Clinical Sensor-Based Fall Risk Assessment at an Orthopedic Clinic: A Case Study of the Staff's Views on Utility and Effectiveness.

In-hospital falls are a serious threat to patient security and fall risk assessment (FRA) is important to identify high-risk patients. Although sensor-based FRA (SFRA) can provide objective FRA, its clinical use is very limited and research to identify meaningful SFRA methods is required. This study aimed to investigate whether examples of SFRA methods might be relevant for FRA at an orthopedic clinic. Situations where SFRA might assist FRA were identified in a focus group interview with clinical staff. Thereafter, SFRA methods were identified in a literature review of SFRA methods developed for older adults. These were screened for potential relevance in the previously identified situations. Ten SFRA methods were considered potentially relevant in the identified FRA situations. The ten SFRA methods were presented to staff at the orthopedic clinic, and they provided their views on the SFRA methods by filling out a questionnaire. Clinical staff saw that several SFRA tasks could be clinically relevant and feasible, but also identified time constraints as a major barrier for clinical use of SFRA. The study indicates that SFRA methods developed for community-dwelling older adults may be relevant also for hospital inpatients and that effectiveness and efficiency are important for clinical use of SFRA.

Gray Literature in Evaluating Effectiveness in Digital Health and Health and Welfare Technology: A Source Worth Considering.

BACKGROUND: The need to assess the effectiveness and value of interventions involving digital health and health and welfare technologies is becoming increasingly important due to the rapidly growing development of these technologies and their areas of application. Systematic reviews of scientific literature are a mainstay of such assessment, but publications outside the realm of traditional scientific bibliographic databases-known as gray literature-are often not included. This is a disadvantage, particularly apparent in the health and welfare technology (HWT) domain. OBJECTIVE: The aim of this article is to investigate the significance of gray literature in digital health and HWT when reviewing literature. As an example, the impact of including gray literature to the result of two systematic reviews in HWT is examined. METHODS: In this paper, we identify, discuss, and suggest methods for including gray literature sources when evaluating effectiveness and appropriateness for different review types related to HWT. The analysis also includes established sources, search strategies, documentation, and reporting of searches, as well as bias and credibility assessment. The differences in comparison to scientific bibliographic databases are elucidated. We describe the results, challenges, and benefits of including gray literature in 2 examples of systematic reviews of HWT. RESULTS: In the 2 systematic reviews described in this paper, most included studies came from context-specific gray literature sources. Gray literature contributed to the overall result of the reviews and corresponded well with the reviews' aims. The assessed risk of bias of the included studies derived from gray literature was similar to the included studies from other types of sources. However, because of less standardized publication formats, assessing and extracting data from gray literature studies were more time-consuming and compiling statistical results was not possible. The search process for gray literature required more time and the reproducibility of gray literature searches were less certain due to more unstable publication platforms. CONCLUSIONS: Gray literature is particularly relevant for digital health and HWT but searches need to be conducted systematically and reported transparently. This way gray literature can broaden the range of studies, highlight context specificity, and decrease the publication bias of reviews of effectiveness of HWT. Thus, researchers conducting systematic reviews related to HWT should consider including gray literature based on a systematic approach.

Mobile Safety Alarms Based on GPS Technology in the Care of Older Adults: Systematic Review of Evidence Based on a General Evidence Framework for Digital Health Technologies.

