拟南芥DUF647家族成员RUS4植物表达载体的构建及亚细胞定位

ROOT UV-B SENSITIVE4 (RUS4)是拟南芥DUF647蛋白家族的一个功能未知的成员。RUS1和RUS2曾经报道和根UV-B-感应途径相关联,并在拟南芥早期幼苗形态发生和发育中发挥重要作用。为了探究RUS4的分子功能,本研究对RUS4蛋白进行了亚细胞定位分析。首先,我们通过Gateway TOPO载体系统,构建了融合蛋白表达载体pMDC83-RUS4;然后,通过农杆菌介导的花苞转化法,获得了转化pMDC83-RUS4的转基因拟南芥株系;对转基因植物叶肉原生质体的荧光显微镜观察显示,RUS4-GFP信号与叶绿体的自发荧光共定位,说明RUS4蛋白定位叶绿体中。该结果为进一步研究RUS4的分子功能奠定了良好的基础。     

三种融合标签对大熊猫轮状病毒结构蛋白VP6-VP7可溶性表达的影响

大熊猫轮状病毒(giant panda rotavirus,GPRV)是引起幼龄大熊猫腹泻的主要病原,对圈养大熊猫产生了较大的危害。轮状病毒结构蛋白VP6是一种载体蛋白,可介导黏膜免疫反应。VP7是轮状病毒结构蛋白中主要的中和抗原。因此,VP6-VP7的融合表达作为候选抗原对该病的防治具有重要的意义。传统大肠埃希菌原核表达存在表达量低、可溶性差以及纯度低等弊端。本研究使用醛缩酶(EDA)、谷胱甘肽S-转移酶(GST)、麦芽糖结合蛋白(MBP)3种融合标签,以实现获得表达量高和纯度高的GPRV-VP6-VP7重组表达蛋白。将扩增的VP6、VP7基因片段利用同源重组酶构建到含3种融合标签的表达载体pET21b上,将重组质粒转化至大肠埃希菌Rosetta(DE3)感受态细胞中进行低温诱导表达。用Ni-柱亲和层析法纯化目的蛋白,SDS-PAGE和Image J分析蛋白表达量和可溶性,Western blot分析得到表达的重组表达蛋白正确且具有蛋白活性。实验结果证明,EDA标签能显著促进VP6-VP7蛋白的原核可溶性表达,提高VP6-VP7蛋白表达量。     

Detection and classification of Lepeophterius salmonis (Krøyer, 1837) using underwater hyperspectral imaging

Salmon louse, or sea lice, (Lepoptherius salmonis) represents practical, economical and fish welfare challenges for salmon farming (Hamre et al., 2013) and for the free-living stocks of salmon. There is an urgent need in the industry for a system that provides reliable numbers of louse on farmed salmon. Underwater hyperspectral imaging represents a potential new technique for louse counting in sea cages. In laboratory studies, the UHI technology could detect and classify pre-adults (pre-adult I and II), adult males and adult females (ovigerous) of sea lice based on the difference in their spectral characteristics. A model was built for detection of lice on the salmon and the UHI had a detection success ranging from 67 to 100 % with an average of 82%. A classification of the detected lice was performed for pre-adults, adult males and ovigerous lice and had a prediction accuracy of 85% when lice were divided into three groups (pre-adults, adult male and ovigerous lice) and 93% when lice were divided in two groups, ovigerous lice and all the other mobile lice (pre-adults and adult male). An automatic procedure for in situ measurements of louse infected salmon could deliver a data basis several times higher than the traditional counting system. The next generation of UHI louse detector should be developed with a higher spatial resolution to be able to detect also the sessile stages of lice. For succeeding with in situ classification of L. salmonis, correction algorithms to compensate for the impact of water between the UHI and lice need also to be developed.     

