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Human efferent adaptation of DPOAEs in the L1,L2 space.

The adaptive properties of distortion product otoacoustic emissions (DPOAEs) at 2f(1)-f2 were investigated in 12 ears of normally hearing adults aged 18-30 years using long-lasting 1-s primary-tone on-times. In this manner, DPOAE adaptation at a single f2 of 1.55 kHz (f2/f1=1.21) was evaluated as a function of the levels of the primary tones in a matrix of L1, L2 settings, which varied from 45 to 80 dB SPL, in 5-dB steps. DPOAEs were elicited under both monaural and binaural stimulus-presentation conditions. Adaptation was defined as the difference in DPOAE levels between the initial 92-ms baseline measure using a standard protocol and one obtained during the final 92 ms of the prolonged 1-s primary-tones. These differences were averaged across subjects to create contour plots of mean adaptation in the L1,L2 space. The 2f(1)-f2 DPOAE revealed consistent regions of suppression (-0.5 dB difference) or enhancement (+0.5 dB difference) with respect to baseline measures within the L(1),L(2) matrix for both acoustic-stimulation conditions. Specifically, 2f(1)-f2 DPOAE suppressions of 1-2 dB occurred for both monaural and binaural presentations, typically at level combinations in which L1>L2. In contrast, larger 2f(1)-f2 DPOAE enhancements of 3-4 dB occurred for only the binaural condition, at primary-tone level combinations where L1<L2. Although adaptation activity was also evaluated for the DPOAEs at f(2)-f1, 2f(2)-f1, and 3f(1)-2f2, these emissions were either immeasurable (e.g., f(2)-f1) or only present in a subset of subjects over a narrow range of primary-tone frequencies and levels that did not support a systematic analysis. In summary, the 2f(1)-f2 results suggest that a potentially important area for adaptation measures exists in the L1,L2 space, when L1 is lower than L2. This combination of primary-tone levels can lead to large DPOAE adaptation effects that may be related to a notch in the DPOAE response/growth or input/output (I/O) function.

Acoustic Stimulation↗

Repertory and likelihood ratio: time for structural changes.

If the likelihood ratio (LR) method is introduced, the repertory will gradually change as more symptoms are assessed. It will also change the use of the repertory: the most important medicines of each symptom rubric can be identified and relied on, even in large rubrics. This is also a good opportunity to correct structural shortcomings of the repertory, for instance, entries should be based on systematic analysis of materia medica instead of casual observations.

Bayes Theorem↗

Acute cellular rejection grading scheme for human gastric allografts.

The control of acute cellular rejection (ACR) in multivisceral transplantation improves long-term survival, but monitoring this process can be challenging because different allografts can display varying forms and degrees of rejection. Criteria for ACR of small bowel and liver have been established, but a systematic analysis for ACR in stomach is lacking. For this reason we have developed a comprehensive grading scheme for the evaluation of gastric allograft rejection. The grading scheme was designed to individually grade a variety of changes in the surface epithelium, lamina propria, and glandular structures. The individual values are cumulated, and the final score determines assignment of the rejection grade. The ACR grades range from no evidence of acute cellular rejection to severe rejection. We performed a retrospective study based on 70 gastric allograft biopsies from 20 patients who received multivisceral transplantation from 1995 to 2001. We found that the scoring system showed no significant interobserver variability and allowed for an accurate designation of the ACR grade to the gastric allografts. We found with this grading system that neither clinical symptoms nor gastric endoscopic findings could serve as specific indicators of gastric ACR. Our results also showed that there were differences in the occurrence and intensity of acute rejection between the stomach and other transplanted organs, suggesting that ACR can occur independently among different allografts of the same host. In conclusion, we find that this scheme for grading ACR in gastric transplants is objective and reproducible. This grading system will likely allow for improved correlation between gastric ACR grade and clinical symptoms, as well as improve interobserver uniformity within and between institutions.

Acute Disease↗

Risk assessment and disease prevention in travelers visiting friends and relatives.

