Targeted functional experiments, largely based on designable DNA-binding proteins, have begun to define the major architectural proteins required to establish and maintain appropriate genome regulation. Here, we focus on the accessible and well-characterized system of pluripotent cells to review the functional role of chromatin organization in regulating pluripotency, differentiation and reprogramming.Translating the vast amounts of genomic and epigenomic information accumulated on the linear genome into three-dimensional models of nuclear organization is a current major challenge. In response to this challenge, recent technological innovations based on chromosome conformation capture methods in combination with increasingly powerful functional approaches have revealed exciting insights into key aspects of genome regulation. These findings have led to an emerging model where the genome is folded and compartmentalized into highly conserved topological domains that are further divided into functional subdomains containing physical loops that bring  Three-dimensional nuclear structure is established and maintained by complex layers of regulatory information superimposed on the genome. Compartmentalization of functional chromosome domains and their correct spatial distribution within the nucleus are required for several important nuclear functions including transcription, repair and replication . In addiPluripotent cells, which consist of embryonic stem (ES) cells and their reprogrammed counterparts, induced pluripotent stem (iPS) cells, are one of the most well-characterized cellular systems to study the connection between chromatin organization and cell identity . ExtensiHere, we highlight several emerging technologies that enable the study and functional analysis of chromatin organization, with a particular focus on their application to pluripotent cells. We review how these recent findings have contributed to our understanding of genome function and discuss potential future applications of these novel approaches.Microscopy-based techniques were the first to reveal large-scale structural organization in the genome, when Emil Heitz observed differences in chromosome condensation patterns within interphasic nuclei . Subsequin situ hybridization (FISH) against major satellite repeats that occur within pericentromeric heterochromatin [More recent microscopy-based techniques have advanced our understanding of nuclear organization and how this impacts on cell regulation and function. In particular, an unusual chromatin organization has been observed in mouse ES cells and fully reprogrammed iPS cells, which may be linked to their pluripotent status. For example, visualization of chromatin with DNA-binding dyes or indirectly with antibodies against heterochromatin-binding protein HP1 or heterochromatin-associated histone modifications, such as histone H3 lysine 9 trimethylation (H3K9me3), have shown that mouse ES cells have fewer but larger heterochromatin foci , 14. Thehromatin . Furtherhromatin . Excitinhromatin .in vitro and in vivo cell differentiation [cis-regulatory elements by probing the colocalization of long-range enhancers and their target genes [HoxB or uPA) can loop out of their chromosome territories (CT) on gene activation [FISH studies have further revealed sub-megabase changes in chromatin condensation at specific loci that occur on ntiation . Functioet genes . FISH hativation , 23. Inttivation . Despitetivation   are known architectural proteins that promote and stabilize chromatin looping between distal regulatory elements, including enhancers and promoters , 55. TheNanog and Oct4 promoters [Nanog locus [Key pluripotent transcription factors have also been implicated in chromatin looping in ES and iPS cells. Depletion in ES cells of transcription factors OCT4, KLF4 or SOX2 leads to the transcriptional down-regulation of genes associated with super-enhancers, disturbs enhancer–promoter loops and promotes differentiation , 63–65. romoters , 66, andromoters , 67, 68.og locus .Nanog-promoter is re-established during iPS cell reprogramming, and this is dependent on transcription factor and architectural protein occupancy [OCT4, SOX2 and NANOG loci is specific to iPS cells and correlated with complete reprogramming and active transcription of these endogenous loci [Chromatin loops are also associated with changes in chromatin organization that occur on cell differentiation and reprogramming. For example, differentiated cells have distinct interactomes at several key loci when compared with their originating ES cells , suggestccupancy . During ous loci . Furtherous loci , 70. Theous loci .Supporting a model where cell-identity-specific transcription factors play a dominant role in chromatin organization, a study applied ChIA-PET and chromatin immunoprecipitation to identify RNA polymerase II-mediated chromatin interactions in ES cells and B-cells and found a correlation for cell-specific transcription factors in the establishment of chromatin structure . AstoundExperiments aimed at determining the causative role of genome organization in controlling nuclear function and gene regulation have been challenging because many of the available approaches result in genome-wide perturbation, and can therefore lead to difficulties in distinguishing between cause and consequence. Nevertheless, as described above, many landmark studies have successfully used gain and loss of function studies to define the major factors required for genome organization in stem cells and development including architectural proteins, chromatin regulators and pluripotency transcription factors . AlthougThe new classes of DNA-binding factors include proteins within the zinc-finger family, transcription activator-like effectors (TALE) and clustered regularly interspaced short palindromic repeats (CRISPR). All three protein classes have important and diverse biological roles, but their uptake as sequence-specific genome engineering tools has been driven by rapid technological developments that have repurposed their biological function . DNA-binA recent methodological advance is the design of systems that enable live imaging of nuclear organization and sub-nuclear compartments in individual cells, which is attractive for studying dynamic processes such as the cell cycle and chromatin structure. Endogenous repetitive genomic sequences, including centromeres, telomeres and pericentric heterochromatin, can be visualized through sequence-specific TALEs fused to monomeric green fluorescent protein (GFP) A 83, 8484. When A related approach using an optimized CRISPR/Cas system to visualize specific DNA loci in live cells was recently described , 87. HerThe TALE and Cas9 approaches open up an exciting new area of cell biology, enabling real-time experiments to track the movement of chromatin and specific DNA regions, as well as imaging through cell division and other cell processes. The technology is remarkably flexible and customizable, ensuring their wide adoption and use in the field. Several technical challenges remain. One is that the targeting of the TALE and Cas9 proteins must be carefully designed to minimize the likelihood of off-target effects caused by transcriptional changes and/or blocking access of other DNA-binding elements to their regulatory sequences. A second challenge is that some background signal was detected in the TALE and Cas9 studies, presumably owing to unbound TALE-GFP or dCas9-GFP proteins. Further technical developments are therefore required to improve the signal to noise ratio, and this should also lead to improvements in the ability to visualize non-repetitive sequences.As described earlier, the position of gene loci within the nucleus is correlated with transcriptional regulation, with localization at the nuclear periphery associated with transcriptional down-regulation , 88–90. cis-regulatory region, in line with the conclusion that the CTCF sites can form boundaries that insulate genes in one neighbourhood from another [Hox clusters in mouse ES cells [Hox genes on cell differentiation. These results reveal that CTCF helps establish functional domains that control developmental gene regulation. Our understanding of chromosome boundaries is still far from complete, however, as it is not possible to predict which genes will be transcriptionally altered on loss of boundary and other structural elements. Nevertheless, the combination of DNA interaction methods and functional analysis of target regions will ensure exciting progress towards these goals.Gene loops can be identified from genome-wide DNA proximity ligation assays and are often anchored at either end by architectural components such as mediator, cohesin and CTCF , 59–61.  another . A recenES cells . Excitincis-regulatory elements using chromatin signatures, 3C and functional approaches is now well established [Identification of ablished . One emecis-regulatory elements  with the predicted decrease in histone modifications associated with active enhancers. For several targets , transcription of a nearby gene was also altered, suggesting that regulatory function was perturbed. This is an exciting first step in using epigenomic engineering to alter cis-regulatory element function, and further refinements to the technology, such as combinatorial approaches, will provide a powerful platform to push this work forward.In addition to enhancer deletions, another elegant way to test the function of enhancer . The autThe combination of microscopy, 3C technologies and functional approaches has tremendously advanced our knowledge of genome organization. Identification of the general principles that govern chromatin compartmentalization and physical interactions between regulatory elements have led to a better understanding of cell identity and potentially mechanisms to control cell state changes during pluripotent cell differentiation and reprogramming.Despite the exciting progress in this area, several methodological challenges remain to be overcome. First, because interaction data sets are created from static populations of cells, they represent only the topological tendency of the population. Nagano and colleagues have provided the first attempt to bypass this population-biased limitation by developing a single-cell Hi-C technique , and furIn summary, methods to study and perturb genome organization are enabling exciting new insights into nuclear function. Progress in this area will better define how chromosomes are compartmentalized into functional domains, and how physical interactions between regulatory sequences are formed and regulated, which together will further our understanding of the relationship between three-dimensional organization and genome function.Key PointsThree-dimensional nuclear organization contributes to genome folding, chromosome compartmentalization and the formation of gene regulatory interactions, ensuring appropriate genome function.Recent technological innovations including chromosome conformation capture approaches have revealed the presence of major structural and functional domains within the genome.Architectural proteins and master transcription factors coordinate aspects of genome organization to ensure transcriptional states and appropriate cell identity.Designable DNA-binding proteins have been repurposed to enable the visualization and targeted perturbation of genome organization, thereby providing new functional insights.


