Prolonged voluntary wheel running reveals unique adaptations in mdx mice treated with microdystrophin constructs ± the nNOS-binding site.

We tested the effects of prolonged voluntary wheel running on the muscle function of mdx mice treated with one of two different microdystrophin constructs. At 7 weeks of age mdx mice were injected with a single dose of AAV9-CK8-microdystrophin with (gene therapy 1, GT1) or without (gene therapy 2, GT2) the nNOS-binding domain and were assigned to one of four gene therapy treated groups: mdxRGT1 (run, GT1), mdxGT1 (no run, GT1), or mdxRGT2 (run,GT2), mdxGT2 (no run, GT2). There were two mdx untreated groups injected with excipient: mdxR (run, no gene therapy) and mdx (no run, no gene therapy). A third no treatment group, Wildtype (WT) received no injection and did not run. mdxRGT1, mdxRGT2 and mdxR performed voluntary wheel running for 52 weeks; WT and remaining mdx groups were cage active. Robust expression of microdystrophin occurred in diaphragm, quadriceps, and heart muscles of all treated mice. Dystrophic muscle pathology was high in diaphragms of non-treated mdx and mdxR mice and improved in all treated groups. Endurance capacity was rescued by both voluntary wheel running and gene therapy alone, but their combination was most beneficial. All treated groups increased in vivo plantarflexor torque over both mdx and mdxR mice. mdx and mdxR mice displayed ∼3-fold lower diaphragm force and power compared to WT values. Treated groups demonstrated partial improvements in diaphragm force and power, with mdxRGT2 mice experiencing the greatest improvement at ∼60% of WT values. Evaluation of oxidative red quadriceps fibers revealed the greatest improvements in mitochondrial respiration in mdxRGT1 mice, reaching WT levels. Interestingly, mdxGT2 mice displayed diaphragm mitochondrial respiration values similar to WT but mdxRGT2 animals showed relative decreases compared to the no run group. Collectively, these data demonstrate that either microdystrophin construct combined with voluntary wheel running increased in vivo maximal muscle strength, power, and endurance. However, these data also highlighted important differences between the two microdystrophin constructs. GT1, with the nNOS-binding site, improved more markers of exercise-driven adaptations in metabolic enzyme activity of limb muscles, while GT2, without the nNOS-binding site, demonstrated greater protection of diaphragm strength after chronic voluntary endurance exercise but decreased mitochondrial respiration in the context of running.

Predictive index for adverse perinatal outcome in pregnancies complicated by fetal growth restriction.

