Cleavage of roquin and regnase-1 by the paracaspase MALT1 releases their cooperatively repressed targets to promote T(H)17 differentiation.

Humoral autoimmunity paralleled by the accumulation of follicular helper T cells (T(FH) cells) is linked to mutation of the gene encoding the RNA-binding protein roquin-1. Here we found that T cells lacking roquin caused pathology in the lung and accumulated as cells of the T(H)17 subset of helper T cells in the lungs. Roquin inhibited T(H)17 cell differentiation and acted together with the endoribonuclease regnase-1 to repress target mRNA encoding the T(H)17 cell-promoting factors IL-6, ICOS, c-Rel, IRF4, IκBNS and IκBζ. This cooperation required binding of RNA by roquin and the nuclease activity of regnase-1. Upon recognition of antigen by the T cell antigen receptor (TCR), roquin and regnase-1 proteins were cleaved by the paracaspase MALT1. Thus, this pathway acts as a 'rheostat' by translating TCR signal strength via graded inactivation of post-transcriptional repressors and differential derepression of targets to enhance T(H)17 differentiation.

Exploring experiential learning within interprofessional practice education initiatives for pre-licensure healthcare students: a scoping review.

BACKGROUND: Interprofessional collaborative team-based approaches to care in health service delivery has been identified as important to health care reform around the world. Many academic institutions have integrated interprofessional education (IPE) into curricula for pre-licensure students in healthcare disciplines, but few provide formal initiatives for interprofessional practice (IPP). It is recognized that experiential learning (EL) can play a significant role supporting IPP education initiatives; however, little is known of how EL is used within education for IPP in healthcare settings. METHODS: We conducted a scoping review to map peer-reviewed literature describing IPP education initiatives involving EL for pre-licensure students in healthcare disciplines. A literature search was executed in MEDLINE, CINAHL, EMBASE, ERIC, PsycINFO, Scopus, and Social Services Abstracts. After deduplication, two independent reviewers screened titles and abstracts of 5664 records and then 252 full-text articles that yielded 100 articles for data extraction. Data was extracted using an Excel template, and results synthesized for presentation in narrative and tabular formats. RESULTS: The 100 included articles represented 12 countries and IPP education initiatives were described in three main typologies of literature - primary research, program descriptions, and program evaluations. Forty-three articles used a theory, framework, or model for design of their initiatives with only eight specific to EL. A variety of teaching and learning strategies were employed, such as small interprofessional groups of students, team huddles, direct provision of care, and reflective activities, but few initiatives utilized a full EL cycle. A range of perspectives and outcomes were evaluated such as student learning outcomes, including competencies associated with IPP, impacts and perceptions of the IPP initiatives, and others such as client satisfaction. CONCLUSION: Few educational frameworks specific to EL have been used to inform EL teaching and learning strategies to consolidate IPE learning and prepare students for IPP in healthcare settings. Further development and evaluation of existing EL frameworks and models would be beneficial in supporting robust IPP educational initiatives for students in healthcare disciplines. Intentional, thoughtful, and comprehensive use of EL informed by theory can contribute important advances in IPP educational approaches and the preparation of a future health care workforce.

Pilot of a community-based interprofessional "student-infused" pulmonary rehabilitation program in Saint John, New Brunswick.

