Quantifying Changes in Gap Junction Structure as a Function of Lens Fiber Cell Differentiation

The ocular lens is an ideal model system for studying gap junction structure-function relationships. Here we apply novel methods to quantitatively compare connexin expression over macroscopic distances while simultaneously resolving the intracellular distribution of gap junctions in sub-micron detail. Our approach has identified three distinct zones of connexin density and allowed changes in gap junction plaque size, number and dispersion to be quantified. Our analysis is the first to precisely correlate changes in gap junction plaque structure with the reported changes in gap junction function that occur as a consequence of fiber cell differentiation.     

基于无人机和决策树算法的榆树疏林草原植被类型划分和覆盖度生长季动态估计

植被覆盖度是评估生态环境质量与植被生长的重要指标,也是全球众多陆面过程模型和生态系统模型中表达植被动态的重要参数。卫星遥感和地面测量是估算植被覆盖度的常见方法。然而,如何精确估计榆树疏林草原上木本、草本不同类型植被的覆盖度仍然具有挑战性。无人机飞行系统有效的补充了区域尺度低空间分辨率的卫星遥感影像与样地尺度实地调查之间的缺口,为精确的监测、评估疏林草原的植被动态提供了新途径。利用无人机监测平台和决策树算法构建了一套快速、准确、自动获取景观尺度植被类型和估算植被覆盖度的自动化工具,以浑善达克沙地榆树疏林草原为对象,应用无人机监测平台对榆树疏林草原长期定位监测大样地2017年生长季植被状况进行7次监测。结果表明:1)无人机植被监测平台数据飞行高度100 m,获取的样地数字正射影像空间分辨率为2.67 cm/像元,远高于高分卫星影像,利用决策树算法基于数字正射影像可以实现自动划分榆树疏林草原木本和草本植被类型和估算植被覆盖度; 2)生长季内榆树疏林草原木本植被覆盖度为(19±2)%,草本植被覆盖度为(50±8)%,植被总覆盖度为(69±9)%,相对于木本植被,草本植被生长季内盖度变幅较大; 3)在整个生长季中,木本植被和草本植被对植被总覆盖度的平均贡献率分别为27%和73%,草本植被对植被总盖度的贡献远大于木本植被,榆树疏林草原植被的盖度主要受草本植被的影响。本研究证明无人机监测平台是一种高效、准确的植被监测工具,结合机器学习算法,实现了景观尺度植被类型的自动划分和不同类型植被覆盖度快速获取;在浑善达克沙地榆树疏林草原地区首次获取了木本植被和草本植被覆盖度的生长季动态。该平台未来可应用于各种类型生态系统植被类型划分、监测和评估。     

基于微滴式数字PCR检测肠癌病人游离环状DNA KRAS突变的新方法

基于微滴式数字聚合酶链式反应(Droplet digital polymerase chain reaction,dd PCR)设计一种检测肠癌游离循环DNA(Circulating cell free DNA,cf DNA)中KRAS(V-Ki-ras2 Kirsten ratsarcoma viral oncogene homolog)基因突变的新方法并评估其灵敏度和准确性。根据肠癌病人KRAS基因的突变类型设计并合成,采用dd PCR扩增并评估其灵敏度和准确性;根据AMRS-PCR引物设计原理设计KRAS基因的实时定量PCR扩增引物并评估其准确性,进而比较dd PCR和q PCR二者之间的优缺点;最后针对52例肠癌病人的cf DNA采用dd PCR进行检测,研究dd PCR在cf DNA KRAS基因突变检测的应用。成功使用dd PCR和q PCR两种方法对KRAS野生型及7种突变型建立检测方法,使用质粒标准品及实际样品验证该两种方法可行并对其假阳性率、线性范围及检测下限等性能进行了评价,最后成功对52例临床患者和20例正常人的血浆cf DNA样本进行检测,临床灵敏度为97.64%,临床特异性为81.43%。dd PCR的检测性能优于q PCR,LOD达到个位数DNA拷贝,最低可确认突变浓度达到0.01%–0.04%。样本提取效率在方法学建立中也十分重要,直接影响到灵敏度和Cut Off值的判定。临床患者检测结果显示其KRAS突变率接近报道水平。     