黄瓜低霜霉威残留性相关基因SDH克隆及表达

通过Solexa技术和比较基因组学方法鉴定霜霉威胁迫下黄瓜果实差异表达基因,筛选黄瓜低霜霉威残留性相关基因SDH。研究利用PCR克隆获得SDH基因全长,命名为Cs SDH。构建亚细胞定位表达载体,利用农杆菌浸润法注射烟草叶片后激光共聚焦显微镜下观察;分别用荧光定量RT-PCR方法和酶联免疫法研究高、低农残品系D9320和D0351中Cs SDH基因在农药霜霉威处理后,不同时间点、不同组织部位时空表达特征和琥珀酸脱氢酶活性。结果表明,Cs SDH基因在烟草叶片细胞中被定位在细胞膜上。黄瓜低农残品种D0351中,Cs SDH基因在果实受霜霉威胁迫后反应迅速,主要在处理前期响应表达强烈,琥珀酸脱氢酶活性显著增加,且该基因表达量显著高于其在高农残品种D9320中表达量。Cs SDH果、叶中表达量较茎中高,且各组织部位表达量高低顺序稳定,为叶果茎。黄瓜高农残品种D9320中,Cs SDH基因表达量在0.5、1、3、12 h呈现上调表达,6、9 h基因表达量差异不显著,48 h出现上调表达高峰。说明克隆得到的Cs SDH基因属农药处理前期响应基因,且该基因可能在降低黄瓜农药残留过程中发挥重要作用。试验通过研究黄瓜琥珀酸脱氢酶基因(SDH)在霜霉威胁迫下表达情况,为挖掘黄瓜低农药残留性功能基因以及探明其作用分子机制奠定基础。     

被征地农民的群体分化与社区融合——以重庆为例

在对重庆市进行实地调研的基础上，从群体、社区以及社会工作者3个方面来探寻影响被征地农民社区融合的原因，在分析不同类型被征地农民社区融合的需求的基础上，探讨不同层次的社会工作介入被征地农民社区融合的路径。     

间接免疫荧光染色检测鸭源副黏病毒及在鸭体内的抗原定位

以抗新城疫病毒F蛋白的单克隆抗体为一抗,建立了间接免疫荧光染色法(IFA)检测石蜡切片中鸭副黏病毒(DPMV)的方法。以建立的IFA对DPMV人工感染鸭的各组织器官进行检测,结果显示:各组织器官均能检测到DPMV,但不同时间取样,阳性信号分布的器官不同。脾脏、胸腺、法氏囊、肠道、肝脏、肺脏DPMV的阳性检出率较高,表明这些器官为DPMV的主要靶器官。在所检测的阳性组织中,病毒抗原分布在细胞浆中。IFA检测石蜡切片中的DPMV具有直观、特异性强的优点,是对DPMV进行检测和抗原定位的良好方法。     

Evaluation of left ventricular function with cardiac magnetic resonance imaging and echocardiography after administration of dobutamine and esmolol in healthy beagle dogs

Unlike echocardiography, cardiac magnetic resonance imaging (cardiac MRI) results in a near-exact assessment of cardiac structures and function. However, most veterinary studies have focused on dogs with normal cardiac function. We hypothesized that there would be significant differences in cardiac measurements between cardiac MRI and echocardiography when left ventricular (LV) function was abnormal. This study was undertaken to compare measurements of LV function produced by cardiac MRI and echocardiography in dogs whose LV function was altered by pharmacological agents. This study was conducted with six healthy beagle dogs. We increased left ventricular contractility by administration of dobutamine; we decreased cardiac contractility with esmolol. Stroke volume measurements were made by using both cardiac MRI and echocardiography under seven different conditions with general anesthesia: control, three doses of esmolol (100, 200, and 500 µg/kg/min), and three doses of dobutamine (10, 20, and 50 µg/kg/min). Experiments involving each condition were conducted at least 1 week apart. When LV contractility was normal, ejection fraction (EF) and stroke volume (SV), as measured by echocardiography and cardiac MRI, were not significantly different. However, when contractility was changed by pharmacological agents, EF and SV were overestimated by echocardiography, compared to MRI. Evaluation of cardiac function in patients treated with pharmacological agents should be conducted carefully because EF and SV measured by echocardiography can be overestimated, compared with EF and SV obtained by cardiac MRI.     