Background: With the increasing prevalence of chronic pulmonary conditions in New Brunswick and Canada it is necessary to consider innovative interventions to improve access to rehabilitation and supportive care for affected clients. In Fall 2018 we piloted a pulmonary rehabilitation (PR) program for persons with moderate to severe chronic obstructive pulmonary disease (COPD) to demonstrate a novel approach of bridging interprofessional education of students in health care fields with provision of care in a community setting. Methods: An 8-week PR program was implemented and evaluated using a quasi-experimental design with pre- and post-testing to measure the effects of the program's exercise and educational interventions on persons with COPD. Participants were assessed using the 6-Minute Walk Test (6MWT), the St. George's Respiratory Questionnaire (SGRQ), and a custom questionnaire that rated the participants' activities of daily living and the PR program. Results: Seven participants completed our PR program. Following the intervention, participants' self-reported health demonstrated a statistically significant improvement. Even though changes on the 6MWT and SGRQ were not shown to be statistically significant, there was evidence of clinically meaningful improvements in those measures. On average, participants walked 25 m further postintervention and showed clinically meaningful improvements on the SGRQ. Conclusions: This pilot project demonstrated that a community-based PR program with active involvement of students from multiple health care programs can have positive outcomes for clients with COPD. It also illustrated how educational programs can provide an innovative means for increasing access to rehabilitation and supportive care for clients in the community.

A concept analysis of children with complex health conditions: implications for research and practice.

BACKGROUND: This concept analysis aimed to clarify the meaning of "children with complex health conditions" and endorse a definition to inform future research, policy, and practice. METHODS: Using Walker and Avant's (2011)'s approach, we refined the search strategy with input from our team, including family representatives. We reviewed the published and grey literature. We also interviewed 84 health, social, and educational stakeholders involved in the care of children with complex health conditions about their use/understanding of the concept. RESULTS: We provided model, borderline, related, and contrary cases for clarification purposes. We identified defining attributes that nuance the concept: (1) conditions and needs' breadth; (2) uniqueness of each child/condition; (3) varying extent of severity over time; 4) developmental age; and (5) uniqueness of each family/context. Antecedents were chronic physical, mental, developmental, and/or behavioural condition(s). There were individual, family, and system consequences, including fragmented services. CONCLUSIONS: Building on previous definitions, we proposed an iteration that acknowledges the conditions' changing trajectories as involving one or more chronic condition(s), regardless of type(s), whose trajectories can change over time, requiring services across sectors/settings, oftentimes resulting in a lower quality of life. A strength of this paper is the integration of the stakeholders'/family's voices into the development of the definition.

Dynamical coring of Markov state models.

The accurate definition of suitable metastable conformational states is fundamental for the construction of a Markov state model describing biomolecular dynamics. Following the dimensionality reduction in a molecular dynamics trajectory, these microstates can be generated by a recently proposed density-based geometrical clustering algorithm [F. Sittel and G. Stock, J. Chem. Theory Comput. 12, 2426 (2016)], which by design cuts the resulting clusters at the energy barriers and allows for a data-based identification of all parameters. Nevertheless, projection artifacts due to the inevitable restriction to a low-dimensional space combined with insufficient sampling often leads to a misclassification of sampled points in the transition regions. This typically causes intrastate fluctuations to be mistaken as interstate transitions, which leads to artificially short life times of the metastable states. As a simple but effective remedy, dynamical coring requires that the trajectory spends a minimum time in the new state for the transition to be counted. Adopting molecular dynamics simulations of two well-established biomolecular systems (alanine dipeptide and villin headpiece), dynamical coring is shown to considerably improve the Markovianity of the resulting metastable states, which is demonstrated by Chapman-Kolmogorov tests and increased implied time scales of the Markov model. Providing high structural and temporal resolution, the combination of density-based clustering and dynamical coring is particularly suited to describe the complex structural dynamics of unfolded biomolecules.

Pharmacological inhibition of MALT1 protease activity protects mice in a mouse model of multiple sclerosis.

