Mitochondrial aminoacyl-tRNA synthetases trigger unique compensatory mechanisms in neurons.

Mitochondrial aminoacyl-tRNA synthetase (mt-ARS) mutations cause severe, progressive, and often lethal diseases with highly heterogeneous and tissue-specific clinical manifestations. This study investigates the molecular mechanisms triggered by three different mt-ARS defects caused by biallelic mutations in AARS2, EARS2, and RARS2, using an in vitro model of human neuronal cells. We report distinct molecular mechanisms of mitochondrial dysfunction among the mt-ARS defects studied. Our findings highlight the ability of proliferating neuronal progenitor cells (iNPCs) to compensate for mitochondrial translation defects and maintain balanced levels of oxidative phosphorylation (OXPHOS) components, which becomes more challenging in mature neurons. Mutant iNPCs exhibit unique compensatory mechanisms, involving specific branches of the integrated stress response, which may be gene-specific or related to the severity of the mitochondrial translation defect. RNA sequencing revealed distinct transcriptomic profiles showing dysregulation of neuronal differentiation and protein translation. This study provides valuable insights into the tissue-specific compensatory mechanisms potentially underlying the phenotypes of patients with mt-ARS defects. Our novel in vitro model may more accurately represent the neurological presentation of patients and offer an improved platform for future investigations and therapeutic development.

Metabolic shift underlies recovery in reversible infantile respiratory chain deficiency.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.

Impact of setup errors on the robustness of linac-based single-isocenter coplanar and non-coplanar VMAT plans for multiple brain metastases.

PURPOSE: Patient setup errors have been a primary concern impacting the dose delivery accuracy in radiation therapy. A robust treatment plan might mitigate the effects of patient setup errors. In this reported study, we aimed to evaluate the impact of translational and rotational errors on the robustness of linac-based, single-isocenter, coplanar, and non-coplanar volumetric modulated arc therapy treatment plans for multiple brain metastases. METHODS: Fifteen patients were retrospectively selected for this study with a combined total of 49 gross tumor volumes (GTVs). Single-isocenter coplanar and non-coplanar plans were generated first with a prescribed dose of 40 Gy in 5 fractions or 42 Gy in 7 fractions to cover 95% of planning target volume (PTV). Next, four setup errors (+1  and +2 mm translation, and +1° and +2° rotation) were applied individually to generate modified plans. Different plan quality evaluation metrics were compared between coplanar and non-coplanar plans. 3D gamma analysis (3%/2 mm) was performed to compare the modified plans (+2 mm and +2° only) and the original plans. Paired t-test was conducted for statistical analysis. RESULTS: After applying setup errors, variations of all plan evaluation metrics were similar (p > 0.05). The worst case for V100% to GTV was 92.07% ± 6.13% in the case of +2 mm translational error. 3D gamma pass rates were > 90% for both coplanar (+2 mm and +2°) and the +2 mm non-coplanar groups but was 87.40% ± 6.89% for the +2° non-coplanar group. CONCLUSION: Translational errors have a greater impact on PTV and GTV dose coverage for both planning methods. Rotational errors have a greater negative impact on gamma pass rates of non-coplanar plans. Plan evaluation metrics after applying setup errors showed that both coplanar and non-coplanar plans were robust and clinically acceptable.

De Novo and Bi-allelic Pathogenic Variants in NARS1 Cause Neurodevelopmental Delay Due to Toxic Gain-of-Function and Partial Loss-of-Function Effects.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.

NCAM1 and GDF15 are biomarkers of Charcot-Marie-Tooth disease in patients and mice.

