GAP,an aequorin-based fluorescent indicator for imaging Ca2+ in organelles

Genetically encoded calcium indicators allow monitoring subcellular Ca²⁺ signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca²⁺ sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg²⁺, tunable Ca²⁺ affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca²⁺ indicators for organelles and becomes a valuable tool for in vivo Ca²⁺ imaging applications.Ca²⁺ is involved in the regulation of many intracellular processes that take place both in the cytosol and inside organelles (1–3). Therefore, accurate measurement of the calcium concentration ([Ca²⁺]) inside organelles is essential to discriminate discrete Ca²⁺ signals between the different compartments. Although synthetic Ca²⁺ indicators can be loaded into organelles, the signal has poor selectivity, as the dye is also present in the cytosol and must be carefully removed before measurements (4). The main advantage of Genetically Encoded Ca²⁺ Indicators (GECIs) is their ability to be targeted to specific intracellular locations. Both bioluminescent and fluorescent proteins have been successfully used to measure subcellular [Ca²⁺]. The photoprotein aequorin (5), purified from the jellyfish Aequorea victoria, was the first protein-based Ca²⁺ indicator, injected into cells in the early 1970s (6). After cloning of its cDNA (7), recombinant aequorin became the most frequently used probe to measure Ca²⁺ in organelles, including mitochondria (8), the endoplasmic reticulum (ER) (9), the nucleus (10), the Golgi apparatus (11), or secretory vesicles (12).Fluorescent GECIs achieve a better spatial resolution than bioluminescent sensors. They are generally composed of one or two fluorescent proteins, most of them variants of GFP, fused to a Ca²⁺-binding protein (13). Recently, a single EF-hand motif has been inserted in the GFP moiety to generate a Ca²⁺ fluorescent probe (14). Since the first cameleon based on FRET (15), the number of GECIs has exponentially increased, attempting optimization of critical features such as adequate expression, signal strength, or dynamic range. However, the in vivo use in mammals, one of the main applications of GECIs, has grown more slowly and has disclosed severe limitations (16, 17). Transgenic sensors usually showed a low expression, often resulting in its inactivation or reduced dynamic range. With the exception of troponin derivatives, most of the available GECIs, namely cameleons, camgaroos, pericams, or GCaMPs (circularly permutated EGFP-based Ca²⁺ sensors), are based on calmodulin, a highly regulated ubiquitous protein that binds a large number of targets (13). Although the interference with endogenous calmodulin has been reduced in the improved cameleons (18), the interaction with other cellular proteins cannot be ruled out. Thus, the loss of Ca²⁺ sensitivity observed in vivo may reflect the interaction of the probe with endogenous partners, which may disturb cellular functions.The jellyfish aequorin exhibits a number of advantages over mammalian EF-hand proteins. It is not toxic and appears not to interfere with other intracellular Ca²⁺-binding molecules, even when microinjected at high concentrations in mammalian cells. Moreover, the use of aequorin as a bioluminescence sensor has been extensively reported, ranging from subcellular Ca²⁺ measurements in many different cell types up to whole organisms, including transgenic animals (19–21).Here we describe a family of fluorescent Ca²⁺ sensors based on the fusion of two jellyfish proteins, GFP and apoaequorin. This Ca²⁺ probe shows a larger dynamic range compared with other GECIs and a robust photonic and thermal stability. It can be targeted to distinct compartments such as the nucleus, cytosol, or mitochondria, where it selectively and accurately monitors dynamic Ca²⁺ changes. In addition, we have generated a variant with a lower Ca²⁺ affinity suited for imaging Ca²⁺ changes in organelles with high resting [Ca²⁺] such as the ER or the Golgi apparatus. Finally, we demonstrate its in vivo applicability by generating transgenic mice where the Ca²⁺ biosensor maintained its in vitro features.     

