Regulation of MMP-2 and TIMP-3 expression by uPA signal transduction system in human bone giant cell tumor

AIM: To study effects of urokinase-type plasminogen activator (uPA) signal transduction on expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) in giant cell tumor of bone (GCT). METHODS: Expression of uPAR, MMP-2 and TIMP-3 in GCT tissue was detected by immunohistochemistry. Phosphorylation level of mitogen-activated protein kinase (p44) in uPA/uPAR signal pathway in cultured GCT cells was detected by immunoprecipitation. The expression of MMP-2 and TIMP-3 in cultured cells after treatment with uPA-ATF or anti-uPAR antibody was also detected by Western blotting. RESULTS: 1) Urokinase-type plasminogen activator receptor (uPAR) was positive on the cell membrane and in cytoplasm of some mononuclear stromal cells (MSCs) and multinucleated giant cells (MGCs); 2) MMP-2 was positive in the cytoplasm and on the cell membrane of almost all of MSCs and some of MGCs. The polar distribution of MMP-2 in the cytoplasm of MGCs was especially obvious; 3) The expression of TIMP-3 of some MSCs and MGCs in GCT was much lower than MMP-2. The positive signal also showed a prominent polarity; 4) After treatment with uPA-ATF, the phosphorylation level of p44 in GCT cultured cells was much higher than the control. Addition of anti-uPAR antibody in the cells remarkably down-regulated the phosphorylation level of p44 as compared with the control group, suggesting that uPA-ATF participates cell signal transduction and this reaction can be inhibited by anti-uPAR antibody; 5) uPA-ATF cell signal pathway up-regulated expression of MMP-2 and TIMP-3, while anti-uPAR antibody down-regulated the expression of MMP-2 and TIMP-3. CONCLUSION: These results demonstrate for the first time that uPA-ATF directly regulates the expression of MMP-2 and TIMP-3 by signal transduction pathway, and the over-expression of MMP-2 and TIMP-3 may play an important role in local osteolysis of GCT.     

Improved milk production through PG–PL system by provision of in‐house shelter management in lactating Murrah buffaloes during winter season

Comprehensive information on the role of β‐casein and plasminogen–plasmin (PG–PL) system in milk secretion of Murrah buffaloes during winter season is lacking, although effects of cold stress can be ameliorated to an extent by altering microclimate at farm level. Hence, this study was aimed to determine the changes in productivity along with PG–PL system of milk, plasma hormones and metabolites of buffaloes during winter (December–January) season under two different management systems. Average minimum temperature and wind chill index during this season were 7.02 and 12.74 °C respectively. Buffaloes were divided in two groups of six animals each: control and treatment, where treatment group animals were placed in‐house with floor bedding of paddy straw and the control group animals in loose housing system without straw bedding. Physiological responses were recorded, and milk and blood samples were collected at weekly intervals for six‐week experimental period. Under in‐house management system, buffaloes experienced better comfort by alleviating environmental stress as their physiological responses such as respiration rate and pulse rate were significantly reduced (p < 0.01) as compared to the control, which subsequently resulted higher milk yield by 9.92% (p < 0.05). Analysis of milk samples revealed higher concentration of plasminogen (10.6 vs. 8.05 μg/ml; p < 0.01) and β‐casein (p < 0.05), and lower plasmin level (0.299 vs. 0.321 μg/ml; p < 0.05) in buffaloes under treatment group. It was also found that plasma cortisol, glucose and non‐esterified fatty acids levels were higher (p < 0.01) in control group as compared to the treatment animals by 13.6%, 8.14% and 12.6% respectively. However, milk composition, growth hormone, epinephrine and norepinephrine level in plasma were similar in both the groups. Hence, it may be concluded that provision of in‐house shelter management with floor bedding of paddy straw during winter was effective to minimize environmental stress and improved milk production through manipulation of PG–PL system in buffaloes.     

