patients were enrolled. We found a significant association between PAD and all-cause mortality (hazard ratio [HR], 1.42; 95% confidence interval [CI], 1.25-1.62; p < .001), CV mortality (HR, 1.64; 95% CI, 1.32-2.05; p < .001) and MACE (HR, 1.75; 95% CI, 1.38-2.22; p < .001) in patients with AF. No significant relationship was found in major bleeding (HR, 1.22; 95% CI, 0.95-1.57; p = 0.118), myocardial infarction (MI) (HR, 2.07; 95% CI, 1.17-3.67; p = .038), and stroke (HR, 1.14; 95% CI, 0.87-1.50, p = 0.351). CONCLUSIONS: PAD is associated with an increased risk of all-cause mortality, CV mortality, and MACE in patients with AF. However, no significant association was found with major bleeding, MI, and stroke.

Hulless Black Barley as a Carrier of Probiotics and a Supplement Rich in Phenolics Targeting Against H2O2-Induced Oxidative Injuries in Human Hepatocarcinoma Cells.

Lactic acid bacteria can provide benefits to human beings and transform phenolic substances to improve their potential functionality. It was of interest to develop black barley as a carrier of probiotics and nutraceutical supplement rich in more antioxidants. Due to fermentation, bacterial counting and free phenolic content in black barley increased to 9.54 ± 0.22 log cfu/mL and 5.61 ± 0.02 mg GAE/mL, respectively. Eleven phenolic compounds, including nine isoflavones and two nitrogenous compounds were characterized using UPLC-QTOF-MS, among which epicatechin, hordatine, and pelargonidin aglycone were largely enriched. Moreover, free phenolic extracts from fermented barley (F-BPE) played a greater role in scavenging DPPH radicals, reducing Fe3+ to Fe2+, and increasing oxygen radical absorbance capacity, compared phenolic extracts from unfermented barley [UF-BPE (1.94-, 1.71-, and 1.35-fold at maximum for F-BPE vs. UF-BPE, respectively)]. In hepatocarcinoma cells, F-BPE also better inhibited ROS production and improved cell viability, cell membrane integrity, SOD activity, and non-enzymatic antioxidant GSH redox status (2.85-, 3.28-, 2.05-, 6.42-, and 3.99-fold at maximum for F-BPE vs. UF-BPE, respectively).

Effects of lactic acid bacteria fermented yellow whey on the protein coagulation and isoflavones distribution in soymilk.

This study investigated the soymilk coagulation induced by fermented yellow whey (FYW), which is extensively used as a natural tofu coagulant in China. The aggregations involving proteins and isoflavone particles caused by FYW were analyzed using the proteomic technology and high-performance liquid chromatography, respectively. As indicated, the FYW-induced coagulation of soy proteins mainly occurred at pH 5.80-5.90. When the pH of soymilk decreased, the 7S ß, 11S A3 and some of 11S A1a subunits and SBP, Bd, lectin and TA aggregated the earliest, and later did the 11S A4, other 11S A1a, 11S A2 and 11S A1b subunits. The 7S α and α' subunits and TB showed an obvious delay in aggregation. Moreover, isoflavones in the form of aglycones were more likely to coprecipitate with proteins, compared with glycosides. These results could provide an important reference and assistance for future research on the development of traditional FYW-tofu.

[Effects of atorvastatin calcium on osteopontin in renovascular hypertensive rats].