Both phenomena are characterized by a regulated suppression of metabolic rate, a slowing of many physiological processes , although considerable work has also been done on bats, some small marsupials and a few others Tb values, such as typically occurring during rodent hibernation, actually necessary for the preservation applications envisioned for humans or their isolated organs? The latter question is actually relevant in that some recent studies are showing that warm perfusion of excised human organs during transfer from donor to recipient is an effective and potentially less injurious option than the cold ischemia (packing in ice) that has been the standard for many years Long-standing models of torpor and hibernation are mainly rodents (Microcebus murinus and M. griseorufus) and the dwarf lemurs  We believe that the ultimate model of relevance to human biomedical concerns would be a primate species that exhibits natural torpor/hibernation. Such species exist; studies in recent years have documented both daily torpor and seasonal hibernation among several Afrotropical mammals M. murinus)  , a smallM. murinus has been maintained in Brunoy, France for more than 40 years and the animals have been used as a non-human primate model for many types of studies. In particular, as a relatively long-lived species (for its body mass), mouse lemurs are extensively studied as a primate model to understand the aging process, age-associated pathologies , and factors that could help to delay aging, such as calorie restriction A large breeding colony of e.g., hibernation in cold climates, estivation in hot arid environments, anoxic tolerance, and life in a frozen state. As a result, we have identified conserved metabolic mechanisms that are used across the animal kingdom to coordinate and regulate metabolic rate depression, reprioritize ATP expenditures to conserve energy, stabilize macromolecules, and maximally extend viability in the hypometabolic state.Research in my laboratory centers on hypometabolism with a major focus on elucidating and exploring the biochemical mechanisms that regulate metabolic rate depression, including studies of gene and protein expression, enzyme regulation, posttranslational modification, signal transduction, microRNA control of mRNA transcripts, antioxidant defenses, chaperones, and intermediary energy metabolism e.g., insulin signaling, AMP-dependent protein kinase (AMPK), glycogen synthase kinase 3 (GSK3), and mitogen-activated protein kinase (MAPK) pathways], coordinated regulation of multiple enzymes and proteins via reversible protein phosphorylation to suppress both anabolic functions  and energy-expensive activities , as well as selective gene/protein expression to produce cytoprotective proteins . From this, we have designed what we call a “toolkit” approach to evaluate the crucial molecular signatures of hypometabolism e.g., 96-well microplates) that permit rapid, consistent, and sensitive analyses allowing multiple tissues and multiple experimental conditions to be compared on a single plate using technologies of PCR, ELISA, or Luminex. These technologies are particularly well-suited for making maximal use of very small tissue samples.Based on these wide-ranging studies of metabolic regulation in diverse systems of hypometabolism, we have been able to discern the central features and regulatory requirements of hypometabolism across the animal kingdom. These include factors such as altered patterns of intracellular signaling by protein kinase cascades [In the group of papers that follows, we put our toolkit of metabolic markers and our multiplex assay methods to use for a broad-based and comprehensive analysis of the biochemical responses to daily torpor in six organs of gray mouse lemurs. The studies include analysis of torpor-responsive changes in cell signaling pathways  KBS wrote and edited this manuscript.The author has declared no competing interests.


Dental caries is the most common chronic disease of adult and childhood, a largely preventable yet widespread, costly public health problem. This study identified patient-, organization-, and system-level factors influencing routine delivery of recommended care for prevention and management of caries in primary dental care.A convergent mixed-methods design assessed six guidance-recommended behaviours to prevent and manage caries . A diagnostic questionnaire assessing current practice, beliefs, and practice characteristics was sent to a random sample of 651 dentists in National Health Service (NHS) Scotland. Eight in-depth case studies comprising observation of routine dental visits and dental team member interviews were conducted. Patient feedback was collected from adult patients with recent checkups at case study practices. Key informant interviews were conducted with decision makers in policy, funding, education, and regulation. The Theoretical Domains Framework within the Behaviour Change Wheel was used to identify and describe patient-, organization-, and system-level barriers and facilitators to care. Findings were merged into a matrix describing theoretical domains salient to each behaviour. The matrix and Behaviour Change Wheel were used to prioritize behaviours for change and plan relevant intervention strategies.N-196), case studies , and patient feedback (N = 19). Using the study matrix, key stakeholders identified priority behaviours  and strategies  to improve guidance implementation. Proposed strategies were assessed as appropriate for immediate implementation and suitable for development with remaining behaviours.Theoretical domains associated with best practice were identified from the questionnaire  contains supplementary material, which is available to authorized users. Despite being highly preventable, dental caries is a widespread and costly public health problem . GloballPopulation- and clinical-level initiatives have been developed to promote oral health in Scotland. National guidance on oral health assessment in adults  and cariGuidance implementation relies on strategies that consider the needs and experiences of users across multiple levels, the context and culture of care, and applicable, practical change strategies tailored to relevant barriers and facilitators –25. To fThe Behaviour Change Wheel was selected as the overarching theoretical framework for this study given the wheel's direct path from behavioural assessment to selection and development of theory-based intervention strategies , 28 and This study used a convergent mixed-methods design –34 incorCaries prevention and management encompasses a range of behaviours, and prioritization was necessary to target our assessment. Behaviour Change Wheel and TDF protocols recommend balancing the number of behaviours assessed to improve targeting and specificity of interventions and so their likelihood of success . A priorN = 501) were selected from a publicly available list published by Practitioner Services Division of NHS National Services for Scotland [N = 150) were selected from Information Services Division of NHS National Services for Scotland database of Childsmile demonstration practices. Sample size was calculated based on a standard response rate of 50 % as in previous questionnaires conducted by PMC study team members [The initial study sample included 651 dentists in general dental practice providing care through NHS Scotland; 150 of whom participated in Childsmile prior to October 2011 when a fee for fluoride varnish was introduced and Childsmile became a universal programme. Dentists providing Childsmile before October 2011 were oversampled to enable comparison of recommended PMC by early participation in the initial phase of the programme. Using computer-generated random numbers at the practice and dentist level, general dental practitioners  was identified from questionnaire respondents. Case study practices were selected from the highest and lowest performing quartiles of self-reported guidance-recommended practice. Selection criteria also included Childsmile enrolment, service division , and Scottish Index of Multiple Deprivation (SIMD) quintile ranking as characteristics known to influence caries burden and preventive care delivery [A purposive, theoretical sample of case study practices  were identified by the PMC study team and TRiaDS methodology group for their expertise, involvement, or leadership in PMC and preventive oral health. Informants represented patient, practice, and policy interests at the system  level. The sample was small as informants were being asked to provide general PMC feedback, not detailed, behaviour-specific assessment as in the diagnostic questionnaire or case studies.System-level informants , mixed, and adult dentition types. The timing of routine visits was not specified as intervals should be risk-based and therefore variable , 17.nature of the behaviour was eliminated a priori consistent with other quantitative applications of the TDF [not reasonably expected to be associated with each behaviour were eliminated. For universal behaviours , all prioritized domains   as a comN = 5) of dental professionals and oral health researchers.The questionnaire , only adult exams were observed. A structured observation guide based on the TDF and CFIR was developed.N = 8) and up to three other dental team members  (N = 23) assessed guidance use and barriers and facilitators to recommended PMC. Questions were not domain specific in order to allow domains excluded from the questionnaire to emerge and to elicit more contextual detail. Interview schedules were mapped to the TDF to ensure coverage of all domains. The interview schedule was reviewed for comprehension and face validity by the same convenience sample (N = 5) as the questionnaire.Semi-structured interviews with respondent dentists (Patient feedback was planned through a series of focus groups embedded in four case study practices. The focus group schedule emphasized patient experiences of PMC (nature of the behaviour), barriers and facilitator to PMC (beliefs about consequences), and patient expectations . As focus group attendance was low, all practices were invited to provide patient feedback through structured telephone interviews. Interviews were based on the focus group schedule, emphasizing the same domains but using a less open-ended approach to reduce time required for participation. Patient representatives from the study management team provided in-depth feedback and guidance in recruitment and conduct of patient focus groups and interviews. Patients were provided a £10 voucher in appreciation of their participation.Semi-structured interviews were conducted with key informants in policy, service delivery, and service development. Informants were identified by the study team based on their expertise and involvement in oral health policy and delivery systems. Interview schedules were developed using the COM-B and intervention and policy categories in the Behaviour Change Wheel . Intervip ≤ 0.05) variables from univariate analyses were entered into a logistic regression by best/not best practice and dentition type. A forward stepwise (Likelihood ratio) method was used to manage co-linearity issues with TDF scales as sample sizes were restricted to respondents in each questionnaire version.Reliability analyses  of TDF domains assessed for each behaviour were conducted. In domain items with alpha >0.60, items were combined into single scales. Responses