OBJECTIVES: To develop and validate an index predictive of adverse perinatal outcome (APO) in pregnancies meeting the consensus-based criteria for fetal growth restriction (FGR) endorsed by the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG). METHODS: This was a retrospective analysis of consecutive singleton non-anomalous gestations meeting the ISUOG-endorsed criteria for FGR at a single tertiary care center from November 2010 to August 2020. The dataset was divided randomly into a development set (two-thirds) and a validation set (one-third). The primary composite APO comprised one or more of: perinatal demise, Grade III-IV intraventricular hemorrhage (IVH), periventricular leukomalacia (PVL), seizures, hypoxic ischemic encephalopathy (HIE), necrotizing enterocolitis (NEC), sepsis, bronchopulmonary dysplasia (BPD) and length of stay in the neonatal intensive care unit (NICU) > 7 days. Regression analysis incorporated clinical factors readily available at the time of FGR diagnosis. The sum of ß coefficient-based weights yielded an index score, the performance of which was assessed in the validation set. Score cut-offs were selected to identify 'high-risk' and 'low-risk' ranges for which positive (PPV) and negative (NPV) predictive values and positive (LR+) and negative (LR-) likelihood ratios were calculated. RESULTS: Of the 875 consecutive pregnancies that met the criteria for FGR and were included in the study cohort, 405 (46%) were complicated by one or more components of the composite APO, including 54 (6%) perinatal deaths, 22 (3%) neonates with Grade III-IV IVH and/or PVL, nine (1%) with seizures and/or HIE, 91 (10%) with BPD, 57 (7%) with sepsis, 21 (2%) with NEC, and 361 (41%) who remained in the NICU > 7 days. In addition, 270 (31%) pregnancies were delivered by Cesarean section for non-reassuring fetal status, 43 (5%) were admitted to the NICU for < 7 days, 79 (9%) had 5-min Apgar score < 7, 125/631 (20%) had a cord gas pH ≤ 7.1 and 35/631 (6%) had a base excess ≥ 12 mmol/L. The predictive index we developed included seven factors available at the time of FGR diagnosis: hypertensive disorder of pregnancy (HDP) (+8 points), chronic hypertension without HDP (+4 points), gestational age ≤ 32 weeks (+5 points), absent or reversed end-diastolic flow in the umbilical artery (+8 points), prepregnancy body mass index ≥ 35 kg/m2 (+3 points), isolated abdominal circumference < 3rd percentile (-4 points) and non-Hispanic black race (-2 points). The bias-corrected bootstrapped (1000 replicates) area under the receiver-operating-characteristics curve (AUC) of the predictive index for composite APO in the validation group was 0.88 (95% CI, 0.84-0.92), which was similar to that in the development group (AUC, 0.86 (95% CI, 0.82-0.89); P = 0.34). In the total cohort, 40% of pregnancies had a low-risk index score (≤ 2), associated with a NPV of 85% (95% CI, 81-88%) and a LR- of 0.21 (95% CI, 0.16-0.27), and 23% had a high-risk index score (≥ 10), associated with a PPV of 96% (95% CI, 93-98%) and a LR+ of 27.36 (95% CI, 14.33-52.23). Of the remaining pregnancies that had an intermediate-risk score, 50% were complicated by composite APO. CONCLUSION: An easy-to-use index incorporating seven clinical factors readily available at the time of FGR diagnosis is predictive of APO and may prove useful in counseling and management of pregnancies meeting the ISUOG-endorsed criteria for FGR. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

Cost of intervention delivery in a lifestyle weight loss trial in type 2 diabetes: results from the Look AHEAD clinical trial.

OBJECTIVE: The Action for Health in Diabetes (Look AHEAD) trial was a randomized controlled clinical trial to compare the effects of 10 years of intensive lifestyle intervention (ILI) with a control condition of diabetes support and education (DSE) on health outcomes in over 5,000 participants with type 2 diabetes. The ILI had significantly greater weight losses than DSE throughout the trial. The goal of this analysis is to describe the cost of delivering the intervention. METHODS: The ILI was designed to promote weight loss and increase physical activity. It involved a combination of group plus individual intervention sessions, with decreasing frequency of contact over the 10 years. The intervention incorporated a variety of strategies, including meal replacement products, to improve weight loss outcomes. The costs of intervention delivery were derived from staff surveys of effort and from records of intervention materials from the 16 US academic clinical trial sites. Costs were calculated from the payer perspective and presented in 2012 dollars. RESULTS: During the first year, when intervention delivery was most intensive, the annual cost of intervention delivery, averaged (standard deviation) across clinical sites, was $2,864.6 ($513.3) per ILI participant compared with $202.4 ($76.6) per DSE participant. As intervention intensity declined, costs decreased, such that from years 5 to 9 of the trial, the annual cost of intervention was $1,119.8 ($227.7) per ILI participant and $102.9 ($33.0) per DSE participant. Staffing accounted for the majority of costs throughout the trial, with meal replacements and materials to promote adherence accounting for smaller shares. CONCLUSIONS: The sustained weight losses produced by the Look AHEAD intervention were supported by intervention costs that were within the range of other weight loss programmes. Future work will include an evaluation of the cost-effectiveness of the ILI and will contain additional follow-up data.

Large duplication in MTM1 associated with myotubular myopathy.