Molecular markers scalable for clinical use are critical for the development of effective treatments and the design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We collected serum from mouse models of CMT1A (C61 het), CMT2D (GarsC201R, GarsP278KY), CMT1X (Gjb1-null), CMT2L (Hspb8K141N) and from CMT patients with genotypes including CMT1A (PMP22d), CMT2D (GARS), CMT2N (AARS) and other rare genetic forms of CMT. The severity of neuropathy in the patients was assessed by the CMT Neuropathy Examination Score (CMTES). We performed multitargeted proteomics on both sample sets to identify proteins elevated across multiple mouse models and CMT patients. Selected proteins and additional potential biomarkers, such as growth differentiation factor 15 (GDF15) and cell free mitochondrial DNA, were validated by ELISA and quantitative PCR, respectively. We propose that neural cell adhesion molecule 1 (NCAM1) is a candidate biomarker for CMT, as it was elevated in Gjb1-null, Hspb8K141N, GarsC201R and GarsP278KY mice as well as in patients with both demyelinating (CMT1A) and axonal (CMT2D, CMT2N) forms of CMT. We show that NCAM1 may reflect disease severity, demonstrated by a progressive increase in mouse models with time and a significant positive correlation with CMTES neuropathy severity in patients. The increase in NCAM1 may reflect muscle regeneration triggered by denervation, which could potentially track disease progression or the effect of treatments. We found that member proteins of the complement system were elevated in Gjb1-null and Hspb8K141N mouse models as well as in patients with both demyelinating and axonal CMT, indicating possible complement activation at the impaired nerve terminals. However, complement proteins did not correlate with the severity of neuropathy measured on the CMTES scale. Although the complement system does not seem to be a prognostic biomarker, we do show complement elevation to be a common disease feature of CMT, which may be of interest as a therapeutic target. We also identify serum GDF15 as a highly sensitive diagnostic biomarker, which was elevated in all CMT genotypes as well as in Hspb8K141N, Gjb1-null, GarsC201R and GarsP278KY mouse models. Although we cannot fully explain its origin, it may reflect increased stress response or metabolic disturbances in CMT. Further large and longitudinal patient studies should be performed to establish the value of these proteins as diagnostic and prognostic molecular biomarkers for CMT.

From burden to benefit: a multi-site study of the impact of allied health work-based learning placements on patient care quality.

Allied health clinical placements take place within an increasingly overstretched health care system where demand for services often exceeds availability of resources. Within this environment, student placements are often perceived as an additional burden to an already overwhelmed workforce. This study explored whether the quality of patient care was enhanced when services were re-designed using a collaborative partnership approach to more purposefully integrate students into delivery of care. Using an embedded multiple case study design, data were collected through focus groups and interviews, patient experience surveys, and secondary administrative data sources. Cases were across physiotherapy and occupational therapy in six different hospital settings. Perception of care provided by students was viewed positively by all stakeholders, including patients. Perceived health outcomes of faster improvement of health condition, improved mobility and function identified through our qualitative findings were supported by quantitative service delivery markers such as increased therapy sessions, more patients being discharged home instead of to other care facilities and reduced length of stay. Health care providers and students alike perceived improvements in service efficiencies whilst maintaining high quality care. This study has provided preliminary evidence towards improved patient care when a partnering approach is adopted whereby students are intentionally integrated into services that otherwise might not have been delivered. Furthermore, it has shifted the associated narrative from students as additional burden to students as benefit.

Closing the health gap in Central Australia: reduction in Indigenous Australian inpatient self-discharge rates following routine collaboration with Aboriginal Health Workers.