were analysed using simple descriptive statistics, univariate analyses, and logistic regression in SPSS 22. Comparisons and regressions were conducted for primary, mixed, and adult dentition types based on self-reported current practice. For each of the six behaviours, TDF domain scales and demographic data  were compared by self-reported provision of guidance-recommended care. Significant  as barriers or facilitators to recommended care. Responses were coded into themes by policy, system, and intervention characteristics in the Behaviour Change Wheel and then mapped to the COM-B to enable later integration with questionnaire and case study data. Data were managed in Excel.oral health advice was added as an additional behaviour to the matrix. Third, associated domains in the matrix were prioritized into salient domains based on critical appraisal of frequency within and across study components, degree to which respondents and components agreed or disagreed about the domain, and evidence of strong beliefs impacting the behaviour [A day-long workshop with study team members was held to integrate findings from individual study components, identify associated domains (i.e. any domain assessed as influencing behaviour), and appraise salient domains . Study team members familiarized themselves with findings using questionnaire, case study, patient feedback, and system-level informant interview summary reports. Reports included methods, in-depth analyses, summary findings of behaviour-specific beliefs, associated domains, and domain function as a barrier or facilitator. The workshop followed a three-step process. First, associated domains from each study component were reviewed individually for clarification, discussion of unexpected findings, and consideration of differences between study components. Second, associated domains were entered into a matrix describing barriers and facilitators across study components for the six key behaviours. Based on emergent themes around information exchange and advice from the case studies  and patient feedback , ehaviour –46.Salient domains in the finalized matrix were mapped to COM-B categories, theoretically relevant intervention strategies in the Behaviour Change Wheel, and associated behaviour change techniques. Behaviour change techniques were defined using the Behaviour Change Technique Taxonomy, version 1 , 47.N = 9) comprised patient-, practice-, and policy-level representatives. Stakeholders familiarized themselves with study findings through brief behavioural summaries (including pertinent quotes and routine data where available) and the barrier/facilitator matrix. Intervention planning followed a three-step nominal group technique [Key stakeholders met in a half-day workshop to review findings, intervention strategies, and behaviour change techniques and prioritize approaches to improve preventive care delivery in primary dental care. Stakeholders , 59 declined taking part, and 323 questionnaires were not returned. A total of 196 dentists responded (34 % response rate). Response rates did not differ between the three belief assessment sections. Responders and non-responders did not differ by health board, SIMD, or Childsmile enrolment before October 2011.Individual respondent characteristics are summarized in Table Case study practices were selected by best practice (highest quartile for guidance-recommended care delivery) and not best practice (lowest quartile). Two practices were Childsmile practices before October 2011; one practice did not identify as a Childsmile practice. All practices were in the General Dental Service with at least 60 % of patients seen on NHS. Practices spanned all SIMD quintiles; five were SIMD 3 or below. Twenty-nine team members were interviewed across the eight practices. In each case, interviewees included the dentist who completed the questionnaire and one dental nurse. Other team members interviewed included additional dentists and dental nurses, three practice managers, two extended-duty nurses, two vocational trainees, one receptionist, and one hygienist.Of the 19 patients who took part, most (73.7 %) were female, 30–50 years old (52.6 %). Just under half (47.4 %) had children under the age of 16. Patients attended seven of the eight case study practices.N = 4) included representation from the Scottish Government, NHS Education for Scotland, British Dental Association, dental public health, Childsmile, guidance development, general and salaried dental services, patient representatives, and general dental practitioners.Key informants , prevention as integral but underutilized skill set in their role , and a desire to provide more preventive care. Respondents had positive attitudes toward guidance but emphasized guidance as often too long, complicated, and not universally applicable or practical.Across cases, all team members thought preventive care is important, benefits patient oral health, and is part of every team member’s role. Within individual cases, dental teams reflected the dentist’s beliefs about barriers and facilitators to PMC behaviours . Team members’ beliefs were more divergent on the emergent theme of Barriers and facilitators to PMC behaviours and general preventive care differed little between high- and low-performing practices. Low-performing practices tended to emphasize barriers such as patient expectations , lack of time , and few perceived benefits (beliefs about consequences) and did not identify preventive oral health as strongly within their roles . The same barriers were identified to a lesser extent by high-performing practices. Few domains acted exclusively as a facilitator or barrier to PMC behaviours. Most domains  functioned simultaneously as barriers (e.g. parents refuse fluoride varnish) and facilitators (parents request fluoride varnish) to PMC behaviours. Duality applied across dentition types, even when some feature of the domain (e.g. a child versus an adult patient) changed. Given the consistency of domains influencing behaviour in both high- and low-performing practices and often dual function of those domains as barriers and facilitators, associated domains were described across all cases rather than by best practice/not best practice. Domains associated with PMC behaviours and general preventive care are summarized in Additional file oral health advice and oral health management themes. Associated domains are summarized in Additional file Patients identified the most important factors for preventive oral health as tooth brushing and cleaning for adults and supervising tooth brushing and limiting sugar intake for children. The discussion of barriers and facilitators to PMC was framed by patient’s experiences, including self-care , advice and information from their dental team, and other sources of information (e.g. media). For both adults and children, patients identified multiple long-term benefits of preventive oral health care but were unsure about the efficacy of their self-care techniques, were anxious about dental appointments, and struggled with care (particularly brushing) of children’s teeth. As few patients had experienced hands-on demonstrations of oral health maintenance, few expected this type of instruction but nearly all of them anticipated it as highly beneficial and were eager to receive such care. Patient experiences and expectations centred primarily on informational exchange and were categorized in emergent System-level informants emphasized improvements in preventive oral health made in recent years and were optimistic about narrowing the gap between patients in high- and low-deprivation areas. Persistent concern about caries, PMC, and disproportionate burden of disease among higher deprivation populations was common. Gains in PMC were attributed to community-based preventive care programmes (particularly in primary schools) and increased delivery of oral health advice in dental practices. Eighteen themes emerged as barriers, facilitators, or both  were common across behaviours. Available behaviour change techniques were identified based on theoretically relevant intervention strategies, available evidence summarizing effective behaviour change techniques [Domains from the conceptual framework were mapped through the COM-B Fig.  to interchniques , the Behchniques , 47, andStakeholders reviewed the study matrix and resolved that while guidance recommendations should be based on dentition type, interventions should be developed by patient age group given current service delivery and remuneration structures. Using these parameters, stakeholders prioritized the 6–12-year-old age group  for initial development of interventions to support improved PMC practice. Stakeholders next prioritized study behaviours for 6–12-year-olds using established criteria of appropriateness, relevance, feasibility, and potential impact –51. CritIn the final stage of the workshop, theoretically relevant intervention strategies to improve fissure sealant placement were discussed. Two interventions were recommended for future implementation: audit and feedback at the provider level  and a preventive oral health awareness campaign at the patient level (enablement). In APEASE appraisal, equity did not score well as interventions were practice based and patients who rarely or never attend the dentist would not benefit. However, given other criteria and continuing increases in registration rates, stakeholders were unanimous that both interventions be developed for implementation. Behaviour change techniques agreed for the audit and feedback intervention were action planning, feedback on behaviour and outcomes, goal setting, problem solving, review of goals and outcomes, and self-monitoring. The primary technique in the patient intervention would be social support to encourage patients (parents) to discuss fissure sealants and fluoride varnish with dental care providers and enable patient requests for preventive care or additional oral health advice.The mixed-methods approach identified well-defined barriers and facilitators to preventive care; consistent application of the theoretical framework through each component to data integration and intervention planning resulted in specific, targeted interventions suitable for implementation.The questionnaire quantified beliefs of dentists following best practice versus not best practice and identified high- and low-performing practices for case study selection. At the time of writing the questionnaire, the TDF had only been applied in two quantitative studies , 41 and Consistent with case study methodology , 55, casInclusion of patient feedback emphasized differences between patient and dentist expectations of preventive care and identified patients’ social influence as an intervention approach to improving practitioner behaviour. This finding in particular highlights the potential benefits of developing interventions to encourage and empower patients to influence the delivery and content of preventive care.System-level informant interviews provided a broad policy context which helped identify salient domains and integrate intervention planning with ongoing initiatives including PMC guidance, audit, and service development. While the number of interviews was limited, interventions in all three categories of the COM-B were clearly identified. Improvement strategies emerging from the interviews (particularly audit) were consistent with those discussed in the