非小细胞肺癌患者中EGFR突变、ALK和ROS1融合基因表达变化及其临床病理学意义

目的探讨非小细胞肺癌(non-small cell lung cancer,NSCLC)患者中表皮生长因子受体(epidermic growth factor receptor,EGFR)突变、间变性淋巴瘤激酶(anaplastic lymphoma kinase,ALK)和ROS1融合基因的表达情况及其与临床病理特征的关系。方法应用ARMS法检测379例非小细胞肺癌患者中EGFR突变、ALK和ROS1融合基因的表达情况,并分析其与患者临床病理特征的关系。结果 379例非小细胞肺癌患者组织中,EGFR突变率为36.15%(137/379),19del和L858R突变为其主要突变类型,同时检出L858R和T790双突变4例,L858R和19del双突变2例;EGFR突变人群主要是女性、腺癌、非吸烟患者(P<0.05)。ALK融合基因阳性率为3.43%(13/379),其中ALK-M1融合基因型4例,ALK-M2融合基因型3例,ALK-M3融合基因型3例,ALK-M4融合基因型1例,ALK-M6融合基因型2例。ROS1融合基因阳性率为3.17%(12/379),主要为ROS1-M8融合基因型(8例),存在1例ROS1-M3和ROS1-M8融合基因型双融合。不同性别、年龄、组织学和吸烟状况的NSCLC患者ALK和ROS1基因突变率无统计学差异。结论 EGFR基因在NSCLC患者中存在较高的突变率,而ALK、ROS1融合基因在NSCLC患者中突变率较低,但代表了非小细胞肺癌的特点分子亚型,为指导临床靶向治疗提供依据。     

Cosupplementation with a synthetic, lipid-soluble polyphenol and vitamin C inhibits oxidative damage and improves vascular function yet does not inhibit acute renal injury in an animal model of rhabdomyolysis

We investigated whether cosupplementation with synthetic tetra-tert-butyl bisphenol (BP) and vitamin C (Vit C) ameliorated oxidative stress and acute kidney injury (AKI) in an animal model of acute rhabdomyolysis (RM). Rats were divided into groups: Sham and Control (normal chow), and BP (receiving 0.12% w/w BP in the diet; 4 weeks) with or without Vit C (100mg/kg ascorbate in PBS ip at 72, 48, and 24h before RM induction). All animals (except the Sham) were treated with 50% v/v glycerol/PBS (6 mL/kg injected into the hind leg) to induce RM. After 24h, urine, plasma, kidneys, and aortae were harvested. Lipid oxidation (assessed as cholesteryl ester hydroperoxides and hydroxides and F(2)-isoprostanes accumulation) increased in the kidney and plasma and this was coupled with decreased aortic levels of cyclic guanylylmonophosphate (cGMP). In renal tissues, RM stimulated glutathione peroxidase (GPx)-4, superoxide dismutase (SOD)-1/2 and nuclear factor kappa-beta (NFκβ) gene expression and promoted AKI as judged by formation of tubular casts, damaged epithelia, and increased urinary levels of total protein, kidney-injury molecule-1 (KIM-1), and clusterin. Supplementation with BP±Vit C inhibited the two indices of lipid oxidation, down-regulated GPx-4, SOD1/2, and NF-κβ gene responses and restored aortic cGMP, yet renal dysfunction and altered kidney morphology persisted. By contrast, supplementation with Vit C alone inhibited oxidative stress and diminished cast formation and proteinuria, while other plasma and urinary markers of AKI remained elevated. These data indicate that lipid- and water-soluble antioxidants may differ in terms of their therapeutic impact on RM-induced renal dysfunction.     

Revealing genetic diversity of tree peonies at micro-evolution level with hyper-variable chloroplast markers and floral traits

Key message

Highly variable regions of chloroplast genome were found to be useful in the detection of plant genetic diversity at micro-evolution level. Our methodology will improve understanding and conservation of plant diversity.

