Development and validity testing of a matrix to evaluate maturity of clinical pathways: a case study in Saskatchewan, Canada.

BACKGROUND: Healthcare systems are transforming into learning health systems that use data-driven and research-informed approaches to achieve continuous improvement. One of these approaches is the use of clinical pathways, which are tools to standardize care for a specific population and improve healthcare quality. Evaluating the maturity of clinical pathways is necessary to inform pathway development teams and health system decision makers about required pathway revisions or implementation supports. In an effort to improve the development, implementation, and sustainability of provincial clinical pathways, we developed a clinical pathways maturity evaluation matrix. To explore the initial content and face validity of the matrix, we used it to evaluate a case pathway within a provincial health authority in Saskatchewan, Canada. METHODS: By using iterative consensus-based processes, we gathered feedback from stakeholders including patient and family partners, policy makers, clinicians, and quality improvement specialists, to rank, retain, or remove enablers and sub-enablers of the draft matrix. We tested the matrix on the Chronic Pain Pathway (CPP) for primary care in a local pilot area and revised the matrix based on feedback from the CPP development team leader. RESULTS: The final matrix contains five enablers (i.e., Design, Ownership and Performer, Infrastructure, Performance Management, and Culture), 20 sub-enablers, and three trajectory definitions for each sub-enabler. Supplemental documents were created for six sub-enablers. The CPP scored 15 out of 40 possible points of maturity. Although the pathway scored highest in the Design enabler (10/12), it requires more attention in several areas, specifically the Ownership and Performer and the Performance Management enablers, each of which scored zero. Additionally, the Infrastructure and Culture enablers scored 2/4 and 3/8 points, respectively. These areas of the CPP are in need of improvement in order to enhance the overall maturity of the CPP. CONCLUSIONS: We developed a clinical pathways maturity matrix to evaluate the various dimensions of clinical pathways' development and implementation. The goals of this initial work were to develop and validate a tool to assess the maturity and readiness of new or existing pathways and to track pathways' revisions and improvements.

Development, implementation and impact of an immunoglobulin stewardship programme in Saskatchewan, Canada.

BACKGROUND AND OBJECTIVES: Intravenous immunoglobulin (IVIG) is one of the most costly and limited-supply blood products. Judicious use of this therapy is important to ensure a continued supply is available for patients in need. The Saskatchewan IG Stewardship Program was initiated to monitor and reduce inappropriate IG use. MATERIALS AND METHODS: The Program was developed and implemented through the collaborative efforts of a multidisciplinary, inter-organizational team. Funding was provided from provincial organizations to create new positions within the Program and to support stakeholder engagement throughout the process of implementation. Data were collected from local and national databases regarding the amount of IVIG used and appropriateness of orders based on published criteria. RESULTS: Over 20 months, the Program helped to reduce unnecessary IVIG use from pre-intervention levels by more than 20%. Interventions from nurse navigators alone reduced inappropriate IVIG use by 2.6%. During the 20-month period following Program initiation, more than 4 million CAD less was spent on IVIG compared with the previous 20 months. CONCLUSION: The Saskatchewan IG Stewardship Program has led to more appropriate IVIG use across the province, more effective preservation of this limited healthcare resource, and cost savings that more than cover the cost of administering the Program.

Development and implementation of a clinician report to reduce unnecessary urine drug screen testing in the ED: a quality improvement initiative.

BACKGROUND: Unnecessary testing is a problem-facing healthcare systems around the world striving to achieve sustainable care. Despite knowing this problem exists, clinicians continue to order tests that do not contribute to patient care. Using behavioural and implementation science can help address this problem. Locally, audit and feedback are used to provide information to clinicians about their performance on relevant metrics. However, this is often done without evidence-based methods to optimise uptake. Our objective was to improve the appropriate use of laboratory tests in the ED using evidence-based audit and feedback and behaviour change techniques. METHODS: Using the behaviour change wheel, we implemented an audit and feedback tool that provided information to ED physicians about their use of laboratory tests; specifically, we focused on education and review of the appropriate use of urine drug screen tests. The report was designed in collaboration with end users to help maximise engagement. Following development of the report, audit and feedback sessions were delivered over an 18-month period. RESULTS: Data on urine drug screen testing were collected continually throughout the intervention period and showed a sustained decrease among ED physicians. Test use dropped from a monthly departmental average of 26 urine drug screen tests per 1000 patient visits to only eight tests per 1000 patient visits following the initiation of the audit and feedback intervention. CONCLUSION: Audit and feedback reduced unnecessary urine drug screen testing in the ED. Regular feedback sessions continuously engaged physicians in the audit and feedback intervention and allowed the implementation team to react to changing priorities and feedback from the clinical group. It was important to include the end users in the design of audit and feedback tools to maximise physician engagement. Inclusion in this process can help ensure physicians adopt a sense of ownership regarding which metrics to review and provides a key component for the motivation aspect of behaviour change. Departmental leadership is also critical to the process of implementing a successful audit and feedback initiative and achieving sustained behaviour change.