BACKGROUND: The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is crucial for lymphocyte activation through signaling to the transcription factor NF-κB. Besides functioning as a scaffold signaling protein, MALT1 also acts as a cysteine protease that specifically cleaves a number of substrates and contributes to specific T cell receptor-induced gene expression. Recently, small molecule inhibitors of MALT1 proteolytic activity were identified and shown to have promising anticancer properties in subtypes of B cell lymphoma. However, information on the therapeutic potential of small compound inhibitors that target MALT1 protease activity in autoimmunity is still lacking. METHODS: The present study aimed to elucidate whether MALT1 protease inhibitors are also useful in the treatment of lymphocyte-mediated autoimmune pathologies such as multiple sclerosis (MS). For this, we studied the therapeutic potential of a recently identified inhibitor of MALT1 protease activity, the phenothiazine derivative mepazine, in the context of experimental autoimmune encephalomyelitis (EAE), the main animal model for MS. RESULTS: We demonstrate that administration of mepazine prophylactically or after disease onset, can attenuate EAE. Importantly, while complete absence of MALT1 affects the differentiation of regulatory T (Treg) cells in vivo, the MALT1 protease inhibitor mepazine did not affect Treg development. CONCLUSIONS: Altogether, these data indicate that small molecule inhibitors of MALT1 not only hold great promise for the treatment of B cell lymphomas but also for autoimmune disorders such as MS.

Critical role of PI3K signaling for NF-kappaB-dependent survival in a subset of activated B-cell-like diffuse large B-cell lymphoma cells.

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) represents a very aggressive human lymphoma entity. Constitutive NF-κB activation caused by chronic active B-cell receptor (BCR) signaling is common feature of many ABC DLBCL cells; however, the pathways linking BCR signaling to the NF-κB prosurvival network are largely unknown. Here we report that constitutive activity of PI3K and the downstream kinase PDK1 are essential for the viability of two ABC DLBCL cell lines that carry mutations in the BCR proximal signaling adaptor CD79B. In these cells, PI3K inhibition reduces NF-κB activity and decreases the expression of NF-κB target genes. Furthermore, PI3K and PDK1 are required for maintaining MALT1 protease activity, which promotes survival of the affected ABC DLBCL cells. These results demonstrate a critical function of PI3K-PDK1 signaling upstream of MALT1 protease and NF-κB in distinct ABC DLBCL cells and provide a rationale for the pharmacologic use of PI3K inhibitors in DLBCL therapy.

Remote symptom support training programs for oncology nurses in Canada: an environmental scan.

The overall aim of this study was to explore current remote symptom support training programs provided to nurses in ambulatory oncology programs across Canada, using a survey-methods environmental scan. Of 36 delivered invitations, 28 programs responded (77.8%) representing 10 provinces. Of 25 programs that offer telephone symptom support, 17 provide symptom support training, seven do not, and one did not say. Seven programs shared training materials with the investigators and elements of training included: symptom management guidelines (n = 6), telephone triage process/principles (n = 5), competent telephone practices (n = 4), documentation (n = 4), professional standards (n = 3), role-play (n = 3), communication skills/etiquette (n = 3), and monitoring quality (n = 1). Formats included: presentations (n = 3), paper-based resources (n = 3), or e-learning modules (n = 1). No training programs were rigorously evaluated. Training in telephone nursing symptom support across oncology programs is variable. Opportunities exist to identify core competencies and evaluate if training programs enhance delivery of remote cancer symptom support.

'Snapshot in time': a cross-sectional study exploring stakeholder experiences with environmental scans in health services delivery research.

OBJECTIVE: To describe stakeholder characteristics and perspectives about experiences, challenges and information needs related to the use of environmental scans (ESs). DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: A web-based survey platform was used to disseminate an online survey to stakeholders who had experience with conducting ESs in a health services delivery context (eg, researchers, policy makers, practitioners). Participants were recruited through purposive and snowball sampling. The survey was disseminated internationally, was available in English and French, and remained open for 6 weeks (15 October to 30 November 2022). ANALYSIS: Descriptive statistics were used to describe the characteristics and experiences of stakeholders. Thematic analysis was used to analyse the open-text questions. RESULTS: Of 47 participants who responded to the survey, 94% were from Canada, 4% from the USA and 2% from Australia. Respondents represented academic institutions (57%), health agency/government (32%) and non-government organisations or agencies (11%). Three themes were identified: (a) having a sense of value and utility; (b) experiencing uncertainty and confusion; and (c) seeking guidance. The data suggest stakeholders found value and utility in ESs and conducted them for varied purposes including to: (a) enhance knowledge, understanding and learning about the current landscape or state of various features of health services delivery (eg, programmes, practices, policies, services, best practices); (b) expose needs, service barriers, challenges, gaps, threats, opportunities; (c) help guide action for planning, policy and programme development; and (d) inform recommendations and decision-making. Stakeholders also experienced conceptual, methodological and practical barriers when conducting ESs, and expressed a need for methodological guidance delivered through published guidelines, checklists and other means. CONCLUSION: ESs have value and utility for addressing health services delivery concerns, but conceptual and methodological challenges exist. Further research is needed to help advance the ES as a distinct design that provides a systematic approach to planning and conducting ESs.

