Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition

The recent development of mutant-selective inhibitors for the oncogenic KRAS^G12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRAS^G12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pK_a of the C12 thiol is depressed (pK_a ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRAS^Y137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRAS^G12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, K_d = 36.0 ± 0.7 μM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pK_a of the C12 thiol, we found that reversible and irreversible oxidation of KRAS^G12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRAS^G12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRAS^G12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRAS^G12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.     

基于森林清查资料的河南省森林植被碳储量动态变化

利用1994—1998年、1999—2003年、2004—2008年、2009—2013年河南省4期森林资源清查数据,运用生物量转换因子连续函数法和平均生物量法,估算了1998—2013年河南省森林植被的碳储量和碳密度变化。研究结果表明,河南省森林植被碳储量由1998年的45.57 Tg增加到2013年的107.98 Tg,年均碳汇量为4.16 Tg/a。乔木林碳储量和碳密度分别由1998年的33.54 Tg和22.39 Mg/hm~2增加到2013年的97.11 Tg和31.80 Mg/hm~2。乔木林碳储量在所有植被类型中占主体,4个森林清查时期乔木林碳储量占森林植被总碳储量的比例分别为73.60%、79.22%、85.63%和89.93%。2013年森林清查时,乔木林中杨树和栎类碳储量最大,分别占总碳储量的37.61%和25.22%,各龄组乔木林碳密度大小顺序依次为成熟林近熟林中龄林过熟林幼龄林。阔叶林面积、碳储量、碳密度均高于针叶林,阔叶林是河南省森林碳汇的主要贡献者。人工林面积、碳储量、碳密度增加幅度都要高于天然林,人工林碳储量由1998年的9.62 Tg增加到2013年的55.67 Tg,占乔木林碳储量总增量的77.15%,人工林碳密度由1998年的17.86 Mg/hm~2提高到2013年的32.01 Mg/hm~2,人工林在河南省森林碳汇中逐步发挥重要的作用,逐渐成为河南省森林碳汇的主体,随着人工林生长为具有较高碳密度的成熟林,河南省乔木林将具有较大的碳汇潜力。     

几种耐热戴氏霉对秸秆的降解效果

【目的】测定6株戴氏霉(Taifanglania)的生长温度特性及其对秸秆的降解效果。【方法】通过定时测定不同培养温度下的菌落直径绘制6株戴氏霉菌株的生长曲线;采用苯胺蓝法、愈创木酚法和木质素磺酸钙降解试验测定其木质素降解能力;用羧甲基纤维素钠水解圈测定法和胞外酶活测定法判定其对纤维素的降解能力;以失重法和范氏洗涤剂法检测其对水稻秸秆的降解效果。【结果】所试的耐热戴氏霉菌株均能耐受50 °C的高温,并能产生纤维素酶,但不同菌株产生的木质素降解酶有所差异;均具有降解秸秆的能力,其中合川戴氏霉(T. hechuanensis) H08.1菌株降解能力最强,其次是灰戴氏霉(T. cinerea) H57.1菌株,其秸秆降解率分别为50.2%和42.2%。【结论】合川戴氏霉H08.1菌株和灰戴氏霉H57.1菌株在秸秆的降解利用上具有潜在开发价值。     

高、低氮浓度对2株真眼点藻的生长和油脂积累的影响

【目的】研究氮浓度对真眼点藻纲(Eustigmatophyceae)的2株高产油微藻大真眼点藻(Eustigmatos magnus,EM)和波氏真眼点藻(Eustigmatos polyphem,EP)的细胞形态、生长、总脂含量、脂质组成和脂肪酸组成与含量的时序变化规律。【方法】利用高氮(18.0 mmol/L NO3?-N)和低氮(3.6 mmol/L NO3?-N)浓度培养微藻。【结果】形态观察结果表明,大真眼点藻(E. magnus)和波氏真眼点藻(E. polyphem)营养细胞具有1个周生的裂叶状叶绿体,细胞质中有液泡,内含能够振动的颗粒物,以及一个较为明显的红色色素体;生殖方式通过形成2个D形或4个四角形的似亲孢子;随着培养周期的延伸和营养盐的消耗,细胞中油体逐步形成,其数量不断增加,体积不断增大。实验结果表明,初始氮浓度对2种微藻的总脂积累及生长均有显著影响(P<0.05),低氮浓度下2种微藻的生物质浓度分别为9.0 g/L和8.5 g/L,均低于高氮浓度下的生物质浓度。而低氮浓度下2种微藻的总脂、中性脂和总脂肪酸的含量以及总脂、中性脂与总脂肪酸的单位体积产率均明显高于高氮浓度组,其最高值分别为：59.10%、51.90%、46.95%和0.28、0.24、0.22 g/(L·d) (EM);64.20%、56.80%、50.01%和0.32、0.28、0.25 g/(L·d) (EP)。脂肪酸分析结果表明,两种微藻的脂肪酸主要成分均为棕榈酸(C16:0)、棕榈油酸(C16:1)、油酸(C18:1)和二十碳五烯酸(C20:5,EPA),四者的总含量(占总脂肪酸)分别达到85.83%和85.48%,其中棕榈油酸的含量最高。【结论】低氮浓度胁迫有利于大真眼点藻和波氏真眼点藻细胞内油脂的积累,两种微藻均为适合于生产生物柴油的油脂生产藻株。     

Single-cell transcriptomic atlas of primate cardiopulmonary aging

Aging is a major risk factor for many diseases,especially in highly prevalent cardiopulmonary comorbidities and infectious diseases including Coronavirus Diseas...     