应用液滴数字PCR技术检测rs6983267三种多态性位点

核苷酸的多态性检测在临床以及基础生命科学研究中占据着重要地位.目前基于PCR的探针法应用最为广泛.由于在核苷酸上微小差异,探针的设计往往带有交叉活性反应,这阻碍了qPCR方法的推广.数字PCR(dPCR)是近年来已成功实现商业化的基于单分子分析的核酸检测技术.通过对条件的优化,dPCR可以消除探针的交叉活性,不过目前商业化的dPCR一般只有2个通道,对于同时检测3个多态性需要更加细致的优化.本研究以rs6983267位点的CCAT2基因3种多态性检测为例,利用探针的交叉活性反应检测其3个多态性位点.检测涉及到3个探针:2个针对多态性位点,另1个位于多态性位点外侧作为参照探针.结果表明,成功地区分了3个包含多态性位点基因片段的簇.在本研究中交叉活性反应可以为dPCR留出白空间,利于多样品的检测.     

Comparative analysis of high butanol tolerance and production in clostridia

2016, was the 100 years anniversary from launching of the first industrial acetone-butanol-ethanol (ABE) microbial production process. Despite this long period and also revival of scientific interest in this fermentative process over the last 20 years, solventogenic clostridia, mainly Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum and Clostridium pasteurianum, still have most of their secrets. One such poorly understood mechanism is butanol tolerance, which seems to be one of the most significant bottlenecks obstructing industrial exploitation of the process because the maximum achievable butanol concentration is only about 21 g/L. This review describes all the known cellular responses elicited by butanol, such as modifications of cell membrane and cell wall, formation of stress proteins, extrusion of butanol by efflux pumps, response of regulatory pathways, and also maps both random and targeted mutations resulting in high butanol production phenotypes. As progress in the field is inseparably associated with emerging methods, enabling a deeper understanding of butanol tolerance and production, progress in these methods, including genome mining, RNA sequencing and constructing of genome scale models are also reviewed. In conclusion, a comparative analysis of both phenomena is presented and a theoretical relationship is described between butanol tolerance/high production and common features including efflux pump formation/activity, stress protein production, membrane modifications and biofilm growth.     

Multiscale landscape genomic models to detect signatures of selection in the alpine plant Biscutella laevigata

Plant species are known to adapt locally to their environment, particularly in mountainous areas where conditions can vary drastically over short distances. The climate of such landscapes being largely influenced by topography, using fine‐scale models to evaluate environmental heterogeneity may help detecting adaptation to micro‐habitats. Here, we applied a multiscale landscape genomic approach to detect evidence of local adaptation in the alpine plant Biscutella laevigata. The two gene pools identified, experiencing limited gene flow along a 1‐km ridge, were different in regard to several habitat features derived from a very high resolution (VHR) digital elevation model (DEM). A correlative approach detected signatures of selection along environmental gradients such as altitude, wind exposure, and solar radiation, indicating adaptive pressures likely driven by fine‐scale topography. Using a large panel of DEM‐derived variables as ecologically relevant proxies, our results highlighted the critical role of spatial resolution. These high‐resolution multiscale variables indeed indicate that the robustness of associations between genetic loci and environmental features depends on spatial parameters that are poorly documented. We argue that the scale issue is critical in landscape genomics and that multiscale ecological variables are key to improve our understanding of local adaptation in highly heterogeneous landscapes.     

Whole‐field macro‐ and micro‐deformation characteristic of unbound water‐loss in dentin hard tissue

High‐resolution deformation measurements in a functionally graded hard tissue such as human dentin are essential to understand the unbound water‐loss mediated changes and their role in its mechanical integrity. Yet a whole‐field, 3‐dimensional (3D) measurement and characterization of fully hydrated dentin in both macro‐ and micro‐scales remain to be a challenge. This study was conducted in 2 stages. In stage‐1, a stereo‐digital image correlation approach was utilized to determine the water‐loss and load‐induced 3D deformations of teeth in a sagittal section over consecutively acquired frames, from a fully hydrated state to nonhydrated conditions for a period up to 2 hours. The macroscale analysis revealed concentrated residual deformations at the dentin‐enamel‐junction and the apical regions of root in the direction perpendicular to the dentinal tubules. Significant difference in the localized deformation characteristics was observed between the inner and outer aspects of the root dentin. During quasi‐static loadings, further increase in the residual deformation was observed in the dentin. In stage‐2, dentin microstructural variations induced by dynamic water‐loss were assessed with environmental scanning electron microscopy and atomic force microscopy (AFM), showing that the dynamic water‐loss induced distention of dentinal tubules with concave tubular edges, and concurrent contraction of intertubular dentin with convex profile. The findings from the current macro‐ and micro‐scale analysis provided insight on the free‐water‐loss induced regional deformations and ultrastructural changes in human dentin.      