BACKGROUND: Indigenous Australians experience significant socioeconomic disadvantage and healthcare disparity compared to non-Indigenous Australians. A retrospective cohort study to describe the association between rates of self-discharge in Indigenous orthopaedic patients and the introduction of routine Aboriginal Liaison Officers (ALO) within the Orthopaedic multi-disciplinary team (MDT) was performed. METHODS: ALO were introduced within our routine Orthopaedic MDT on the 22nd of February 2021. Two patient cohorts were analysed, Group 1; patients admitted in the 9-months prior to inclusion of ALO, and Group 2; patients admitted within 9-months thereafter. The primary outcome of interest was the rate of self-discharge among Indigenous patients. Secondary outcomes of interest were the stage of treatment when patients self-discharged, recurrent self-discharge, risk factors for self-discharge and association between self-discharge and length of hospital stay. RESULTS: Introduction of ALO within routine Orthopaedic MDT was associated with a significant 37% reduced risk of self-discharge among Indigenous patients (p = 0·009), and significantly fewer self-discharges before their definitive surgical and medical treatment (p = 0·0024), or before completion of postoperative intravenous antibiotic treatment (p = 0·030). There was no significant change in the risk of recurrent self-discharge (p = 0·557). Risk factors for self-discharge were younger age; pensioners or unemployed; residents of Alice Springs Town-Camps or of communities within 51 to 100 km of Alice Springs; and those diagnosed with lacerations of the upper limb, but without tendon injury, wound and soft tissue infections or osteomyelitis. In Group 2, the odds of self-discharge decreased with increased length of hospital stay (p = 0·040). CONCLUSIONS: Routine inclusion of ALO within the Orthopaedic MDT reduced the risk of self-discharge in Indigenous patients. Those who self-discharged did so only after critical aspects of their care were met.

The feasibility of implementing a cultural mentoring program alongside pain management and physical rehabilitation for chronic musculoskeletal conditions: results of a controlled before-and-after pilot study.

BACKGROUND: Culturally diverse communities face barriers managing chronic musculoskeletal pain conditions including navigation challenges, sub-optimal healthcare provider engagement and difficulty adopting self-management behaviours. OBJECTIVES: To explore the feasibility and trends of effectiveness of implementing a cultural mentoring program alongside clinical service delivery. METHODS: This quasi-experimental controlled before-and-after multiple case study was conducted in three hospital-based services that provide treatment for patients with musculoskeletal pain. Two prospective cohorts, a pre-implementation and a post-implementation cohort, of adults with chronic musculoskeletal pain who attended during the 6-month recruitment phase, were eligible if they self-identified with one of the cultures prioritised for mentoring by the clinic. The pre-implementation cohort received routine care for up to 3-months, while the post-implementation cohort received up to 3-months of cultural mentoring integrated into routine care (3 to 10 sessions), provided by a consumer (n = 6) with lived experience. Feasibility measures (recruitment and completion rates, attendance, satisfaction), and trends of effectiveness (Patient Activation Measure and Health Literacy Questionnaire items one and six) were collated over 3-months for both cohorts. Outcomes were presented descriptively and analysed using Mann-Whitney U-tests for between-group comparisons. Translation and transcription of post-treatment semi-structured interviews allowed both cohorts' perspectives of treatment to be analysed using a Rapid Assessment Process. RESULTS: The cultural mentor program was feasible to implement in clinical services with comparable recruitment rates (66% pre-implementation; 61% post-implementation), adequate treatment attendance (75% pre-implementation; 89% post-implementation), high treatment satisfaction (97% pre-implementation; 96% post-implementation), and minimal participant drop-out (< 5%). Compared to routine care (n = 71), patients receiving mentoring (n = 55) achieved significantly higher Patient Activation Measure scores (median change 0 vs 10.3 points, p < 0.01) at 3-months, while Health Literacy Questionnaire items did not change for either cohort over time. Three themes underpinned participant experiences and acceptability of the mentoring intervention: 'expectational priming', 'lived expertise' and 'collectivist orientation' to understand shared participant experiences and explore the potential differential effect of the mentoring intervention. CONCLUSION: Participant experiences and observations of improved patient activation provide support for the acceptability of the mentoring intervention integrated into routine care. These results support the feasibility of conducting a definitive trial, while also exploring issues of scalability and sustainability.

Prevalence of Obstructive Sleep Apnea With Floppy Eyelid Syndrome: A Systematic Review and Meta-analysis.

