关键词： Antimicrobial peptide   Persulcatusin   Molecular farming   Protein production   Rice  

摘要： Antimicrobial proteins and peptides are potential alternatives to antibiotics. Persulcatusin is an antimicrobial peptide found in the taiga tick Ixodes persulcatus. We constructed fusion genes that encode, from the N-terminus to the C-terminus, a signal sequence of rice alpha-amylase 3D, mouse calmodulin, a target sequence of TEV protease of tobacco etch virus, and persulcatusin with or without a His tag at the N-terminus of the mature fusion protein. These fusion genes were then introduced into rice. Western blot analysis detected persulcatusin fusion proteins in transgenic calli, suspension cells, and their culture medium. Antimicrobial activity against Staphylococcus aureus was detected in the protein extracts prepared from the transgenic callus but not from the non-transgenic wild-type callus, and TEV protease treatment to release persulcatusin from the fusion protein enhanced antimicrobial activity. The growth of the transgenic rice plants was unaffected. Our results indicate that functional persulcatusin can be produced in rice cells. This provides a basis for the mass production of persulcatusin for therapeutic use against bacterial infectious diseases in humans and livestock.

关键词： p53   MDM2   Protein-protein interaction   Mutant p53   Gene therapy   PROTAC   Degrader  

摘要： p53, encoded by the TP53 gene, is a critical tumor suppressor, and its pharmacological activation has been clinically validated as an effective strategy for the treatment of human malignancies. In recent years, numerous novel approaches have emerged to activate both wild-type and mutant p53, including MDM2 degraders, p53 stabilizers, gene therapies targeting TP53, and inhibitors of the MDM2-p53 protein-protein interaction. Since 2020, drug discovery efforts in these areas have advanced rapidly, leading to several notable breakthroughs. This review provides a comprehensive overview of recently reported agents that enhance p53 function, with particular emphasis on those that have progressed to clinical trials or are capable of restoring p53 activity in cancers harboring TP53 mutations. We aim to offer an up-to-date snapshot of the evolving landscape of p53targeted therapies and to serve as a reference for future drug discovery endeavors in this important field.

关键词： 肌筋膜触发点   干针   膝关节疼痛   视觉模拟评分   Meta分析   康复   随机对照   功能障碍   工程化组织构建  

摘要： 目的:干针治疗被广泛应用于各种肌筋膜疼痛综合征,该文旨在系统评价干针治疗膝关节疾病的临床效果。方法:检索PubMed、EBSCO、ScienceDirect、Web of Science、CINAHL、Cochrane Library、中国知网等数据库中发表的文献,选取随机对照试验且使用干针作为主要治疗手段,并纳入诊断为膝关节疾病的患者。由2位评估人独立筛选文章,对方法学质量进行评分,并提取数据。主要指标有目测类比评分,次要指标主要有西安大略和麦克马斯特大学骨关节炎指数(WOMAC)评分、压痛阈值、膝关节活动度和Kujala评分(膝关节功能评分量表)。结果:Meta分析共纳入15项随机对照试验,涉及698例患者。Meta分析结果显示,与非干针治疗组相比,干针治疗组目测类比评分、WOMAC疼痛评分与WOMAC僵硬度评分存在明显优势(MD=-0.63,95%CI:-1.06至-0.19,P=0.005;MD=-0.74,95%CI:-1.32至-0.17,P=0.01;MD=-0.43,95%CI:-0.77至-0.09,P=0.01),WOMAC总评分、WOMAC功能评分、压痛阈值、膝关节活动度、Kujala评分没有明显优势。结论:干针可以有效治疗膝关节疼痛和僵硬度,然而在改善其他膝关节功能障碍方面的临床优势及随访效果证据不足。所以,对于一些年龄较大患有膝关节慢性疼痛或关节僵硬的患者,可以谨慎考虑使用干针治疗。