Assessment of Induced Sputum Cellularity in COPD Patients Belonging to Two Different Classes of Air Pollution Exposure

IntroductionSeveral studies have previously demonstrated that long-term exposure to outdoor pollution present airway inflammation in term of an increase of sputum neutrophils.Aim and methodsThe aim of our study was to evaluate the level of airway inflammation by induced sputum in a group of 15 non-professionally exposed population of well-characterized COPD patients, residing in urban areas with high rate of outdoor pollution, compared to a control group of 13 individuals with COPD, living in rural areas with a low pollution rate. All participants underwent spirometry and sputum induction.ResultsA statistically significant increase in the percentage of neutrophil cell count was found among the residents in urban areas compared to those living in rural regions (89.1 vs 79.0, p < 0.05)ConclusionsIn conclusion, we showed that non-professionally exposed patients with COPD residing in highly-polluted urban areas had greater airway inflammation in terms of sputum neutrophils compared to a population with very similar characteristics, living in rural areas with lower outdoor pollution. The results of this pilot study may be relevant for the long term effect of environmental outdoor pollution in vulnerable patients like those with COPD.     

A new method for muscle fatigue assessment: Online model identification techniques

Introduction: The purpose of this study was to propose a method that allows extraction of the current muscle state under electrically induced fatigue. Methods: The triceps surae muscle of 5 subjects paralyzed by spinal cord injury was fatigued by intermittent electrical stimulation (5 × 5 trains at 30 Hz ). Classical fatigue indices representing muscle contractile properties [peak twitch (Pt) and half‐relaxation time (HRT)] were assessed before and after each 5‐train series and were used to identify 2 relevant parameters (F_m, U_r) of a previously developed mathematical model using the Sigma‐Point Kalman Filter. Results: Pt declined significantly during the protocol, whereas HRT remained unchanged. Identification of the model parameters with experimental data yielded a model‐based fatigue assessment that gave a more stable evaluation of fatigue than classical parameters. Conclusions: This work reinforces clinical research by providing a tool that clinicians can use to monitor fatigue development during stimulation. Muscle Nerve 50: 556–563, 2014     

COVID-19疫情中的生物力学问题

新型冠状病毒肺炎自爆发以来,已成为全球关注的焦点。由于疫情来势迅猛,给医疗制度体系、医疗资源、应急处置、医疗器械等提出了严峻的挑战,也暴露出其中诸多不足。以病毒的体外活性规律研究、气溶胶传播的生物力学问题、呼吸性疾病对心血管疾病影响的研究、公共交通和公共场所中通风方式的改进、医疗器械的生物力学研究为例,应用生物力学的原理、观点和方法,认识和剖析其中所存在和亟待改进的问题,期望生物力学能在疫情防控中发挥学科优势和力量,以此促进生物力学学科发展。     

痰培养致病菌构成与耐药性分析

目的调查本院呼吸科患者痰培养病原菌分布及耐药性,指导临床合理使用抗菌药物。方法对2011年7月至2013年6月呼吸科的住院患者痰标本进行细菌分离培养、鉴定和药敏试验,利用瑞美数据管理系统,收集阳性结果进行统计分析。结果 1 640株阳性病原菌中,革兰氏阴性杆菌800株,占48.8%,其中铜绿假单胞菌200株,占12.2%,克雷伯菌属198株,占12.0%,流感嗜血杆菌108株,占6.6%,大肠埃希菌98株,占6.0%;检出的主要革兰氏阴性杆菌对阿米卡星、环丙沙星、亚胺培南、左氧氟沙星、哌拉西林/他唑巴坦、替卡西林/棒酸的敏感性较高,大肠埃希菌和克雷伯菌属产超广谱β-内酰胺酶（ESBLs）的比率分别是59.1%和28.9%,且呈多药耐药性;革兰氏阳性球菌138株,占8.4%,其中金黄色葡萄球菌56株,占3.4%,凝固酶阴性葡萄球菌30株,占1.9%,耐甲氧西林的葡萄球菌占44.2%;检出的主要的革兰阳性球菌对万古霉素、利福平较为敏感。真菌感染呈上升趋势,检出702株,占42.8%,以白色假丝酵母菌为主。结论呼吸道感染以革兰氏阴性杆菌和白色假丝酵母菌为主,其中产ESBLs的大肠埃希菌的比率较高,耐药现象较严重;耐甲氧西林葡萄球菌的感染应引起临床医生的高度重视,病原菌的耐药监测对临床正确合理应用抗生素具有重要的指导作用。     