PURPOSE: This study investigated the prevalence of obstructive sleep apnea (OSA) in floppy eyelid syndrome (FES) patients and evaluated the severity of OSA with FES prevalence. METHODS: Cochrane CENTRAL, Medline, Science Direct, Google Scholar, and PubMed databases were searched for studies on FES patients and its association with OSA syndrome, of any design, published from January 1, 1997, to January 1, 2022. A random-effects model that weighted the studies was used when there was heterogeneity between studies ( p < 0.10) and if I 2 values were more than 50%. All p values were 2-tailed and considered statistically significant if <0.05. RESULTS: A total of 12 studies comprising 511 patients were included in this meta-analysis. Of these, 368 were male (77.6%) and the average age was 55.10 years. The overall prevalence of OSA in FES patients was 57.1% (95% CI: 46.5-74.8%), M:F ratio was 48:1 (98% male), and 69.1% of patients received their OSA diagnosis at the time of the study. Of those with FES, tear film abnormalities were the most common ocular comorbidity (78.9%) followed by keratoconus (20.6%), glaucoma (9.8%), and lower eyelid ectropion (4.6%). Obesity was the most common systemic morbidity (43.7%) followed by hypertension (34.0%) and diabetes mellitus (17.9%). CONCLUSION: This meta-analysis demonstrates OSA is a common comorbidity in the FES population. Ophthalmologists are often the first to evaluate patients with FES, and considering this coincidence, routine screens for sleep apnea symptoms in at-risk FES patients should be undertaken. Large case-control studies are required to better elucidate the exact prevalence of OSA and other morbidities in patients with FES, and to better understand the etiology of FES.

AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy.

Mutations in the GJB1 gene, encoding the gap junction (GJ) protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We developed a gene therapy approach for CMT1X using an AAV9 vector to deliver the GJB1/Cx32 gene under the myelin protein zero (Mpz) promoter for targeted expression in Schwann cells. Lumbar intrathecal injection of the AAV9-Mpz.GJB1 resulted in widespread biodistribution in the peripheral nervous system including lumbar roots, sciatic and femoral nerves, as well as in Cx32 expression in the paranodal non-compact myelin areas of myelinated fibers. A pre-, as well as post-onset treatment trial in Gjb1-null mice, demonstrated improved motor performance and sciatic nerve conduction velocities along with improved myelination and reduced inflammation in peripheral nerve tissues. Blood biomarker levels were also significantly ameliorated in treated mice. This study provides evidence that a clinically translatable AAV9-mediated gene therapy approach targeting Schwann cells could potentially treat CMT1X.

Synergism between Rifampicin and Cationic Polyurethanes Overcomes Intrinsic Resistance of Escherichia coli.

Antibiotic-resistant Gram-negative bacteria are emergent pathogens, causing millions of infections worldwide. While there are several classes of antibiotics that are effective against Gram-positive bacteria, the outer membrane (OM) of Gram-negative bacteria excludes high-molecular-weight hydrophobic antibiotics, making these species intrinsically resistant to several classes of antibiotics, including polyketides, aminocoumarins, and macrolides. The overuse of antibiotics such as ß-lactams has also promoted the spread of resistance genes throughout Gram-negative bacteria, including the production of extended spectrum ß-lactamases (ESBLs). The combination of innate and acquired resistance makes it extremely challenging to identify antibiotics that are effective against Gram-negative bacteria. In this study, we have demonstrated the synergistic effect of outer membrane-permeable cationic polyurethanes with rifampicin, a polyketide that would otherwise be excluded by the OM, on different strains of E. coli, including a clinically isolated uropathogenic multidrug-resistant (MDR) E. coli. Rifampicin combined with a low-dose treatment of a cationic polyurethane reduced the MIC in E. coli of rifampicin by up to 64-fold. The compositions of cationic polyurethanes were designed to have low hemolysis and low cell cytotoxicity while maintaining high antibacterial activity. Our results demonstrate the potential to rescue the large number of available OM-excluded antibiotics to target normally resistant Gram-negative bacteria via synergistic action with these cationic polyurethanes, acting as a novel antibiotic adjuvant class.

