经支气管镜气管插管与常规气管插管救治呼吸衰竭对比分析

目的：回顾性分析呼吸机辅助呼吸救治危重呼吸衰竭患者气管插管方式对于救治成功率的影响。方法：我科收治的各种呼吸衰竭患者94例,回顾分析插管方式对于救治成功率、引发心跳骤停及其对心肺复苏效果的影响。结果：经纤维支气管镜经口气管插管（35例）与经直接喉镜经口（59例）引发心跳骤停有明显统计学差异（X^2=11．5,v=1,t〈0．001）。经直接喉镜经口气管插管在术前用药与否对引发心跳骤停并无统计学意义,但是对于引发心跳骤停后心肺复苏成功率确有明显统计学意义。结论：经纤维支气管镜气管插管具有较高的安全性,在经直接喉镜气管插管是选择镇静药或浅麻醉药物应慎重,尽力避免心跳骤停和心肺复苏失败。     

Complex Effects of Prescribed Fire on Restoring the Soil Water Content in a High-Elevation Riparian Meadow, Arizona

One of the largest and rarest Bebb willow (Salix bebbiana) communities in the United States occurs at Hart Prairie, Arizona. Low recruitment of the willow over the past several decades has been linked to inadequate soil water content for seed germination and seedling establishment. We tested a hypothesis that a prescribed burn would reduce biomass of and evapotranspiration by herbaceous plants, thereby increasing soil water content. Three treatments (unburned control, early‐growing season burned, late‐growing season burned) were applied in year 2001 to replicated plots in fern‐ and grass‐dominated herbaceous communities. Soil water content (0–30 cm) was measured weekly in plots during the 2001, 2002, and 2003 growing seasons. Both early‐ and late‐season burning reduced herbaceous biomass in the fern‐dominated community in 2002 and 2003 and reduced biomass in the grass‐dominated community in 2002 but not in 2003. Soil water content increased for approximately four weeks in 2001 following the early‐season burn, but the early‐season and late‐season burns reduced soil water content in both communities over much of the 2002 and 2003 growing seasons. Thus, early‐season burning may benefit willow seed germination by increasing soil water content immediately following burning but be detrimental to germination in the second and third growing seasons after burning because of drier soil. Large temporal variation in the effect of prescribed burning on soil water content will complicate the use of fire as a restoration tool to manage soil water available for threatened plants such as Bebb willow and for recharge of groundwater.     

Novel tricyclic pyrazole BRAF inhibitors with imidazole or furan central scaffolds

V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine-specific protein kinase that is mutated with high frequency in cutaneous melanoma, and many other cancers. Inhibition of mutant BRAF is an attractive therapeutic approach for the treatment of melanoma. A triarylimidazole BRAF inhibitor bearing a phenylpyrazole group (dimethyl-[2-(4-{5-[4-(1H-pyrazol-3-yl)-phenyl]-4-pyridin-4-yl-1H-imidazol-2-yl}-phenoxy)-ethyl]-amine, 1a) was identified as an active BRAF inhibitor. Based on this starting point, we synthesized a series of analogues leading to the discovery of 6-{2-[4-(4-methyl-piperazin-1-yl)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-2,4-dihydro-indeno[1,2-c]pyrazole (1j), with nanomolar activity in three assays: inhibition of purified mutant BRAF activity in vitro; inhibition of oncogenic BRAF-driven extracellular regulated kinase (ERK) activation in BRAF mutant melanoma cell lines; and inhibition of proliferation in these cells.     

VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature

Unlike during development, blood vessels in the adult are generally thought not to require VEGF for normal function. However, VEGF is a survival factor for many tumor vessels, and there are clues that some normal blood vessels may also depend on VEGF. In this study, we sought to identify which, if any, vascular beds in adult mice depend on VEGF for survival. Mice were treated with a small-molecule VEGF receptor (VEGFR) tyrosine kinase inhibitor or soluble VEGFRs for 1-3 wk. Blood vessels were assessed using immunohistochemistry or scanning or transmission electron microscopy. In a study of 17 normal organs after VEGF inhibition, we found significant capillary regression in pancreatic islets, thyroid, adrenal cortex, pituitary, choroid plexus, small-intestinal villi, and epididymal adipose tissue. The amount of regression was dose dependent and varied from organ to organ, with a maximum of 68% in thyroid, but was less in normal organs than in tumors in RIP-Tag2-transgenic mice or in Lewis lung carcinoma. VEGF-dependent capillaries were fenestrated, expressed high levels of both VEGFR-2 and VEGFR-3, and had normal pericyte coverage. Surviving capillaries in affected organs had fewer fenestrations and less VEGFR expression. All mice appeared healthy, but distinct physiological changes, including more efficient blood glucose handling, accompanied some regimens of VEGF inhibition. Strikingly, most capillaries in the thyroid grew back within 2 wk after cessation of treatment for 1 wk. Our findings of VEGF dependency of normal fenestrated capillaries and rapid regrowth after regression demonstrate the plasticity of the adult microvasculature.     

Discovery and optimization of potent and selective triazolopyridazine series of c-Met inhibitors

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. We previously showed that O-linked triazolopyridazines can be potent inhibitors of c-Met. Herein, we report the discovery of a related series of N-linked triazolopyridazines which demonstrate nanomolar inhibition of c-Met kinase activity and display improved pharmacodynamic profiles. Specifically, the potent time-dependent inhibition of cytochrome P450 associated with the O-linked triazolopyridazines has been eliminated within this novel series of inhibitors. N-linked triazolopyridazine 24 exhibited favorable pharmacokinetics and displayed potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver PD model. Once-daily oral administration of 24 for 22 days showed significant tumor growth inhibition in an NIH-3T3/TPR-Met xenograft mouse efficacy model.     