BACKGROUND: GPS alarms aim to support users in independent activities. Previous systematic reviews have reported a lack of clear evidence of the effectiveness of GPS alarms for the health and welfare of users and their families and for social care provision. As GPS devices are currently being implemented in social care, it is important to investigate whether the evidence of their clinical effectiveness remains insufficient. Standardized evidence frameworks have been developed to ensure that new technologies are clinically effective and offer economic value. The frameworks for analyzing existing evidence of the clinical effectiveness of GPS devices can be used to identify the risks associated with their implementation and demonstrate key aspects of successful piloting or implementation. OBJECTIVE: The principal aim of this study is to provide an up-to-date systematic review of evidence based on existing studies of the effects of GPS alarms on health, welfare, and social provision in the care of older adults compared with non-GPS-based standard care. In addition, the study findings were assessed by using the evidence standards framework for digital health technologies (DHTs) established by the National Institute for Health and Care Excellence (NICE) in the United Kingdom. METHODS: This review was conducted according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Primary studies published in peer-reviewed journals and gray literature from January 2005 to August 2020 were identified through searches in 13 databases and several sources of gray literature. Included studies had individuals (aged ≥50 years) who were receiving social care for older adults or for persons with dementia; used GPS devices as an intervention; were performed in Canada, the United States, European Union, Singapore, Australia, New Zealand, Hong Kong, South Korea, or Japan; and addressed quantitative outcomes related to health, welfare, and social care. The study findings were analyzed by using the NICE framework requirements for active monitoring DHTs. RESULTS: Of the screened records, 1.6% (16/986) were included. Following the standards of the NICE framework, practice evidence was identified for the tier 1 categories Relevance to current pathways in health/social care system and Acceptability with users, and minimum evidence was identified for the tier 1 category Credibility with health, social care professionals. However, several evidence categories for tiers 1 and 2 could not be assessed, and no clear evidence demonstrating effectiveness could be identified. Thus, the evidence required for using DHTs to track patient location according to the NICE framework was insufficient. CONCLUSIONS: Evidence of the beneficial effects of GPS alarms on the health and welfare of older adults and social care provision remains insufficient. This review illustrated the application of the NICE framework in analyses of evidence, demonstrated successful piloting and acceptability with users of GPS devices, and identified implications for future research.

Performance and Characteristics of Wearable Sensor Systems Discriminating and Classifying Older Adults According to Fall Risk: A Systematic Review.

Sensor-based fall risk assessment (SFRA) utilizes wearable sensors for monitoring individuals' motions in fall risk assessment tasks. Previous SFRA reviews recommend methodological improvements to better support the use of SFRA in clinical practice. This systematic review aimed to investigate the existing evidence of SFRA (discriminative capability, classification performance) and methodological factors (study design, samples, sensor features, and model validation) contributing to the risk of bias. The review was conducted according to recommended guidelines and 33 of 389 screened records were eligible for inclusion. Evidence of SFRA was identified: several sensor features and three classification models differed significantly between groups with different fall risk (mostly fallers/non-fallers). Moreover, classification performance corresponding the AUCs of at least 0.74 and/or accuracies of at least 84% were obtained from sensor features in six studies and from classification models in seven studies. Specificity was at least as high as sensitivity among studies reporting both values. Insufficient use of prospective design, small sample size, low in-sample inclusion of participants with elevated fall risk, high amounts and low degree of consensus in used features, and limited use of recommended model validation methods were identified in the included studies. Hence, future SFRA research should further reduce risk of bias by continuously improving methodology.

User-centered requirements engineering to manage the fuzzy front-end of open innovation in e-health: A study on support systems for seniors' physical activity.