A Mixed-Methods Investigation into Patients' Decisions to Attend an Emergency Department for Chronic Pain.

OBJECTIVE: This study explored factors that underpin decisions to seek emergency department (ED) care for chronic noncancer pain in patients identifying as culturally and linguistically diverse (CALD) or Australian born. DESIGN AND METHODS: This mixed-methods study was underpinned by the Behavioral Model of Health Services Use conceptual framework. Consenting consecutive patients attending the ED for a chronic pain condition were recruited to a CALD (n = 45) or Australian-born (n = 45) cohort. Statistical comparisons compared the demographic, pain, health literacy, and episode of care profiles of both cohorts. Twenty-three CALD and 16 Australian-born participants consented to an audio-recorded semi-structured interview (n = 24) or focus group (n = 5 focus groups) conducted in their preferred language. Interviews were translated and transcribed into English for analysis using applied thematic analysis, guided by the conceptual framework. Data were triangulated to investigate the patterns of ED utilization and contributing factors for both cohorts. RESULTS: ED attendance was a product of escalating distress, influenced by the degree to which participants' perceived needs outweighed their capacity to manage their pain. This interaction was amplified by the presence of predisposing factors, including constrained social positions, trauma exposure, and biomedical health beliefs. Importantly, experiences varied between the two cohorts with higher degrees of pain catastrophizing, lower health literacy, and greater social challenges present for the CALD cohort. CONCLUSION: This study highlights the role contextual factors play in amplifying pain-related distress for CALD and Australian-born patients with chronic pain. The findings support a need for health care providers to recognize features of higher vulnerability and consider streamlining access to available support services.

Moving beyond solutionism: Re-imagining placements through an activity systems lens.

INTRODUCTION: Clinical placements are central to the process of preparing future health professionals for practice. Health care environments are increasingly complex and demanding with clinical placements often being perceived as a burden on busy health professionals giving rise to a service-education tension. This tension creates a situation ripe for simplistic solutionist approaches. For example, characterising the problem of clinical placements as students negatively impacting on service productivity results in a reductionist solution such as universities compensating health services for student education. Challenges faced by placement seekers and placement providers are multifaceted and complex requiring a more sophisticated understanding and response to the challenges of involving students in the workplace to prepare them for the future workforce. RE-CONCEPTUALISATION: We argue that the health and education systems have become de-coupled. Learning and working are seen as distinct activities that are at odds with one another. Re-imagining the purposes and practices of clinical placements for the mutual benefit of patients, health services and students may fruitfully address this disconnect. WORKED EXAMPLE: We present a worked example using the conceptual and analytical tools of cultural-historical activity theory to articulate what we have learnt about this health-education disconnect. Our worked example draws on research involving a series of clinical education case studies within acute care contexts. CONCLUSION: Through the lens of cultural-historical activity theory, we highlight that solutionist approaches are entrenched in a de-coupling of health from education where the shared object of preparing the future workforce is fragmented. Successful re-coupling requires a partnership that is founded on a shared commitment to preparing the future workforce; recognises that learning and practice are inseparable; and understands that both activity systems are fluid and that collaboration to stay focused on the shared object of preparing the future workforce is complex, challenging and ongoing work.

Effectiveness of a coordinated support system linking public hospitals to a health coaching service compared with usual care at discharge for patients with chronic low back pain: protocol for a randomised controlled trial.