OBJECTIVE: To investigate the effects of atorvastatin calcium on development of vascular remodeling and osteopontin (OPN) in renovascular hypertensive rats. METHODS: The 2-kidney, 1-clip hypertensive rats were prepared with SD rats. Twenty-one rats were randomly selected seven rats as the control group (n=7), the remaining rats were randomly divided into hypertension group (n=7) and the statins group (n=7) after the establishment of model. Statins group was treated with atorvastatin calcium 30 mg/kg/d four weeks after model construction and then given through lavage for 8 consecutive weeks. All rats were evaluated for caudal systolic blood pressure (SBP) and blood lipid, microscopic changes in vascular structure and ratio of intima media thickness to lumen diameter (MT/LD) of thoracic aorta. Expressions of OPN were assessed with immunohistochemical staining, and expressions of OPN mRNA with fluorescence quantitative PCR. RESULTS: Compared with control group, hypertension group showed significant increase in SBP, MT/LD, and the expression of OPN and OPN mRNA, and significant decrease in HDL level. As compared with hypertension group, MT/LD and the expression of OPN and OPN mRNA of statins group were significantly decreased, while its HDL level significantly increased. CONCLUSION: Hypertension can lead to high expression of artery OPN, atorvastatin calcium was shown to improve the development of vascular remodeling in rats, possibly associated with decreased expression of OPN.

[Clinical application of bridge-link combined fixation system for adult mid-shaft clavicle fractures].

OBJECTIVE: To explore clinical efficacy of bridge-link combined fixation system for adult mid-shaft clavicle fractures. METHODS: From January 2016 to August 2016, 28 patients with mid-shaft clavicle fractures were treated by bridge-link combined fixation system, including 15 males and 13 females, aged from 27 to 82 years old with an average of(48.50±15.34) years old; the courses of disease was for 13 to 15 months with an average of (14.17±0.77) months. Fracture healing time and complication were observed, postoperative and postoperative Constant function score of shoulder joint at 1, 3 and 13 months and were compared. RESULTS: All patients were followed up for 13 to 15 months with an average of (14.17±0.77) months. Twenty-eight patients achieved clinical fracture healing without infection, bone un-union, delayed union, breakage of internal fixation and re-fracture after removing of the internal fixation occurred. Fracture healing time ranged from 2.5 to 4 months with (3.05±0.44) months. Postoperative Constant score at 1, 3 and 13 months were 76.57±4.70, 90.75±3.62, 96.07±2.40 respectively, and had significant difference compared with before operation(58.36±4.98). CONCLUSIONS: Bridge-link combined fixation system could be used as a new internal fixation for adult mid-shaft clavicle fractures, which has advantages of rapid recovery, less complications, and could reduce incidence of breakage of internal fixation, osteoporosis, re-fractures after removing the internal fixation.

Protective Effects of Blueberry Anthocyanins against H2O2-Induced Oxidative Injuries in Human Retinal Pigment Epithelial Cells.

Blueberry anthocyanins are considered protective of eye health because of their recognized antioxidant properties. In this study, blueberry anthocyanin extract (BAE), malvidin (Mv), malvidin-3-glucoside (Mv-3-glc), and malvidin-3-galactoside (Mv-3-gal) all reduced H2O2-induced oxidative stress by decreasing the levels of reactive oxygen species and malondialdehyde and increasing the levels of superoxide dismutase, catalase, and glutathione peroxidase in human retinal pigment epithelial cells. BAE and the anthocyanin standards enhanced cell viability from 63.69 ± 3.36 to 86.57 ± 6.92% (BAE), 115.72 ± 23.41% (Mv), 98.15 ± 9.39% (Mv-3-glc), and 127.97 ± 20.09% (Mv-3-gal) and significantly inhibited cell apoptosis (P < 0.01 for all). Mitogen-activated-protein-kinase pathways, including ERK1/2 and p38, were involved in the bioactivities. In addition, the anthocyanins decreased vascular-endothelial-cell-growth-factor levels and activated Akt-signal pathways. These combined results supported the hypothesis that blueberry anthocyanins could inhibit the induction and progression of age-related macular degeneration (AMD) through antioxidant mechanisms.

[Study of altered expression of annexin IV and human endometrial receptivity].