Abstract

Tree peonies are famous flowers with about 2,000 cultivars in the world, belonging to Paeonia sect. Moutan of the Paeoniaceae. They are traditionally classified based on flower forms and colors. Due to the limited number of DNA and morphological markers, and the existence of synonyms and homonyms, evaluation on genetic diversity of so many cultivars remains a challenge. In most cases, it is difficult and even impossible to discriminate tree peony cultivars when they are not in flower. In this study, single nucleotide polymorphism detected from the hyper-variable regions of chloroplast genome was employed to separate tree peony cultivars into different maternal lineages which can be expressed briefly by a nucleotide molecular formula. Our approach enabled a much higher resolution of cultivar identification and classification that has not been obtained before. The newly developed hyper-variable chloroplast markers, as an independent source of taxonomic characteristics, provided novel evidences and higher resolution ability that are helpful in building an effective classification system for evaluation, conservation, and utilization of the tree peony germplasm resources at cultivar level.     

GRK5 Deficiency Leads to Reduced Hippocampal Acetylcholine Level via Impaired Presynaptic M2/M4 Autoreceptor Desensitization

G protein-coupled receptor kinase 5 (GRK5) deficiency has been linked recently to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may contribute to AD pathogenesis remains elusive. Here we report that overexpression of dominant negative mutant of GRK5 (dnGRK5) in a cholinergic neuronal cell line led to decreased acetylcholine (ACh) release. This reduction was fully corrected by pertussis toxin, atropine (a nonselective muscarinic antagonist), or methoctramine (a selective M2/M4 muscarinic receptor antagonist). Consistent with results in cultured cells, high potassium-evoked ACh release in hippocampal slices from young GRK5 knock-out mice was significantly reduced compared with wild type littermates, and this reduced ACh release was also fully corrected by methoctramine. In addition, following treatment with the nonselective muscarinic agonist oxotremorine-M, M2, and M4 receptors underwent significantly reduced internalization in GRK5KO slices compared with wild type slices, as assessed by plasma membrane retention of receptor immunoreactivity, whereas M1 receptor internalization was not affected by loss of GRK5 expression. Moreover, Western blotting revealed no synaptic or cholinergic degenerative changes in young GRK5 knock-out mice. Altogether, these results suggest that GRK5 deficiency leads to a reduced hippocampal ACh release and cholinergic hypofunction by selective impairment of desensitization of presynaptic M2/M4 autoreceptors. Because this nonstructural cholinergic hypofunction precedes the hippocampal cholinergic hypofunction associated with structural cholinergic degeneration and cognitive decline in aged GRK5 knock-out mice, this nonstructural alteration may be an early event contributing to cholinergic degeneration in AD.G protein-coupled receptor kinase-5 (GRK5)² is one of the seven GRK family members whose primary function is to desensitize G protein-coupled receptors (GPCRs) (1, 2). We recently reported that increased soluble β-amyloid decreases membrane (functional) levels of GRK5 in vitro, and this membrane GRK5 deficiency occurs in vivo as well in an Alzheimer disease (AD) transgenic model (3) and in postmortem human AD brain samples (4). Moreover, the aged GRK5 knock-out (GRK5KO) mouse, which models this GRK5 deficiency in the absence of exogenous mutant human β-amyloid precursor protein (β-APP) or any other known AD-related genes (i.e. presenilins or tau), develops axonal defects and mild cholinergic degeneration with associated amnestic mild cognitive impairment (5). When Swedish mutant βAPP is overexpressed in the GRK5KO mice by cross-breeding with Swedish APP transgenic mice, the aged double mutant mice display significantly exaggerated brain inflammation (6). These accumulating data strongly suggest that GRK5 deficiency significantly contributes to AD pathogenesis, although the precise molecular mechanisms remain to be delineated.Mounting evidence indicates that the substrate spectrum of broadly expressed GRKs (i.e. GRK2/3/5/6) can significantly overlap for some receptors, suggesting that a lack of one of these members may have only a limited impact on GPCR regulation (2). On the other hand, compensation for loss of a particular GRK member by others in vivo can be incomplete or selective for other receptor types. For example, GRK2KO and GRK6KO mice have been shown to display selective impairments of adrenergic and dopaminergic receptor desensitization, respectively (7, 8). Findings from different GRK isoform-targeted animals strongly support the conclusion that although redundancy exists between GRK isoforms, each isoform has its own selective substrates; should one GRK be deficient or inactivated, desensitization of its selective substrates will be impaired (1). For GRK5 in particular, previous studies have demonstrated that GRK5KO mice display selectively impaired desensitization of muscarinic acetylcholine receptors (mAChRs) (9, 10).To date, five mAChR subtypes have been identified, with M1, M3, and M5 receptors being G_q/11-coupled, and M2 and M4 receptors being G_i/o-coupled (11). In hippocampal memory circuits, M2 receptor (M2R) is primarily a presynaptic autoreceptor that inhibits ACh release (12, 13), whereas M1R is postsynaptic and is believed to be critical in memory processes involving an interaction between the cerebral cortex and hippocampus (11). In AD, there is a selective loss of cholinergic neurons that leads to a cholinergic hypofunction, primarily a hypoactivity of postsynaptic nicotinic and M1 muscarinic receptors (14). GRK5KO mice, when challenged with nonselective muscarinic agonists, display augmented hypothermia, hypoactivity, tremor, and salivation, as well as antinociceptive changes (9). These behavioral changes are typical M2 and/or M4 receptor-mediated functions, according to the findings from muscarinic receptor subtype knock-out mice (11, 15). Therefore, GRK5 deficiency in vivo may selectively impair M2/M4R desensitization. If so, the resulting presynaptic M2/M4R hyperactivity would overly inhibit ACh release from cholinergic neurons and eventually compromise the learning and memory function. This study was undertaken to investigate the impact of GRK5 deficiency on ACh release and desensitization of mAChR subtypes using GRK5-deficient models both in vitro and in hippocampal slices from the GRK5KO mice.     