intervention planning workshop.Application of the Behaviour Change Wheel as our single theoretical framework provided a common language across study component findings and a logical structure for data integration and intervention planning. Component findings converged clearly, and qualitative findings emphasized and explain barriers and facilitators within each domain. Using the wheel, salient domains were easily mapped to appropriate, evidence-based intervention techniques framed in policy terms familiar to stakeholders. The practical, direct path from barriers and facilitators to interventions greatly helped to parse the diverse data collected and identify specific techniques to improve care.While six target behaviours were prioritized from recommended practices, determinants varied by patient dentition types . This complexity necessitated design adaptations including belief assessment of only two behaviours per questionnaire respondent and discussion of preventive care in general, using the six behaviours as specific examples or instances, in the case studies. These were practical compromises for initial identification of barrier and facilitator PMC in general dental practice; in the future, we would recommend focusing on a narrower range of behaviours or a single patient age group to improve comparability of findingsThe response rate in our questionnaire (34 %) was lower than the anticipated 50 %. Efforts to improve the response rate  helped produce a further 69 responses after the second reminder but did not achieve the desired rate. Responses were sufficient for statistical analyses and varied enough to identify case study practices but make the sample more vulnerable to self-selection bias.Assessing PMC practice by self-report revealed higher than expected rates of recommended care delivery given nationally reported rates of fluoride varnish application to 23 % of 2–5-year-olds  and prevThe Behaviour Change Wheel helped structure our study but presented some challenges and limitations in application. First, operationalizing the framework within a multi-disciplinary study team, particularly familiarization with the TDF and COM-B model, took concentrated effort and structured communication among team members. Discriminating between TDF domains necessitated the development of clear guidelines for team members conducting analysis and interpretation of data. Second, once salient domains were mapped to relevant intervention strategies, many of the interventions and behaviour change techniques overlapped raising the question whether detailed assessment of TDF or even COM-B categories was necessary. Reflecting upon this with the TRiaDS group, the study team generally felt that while the interventions were not discrete to individual behaviours, the theoretical grounding of interventions and potential to adapt interventions to multiple behaviours outweighed crossover of intervention strategies within the framework. Third, organizational characteristics influencing PMC practice did not emerge as strongly as expected. While efforts to improve organizational assessment through the CFIR were made, the depth in which organizational characteristics were evaluated was practically limited by the number of behavioural domains assessed. Mapping the CFIR to the TDF helped remediate this issue and identified several specific characteristics  common to practices delivering recommended care. Focus on fewer behaviours may have enabled a more detailed assessment of organizational climate and culture and identification of practice-level influences on PMC. However, practical compromises were necessary to provide sufficient breadth in this initial assessment of PMC and it is possible that organizational factors were not as influential as expected.Key stakeholders and study team members coproduced specific, theoretically based, testable interventions representative of interests within and across patient, practice, and policy groups. Interventions have been favourably appraised by all stakeholders as implementation ready, complementary to ongoing national guidance and professional development initiatives, and likely to improve translation of recommended care into routine primary practice—reducing both the incidence and prevalence of caries in children and adults. Across mixed-methods components, our findings emphasize the duality of domains as barriers and facilitators, the strength of patient influence on practitioner behaviour, and the benefit of integrating multiple levels of interest when planning interventions in a dynamic yet resource-constrained preventive care delivery system. The development of simultaneous patient- and practice-focussed implementation strategies to improve preventive services in primary care is highly relevant to translational research across health services.Findings are actively being used to review, update, and develop implementation tools accompanying the revised Scottish Dental Clinical Effectiveness guidance on child caries to be published in 2016. In collaboration with NHS Education for Scotland and Childsmile, TRiaDS has developed and piloted pre-approved national audits for fluoride varnish and fissure sealants which will be available with publication of the SDCEP guidance. Future plans include testing the use of audit and feedback interventions for preventive care  and a practice-based campaign to promote patient awareness, support, and requests for PMC.Additional file 1:Prioritized domains included in diagnostic questionnaire. TDF domains assessed for target behaviours.Additional file 2:Diagnostic questionnaire. Assessment of current practice, beliefs, and demographic information.Additional file 3:Case study and patient feedback data collection tools. Qualitative data collection tools.Additional file 4:Dental team member interview and patient feedback coding guides. Qualitative TDF coding guides.Additional file 5:Associated TDF domains identified in individual study components.Additional file 6:Salient domains matrix. Annotated domains influencing targeted PMC behaviours.


As with in vivo toxicity testing, an important question for in vitro results is whether there are thresholds for adverse cellular responses. Empirical evaluations may show consistency with thresholds, but the main evidence has to come from mechanistic considerations.Background: Increasingly, there is a move toward using in vitro testing data.Objectives: Cellular response behaviors depend on the molecular pathway and circuitry in the cell and the manner in which chemicals perturb these circuits. Understanding circuit structures that are inherently capable of resisting small perturbations and producing threshold responses is an important step towards mechanistically interpreting Methods: Here we have examined dose–response characteristics for several biochemical network motifs. These network motifs are basic building blocks of molecular circuits underpinning a variety of cellular functions, including adaptation, homeostasis, proliferation, differentiation, and apoptosis. For each motif, we present biological examples and models to illustrate how thresholds arise from specific network structures.in vitro cellular assays.Discussion and Conclusion: Integral feedback, feedforward, and transcritical bifurcation motifs can generate thresholds. Other motifs produce responses where the slope in the low-dose region is small and stays close to the baseline. Feedforward control may lead to nonmonotonic or hormetic responses. We conclude that network motifs provide a basis for understanding thresholds for cellular responses. Computational pathway modeling of these motifs and their combinations occurring in molecular signaling networks will be a key element in new risk assessment approaches based on http://dx.doi.org/10.1289/ehp.1408244Citation: Zhang Q, Bhattacharya S, Conolly RB, Clewell HJ III, Kaminski NE, Andersen ME. 2014. Molecular signaling network motifs provide a mechanistic basis for cellular threshold responses. Environ Health Perspect 122:1261–1270;  Quantitative human health risk assessment for environmental toxicants requires accurate dose–response information on relevant end points. Dose–response studies using animals, tissues, and cells can examine diverse end points. However, scientists in the toxicology and risk assessment community are still at odds over the issue of whether threshold doses exist for adverse responses . The debin vivo and in vitro toxicity data  monotonically increasing but staying close to the control baseline (b) superimposed on the baseline, representing a true threshold response (c) nonmonotonic [i.e., first decreasing then increasing  superesponse ; or c) nAlthough empirical dose–response curves are essential for examining low-dose behaviors, optimally, evaluation of the evidence for thresholds also needs to take into account the primary biological mechanisms governing both control and treated responses . To do tin vitro cellular systems based on perturbations of toxicity pathways. Toxicity pathways—preexisting protein and gene networks that, when sufficiently perturbed by chemicals, can lead to adverse health outcomes—can be examined in great detail with modern high-throughput molecular techniques. These toxicity pathways link molecular initiating events to modes of action and adverse outcome pathways  pathways. These motifs are more than theoretical concepts. They are ubiquitous in all biological signaling and control systems, and they are necessary for the integrity and normal functioning of cells in response to physiological signals and in the face of environmental fluctuations. These motifs form the basis of the circuits underpinning integrated cellular functions, including adaptation, homeostasis, proliferation, differentiation, and apoptosis . In thisHuman intuition for thresholds comes primarily from our everyday experience with the surrounding physical world . In contrast to mechanical thresholds, the manner in which thresholds arise for the molecular signaling pathways and networks that underlie cellular responses is less intuitive. According to mass action, the strength of initial interaction between a chemical and its direct molecular target is proportional to the free concentrations of the reactants. A suite of subsequent biochemical processes can transform these linear, molecular initiating events into responses that may or may not have thresholds.Increasingly, biologists and biomedical engineers regard biological responses in terms of systems-level behaviors of dynamic networks. In this section, we discuss network motifs that generate dose–response profiles similar to those shown in One of the most vital properties of biological organisms is homeostasis . Homeostasis takes place over various levels of biological hierarchy: in cells, tissues, organs, and the whole organism. Adaptation and homeostasis are the mechanisms most frequently cited in the literature in support of thresholds in biological organisms . Below wNegative feedback control. In negative feedback, an undesirable change brought about by a stressor functions as a signal that works to restore the system to its original condition. Negative feedback loops are frequently encountered in molecular circuits underlying signal attenuation, metabolic control, and most relevant to toxicologists, maintenance of cellular homeostasis (2), DNA damage, protein folding, metal ions, or osmolarity  is proportional to the error signal . In