Developing a patient-oriented realist evaluation for COVID-19 vaccine implementation in Saskatchewan: a methodologic framework.

BACKGROUND: There is an urgent need to inform decision-making and safe delivery of vaccines in a timely manner. Our objective is to describe the methods we used to perform a patient-oriented realist evaluation of COVID-19 vaccination implementation in Saskatchewan, Canada, in order to understand the underlying mechanisms and contexts of vaccination implementation and vaccine uptake. METHODS: This methodology paper describes a patient-oriented, realist, mixed-method evaluation to assess COVID-19 vaccination implementation in Regina, Saskatoon and Prince Albert, Saskatchewan. The study comprised 3 iterative phases guided by Realist And Meta-narrative Evidence Synthesis: Evolving Standards II (RAMESES II). In phase 1 (January-February 2021), we developed the initial program theory, in phase 2 (March-May 2021), we tested and refined the initial program theory, and in phase 3 (June-July 2021), we established the final program theory. Three patient and family partners with different backgrounds and experiences were selected purposively from various locations (urban and rural) in Saskatchewan to engage collaboratively in the evaluation. Data analysis and synthesis occurred at all 3 phases of the project. We analysed qualitative data from phases 2 and 3 using a "retroductive" approach. We used quantitative data to compare outcomes from the 3 sites. INTERPRETATION: This protocol describes how we developed a final program theory for COVID-19 vaccination implementation with patient and family partners to show for whom, under what circumstances, how and why Saskatchewan's COVID-19 vaccination program has led to vaccine uptake. With patient and family partners' engagement, the evaluation findings will be shared with the Saskatchewan Health Authority and provincial government policy-makers and communications departments, published in peer-reviewed journals, presented at provincial or national conferences, and disseminated through any additional media identified by the patient and family partners.

Development of a Patient-Oriented Intervention to Support Patient-Provider Conversations about Unnecessary Lower Back Pain Imaging.

BACKGROUND: despite the efforts of multiple stakeholders to promote appropriate care throughout the healthcare system, studies show that two out of three lower back pain (LBP) patients expect to receive imaging. We used the Choosing Wisely Canada patient-oriented framework, prioritizing patient engagement, to develop an intervention that addresses lower back pain imaging overuse. METHODS: to develop this intervention, we collaborated with a multidisciplinary advisory team, including two patient partners with lower back pain, researchers, clinicians, healthcare administrators, and the Choosing Wisely Canada lead for Saskatchewan. For this qualitative study, data were collected through two advisory team meetings, two individual interviews with lower back pain patient partners, and three focus groups with lower back pain patient participants. A lower back pain prescription pad was developed as an outcome of these consultations. RESULTS: participants reported a lack of interactive and informative communication was a significant barrier to receiving appropriate care. The most cited content information for inclusion in this intervention was treatments known to work, including physical activity, useful equipment, and reliable sources of educational material. Participants also suggested it was important that benefits and risks of imaging were explained on the pad. Three key themes derived from the data were also used to guide development of the intervention: (a) the role of imaging in LBP diagnosis; (b) the impact of the patient-physician relationship on LBP diagnosis and treatment; and (c) the lack of patient awareness of Choosing Wisely Canada and their recommendations. CONCLUSIONS: the lower back pain patient-developed prescription pad may help patients and clinicians engage in informed conversations and shared decision making that could support reduce unnecessary lower back pain imaging.

Identification of Staphylococci by Polymerase Chain Reaction Directly from a Positive Blood Culture and Effect on Patient Care.