关键词： 盐酸青藤碱   急性T淋巴细胞白血病   CEM细胞   细胞凋亡   RNA-seq测序   增殖抑制   工程化细胞  

摘要： 背景:盐酸青藤碱有抗多种肿瘤的作用,但目前尚不清楚盐酸青藤碱对急性T淋巴细胞白血病的作用。目的:探讨盐酸青藤碱对急性T淋巴细胞白血病CEM细胞的抑制作用。方法:应用不同浓度(0.5,1,2,4 mmol/L)盐酸青藤碱处理CEM细胞,CCK-8检测细胞增殖抑制率并计算IC50;倒置显微镜和吉姆萨染色观察CEM细胞形态变化;利用RNA-Seq测序分析差异基因表达并进行生物信息学分析。结合转录组测序结果,流式细胞术检测不同浓度(1,2,4 mmol/L)盐酸青藤碱作用后CEM细胞凋亡率;Western blot检测不同浓度(1,2,4 mmol/L)盐酸青藤碱作用后CEM细胞中Bcl-2、Bax、Caspase-9蛋白的表达。结果与结论:①盐酸青藤碱呈剂量和时间依赖性地抑制CEM细胞生长;②盐酸青藤碱干预后CEM细胞数量下降,核固缩;③RNA-seq测序筛出53个异常表达基因,基因本体分析主要富集在细胞过程、细胞解剖实体与粘连关系等,信号通路分析与肿瘤相关的是细胞凋亡;④盐酸青藤碱呈剂量依赖性地促进CEM细胞凋亡;⑤盐酸青藤碱上调CEM细胞中Bax、Caspase-9蛋白表达,下调Bcl-2蛋白表达。由此可见,盐酸青藤碱可诱导CEM细胞凋亡从而抑制细胞增殖,可能与其上调Bax和Caspase-9蛋白表达,以及下调Bcl-2蛋白表达有关。

关键词： 人脐带间充质干细胞   Toll样受体4   Caspase-11   GSDMD   急性肺损伤   急性呼吸窘迫综合征   细胞焦亡   脓毒症   工程化干细胞  

摘要： 背景:急性肺损伤是脓毒症患者的常见严重并发症,其发病机制复杂,目前尚无有效的药物治疗方案。人脐带间充质干细胞通过抑制细胞过度焦亡从而改善脓毒症急性肺损伤的作用机制愈发受到重视。目的:探索人脐带间充质干细胞对脓毒症所致急性肺损伤小鼠肺组织细胞焦亡的影响及机制。方法:48只Balb/c雄性小鼠按随机数字法分为假手术组、模型组、治疗组(n=16)。模型组、治疗组行盲肠结扎穿孔建立脓毒症所致急性肺损伤模型,假手术组仅开腹后缝合。治疗组术后6 h尾静脉注射200μL人脐带间充质干细胞悬液(5×105个细胞),模型组及假手术组术后6 h尾静脉注射200μL生理盐水。术后28 h,各组随机取5只小鼠予以尾静脉注射伊文思蓝检测肺血管通透性;收集各组剩余小鼠肺泡灌洗液和肺组织,ELISA检测肺泡灌洗液中白细胞介素1β、白细胞介素18水平,计数肺泡灌洗液中细胞总数及巨噬细胞数;苏木精-伊红染色观察肺组织病理学形态,TUNEL染色观察肺组织细胞焦亡情况,RT-PCR、Western blot检测肺组织中Toll样受体4、GSDMD及Caspase-11的mRNA和蛋白表达。结果与结论:与模型组相比,治疗组术后28 h肺血管通透性显著下降(P<0.05),肺泡灌洗液中白细胞介素1β、白细胞介素18水平降低(P<0.05),细胞总数及巨噬细胞数降低(P<0.05),肺损伤程度减轻,肺组织细胞焦亡指数降低(P<0.05),肺组织Toll样受体4、GSDMD、GSDMD-N及Caspase-11 mRNA及蛋白表达水平均显著下降(P<0.05)。结果表明,人脐带间充质干细胞可能通过抑制Toll样受体4/Caspase-11/GSDMD信号通路活化,改善肺组织细胞焦亡,从而在一定程度上改善脓毒症小鼠肺损伤的严重程度。

关键词： 骨髓间充质干细胞   端粒酶卡哈尔体蛋白1   衰老   过表达   干扰   细胞增殖   细胞凋亡   工程化干细胞  

摘要： 背景:随着年龄的增长,骨髓间充质干细胞的功能逐渐降低,延缓骨髓间充质干细胞自身衰老成为一项重要课题。目的:通过改变端粒酶卡哈尔体蛋白1(telomerase cajal body protein 1,TCAB1)基因的表达,探讨延缓骨髓间充质干细胞衰老的途径。方法:细胞贴壁法培养小鼠骨髓间充质干细胞,应用重组慢病毒技术过表达或干扰骨髓间充质干细胞中的TCAB1基因,采用qPCR检测衰老相关基因P16、P21、P53、P27的表达,qPCR检测Telomere的表达判断端粒的相对长度;Western blot检测衰老蛋白P16、P21、P53、P27的表达;CCK-8法检测细胞增殖情况;Annexin V-PE/7-AAD细胞凋亡试剂盒检测细胞凋亡情况。结果与结论:过表达TCAB1基因骨髓间充质干细胞中衰老相关基因及蛋白的表达量下降,端粒相对长度增加,细胞增殖能力较强,细胞凋亡数量较少,细胞呈现年轻状态;干扰TCAB1基因骨髓间充质干细胞中衰老相关基因及蛋白的表达量上升,端粒相对长度缩短,细胞增殖能力较弱,细胞凋亡数量较多,细胞呈现衰老状态。以上结果表明,在适当范围内增加TCAB1的表达量,可以延缓细胞衰老的速度。