Myotubular myopathy is a subtype of centronuclear myopathy with X-linked inheritance and distinctive clinical and pathologic features. Most boys with myotubular myopathy have MTM1 mutations. In remaining individuals, it is not clear if disease is due to an undetected alteration in MTM1 or mutation of another gene. We describe a boy with myotubular myopathy but without mutation in MTM1 by conventional sequencing. Array-CGH analysis of MTM1 uncovered a large MTM1 duplication. This finding suggests that at least some unresolved cases of myotubular myopathy are due to duplications in MTM1, and that array-CGH should be considered when MTM1 sequencing is unrevealing.

Functional muscle analysis of the Tcap knockout mouse.

Autosomal recessive limb-girdle muscular dystrophy type 2G (LGMD2G) is an adult-onset myopathy characterized by distal lower limb weakness, calf hypertrophy and progressive decline in ambulation. The disease is caused by mutations in Tcap, a z-disc protein of skeletal muscle, although the precise mechanisms resulting in clinical symptoms are unknown. To provide a model for preclinical trials and for mechanistic studies, we generated knockout (KO) mice carrying a null mutation in the Tcap gene. Here we present the first report of a Tcap KO mouse model for LGMD2G and the results of an investigation into the effects of Tcap deficiency on skeletal muscle function in 4- and 12-month-old mice. Muscle histology of Tcap-null mice revealed abnormal myofiber size variation with central nucleation, similar to findings in the muscles of LGMD2G patients. An analysis of a Tcap binding protein, myostatin, showed that deletion of Tcap was accompanied by increased protein levels of myostatin. Our Tcap-null mice exhibited a decline in the ability to maintain balance on a rotating rod, relative to wild-type controls. No differences were detected in force or fatigue assays of isolated extensor digitorum longus (EDL) and soleus (SOL) muscles. Finally, a mechanical investigation of EDL and SOL indicated an increase in muscle stiffness in KO animals. We are the first to establish a viable KO mouse model of Tcap deficiency and our model mice demonstrate a dystrophic phenotype comparable to humans with LGMD2G.

Specific unwillingness to donate eyes: the impact of disfigurement, knowledge and procurement on corneal donation.

Although willingness, attitudes and beliefs surrounding solid-organ donation have been extensively investigated, much less is known about corneal donation. Despite evidence that a substantial number of families who agree to multiorgan donation also specifically refuse corneal donation, it is unclear why this occurs and what can be done to increase rates of corneal donation. We conducted a survey of 371 Australian adults regarding their views on corneal donation. Although willingness to donate corneas generally reflected a person's willingness to donate all of one's organs, unwillingness to donate corneas appeared to be due to other factors. Specifically, decisions not to donate appear to be driven by a range of concerns surrounding disfigurement. The survey also provides eye banks with reassurance about the acceptability of whole globe procurement, and recognition that research into blindness is a highly valued part of corneal donation. Finally, the survey identifies that many individuals see benefit in having their family engaged in the decision-making process, suggesting that decisions about donation are more complex than a simple appeal to the autonomy of the deceased.

An interesting case of adenoviral hepatitis in a cardiac transplant recipient.

Adenoviral infections are commonly described in pediatric transplant populations. However, much less information is available regarding the incidence of infection and clinical spectrum of disease in adult transplant recipients. Moreover, this infection usually manifests as involvement of the transplanted organ in one pathologic form or the other, in addition to other systemic manifestations. We present a case of adenoviral infection of a nontransplanted organ in a solid organ transplant recipient.

Registering wishes about organ and tissue donation: personal discussion during licence renewal may be superior to online registration.

Consent to organ and tissue donation is higher when the deceased has indicated a wish to donate. The Australian Organ Donor Register (AODR) is the national register of preferences regarding donation. The AODR has a number of limitations; it has no mechanism for requiring individuals to register their wishes, while the online format both raises concerns about the validity of the consent obtained and precludes personal discussion of fears and concerns about donation. A solution to these limitations is to utilize state-based agencies that administer driving licences. This strategy ties the donation decision to an existing task (renewal of driving licences), and provides an opportunity for a personalized intervention at the time the decision is being made.