BACKGROUND: As one of the most common bloodstream infections worldwide, Staphylococcus aureus bacteremia places a major burden on health care. Implementation of a rapid, genetic-based diagnostic test may have important implications in the clinical management of patients with S. aureus bacteremia. OBJECTIVES: The primary objective was to assess concordance between testing based on polymerase chain reaction (PCR) and the current gold standard, culture and sensitivity testing; the secondary objective was to assess the impact of this technology on patient care. METHODS: A pre-post intervention retrospective chart review was used to document the hospital course of patients with a diagnosis of S. aureus bacteremia before and after implementation of the PCR-based diagnostic system. Laboratory results from all patient samples subjected to PCR-based analysis following implementation of this system were compared with culture and sensitivity data for the same samples to determine accuracy of the new system. In addition, time to optimal therapy for each patient was calculated as the interval between the initiation of empiric and terminal therapies. The appropriateness of antimicrobial treatment was characterized as guideline-concordant, nonconcordant with the guidelines, or nonconcordant yet still clinically appropriate. RESULTS: In total, 98 and 99 patients met the inclusion criteria before and after implementation of the PCR-based diagnostic system, respectively. PCR-based results displayed 99.8% concordance (440/441 total samples) with results from culture and sensitivity testing. The time to optimal therapy was significantly shorter after implementation, by a mean of 22.8 h (p < 0.001). Overall, 97% of empiric and 99% of terminal antimicrobial regimens were either guideline-concordant or clinically appropriate for treatment of S. aureus bacteremia; 3% of empiric and 1% of terminal antimicrobial regimens were nonconcordant with clinical guidelines without any explanation based on other clinical considerations. CONCLUSIONS: The study findings support the utility of using a direct-from-positive-blood-culture PCR-based diagnostic tool as the primary method of identifying S. aureus bacteremia in patients, as well as the acceptance of and acting upon the new assay's results by our local clinicians. PCR-based assays can help reduce the time to optimal terminal therapy for patients with bacteremia.

CONTEXTE: La bactériémie à Staphylococcus aureus (BAC-SA), qui est l'une des infections du sang les plus répandues dans le monde, fait peser une lourde charge sur les soins de santé. La mise en place d'un test diagnostique génétique rapide pourrait avoir des retombées importantes sur la gestion clinique des patients présentant une BAC-SA. OBJECTIFS: L'objectif principal consistait à évaluer la concordance entre les tests basés sur la réaction en chaîne par polymérase (PCR) et le test de sensibilité et de culture, qui est la référence absolue actuelle; l'objectif secondaire consistait à évaluer l'impact de cette technologie sur les soins des patients. MÉTHODES: Un examen rétrospectif des dossiers pré-et post-intervention a servi à décrire le séjour à l'hôpital des patients ayant reçu un diagnostic de BAC-SA avant et après la mise en place du système de diagnostic de la PCR. Les résultats de laboratoire de tous les échantillons des patients soumis à une analyse de la PCR à la suite de la mise en place de ce système ont été comparés avec les données relatives à la culture et à la sensibilité de ces mêmes échantillons afin de déterminer la précision du nouveau système. De plus, l'évaluation du délai d'atteinte du traitement optimal de chaque patient repose sur le calcul de l'intervalle entre le début des thérapies empiriques et terminales. La pertinence du traitement antimicrobien était caractérisée comme suit : concordance avec les lignes directrices, non-concordance avec les lignes directrices ou non-concordance mais encore approprié d'un point de vue clinique. RÉSULTATS: Au total, 98 et 99 patients ont satisfait au critère d'inclusion respectivement avant et après la mise en place du système de diagnostic de la PCR. Les résultats basés sur la PCR affichaient une concordance de 99,8 % (440/441 échantillons au total) avec les résultats des tests de sensibilité et de culture. La diminution du délai d'atteinte du traitement optimal était importante après la mise en place du système, puisqu'elle atteignait en moyenne 22,8 h (p < 0,001). De manière générale, 97 % des régimes antimicrobiens empiriques et 99 % des régimes antimicrobiens terminaux concordaient avec les lignes directrices ou étaient cliniquement appropriés pour le traitement de la BAC-SA; 3 % des régimes antimicrobiens empiriques et 1 % des régimes antimicrobiens terminaux n'étaient pas conformes aux lignes directrices cliniques sans qu'aucune explication basée sur d'autres considérations cliniques n'ait été donnée. CONCLUSIONS: Les résultats de l'étude confirment la nécessité d'utiliser un outil diagnostique basé sur la PCR directement de l'hémoculture positive en guise de méthode principale pour déterminer la présence de BAC-SA chez les patients ainsi que l'acceptation et l'utilisation des nouveaux résultats du test par nos cliniciens locaux. Les tests basés sur la PCR peuvent aider à réduire le délai d'attente du traitement optimal pour les patients atteints de BAC-SA.