a biological setting, such a definition can be sometimes confusing because Y may activate T or G in a nonlinear manner . Regardl2 occurs through multistep signaling (protein stabilization and transactivation) and molecular titration . Bistable switching allows cells to undergo discontinuous, sometimes irreversible, changes in phenotypic state. Such switches drive integrated cellular functions such as cell cycle progression, metabolic switching, lineage specification, and differentiation (ntiation . The netntiation  consistsntiation . Here G1ntiation . This bentiation . The thrp-dioxin) appears to disrupt the bistable switch triggered by antigens, leading to all-or-none suppression of B-cell differentiation  activation of the extrinsic apoptosis pathway by tumor necrosis factor (b) the phase transition between proliferation and extinction of RNA virus in response to variations in RNA proliferation mode and RNA strand degradation (c) liver damage induced by HIV infection (d) switching of excitability of neurons expressing both restorative and regenerative ion channels (e) the transition from the quiescent to persistent firing states in neuronal networks when the network connectivity exceeds a threshold level  the radation ; c) livenfection ; d) switchannels ; and e) ld level . EnvironUltrasensitivity is a common type of nonlinear signal processing in molecular signaling networks where a small fractional change in the input generates a much larger fractional change in the output. These ultrasensitive motifs often produce sigmoidal dose–response curves , 1984. AXenopus oocytes, also exhibited an ultrasensitive response  hydroxylates HIF-1α but also has a range of ankyrin-repeat domain (ARD)-containing proteins as substrate . The molresponse .Dose–response relationships are at the core of quantitative toxicology and chemical risk assessment. Determining the shape of dose–response curves in the low-dose region to assess evidence for thresholds based on statistical analyses has proven difficult experimentally and unattainable theoretically. Due to biological and experimental variability, a statistical threshold does not necessarily indicate the existence of a real biological threshold, nor does a real biological threshold necessarily lead to an observable statistical threshold. Mechanistic knowledge of relevant biological networks and toxicity pathways perturbed by chemicals of health concern, as we show here, will be increasingly important in providing the biological underpinning for threshold responses.Threshold effects can arise from simple network structures, commonly referred to as network motifs. Here, we have reviewed a number of network motifs that can underlie biological threshold responses at the cellular level. We have also described simple mathematical models for the motifs and examined their dose–response behaviors  describe the processes from molecular initiating events through toxicity pathways and cellular and organ responses, resulting in apical responses in exposed individuals . MoleculStress pathway function appears to involve both rapid, posttranslational signaling for smaller, transient deviations from basal function and slower activation of transcriptional responses for more persistent, higher-level stresses . The losPutting knowledge of network motifs into practical use for low-dose extrapolations requires an understanding of the primary network structure of the toxicity pathway affected by the chemical stressor. Some quantitative measures of pathway biomarkers  may be needed to determine the dominant interactions in specific feedback or feedforward motifs. With this information in hand, the structure of the primary network would then support inferences about expected dose–response behaviors, including thresholds. Additional experimental data might be needed to differentiate possibilities for specific variations of some of the motifs, for example, in distinguishing between proportional and integral feedback controls or among feedforward controls with different signaling strengths in the feedforward arm. Many cellular signaling pathways utilize combinations of motifs. For example, in stress-response pathways, rapid, robust adaptation often arises by coupling negative feedback and incoherent feedforward loops . In thesAlthough the threshold motifs presented here primarily function at the level of intracellular molecular networks, the overall concepts will also be applicable to higher-level biological hierarchies in which cells, tissues, and organs are the interacting components, which are connected by paracrine, endocrine, and neural signals into large-scale biological networks. A clear understanding of the network motif context in which biological responses are examined will help in predicting the shape of the responses in low-dose regions. Nevertheless, biological variability existing among individuals  complicates estimation of the shape of dose–response curves and the specific doses associated with thresholds. These variations will result in heterogeneous responses to identical chemical perturbations among individuals. In theory, if each individual has a non-zero, different threshold for a particular stressor, then the population-averaged response should exhibit a threshold comparable to the lowest individual threshold values. The robust nature of biological systems can ensure that healthy individuals respond in a qualitatively similar fashion despite large parameter variations in the underlying components . Howeverin vitro test results for conducting risk and safety assessments  PDFClick here for additional data file.


Stroke remains a leading cause of disability worldwide, with a majority of survivors experiencing long term decrements in motor function that severely undermine quality of life. While many treatment approaches and adjunctive strategies exist to remediate motor impairment, many are only efficacious or feasible for survivors with active hand and wrist function, a population who constitute only a minority of stroke survivors. Transcranial direct current stimulation (tDCS), a type of non-invasive brain stimulation, has been increasingly utilized to increase motor function following stroke as it is able to be used with stroke survivors of varying impairment levels, is portable, is relatively inexpensive and has few side effects and contraindications. Accordingly, in recent years the number of studies investigating its efficacy when utilized as an adjunct to motor rehabilitation regimens has drastically increased. While many of these trials have reported positive and promising efficacy, methodologies vary greatly between studies, including differences in stimulation parameters, outcome measures and the nature of physical practice. As such, an urgent need remains, centering on the need to investigate these methodological differences and synthesize the most current evidence surrounding the application of tDCS for post-stroke motor rehabilitation. Accordingly, the purpose of this paper is to provide a detailed overview of the most recent tDCS literature (published 2014-2015), while highlighting these variations in methodological approach, as well to elucidate the mechanisms associated with tDCS and post-stroke motor re-learning and neuroplasticity. Many stroke survivors exhibit hemiparesis, which undermines independence and quality of life , with an emphasis on methodological approach, as well a description of the mechanisms associated with tDCS and post-stroke motor re-learning and neuroplasticity.tDCS involves the application of a low-intensity direct current (DC) , typically delivered through two electrodes situated inside saline-soaked sponges. These currents can promote subthreshold depolarization or hyperpolarization of underlying tissue using anodal or cathodal approaches, respectively. This subthreshold modulation is achieved through electrode placement based on EEG mapped sites, with current flowing in from the anodal electrode and out via the cathodal electrode. Despite only a portion of the current actually penetrating the scalp into the cortex, tDCS induces cortical excitability changes that outlast the stimulus period  and calcium (Ca2+) ion channels, NMDA receptors and Gamma-Aminobutyric acid (GABA)-ergic interneurons  (long-term increase in synaptic efficiency) and long-term depression (LTD) (long-term synaptic weakening)-like mechanisms are strongly implicated as the primary factors underlying learning and plasticity in the motor cortex and may explain tDCS aftereffects  reported positive results, supporting the hypothesis that tDCS may be most effective and motor recovery most pronounced when it is combined with physical practice. One exception to this finding is the results of a trial administering tDCS concurrent to robotic training  , and subject characteristics . It is likely that a number of therapeutic approaches will be effective when combined with tDCS. Nonetheless, standardizing the UE training and minimizing individual differences in the sample is necessary in future work to permit scientists to discern the most important TX ingredients. For example UE robotics enable the TX to be well-defined and consistent across subjects. Additionally, the combination of tDCS with this efficacious form of UE motor training (Lo et al., In addition to motor tasks, the integration of cognitively-loaded behavioral tasks alongside motor training has positively affected UE outcomes. For instance, a brain-computer interface with which subjects plan and execute movements while incorporating attention, pre-frontal decision-making and problem solving significantly improves UE outcomes (Kasashima-Shindo et al., In addition to modulating TX, methods for harmonizing the targeting of cortical areas are currently under investigation. For example, pathophysiology studies suggest that current logic for altering excitability in stroke survivors may be over-simplified, with a need to prescribe stimulation according to individualized neurophysiological or neuroanatomical factors (Stinear et al., tDCS constitutes a promising adjunctive tool to increase post-strike UE motor function. More research is needed to refine variables such as targeting, dosing, individualized pathophysiological bases, optimal training, and optimal timing of training, which may account for subject response variability. Elucidation of these variables could allow for safe, efficacious and potentially widespread integration of tDCS into clinical rehabilitative therapies.