OBJECTIVE: To explore the association of altered expression of annexin IV in human endometrium during the implantation window and endometrial receptivity. METHODS: A comparative proteomic strategy, in a combination of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), was adopted to search for proteome alternations of pre-receptive (day LH + 2) versus receptive (LH + 7) endometria. The location and abundance of the identified differentially expressed protein- annexin IV were analyzed by immunostaining and western blot. RESULTS: By comparing protein profiles of LH + 2 and LH + 7 samples, we found a protein up-regulated 2.12 times in LH + 7 samples, with a relative molecular weight of 36,000 and an isoelectric point near pH 5.8. It was characterized using mass spectrometry and was identified as annexin IV. Immunohistochemical analysis revealed an altered localization of annexin IV--in the epithelia on day LH + 2, and both in the epithelia and stroma cells on day LH + 7. Protein levels of annexin IV were up-regulated on day LH + 7 compared with that on day LH + 2 by Western blot. Integrated optical density of the object (OPTDI) was 46.249 +/- 32.376 and 249.507 +/- 31.959, respectively (P = 0.004). CONCLUSIONS: Our study indicates endometrial samples obtained by microbiopsy are available for proteomics studies. It seems possible that the increased expression of annexin IV during the implantation window plays an important role in the morphological differentiation of the uterus to the receptive state.

[Effect of Erigeron breviscapus injection on ventricular and vascular remodeling in spontaneous hypertension rats].

OBJECTIVE: To observe the ventricular and vascular remodeling reversal effect of Erigeron breviscapus injection (EBI), a protein kinase C inhibitor, in spontaneous hypertension rats (SHR). METHODS: Twenty-four SHR were divided into 4 groups, they were treated respectively with EBI, Fosinopril, Enalapril and normal saline 10 mg/kg per day by intraperitoneal injection for 8 weeks. Systolic blood pressure (SBP), ventricular weight index (VWI) and protein kinase C (PKC) activity in myocardial cells were determined, ultrastructural changes of heart and vessel were observed by polaroscope and transmission electron microscope, and the area and content of myocardial interstitial collagen (MIC) were determined by image analyzer system. RESULTS: The left ventricular hypertrophy was regressed to certain degree after EBI, Fosinopril and Enalapril treatment, but no significant change in heart rate and right VWI was found. Fosinopril and Enalapril were superior to EBI in lowering SBP and left VWI, and EBI was more obvious in improving myocardial ultrastructure such as hypertrophy and degeneration. All the 3 drugs could improve the MIC and vascular remodeling, the MIC area, content and collagen volume fraction in the EBI group were lowered after treatment, as compared with those in the control group, but comparison between the three groups showed no significant difference. The 3 drugs could reduce the PKC activity in myocardial cell membrane, and EBI showed the effect more significant than that of the other two (P < 0.05). CONCLUSION: EBI could reverse the myocardial, interstitial and vascular remodeling, improve the rigidness of cardiac muscle, thus, has protective effect on heart.

[Effects of angiotensin-(1-7) on remodeling of myocardial collagen network in pressure-overloaded rats].

OBJECTIVE: To investigate the effects of angiotensin-(1-7) on remodeling of myocardial collagen network in pressure-overloaded rats. METHODS: A rat model of pressure-overloaded heart was induced by constriction of abdominal aorta. Seventy-five male Sprague Dawley rats were randomized to sham-operated group, model control group and angiotensin-(1-7) group. The rats of the latter two groups were treated with intravenous infusion of either angiotensin-(1-7) (25 microg x kg(-1) x h(-1)) or saline after operation. Ratio of left ventricular weight to body weight (LVW/BW) and myocardial collagen volume fraction (CVF) were determined at 1 and 4 weeks after operation. And types I and III collagen mRNA expressions were assessed with reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: At 1 week after the operation, types I and III collagen mRNA expressions of the left ventricular myocardium in model group and angiotensin-(1-7) group were increased significantly compared with those in sham-operated group. But those in angiotensin-(1-7) group were significantly lower than that in model group. At 4 weeks after the operation, LVW/BW and CVF were increased significantly in model group and angiotension-(1-7) group compared with those in sham-operated group, but those in angiotension-(1-7) group were significantly lower than that of model group. CONCLUSION: Angiotensin -(1-7) can attenuate remodeling of myocardial collagen network in pressure-overloaded rats.