BACKGROUND: Although many people with chronic low back pain (LBP) improve following conservative treatment, one in five will experience worsening symptoms after discharge from treatment and seek health care again. The current LBP clinical care pathway in many health services lacks a well-integrated, systematic approach to support patients to remain physically active and self-manage their symptoms following discharge from treatment. Health coaching can support people to improve physical activity levels and may potentially reduce health care utilisation for LBP. The primary aim of this study is to evaluate the effect of introducing a coordinated support system (linking hospital outpatient physiotherapy services to a public health coaching service) at discharge from LBP treatment, on the future use of hospital, medical, and health services for LBP, compared with usual care provided at discharge. METHODS: Three hundred and seventy-four adults with chronic non-specific LBP will be recruited from the outpatient physiotherapy departments of public hospitals in New South Wales, Australia. Participants will be individually randomised to a support system (n = 187) or usual care group (n = 187). All participants will receive usual care provided at discharge from treatment. Participants allocated to the support system will also receive up to 10 telephone-based health coaching sessions, delivered by the Get Healthy Service®, over a 6-month period. Health coaches will monitor and support participants to improve physical activity levels and achieve personal health-related goals. The primary outcome is the total number of encounters with hospital, medical, and health services for LBP, at 12 months from baseline. A within-trial economic evaluation will quantify the incremental costs and benefits of the support system from a health system perspective, to support reimbursement decision making. DISCUSSION: This study will establish the effect of a coordinated support system, introduced at discharge from treatment, on the future use of hospital, medical, and health services for LBP and various health outcomes. CONCLUSION: Innovative community-driven solutions to support people with chronic LBP after discharge from treatment are urgently needed. Study findings will help inform health care policy and clinical practice in Australia. TRIAL REGISTRATION: Prospectively registered on the Australian New Zealand Clinical Trials Registry ( ACTRN12620000889954 ) on 10/09/2020.

Integrating Mobile-health, health coaching, and physical activity to reduce the burden of chronic low back pain trial (IMPACT): a pilot randomised controlled trial.

BACKGROUND: Low back pain is one of the most prevalent musculoskeletal conditions and the highest contributor to disability in the world. It is characterized by frequent relapses leading to additional care-seeking. Engagement in leisure physical activity is associated with lower recurrences and better prognosis and potentially reduced care-seeking. Our aim was to investigate the feasibility and preliminary efficacy of a patient-centred physical activity intervention, supported by health coaching and mobile health, to reduce care-seeking, pain and disability in patients with chronic low back pain after treatment discharge. METHODS: We conducted a pilot randomised controlled trial with blinded outcome assessment. Sixty-eight participants were recruited from four public outpatient physiotherapy departments and the general community in Sydney. The intervention group received a physical activity information booklet, plus one face-to-face and 12 telephone-based health coaching sessions. The intervention was supported by an internet-based application and an activity tracker (Fitbit). Control group (standard care) received the physical activity information booklet and advice to stay active. Feasibility measures included recruitment rate, intervention compliance, data completeness, and participant satisfaction. Primary outcomes were care-seeking, pain levels and activity limitation. Outcomes were assessed at baseline, 6-month follow-up and weekly for 6 months. RESULTS: Ninety potential participants were invited over 15 months, with 68 agreeing to take part (75%). Overall, 903 weekly questionnaires were answered by participants from a total of 1107 sent (89%). Participants were largely satisfied with the intervention (mean = 8.7 out of 10 on satisfaction scale). Intervention group participants had a 38% reduced rate of care-seeking (Incidence Rate Ratio (IRR): 0.62, 95% CI: 0.32 to 1.18, p = 0.14, using multilevel mixed-effects Poisson regression analysis) compared to standard care, although none of the estimates was statistically significant. No between groups differences were found for pain levels or activity limitation. CONCLUSION: The health coaching physical activity approach trialed here is feasible and well accepted by participants and may reduce care-seeking in patients with low back pain after treatment discharge, although further evaluation with an adequately powered trial is needed. TRIAL REGISTRATION: Australian and New Zealand Trial Registry ACTRN12615000189527 . Registered prospectively on 26-02-2015.

PFN2 and GAMT as common molecular determinants of axonal Charcot-Marie-Tooth disease.