BACKGROUND: Although e-health potentials for improving health systems in their safety, quality and efficiency has been acknowledged, a large gap between the postulated and empirically demonstrated benefits of e-health technologies has been ascertained. E-health development has classically been technology-driven, often resulting in the design of devices and applications that ignore the complexity of the real-world setting, thus leading to slow diffusion of innovations to care. Therefore, e-health innovation needs to consider the mentioned complexity already from the start. The early phases of innovation, fuzzy front-end (FFE) defined as "the period between when an opportunity is first considered and when an idea is judged ready for development" has been identified to have the highest impact on the innovation process and its outcome. The FFE has been recognized as the most difficult stage to manage in the innovation process as it involves a high degree of uncertainty. Such a phase becomes even more difficult when different sectors and organizations are involved. Therefore, effective methods for involving different organizations and user groups in the FFE of innovation are needed. OBJECTIVE: The aim of this study was to manage the FFE of a collaborative, open innovation (OI) process, to define a software system supporting seniors' physical activity (PA) by applying a framework of methods from software requirements engineering (RE) to elicit and analyze needs and requirements of users and stakeholders, as well as the context in which the system should be used. METHODS: Needs and requirements of three future user groups were explored through individual- and focus group interviews. Requirements were categorized and analyzed in a workshop with a multidisciplinary team: a system overview was produced by conceptual modelling using elicited functional requirements; high-level non-functional requirements were negotiated and prioritized. Scenario descriptions of system's supportive roles in different phases of a behavioral change process were developed. RESULTS: User-centered RE methods were successfully used to define a system and a high-level requirements description was developed based on needs and requirements from three identified user groups. The system aimed to support seniors' motivation for PA and contained four complementary sub-systems. The outcome of the study was a Concept of Operations (ConOps) document that specified the high-level system requirements in a way that was understandable for stakeholders. This document was used both to identify and recruit suitable industrial partners for the following open innovation development and to facilitate communication and collaboration in the innovation process. CONCLUSIONS: Applying software RE methods and involving user groups in the early phases of OI can contribute to the development of new concepts that meet complex real-world requirements. Different user groups can complement each other in conveying needs and requirements from which systems can be designed. Empirical studies applying and exploring different methods used to define new e-health solutions can contribute with valuable knowledge about handling innovation FFE.

Nocturnal digital surveillance in aged populations and its effects on health, welfare and social care provision: a systematic review.

BACKGROUND: Nocturnal digital surveillance technologies are being widely implemented as interventions for remotely monitoring elderly populations, and often replace person-based surveillance. Such interventions are often placed in care institutions or in the home, and monitored by qualified personnel or relatives, enabling more rapid and/or frequent assessment of the individual's need for assistance than through on-location visits. This systematic review summarized the effects of these surveillance technologies on health, welfare and social care provision outcomes in populations ≥ 50 years, compared to standard care. METHOD: Primary studies published 2005-2020 that assessed these technologies were identified in 11 databases of peer-reviewed literature and numerous grey literature sources. Initial screening, full-text screening, and citation searching steps yielded the studies included in the review. The Risk of Bias and ROBINS-I tools were used for quality assessment of the included studies. RESULT: Five studies out of 744 identified records met inclusion criteria. Health-related outcomes (e.g. accidents, 2 studies) and social care outcomes (e.g. staff burden, 4 studies) did not differ between interventions and standard care. Quality of life and affect showed improvement (1 study each), as did economic outcomes (1 study). The quality of studies was low however, with all studies possessing a high to critical risk of bias. CONCLUSIONS: We found little evidence for the benefit of nocturnal digital surveillance interventions as compared to standard care in several key outcomes. Higher quality intervention studies should be prioritized in future research to provide more reliable evidence.

Integrating Key User Characteristics in User-Centered Design of Digital Support Systems for Seniors' Physical Activity Interventions to Prevent Falls: Protocol for a Usability Study.