Consent for corneal donation: the effect of age of the deceased, registered intent and which family member is asked about donation.

AIM: To determine whether consent to corneal donation is related to which next of kin is asked to consent, the age of the potential donor and the indication about donation made by the deceased on their driving licence. METHOD: The Lions New South Wales Eye Bank (Sydney, New South Wales, Australia) provides the corneal transplantation service for Australia's most populous state. Over the 18-month period from 1 July 2004 to 31 December 2005 for all requests for donation, records were kept of which next of kin was asked for consent, the age of the deceased and the indication about donation by the deceased on their driving licence. RESULTS: Over the 18-month study period, 841 people were approached about corneal donation. 63.2% of those people approached gave their consent to donation. Increasing age of the deceased was significantly positively associated with consent to donation (p = 0.006). Multivariable univariate analysis adjusting for age of deceased showed that relative type was strongly associated with consent (p<0.001), with mothers and fathers more likely to donate than siblings, and siblings more likely to donate than children and spouses. An indication of willingness to donate on a driving licence was strongly associated with consent (p<0.001). CONCLUSIONS: Higher consent rates from older donors have implications for policies to maximise corneal procurement. The decision to donate on behalf of a deceased family member is complex and influenced by social context. Research should investigate individualised strategies to be used when seeking consent from particular categories of next of kin.

Bronchitis symptoms in young teenagers who actively or passively smoke cigarettes.

This study was undertaken to examine the prevalence of bronchitis (cough with phlegm) symptoms in teenagers who either smoked cigarettes on a regular basis (active smokers) or were non-smokers but who are exposed to passive smoking (passive smokers) in the home. The study was undertaken in 1995 and repeated in 1998. The 1995 study was a cross sectional questionnaire survey of smoking habits in secondary school children aged 13-14 years and was undertaken as part of the ISAAC questionnaire survey. Thirty representative and randomly selected schools from throughout the Republic of Ireland took part in the study. In the 1995 study, 3066 students completed a questionnaire on their current smoking habits and symptoms of cough and phlegm. We found that 634 (20.7%) of these young teenagers actively smoked cigarettes with significantly more females smoking than males with 23.3% of girls compared to 17.6% boys (p = 0.0001). We found that 46.3% of non-smoking children were exposed to smoking in the home (passive smokers) with parental smoking accounting for most of the passive smoking. Bronchitis symptoms were more commonly reported in active smokers compared to non-smokers with an odds ratio of 3.02 (95% CI 2.34-3.88) (p < 0.0001) or in passive smokers compared to those not exposed to smoking with odds ratio of 1.82 (95% CI 1.32-2.52) (p < 0.0001). The 1998 study showed similar results for smoking habits, passive smoking and prevalence of bronchitis symptoms as with the 1995 study. These results document that increased bronchitis symptoms occur in teenagers exposed to active or passive smoking.

PKB/Akt: a key mediator of cell proliferation, survival and insulin responses?

The serine/threonine protein kinase PKB (also known as Akt) is thought to be a key mediator of signal transduction processes. The identification of PKB substrates and the role PKB phosphorylation plays in regulating these molecules have been a major focus of research in recent years. A recently developed motif-profile scoring algorithm that can be used to scan the genome for potential PKB substrates is therefore a useful tool, although additional considerations, such as the evolutionary conservation of the phosphorylation site, must also be taken into account. Recent evidence indicates that PKB plays a key role in cancer progression by stimulating cell proliferation and inhibiting apoptosis and is also probably a key mediator of insulin signalling. These findings indicate that PKB is likely to be a hot drug target for the treatment of cancer, diabetes and stroke. There are, however, a number of pitfalls of methodologies currently employed to study PKB function, and therefore caution should be used in interpretation of such experiments.

Serial prealbumin levels as predictors of outcomes in a retrospective cohort of peritoneal and hemodialysis patients.

