The B-RafV600E inhibitor dabrafenib selectively inhibits RIP3 and alleviates acetaminophen-induced liver injury

Receptor-interacting protein (RIP)3 is a critical regulator of necroptosis and has been demonstrated to be associated with various diseases, suggesting that its inhibitors are promising in the clinic. However, there have been few RIP3 inhibitors reported as yet. B-Raf^V600E inhibitors are an important anticancer drug class for metastatic melanoma therapy. In this study, we found that 6 B-Raf inhibitors could inhibit RIP3 enzymatic activity in vitro. Among them, dabrafenib showed the most potent inhibition on RIP3, which was achieved by its ATP-competitive binding to the enzyme. Dabrafenib displayed highly selective inhibition on RIP3 over RIP1, RIP2 and RIP5. Moreover, only dabrafenib rescued cells from RIP3-mediated necroptosis induced by the necroptosis-induced combinations, that is, tumor necrosis factor (TNF)α, TNF-related apoptosis-inducing ligand or Fas ligand plus Smac mimetic and the caspase inhibitor z-VAD. Dabrafenib decreased the RIP3-mediated Ser358 phosphorylation of mixed lineage kinase domain-like protein (MLKL) and disrupted the interaction between RIP3 and MLKL. Notably, RIP3 inhibition of dabrafenib appeared to be independent of its B-Raf inhibition. Dabrafenib was further revealed to prevent acetaminophen-induced necrosis in normal human hepatocytes, which is considered to be mediated by RIP3. In acetaminophen-overdosed mouse models, dabrafenib was found to apparently ease the acetaminophen-caused liver damage. The results indicate that the anticancer B-Raf^V600E inhibitor dabrafenib is a RIP3 inhibitor, which could serve as a sharp tool for probing the RIP3 biology and as a potential preventive or therapeutic agent for RIP3-involved necroptosis-related diseases such as acetaminophen-induced liver damage.Necroptosis, also known as programmed necrosis, is a kind of programmed cell death that occurs at conditions that result in blocking the execution of apoptosis.^{1, 2} The protein kinase receptor-interacting protein (RIP)3 is a serine/threonine protein kinase that has recently been demonstrated to be the critical regulator that switches cells from apoptosis to necroptosis.^{3, 4, 5, 6} The death receptor ligands, such as tumor necrosis factor (TNF)α, Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL), are classical inducers of apoptosis or necroptosis. By binding to their respective receptors, they lead to activation of functional caspase-8, which results in apoptosis by activating the effector caspases such as caspase-3 but inactivating the necroptic kinases such as RIP3. When caspase-8 is absent or inhibited by caspase inhibitors such as z-VAD, those death receptor ligands cause necroptosis, which can be augmented by Smac mimetic that promotes degradation of inhibitor of apoptosis proteins.^{3, 4, 5, 6}RIP3 is widely involved in physiological processes and pathological states.⁶ RIP3 deficiency not only rescues the lethality of caspase-8^−/− and FADD^−/− mice⁷ and restores normal proliferation of their T cells,⁶ but also protects hepatocytes from ethanol-induced injury and steatosis,⁸ rescues caspase-8 or FADD deficiency-induced massive inflammation in epithelium,⁹ prevents cerulean-induced acute necrotizing pancreatitis,^{3, 4} inhibits photoreceptor and cone cell death^{10, 11} and alleviates macrophage necrosis in advanced atherosclerosis lesions.¹² Acetaminophen is an extensively used analgesic and antipyretic. When taken in overdose, its most frequent toxicity is hepatotoxicity including fatal centrilobular hepatic necrosis.^{13, 14} Acetaminophen overdose is the most common cause of acute liver failure in the United States and the United Kingdom.¹⁵ It also causes 11.86% of acute liver failure in China.¹⁶ Enhanced levels of high-mobility group box-1 and necrosis keratin-18 marked occurrence of hepatic necrosis.¹⁴ Necrosis has been considered as the predominant mode of cell death in this case, for which RIP3 has been shown to be responsible.¹⁷ In addition, RIP3 might also be associated with carcinogenesis and tumor drug resistance to chemotherapeutics.^{18, 19} These lines of evidence suggest potential extensive uses of small-molecule RIP3 inhibitors in medical prevention or therapy.However, few RIP3 inhibitors have been reported²⁰ and no small-molecule RIP3 inhibitors have been investigated for the potential medical uses. One possible cause is that there lacks a proper RIP3 kinase assay for screening for its inhibitors at molecular levels, which should be highly sensitive, free of radioisotopes, and high throughput. We thus established a non-radioactive luminescent RIP3 kinase assay in this study. By using this assay, we found that 6 B-Raf inhibitors inhibited the RIP3 enzymatic activity in vitro. But only dabrafenib could rescue cells from RIP3-mediated necroptosis induced by TNFα, TRAIL or Fas ligand plus Smac mimetic and the caspase inhibitor z-VAD. Dabrafenib directly and ATP-competitively bound to RIP3 protein and caused highly selective inhibition on RIP3 over RIP1, RIP2 and RIP5. Dabrafenib was demonstrated to ease acetaminophen-induced necrosis in normal human hepatocytes and to prevent acetaminophen-induced liver injury in mice. Our study raises a possibility that the medical indications of the B-Raf^V600E inhibitor dabrafenib might be extended from cancers to RIP3-involved diseases.     