BACKGROUND: The goal of user-centered design (UCD) is to understand the users' perspective and to use that knowledge to shape more effective solutions. The UCD approach provides insight into users' needs and requirements and thereby improves the design of the developed services. However, involving users in the development process does not guarantee that feedback from different subgroups of users will shape the development in ways that will make the solutions more useful for the entire target user population. OBJECTIVE: The aim of this study was to describe a protocol for systematic analysis and prioritization of feedback from user subgroups in the usability testing of a digital motivation support for fall-preventive physical activity (PA) interventions in seniors (aged 65 years and older). This protocol can help researchers and developers to systematically exploit feedback from relevant user subgroups in UCD. METHODS: Gender, PA level, and level of technology experience have been identified in the literature to influence users' experience and use of digital support systems for fall-preventive PA interventions in seniors. These 3 key user characteristics were dichotomized and used to define 8 (ie, 23) possible user subgroups. The presented method enables systematic tracking of the user subgroups' contributions in iterative development. The method comprises (1) compilation of difficulties and deficiencies in the digital applications identified in usability testing, (2) clustering of the identified difficulties and deficiencies, and (3) prioritization of deficiencies to be rectified. Tracking user subgroup representation in the user feedback ensures that the development process is prioritized according to the needs of different subgroups. Mainly qualitative data collection methods are used. RESULTS: A protocol was developed to ensure that feedback from users representing all possible variants of 3 selected key user characteristics (gender, PA level, and level of technology experience) is considered in the iterative usability testing of a digital support for seniors' PA. The method was applied in iterative usability testing of two digital applications during spring/summer 2018. Results from the study on the users' experiences and the iterative modification of the digital applications are expected to be published during 2021. CONCLUSIONS: Methods for systematic collection, analysis, and prioritization of feedback from user subgroups might be particularly important in heterogenous user groups (eg, seniors). This study can contribute to identifying and improving the understanding of potential differences between user subgroups of seniors in their use and experiences of digital support for fall-preventive PA interventions. This knowledge may be relevant for developing digital support systems that are appropriate, useful, and attractive to users and for enabling the design of digital support systems that target specific user subgroups (ie, tailoring of the support). The protocol needs to be further used and investigated in order to validate its potential value. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR1-10.2196/20061.

Technology-Based Motivation Support for Seniors' Physical Activity-A Qualitative Study on Seniors' and Health Care Professionals' Views.

This paper investigates seniors' and health care professionals' (HCPs) perceptions on needed contributions and qualities of digital technology-based motivation support for seniors' physical activity (PA). Seniors and HCPs expressed their views in focus groups, which were analyzed separately by inductive content analysis. Similarities and differences in seniors' and HCPs' views were identified through thematic analysis of qualitative results from both focus groups. This article's main findings are that both seniors and HCPs believed digital technology should support and make PA more enjoyable in ways to strengthen seniors' control and well-being. However, seniors emphasized support for social interaction, while HCPs also requested support for increasing seniors' insight into PA and for facilitating their dialogue with seniors. Conclusions to be drawn are that seniors and HPCs shared overall views on digital technology's main contributions but had different perspectives on how those contributions could be obtained. This highlights the importance of the early identification of user groups and exploration of their different needs when developing new solutions. Moreover, seniors' and HCPs' perceptions included aspects relevant for personal motivation, technology acceptance, and PA behavioral change according to self-determination theory, unified theory of acceptance and use of technology, and behavioral change techniques for increasing PA.

Activity Monitors as Support for Older Persons' Physical Activity in Daily Life: Qualitative Study of the Users' Experiences.

BACKGROUND: Falls are a major threat to the health and independence of seniors. Regular physical activity (PA) can prevent 40% of all fall injuries. The challenge is to motivate and support seniors to be physically active. Persuasive systems can constitute valuable support for persons aiming at establishing and maintaining healthy habits. However, these systems need to support effective behavior change techniques (BCTs) for increasing older adults' PA and meet the senior users' requirements and preferences. Therefore, involving users as codesigners of new systems can be fruitful. Prestudies of the user's experience with similar solutions can facilitate future user-centered design of novel persuasive systems. OBJECTIVE: The aim of this study was to investigate how seniors experience using activity monitors (AMs) as support for PA in daily life. The addressed research questions are as follows: (1) What are the overall experiences of senior persons, of different age and balance function, in using wearable AMs in daily life?; (2) Which aspects did the users perceive relevant to make the measurements as meaningful and useful in the long-term perspective?; and (3) What needs and requirements did the users perceive as more relevant for the activity monitors to be useful in a long-term perspective? METHODS: This qualitative interview study included 8 community-dwelling older adults (median age: 83 years). The participants' experiences in using two commercial AMs together with tablet-based apps for 9 days were investigated. Activity diaries during the usage and interviews after the usage were exploited to gather user experience. Comments in diaries were summarized, and interviews were analyzed by inductive content analysis. RESULTS: The users (n=8) perceived that, by using the AMs, their awareness of own PA had increased. However, the AMs' impact on the users' motivation for PA and activity behavior varied between participants. The diaries showed that self-estimated physical effort varied between participants and varied for each individual over time. Additionally, participants reported different types of accomplished activities; talking walks was most frequently reported. To be meaningful, measurements need to provide the user with a reliable receipt of whether his or her current activity behavior is sufficient for reaching an activity goal. Moreover, praise when reaching a goal was described as motivating feedback. To be useful, the devices must be easy to handle. In this study, the users perceived wearables as easy to handle, whereas tablets were perceived difficult to maneuver. Users reported in the diaries that the devices had been functional 78% (58/74) of the total test days. CONCLUSIONS: Activity monitors can be valuable for supporting seniors' PA. However, the potential of the solutions for a broader group of seniors can significantly be increased. Areas of improvement include reliability, usability, and content supporting effective BCTs with respect to increasing older adults' PA.