Although VFR travelers are at risk for acquiring infections and experiencing illness while traveling, many of these diseases are preventable. A comprehensive approach to decreasing their travel-related morbidity requires continued surveillance, data collection, systematic analysis, and action. A review of the literature provides few examples of interventions designed specifically to address VFR travel needs. Given the geographic and cultural diversity of these populations, models grounded in health behavior theory provide the best potential for clinically relevant replication. Outreach aimed at improving knowledge and care-seeking behaviors among VFR travelers may be facilitated through community-based campaigns in areas with large foreign-born populations. In developed countries, policies must be reviewed to ensure that travel-related services are accessible, affordable, and appropriate for these diverse populations. In the clinical setting, providers must develop culturally appropriate methods of communicating with traveling populations to influence behavior. In particular, primary care providers should take an active approach through screening for high-risk travel, and increasing their competency in travel medicine. Special attention should be given to illness that is prevented by routine childhood immunization (eg, varicella, measles, and hepatitis B); by disease prevented by travel vaccines (eg, typhoid fever and hepatitis A); and disease that can be prevented by careful avoidance measures or compliance with preventive medication (eg, malaria and tuberculosis). With increased immigration from developing to developed regions and widely affordable travel, the number of VFR travelers is expected to increase. As such, increased efforts to prevent VFR traveler morbidity serve the individual while also contributing to global public health.

Chickenpox↗

Spacer-engineered donor DNA enhances CRISPR-Cas9-mediated knockin to establish a chemical knockdown platform for endogenous proteins.

Precise installation of functional protein domains at endogenous loci is a powerful approach for interrogating protein functions, but its broad application is limited by the low efficiency of homology-directed repair (HDR)-mediated knockin during Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene editing. Here, we investigated a simple donor DNA engineering strategy that enhances HDR-mediated gene knockin by appending additional gRNA-recognizable spacer sequences to donor templates. Systematic analysis of linear dsDNA and plasmid donors showed that spacer position, length, and orientation influenced HDR efficiency, and that spacer-containing donors improved knockin across multiple genomic loci, insertion sizes, cell types and delivery modalities. Mechanistic analyses revealed that spacer-containing donors formed stable complexes with Cas9/gRNA and showed increased nuclear localization, supporting nuclear delivery as a key contributor to improved editing outcomes. We then applied this gene-editing strategy to establish a chemical knockdown platform by installing drug-responsive degrons at endogenous loci, generating cell lines in which GSK3&#x3b2; or Lin28A protein could be rapidly, potently and reversibly depleted by drug treatment. These platforms enable selective modulation of endogenous proteins and reveal cellular responses that may differ from those obtained using conventional genetic perturbation. Together, this work establishes a readily implementable framework that integrates improved gene editing with on-demand chemical knockdown of endogenous proteins.

CRISPR-Cas9↗

Positive pressure ventilation in the management of acute and chronic cardiac failure: a systematic review and meta-analysis.

Chronic heart failure (CHF) is a common condition and is associated with excess morbidity and mortality, in spite of the many advances in its treatment. Chronic stable heart failure is also associated with an increased incidence of sleep-related breathing disorders, such as central sleep apnoea (CSA) and Cheyne Stokes respiration (CSR). Continuous positive airways pressure (CPAP) has been shown to alleviate the symptoms of CHF, improve left ventricular function and oxygenation. To a certain extent, CPAP also abolishes sleep-related breathing disorders in patients with chronic heart failure. In patients with acute pulmonary oedema, the use of positive pressure ventilation improves cardiac haemodynamic indices, as well as symptoms and oxygenation, and is associated with a lower need for intubation. However, some studies have cast doubts about its safety and suggest a higher rate of myocardial infarction associated with its use. In our opinion, non-invasive positive pressure ventilation and CPAP offers an adjunctive mode of therapy in patients with acute pulmonary oedema and chronic heart failure, who may not be suitable for intubation and in those not responsive to conventional therapies. Non-invasive ventilation also helps to improve oxygenation in those patients with exhaustion and respiratory acidosis. Many trials are still ongoing and the results of these studies would throw more light on the present role of non-invasive ventilation in the management of CHF.

Acute Disease↗

Metabolic syndrome and coronary artery disease: is there a gender specific effect?