关键词： Ovarian cancer   Endometrial cancer   HO-3867   APR-246   Prima-1met   Olaparib   P53   BRCA  

摘要： Ovarian and endometrial cancers frequently harbor a mutation in the tumor suppressor gene TP53 , which occurs in over 90 % of ovarian cancers and in the most aggressive endometrial cancers. The normal tumor suppressive functions of p53 are disrupted, resulting in unregulated cell growth and therapeutic resistance to standard treatments including chemotherapy and PARP inhibitors. Hence, a novel therapeutic strategy is urgently needed for p53 mutant gynecologic cancers, and we propose that converting mutant p53 to a wild type conformation and restoring its tumor suppressive functions has the potential to greatly improve treatment. In this study, we investigated the effects of two purported p53 reactivators, HO-3867 and APR-246, on cell proliferation via half-maximal inhibitory concentration (IC50) analyses using CyQUANT DNA measurements, tumor growth in vivo and gene expression by bulk RNA sequencing in gynecologic cancer cell lines that harbor oncogenic mutations in p53. We also tested these compounds in combination with the PARP inhibitor olaparib. We found that HO-3867 was very effective in inhibiting growth, with IC50 values in the low micromolar range. Importantly, HO-3867 was synergistic with olaparib treatment in five cell lines studied in vitro as well as in vivo in a xenograft model of high grade serous ovarian cancer. RNA sequencing data suggest that HO-3867 is acting through both p53-independent and p53-dependent pathways resulting in inhibition of DNA repair pathways including homologous recombination in p53 mutant cancer cells. Significance: The development of resistance to PARP inhibitors is a major problem and a cause of treatment failures in advanced gynecologic cancers, and we show that adding a p53 reactivator such as HO-3867 enhances the efficacy of PARP inhibitors in p53-mutant cancer models.

关键词： 枸杞多糖   阿尔茨海默病   SH-SY5Y细胞   线粒体自噬   β-淀粉样蛋白   细胞凋亡   工程化细胞  

摘要： 背景:神经退行性疾病与线粒体自噬调节失衡密切相关。课题组前期研究表明枸杞多糖具有神经保护作用,但其能否通过调控线粒体自噬来改善β-淀粉样蛋白1-42诱导的SH-SY5Y细胞损伤尚不明确。目的:探讨枸杞多糖对β-淀粉样蛋白1-42诱导的SH-SY5Y细胞损伤的保护作用及机制。方法:通过β-淀粉样蛋白1-42诱导SH-SY5Y细胞建立阿尔茨海默病细胞模型,并用枸杞多糖进行干预。将SH-SY5Y细胞分为3组:对照组,β-淀粉样蛋白1-42组(20μmol/Lβ-淀粉样蛋白1-42干预24 h),枸杞多糖组(先提前1 h加入1 g/L枸杞多糖形成保护作用,然后再加入20μmol/Lβ-淀粉样蛋白1-42与枸杞多糖共同干预24 h)。CCK-8法检测细胞活力;JC-1检测线粒体膜电位;TUNEL染色检测细胞凋亡;免疫荧光和Western blot检测突触、凋亡和线粒体自噬相关指标的表达。结果与结论:①与对照组比较,β-淀粉样蛋白1-42组细胞活力下降(P<0.05),细胞凋亡率上升(P<0.05),线粒体膜电位下降(P<0.05),促凋亡蛋白Bax、Caspase-3表达升高(P<0.05),抗凋亡蛋白Bcl-2表达降低(P<0.05),突触相关蛋白Syn、PSD-95表达降低(P<0.05),线粒体自噬相关蛋白Pink1、LC3A/B、Parkin、Beclin-1表达降低(P<0.05),P62表达升高(P<0.05);②与β-淀粉样蛋白1-42组比较,枸杞多糖组细胞活力升高(P<0.05),细胞凋亡率降低(P<0.05),线粒体膜电位上升(P<0.05),Bax和Caspase-3表达降低(P<0.05),Bcl-2表达升高(P<0.05),Syn和PSD-95表达升高(P<0.05),Pink1、LC3A/B、Parkin、Beclin-1表达升高(P<0.05),P62表达降低(P<0.05)。结果表明:枸杞多糖可能通过调控线粒体自噬来抑制β-淀粉样蛋白1-42诱导的SH-SY5Y细胞损伤,减少细胞凋亡,增加神经元突触可塑性。