An interactive distance solution for stroke rehabilitation in the home setting - A feasibility study.

BACKGROUND: In this study an interactive distance solution (called the DISKO tool) was developed to enable home-based motor training after stroke. OBJECTIVES: The overall aim was to explore the feasibility and safety of using the DISKO-tool, customized for interactive stroke rehabilitation in the home setting, in different rehabilitation phases after stroke. METHODS: Fifteen patients in three different stages in the continuum of rehabilitation after stroke participated in a home-based training program using the DISKO-tool. The program included 15 training sessions with recurrent follow-ups by the integrated application for video communication with a physiotherapist. Safety and feasibility were assessed from patients, physiotherapists, and a technician using logbooks, interviews, and a questionnaire. Qualitative content analysis and descriptive statistics were used in the analysis. RESULTS: Fourteen out of 15 patients finalized the training period with a mean of 19.5 minutes spent on training at each session. The DISKO-tool was found to be useful and safe by patients and physiotherapists. CONCLUSIONS: This study demonstrates the feasibility and safety of the DISKO-tool and provides guidance in further development and testing of interactive distance technology for home rehabilitation, to be used by health care professionals and patients in different phases of rehabilitation after stroke.

Users perspectives on interactive distance technology enabling home-based motor training for stroke patients.

The aim of this work has been to develop a technical support enabling home-based motor training after stroke. The basis for the work plan has been to develop an interactive technical solution supporting three different groups of stroke patients: (1) patients with stroke discharged from hospital with support from neuro team; (2) patients with stroke whose support from neuro team will be phased out and (3) patients living with impaired motor functions long-term. The technology has been developed in close collaboration with end-users using a method earlier evaluated and described [12]. This paper describes the main functions of the developed technology. Further, results from early user-tests with end-users, performed to identify needs for improvements to be carried out during further technical development. The developed technology will be tested further in a pilot study of the safety and, usefulness of the technology when applied as a support for motor training in three different phases of the post-stroke rehabilitation process.

Structure of the synthetase domain of human CTP synthetase, a target for anticancer therapy.

Cytidine triphosphate synthetase (CTPS) is a key enzyme in nucleic acid and phospholipid biosynthesis and its activity is increased in certain human cancers, making it a promising drug target. The crystal structure of the synthetase domain of human CTPS, which represents the first structure of a CTPS from an eukaryote, has been determined. The structure is homotetrameric and each active site is formed by three different subunits. Sulfate ions bound to the active sites indicate the positions of phosphate-binding sites for the substrates ATP and UTP and the feedback inhibitor CTP. Together with earlier structures of bacterial CTPS, the human CTPS structure provides an extended understanding of the structure-function relationship of CTPS-family members. The structure also serves as a basis for structure-based design of anti-proliferative inhibitors.

The crystal structure of a human PP2A phosphatase activator reveals a novel fold and highly conserved cleft implicated in protein-protein interactions.