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum. However, the parent compound quinine, as well as its more recent counterparts, suffers from an incidence of adverse neuropsychiatric side effects ranging from mild mood disturbances and anxiety to hallucinations, seizures, and psychosis. This review considers how the pharmacology, cellular neurobiology, and membrane channel kinetics of mefloquine could lead to the significant and sometimes life-threatening neurotoxicity associated with mefloquine exposure. A key role for mefloquine blockade of ATP-sensitive potassium channels and connexins in the substantia nigra is considered as a unifying hypothesis for the pathogenesis of severe neuropsychiatric events after mefloquine exposure in humans.The alkaloid toxin quinine and its derivative compounds have been used for many centuries as effective medications for the prevention and treatment of malaria. More recently, synthetic derivatives, such as the quinoline derivative mefloquine (bis(trifluoromethyl)-(2-piperidyl)-4-quinolinemethanol), have been widely used to combat disease caused by chloroquine-resistant strains of the malaria parasite,  Plasmodium falciparum during its intraerythrocytic phase, quinine suffers from a number of contraindications which have made it problematic as an effective therapeutic including a low therapeutic index and high incidence of adverse side effects [Quinine is an alkaloid toxin found in the bark of the South American cinchona (quina-quina) tree . Quinine effects . Despite effects .By the 1950s, increasing levels of chloroquine resistance necessitated a push for the discovery of novel compounds for malarial chemoprophylaxis , an effeOver the next twenty years mefloquine was widely advocated as the drug of choice for travellers to areas known to be endemic for chloroquine-resistant malaria  such as This “toxidrome,” a collection of significant neurological symptoms affecting balance, vision, hearing, memory, personality, and emotional status, has now been described as a limbic encephalopathy with central vestibulopathy , an overThe incidence of adverse reactions to mefloquine treatment and/or prophylaxis has long been a point of controversy. Early studies suggested that patients did not experience the very severe neuropsychiatric side effects that had been reported with chloroquine –42 but aSome of the pharmacological properties of mefloquine, which contribute to its efficacy as an antimalarial, may also contribute to its neurotoxicity. Mefloquine has a long plasma half-life (13–28 days), which contributes to its efficacy as a prophylactic treatment achievable by easy weekly dosing , 48. MefOne mechanism likely to cause increased retention of mefloquine in the CNS is via inhibition of the membrane efflux pump P-glycoprotein. P-gp , encoded by the Multi-Drug Resistance gene 1 (MDR1), is a transmembrane protein found lining the brain capillary endothelium that plays a specific role in central neuroprotection by restricting access of lipophilic molecules across the BBB . The nor P. falciparum malaria [Effectiveness of the CYP450 enzyme superfamily in oxidative enzymatic degradation of common pharmaceuticals, including mefloquine, is likely to also play a significant role in the presentation and severity of neuropsychiatric side effects as genetic polymorphisms in the enzymes CYP2D6, CYP2C19, CYP3A4, and CYP1A2  have all malaria  where mu malaria , symptom in vitro [ Plasmodium falciparum [Mutations in genes associated with cellular metabolism of toxic compounds are not the only polymorphisms implicated in increased risk of adverse neuropsychiatric events associated with quinoline therapy. Mutations at the MNDR1 locus have also been shown to be associated with a heightened sensitivity to mefloquinein vitro  as well in vitro . The mallciparum , 64 suggThe highly lipophilic nature of mefloquine is also likely to be a contributory factor to the variable presentation of adverse neuropsychiatric events in humans. It has been shown that travellers with a low body weight index, taking mefloquine for malarial prophylaxis, showed an increased likelihood of adverse reactions than those taking chloroquine or the combination therapy chloroquine and proguanil . This isTogether, these findings suggest that variability in plasma half-life, activity of efflux pumps of the CNS/vascular interface, and compartmentalisation, as well as underlying genetics regulating neuroactive drug sensitivity and metabolism, may all contribute to toxic loading of mefloquine in the CNS and therefore the highly variable presentation of adverse events in a subpopulation of mefloquine users. Thus, multiple factors affecting drug retention and concentration in the CNS make accurate prediction of adverse events in patients exposed to mefloquine highly problematic.It has been well described that toxic loading of mefloquine in the CNS can be subject to significant interpersonal variation; however, this does not fully explain the highly varied expression of adverse side effects reported by individuals exposed to therapeutic levels of mefloquine. At toxic doses, mefloquine has been widely reported to cause neuronal dysfunction, axonal degeneration, and neuronal cell death in a variety of cell types of the central nervous system –70, yet ATP) channels. KATP channels are found in a wide range of tissues, including cardiomyocytes, smooth muscle, hormone secreting cells of the pancreas, and neurons of the CNS [ATP channels contain two components, a pore-forming Kir subunit and a sulfonylurea receptor (SUR) subunit, members of the ATP-binding cassette superfamily [ATP channels have been shown to play a fundamental role in glucose homeostasis via their activity in insulin-secreting islet cells of the pancreas [γ-amino-butyric acid (GABA) inhibitory neurons of the cerebral cortex, substantia nigra pars reticulata (SNr), pars compacta (SNc), and cerebellum [One family of neuronal cell membrane channels likely to play a key role in mefloquine neurotoxicity are the octomeric ATP-sensitive potassium , pars compacta (SNc), and cerebellum [ATP channels, cells which are known to play a key role in maintenance of normal spontaneous firing activity in the brain [Krebellum , 82. Of he brain , 90. Thihe brain .ATP channels in the SNr are closed and cells exhibit a high level of spontaneous activity, suppressing seizure activity by release of GABA onto postsynaptic terminals  play an important role in neuronal metabolism and homeostasis by controlling movement of ions, metabolites, and other molecules between adjacent cells of the CNS. Gap junctions establish electrical coupling by allowing intercellular exchange of ions and metabolic support by transport of ADP, glucose, glutamate, and glutathione, as well as movement of second messengers such as cyclic AMP. A wide variety of pharmacological agents have been shown to influence their activity [The connection between mefloquine disruptions of interneuronal communication via blockade of connexins channels, a family of gap junction family proteins, is now well established . Like KAactivity  and dysfactivity , vestibuactivity , and epiactivity .ATP channels in states of neuropsychiatric abnormality. It has been shown that KATP channels regulate the expression of Cx43 and Cx45 in the epileptic hippocampus [ATP channels in these regions. Both mefloquine and quinine have been shown to selectively block the activity of Cx36 and Cx50 channels in vitro, mefloquine with significantly higher potency than its parent molecule [There is a known functional connection between connexins and Kpocampus . Cx36 ispocampus , 133. Inpocampus , 135, agmolecule , 137 andmolecule .ATP and connexin membrane channels (Figures A connection between mefloquine, connexins, and altered neurosensory function has also been identified. Mefloquine inhibits Cx26, dominant mutations of which have been shown to cause neurosensory deafness as well as attenuating increased membrane currents in primary cells expressing a dominant negative human Cx26 channel . Cx26 is Figures , giving ATP channels regulating intracellular metabolism, in the pathogenesis of mefloquine toxicity via multiplex membrane channel blockade. Variation in the sensitivity of either or both membrane channels, due to age, genetic variation, interaction with other gap junction blockers, or channel antagonists, could explain the extreme variation in neuropsychiatric symptoms presenting in patients exposed to supposedly “safe” levels of mefloquine.Together, these studies suggest a synergistic role for connexins regulating intercellular excitability, and KATP channels both implicated in this process. A diagrammatic representation of the complex interrelationship between risk and severity of adverse events, pharmacogenetics, comorbidity with other neuropsychiatric disorders, and dose/length of exposure is illustrated in Significant evidence now exists for a primary role for membrane channel blockade in the presentation and severity of adverse neuropsychiatric reactions in patients exposed to mefloquine at normal prophylactic or treatment levels. How these complex cellular interactions manifest as neuroelectrophysiological and neurochemical changes, synaptic dysfunction, or neuronal cell death is still not clear but it seems likely that the delicate balance between excitation and inhibition caused by mefloquine exposure, both intra- and intercellularly, is likely to play a central role with connexins and KATP and connexin channels on exposure to mefloquine, could begin to elucidate the delicate interplay between excitation and inhibition in cases of mefloquine toxicoses. A hypothesis of dysfunction of the central pacemaker, giving rise to mesolimbic dysregulation, could also provide novel treatment options for patients suffering from adverse reactions to mefloquine exposure in the future.Further studies, including functional and structural imaging of deep brain regions in patients suffering from mefloquine toxicity and examination of electrophysiological changes in cells of the substantia with mutation or variation in both K


Clostridium difficile are limited, and some patients have developed untreatable infections. Evidence-supported interventions are available, but coordinated approaches to interrupt the spread of HAIs could have a greater impact on reversing the increasing incidence of these infections than independent facility-based program efforts.Treatments for health care–associated infections (HAIs) caused by antibiotic-resistant bacteria and C. difficile both with and without a large scale national intervention that would include interrupting transmission and improved antibiotic stewardship. As an example, the impact of reducing transmission of one antibiotic-resistant infection (carbapenem-resistant Enterobacteriaceae [CRE]) on cumulative prevalence and number of HAI transmission events within interconnected groups of health care facilities was modeled using two distinct approaches, a large scale and a smaller scale health care network.Data from CDC’s National Healthcare Safety Network and Emerging Infections Program were analyzed to project the number of health care–associated infections from antibiotic-resistant bacteria or Pseudomonas aeruginosa, invasive methicillin-resistant Staphylococcus aureus (MRSA), or C. difficile. Compared with independent efforts, a coordinated response to prevent CRE spread across a group of inter-connected health care facilities resulted in a cumulative 74% reduction in acquisitions over 5 years in a 10-facility network model, and 55% reduction over 15 years in a 102-facility network model.Immediate nationwide infection control and antibiotic stewardship interventions, over 5 years, could avert an estimated 619,000 HAIs resulting from CRE, multidrug-resistant With effective action now, more than half a million antibiotic-resistant health care–associated infections could be prevented over 5 years. Models representing both large and small groups of interconnected health care facilities illustrate that a coordinated approach to interrupting transmission is more effective than historical independent facility-based efforts.C. difficile than independent facility–based efforts.Public health–led coordinated prevention approaches have the potential to more completely address the emergence and dissemination of these antibiotic-resistant organisms and  C. difficile infections (CDIs), which