Identification and analysis of the regulation of a prestalk cell-surface antigen of Dictyostelium discoideum

The properties of two differentiation antigens, rsa 4.2 and rsa 3.1, were examined. Both appear on the cell surface early in differentiation, but they differ with respect to their cell-type specificity. rsa 4.2 appears 1-2 h after differentiation has begun and is present on all cells during all stages of differentiation. In contrast, rsa 3.1 appears after 1-2 h on all aggregating amebae and later becomes restricted to prestalk cells. The pattern of regulation of rsa 3.1 indicates that this prestalk antigen appears on all cells early in differentiation but disappears in cells that differentiate along the prespore pathway. As a result, only cells in the anterior of migrating slugs carry this antigen. Predictions of two competing models of Dictyostelium pattern formation, i.e., position-dependent differentiation and random, position-independent differentiation, were tested by flow cytometry and immunochemical staining of sections of cells at the mound and mound-with-tip stages. Our results do not rule out either model, although they are incompatible with the simplest interpretation of the model for position-independent differentiation. The results clearly indicate that cells that ultimately differentiate along the spore pathway pass through an earlier cell state that includes the presence of a prestalk cell-surface antigen identified as rsa 3.1.     

Life cycle assessment capacity roadmap (section 1): decision-making support using LCA

When life cycle assessment (LCA) results do not show a clear and certain environmental preference of one choice over one or several alternatives, current methods are limited in their ability to inform decision-makers. To address this and related cross-cutting issues, a group of LCA practitioners has been working on a roadmap for capacity development in LCA. The roadmap is identifying common needs for development in LCA, which can then be addressed by the broader LCA community. The roadmap document on decision-making support, having undergone a public comment period, outlines the current state as well as needs and milestones to ensure progress continues apace. The roadmap document, available for download, covers five main areas of development: (1) performance measures of confidence, which identify the acceptable uncertainty for study results, while minimizing expenditures; (2) selection of impact categories, an area with multiple existing methods. The roadmap suggests codifying these methods and identifying their suitability to various applications; (3) normalization; while several methods of normalization are in use, the method with the greatest acceptance in the LCA community (i.e., relying on total or per capita regional emissions/extractions) has a number of methodological drawbacks; (4) weighting, which is a form of multi-criteria decision analysis (MCDA). The broader MCDA field can enrich LCA by providing studied methods of assessing trade-offs; and (5) visualization of results. Many other LCA capacity needs would benefit from documentation. These include but are not limited to the following: addressing ill-characterized uncertainty, life cycle inventory data needs, data format needs, and tool capabilities. Other roadmapping groups are forming and are looking for practitioners to support the effort.     

Water and Food Consumption Patterns of U.S. Adults from 1999 to 2001

Objective: High water consumption has been proposed as an aid to weight control and as a means of reducing the energy density of the diet. This study examines the relationship between water consumption and other drinking and eating patterns. Research Methods and Procedures: The National Health and Nutrition Examination Survey 1999 to 2001, with responses from 4755 individuals ≥18 years of age, provides the data for this cross‐sectional analysis. A cluster analysis was performed using z‐scores of specific food and beverage consumption to examine patterns. A multinomial logit analysis was used to examine sociodemographic characteristics of each dietary pattern and to examine the effects of water consumption on the likelihood of consuming a non–dairy caloric beverage. All results were weighted to be nationally representative and controlled for design effects. Results: Within the sample, 87% consumed water, with an average daily consumption of 51.9 oz (1.53 liters) per consumer. Water consumers drank fewer soft/fruit drinks and consumed 194 fewer calories per day. Water consumers (potentially a self‐selected sample) consumed more fruits, vegetables, and low‐ and medium‐fat dairy products. Four distinct unhealthy dietary patterns were found that included little or no water consumption. Older and more educated persons used healthier food patterns. Mexican dietary patterns were much healthier than dietary patterns of blacks. Discussion: Water consumption potentially is a dietary component to be promoted, but much more must be understood about its role in a healthy diet. Because high water consumption is linked with healthier eating patterns—patterns more likely to be followed by higher‐educated, older adults—the target of water promotion and healthy diet options should focus on younger and less educated adults.     

A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells

We describe a pipeline for the rapid production of recombinant Fabs derived from mouse monoclonal antibodies suitable for use in structural studies. The pipeline is exemplified by the production of three Fabs derived from the monoclonal antibodies OX108 (anti-CD200 receptor), OX117 and OX119 (anti-SIRPgamma). Heavy and light chain variable domains were inserted into separate expression vectors containing resident constant regions using In-Fusion PCR cloning. Following transient co-expression in HEK 293T cells, secreted Fab fragments were purified by metal chelate chromatography and gel filtration using an automated procedure with yields of up to 4mg/L of cell culture. Following crystallization trials, diffracting crystals were obtained for the recombinant Fabs of OX108 and OX117, and their structures solved to 2.3A and 2.4A, respectively.