Cerebrospinal fluid flow in normal beagle dogs analyzed using magnetic resonance imaging

BackgroundDiseases related to cerebrospinal fluid flow, such as hydrocephalus, syringomyelia, and Chiari malformation, are often found in small dogs. Although studies in human medicine have revealed a correlation with cerebrospinal fluid flow in these diseases by magnetic resonance imaging, there is little information and no standard data for normal dogs.ObjectivesThe purpose of this study was to obtain cerebrospinal fluid flow velocity data from the cerebral aqueduct and subarachnoid space at the foramen magnum in healthy beagle dogs.MethodsSix healthy beagle dogs were used in this experimental study. The dogs underwent phase-contrast and time-spatial labeling inversion pulse magnetic resonance imaging. Flow rate variations in the cerebrospinal fluid were observed using sagittal time-spatial labeling inversion pulse images. The pattern and velocity of cerebrospinal fluid flow were assessed using phase-contrast magnetic resonance imaging within the subarachnoid space at the foramen magnum level and the cerebral aqueduct.ResultsIn the ventral aspect of the subarachnoid space and cerebral aqueduct, the cerebrospinal fluid was characterized by a bidirectional flow throughout the cardiac cycle. The mean ± SD peak velocities through the ventral and dorsal aspects of the subarachnoid space and the cerebral aqueduct were 1.39 ± 0.13, 0.32 ± 0.12, and 0.76 ± 0.43 cm/s, respectively.ConclusionsNoninvasive visualization of cerebrospinal fluid flow movement with magnetic resonance imaging was feasible, and a reference dataset of cerebrospinal fluid flow peak velocities was obtained through the cervical subarachnoid space and cerebral aqueduct in healthy dogs.     

CLINICAL AND MAGNETIC RESONANCE IMAGING FEATURES OF INFLAMMATORY VERSUS NEOPLASTIC MEDIAL RETROPHARYNGEAL LYMPH NODE MASS LESIONS IN DOGS AND CATS

Medial retropharyngeal lymph node (MRLN) mass lesions are a common cause of cranial cervical masses in dogs and cats, and are predominantly due to metastatic neoplasia, primary neoplasia, or inflammatory lymphadenitis. The purpose of this retrospective cross‐sectional study was to test the hypothesis that clinical and magnetic resonance imaging (MRI) characteristics for dogs and cats with MRLN mass lesions would differ for inflammatory vs. neoplastic etiologies. Dogs and cats with MRLN mass lesions that had undergone MRI and had a confirmed cytological or histopathological diagnosis were recruited from medical record archives. Clinical findings were recorded by one observer and MRI characteristics were recorded by two other observers who were unaware of clinical findings. A total of 31 patients were sampled, with 15 in the inflammatory lymphadenitis group and 16 in the neoplasia group. Patients with inflammatory lymphadenitis were more likely to be younger and present with lethargy (P = 0.001), pyrexia (P = 0.000), and neck pain (P = 0.006). Patients with inflammatory lymphadenitis were also more likely to have a leukocystosis (P = 0.02) and segmental neutrophilia (P = 0.001). Inflammatory masses were more likely to have moderate or marked MRI perinodal contrast enhancement (P = 0.021) and local muscle contrast enhancement (P = 0.03) whereas the neoplastic masses were more likely to have greater MRI width (P = 0.002) and height (P = 0.009). In conclusion, findings indicated that some clinical and MRI characteristics differed for dogs and cats with inflammatory vs. neoplastic medial retropharyngeal lymph node masses. Although histopathological or cytological diagnosis remains necessary for confirmation, these findings may help with the ranking of differential diagnoses of future cases.