Identification of therapeutics that target eEF1A2 and upregulate utrophin A translation in dystrophic muscles.

Up-regulation of utrophin in muscles represents a promising therapeutic strategy for the treatment of Duchenne Muscular Dystrophy. We previously demonstrated that eEF1A2 associates with the 5'UTR of utrophin A to promote IRES-dependent translation. Here, we examine whether eEF1A2 directly regulates utrophin A expression and identify via an ELISA-based high-throughput screen, FDA-approved drugs that upregulate both eEF1A2 and utrophin A. Our results show that transient overexpression of eEF1A2 in mouse muscles causes an increase in IRES-mediated translation of utrophin A. Through the assessment of our screen, we reveal 7 classes of FDA-approved drugs that increase eEF1A2 and utrophin A protein levels. Treatment of mdx mice with the 2 top leads results in multiple improvements of the dystrophic phenotype. Here, we report that IRES-mediated translation of utrophin A via eEF1A2 is a critical mechanism of regulating utrophin A expression and reveal the potential of repurposed drugs for treating DMD via this pathway.

The viral prescription pad - a mixed methods study to determine the need for and utility of an educational tool for antimicrobial stewardship in primary health care.

BACKGROUND: In order to combat rising rates of antimicrobial resistant infections, it is vital that antimicrobial stewardship become embedded in primary health care (PHC). Despite the high use of antimicrobials in PHC settings, there is a lack of data regarding the integration of antimicrobial stewardship programs (ASP) in non-hospital settings. Our research aimed to determine which antimicrobial stewardship interventions are optimal to introduce into PHC clinics beginning to engage with an ASP, as well as how to optimize those interventions. This work became focused specifically around management of viral upper respiratory tract infections (URTIs), as these infections are one of the main sources of inappropriate antibiotic use. METHODS: This mixed methods study of sequential explanatory design was developed through three research projects over 3 years in Regina, Saskatchewan, Canada. First, a survey of PHC providers was performed to determine their perceived needs from a PHC-based ASP. From this work, a "viral prescription pad" was developed to provide a tool to help PHC providers engage in patient education regarding appropriate antimicrobial use, specifically for URTIs. Next, interviews were performed with family physicians to discuss their perceived utility of this tool. Finally, we performed a public survey to determine preferences for the medium by which information is received regarding symptom management for viral URTIs. RESULTS: The majority of PHC providers responding to the initial survey indicated they were improperly equipped with tools to aid in promoting conversations with patients and providing education about the appropriate use of antimicrobials. Following dissemination of the viral prescription pad and semi-structured interviews with family physicians, the viral prescription pad was deemed to be a useful educational tool. However, about half of the physicians interviewed indicated they did not actually provide a viral prescription to patients when providing advice on symptom management for viral URTIs. When asked about their preferences, 76% of respondents to the public survey indicated they would prefer to receive written or a combination of verbal and written information in this circumstance. CONCLUSIONS: PHC providers indicated a need for educational tools to promote conversations with patients and provide education about the appropriate use of antimicrobials. Viral prescription pads were regarded by family physicians and patients as useful tools in facilitating discussion on the appropriate use of antimicrobials. PHC providers should exercise caution in opting out of providing written forms of information, as many respondents to the general public survey indicated their preference in receiving both verbal and written information.

Educational intervention to reduce treatment of asymptomatic bacteriuria in long-term care.

OBJECTIVE: To determine if an educational intervention can decrease the inappropriate antibiotic treatment of long-term care (LTC) residents with asymptomatic bacteriuria (ASB). DESIGN: Prospective chart audit between May and July 2017. SETTING: Seven LTC facilities in Regina, Saskatchewan, Canada. PARTICIPANTS: Chart audits were performed on all LTC residents over 18 years of age with a positive urine culture. Educational sessions and tools were available to all clinical staff at participating LTC facilities. INTERVENTION: Fifteen-minute educational sessions were provided to LTC facility staff outlining the harms of unnecessary antibiotic use, antibiotic resistance and the diagnostic criteria of a urinary tract infection (UTI). Educational sessions were complimented with posters and pocket cards that summarised UTI diagnostic criteria. MAIN OUTCOME MEASURE: The primary outcome measure was the number of residents who received inappropriate antibiotic treatment for ASB. Secondary outcome measures included the appropriateness of urine culture tests, number of tests and cost associated with inappropriate treatments. RESULTS: In the preintervention period, 172 urine culture and sensitivity (UC&S) tests were performed, 62 (36.0%) were positive and 50/62 (80.6%) residents had ASB based on chart review. In the postintervention period, 151 UC&S tests were performed, 50 (33.1%) were positive and 35/50 (70.0%) residents had ASB. There was a statistically significant decrease in the number of residents treated with antibiotics for ASB, from 45/50 (90%) preintervention to 22/35 (62.9%) postintervention (χ2=9.087, p=0.003). CONCLUSIONS: An educational intervention was associated with a statistically significant decrease in inappropriate antibiotic treatment of LTC residents with ASB.

