A removable virus vector suitable for plant genome editing

Plant genome editing is achieved by the expression of sequence‐specific nucleases (SSNs). RNA virus vector‐mediated expression of SSNs is a promising approach for transgene integration‐free targeted mutagenesis in plants. However, the removal of virus vectors from infected plants is challenging because no antiviral drugs are available against plant viruses. Here, we developed a removable RNA virus vector that carries the target site of tobacco microRNA398 (miR398) whose expression is induced during shoot regeneration. In the inoculated leaves in which expression of miR398 is not induced, insertion of the miR398 target site did not affect the practicability of the virus vector. When shoots were regenerated from the infected leaves, miR398 was expressed and viral RNA was eliminated. The virus vector successfully expressed SSNs in inoculated leaves, from which virus‐free genome‐edited plants were regenerated via tissue culture.     

基于多源遥感数据的面向对象林分类型识别

林分类型的识别是森林资源监测的核心问题之一.为研究多源遥感数据协同的面向对象林分类型分类识别,采用Radarsat-2数据和QuickBird遥感影像协同进行面向对象分类.在面向对象分类过程中,采用3种分割方案:单独使用QuickBird遥感影像分割;单独使用Radarsat-2数据分割;Radarsat-2&QuickBird协同分割.3种分割方案均采用10种分割尺度(25～250,步长25),应用修正的欧式距离3指标评价不同分割方案的分割结果,确定最优分割方案及最优分割尺度.在最优分割结果的基础上,基于地形、高度、光谱及共同特征的不同特征组合,应用带有径向基(RBF)核函数的支持向量机(SVM)分类器进行杉木林、马尾松林、阔叶林3种林分类型识别.结果表明:与单独使用一种数据相比,Radarsat-2数据和QuickBird遥感影像协同方案在面向对象林分类型分类方面具有优势.Radarsat-2&QuickBird协同分割方案,以最优尺度参数100进行分割时,分割结果最好.在最优分割结果的基础上,应用两种数据源提取的全部特征进行面向对象林分类型识别的精度最高(总精度为86%,Kappa值为0.86).本研究结果不仅可为多源遥感数据结合进行林分类型识别提供参考和借鉴,而且对于森林资源调查和监测有现实意义.     

Inter- and intra-rater reliability of 3D kinematics during maximum mouth opening of asymptomatic subjects

Previous studies evaluated 3D human jaw movements using kinematic analysis systems during mouth opening, but information on the reliability of such measurements is still scarce. The purpose of this study was to analyze within- and between-session reliabilities, inter-rater reliability, standard error of measurement (SEM), minimum detectable change (MDC) and consistency of agreement across raters and sessions of 3D kinematic variables during maximum mouth opening (MMO). Thirty-six asymptomatic subjects from both genders were evaluated on two different days, five to seven days apart. Subjects performed three MMO movements while kinematic data were collected. Intraclass correlation coefficient (ICC), SEM and MDC were calculated for all variables, and Bland-Altman plots were constructed. Jaw radius and width were the most reproducible variables (ICC > 0.81) and demonstrated minor error. Incisor displacement during MMO and angular movements in the sagittal plane presented good reliability (ICC from 0.61 to 0.8) and small errors and, consequently, could be used in future studies with the same methodology and population. The variables with smaller amplitudes (condylar translations during mouth opening and closing and mandibular movements on the frontal and transversal planes) were less reliable (ICC < 0.61) and presented larger SEM and MDC. Although ICC, SEM and MDC showed less between-session reproducibility than within-session and inter-rater, the limits of agreement were larger in inter-rater comparisons. In future studies care must be taken with variables collected on different days and with mandibular movements in the frontal and transversal planes.     

Observations of marine wildlife tourism effects on a non‐focal species

A radio‐acoustic positioning system was used to assess the effects of shark cage‐diving operators (SCDO) on the fine‐scale movements of a non‐focal species, the smooth stingray Bathytoshia brevicaudata. The results revealed that the time spent in the array was individually variable, but generally increased when SCDO were present and that the presence of SCDO may have the capacity to elicit changes in the space use of B. brevicaudata. These results indicate that the effects of marine wildlife tourism may extend beyond the focal species of interest.     

