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Messenger RNA (mRNA) therapeutics delivered via lipid nanoparticles hold the potential to treat metabolic diseases caused by protein deficiency, including propionic acidemia (PA), methylmalonic acidemia (MMA), and phenylketonuria (PKU). Herein we report results from multiple independent preclinical studies of mRNA-3927 (an investigational treatment for PA), mRNA-3705 (an investigational treatment for MMA), and mRNA-3210 (an investigational treatment for PKU) in murine models of each disease. All 3 mRNA therapeutics exhibited pharmacokinetic/pharmacodynamic (PK/PD) responses in their respective murine model by driving mRNA, protein, and/or protein activity responses, as well as by decreasing levels of the relevant biomarker(s) when compared to control-treated animals. These preclinical data were then used to develop translational PK/PD models, which were scaled allometrically to humans to predict starting doses for first-in-human clinical studies for each disease. The predicted first-in-human doses for mRNA-3927, mRNA-3705, and mRNA-3210 were determined to be 0.3, 0.1, and 0.4 mg/kg, respectively., (© 2024. The Author(s).)

Background: In academic research and the pharmaceutical industry, in vitro cell lines and in vivo animal models are considered as gold standards in modelling diseases and assessing therapeutic efficacy. However, both models have intrinsic limitations, whilst the use of precision-cut tissue slices can bridge the gap between these mainstream models. Precision-cut tissue slices combine the advantage of high reproducibility, studying all cell sub-types whilst preserving the tissue matrix and extracellular architecture, thereby closely mimicking a mini-organ. This approach can be used to replicate the biological phenotype of liver monogenic diseases using mouse models., Methods: Here, we describe an optimised and easy-to-implement protocol for the culture of sections from mouse livers, enabling its use as a reliable ex-vivo model to assess the therapeutic screening of inherited metabolic diseases., Results: We show that precision-cut liver sections can be a reliable model for recapitulating the biological phenotype of inherited metabolic diseases, exemplified by common urea cycle defects such as citrullinemia type 1 and argininosuccinic aciduria, caused by argininosuccinic synthase (ASS1) and argininosuccinic lyase (ASL) deficiencies respectively., Conclusions: Therapeutic response to gene therapy such as messenger RNA replacement delivered via lipid nanoparticles can be monitored, demonstrating that precision-cut liver sections can be used as a preclinical screening tool to assess therapeutic response and toxicity in monogenic liver diseases., Competing Interests: Competing interests: The mRNA lipid nanoparticles were provided by Moderna. JB is receiving research funding from Moderna Therapeutics, (Copyright: © 2024 Perocheau D et al.)

Introduction: Experts frequently assess competency in criminal settings where the rate of feigning cognitive deficit is demonstrably elevated. We describe the construction and validation of the Denney Competency Related Test (D-CRT) to assess feigned incompetency of defendants in the criminal adjudicative setting. It was expected the D-CRT would prove effective at identifying feigned incompetence based on its two alternative, forced-choice and performance curve characteristics., Method: Development and validation of the D-CRT occurred in described phases. Items were developed to measure competency based upon expert review. Item analysis and adjustments were completed with 304 young teenage volunteers to obtain a proper spread of item difficulty in preparation for eventual performance curve analysis (PCA). Test-retest reliability was assessed with 44 adult community volunteers. Validation included an analog simulation design with 101 jail detainees using MacArthur Competency Assessment Test-Criminal Adjudication and Word Memory Test as criterion measures. Effects of racial/ethnic demographic differences were examined in a separate study of 208 undergraduate volunteers. D-CRT specificity was identified with 46 elderly clinic referrals diagnosed with mild cognitive impairment and dementia., Results: Item development, adjustment, and repeat analysis resulted in item probabilities evenly spread from .28 to 1.0. Test-retest correlation was good (.83). Internal consistency of items was excellent (KR-20 > .91). D-CRT demonstrated convergent validity in regard to measuring competency related information and as well as malingering. The test successfully differentiated between jail inmates asked to perforfm their best and inmates asked to simulate incompetency (AUC = .945). There were no statistically significant differences found in performance across racial/ethnic backgrounds. D-CRT specificity remained excellent among elderly clinic referrals with significant cognitive compromise at the recommended total score cutoff., Conclusions: D-CRT is an effective measure of feigned criminal incompetency in the context of potential cognitive deficiency, and PCA is assistive in the determination. Additional validation using knowns groups designs with various mental health-related conditions are needed.