Diet drives convergent evolution of gut microbiomes in bamboo-eating species

Gut microbiota plays a critical role in host physiology and health. The coevolution between the host and its gut microbes facilitates animal adaptation to its specific ecological niche. Multiple factors such as host diet and phylogeny modulate the structure and function of gut microbiota. However, the relative contribution of each factor in shaping the structure of gut microbiota remains unclear. The giant(Ailuropoda melanoleuca) and red(Ailurus styani) pandas belong to different families of order Carnivora. They have evolved as obligate bamboo-feeders and can be used as a model system for studying the gut microbiome convergent evolution. Here, we compare the structure and function of gut microbiota of the two pandas with their carnivorous relatives using 16S rRNA and metagenome sequencing. We found that both panda species share more similarities in their gut microbiota structure with each other than each species shares with its carnivorous relatives. This indicates that the specialized herbivorous diet rather than host phylogeny is the dominant driver of gut microbiome convergence within Arctoidea.Metagenomic analysis revealed that the symbiotic gut microbiota of both pandas possesses a high level of starch and sucrose metabolism and vitamin B12 biosynthesis. These findings suggest a diet-driven convergence of gut microbiomes and provide new insight into host-microbiota coevolution of these endangered species.     

LRP6 downregulation promotes cardiomyocyte proliferation and heart regeneration

The adult mammalian heart is thought to be a terminally differentiated organ given the postmitotic nature of cardiomyocytes. Consequently, the potential for cardiac repair through cardiomyocyte proliferation is extremely limited. Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor that is required for embryonic heart development. In this study we investigated the role of LRP6 in heart repair through regulation of cardiomyocyte proliferation. Lrp6 deficiency increased cardiomyocyte cell cycle activity in neonatal, juvenile and adult mice. Cardiomyocyte-specific deletion of Lrp6 in the mouse heart induced a robust regenerative response after myocardial infarction (MI), led to reduced MI area and improvement in left ventricular systolic function. In vivo genetic lineage tracing revealed that the newly formed cardiomyocytes in Lrp6-deficient mouse hearts after MI were mainly derived from resident cardiomyocytes. Furthermore, we found that the pro-proliferative effect of Lrp6 deficiency was mediated by the ING5/P21 signaling pathway. Gene therapy using the adeno-associated virus (AAV)9 miRNAi-Lrp6 construct promoted the repair of heart injury in mice. Lrp6 deficiency also induced the proliferation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our study identifies LRP6 as a critical regulator of cardiomyocyte proliferation, which may lead to the development of a novel molecular strategy to promote myocardial regeneration and repair.Subject terms: Cell-cycle exit, Cytokinesis     

Oxidized high-density lipoprotein promotes CD36 palmitoylation and increases lipid uptake in macrophages

Oxidized high-density lipoprotein (oxHDL) reduces the ability of cells to mediate reverse cholesterol transport and also shows atherogenic properties. Palmitoylation of cluster of differentiation 36 (CD36), an important receptor mediating lipoprotein uptake, is required for fatty acid endocytosis. However, the relationship between oxHDL and CD36 has not been described in mechanistic detail. Here, we demonstrate using acyl-biotin exchange analysis that oxHDL activates CD36 by increasing CD36 palmitoylation, which promotes efficient uptake in macrophages. This modification increased CD36 incorporation into plasma lipid rafts and activated downstream signaling mediators, such as Lyn, Fyn, and c-Jun N-terminal kinase, which elicited enhanced oxHDL uptake and foam cell formation. Furthermore, blocking CD36 palmitoylation with the pharmacological inhibitor 2-bromopalmitate decreased cell surface translocation and lowered oxHDL uptake in oxHDL-treated macrophages. We verified these results by transfecting oxHDL-induced macrophages with vectors expressing wildtype or mutant CD36 (mCD36) in which the cytoplasmic palmitoylated cysteine residues were replaced. We show that cells containing mCD36 exhibited less palmitoylated CD36, disrupted plasma membrane trafficking, and reduced protein stability. Moreover, in ApoE^−/−CD36^−/− mice, lipid accumulation at the aortic root in mice receiving the mCD36 vector was decreased, suggesting that CD36 palmitoylation is responsible for lipid uptake in vivo. Finally, our data indicated that palmitoylation of CD36 was dependent on DHHC6 (Asp-His-His-Cys) acyltransferase and its cofactor selenoprotein K, which increased the CD36/caveolin-1 interaction and membrane targeting in cells exposed to oxHDL. Altogether, our study uncovers a causal link between oxHDL and CD36 palmitoylation and provides insight into foam cell formation and atherogenesis.