Heterogenous nuclear ribonucleoprotein Q increases protein expression from HIV-1 Rev-dependent transcripts.

BACKGROUND: Heterogenous nuclear ribonucleoproteins (hnRNPs) control many processes of the gene expression machinery including mRNA transcription, splicing, export, stability and translation. Recent data show interaction of the HIV-1 Rev regulatory protein with a subset of hnRNP proteins, that includes hnRNP Q, suggesting that hnRNPs can contribute to regulation of HIV-1 gene expression by Rev. FINDINGS: In this work we address the effect of hnRNP Q on Rev-dependent gene expression. We show that hnRNP Q overexpression increased levels of proteins produced from a Rev-dependent reporter gene in the presence of Rev. Increased protein levels did not correlate with changes in either the levels or the nucleocytoplasmic distribution of Rev-dependent reporter mRNAs. Similar observations were made in persistently HIV-1 infected HeLa cells. In these cells, hnRNP Q overexpression increased levels of the HIV-1 Gag-p24 protein, while levels of viral Rev-dependent mRNAs were not affected. CONCLUSION: Our data indicate that hnRNP Q can stimulate the protein production of Rev-dependent mRNAs without changing mRNA levels and mRNA export, respectively. This suggests that hnRNP Q can boost HIV gene expression at the level of protein production.

Toward a Benchmark for Markov State Models: The Folding of HP35.

Adopting a 300 µs long MD trajectory of the folding of villin headpiece (HP35) by D. E. Shaw Research, we recently constructed a Markov state model (MSM) based on inter-residue contacts. The model reproduces the folding time and predicts that the native basin and unfolded region consist of metastable substates that are structurally well-characterized. Recognizing the need to establish well-defined benchmark problems, we study to what extent and in what sense this MSM can be employed as a reference model. Hence, we test the robustness of the MSM by comparing it to models that use alternative combinations of features, dimensionality reduction methods, and clustering schemes. The study suggests some main characteristics of the folding of HP35 that should be reproduced by other competitive models. Moreover, the discussion reveals which parts of the MSM workflow matter most for the considered problem and illustrates the promises and pitfalls of state-based models for the interpretation of biomolecular simulations.

Selecting Features for Markov Modeling: A Case Study on HP35.

Markov state models represent a popular means to interpret molecular dynamics trajectories in terms of memoryless transitions between metastable conformational states. To provide a mechanistic understanding of the considered biomolecular process, these states should reflect structurally distinct conformations and ensure a time scale separation between fast intrastate and slow interstate dynamics. Adopting the folding of villin headpiece (HP35) as a well-established model problem, here we discuss the selection of suitable input coordinates or "features", such as backbone dihedral angles and interresidue distances. We show that dihedral angles account accurately for the structure of the native energy basin of HP35, while the unfolded region of the free energy landscape and the folding process are best described by tertiary contacts of the protein. To construct a contact-based model, we consider various ways to define and select contact distances and introduce a low-pass filtering of the feature trajectory as well as a correlation-based characterization of states. Relying on input data that faithfully account for the mechanistic origin of the studied process, the states of the resulting Markov model are clearly discriminated by the features, describe consistently the hierarchical structure of the free energy landscape, and─as a consequence─correctly reproduce the slow time scales of the process.