Semi-Automated Digital Image Analysis of Pick’s Disease and TDP-43 Proteinopathy

Digital image analysis of histology sections provides reliable, high-throughput methods for neuropathological studies but data is scant in frontotemporal lobar degeneration (FTLD), which has an added challenge of study due to morphologically diverse pathologies. Here, we describe a novel method of semi-automated digital image analysis in FTLD subtypes including: Pick’s disease (PiD, n=11) with tau-positive intracellular inclusions and neuropil threads, and TDP-43 pathology type C (FTLD-TDPC, n=10), defined by TDP-43-positive aggregates predominantly in large dystrophic neurites. To do this, we examined three FTLD-associated cortical regions: mid-frontal gyrus (MFG), superior temporal gyrus (STG) and anterior cingulate gyrus (ACG) by immunohistochemistry. We used a color deconvolution process to isolate signal from the chromogen and applied both object detection and intensity thresholding algorithms to quantify pathological burden. We found object-detection algorithms had good agreement with gold-standard manual quantification of tau- and TDP-43-positive inclusions. Our sampling method was reliable across three separate investigators and we obtained similar results in a pilot analysis using open-source software. Regional comparisons using these algorithms finds differences in regional anatomic disease burden between PiD and FTLD-TDP not detected using traditional ordinal scale data, suggesting digital image analysis is a powerful tool for clinicopathological studies in morphologically diverse FTLD syndromes.     

Establishment and evaluation of detection methods for process-specific residual host cell protein and residual host cell DNA in biological preparation

To establish accurate detection methods of process-specific Escherichia coli residual host cell protein (HCP) and residual host cell DNA (rcDNA) in recombinant biological preparations. Taking the purification process of GLP expressed by E. coli as a specific-process model, the HCP of empty E. coli was intercepted to immunize mice and rabbits. Using IgG from immunized rabbits as the coating antibody and mouse immune serum as the second sandwich antibody, a process-specific enzyme-linked immunosorbent assay (ELISA) for E. coli HCP was established. Targeting the 16S gene of E. coli, ddPCR was used to obtain the absolute copies of rcDNA in samples. Non-process-specific commercial ELISA kit and the process-specific ELISA established in this study were used to detect the HCP in GLP preparation. About 62% of HCPs, which should be process-specific HCPs, could not be detected by the non-process-specific commercial ELISA kit. The sensitivity of established ELISA can reach 338 pg/mL. The rcDNA could be absolutely quantitated by ddPCR, for the copies of rcDNA in three multiple diluted samples showed a reduced gradient. While the copies of rcDNA in three multiple diluted samples could not be distinguished by the qPCR. Process-specific ELISA has high sensitivity in detecting process-specific E. coli HCP. The absolutely quantitative ddPCR has much higher accuracy than the relatively quantitative qPCR, it is a nucleic acid quantitative method that is expected to replace qPCR in the future.     

From Data to Decisions: Helping Crop Producers Build Their Actionable Knowledge

Recent advances in distributed information technologies are providing the means to capture and process abundant data, and to reveal associations between variables describing the crop-environment-management interaction. This review describes the determinants and moderating factors influencing how much value a crop producer and his or her advisor can derive from data, and information derived from data. We describe the social, technological, and entrepreneurial processes needed to progress the nonlinear pathway from data to an on-farm decision, and explore the meaning of actionable knowledge; that is, knowledge that can be acted upon and applied to solve a real-world problem. We argue that effective decision support is also a system that supports the learning needs of crop producers and their transactions with trusted advisors. Crop protection, the sub-set of crop management used to mitigate crop loss, is used to illustrate current approaches and technologies to support farmers' decisions. We describe how situational awareness and actionable knowledge could be improved through use of emerging platform technologies, advances in artificial intelligence, consideration of farmer decision style, knowledge capture and maintenance, and embedding technology in human-centered services. Implications for the conduct of research and development are discussed.