Structural Evidence for Loose Linkage between Ligand Binding and Kinase Activation in the Epidermal Growth Factor Receptor

The mechanisms by which signals are transmitted across the plasma membrane to regulate signaling are largely unknown for receptors with single-pass transmembrane domains such as the epidermal growth factor receptor (EGFR). A crystal structure of the extracellular domain of EGFR dimerized by epidermal growth factor (EGF) reveals the extended, rod-like domain IV and a small, hydrophobic domain IV interface compatible with flexibility. The crystal structure and disulfide cross-linking suggest that the 7-residue linker between the extracellular and transmembrane domains is flexible. Disulfide cross-linking of the transmembrane domain shows that EGF stimulates only moderate association in the first two α-helical turns, in contrast to association throughout the membrane over five α-helical turns in glycophorin A and integrin. Furthermore, systematic mutagenesis to leucine and phenylalanine suggests that no specific transmembrane interfaces are required for EGFR kinase activation. These results suggest that linkage between ligand-induced dimerization and tyrosine kinase activation is much looser than was previously envisioned.Fundamental to cellular physiology is the ability to transmit extracellular signals across the cell membrane to trigger intracellular responses. Although the extracellular and intracellular portions of cell surface receptors are responsible for detecting ligands and initiating signal cascades, respectively, transmembrane (TM) domains are thought to play critical roles by specifically associating and propagating signals across the phospholipid bilayer. However, the mechanisms by which single-pass TM domains associate and conduct signals are poorly understood.The epidermal growth factor receptor (EGFR) is the prototypical type I TM receptor tyrosine kinase. EGFR and related members of the ErbB family—ErbB2, ErbB3, and ErbB4—contain a glycosylated extracellular ligand binding domain; a single-pass TM domain; and intracellular juxtamembrane, tyrosine kinase, and autophosphorylation domains. The extracellular domain of EGFR binds polypeptide growth factor ligands, such as epidermal growth factor (EGF), to stimulate an array of intracellular signaling cascades that regulate normal and oncogenic cellular growth and proliferation (3, 17, 36). In one model of growth factor-dependent EGFR activation, ligand binding promotes receptor dimerization and activation of intracellular protein tyrosine kinase activity (35); other models suggest that receptors are predimerized on the cell surface and ligand binding alters the equilibrium between inactive and active dimeric (or higher-order oligomeric) configurations (9, 29).Structural mechanisms of growth factor-mediated receptor dimerization and allosteric kinase domain activation have been proposed from recent crystal structures of isolated extracellular ligand binding domains (7) and intracellular tyrosine kinase domains (37). The orientation between the four extracellular domains is dramatically altered upon ligand binding, which frees interfaces that are masked in tethered, unliganded monomers to mediate dimer formation (7). Furthermore, an unusual asymmetric interface between two kinase domain monomers is linked to rearrangement of the kinase site to the active conformation (37). However, neither the position of the last extracellular domain, domain IV, nor association between the TM domains is well-defined experimentally in liganded receptors. The approximate location of domain IV has been suggested by models based on the orientation between domains III and IV in unliganded monomers (7, 12) and two-dimensional negative-stain electron microscopy (EM) averages (27); however, the position of domain IV in the liganded dimer has not been determined in previous crystal structures (13, 30). Thus, it is not known how the extracellular domain positions the TM domains for transmembrane signaling.Several lines of evidence suggest that the TM domain contributes directly to receptor dimerization and signaling. The neu oncogene encodes a Val → Glu substitution in the TM domain of ErbB2 that results in constitutive activation (34). Recombinant EGFR fragments consisting of the extracellular and TM domains have a 10⁵-fold higher affinity for dimerization than the isolated soluble extracellular domains (31). The TM domains of all four ErbB family members self-associate when expressed in bacterial inner membranes (26). A dimeric structure for isolated ErbB2 TM peptides in bicelles has been defined by nuclear magnetic resonance (NMR) imaging (4). However, ErbB2 does not bind ligand and does not physiologically homodimerize (17). Moreover, different ErbB family member TM domains utilize potentially distinct GxxxG sequence motifs to dimerize, as shown with fusion proteins in bacterial membranes (26). However, it is not clear how the TM domains contribute to dimerization and signaling in intact receptors on the cell surface.Here, we characterize the structural basis for EGFR transmembrane signaling. An improved crystal structure of the EGF-bound EGFR extracellular domain resolves domain IV in electron density maps and identifies a small domain IV dimerization interface, the mutation of which does not abolish signaling. The crystal structure and disulfide cross-linking demonstrate a flexible, dimeric linker between the extracellular and transmembrane domains. EGF-induced dimerization of the TM domains involves an interface far less extensive than that found in two receptors that dimerize in the absence of activation. Furthermore, mutagenesis shows that no unique interface is required for transmembrane signaling. Thus, we propose that signal transmission through the EGFR is communicated much more loosely than was previously thought.     

Epigenetic features drastically impact CRISPR–Cas9 efficacy in plants

CRISPR–Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR–Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR–Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR–Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR–Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes.

Epigenetic features substantially influence genome editing efficiency and DNA repair outcomes.