关键词： Deep learning   Dynamical network analysis   Protein allostery   p53 protein  

摘要： Protein allostery is a critical regulatory mechanism in various biological processes, representing a challenging aspect of biological research. Dynamical network analysis serves as a foundational computational methodology for studying allosteric effects, while the recent emergence of neural relational inference (NRI) model has introduced novel insights into understanding it. In this study, we modified NRI model by integrating the multi-head self-attention module of transformer, and then compared the variant model with dynamical network analysis and initial NRI model for p53-DNA allosteric interactions. Our results show that the variant model is more focused on long-range allosteric than dynamical network in predicting p53-DNA allosteric pathways, and enhances superior accuracy and comprehensiveness compared to initial NRI model. Moreover, divergent allosteric pathways in wild-type (WT) versus mutant (MT) p53 may underlie their substantially different DNA recognition and binding behaviors. Finally, we found that even with undefined ending nodes, allosteric pathways consistently propagate from mutation sites toward DNA, and the length of pathways in MT p53 is significantly longer than that of WT p53. These suggest that mutation sites impair long-range allosteric communication, potentially disrupting signal transmission efficiency. Our results offer novel insights for a deep understanding of protein allosteric pathways through different methods.

关键词： 脊髓损伤   炎症因子   小胶质细胞   尼氏体   二氢槲皮素   神经元   活性氧   抗炎因子  

摘要： 背景:小胶质细胞诱导的“炎症风暴”是导致脊髓损伤后神经元细胞死亡的重要病理因素,极其不利于脊髓损伤后的恢复。二氢槲皮素属于维生素P族,有极好的生物活性,对脊髓损伤具有良好的抗炎、抗脂质过氧化反应以及神经保护作用,需进一步明确其促进脊髓损伤修复的作用机制。目的:探究二氢槲皮素是否能够通过其抗炎、抗氧化性能改善脊髓损伤后诱发的炎症状态,达到保护脊髓损伤的目的。方法:将48只SD大鼠按随机数字表法随机分为假手术组、脊髓损伤组和低剂量(30 mg/kg)二氢槲皮素组、高剂量(50 mg/kg)二氢槲皮素组。假手术组仅行椎板切除术,术后给予生理盐水腹腔注射;后3组制备脊髓损伤模型,术后分别给予生理盐水、30 mg/kg及50 mg/kg二氢槲皮素腹腔注射,术后4周取材。采用BBB肢体运动功能评分及机械缩足反应阈值评价大鼠肢体运动功能以及痛觉恢复情况,苏木精-伊红染色观察脊髓组织病理学变化,尼氏染色观察尼氏体数量及神经元形态,检测脊髓组织超氧化物歧化酶活性,免疫荧光检测活性氧表达及小胶质细胞Iba1的表达,Western blot检测脊髓中Iba1及白细胞介素1β、白细胞介素6、白细胞介素10、肿瘤坏死因子α、p38丝裂原活化蛋白激酶的蛋白表达情况。结果与结论:①行为学测试:自术后14 d起,高剂量二氢槲皮素组大鼠BBB肢体运动功能评分显著高于脊髓损伤组(P<0.01);在术后21 d时,高剂量二氢槲皮素组的机械缩足反应阈值显著低于脊髓损伤组(P<0.05),表现出更出色的运动功能;②苏木精-伊红染色及尼氏染色:与脊髓损伤组相比,高剂量二氢槲皮素组脊髓组织可见神经元形态稍肿胀但结构完整较清晰,尼氏体数量增多;③超氧化物歧化酶检测:与脊髓损伤组相比,高剂量二氢槲皮素组大鼠脊髓组织超氧化物歧化酶活性显著升高(P<0.05);④活性氧免疫荧光检测:与脊髓损伤组相比,高剂量二氢槲皮素的治疗能显著减轻脊髓损伤后活性氧的堆积(P<0.05);⑤小胶质细胞标志物Iba1的检测:与脊髓损伤组相比,高剂量二氢槲皮素组Iba1的蛋白表达水平显著降低(P<0.01);⑥Western blot检测:与脊髓损伤组相比,高剂量二氢槲皮素组的炎性因子(白细胞介素1β、白细胞介素6和肿瘤坏死因子α)及磷酸化P38丝裂原活化蛋白激酶的表达显著减少(P<0.01),而抗炎因子白细胞介素10则显著增加(P<0.01);⑦提示二氢槲皮素能有效逆转大鼠脊髓损伤后诱发的多种炎症标志物的表达变化,从而发挥神经保护作用,改善运动功能。