INTRODUCTION: Clustering of cardiovascular risk factors is observed in metabolic syndrome (MS), but the relative contribution of different factors to determine outcomes remains largely unknown. We investigated the influence of gender in the effect of MS in coronary vascular lesions in 385 patients who underwent first time coronary artery disease (CAD) assessment through coronary angiography. MATERIALS AND METHODS: Patients were stratified in two groups: metabolic syndrome (MS, N = 190) and non-metabolic syndrome (NMS, N = 195), according to standard criteria. Two vascular scores related to extension and severity of vascular lesions were determined by systematic analysis of 20 coronary segments. RESULTS AND CONCLUSIONS: MS was more prevalent in females (58.7 vs. 39.7%, p = 0.0005) and hypertension and low-HDL were the leading prevalent factors in the MS group. Scores for extension and severity of lesions were higher in the MS than in the NMS group (2.10 +/- 1.75 vs. 1.75 +/- 1.58; p = 0.01, and 1.49 +/- 1.29 vs. 1.25 +/- 1.15; p = 0.03, respectively). The impact of MS in CAD scores was more pronounced in females (OR 2.2, 95% CI 1.1-4.3, p = 0.02) than in males (OR 1.5, 95% CI 0.8-2.7, p = 0.17). Non-HDL cholesterol was the only risk factor influencing the scores in a gender specific manner, showing an increased impact in the female gender. Normal coronary angiograms were more frequent in females (p = 0.0001), especially in NMS. Collectively, these data suggest that MS is related to higher extension and severity of vascular lesions in this population regardless of gender but lipids and anthropometric parameters may differentially modulate the MS effect in a particular gender.

Brazil↗

Electronic implementation of guidelines in the EsPeR system: a knowledge specification method.

Despite initiatives to standardize methods for the development of clinical guidelines, several barriers hinder their integration in daily clinical practice: failure to fulfil quality criteria, poor effectiveness of their dissemination. Computerization of guidelines can favor their dissemination. The initial step of computerization is the knowledge specification from the text of the guideline. We describe the method of knowledge specification, which is used in EsPeR (Personalized Estimate of Risks), a web-based decision support system in preventive medicine, which allows, for a given person, to estimate risks and access recommendations, based on clinical profile. This method is based on a structured and systematic analysis of text allowing detailed specification of a decision tree. We use decision tables to validate the decision algorithm and decision trees to specify this algorithm, along with elementary messages of recommendation. Editing tools are used to facilitate the process of validation and the workflow between expert physicians and computer scientists. Applied to eleven different guidelines, the method allows a quick and valid computerization and integration in the EsPeR system. The method used for computerization could help to define a framework usable at the initial step of guideline development in order to produce guidelines ready for electronic implementation.

Decision Support Systems, Clinical↗

Service user involvement in nursing, midwifery and health visiting research: a review of evidence and practice.

OBJECTIVES: In the UK policy recommends that service users (patients, carers and the public) should be involved in all publicly funded health and social care research. However, little is known about which approaches work best in different research contexts and why. The purpose of this paper is to explain some of the theoretical limitations to current understandings of service user involvement and to provide some suggestions for theory and methods development. This paper draws upon findings from a review of the research 'evidence' and current practice on service user involvement in the design and undertaking of nursing, midwifery and health visiting research. DESIGN: A multi-method review was commissioned by the NHS Service Delivery and Organisation (SDO) Research and Development Programme. The timeframe was April 2004-March 2005. The full report (Ref: SDO/69/2003) and supplementary bibliography are available from: http://www.sdo.lshtm.ac.uk. REVIEW METHODS/DATA: Initial searches of the health and social care literature and consultations with researchers were used to develop a broad definition of the topic area. A service user reference group (26 members) worked with the project team to refine the scope of the review, to set inclusion criteria and develop a framework for the analysis. Systematic searches of the literature were undertaken online and through library stacks (345 relevant documents were identified). Ongoing and recently completed studies that had involved service users were identified through online databases (34 studies) and through a national consultation exercise (17 studies). Selected studies were followed up using telephone interviews (n=11). Members of the service user reference group worked with the research team to advise on key messages for dissemination to different audiences. RESULTS: Information was gained about contextual factors, drivers, concepts, approaches and outcomes of service user involvement in nursing, midwifery and health visiting research, as well as developments in other research fields. Synthesis of this information shows that there are different purposes and domains for user involvement, either as part of researcher-led or user-led research, or as part of a partnership approach. A number of issues were identified as being important for future research. These include: linking different reasons for service user involvement with different outcomes; understanding the relationship between research data and service user involvement, and developing conceptualisations of user involvement that are capable of accommodating complex research relationships. Suggestions for the development of practice include: consideration of diversity, communication, ethical issues, working relationships, finances, education and training. CONCLUSIONS: Because research is undertaken for different reasons and in different contexts, it is not possible to say that involving service users will, or should, always be undertaken in the same way to achieve the same benefits. At a research project level uniqueness of purpose is a defining characteristic and strength of service user involvement.