are typically associated with antibiotic use  and Emerging Infections Program (EIP). Four particularly problematic health care–associated infections (HAIs) were included: CRE, multidrug-resistant pathogen . Projectpathogen –12.Two independently developed and complementary agent-based mathematical simulation models were used to measure the impact of a coordinated approach to prevent the spread of antibiotic-resistant organisms within a group of health care facilities interconnected through patient sharing , using CRE as a test case. These agent-based models are computer simulations that represent hospitalized patients as “agents” and track their dynamic interactions with other patients and CRE status throughout the health care system. The first model assessed the impact of the coordinated approach in a simulated network of 10 health care facilities consisting of four acute care hospitals , and six free-standing nursing homes serving adult patients. Transfer of patients between facilities was calibrated based on actual transfer data from the U.S. Department of Veterans Affairs, supplemented with state inpatient database data . The perWith each model, the spread of CRE was simulated under three hypothetical scenarios (1): infection control activity currently in common use  (2), augmented efforts implemented independently at individual subsets of facilities (independent efforts), and (3) coordinated augmented approach across a health care network (coordinated approach). Baseline activity simulations assumed that facilities applied contact precautions only to colonized or infected patients identified through routine tests. The independent efforts allowed for up to 15% of hospitals to begin active detection  and isolation of CRE-colonized patients after a predetermined number of patients had been identified through routine clinical tests at each individual hospital. The coordinated approach allowed for all health care facilities to share CRE test results with a central public health authority, which used that information to strategically target prevention activity across multiple facilities. Notification of patient status as CRE-colonized or CRE-infected to facilities receiving a patient upon inter-facility transfer varied by model, and increased in frequency from independent efforts to coordinated approaches.Both models simulated the movement of patients within and between different health care facilities and transmission of CRE in a health care network based upon key parameter estimates that included inter-facility patient movement, the proportion of colonized patients recognized by routine clinical tests, and effectiveness of barrier precautions in preventing transmission. Models were parameterized based on published data or calibrated to published estimates of CRE incidence and prevalence at acute care hospitals, long-term acute care hospitals, and nursing homes in regions where CRE outbreaks have occurred. Mean values for number of acquisitions and cumulative prevalence were calculated from simulations.*P. aeruginosa, invasive MRSA, or CDI occurred in the United States. Based on current trends, in 5 years the number of infections caused by these pathogens is estimated to increase by approximately 10%, to 340,000 per year, unless additional interventions are implemented. With immediate implementation of national interventions combining infection control and antibiotic stewardship and, assuming similar effectiveness to that reported in other countries, an estimated 619,000 health care–associated infections and 37,000 deaths could be averted in 5 years , the prevalence of health care–associated CRE infection or colonization after 5 years could be 12.2% with 2,141 patients acquiring CRE . With in†Using the 102-facility model of Orange County simulations over 15 years, the model estimated that the average network prevalence of CRE after 15 years would be 15% with 35,159 patients acquiring CRE . With inWith effective action now, including nationwide antibiotic stewardship efforts and interventions to prevent spread of antibiotic-resistant infections, an estimated 619,000 infections caused by three problematic antibiotic-resistant HAIs or CDIs, and 37,000 deaths among infected patients might be averted nationally over the next 5 years. When considering published estimates of costs related to these four infections in the projections ,18, an eSeveral methods exist to coordinate prevention of antibiotic resistant HAIs; however, public health departments, particularly large local or state health departments, are uniquely suited to facilitate and accelerate this approach. Health departments are able to work with facilities within their jurisdiction in ways that amplify ongoing efforts of individual facilities or health systems. Because health departments possess substantial expertise in surveillance and prevention, they are well equipped to partner with multiple stakeholders, including hospitals, corporate and academic institutions, hospital associations, professional organizations, quality improvement organizations, and federal partners. Such state-based HAI antibiotic-resistance prevention programs can enable communities to locate the threat by sharing antibiotic resistance data and promoting accurate testing. Such programs also can respond quickly to prevent spread by identifying and rapidly responding to clusters, implementing a regionally coordinated response that includes opening lines of communication between facilities, helping individual hospitals improve infection prevention practices, and strategically targeting resources to prevent spread and maximize community impact.Key PointsClostridium difficile infections, which are quite contagious, especially in health care facilities.Antibiotic use can cause germs to become resistant to antibiotics. Their use can also cause About 2 million illnesses and 23,000 deaths are caused by antibiotic resistant infections in the United States annually.C. difficile infections annually, typically caused by antibiotic use.About 250,000 people are hospitalized for If best infection control practices and antibiotic stewardship were nationally adopted, more than 600,000 infections and 37,000 deaths could be prevented over 5 years.If health care sites coordinated their patient infection information to guide interventions, an estimated 74% fewer patients would be infected by highly-resistant carbapenem-resistant Enterobacteriaceae over 5 years.http://www.cdc.gov/vitalsigns.Additional information is available at Sharing the responsibilities to establish a coordinated program among communities of health care institutions with leadership by local health authorities will bring about the collective, shared benefits of coordination. Shifting the current culture to one of sharing information and sharing responsibility in prevention will require local leadership and commitment across various sectors. Developing a plan to share facility-level information regarding the presence and incidence of important antibiotic-resistant infections in ways that acknowledge the importance of protecting personally identifiable and other sensitive information, as occurred with facility-specific disclosure of HAI rates over the past decade, will be essential. Several key steps need to be taken to begin a coordinated approach. Health care facility leaders can take action to accelerate efforts to improve infection control practices within their own facilities and assure accurate and timely detection and reporting of antibiotic-resistant infections. In addition to augmented efforts, facilities can alert one another when enhanced infection control is needed for transferred patients who are colonized or infected with resistant organisms. Facility leadership should work with their respective health departments to determine best data sharing practices. Such steps improve access by public health departments to an established flow of HAI data, including those reported from hospitals to CDC’s NHSN. CDC is working to better assist health departments and health care facilities to collect, access, and respond to their HAI-related data, thereby enabling more efficient use of staff time and resources to implement effective prevention efforts.A number of states have begun to develop programmatic capacity and experience in a coordinated approach for action to prevent antibiotic-resistant infections in health care settings. For example, the South Dakota Department of Health identified CRE in a region of the state, and in response, implemented a comprehensive program that included the introduction of mandatory reporting of CRE in 2013. The educational program was developed to increase CRE prevention knowledge among health care providers, and, with the two main hospital systems in the state, develop and implement interventions to reduce transmission. The program determines extent of spread and has worked with neighboring states to prevent cross-border transmission. This coordinated approach in oversight and rapid and efficient response resulted in a statewide decrease in CRE infections from 24 in 2012, to four in 2014. In Tennessee, the Department of Health has begun accessing data reported to NHSN and using analytic methods similar to The Targeted Assessment for PreventionThe findings in this report are subject to at least five limitations. First, estimates of the projected number of infections and the impact of interventions are based on the assumption that rates will rise yearly according to current trends and that effective interventions will reduce annual rates of infections by 30%–50%. Second, reductions in infections with these four pathogens over the next 5 years might not translate into fewer HAIs overall; however, even if the infections prevented with these four pathogens are replaced by infections caused by less resistant organisms, such infections would be easier to treat. Third, the models were focused on interventions that are designed for interrupting transmission within and between health care facilities. Antibiotic resistant pathogens, such as MRSA, can also be spread in community settings; parallel efforts to prevent AR in the community are also of great importance. Fourth, illustration of the impact of coordinated approaches to preventing transmission as presented here is based on current understanding of CRE transmission within facilities and inter-facility transfer patterns, and some of the simplifying assumptions used in the simulations might bias the results. The use of Veterans Affairs data in the 10-facility model made it feasible to represent dependencies between lengths of stay, probabilities of readmission, and infection status. These are relationships that, in their basic form, likely are generalizable across health systems, and other models using different assumptions have suggested a similar advantage to regionally coordinated interventions involving other pathogens ,5. AlthoThe threat of antibiotic-resistant infections and CDI is not limited to certain areas or types of health care facilities. The current threat of antibiotic resistance in health care settings suggests that historical independent institution-based efforts to prevent transmission have been inadequate. Coordinated prevention approaches led by public health agencies, when coupled with intensified facility-based prevention programs, have the potential to more completely address the emergence and dissemination of these organisms.