Spinocerebellar ataxia type 29 due to mutations in ITPR1: a case series and review of this emerging congenital ataxia.

BACKGROUND: Spinocerebellar ataxia type 29 (SCA29) is an autosomal dominant, non-progressive cerebellar ataxia characterized by infantile-onset hypotonia, gross motor delay and cognitive impairment. Affected individuals exhibit cerebellar dysfunction and often have cerebellar atrophy on neuroimaging. Recently, missense mutations in ITPR1 were determined to be responsible. RESULTS: Clinical information on 21 individuals from 15 unrelated families with ITPR1 mutations was retrospectively collected using standardized questionnaires, including 11 previously unreported singletons and 2 new patients from a previously reported family. We describe the genetic, clinical and neuroimaging features of these patients to further characterize the clinical features of this rare condition and assess for any genotype-phenotype correlation for this disorder. Our cohort consisted of 9 males and 12 females, with ages ranging from 28 months to 49 years. Disease course was non-progressive with infantile-onset hypotonia and delays in motor and speech development. Gait ataxia was present in all individuals and 10 (48%) were not ambulating independently between the ages of 3-12 years of age. Mild-to-moderate cognitive impairment was present in 17 individuals (85%). Cerebellar atrophy developed after initial symptom presentation in 13 individuals (72%) and was not associated with disease progression or worsening functional impairment. We identified 12 different mutations including 6 novel mutations; 10 mutations were missense (with 4 present in >1 individual), 1 a splice site mutation leading to an in-frame insertion and 1 an in-frame deletion. No specific genotype-phenotype correlations were observed within our cohort. CONCLUSIONS: Our findings document significant clinical heterogeneity between individuals with SCA29 in a large cohort of molecularly confirmed cases. Based on the retrospective observed clinical features and disease course, we provide recommendations for management. Further research into the natural history of SCA29 through prospective studies is an important next step in better understanding the condition.

Detailed Biochemical and Bioenergetic Characterization of FBXL4-Related Encephalomyopathic Mitochondrial DNA Depletion.

Mutations of FBXL4, which encodes an orphan mitochondrial F-box protein, are a recently identified cause of encephalomyopathic mtDNA depletion. Here, we describe the detailed clinical and biochemical phenotype of a neonate presenting with hyperlactatemia, leukoencephalopathy, arrhythmias, pulmonary hypertension, dysmorphic features, and lymphopenia. Next-generation sequencing in the proband identified a homozygous frameshift, c.1641_1642delTG, in FBXL4, with a surrounding block of SNP marker homozygosity identified by microarray. Muscle biopsy showed a paucity of mitochondria with ultrastructural abnormalities, mitochondrial DNA depletion, and profound deficiency of all respiratory chain complexes. Cell-based mitochondrial phenotyping in fibroblasts showed mitochondrial fragmentation, decreased basal and maximal respiration, absence of ATP-linked respiratory and leak capacity, impaired survival under obligate aerobic respiration, and reduced mitochondrial inner membrane potential, with relative sparing of mitochondrial mass. Cultured fibroblasts from the patient exhibited a more oxidized glutathione ratio, consistent with altered cellular redox poise. High-resolution respirometry of permeabilized muscle fibers showed marked deficiency of oxidative phosphorylation using a variety of mitochondrial energy substrates and inhibitors. This constitutes the fourth and most detailed report of FBXL4 deficiency to date. In light of our patient's clinical findings and genotype (homozygous frameshift), this phenotype likely represents the severe end of the FBXL4 clinical spectrum.

DNM1L-related mitochondrial fission defect presenting as refractory epilepsy.