Community Health Nursing↗

Characterization of carbonic anhydrase IX (CA IX) as an endogenous marker of chronic hypoxia in live human tumor cells.

PURPOSE: Published clinical studies provide conflicting data regarding the prognostic significance of carbonic anhydrase IX (CA IX) overexpression as an endogenous marker of tumor hypoxia and its comparability with other methods of hypoxia detection. We performed a systematic analysis of CA IX protein levels under various in vitro conditions of tumor hypoxia in HT 1080 human fibrosarcoma and FaDu human pharyngeal carcinoma cells. Because sorting of live CA IX positive cells from tumors provides a tool to study the radiosensitivity of chronically hypoxic cells, we modified and tested a CA IX flow cytometry protocol on mixed hypoxic/aerobic suspensions of HT 1080 and FaDu cells. METHODS AND MATERIALS: HT 1080 and FaDu cells were treated with up to 24 h of in vitro hypoxia and up to 96 h of reoxygenation. To test the effect of nonhypoxic stimuli, glucose and serum availability, pH and cell density were modified. CA IX protein was quantified in Western blots of whole-cell lysates. Mixed suspensions with known percentages of hypoxic cells were prepared for CA IX flow cytometry. The same mixtures were assayed for clonogenic survival after 10 Gy. RESULTS: Hypoxia-induced CA IX protein expression was seen after >6 h at < or =5% O(2), and protein was stable over 96 h of reoxygenation in both cell lines. Glucose deprivation abolished the hypoxic CA IX response, and high cell density caused CA IX induction under aerobic conditions. Measured percentages of CA IX-positive cells in mixtures closely reflected known percentages of hypoxic cells in HT 1080 and were associated with radioresistance of mixtures after 10 Gy. CONCLUSION: CA IX is a stable marker of current or previous chronic hypoxia but influenced by nonhypoxic stimuli. Except the time course of accumulation, all properties of this marker resembled our previous findings for hypoxia-inducible factor-1alpha. A modified flow cytometry protocol provided good separability of CA IX-negative and -positive cells in vitro and can be applied to sort chronically hypoxic cells from tumors.

Antigens, Neoplasm↗

Regulators of the Toll and Imd pathways in the Drosophila innate immune response.

The innate immune response is the first line of defense against microbial infections in both insects and mammals. Systematic analysis of the innate immune response in the model organism Drosophila melanogaster has provided important insights into the mechanisms of pathogen recognition and host response. Recognition of pathogen-associated molecules, such as peptidoglycans, stimulates the Toll and immune deficiency (Imd) pathways to induce antimicrobial responses. The Toll and Imd pathways are homologous to the mammalian Toll-like receptor (TLR) and tumor necrosis factor receptor (TNFR) signaling pathways, respectively, and are essential for Drosophila to survive infection. In this Review, we will discuss the recent genetic, genomic and RNA interference analyses that have unveiled additional intricacy in the Toll and Imd pathways.

Animals↗

Angiotensin-converting enzyme inhibitors in coronary artery disease and preserved left ventricular systolic function: a systematic review and meta-analysis of randomized controlled trials.

OBJECTIVES: This study sought to assess the efficacy of angiotensin-converting enzyme inhibitors (ACEIs) in patients with coronary heart disease and preserved left ventricular (LV) function. BACKGROUND: The ACEIs have been shown to improve outcomes in patients with heart failure and myocardial infarction (MI). However, there is conflicting evidence concerning the benefits of ACEIs in patients with coronary artery disease (CAD) and preserved LV systolic function. METHODS: An extensive search was performed to identify randomized, placebo-controlled trials of ACEI use in patients with CAD and preserved LV systolic function. Of 61 potentially relevant articles screened, 6 trials met the inclusion criteria. They were reviewed to determine cardiovascular mortality, nonfatal MI, all-cause mortality, and revascularization rates. We performed random-effect model meta-analyses and quantified between-studies heterogeneity with I(2). RESULTS: There were 16,772 patients randomized to ACEI and 16,728 patients randomized to placebo. Use of ACEIs was associated with a decrease in cardiovascular mortality (relative risk [RR] 0.83, 95% confidence interval [CI] 0.72 to 0.96, p = 0.01), nonfatal MI (RR 0.84, 95% CI 0.75 to 0.94, p = 0.003), all-cause mortality (RR 0.87, 95% CI 0.81 to 0.94, p = 0.0003), and revascularization rates (RR 0.93, 95% CI 0.87 to 1.00, p = 0.04). There was no significant between-studies heterogeneity. Treatment of 100 patients for an average duration of 4.4 years prevents either of the adverse outcomes (one death, or one nonfatal myocardial infarction, or one cardiovascular death or one coronary revascularization procedure). CONCLUSIONS: The cumulative evidence provided by this meta-analysis shows a modest favorable effect of ACEIs on the outcome of patients with CAD and preserved LV systolic function.