Sonneratia ovata Backer–A genetically depauperate mangrove species

It is expected that geographically widespread and outcrossing tree species would have high level of genetic variation. Three chloroplast regions and six nuclear genes were sequenced to assess the genetic variation of a species of mangroves, Sonneratia ovata, from China and Thailand. No nucleotide polymorphism was found in the three chloroplast regions and in the six nuclear genes from all of the four populations examined. The depauperation of polymorphism in S. ovata in comparison to moderate polymorphisms of other congeneric species is surprising, particularly considering high level of polymorphism in the past and relatively wide geographic distribution of the species. Since multiple independent loci were surveyed in this study, the most plausible explanation for our observation is that S. ovata has experienced severe demographic bottlenecks in the Pleistocene glaciation, followed by subsequent recolonization by sea currents in China and Southeast Asia regions. The lack of polymorphism may also be attributable to its small population size, despite its wide geographic distribution.     

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.Download video file.^{(125M, mp4)}     

子宫内膜异位症患者血清可溶性B7-H4的水平及意义

目的:探讨子宫内膜异位症患者血清可溶性B7-H4(sB7-H4)的水平及其临床意义。方法:用ELISA夹心法检测43例子宫内膜异位症患者术前血清sB7-H4的水平及40例子宫内膜异位症患者术后血清sB7-H4的水平,同时选取30例体检健康妇女血清sB7-H4水平作为对照。结果:子宫内膜异位症患者血清sB7-H4水平为(36.23±5.67)μg/L,体检健康者血清sB7-H4水平为(31.24±4.56)μg/L,两者比较差异有统计学意义(P0.01)。手术前,子宫内膜异位症患者血清sB7-H4水平为(36.23±5.67)μg/L,明显高于术后(32.54±4.27)μg/L(P0.05)。子宫内膜异位症患者血清sB7-H4水平与CA125水平呈显著正相关(r=0.531,P0.01)。结论:血清可溶性B7-H4可能与子宫内膜异位症的发病有关,检测血清中可溶性B7-H4水平对内异症的辅助诊断和疗效观察可能具有一定的临床意义。