Protein phosphatase 2A (PP2A) is a heterotrimeric Ser/Thr phosphatase that is involved in regulating a plethora of signaling pathways in the cell, making its regulation a critical part of the well being of the cell. For example, three of the non-catalytic PP2A subunits have been linked to carcinogenic events. Therefore, the molecular basis for the complicated protein-protein interaction pattern of PP2A and its regulators is of special interest. The PP2A phosphatase activator (PTPA) protein is highly conserved from humans to yeast. It is an activator of PP2A and has been shown to be essential for a fully functional PP2A, but its mechanism of activation is still not well defined. We have solved the crystal structure of human PTPA to 1.6A. It reveals a two-domain protein with a novel fold comprised of 13 alpha-helices. We have identified a highly conserved cleft as a potential region for interaction with peptide segments of other proteins. Binding studies with ATP and its analogs are not consistent with ATP being a cofactor/substrate for PTPA as had previously been proposed. The structure of PTPA can serve as a basis for structure-function studies directed at elucidating its mechanism as an activator of PP2A.

Pyrosequencing: history, biochemistry and future.

BACKGROUND: Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle. METHODS: The technique is built on a 4-enzyme real-time monitoring of DNA synthesis by bioluminescence using a cascade that upon nucleotide incorporation ends in a detectable light signal (bioluminescence). The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA-strand. Thereby, the signal can be quantitatively connected to the number of bases added. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis and genotyping. Mutation detection and single-nucleotide polymorphism genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. In order to expand the field for pyrosequencing, the read length needs to be improved. CONCLUSIONS: Th pyrosequencing system is based on an enzymatic system. There are different current and future applications of this technique.

Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins.

Mutation detection and single-nucleotide polymorphism genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technology based on the sequencing-by-synthesis principle. Currently, the technique is limited to simultaneous analysis of 96 or 384 samples. Earlier, attempts to increase the sample capacity were made using micromachined filter chamber arrays where parallel analyses of nanoliter samples could be monitored in real time. We have developed a strategy for specific immobilization of the light-producing enzyme luciferase to the DNA template within a reaction chamber. By this approach, luciferase is genetically fused to a DNA-binding protein (Klenow polymerase or Escherichia coli single-stranded DNA-binding (SSB) protein) and to a purification handle (Z(basic)). The proteins are produced in E. coli and purified using cation and anion exchange chromatography with removal of Z(basic). The produced proteins have been analyzed using an assay for complete primer extension of DNA templates immobilized on magnetic beads detected by pyrosequencing chemistry. Results from these experiments show that the proteins bind selectively to the immobilized DNA and that their enzymatic domains were active. Z(basic)-SSB-luciferase produced the highest signal in this assay and was further exploited as enzymatic reagent for DNA sequencing.

Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum).

ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.

Escherichia coli single-stranded DNA-binding protein, a molecular tool for improved sequence quality in pyrosequencing.

Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.

Gene expression analysis by signature pyrosequencing.

We describe a novel method for transcript profiling based on high-throughput parallel sequencing of signature tags using a non-gel-based microtiter plate format. The method relies on the identification of cDNA clones by pyrosequencing of the region corresponding to the 3'-end of the mRNA preceding the poly(A) tail. Simultaneously, the method can be used for gene discovery, since tags corresponding to unknown genes can be further characterized by extended sequencing. The protocol was validated using a model system for human atherosclerosis. Two 3'-tagged cDNA libraries, representing macrophages and foam cells, which are key components in the development of atherosclerotic plaques, were constructed using a solid phase approach. The libraries were analyzed by pyrosequencing, giving on average 25 bases. As a control, conventional expressed sequence tag (EST) sequencing using slab gel electrophoresis was performed. Homology searches were used to identify the genes corresponding to each tag. Comparisons with EST sequencing showed identical, unique matches in the majority of cases when the pyrosignature was at least 18 bases. A visualization tool was developed to facilitate differential analysis using a virtual chip format. The analysis resulted in identification of genes with possible relevance for development of atherosclerosis. The use of the method for automated massive parallel signature sequencing is discussed.

Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner.

To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained alpha-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI approximately/= 5.8) and ZZ-Cutinase-Zbasic (pI approximately/= 7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.