Angiotensin-Converting Enzyme Inhibitors↗

More normal than not: a qualitative assessment of the developmental experiences of gay male youth.

PURPOSE: To examine gay youth experiences within the context of normal adolescent development. METHODS: Thematic analyses of interviews with 13 self-identified gay male youth, aged 16-22 years, each reporting minimal sexual identity distress, were completed. Interviews focused on: (a) descriptions of developmental changes perceived to occur for all adolescents, (b) descriptions of the participants' developmental experience, and (c) participants' direct comparisons of their perceptions of gay and nongay developmental experience. Data were analyzed by two investigators who, after initial review of the interview transcripts, developed a unified coding template to permit systematic analysis of the transcripts for recurrent themes. RESULTS: (a) Few (2 of 13) participants reported overall developmental experience markedly different from nongay peers. (b) Peer interaction was seen as the domain most different from that of nongay peers. (c) Open gay self-identification altered, generally positively, all peer interaction. (d) Increased peer interaction enhanced maturity in other domains. (e) Family dynamics were not substantively altered by open gay self-identification. (f) Middle and high school were identified as relatively hostile environments in which to openly identify as gay, affecting the timing and the extent of self-disclosure. (g) Developmental progress showed asynchrony across developmental domains. CONCLUSION: General developmental dysfunction is not inevitable for gay adolescents, nor is identifiable personal or family pathology directly related to sexual identity.

Adaptation, Psychological↗

Education: an antidote for the spread of HIV/AIDS.

There are growing fears about the rate at which AIDS is spreading across the globe today, particularly in Africa. In 20 years, AIDS has become the greatest pandemic ever, and indications are that it will continue to increase. Because there is no universally accepted curative drug for the treatment of the epidemic, this report describes an attempt at providing education as a preventive method for the spread of the deadly disease. It is also an attempt to rise to this challenge by a systematic analysis of appropriate theories that can lead to insight into the epidemic. An empirical approach will further add to scholarly understanding and much-desired solutions. Suggestions are offered on how to achieve these goals through integration of HIV/AIDS education into school curricula, and how nursing practice can help in the education about and treatment of the disease. This report is the first part of a two-phased study from a sociologic point of view of an emergent universal health problem.

Africa↗

Importance of hepatitis C coinfection in the development of QT prolongation in HIV-infected patients.

BACKGROUND: Case reports and unblinded studies suggest that human immunodeficiency virus (HIV) disease is associated with QT prolongation and torsade de pointes ventricular tachycardia. Hepatitis C coinfection is common in patients with HIV disease, and cirrhosis is also associated with QT prolongation. We therefore undertook a systematic analysis of the role of liver injury, nutritional state, and coinfection with hepatitis C in the etiology of QT prolongation in HIV disease. METHODS: We performed a blinded, controlled retrospective cohort study of 1648 patients over a 3-year period at a university-affiliated municipal hospital. All electrocardiograms were included if patients with HIV disease had measurements of CD4 count and viral load within 3 months and serum electrolytes within 30 days (n = 816). Control subjects were chosen randomly from the general medicine service (n = 832). QT interval was measured in lead II and corrected for heart rate by Bazett's formula (QTc). RESULTS: QTc was slightly but significantly longer in patients with HIV disease than in controls (443 +/- 37 vs 436 +/- 36 milliseconds, P < .001). Patients with hepatitis C had more pronounced QTc prolongation (452 +/- 41 vs 437 +/- 35 milliseconds, P < .001). CD4 count, HIV viral load, and HIV medications had no effect on QTc. When patients with hepatitis C were excluded from the analysis, there was no statistical difference between patients with HIV disease and controls (438 +/- 34 vs 436 +/- 36 milliseconds, P = .336). Multiple linear regression revealed that both HIV and hepatitis C infection predicted QTc prolongation, as did age, female sex, history of hypertension, use of opiates, low serum K+ and albumin, and high AST. Hepatitis C coinfection nearly doubled the risk of QTc of 470 milliseconds or greater in patients with HIV disease (29.6% vs 15.8%, P < .001). CONCLUSIONS: Human immunodeficiency virus and hepatitis C infections both independently prolong QTc. Coinfection with hepatitis C greatly increases the likelihood of clinically significant QTc prolongation in patients with HIV disease.

