How to copy and paste DNA microarrays.

Analogous to a photocopier, we developed a DNA microarray copy technique and were able to copy patterned original DNA microarrays. With this process the appearance of the copied DNA microarray can also be altered compared to the original by producing copies of different resolutions. As a homage to the very first photocopy made by Chester Charlson and Otto Kornei, we performed a lookalike DNA microarray copy exactly 80 years later. Those copies were also used for label-free real-time kinetic binding assays of apo-dCas9 to double stranded DNA and of thrombin to single stranded DNA. Since each DNA microarray copy was made with only 5 µl of spPCR mix, the whole process is cost-efficient. Hence, our DNA microarray copier has a great potential for becoming a standard lab tool.

Improving health status and reduction of institutionalization in long-term care--Effects of the Resident Assessment Instrument-Home Care by degree of implementation.

A cluster randomized controlled trial showed that the Resident Assessment Instrument (RAI) could not improve or stabilize the health status of people in need of long-term care or reduce the rate of institutionalization in Germany among clients of home care agencies. The aim of this article is to investigate whether the effect of RAI depends on the degree of implementation. A factor analysis was used to distinguish between those agencies that implemented RAI intensively and those that did not. The clients of home care agencies working intensively with RAI were significantly less hospitalized (P = 0.0284) and fared slightly better according to activities of daily living (ADL, instrumental ADL (IADL)), cognitive skills (Mini-Mental Status Test (MMST)) and quality of life (EuroQol (EQ-5D)) compared with the control group. In contrast, those not working intensively with RAI had worse outcomes (IADL, MMST, EQ-5D) than the control group (not significant). It is important to guarantee a successful implementation of RAI.

The long and bumpy road to outcome-oriented management of long-term care in Germany: implementation of the Resident Assessment Instrument in home-care services.

OBJECTIVE: Although the quality of long-term care has improved, many problems still remain, and better processes seem to be necessary. Hence, outcome-oriented management is of particular importance. The Resident Assessment Instrument (RAI) is a tool that has been used successfully in many countries to improve quality of care. However, there are problems of implementation and it lacks information on the conditions of successful or failing information of the RAI. The aim of this article is to find out to what extent technical/qualification requirements help to introduce or lead to failure of the implementation of an assessment instrument like RAI. METHODS: Therefore, a cluster randomized controlled trial showed services using RAI intensively tend to have better outcomes after 12 months. But the effects depend on the success of the implementation. Using a factor analysis, an index was built to divide the care providers into "optimal" and "suboptimal" RAI users. RESULTS: Some factors that seem to lead to a rather successful implementation could be detected: A higher proportion of qualified staff, a lower perceived quantitative workload, a small size of care providers, the type of ownership (for-profit) and a late entry in study [Correction made here after initial online publication.]. CONCLUSION: The success or failure of the implementation of an outcome-oriented control instrument is determined by professional, organizational restrictions. The results show that a better implementation leads to better outcomes for clients.

A quantitative assessment of costimulation and phosphatase activity on microclusters in early T cell signaling.

T cell signaling is triggered through stimulation of the T cell receptor and costimulatory receptors. Receptor activation leads to the formation of membrane-proximal protein microclusters. These clusters undergo tyrosine phosphorylation and organize multiprotein complexes thereby acting as molecular signaling platforms. Little is known about how the quantity and phosphorylation levels of microclusters are affected by costimulatory signals and the activity of specific signaling proteins. We combined micrometer-sized, microcontact printed, striped patterns of different stimuli and simultaneous analysis of different cell strains with image processing protocols to address this problem. First, we validated the stimulation protocol by showing that high expression levels CD28 result in increased cell spreading. Subsequently, we addressed the role of costimulation and a specific phosphotyrosine phosphatase in cluster formation by including a SHP2 knock-down strain in our system. Distinguishing cell strains using carboxyfluorescein succinimidyl ester enabled a comparison within single samples. SHP2 exerted its effect by lowering phosphorylation levels of individual clusters while CD28 costimulation mainly increased the number of signaling clusters and cell spreading. These effects were observed for general tyrosine phosphorylation of clusters and for phosphorylated PLCγ1. Our analysis enables a clear distinction between factors determining the number of microclusters and those that act on these signaling platforms.

Mediator probe PCR: a novel approach for detection of real-time PCR based on label-free primary probes and standardized secondary universal fluorogenic reporters.

BACKGROUND: The majority of established techniques for monitoring real-time PCR amplification involve individual target-specific fluorogenic probes. For analysis of numerous different targets the synthesis of these probes contributes to the overall cost during assay development. Sequence-dependent universal detection techniques overcome this drawback but are prone to detection of unspecific amplification products. We developed the mediator probe PCR as a solution to these problems. METHODS: A set of label-free sequence-specific primary probes (mediator probes), each comprising a target-specific region and a standardized mediator tag, is cleaved upon annealing to its target sequence by the polymerases' 5' nuclease activity. Release of a mediator triggers signal generation by cleavage of a complementary fluorogenic reporter probe. RESULTS: Real-time PCR amplification of human papillomavirus 18 (HPV18), Staphylococcus aureus, Escherichia coli, and Homo sapiens DNA dilution series showed exceptional linearity when detected either by novel mediator probes (r(2) = 0.991-0.999) or state-of-the-art hydrolysis probes (TaqMan probes) (r(2) = 0.975-0.993). For amplification of HPV18 DNA the limits of detection were 78.3 and 85.1 copies per 10-µL reaction when analyzed with the mediator probe and hydrolysis probe, respectively. Duplex amplification of HPV18 target DNA and internal standard had no effects on back calculation of target copy numbers when quantified with either the mediator probe PCR (r(2) = 0.998) or the hydrolysis probe PCR (r(2) = 0.988). CONCLUSIONS: The mediator probe PCR has equal performance to hydrolysis probe PCR and has reduced costs because of the use of universal fluorogenic reporters.