HBV感染慢性肝病患者血清GP73浓度对肝纤维化程度的诊断意义

目的 探讨血清GP73浓度与慢性HBV肝病患者肝纤维化程度之间的关系,以评估GP73在慢性HBV患者中的临床诊断价值.方法 选取慢性HBV感染者761名,分别进行FibroScan检测和血清GP73测试.结果 血清GP73浓度与肝硬度相关（r=0.601）,ROC曲线下面积为0.76.GP73用于诊断显著肝纤维化（≥F2）的敏感性和特异性分别为62.81％和80.05％（cut off值：76.6ng/ml）.结论 GP73可能是一种诊断慢性HBV感染者显著肝纤维化的新标志物.     

高尔基体蛋白73在原发性肝癌预后判断中的应用价值

目的 探讨血清高尔基体蛋白73（GP73）在原发性肝癌预后判断中的价值.方法 酶联免疫吸附试验定量检测血清GP73,分别测定63例原发性肝癌患者术前、术后（2周、4周）以及术后3～12个月复发的21例重新入院患者的GP73的浓度.结果 原发性肝癌患者术前GP73平均水平为（240.5±81.4） ng/ml,手术治疗后2周GP73平均浓度为（114.4±34.1）ng/ml,治疗后4周平均浓度为（57.6±23.3） ng/ml,治疗前浓度显著高于治疗后浓度（P＜0.05）.肝癌复发的患者重入院后GP73浓度[（181.2±72.4） ng/ml]明显高于GP73术后的浓度（P＜0.05）.结论 GP73作为一个新型肝癌血清标记物在判断肝癌预后中具有较好的应用价值.     

口腔科常用医疗器械清洗消毒效果的评价

目的评价口腔科医疗器械常规清洗消毒的效果。方法选择2013年7月～12月口腔科使用过的非一次性医疗器械（拔牙钳、车针）共36669件,消毒供应室清洗消毒处理前后,对物体表面口腔常见的变形链球菌、白色念珠菌、金黄色葡萄球菌以及乙肝病毒进行检测,并用目测法分析清洗的质量,测评临床的满意度。结果（1）器械清洗消毒质量的合格数为35395件,合格率达到96．52％;（2）细菌和病毒检测结果显示,除了工作室桌子有10次细菌不达标以外,其余均未检出细菌和病毒;（3）紫外照射与高压的消毒效果差距较大,未高压的灭菌消毒效果低于高压的灭菌消毒效果;（4）临床使用满意度达到95．83％。结论经规范化清洗消毒与管理,基本能确保口腔器械的卫生安全,所采用的清洗消毒措施,在一定程度上能有效控制医院感染的发生。     

早期应用吞咽治疗仪治疗脑卒中吞咽障碍的疗效观察与护理

目的分析应用吞咽治疗仪治疗脑卒中吞咽障碍的临床疗效并总结护理要点。方法选取2017年9月-2019年9月我院收治的30例脑卒中吞咽功能障碍患者。按时间先后分为对照组15例及观察组15例。对照组采取综合护理干预,观察组在对照组基础上采取早期应用吞咽治疗仪治疗,比较2组治疗效果。结果治疗护理2周后观察组患者吞咽功能获得明显改善,优于对照组(P<0.05);观察组治疗总有率为93.3%,对照组为66.7%(P<0.05)。结论脑卒中吞咽障碍患者早期给予吞咽治疗仪治疗配合综合护理干预可改善吞咽效果,改善生活质量。