NF-κB essential modulator (NEMO) interaction with linear and lys-63 ubiquitin chains contributes to NF-κB activation.

The IκB kinase (IKK) complex acts as a gatekeeper of canonical NF-κB signaling in response to upstream stimulation. IKK activation requires sensing of ubiquitin chains by the essential IKK regulatory subunit IKKγ/NEMO. However, it has remained enigmatic whether NEMO binding to Lys-63-linked or linear ubiquitin chains is critical for triggering IKK activation. We show here that the NEMO C terminus, comprising the ubiquitin binding region and a zinc finger, has a high preference for binding to linear ubiquitin chains. However, immobilization of NEMO, which may be reminiscent of cellular oligomerization, facilitates the interaction with Lys-63 ubiquitin chains. Moreover, selective mutations in NEMO that abolish association with linear ubiquitin but do not affect binding to Lys-63 ubiquitin are only partially compromising NF-κB signaling in response to TNFα stimulation in fibroblasts and T cells. In line with this, TNFα-triggered expression of NF-κB target genes and induction of apoptosis was partially compromised by NEMO mutations that selectively impair the binding to linear ubiquitin chains. Thus, in vivo NEMO interaction with linear and Lys-63 ubiquitin chains is required for optimal IKK activation, suggesting that both type of chains are cooperating in triggering canonical NF-κB signaling.

Correlation-Based Feature Selection to Identify Functional Dynamics in Proteins.

To interpret molecular dynamics simulations of biomolecular systems, systematic dimensionality reduction methods are commonly employed. Among others, this includes principal component analysis (PCA) and time-lagged independent component analysis (TICA), which aim to maximize the variance and the time scale of the first components, respectively. A crucial first step of such an analysis is the identification of suitable and relevant input coordinates (the so-called features), such as backbone dihedral angles and interresidue distances. As typically only a small subset of those coordinates is involved in a specific biomolecular process, it is important to discard the remaining uncorrelated motions or weakly correlated noise coordinates. This is because they may exhibit large amplitudes or long time scales and therefore will be erroneously considered important by PCA and TICA, respectively. To discriminate collective motions underlying functional dynamics from uncorrelated motions, the correlation matrix of the input coordinates is block-diagonalized by a clustering method. This strategy avoids possible bias due to presumed functional observables and conformational states or variation principles that maximize variance or time scales. Considering several linear and nonlinear correlation measures and various clustering algorithms, it is shown that the combination of linear correlation and the Leiden community detection algorithm yields excellent results for all considered model systems. These include the functional motion of T4 lysozyme to demonstrate the successful identification of collective motion, as well as the folding of the villin headpiece to highlight the physical interpretation of the correlated motions in terms of a functional mechanism.

Understanding Student-Run Health Initiatives in the Context of Community-Based Services: A Concept Analysis and Proposed Definitions.