BACKGROUND: Charcot-Marie-Tooth type 2 (CMT2) neuropathy is characterised by a vast clinical and genetic heterogeneity complicating its diagnosis and therapeutic intervention. Identification of molecular signatures that are common to multiple CMT2 subtypes can aid in developing therapeutic strategies and measuring disease outcomes. METHODS: A proteomics-based approach was performed on lymphoblasts from CMT2 patients genetically diagnosed with different gene mutations to identify differentially regulated proteins. The candidate proteins were validated through real-time quantitative PCR and western blotting on lymphoblast samples of patients and controls, motor neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) and sciatic nerves of CMT2 mouse models. RESULTS: Proteomic profiling of patient lymphoblasts resulted in the identification of profilin 2 (PFN2) and guanidinoacetate methyltransferase (GAMT) as commonly downregulated proteins in different genotypes compared with healthy controls. This decrease was also observed at the transcriptional level on screening 43 CMT2 patients and 22 controls, respectively. A progressive decrease in PFN2 expression with age was observed in patients, while in healthy controls its expression increased with age. Reduced PFN2 expression was also observed in motor neurons differentiated from CMT2 patient-derived iPSCs and sciatic nerves of CMT2 mice when compared with controls. However, no change in GAMT levels was observed in motor neurons and CMT2 mouse-derived sciatic nerves. CONCLUSIONS: We unveil PFN2 and GAMT as molecular determinants of CMT2 with possible indications of the role of PFN2 in the pathogenesis and disease progression. This is the first study describing biomarkers that can boost the development of therapeutic strategies targeting a wider spectrum of CMT2 patients.

Extranucleosomal DNA enhances the activity of the LSD1/CoREST histone demethylase complex.

The promoter regions of active genes in the eukaryotic genome typically contain nucleosomes post-translationally modified with a trimethyl mark on histone H3 lysine 4 (H3K4), while transcriptional enhancers are marked with monomethylated H3K4. The flavin-dependent monoamine oxidase LSD1 (lysine-specific demethylase 1, also known as KDM1) demethylates mono- and dimethylated H3K4 in peptide substrates, but requires the corepressor protein, CoREST, to demethylate nucleosome substrates. The molecular basis for how the LSD1/CoREST complex interacts with its physiological nucleosome substrate remains largely unknown. We examine here the role of extranucleosomal DNA beyond the nucleosome core particle for LSD1/CoREST function. Our studies of LSD1/CoREST's enzyme activity and nucleosome binding show that extranucleosomal DNA dramatically enhances the activity of LSD1/CoREST, and that LSD1/CoREST binds to the nucleosome as a 1:1 complex. Our photocrosslinking experiments further indicate both LSD1 and CoREST subunits are in close contact with DNA around the nucleosome dyad as well as extranucleosomal DNA. Our results suggest that the LSD1/CoREST interacts with extranucleosomal DNA when it productively engages its nucleosome substrate.

Elimination of truncated recombinant protein expressed in Escherichia coli by removing cryptic translation initiation site.

Undesirable truncated recombinant protein products pose a special expression and purification challenge because such products often share similar chromatographic properties as the desired full length protein. We describe here our observation of both full length and a truncated form of a yeast protein (Gcn5) expressed in Escherichia coli, and the reduction or elimination of the truncated form by mutating a cryptic Shine-Dalgarno or START codon within the Gcn5 coding region. Unsuccessful attempts to engineer in a cryptic translation initiation site into other recombinant proteins suggest that cryptic Shine-Dalgarno or START codon sequences are necessary but not sufficient for cryptic translation in E. coli.

What Safe Zone? The Vast Majority of Dislocated THAs Are Within the Lewinnek Safe Zone for Acetabular Component Position.