Chemical chaperone 4-phenylbutyrate prevents endoplasmic reticulum stress induced by T17M rhodopsin

Background

Rhodopsin mutations are associated with the autosomal dominant form of retinitis pigmentosa. T17M mutation in rhodopsin predisposes cells to endoplasmic reticulum (ER) stress and induces cell death. This study aimed to examine whether chemical chaperone 4-phenylbutyrate prevents ER stress induced by rhodopsin T17M.

Results

ARPE-19 cells were transfected with myc-tagged wild-type (WT) and T17M rhodopsin constructs. Turnover of WT and T17M rhodopsin was measured by cycloheximide chase analysis. The activity of ubiquitin-proteasome system was evaluated by GFPU reporter. We found that T17M rhodopsin was misfolded, ubiqutinated and eliminated by ER-associated degradation pathway (ERAD) in ARPE-19 cells. Accumulated T17M rhodopsin induced unfolded protein response, but had no effect on the activity of ubiquitin proteasome system. Moreover, chemical chaperone 4-phenylbutyrate facilitated the turnover of T17M rhodopsin and prevented apoptosis and ER stress induced by T17M rhodopsin.

Conclusions

Chemical chaperone could attenuate UPR signaling and ER stress induced by T17M rhodopsin and has potential therapeutic significance for retinitis pigmentosa.

     

水生哺乳动物信标跟踪记录技术及其应用

水生哺乳动物(主要是鲸类和鳍脚类)分布范围广、活动范围大、行为复杂,且在水下活动时间长,常规的目视观察受时间和空间限制,通常只能获取有限的信。信标跟踪记录(Bio-logging)具有可跟随移动和自主操作的特征,能在很大程度上突破时间和空间限制,实时获取动物及其栖息环境信息。水生哺乳动物的信标跟踪记录始于20世纪60年代1,2,40年来,无论是记录技术,还是应用研究,都有了很大发展。2003年在日本东京国立极地研究所召开的“信标跟踪记录科学国际学术讨论会(International Symposium on Bio-loggingScience)”,收到来自法、美、英、日、澳、德、意、加、南非和中国共152名与会代表的104篇论文。这些论文主要介绍信标跟踪记录技术及其应用的现状和未来趋势。会议以不同研究对象分专题进行交流,共分为鲸类、鳍脚类、类、爬行类、鱼类和其他类6个专题,其中鸟类专题论文最多,其次是鱼类专题。有关水生哺乳动物的研究论文共22篇,除了4篇介绍记录技术外,其他论文主要介绍信标跟踪记录的应用研究,包括潜水行为、捕食策略、能量代谢、栖息地标识和发声策略研究。本文是近年来水生哺乳动物信标跟踪记录研究领域相关论文的综述,除介绍水生哺乳动物信标跟踪记录技术及其应用研究现状外,还对其不足之处和可能的发展趋势进行了讨论。此外,还重点介绍了我国珍稀动物长江江豚(Neophocaena phocaenoides as iaeorientalis)信标跟踪研究的一些进展。       

我国苦草属(Vallisneria L.)植物的生态学研究

苦草是水鳖科(Hydrocharitaceae)苦草属(Vallisneria)沉水草本植物,无直立茎,叶基生,线形或带形,常被称为扁担草、蓼萍草、水韭菜、面条草、扁子草和鸭舌条等,广泛分布于世界各地。本属植物有6～10种〔1,2〕,我国的苦草属植物依《中国植物志》第8卷有3种——苦草(V.natansHara)、刺苦草(V.spinulosaYan)和密刺苦草(V.denseserrulataMakino),而没有V.spiralisL.,VasiaticaMiki和V.giganteaGraebner等种〔1〕。本文苦草属植物的命名以《中国植物志》第8卷为准,引用文献一般尊重原作者的意见。下文中,无具体说明,苦草均为苦草属植物的通…     

退火遗传算法及其应用

将模拟退火方法引入遗传算法中,对非线形问题进行优化。该算法克服了ＳＧＡｓｒ 的过早收敛的问题,并解决了染色体样性要求。最后将该算法应用于解决水资源优化分配的问题中,优化结果同样具备上述特点。     

浙江菌类药资源调查及利用研究初报

本文较全面地了浙江菌类药中药资源。其内容包括开展本项目的方法,研究结果。分别对本省自然环境、野生菌类药中药资源的分布进行了重点论述,简要介绍了菌类药调查结果,重点研究种类,并对本省的菌类药现状和展望作了评述;文后列有本项目研究中公开发表的文献,使本方基本能反映本专题的研究内容。     

HDAC8 drives spindle organization during meiotic maturation of porcine oocytes

ObjectivesHistone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood.Materials and methodsHDAC8‐targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI‐34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ‐tubulin dynamics and acetylation level of α‐tubulin.ResultsWe observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ‐tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI‐34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α‐tubulin.ConclusionsCollectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.     

四种细菌L型的形态定量分析

用抗生素或臭氧的方法诱导出四种细菌和Ｌ型后,用图像分析技术定量检测Ｌ型多形性,表面积、最大直径、周长、等效圆直径和形状因子,并作比较分析。四种细菌Ｌ型及Ｌ型中的巨形体在上述５项形态学参数指标上除形状因子外均较细菌型明显增大,而形状因子参数明显小于细菌型。细菌Ｌ型之间在形态学指标上的差异有显著性（ｐ＜０．０５）。形态定量分析能准确客观地反映形态定性观察的结果以及细菌Ｌ型的形态变异程度,为细菌Ｌ型的形态定量分析提供依据。