Despite the implementation of lifesaving newborn screening programs and a galactose-restricted diet, many patients with classic galactosemia develop long-term debilitating neurological deficits and primary ovarian insufficiency. Previously, we showed that the administration of human GALT mRNA predominantly expressed in the GalT gene-trapped mouse liver augmented the expression of hepatic GALT activity, which decreased not only galactose-1 phosphate (gal-1P) in the liver but also peripheral tissues. Since each peripheral tissue requires distinct methods to examine the biomarker and/or GALT effect, this highlights the necessity for alternative strategies to evaluate the overall impact of therapies. In this study, we established that whole-body galactose oxidation (WBGO) as a robust, noninvasive, and specific method to assess the in vivo pharmacokinetic and pharmacodynamic parameters of two experimental gene-based therapies that aimed to restore GALT activity in a mouse model of galactosemia. Although our results illustrated the long-lasting efficacy of AAVrh10-mediated GALT gene transfer, we found that GALT mRNA therapy that targets the liver predominantly is sufficient to sustain WBGO. The latter could have important implications in the design of novel targeted therapy to ensure optimal efficacy and safety., Competing Interests: The authors declare the following competing interests: X.Y., F.W., J.T., L.C., R.D., S.C., S.M., Y.W., M.Z., J.S., P.G.V.M., and P.F.F. are employees of Moderna and hold equities from the company., (© 2024 The Author(s).)

The urea cycle enzyme argininosuccinate lyase (ASL) enables the clearance of neurotoxic ammonia and the biosynthesis of arginine. Patients with ASL deficiency present with argininosuccinic aciduria, an inherited metabolic disease with hyperammonemia and a systemic phenotype coinciding with neurocognitive impairment and chronic liver disease. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using ( S )-4-(3- 18 F-fluoropropyl)-l-glutamate ([ 18 F]FSPG). Up-regulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. To assess hepatic glutathione dysregulation and liver disease, we present [ 18 F]FSPG PET as a noninvasive diagnostic tool to monitor therapeutic response in argininosuccinic aciduria. Human hASL mRNA encapsulated in lipid nanoparticles improved glutathione metabolism and chronic liver disease. In addition, hASL mRNA therapy corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis. These findings provide mechanistic insights in liver glutathione metabolism and support clinical translation of mRNA therapy for argininosuccinic aciduria.