Positive rate and score discrepancies of myeloperoxidase staining at different temperatures.

Myeloperoxidase (MPO) is a strong marker of acute myeloid leukemia. The aim of this study was to compare MPO staining at different temperatures. Four bone marrow aspirate smears of each case were incubated with MPO staining solution at 4°C, 20°C, 37°C and 50°C, respectively. 30 patients with score <150 at 50°C were group A; 26 patients with score ≥150 at 50°C were group B. The results showed that at 4°C, MPO-positive blasts in two cases of group A were both 2%; at 50°C, MPO-positive blasts in the two cases were 42% and 30%, respectively. At 4°C, 20°C, 37°C and 50°C, the mean positive rates of group A were 30.6%, 42.2%, 53.7% and 56.5%, respectively (P<0.001); their mean scores were 52.2, 75.9, 99.5 and 105.3, respectively (P<0.001); the mean positive rates of group B were 84.1%, 88.7%, 92.3% and 93.7%, respectively (P<0.001); their mean scores were 199.5, 224.7, 243.5 and 244.5, respectively (P<0.001). From this study we can conclude that most MPO staining is effective over a wide temperature range with the highest positive rate at 50°C. MPO staining intensity at low temperature is much lower and may be false negative. Good MPO staining is achieved at 37°C.

[Prokaryotic expression, purification and identification of recombinant staphylokinase-HC fusion protein].

AIM: To construct the prokaryotic expression plasmid pEGX-6P-1-SAK-HC, express it in E.coli, and identify its biological activity. METHODS: The fusion gene (SAK-HC) was obtained by overlap-extension PCR and then inserted into prokaryotic soluble pEGX-6P-1 vector with GST tag to construct expression plasmid (pEGX-6P-1-SAK-HC). GST-SAK-HC was expressed by E.coli B834 (DE3) under the induction of IPTG and purified by Glutathion-Sepharose 4B (GST) affinity chromatography and negative-ion exchange column (DEAE) chromatography. PreScission protease was used to remove the GST tag. The purity of the fusion protein was analyzed by SDS-PAGE and the fibrinolytic activity of SAK-HC in vitro was characterized by soluble fibrin plate method. RESULTS: PCR, sequencing and restriction enzyme digestion analysis demonstrated that the recombinant plasmid was constructed successfully. The fusion protein was expressed in E.coli B834 (DE3), M(r); being 36 000 as shown by SDS-PAGE. After purified by GST affinity and DEAE chromatography, SAK-HC fusion protein of high purity was obtained from the cell supernantants. In vitro experiments showed that the fibrinolytic activity of the recombinant SAK-HC was about 9.4×10();4 IU/mg. CONCLUSION: The SAK-HC fusion protein we obtained was successfully expressed in E.coli and exhibited a fibrinolytic activity as high as the urokinase standard, which offers a base for the identification of immunogenicity of the fusion protein.

Retinoic acids potentiate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells.

BACKGROUND: As one of the least studied bone morphogenetic proteins (BMPs), BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA) signaling is known to play an important role in development, differentiation and bone metabolism. In this study, we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs). METHODOLOGY/PRINCIPAL FINDINGS: Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9, RARalpha and RXRalpha into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as osteopontin (OPN) and osteocalcin (OC). BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants, we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRalpha or RARalpha significantly increases trabecular bone and osteoid matrix formation. CONCLUSION/SIGNIFICANCE: Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore, a combination of BMP9 and retinoic acid (or its agonists) may be explored as effective bone regeneration therapeutics to treat large segmental bony defects, non-union fracture, and/or osteoporotic fracture.