OBJECTIVE: Although earlier research has suggested that baseline prealbumin level is an independent predictor of outcome among dialysis patients, the prognostic importance of serial prealbumin levels is less clear. The present study had 3 objectives: first, to determine if prealbumin (a marker of visceral protein stores with a relatively short half-life) predicts subsequent albumin levels taken at least 1 month later; second, to examine the association between serial prealbumin levels and clinical outcome; and third, to examine the association between changes in prealbumin level and outcome. DESIGN: The prognostic value of serial prealbumin levels was examined by linear regression analysis and Cox hazard models in an observational cohort study using a repeated measures design and time-dependent covariates. SETTING: Patients were followed by a tertiary care center, receiving hemodialysis (HD; at either an in-center dialysis unit or one of several satellite units operated by the hospital) or home peritoneal dialysis (PD). PATIENTS: A retrospective cohort was identified consisting of 268 incident and prevalent chronic HD and PD patients receiving dialysis from June 1998 to September 1999. MAIN OUTCOME: The study examined the association between serial prealbumin measurements and future laboratory and clinical outcomes (albumin, hospitalization, and death). RESULTS: Serial prealbumin values were independent predictors of future albumin levels among HD patients (P =.04), but not PD patients. Independent predictors of hospitalization included diabetes for PD patients (P =.0012) and advanced age for HD patients (P =.0008). Advanced age and diabetes were independent predictors of death for both HD (P =.0001 and P =.0368) and PD patients (P =.0014 and P =.0164). Serial prealbumin values, measured as time-dependent covariates, did not predict hospitalization or death. Further analyses examined the prognostic value of changes in prealbumin and albumin values as time-dependent covariates. The final multivariate analysis identified low baseline albumin level as an independent predictor of hospitalization among HD patients (P =.0282), whereas low baseline prealbumin was an independent predictor of death for HD patients (P =.0001). Interestingly, negative changes in serial prealbumin measurements were also independent predictors of death among HD patients (P =.0025). CONCLUSION: Serial prealbumin measurements predict subsequent albumin values among HD patients. As well, low baseline prealbumin level is an independent predictor of adverse outcome among HD patients. Although repeated prealbumin measurements in and of themselves were of no added prognostic value, falling prealbumin values identified by repeated measurements were additional independent predictors of death. These results support the clinical utility of regular prealbumin monitoring among HD patients.

In vitro and In vivo inhibition of LPS-stimulated tumor necrosis factor-alpha secretion by the gallotannin beta-D-pentagalloylglucose.

The naturally occurring gallotannin beta-D-pentagalloylglucose (beta-PGG) decreases tumor necrosis factor-alpha (TNF-alpha) output from human peripheral blood mononucleocytes exposed to lipopolysaccharide (LPS) by as much as 90% (vs control) at approximately 5 microM concentration. A qualitatively similar but less pronounced effect ( approximately 50% decrease) was observed in the serum of rats dosed with both LPS and beta-PGG. These results may have relevance to therapies that target disease states characterized by an overproduction of TNF-alpha.

Sensory nerve conduction deficit in experimental monoclonal gammopathy of undetermined significance (MGUS) neuropathy.

An emerging body of evidence from in vitro studies and in vivo animal models supports a pathogenic role of antibodies in the development of peripheral neuropathy associated with monoclonal gammopathy of undetermined significance (MGUS). Although the assessment of motor and sensory nerve fiber function is of clinical importance, it is seldom applied experimentally. We describe the application of an electrophysiologic method for the evaluation of motor and sensory nerve fiber function using an experimental model of MGUS neuropathy. Supramaximal stimulation of the tibial nerve elicited an early motor response (M-wave, 1.7 +/- 0.1 ms, n = 10) and a late sensory (H-reflex, 7.8 +/- 0.1 ms, n = 10) response that was recorded from the hind foot of anesthetized rats. Intraneural injection of serum antibodies from a MGUS patient with sensorimotor polyneuropathy, but not from an age-matched control subject, produced a marked attenuation of the H-reflex (P < 0.01, n = 10) without affecting the M-wave. Light and electron microscopy of affected nerve showed myelinoaxonal degeneration with sparing of the smaller unmyelinated nerve fibers. The combined electrophysiologic and morphologic findings presented in this study are consistent with a selective sensory conduction deficit in MGUS neuropathy. Selective injury of afferent nerve fibers by this patient's serum antibodies may result from reactivity to neural antigens uniquely expressed by sensory neurons.