BACKGROUND: Student-run health initiatives in the community setting have been utilized to provide practical experience for undergraduate students to develop professional competencies, gain exposure to diverse populations, and to engage in activities of social accountability. There is much literature on student-run health initiatives; however, there is no consensus on a definition of this concept or a comprehensive synthesis of the literature that describes student-run health initiatives offered by students in pre-licensure healthcare education programs. PURPOSE: To provide a concept analysis of, and propose a definition for, student-run health initiatives that provide community-based services for students during pre-licensure health discipline education. METHODS: A systematic literature search and review process was used to identify and synthesize peer-reviewed articles from 7 academic databases covering a range of pre-licensure health disciplines and education. Walker and Avant's framework for concept analysis was used to guide exploration of attributes, antecedents and consequences of student-run initiatives, and to inform development of a definition for this concept. RESULTS: The review yielded 222 articles for data extraction and represented 17 distinct pre-licensure health disciplines, 18 health-related disciplines, and a range of other baccalaureate and graduate programs. Our analysis revealed 16 definitions, 5 attributes, 6 antecedents, and consequences identified for student-run health initiatives. Attributes were Provision of Service, Service is Free, Target Clientele, Volunteerism, and Student Governance. Antecedents included Purpose/Rationale, Affiliation with Academic Unit, Location and Partnerships, Funding and Resources, Professional Oversight, and Preparation for Student Role. Consequences were improved access to services and outcomes for clients; competency development, personal gains and interprofessional learning for students; and positive outcomes for broader systems, such as decrease of service utilization and cost/benefit. CONCLUSIONS: There was no clear conceptual definition for student-run health initiatives, but many defining characteristics and well-described exemplars in the literature. Given the variations in purpose and scope of these initiatives, particularly to distinguish degree of students' roles in operations and the involvement of academic institutions, we propose 3 distinct conceptual definitions: student-run, student-led, and student-infused health initiatives.

Concept Analysis and Proposed Definition of Community Health Center.

Background: Community health centers (CHCs) have been used for delivery of health services since the 1920s and originally were intended to provide care to underserved populations. CHCs have become an integral part of healthcare systems in many countries; however, the term CHC is used synonymously with other concepts and there is no clear definition for CHC. The purpose of our concept analysis was to determine how CHCs are described in the literature and to develop a concept definition for CHC. Methods: Informed by the 8-step process described by Walker and Avant, we searched for literature spanning disciplines within health, business, and policy. We used a systematic review process to identify a range of peer-reviewed articles that help illustrate the attributes, antecedents, and consequences of CHCs. A total of 102 articles from 7 databases were included in our concept analysis. Results: We distinguished 6 attributes of a CHC: primary care; accessibility; preventative care; defined population; health promotion; and comprehensive and integrated care. About 4 antecedents fundamental to a CHC included: secure funding; vision and support; adequate human resources; and governance structure. Consequences of CHCs are improved health outcomes, efficiency, and cost-effective provision of healthcare services. Conclusions: Our concept analysis revealed core characteristics of CHCs that assisted us in synthesizing a concept definition for CHC. These characteristics and our proposed definition will help provide clarity on the concept of CHC to benefit evaluation, research, and policy development of CHCs.

Use of environmental scans in health services delivery research: a scoping review.

OBJECTIVE: To examine the extent and nature of evidence on the use of the environmental scan (ES) in the health services delivery literature. DESIGN: Scoping review. METHODS: This scoping review followed the five-stage scoping review methodology outlined by Khalil et al. A Peer Review of Electronic Search Strategies was completed. Seven electronic databases and the grey literature were searched. Pairs of researchers independently performed two levels of screening and data extraction. Data were analysed using qualitative content and thematic analysis. RESULTS: Ninety-six studies were included in the scoping review. Researchers conducted ESs for many purposes, the most common being to examine the current state of programmes, services or policies. Recommendations were informed by ESs in 20% of studies. Most common data collection methods were literature review (71%), key informant or semistructured interviews (46%) and surveys (35%). Over half (53%) of the studies used a combination of passive (looking at information eg, literature, policies, guidelines) and active (looking for information eg, surveys, interviews) approaches to data collection. Person sources of data (eg, healthcare stakeholders, community representatives) and non-person sources of data (eg, documents, electronic databases, the web) were drawn on to a similar extent. The thematic analysis of the definitions/descriptions yielded several themes including instrument of discovery, knowledge synthesis, forward-looking and decision making. Research gaps identified included absence of a standard definition, inconsistencies in terminology and lack of guiding frameworks in the health services delivery context. CONCLUSION: ESs were conducted to gather evidence and to help inform decision making on a range of policy and health services delivery issues across the continuum of care. Consistency in terminology, a consensus definition and more guidance on ES design may help provide structure for researchers and other stakeholders, and ultimately advance ES as a methodological approach. A working definition of ES in a health services delivery context is presented.