Mitochondrial fission and fusion are dynamic processes vital to mitochondrial quality control and the maintenance of cellular respiration. In dividing mitochondria, membrane scission is accomplished by a dynamin-related GTPase, DNM1L, that oligomerizes at the site of fission and constricts in a GTP-dependent manner. There is only a single previous report of DNM1L-related clinical disease: a female neonate with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF; OMIM #614388), a lethal disorder characterized by cerebral dysgenesis, seizures, lactic acidosis, elevated very long chain fatty acids, and abnormally elongated mitochondria and peroxisomes. Here, we describe a second individual, diagnosed via whole-exome sequencing, who presented with developmental delay, refractory epilepsy, prolonged survival, and no evidence of mitochondrial or peroxisomal dysfunction on standard screening investigations in blood and urine. EEG was nonspecific, showing background slowing with frequent epileptiform activity at the frontal and central head regions. Electron microscopy of skeletal muscle showed subtle, nonspecific abnormalities of cristal organization, and confocal microscopy of patient fibroblasts showed striking hyperfusion of the mitochondrial network. A panel of further bioenergetic studies in patient fibroblasts showed no significant differences versus controls. The proband's de novo DNM1L variant, NM_012062.4:c.1085G>A; NP_036192.2:p.(Gly362Asp), falls within the middle (oligomerization) domain of DNM1L, implying a likely dominant-negative mechanism. This disorder, which presents nonspecifically and affords few diagnostic clues, can be diagnosed by means of DNM1L sequencing and/or confocal microscopy.

Identification of a pathogenic FTO mutation by next-generation sequencing in a newborn with growth retardation and developmental delay.

BACKGROUND: A homozygous loss-of-function mutation p.(Arg316Gln) in the fat mass and obesity-associated (FTO) gene, which encodes for an iron and 2-oxoglutarate-dependent oxygenase, was previously identified in a large family in which nine affected individuals present with a lethal syndrome characterised by growth retardation and multiple malformations. To date, no other pathogenic mutation in FTO has been identified as a cause of multiple congenital malformations. METHODS: We investigated a 21-month-old girl who presented distinctive facial features, failure to thrive, global developmental delay, left ventricular cardiac hypertrophy, reduced vision and bilateral hearing loss. We performed targeted next-generation sequencing of 4813 clinically relevant genes in the patient and her parents. RESULTS: We identified a novel FTO homozygous missense mutation (c.956C>T; p.(Ser319Phe)) in the affected individual. This mutation affects a highly conserved residue located in the same functional domain as the previously characterised mutation p.(Arg316Gln). Biochemical studies reveal that p.(Ser319Phe) FTO has reduced 2-oxoglutarate turnover and N-methyl-nucleoside demethylase activity. CONCLUSION: Our findings are consistent with previous reports that homozygous mutations in FTO can lead to rare growth retardation and developmental delay syndrome, and further support the proposal that FTO plays an important role in early development of human central nervous and cardiovascular systems.

A differential response to newt regeneration extract by C2C12 and primary mammalian muscle cells.

BACKGROUND: Dedifferentiation, a process whereby differentiated cells lose their specialized characteristics and revert to a less differentiated state, plays a key role in the regeneration process in urodele amphibians such as the red spotted newt, Notophthalmus viridescens. Dedifferentiation of fully mature tissues is generally absent in mammalian cells. Previous studies have shown that mouse C2C12 multinucleated myotubes treated with extract derived from regenerating newt forelimbs can re-enter the cell cycle, fragment into mononucleated cells, and proliferate. However, this response has been difficult to replicate. METHODS: We isolated extract from early newt forelimb regenerates and assessed its effects on differentiation of proliferating primary and C2C12 myoblasts. We also treated fully differentiated primary and C2C12 myotube cultures with extract and assessed cell cycle re-entry and myotube fragmentation. RESULTS: We have confirmed the results obtained in C2C12 cells and expanded these studies to also examine the effects of newt regeneration extracts on primary muscle cells. Newt extract can block differentiation of both C2C12 and primary myoblasts. Once differentiation is induced, treatment with newt extract causes cell cycle re-entry and fragmentation of C2C12 myotubes. Downregulation of p21 and muscle-specific markers is also induced. Primary myotubes also fragment in response to extract treatment, and the fragmented cells remain viable for long periods of time in culture. However, unlike C2C12 cells, primary muscle cells do not re-enter the cell cycle in response to treatment with newt extracts. CONCLUSIONS: Dedifferentiation of fully mature muscle occurs during regeneration in the newt forelimb to contribute cells to the regeneration process. Our study shows that extracts derived from regenerating newt forelimbs can induce dedifferentiation, cell cycle re-entry, and fragmentation of mouse C2C12 cells but can only induce fragmentation in primary muscle cells.