Beyond the unobtrusive observer: reflections on researcher-informant relationships in urban ethnography.

Ethnographic research involves the creation and ongoing renegotiations of relationships between researchers and informants. Prolonged engagement contributes to the complexity as relationships deepen and shift over time and participants accumulate a substantial reservoir of shared experiences. Reflections about the relationships we have co-constructed with informants in several research projects have contributed to our identification of several critical aspects of building and maintaining researcher-informant relationships in cross-cultural research. Aspects of relationship work specifically related to conducting ethnography with children, within the communities in which researchers live, and within the practice of occupational therapy are discussed.

New technologies and therapeutic approaches for the management of pediatric diabetes.

Evolving trends in technology and therapeutic strategies create a very exciting time for the management of diabetes. Technology, like the internet, helps us to keep up with this fast-paced, changing world of diabetes. New therapies most often begin with adult clinical trials; once safety and efficacy are demonstrated, practice recommendations follow for the pediatric population. The Diabetes Research Working Group has defined potentially important new directions and technologies for diabetes research. The group has recommended the creation of regional centers of technologic excellence, if contemporary diabetes research is to succeed. An overview of recent advances in diabetes technology follows covering five main areas: monitoring, telemedicine, insulin analogues, insulin delivery devices, and islet cell transplantation.

Coordinate control of muscle cell survival by distinct insulin-like growth factor activated signaling pathways.

Peptide growth factors control diverse cellular functions by regulating distinct signal transduction pathways. In cultured myoblasts, insulin-like growth factors (IGFs) stimulate differentiation and promote hypertrophy. IGFs also maintain muscle cell viability. We previously described C2 skeletal muscle lines lacking expression of IGF-II. These cells did not differentiate, but underwent progressive apoptotic death when incubated in differentiation medium. Viability could be sustained and differentiation enabled by IGF analogues that activated the IGF-I receptor; survival was dependent on stimulation of phosphatidylinositol 3-kinase (PI3-kinase). We now find that IGF action promotes myoblast survival through two distinguishable PI3-kinase-regulated pathways that culminate in expression of the cyclin-dependent kinase inhibitor, p21. Incubation with IGF-I or transfection with active PI3-kinase led to rapid induction of MyoD and p21, and forced expression of either protein maintained viability in the absence of growth factors. Ectopic expression of MyoD induced p21, and inhibition of p21 blocked MyoD-mediated survival, thus defining one PI3-kinase-dependent pathway as leading first to MyoD, and then to p21 and survival. Unexpectedly, loss of MyoD expression did not impede IGF-mediated survival, revealing a second pathway involving activation by PI3-kinase of Akt, and subsequent induction of p21. Since inhibition of p21 caused death even in the presence of IGF-I, these results establish a central role for p21 as a survival factor for muscle cells. Our observations also define a MyoD-independent pathway for regulating p21 in muscle, and demonstrate that distinct mechanisms help ensure appropriate expression of this key protein during differentiation.

Ellagitannin chemistry. Evolution of a three-component coupling strategy for the synthesis of the dimeric ellagitannin coriariin A and a dimeric gallotannin analogue.

The total synthesis of the dimeric ellagitannin coriariin A is reported. The key reaction to access the dimeric framework was realized early in the synthesis pathway via a bis acylation reaction of a dehydrodigalloyl diacid with 2 equiv of a glucopyranose trichloroacetimidate. The glucose rings were subsequently functionalized, culminating in a double oxidative cyclization to form stereoselectively both (S)-HHDP ester units. This bis acylation strategy was also employed to prepare a gallotannin analogue of coriariin A whose earlier synthesis by orthoquinone dimerization was plagued by yield-limiting side reactions.