BACKGROUND: Numerous factors influence total hip arthroplasty (THA) stability including surgical approach and soft tissue tension, patient compliance, and component position. One long-held tenet regarding component position is that cup inclination and anteversion of 40° ± 10° and 15° ± 10°, respectively, represent a "safe zone" as defined by Lewinnek that minimizes dislocation after primary THA; however, it is clear that components positioned in this zone can and do dislocate. QUESTIONS/PURPOSES: We sought to determine if these classic radiographic targets for cup inclination and anteversion accurately predicted a safe zone limiting dislocation in a contemporary THA practice. METHODS: From a cohort of 9784 primary THAs performed between 2003 and 2012 at one institution, we retrospectively identified 206 THAs (2%) that subsequently dislocated. Radiographic parameters including inclination, anteversion, center of rotation, and limb length discrepancy were analyzed. Mean followup was 27 months (range, 0-133 months). RESULTS: The majority (58% [120 of 206]) of dislocated THAs had a socket within the Lewinnek safe zone. Mean cup inclination was 44° ± 8° with 84% within the safe zone for inclination. Mean anteversion was 15° ± 9° with 69% within the safe zone for anteversion. Sixty-five percent of dislocated THAs that were performed through a posterior approach had an acetabular component within the combined acetabular safe zones, whereas this was true for only 33% performed through an anterolateral approach. An acetabular component performed through a posterior approach was three times as likely to be within the combined acetabular safe zones (odds ratio [OR], 1.3; 95% confidence interval [CI], 1.1-1.6) than after an anterolateral approach (OR, 0.4; 95% CI, 0.2-0.7; p < 0.0001). In contrast, acetabular components performed through a posterior approach (OR, 1.6; 95% CI, 1.2-1.9) had an increased risk of dislocation compared with those performed through an anterolateral approach (OR, 0.8; 95% CI, 0.7-0.9; p < 0.0001). CONCLUSIONS: The historical target values for cup inclination and anteversion may be useful but should not be considered a safe zone given that the majority of these contemporary THAs that dislocated were within those target values. Stability is likely multifactorial; the ideal cup position for some patients may lie outside the Lewinnek safe zone and more advanced analysis is required to identify the right target in that subgroup. LEVEL OF EVIDENCE: Level III, therapeutic study.

Integrating Mobile health and Physical Activity to reduce the burden of Chronic low back pain Trial (IMPACT): a pilot trial protocol.

BACKGROUND: It is well recognised that low back pain is a significant public health problem and engagement in moderate levels of physical activity is associated with positive outcomes. Conservative active care, such as exercise, is effective in reducing pain and disability associated with chronic low back pain. However, a rapid decline in clinical outcomes is commonly seen after discharge from treatment. METHODS/DESIGN: We will conduct a randomised controlled trial to investigate the effectiveness of a mobile health supported physical activity intervention (compared to standard care) in care-seeking, pain and disability in people with chronic low back pain after discharge from treatment. We will recruit 68 patients with chronic low back pain following discharge from an outpatient hospital program, who will be randomly allocated to the physical activity intervention (n = 34) or the standard care group (n = 34) and monitored for 6 months. The physical activity intervention will involve a physical activity advice booklet, a face-to-face health coaching session and 12 fortnightly follow-up telephone-based health coaching sessions. This intervention will be supported by provision of a specifically designed web app and a physical activity monitoring device (FitBit). The standard care group will receive the physical activity advice booklet only. DISCUSSION: This pilot trial will investigate a new model to prevent clinical decline in people following conservative treatment for chronic low back pain. If proven to be effective, this approach will constitute a major advance in the management of low back pain. Chronic patients who experience recurrent pain and disability after treatment are prone to seek additional care in the form of physiotherapy, medication, emergency department attendance, specialist consultation or spinal surgery. This model aims to maintain functional levels and reduce care-seeking empowering patients to self-manage their low back pain by offering them a contemporary patient-centred physical activity program with the support of mobile health technology. The outcomes of this trial will have immediate implications for clinical practice. TRIAL REGISTRATION: ACTRN12615000189527 (26-02-2015).