桥接治疗急性缺血性脑卒中患者的血管再通率及远期神经功能恢复分析

目的 探讨重组组织型纤溶酶原激活物（rtPA）静脉溶栓联合血管内介入治疗（即桥接治疗法）对急性缺血性脑卒中患者的治疗效果。方法 选取我院（2015年1月-2018年1月）收治的100例急性缺血性脑卒中患者进行回顾性分析,根据治疗方法分为观察组47例（桥接治疗法）、对照组53例（rtPA静脉溶栓疗法）,对比两组治疗后的血管再通率、神经功能恢复情况。结果 观察组前循环梗死血管（颈内动脉、大脑前动脉、大脑中动脉）再通率91.49%,对照组前循环梗死血管再通率50.94%,两组比较差异具有统计学意义（P<0.01）;观察组治疗14 d、治疗28 d、治疗3个月,神经功能缺损（NIHSS）评分均显著的低于对照组（P<0.01）;根据mRS标准,观察组的预后良好87.23%,对照组预后良好52.83%,两组比较差异具有统计学意义（P<0.01）;观察组的并发症发生率8.51%,对照组并发症发生率16.98%,两组比较差异无统计学意义（P>0.05）。结论 桥接治疗对缺血性脑卒中患者的效果优于rtPA静脉溶栓疗法,对于患者远期神经功能恢复具有显著效果。     

基于四环素调控系统构建白蛋白启动子调控大鼠uPA转基因表达的慢病毒载体

基于四环素调控系统构建白蛋白(albumin,Alb)启动子调控大鼠uPA(urokinase-type plasminogen activator,ruPA)转基因肝特异性过表达的慢病毒载体pLVX-Alb-TetOne-TRE-ruPA-T2A-CopGFP(pLATTRUTG)。以CTG0875-2-11质粒为模板,PCR扩增大鼠的uPA(ruPA)基因,3’端添加Flag标签,In-Fusion克隆至pLVX-Alb-TetOne-TRE-T2A-CopGFP(pLATTTG)质粒中,得到慢病毒载体pLVX-Alb-TetOne-TRE-ruPA-T2A-CopGFP(pLATTRUTG),所构建质粒经测序和酶切鉴定。将pLATTRUTG瞬时转染293T细胞,转染后24 h倒置荧光显微镜检测CopGFP表达;接着向6孔细胞培养板内加入强力霉素(Doxycycline,Dox),48 h后在倒置荧光显微镜下观察CopGFP表达(包括未加Dox的孔),随后收集细胞以提取总RNA和总蛋白,用于RT-qPCR检测ruPA及报告基因表达和Western blot检测标签蛋白Flag表达。酶切和测序确证我们成功构建了慢病毒载体pLATTRUTG;瞬转293T细胞后,24 h倒置荧光显微镜下可见零星细胞(约占0.1%)发弱的绿色荧光,加Dox 48 h后所有细胞展现强的绿色荧光,而不加Dox的孔内仍然只见到零星细胞(约占0.1%)发弱的绿色荧光。RT-qPCR和Western blot检测结果显示,与不加Dox的细胞相比,加Dox的细胞中ruPA、报告基因CopGFP和Flag表达水平显著升高。结果提示,成功基于四环素调控系统构建Alb启动子调控大鼠uPA转基因表达的慢病毒载体pLATTRUTG,为相关后续实验奠定了基础。     

Emerging therapeutic approaches for canine sarcomas: Pushing the boundaries beyond the conventional

Sarcomas represent a group of genomically chaotic, highly heterogenous tumours of mesenchymal origin with variable mutational load. Conventional therapy with surgery and radiation therapy is effective for managing small, low‐grade sarcomas and remains the standard therapeutic approach. For advanced, high‐grade, recurrent, or metastatic sarcomas, systemic chemotherapy provides minimal benefit, therefore, there is a drive to develop novel approaches. The discovery of “Coley's toxins” in the 19th century, and their use to stimulate the immune system supported the application of unconventional therapies for the treatment of sarcomas. While promising, this initial work was abandoned and treatment paradigm and disease course of sarcomas was largely unchanged for several decades. Exciting new therapies are currently changing treatment algorithms for advanced carcinomas and melanomas, and similar approaches are being applied to advance the field of sarcoma research. Recent discoveries in subtype‐specific cancer biology and the identification of distinct molecular targets have led to the development of promising targeted strategies with remarkable potential to change the landscape of sarcoma therapy in dogs. The purpose of this review article is to describe the current standard of care and limitations as well as emerging approaches for sarcoma therapy that span many of the most active paradigms in oncologic research, including immunotherapies, checkpoint inhibitors, and drugs capable of cellular metabolic reprogramming.