Argininosuccinate lyase (ASL) is integral to the urea cycle detoxifying neurotoxic ammonia and the nitric oxide (NO) biosynthesis cycle. Inherited ASL deficiency causes argininosuccinic aciduria (ASA), a rare disease with hyperammonemia and NO deficiency. Patients present with developmental delay, epilepsy and movement disorder, associated with NO-mediated downregulation of central catecholamine biosynthesis. A neurodegenerative phenotype has been proposed in ASA. To better characterise this neurodegenerative phenotype in ASA, we conducted a retrospective study in six paediatric and adult metabolic centres in the UK in 2022. We identified 60 patients and specifically looked for neurodegeneration-related symptoms: movement disorder such as ataxia, tremor and dystonia, hypotonia/fatigue and abnormal behaviour. We analysed neuroimaging with diffusion tensor imaging (DTI) magnetic resonance imaging (MRI) in an individual with ASA with movement disorders. We assessed conventional and DTI MRI alongside single photon emission computer tomography (SPECT) with dopamine analogue radionuclide 123 I-ioflupane, in Asl-deficient mice treated by hASL mRNA with normalised ureagenesis. Movement disorders in ASA appear in the second and third decades of life, becoming more prevalent with ageing and independent from the age of onset of hyperammonemia. Neuroimaging can show abnormal DTI features affecting both grey and white matter, preferentially basal ganglia. ASA mouse model with normalised ureagenesis did not recapitulate these DTI findings and showed normal 123 I-ioflupane SPECT and cerebral dopamine metabolomics. Altogether these findings support the pathophysiology of a late-onset movement disorder with cell-autonomous functional central catecholamine dysregulation but without or limited neurodegeneration of dopaminergic neurons, making these symptoms amenable to targeted therapy., (© 2023 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Pompe disease is a rare glycogen storage disorder caused by a deficiency in the lysosomal enzyme acid α -glucosidase, which leads to muscle weakness, cardiac and respiratory failure, and early mortality. Alglucosidase alfa, a recombinant human acid α -glucosidase, was the first approved treatment of Pompe disease, but its uptake into skeletal muscle via the cation-independent mannose-6-phosphate (M6P) receptor (CIMPR) is limited. Avalglucosidase alfa has received marketing authorization in several countries for infantile-onset and/or late-onset Pompe disease. This recently approved enzyme replacement therapy (ERT) was glycoengineered to maximize CIMPR binding through high-affinity interactions with ∼7 bis-M6P moieties. Recently, small molecules like the glucosylceramide synthase inhibitor miglustat were reported to increase the stability of recombinant human acid α -glucosidase, and it was suggested that an increased serum half-life would result in better glycogen clearance. Here, the effects of miglustat on alglucosidase alfa and avalglucosidase alfa stability, activity, and efficacy in Pompe mice were evaluated. Although miglustat increased the stability of both enzymes in fluorescent protein thermal shift assays and when incubated in neutral pH buffer over time, it reduced their enzymatic activity by ∼50%. Improvement in tissue glycogen clearance and transcriptional dysregulation in Pompe mice correlated with M6P levels but not with miglustat coadministration. These results further substantiate the crucial role of CIMPR binding in lysosomal targeting of ERTs. SIGNIFICANCE STATEMENT: This work describes important new insights into the treatment of Pompe disease using currently approved enzyme replacement therapies (ERTs) coadministered with miglustat. Although miglustat increased the stability of ERTs in vitro, there was no positive impact to glycogen clearance and transcriptional correction in Pompe mice. However, increasing mannose-6-phosphate levels resulted in increased cell uptake in vitro and increased glycogen clearance and transcriptional correction in Pompe mice, further underscoring the crucial role of cation-independent mannose-6-phosphate receptor-mediated lysosomal targeting for ERTs., (Copyright © 2023 by The Author(s).)

Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart., Competing Interests: Declaration of interests R.J.M., J.E.H., and E.R.P. are co-founders, scientific advisors, and hold equity in Dynomics, a biotechnology company focused on the development of heart failure therapeutics. P.F. and P.G.V.M. are employees of and hold equity in Moderna., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Glycogen storage disease (GSD) type 1a is an inherited autosomal recessive metabolic disease caused by a deficiency in glucose-6-phosphatase activity. The objectives of this research were to systematically review the published literature on the epidemiology of GSD 1a and to assess the performance of reported epidemiology measures in a simulation model. In this systematic literature review 2,539 record titles and abstracts were screened. Of these, only 11 studies contained relevant data on GSD 1a disease epidemiology. Reported disease frequency ranged from 0.085/100,000 to 10.3/100,000 newborns when considering all the GSD literature. When this was narrowed to GSD 1 and GSD 1a, the range was tightened to 0.25-3.02/100,000 and 0.085-4.9/100,000 newborns, respectively. Most of the identified studies counted the number of diagnoses in a defined period and related to the number of births in the same (Dx method) or different time period (DoB method). The simulation model results indicate that in most of the situations, the Dx method provides a closer estimate to the true disease incidence than the DoB method. Despite the scarcity of epidemiology data, the results of this systematic review strongly support that GSD 1a and its parent disease groups (GSD and GSD 1) are rare diseases., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Glucose-6-phosphatase-α (G6Pase-α) catalyzes the hydrolysis of glucose-6-phosphate to glucose and functions as a key regulator in maintaining blood glucose homeostasis. Deficiency in G6Pase-α causes glycogen storage disease 1a (GSD1a), an inherited disorder characterized by life-threatening hypoglycemia and other long-term complications. We have developed a potential mRNA-based therapy for GSD1a and demonstrated that a human G6Pase-α (hG6Pase-α) variant harboring a single serine (S) to cysteine (C) substitution at the amino acid site 298 (S298C) had > twofold increase in protein expression, resulting in improved in vivo efficacy. Here, we sought to investigate the mechanisms contributing to the increased expression of the S298C variant. Mutagenesis of hG6Pase-α identified distinct protein variants at the 298 amino acid position with substantial reduction in protein expression in cultured cells. Kinetic analysis of expression and subcellular localization in mammalian cells, combined with cell-free in vitro translation assays, revealed that altered protein expression stemmed from differences in cellular protein stability rather than biosynthetic rates. Site-specific mutagenesis studies targeting other cysteines of the hG6Pase-α S298C variant suggest the observed improvements in stability are not due to additional disulfide bond formation. The glycosylation at Asparagine (N)-96 is critical in maintaining enzymatic activity and mutations at position 298 mainly affected glycosylated forms of hG6Pase-α. Finally, proteasome inhibition by lactacystin improved expression levels of unstable hG6Pase-α variants. Taken together, these data uncover a critical role for a single amino acid substitution impacting the stability of G6Pase-α and provide insights into the molecular genetics of GSD1a and protein engineering for therapeutic development., (© 2023. The Author(s).)