Identification of a heterogeneous nuclear ribonucleoprotein-recognition region in the HIV Rev protein.

The Rev protein is a key regulator of human immunodeficiency virus type 1 (HIV-1) gene expression. Rev is primarily known as an adaptor protein for nuclear export of HIV RNAs. However, Rev also contributes to numerous other processes by less well known mechanisms. Understanding the functional nature of Rev requires extensive knowledge of its cellular interaction partners. Here we demonstrate that Rev interacts with members of a large family of multifunctional host cell factors called hnRNPs. Rev employs amino acids 9-14 for specific binding to the heterogeneous nuclear ribonucleoproteins (hnRNP) A1, Q, K, R, and U. In addition, Rev interacts with hnRNP E1 and E2 by a different mechanism. The set of hnRNPs recognized by the N terminus of Rev feature RGG boxes. Exemplary testing of hnRNP A1 revealed a critical role of arginine residues within the RGG box for interaction with Rev. Finally, we demonstrate that expression levels of hnRNP A1, Q, K, R, and U influence HIV-1 production by persistently infected astrocytes, linking these hnRNPs to HIV replication. The novel interaction of HIV-1 Rev with functionally diverse hnRNPs lends further support to the idea that Rev is a multifunctional protein and may be involved in coupling HIV replication to diverse cellular processes and promoting virus-host cell interactions.

MSMPathfinder: Identification of Pathways in Markov State Models.

Markov state models represent a popular means to interpret biomolecular processes in terms of memoryless transitions between metastable conformational states. To gain insight into the underlying mechanism, it is instructive to determine all relevant pathways between initial and final states of the process. Currently available methods, such as Markov chain Monte Carlo and transition path theory, are convenient for identifying the most frequented pathways. They are less suited to account for the typically huge amount of pathways with low probability which, though, may dominate the cumulative flux of the reaction. On the basis of a systematic construction of all possible pathways, the here proposed method MSMPathfinder is able to characterize the multitude of unique pathways (say, up to 1010) in a complex system and to quantitatively calculate their correct weights and associated waiting times with predefined accuracy. Adopting the chiral transitions of a peptide helix and the folding of the villin headpiece as model problems, mechanisms and associated waiting times of these processes are discussed using a kinetic network representation. The analysis reveals that the waiting time distribution may yield only little insight into the diversity of pathways, because the measured folding times do typically not reflect the most probable path lengths but rather the cumulative effect of many different pathways.

Requirement of a dopaminergic neuronal phenotype for toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells.

LUHMES cells are conditionally-immortalized non-transformed human fetal cells that can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. After differentiation by GDNF and cyclic adenosine monophosphate, LUHMES were sensitive to 1-methyl-4-phenylpyridinium (MPP(+)) toxicity at < or =5 microM, but resistant to the parental compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The high homogeneity and purity of the cultures allowed the detection of metabolic changes during the degeneration. Cellular ATP dropped in two phases after 24 and 48 h; cellular glutathione (GSH) decreased continuously, paralleled by an increase in lipid peroxidation. These events were accompanied by a time-dependent degeneration of neurites. Block of the dopamine transporter by GBR 12909 or mazindol completely abrogated MPP(+) toxicity. Inhibition of de novo dopamine synthesis by alpha-methyl-l-tyrosine or 3-iodo-l-tyrosine attenuated toxicity, but did not reduce the initial drop in ATP. Inhibition of mixed lineage kinases by CEP1347 completely prevented the MPP(+)-induced loss of viability and intracellular GSH, but failed to attenuate the initial drop of ATP. For the quantitative assessment of neurite degeneration, an automated imaging-based high content screening approach was applied and confirmed the findings made by pharmacological interventions in this study. Our data indicate that inhibition of mitochondrial ATP synthesis is not sufficient to trigger cell death in MPP(+)-treated LUHMES.