Anticoagulation is the mainstay of medical treatment, prevention and reduction of recurrent venous thromboembolism, stroke prevention in patients with non-valvular atrial fibrillation, and it reduces the incidence of recurrent ischemic events and death in patients with acute coronary syndrome. Options for anticoagulation have been steadily increasing. Physicians need to be aware of the clinical profile of anticoagulation agents, reversal agents and treatment strategies in the face of major bleeding.Physicians have numerous anticoagulation treatment options in their arsenal including the traditional injectable indirect factor Xa and IIa (thrombin) inhibitors, which are mediated through an antithrombin dependent mechanism. Examples of injectable indirect factor Xa and IIa inhibitors include unfractionated heparin (UFH), which inhibits factors Xa and to a lesser extent IIa. UFH also inhibits factors XIIa, XIa and IXa. Enoxaparin (Lovenox), a low-molecular-weight-heparin (LMWH), inhibits factors Xa and to a lesser extent IIa. Finally, Fondaparinux (Arixtra) is an exclusive indirect factor Xa inhibitor. Once an injectable indirect factor Xa and IIa inhibitor has been selected, an oral vitamin K antagonist (VKA), traditionally warfarin (Coumadin), is started on the same day. VKAs reduce the synthesis of factors II, VII, IX and X thereby reducing ability to generate active thrombin, rather than inhibition of activated enzymes.Target-specific oral anticoagulants (TSOACs) or direct oral anticoagulants (DOACs) were developed to provide more stable pharmacokinetic and pharmacodynamic options for oral anticoagulation. Examples of DOACs include the following: direct thrombin inhibitors (DTIs) dabigatran (Pradaxa) and direct factor Xa inhibitors (“Xabans”) rivaroxaban (Xarelto), apixaban (Eliquis) and edoxaban . The DOACs act directly upon Factors IIa (DTIs) or Xa (“Xabans”) without using antithrombin as a mediator.2Note that each direct factor Xa inhibitor has the letters “Xa” in its spelling.See The risk of major bleeding with DOACs is low; however, major life-threatening bleeding can occur. Examples of clinically significant major life-threatening bleeding include intracranial, intraspinal, intraocular, retroperitoneal, intraarticular, pericardial, intramuscular with compartment syndrome or a fall in hemoglobin >2g/dL leading to transfusion. Physicians need to have aggressive and comprehensive anticoagulation reversal and treatment strategies in the face of major bleeding.Discontinue anticoagulant  - DOACs have short half-lives (range from 5 to 17 hours), which suggests reversal drugs may not be needed in non-urgent situations; however, in emergency situations such as life-threatening major bleeding or non-elective major surgery anticoagulation reversal strategies should be established.Control active bleeding.Maintain adequate fluid, oxygen and hemodynamic support.Transfuse packed red blood or initiate massive transfusion protocols, if necessary.Consider platelets in patients who are thrombocytopenic or on anti-platelet therapy (aspirin (ASA), clopidogrel (Plavix), prasugrel (Effient), dipyridamole (Persantine) or ticlopidine (Ticlid)Order routine lab tests: complete blood count, basic metabolic panel, liver function tests and disseminated intravascular coagulation panel.Order coagulation testing. (Utility of testing based on anticoagulant is discussed below.)Activated partial thromboplastin time (aPTT) is a measure of the intrinsic pathway. Traditionally it has been useful to determine the anticoagulation status of patients receiving UFH. In patients who are taking DOACs, effects on the aPTT are variable. Prolongation of aPTT occurs in a curvilinear fashion in patients taking both IIa and Xa inhibitors; however, the degree of prolongation is dependent upon the reagent used.5At therapeutic levels of dabigatran, the clinician should expect the aPTT to be prolonged. Recognize that there may still be clinical anticoagulation effects of dabigatran with a normal aPTT; however, the patient’s serum levels would fall below the therapeutic range (<80μg/L).5At therapeutic levels of the direct oral “Xabans,” a PTT will not reliably be prolonged. The test is only useful in patients on Xa inhibitors at supra-therapeutic levels.Prothrombin time (PT) / international normalized ration (INR) is a measure of the extrinsic and common pathway; it is useful to determine the anticoagulation status of patients receiving VKAs. Similar to aPTT, PT/INR is variably affected by the DOACs. The degree of elevation is specific to the reagent as well as the calibration of the laboratory equipment.5At therapeutic levels of dabigatran, an abnormality of the PT/INR is not expected. In a patient taking dabigatran, an elevated INR is an indication of serum levels three to four times the upper limit of normal therapeutic concentrations.4Of the direct oral Xa inhibitors, rivaroxaban has the strongest effect on the PT/INR. At therapeutic serum concentrations, one would expect both rivaroxaban and edoxaban to cause elevation of the PT/INR..4Thrombin time (TT) directly assesses the activity of thrombin. This test is useful in patients receiving dabigatran (Pradaxa). A normal TT excludes dabigatran activity; the degree of elevation of the TT is not a direct correlate of serum levelsChromogenic anti-factor Xa testing measures the concentration of anticoagulants that inhibit factor Xa. This test is useful in patients receiving LMWHs, fondaparinux (Arixtra) and direct oral factor “Xabans”.Anticoagulation reversal and treatment options in major bleeding include protamine, phytonadione (Vitamin K), hemodialysis, oral-activated charcoal, antifibrinolytic agents including tranexamic acid, desmopressin, blood products including packed red blood cells (PRBCs) and platelets, prothrombin complex concentrates (PCCs), and specific reversal agents. See Given most clinicians’ apprehension and relative inexperience with reversal of the DOACs, below we will discuss in detail the risks and benefits of different treatment options.  or four factors . Four-factor (4F-) PCC may be inactive  or active ; this is determined by the Factor VII component.PCCs have been shown to be successful in the reversal of VKAs and are considered first line treatment in patients with major bleeding on VKAs.19In vitro human studies of healthy volunteers who have received oral direct IIa and Xa inhibitors have shown normalization of coagulation testing after receiving both 4F-PCC and 4F-aPCC.in vitro or in animal models and thus, no recommendation can be made regarding its use in bleeding patient on DOACs.in vitro human and bleeding animal model studies have been performed to evaluated the benefit of this therapy in patients on DOACs.Recombinant human Factor VIIa (rFVIIa) activates the coagulation cascade via the extrinsic pathway and has been used off label in the reversal of VKAs. Similar to PCCs, e.g. VKAs) Unlike VKAs, the DOACs do not inhibit overall production of inactive coagulation components, but only bind to specific active factors. Therefore, treatment of significant bleeding in a patient taking DOACs is not aimed at repleting diminished factors. The relative concentration of coagulation factors in FFP is diminutive compared to the PCC products such that one would need over two liters of FFP to approach similar concentrations. The time to thaw and transfuse this volume of FFP also is a disadvantage. For these reasons, FFP is not recommended for reversal of DOACs unless no other agent is available.Fresh frozen plasma (FFP) is derived from whole blood and thus, contains all inactive components of the coagulation cascade in physiologic concentrations. FFP has traditionally been considered an option in patients on oral anticoagulants  is low. Therefore, the recommendation is that TXA should be considered for reversal of bleeding in patients taking DOACs.Desmopressin is a synthetic analogue of vasopressin. It affects thrombosis by stimulating the release of von Willebrand factor (vWF) and increasing production of factor VIII. Similar to TXA, the overall cost and risk of administration of desmopressin is low. Therefore, it should be considered for use in the treatment of significant bleeding in patients on DOACs.Dabigatran excretion is 80–85% renal which makes hemodialysis an option.Direct factor Xa oral inhibitors are mainly protein bound with only 25 to 35% renal excretion, therefore hemodialysis is not an option for the direct factor “Xabans”.10Oral activated charcoal, 100gm PO/NG ×1, is an option to reduce absorption for all DOACs in the appropriate patient. Charcoal can be considered if the dose was taken within eight hours for rivaroxaban, six hours apixaban and within two hours of ingestion for edoxaban and dabigatran.Physicians need to effectively manage major bleeding in patients on anticoagulants. Understanding the clinical profiles of anticoagulants and developing treatment and reversal strategies will help physicians effectively manage this life-threatening emergency. While physicians are comfortable with the use of vitamin K, fresh frozen plasma and protamine, the use of prothrombin complex concentrates and future antidotes hold promise in improving the outcomes of patients who require emergent reversal of their anticoagulant therapy.