Stroke significantly impairs health-related quality of life (HRQL). Stroke survivors with aphasia (SWA) experience lower HRQL than stroke survivors without aphasia (SSA) as a result of poorer communication and social functioning. The extent to which aphasia influences HRQL in African-Americans and the components of social functioning that are most important to HRQL warrants further exploration.There were two main objectives of this paper. The first was to survey HRQL domains of communication, physical, mental/emotional, role, and social functioning in African American SWA and SSA. The second was to examine if social support and social network predicted HRQL in SWA.A total of 39 African American adults (62.4 ± 11.10) participated in this descriptive cross-sectional case control study. Patient-reported outcome measures were used to assess HRQL, perceived social support, and social network in SWA, SSA, and normal-aging healthy controls (NAH). Data analysis included an ANOVA and moderator regression to determine if social support or social network predicted HRQL in SWA.SWA reported a significantly lower overall HRQL (p = <.000) than NAH adults. Communication HRQL was the hallmark difference found between SWA and SSA (p = <.000). Social support and social network were relatively similar among all three groups. However, social support and social network did not predict HRQL in SWA.Findings from this study suggest that social HRQL continues to be significantly lower in SWA; however, social support and social network factors do not drive differences among African-Americans. Moreover, communication HRQL remains the hallmark difference between SWA and SSA.

Background & Aims: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare lethal autosomal recessive liver disorder caused by loss-of-function variations of the ABCB4 gene, encoding a phosphatidylcholine transporter (ABCB4/MDR3). Currently, no effective treatment exists for PFIC3 outside of liver transplantation., Methods: We have produced and screened chemically and genetically modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles (LNPs). We examined their pharmacological effects in a cell-based model and in a new in vivo mouse model resembling human PFIC3 as a result of homozygous disruption of the Abcb4 gene in fibrosis-susceptible BALB/c.Abcb4 -/- mice., Results: We show that treatment with liver-targeted hABCB4 mRNA resulted in de novo expression of functional hABCB4 protein and restored phospholipid transport in cultured cells and in PFIC3 mouse livers. Importantly, repeated injections of the hABCB4 mRNA effectively rescued the severe disease phenotype in young Abcb4 -/- mice, with rapid and dramatic normalisation of all clinically relevant parameters such as inflammation, ductular reaction, and liver fibrosis. Synthetic mRNA therapy also promoted favourable hepatocyte-driven liver regeneration to restore normal homeostasis, including liver weight, body weight, liver enzymes, and portal vein blood pressure., Conclusions: Our data provide strong preclinical proof-of-concept for hABCB4 mRNA therapy as a potential treatment option for patients with PFIC3., Lay Summary: This report describes the development of an innovative mRNA therapy as a potential treatment for PFIC3, a devastating rare paediatric liver disease with no treatment options except liver transplantation. We show that administration of our mRNA construct completely rescues severe liver disease in a genetic model of PFIC3 in mice., Competing Interests: Conflicts of interest JC, JZ, LY, AF, VP, SG, SH, CL, PF, PHG, and PM are employees of, and receive salary, stock, and stock options from, Moderna, Inc. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Glycogen Storage Disease 1a (GSD1a) is a rare, inherited metabolic disorder caused by deficiency of glucose 6-phosphatase (G6Pase-α). G6Pase-α is critical for maintaining interprandial euglycemia. GSD1a patients exhibit life-threatening hypoglycemia and long-term liver complications including hepatocellular adenomas (HCAs) and carcinomas (HCCs). There is no treatment for GSD1a and the current standard-of-care for managing hypoglycemia (Glycosade ® /modified cornstarch) fails to prevent HCA/HCC risk. Therapeutic modalities such as enzyme replacement therapy and gene therapy are not ideal options for patients due to challenges in drug-delivery, efficacy, and safety. To develop a new treatment for GSD1a capable of addressing both the life-threatening hypoglycemia and HCA/HCC risk, we encapsulated engineered mRNAs encoding human G6Pase-α in lipid nanoparticles. We demonstrate the efficacy and safety of our approach in a preclinical murine model that phenotypically resembles the human condition, thus presenting a potential therapy that could have a significant therapeutic impact on the treatment of GSD1a.