Analysis of Variance↗

pKAKA: a protein language model for prioritizing kinase-disrupting variants in diseases.

Protein kinases are pivotal regulators of cellular signaling, and their genetic variations are frequently implicated in diseases. Although numerous kinase mutations have been identified as drivers of altered activity, with a few successfully targeted therapeutically, the functional impact of most variants remains uncharacterized. To bridge this gap, we curate a comprehensive dataset that contains 2553 experimentally validated kinase activity-related key alterations (KAKAs) from the literature. While many mutations outside canonical functional regions are known to affect kinase activity, systematic methods to predict their functional consequences are lacking. Consequently, we develop a computational method to predict potential KAKAs, leveraging transfer learning on the pre-trained protein language model ProtBert. Our model, termed pKAKA, achieves an impressive AUC score of 0.9593 and outperforms the AlphaMissense benchmark in comparative testing. Systematic analysis of kinase missense mutations underscores the critical role of KAKAs in pathogenesis, with highlights including JAK2 V617F in atherosclerotic cardiovascular disease, LRRK2 G2385R in Parkinson's disease, EGFR L858R in lung adenocarcinoma, and EGFR G598V in glioma. Overall, this study significantly advances our understanding of how mutations that influence kinase activity contribute to disease mechanisms.

Humans↗

Main-chain dominated amyloid structures demonstrated by the effect of high pressure.

It has been suggested that, while the globular native forms of proteins are a side-chain-dominated compact structure evolved by pursuing a unique fold with optimal packing of amino acid residues, amyloid fibrils are a main-chain-dominated structure with an extensive hydrogen bond network. To address this issue, the effects of hydrostatic pressure on amyloid fibrils of beta2-microglobulin (beta2-m), involved in dialysis-related amyloidosis, were studied. A systematic analysis at various pressures and concentrations of guanidine hydrochloride conducted by monitoring thioflavin T fluorescence, light-scattering, and tryptophan fluorescence revealed contrasting conformational changes occurring consecutively: first, a pressure-induced reorganization of fibrils and then a pressure-induced unfolding. The changes in volume as well as the observed structural changes indicate that the beta2-m amyloid fibrils under ambient pressure are less tightly packed with a larger number of cavities, consistent with the main-chain-dominated amyloid structure. Moreover, the amyloid structure without optimal packing will enable various isoforms to form, suggesting the structural basis of multiple forms of amyloid fibrils in contrast to the unique native-fold.

Amyloid↗

Local kinetic measures of macromolecular structure reveal partitioning among multiple parallel pathways from the earliest steps in the folding of a large RNA molecule.

At the heart of the RNA folding problem is the number, structures, and relationships among the intermediates that populate the folding pathways of most large RNA molecules. Unique insight into the structural dynamics of these intermediates can be gleaned from the time-dependent changes in local probes of macromolecular conformation (e.g. reports on individual nucleotide solvent accessibility offered by hydroxyl radical (()OH) footprinting). Local measures distributed around a macromolecule individually illuminate the ensemble of separate changes that constitute a folding reaction. Folding pathway reconstruction from a multitude of these individual measures is daunting due to the combinatorial explosion of possible kinetic models as the number of independent local measures increases. Fortunately, clustering of time progress curves sufficiently reduces the dimensionality of the data so as to make reconstruction computationally tractable. The most likely folding topology and intermediates can then be identified by exhaustively enumerating all possible kinetic models on a super-computer grid. The folding pathways and measures of the relative flux through them were determined for Mg(2+) and Na(+)-mediated folding of the Tetrahymena thermophila group I intron using this combined experimental and computational approach. The flux during Mg(2+)-mediated folding is divided among numerous parallel pathways. In contrast, the flux during the Na(+)-mediated reaction is predominantly restricted through three pathways, one of which is without detectable passage through intermediates. Under both conditions, the folding reaction is highly parallel with no single pathway accounting for more than 50% of the molecular flux. This suggests that RNA folding is non-sequential under a variety of different experimental conditions even at the earliest stages of folding. This study provides a template for the systematic analysis of the time-evolution of RNA structure from ensembles of local measures that will illuminate the chemical and physical characteristics of each step in the process. The applicability of this analysis approach to other macromolecules is discussed.

Animals↗