Temperature- and size-dependent growth and movement of the North Pacific giant octopus (Enteroctopus dofleini) in the Bering Sea

Octopus growth and movement occurs during all life stages and have implications for survival, food web dynamics and reproduction. From 2009 to 2011, 1714 North Pacific giant octopus (Enteroctopus dofleini) were tagged and recaptured in the eastern Bering Sea with visible implant elastomers to better understand the ecology of this data-poor species. Over this period, 246 of the tagged individuals were recaptured. In autumn, when temperatures were warmest, E. dofleini had higher growth rates and moved more than in the colder winter months. For both short- and long-term recaptures, small octopus grew faster than large octopus. Movement of octopus over short- and long-term periods was low for very small and very large octopus and high for median-sized octopus, which is likely to be a function of maturity status and reproductive activities. Approximately 80% of recaptures moved less than 2?km from the initial tagging location for time periods up to a year, suggesting long-term site fidelity. As temperature and size may be tightly linked to growth and movement rates for E. dofleini in the eastern Bering Sea, predicted climate warming will likely alter ecological processes for the species and impact their distribution.     

ShRNA knock‐down of CXCR7 inhibits tumour invasion and metastasis in hepatocellular carcinoma after transcatheter arterial chemoembolization

To investigate the effects of lentiviral vector‐mediated shRNA suppressing CXCR7 on tumour invasion and metastasis in hepatocellular carcinoma (HCC) after transcatheter arterial chemoembolization (TACE). HCCLM3 cell lines were cultured and assigned into the CXCR7‐shRNA, negative control (NC) and blank groups. The qRT‐PCR and Western blotting were applied to detect the mRNA and protein expressions of CXCR7, CXCR4 and MMP‐2 in HCCLM3 cells. Cell proliferation and invasion were evaluated by MTT and Transwell assays. A Buffalo rat model of HCC was established. Fifty model rats were divided into the CXCR7‐shRNA + TACE, CXCR7‐shRNA, TACE, NC and control groups. Immunohistochemistry was performed to detect the expressions of CXCR7, MMP‐2, vascular endothelial growth factor (VEGF) and intratumoral CD31‐positive vessel count in tumour tissues of mice. Compared with the blank and NC groups, the mRNA and protein expressions of CXCR7 and MMP‐2 were decreased in the CXCR7‐shRNA group. The cell proliferation and invasion rates of the CXCR7‐shRNA group were lower than the blank and NC groups. At the 4th week after TACE, tumour weight of the CXCR7‐shRNA + TACE group increased continuously. The CXCR7‐shRNA + TACE group showed longer survival time and smaller tumour sizes than other groups. Compared with other groups, the CXCR7‐shRNA + TACE and CXCR7‐shRNA groups had less number of lung metastatic nodules and lower expressions of CXCR7, MMP‐2, VEGF and CD31‐positive vessel count. CXCR7‐shRNA inhibits tumour invasion and metastasis to improve the efficacy of TACE in HCC by reducing the expressions of CXCR7, MMP‐2 and VEGF.     

A hidden Markov movement model for rapidly identifying behavioral states from animal tracks

Electronic telemetry is frequently used to document animal movement through time. Methods that can identify underlying behaviors driving specific movement patterns can help us understand how and why animals use available space, thereby aiding conservation and management efforts. For aquatic animal tracking data with significant measurement error, a Bayesian state‐space model called the first‐Difference Correlated Random Walk with Switching (DCRWS) has often been used for this purpose. However, for aquatic animals, highly accurate tracking data are now becoming more common. We developed a new hidden Markov model (HMM) for identifying behavioral states from animal tracks with negligible error, called the hidden Markov movement model (HMMM). We implemented as the basis for the HMMM the process equation of the DCRWS, but we used the method of maximum likelihood and the R package TMB for rapid model fitting. The HMMM was compared to a modified version of the DCRWS for highly accurate tracks, the DCRWS, and to a common HMM for animal tracks fitted with the R package moveHMM. We show that the HMMM is both accurate and suitable for multiple species by fitting it to real tracks from a grey seal, lake trout, and blue shark, as well as to simulated data. The HMMM is a fast and reliable tool for making meaningful inference from animal movement data that is ideally suited for ecologists who want to use the popular DCRWS implementation and have highly accurate tracking data. It additionally provides a groundwork for development of more complex modeling of animal movement with TMB. To facilitate its uptake, we make it available through the R package swim.     

