Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates

Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ～100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.     

GRASP and IPCEF Promote ARF-to-Rac Signaling and Cell Migration by Coordinating the Association of ARNO/cytohesin 2 with Dock180

ARFs are small GTPases that regulate vesicular trafficking, cell shape, and movement. ARFs are subject to extensive regulation by a large number of accessory proteins. The many different accessory proteins are likely specialized to regulate ARF signaling during particular processes. ARNO/cytohesin 2 is an ARF-activating protein that promotes cell migration and cell shape changes. We report here that protein–protein interactions mediated by the coiled-coil domain of ARNO are required for ARNO induced motility. ARNO lacking the coiled-coil domain does not promote migration and does not induce ARF-dependent Rac activation. We find that the coiled-coil domain promotes the assembly of a multiprotein complex containing both ARNO and the Rac-activating protein Dock180. Knockdown of either GRASP/Tamalin or IPCEF, two proteins known to bind to the coiled-coil of ARNO, prevents the association of ARNO and Dock180 and prevents ARNO-induced Rac activation. These data suggest that scaffold proteins can regulate ARF dependent processes by biasing ARF signaling toward particular outputs.     

Beyond pandas,the need for a standardized monitoring protocol for large mammals in Chinese nature reserves

Monitoring programs are important for effective conservation and management programs. However, most of these programs rely on indirect sign surveys of elusive animals that often leave cryptic signs of their presence. In Sichuan Province, China, sign surveys are oriented mainly toward giant panda (Ailuropoda melanoleuca) populations but also are used to track other nationally listed species. We have developed and tested a monitoring system based on camera-trapping that can detect a wide range of large, terrestrial mammal and bird species within the reserves of Sichuan. This system is embedded within current protected area patrolling activities and relies on a partnership of management agencies, universities and international organizations. The international organizations and national universities primarily provide the training and assist with study design and data analysis. Data management and access is controlled at the regional level by the appropriate state agencies. Limitations to this system include the need for additional training and support to less developed reserves and the long-term availability of funds to support field staff. However, the potential return on investment is a consistent tracking of multiple species across diverse set of reserves, facilitating comparative analysis of results that will assist in adaptive management throughout the region.     

中国的野生动物红外相机监测需要统一的标准

采用及时、可靠的方法对物种开展有效监测是生物多样性保护的基础。红外相机技术可以获得兽类物种的影像、元数据和分布信息, 是监测生物多样性的有效途径。这项技术在野外便于部署, 规程易于标准化, 可提供野生动物凭证标本(影像)以及物种拍摄位置、拍摄日期与时间、拍摄细节(相机型号等)等附属信息。这些特性使得我们可以积累数以百万计的影像资料和野生动物监测数据。在中国, 红外相机技术已得到广泛应用, 众多机构正在使用红外相机采集并存储野生动物影像以及相关联的元数据。目前, 亟需对红外相机元数据结构进行标准化, 以促进不同机构之间以及与外部保护团体之间的数据共享。迄今全球已建立有数个国际数据共享平台, 例如Wildlife Insights, 但他们离不开与中国的合作, 以有效追踪全球可持续发展的进程。达成这样的合作需要3个基础: 共同的数据标准、数据共享协议和数据禁用政策。我们倡议, 中国保护领域的政府主管部门、机构团体一起合作, 共同制定在国内单位之间以及与国际机构之间共享监测数据的政策、机制与途径。     

Temporal population variability in local forest communities has mixed effects on tree species richness across a latitudinal gradient

Among the local processes that determine species diversity in ecological communities, fluctuation‐dependent mechanisms that are mediated by temporal variability in the abundances of species populations have received significant attention. Higher temporal variability in the abundances of species populations can increase the strength of temporal niche partitioning but can also increase the risk of species extinctions, such that the net effect on species coexistence is not clear. We quantified this temporal population variability for tree species in 21 large forest plots and found much greater variability for higher latitude plots with fewer tree species. A fitted mechanistic model showed that among the forest plots, the net effect of temporal population variability on tree species coexistence was usually negative, but sometimes positive or negligible. Therefore, our results suggest that temporal variability in the abundances of species populations has no clear negative or positive contribution to the latitudinal gradient in tree species richness.     

Differential and interacting impacts of invasive plants and white-tailed deer in eastern U.S. forests

Biological Invasions - Forests in eastern North America are experiencing high densities of white-tailed deer (Odocoileus virginianus) and encroachment by invasive plants, both of which threaten...     

Does a temperate ungulate that breeds in summer exhibit rut-induced hypophagia? Analysis of time budgets of male takin (Budorcas taxicolor) in Sichuan,China

Mammals maximize fitness by optimizing time and energy allocation between reproduction and survival. Describing time budgets is a way to understand a species' constraints in energy allocation. We describe a time budget for male takin (Budorcas taxicolor) in Tangjiahe Nature Reserve, China, to better understand rut-induced hypophagia, which is frequently observed in temperate ungulates that breed in autumn or in winter. Observations generally occurred at two elevations (1200-1600m and 2600-3200m), using 20-min focal animal scan sampling from 2007 to 2009. Feeding behaviors accounted for the majority in takin's time budget (61.1%) during daylight hours, relative to the other observed behaviors, such as rest (14.1%), alert behavior (10.2%) and locomotion (6.8%). We found a negative correlation between feeding behavior and rutting behavior during the rutting season. A ratio of feeding time to resting time increased from pre-rut to rut, while resting behavior did not change significantly across seasons. These results suggest the "energy saving" hypothesis could explain reduced foraging in male takin during the rut, but aspects of the species biology suggest that hypotheses for rut-induced hypophagia developed for other temperate ungulates do not apply to takin. We suggest that the unusual summer rutting season of takin releases males from the energy constraints encountered by temperate ungulates that breed in the autumn and has other benefits for offspring survival. Further research should be conducted on ungulates that exhibit rut during the summer and tropical ungulates that might not experience limited food availability following the mating season to improve our understanding on rut-induced hypophagia.     

Sapling growth rates reveal conspecific negative density dependence in a temperate forest

Local tree species diversity is maintained in part by conspecific negative density dependence (CNDD). This pervasive mechanism occurs in a variety of forms and ecosystems, but research to date has been heavily skewed toward tree seedling survival in tropical forests. To evaluate CNDD more broadly, we investigated how sapling growth rates were affected by conspecific adult neighbors in a fully mapped 25.6 ha temperate deciduous forest. We examined growth rates as a function of the local adult tree neighborhood (via spatial autoregressive modeling) and compared the spatial positioning of faster‐growing and slower‐growing saplings with respect to adult conspecific and heterospecific trees (via bivariate point pattern analysis). In addition, to determine whether CNDD‐driven variation in growth rates leaves a corresponding spatial signal, we extended our point pattern analysis to a static, growth‐independent comparison of saplings and the next larger size class. We found that negative conspecific effects on sapling growth were most prevalent. Five of the nine species that were sufficiently abundant for analysis exhibited CNDD, while only one species showed evidence of a positive conspecific effect, and one or two species, depending on the analysis, displayed heterospecific effects. There was general agreement between the autoregressive models and the point pattern analyses based on sapling growth rates, but point pattern analyses based on single‐point‐in‐time size classes yielded results that differed markedly from the other two approaches. Our work adds to the growing body of evidence that CNDD is an important force in temperate forests, and demonstrates that this process extends to sapling growth rates. Further, our findings indicate that point pattern analyses based solely on size classes may fail to detect the process of interest (e.g., neighborhood‐driven variation in growth rates), in part due to the confounding of tree size and age.     

Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which is present within these marine environments.