Obesity is a risk factor for reporting homebound status among community-dwelling older persons

Objective : To test the a priori hypothesis that obesity is a predictor of risk for reporting homebound status. Research Methods and Procedures : A longitudinal cohort study was conducted with 21, 645 community‐dwelling men and women 65 to 97 years old. A nutrition risk screen was administered baseline between 1994 and 1999 and again 3 to 4 years later. Univariate analyses identified baseline variables associated with subsequent reporting of homebound status. Multivariable logistic regression models were created to identify baseline variables that were significant independent predictors of reporting homebound status. Results : At baseline, 24% of the cohort had BMI ≥ 30. There were 12, 834 (45% men) respondents at follow‐up (68% response). Non‐responders at follow‐up differed little from responders except for greater baseline age (72.2 ± 6.2 vs. 71.4 ± 5.6 years, p < 0.001) and reporting of any functional limitations (9.2% vs. 4.9%, p < 0.001). At follow‐up, those who reported homebound status (n = 169) were significantly (p < 0.001) older (80.3 ± 7.3 vs. 75.1 ± 5.5 years) and more likely to report functional limitations (83.4% vs. 10.8%). Univariate analyses identified 16 baseline variables that were eliminated stepwise until five significant independent predictors remained: age ≥ 75 years (2.21, 1.55 to 3.15/odds ratio, 95% confidence interval), BMI ≥ 35 (1.75, 1.04 to 2.96), poor appetite (2.50, 1.29 to 4.86), low income (1.59, 1.00 to 2.56), and any functional limitation (10.67, 7.36 to 15.46). Discussion : Obesity remained a significant independent predictor for reporting homebound status and should be considered in screening of older populations and in the planning, implementation, and evaluation of services for homebound older persons.     

A versatile colony assay based on NADH fluorescence

Direct visualization of the activity of enzymes expressed by bacterial colonies attached to a solid support, often referred to as “filter assay”, is a powerful strategy for the identification of new or improved biocatalysts. In this work we demonstrate the usefulness of NAD⁺/NADH coupled enzymatic reactions as visualization tool in such experimental setups. Dehydrogenases, capable of oxidizing or reducing the reaction product released from the bacterial colony were supplemented to the screening solution, together with the screening substrate and a sufficient amount of NAD⁺ or NADH, respectively. We also examined the screening of directly NAD⁺/NADH coupled reactions. The release or consumption of NADH in the area of colonies was monitored on behalf of its fluorescence at 450 nm. Excitation was achieved by standard “black-light” UV tubes (340–360 nm). The visible fluorescence signal was recorded using a CCD-camera. We got excellent results for the screening of threonine aldolases and esterases and were able to show the principle utility for amidase, nitrilase, nitrile hydratase, hydroxynitrile lyase and benzaldehyde dehydrogenase active colonies.     

Application and evaluation of classification trees for screening unwanted plants

Abstract Risk assessment systems for introduced species are being developed and applied globally, but methods for rigorously evaluating them are still in their infancy. We explore classification and regression tree models as an alternative to the current Australian Weed Risk Assessment system, and demonstrate how the performance of screening tests for unwanted alien species may be quantitatively compared using receiver operating characteristic (ROC) curve analysis. The optimal classification tree model for predicting weediness included just four out of a possible 44 attributes of introduced plants examined, namely: (i) intentional human dispersal of propagules; (ii) evidence of naturalization beyond native range; (iii) evidence of being a weed elsewhere; and (iv) a high level of domestication. Intentional human dispersal of propagules in combination with evidence of naturalization beyond a plants native range led to the strongest prediction of weediness. A high level of domestication in combination with no evidence of naturalization mitigated the likelihood of an introduced plant becoming a weed resulting from intentional human dispersal of propagules. Unlikely intentional human dispersal of propagules combined with no evidence of being a weed elsewhere led to the lowest predicted probability of weediness. The failure to include intrinsic plant attributes in the model suggests that either these attributes are not useful general predictors of weediness, or data and analysis were inadequate to elucidate the underlying relationship(s). This concurs with the historical pessimism that we will ever be able to accurately predict invasive plants. Given the apparent importance of propagule pressure (the number of individuals of an species released), future attempts at evaluating screening model performance for identifying unwanted plants need to account for propagule pressure when collating and/or analysing datasets. The classification tree had a cross‐validated sensitivity of 93.6% and specificity of 36.7%. Based on the area under the ROC curve, the performance of the classification tree in correctly classifying plants as weeds or non‐weeds was slightly inferior (Area under ROC curve = 0.83 ± 0.021 (±SE)) to that of the current risk assessment system in use (Area under ROC curve = 0.89 ± 0.018 (±SE)), although requires many fewer questions to be answered.     

豆豉纤溶酶产生菌的筛选及诱变

从广泛收集的豆豉成品及半成品中筛选到数株菌落形态各异且具有纤溶酶活性的菌株.分别采用亚硝酸和紫外线对豆豉纤溶酶产生菌DC-12进行诱变育种,成功筛选到了3株突变株,其纤溶酶产量较出发菌株分别提高了3.6、3.7和4.75倍.     