Adenovirus vector-mediated macrophage erythroblast attacher (MAEA) overexpression in primary mouse hepatocytes attenuates hepatic gluconeogenesis

Japanese patients with type 2 diabetes mellitus present a different responsiveness in terms of insulin secretion to glucose and body mass index (BMI) from other populations. The genetic background that predisposes Japanese individuals to type 2 diabetes mellitus is under study. Recent genetic studies demonstrated that the locus mapped in macrophage erythroblast attacher (MAEA) increases the susceptibility to type 2 diabetes mellitus in East Asians, including Japanese individuals. MAEA encodes a protein that plays a role in erythroblast enucleation and in the normal differentiation of erythroid cells and macrophages. However, the contribution of MAEA to type 2 diabetes mellitus remains unknown. In this study, to overexpress MAEA in the mouse liver and primary mouse hepatocytes, we generated a MAEA-expressing adenovirus (Ad) vector using a novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-MAEA). Blood glucose and insulin levels in Ad-MAEA-treated mice were comparable to those in control Ad-treated mice. Primary mouse hepatocytes transduced with Ad-MAEA showed lower levels of expression of gluconeogenesis genes than those transduced with the control Ad vector. Hepatocyte nuclear factor-4α (HNF-4α) mRNA expression in primary mouse hepatocytes was also suppressed by MAEA overexpression. These results suggest that MAEA overexpression attenuates hepatic gluconeogenesis, which could potentially lead to improvement of type 2 diabetes mellitus.     

Model acupuncture point:Bone marrow-derived stromal stem cells are moved by a weak electromagnetic field

AIM To show the existence of a structural formative role of magnetic fields(MFs) with respect to biological objects by using our proposed model of an acupoint.METHODS We introduced a magnetised 10-100 μT metal rod(needle) into culture dishes with a negatively charged working surface and observed during 24 h how cells were arranged by MFs and by electrical fields(EFs) when attached. Rat and human bone marrow-derived stromal stem cells(r BMSCs and h BMSCs), human nonadherent mononuclear blood cells, NCTCs and A172 cells, and Escherichia coli(E. coli) were evaluated. The dish containing BMSCs was defined as the model of an acupoint. r BMSCs proliferative activity affected by the needle was investigated. For investigating electromagnetic field structures, we used the gas discharge visualisation(GDV) method.RESULTS During 24 h of incubation in 50-mm culture dishes, BMSCs or the nonadherent cells accumulated into a central heap in each dish. BMSCs formed a torus(central ring) with an inner diameter of approximately10 mm only upon the introduction of the needle in the centre of the dish. The cells did not show these effects in 35- or 90-mm culture dishes or hydrophobic dishes or rectangular cuvettes. NCTCs and A172 cells showed unstable the effects and only up to two weeks after thawing. Moreover, we observed that the appearance of these effects depended on the season. In winter, BMSCs showed no the effects. GDV experiments revealed that the resonant annular illumination gradually formed from 10 to 18-20 s in polar solutions with and without cell suspension of BMSCs, NCTCs and E. coli when using circular 50-mm dishes, stimulation at 115 V and switching of the electrode poles at 1 kH z. All these data demonstrate the resonant nature of the central ring. Significant influence of MFs on the rB MSC proliferation rate was not observed.CONCLUSION BMSCs can be moved by MFs when in the presence of a constant EF and MF, when the cells are in the responsive functional state, and when there is a resonant relationship between them.     

人P2X7真核表达载体的构建及稳定转染细胞株的建立

目的：构建人P2X7基因的真核表达载体,并通过转染获得稳定表达P2X7分子的HEK293细胞株。方法：以人脑组织P2X7cDNA为模板扩增出P2X7基因,插入到真核表达载体pEGFP-N1中,构建重组质粒pEGFP-N1/P2X7。用X-fect试剂盒将重组质粒转染HEK293细胞,通过G418辅助荧光筛选建立稳定表达P2X7-EGFP细胞株。经流式细胞仪、Western blot和激光共聚焦显微镜检测,了解人P2X7在HEK293细胞中的表达水平及细胞内定位。结果：重组质粒pEGFP-N1/P2X7构建正确,建立了稳定表达人P2X7的HEK293细胞系。Western blot和流式细胞仪检测证实,P2X7在HEK293细胞系中成功表达,激光共聚焦显微镜检测显示P2X7-EGFP定位在细胞膜上。结论：重组载体pEGFP-N1/P2X7构建成功并建立了稳定表达人P2X7的HEK293细胞系,为进一步研究P2X7离子通道结构和功能奠定基础。