利用核糖体展示技术筛选功能性蛋白质方法的建立

核糖体展示(ribosomedisplay)是一种体外筛选功能性蛋白质的有力的工具.利用体外转录和翻译偶联系统可以方便而快捷地完成核糖体展示.筛选系统利用一对能够紧密结合的蛋白质:人锚蛋白(ankyrin)和红血球膜带3蛋白细胞质区域(cytoplasmicdomainoferythrocytemembraneproteinBand3,Cdb3)作为模式分子,希望利用cdb3蛋白通过核糖体展示亲和选择得到锚蛋白基因.用于核糖体展示的人锚蛋白基因结构由组装PCR构建,通过PCR技术引入核糖体展示所需的结构元件.在亲和筛选步骤后,只能利用红血球膜带3蛋白筛选得到锚蛋白基因,而不能利用对照牛血清白蛋白(bovineserumalbumin,BSA)筛选得到,从而说明建立的核糖体展示技术能够正常发挥作用.     

基因芯片技术及其在补血中药研究中的应用

补血中药在我国中医药宝库中占有十分重要的地位,中药调控造血的分子机制与细胞因子网络直接相关。多维超高通量药物筛选体系的建立和从分子水平系统说明中药作用的药理学机制,使传统的中药理论与国际通用的医学理论模式接轨,是中药现代化、国际化的迫切要求。由于中药重整体、多靶点、多环节的作用方式,使得传统的研究技术难以完整地阐明其作用机制;而现今发展起来的高密度基因芯片技术,可以同时研究上千种基因的作用模式,进行平行基因分析,因此可以用来检测疾病状态下和中药作用后成千上万个基因的表达模式,并对其进行定性和定量分析,从而使从整体和分子水平上阐明中药作用机制成为可能。基因芯片这种高通量、快速、平行的基因信息处理和分析技术,是实现这一目标的绝佳实验手段。在药物领域对于药物靶标的发现、多靶位同步超高通量药物筛选、药物作用的分子机理、中医药基础理论现代化、药物活性及毒性评价等都有其他方法无可比拟的优越性。因此,建立以基因芯片技术为核心的多靶点、多层次、多水平的中药多维超高通量筛选体系具有极其重要的意义。     

Screening and characterization of a novel esterase from a metagenomic library

Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel ester-hydrolyzing enzyme on Escherichia coli. Stepwise fragmentations and subcloning of the initial insert DNA (30-40 kb) using restriction enzymes selected to exclude already known esterases with subsequent screenings resulted in a positive clone with a 2.5-kb DNA fragment. The cloned sequence included an open reading frame consisting of 1089 bp, designated as est25, encoding a protein of 363 amino acids with a molecular mass of about 38.3 kDa. Amino acid sequence analysis revealed only moderate identity (< or = 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124-127), GxSxG (residues 199-203), and the putative catalytic triad composed of Ser201, Asp303, and His333. Est25 was functionally overexpressed in a soluble form in E. coli with optimal activity at pH 7.0 and 25 degrees C. The purified Est25 exhibited hydrolyzing activity toward p-nitrophenyl (NP)-fatty acyl esters with short-length acyl chains (< or = C6) with the highest activity toward p-NP-acetate (Km=1.0 mM and Vmax = 63.7 U/mg), but not with chain lengths > or = C8, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with Km of 16.4 mM and Vmax of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofen ethyl ester. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzymes from a metagenome.     

Cellular phenotyping by RNAi.

A systematic characterization of genes with unknown function is a key challenge after the sequencing of the human genome and the genomes of many model organisms. High-throughput RNA-interference (RNAi) screenings have become a widely used approach in invertebrate model organisms and also promise to revolutionize cell biology in mammals. Genome-wide RNAi screens in Caenorhabditis elegans and Drosophila, and in a smaller scale in mammalian cells have proven to be a valuable and successful method for the dissection of diverse biological processes. A number of RNAi libraries have become available that rely on different technologies, such as long double-stranded (ds) RNAs, in vitro diced short-interfering (si) RNAs, synthetic siRNAs and short-hairpin (sh) RNAs, which all have specific advantages and disadvantages. In addition, progress in screening technologies and data analysis allows the adaptation of screening methods to analyse more complex cellular processes. This review will summarize strategies in combining genome-scale RNAi libraries, high-throughput screening technologies, integrated high-content data analysis and will discuss future challenges.     

Determining the structural basis for specificity of ligands using crystallographic screening

Crystallographic screening has been used to identify new inhibitors for potential target for drug development. Here, we describe the application of the crystallographic screening to assess the structural basis of specificity of ligands against a protein target. The method is efficient and results in detailed crystallographic information. The utility of the method is demonstrated in the study of the structural basis for specificity of ligands for human purine nucleoside phosphorylase (PNP). Purine nucleoside phosphorylase catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleotides and deoxynucleosides. This enzyme is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. This methodology may help in the future development of a new generation of PNP inhibitors.     

Microbioreactor array for full‐factorial analysis of provision of multiple soluble factors in cellular microenvironments

We report a scalable microbioreactor architecture which uses nested dilution structures to generate a full‐factorial array of cell culture conditions. The proof‐of‐concept microbioreactor array produces all combinations of three concentration levels of two soluble factors (3² = 9 unique conditions in total). The full‐factorial design is especially useful in optimizing soluble factor treatments and elucidating interaction effects between factors which are otherwise difficult to deconvolute. By nesting hierarchical levels of dilution structures, and designing the device purely by resistive flow (no valves are required), suitable diffusive mixing of growth factors up to 40 kDa is achieved such that the nine culture conditions can be generated and maintained from a minimal number of stock solutions. Biotechnol. Bioeng. 2009; 104: 1240–1244. © 2009 Wiley Periodicals, Inc.