Purpose: (1) To survey the employed techniques and the reasons/occasions which adults who had recovered from stuttering after age 11 without previous treatment reported as causal to overcome stuttering, (2) to investigate whether the techniques and causal attributions can be reduced to coherent (inherently consistent) dimensions, and (3) whether these dimensions reflect common therapy components., Methods: 124 recovered persons from 8 countries responded by SurveyMonkey or paper-and-pencil to rating scale questions about 49 possible techniques and 15 causal attributions., Results: A Principal Component Analysis of 110 questionnaires identified 6 components (dimensions) for self-assisted techniques (Speech Restructuring; Relaxed/Monitored Speech; Elocution; Stage Performance; Sought Speech Demands; Reassurance; 63.7% variance explained), and 3 components of perceived causal attributions of recovery (Life Change, Attitude Change, Social Support; 58.0% variance explained)., Discussion: Two components for self-assisted techniques (Speech Restructuring; Elocution) reflect treatment methods. Another component (Relaxed/Monitored Speech) consists mainly of items that reflect a common, non-professional understanding of effective management of stuttering. The components of the various perceived reasons for recovery reflect differing implicit theories of causes for recovery from stuttering. These theories are considered susceptible to various biases. This identification of components of reported techniques and of causal attributions is novel compared to previous studies who just list techniques and attributions., Conclusion: The identified dimensions of self-assisted techniques and causal attributions to reduce stuttering as extracted from self-reports of a large, international sample of recovered formerly stuttering adults may guide the application of behavioral stuttering therapies., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Fabry disease is an X-linked lysosomal storage disease caused by loss of alpha galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of globotriaosylceramide and its analogs in all cells and tissues. Although enzyme replacement therapy (ERT) is considered standard of care, the long-term effects of ERT on renal and cardiac manifestations remain uncertain and thus novel therapies are desirable. We herein report preclinical studies evaluating systemic messenger RNA (mRNA) encoding human α-Gal A in wild-type (WT) mice, α-Gal A-deficient mice, and WT non-human primates (NHPs). The pharmacokinetics and distribution of h-α-Gal A mRNA encoded protein in WT mice demonstrated prolonged half-lives of α-Gal A in tissues and plasma. Single intravenous administration of h-α-Gal A mRNA to Gla-deficient mice showed dose-dependent protein activity and substrate reduction. Moreover, long duration (up to 6 weeks) of substrate reductions in tissues and plasma were observed after a single injection. Furthermore, repeat i.v. administration of h-α-Gal A mRNA showed a sustained pharmacodynamic response and efficacy in Fabry mice model. Lastly, multiple administrations to non-human primates confirmed safety and translatability. Taken together, these studies across species demonstrate preclinical proof-of-concept of systemic mRNA therapy for the treatment of Fabry disease and this approach may be useful for other lysosomal storage disorders., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Traditional journal club models based on didactic presentation sessions followed by group discussion have many limitations. To overcome some of these shortcomings, a virtual journal club (VJC) using social media and e-mail was developed. The aim of this study was to report the initial experience of this novel multimodal e-learning platform to facilitate journal club discussion and promote the development of critical appraisal skills., Methods: Journal articles were discussed monthly via e-mail and social media. After a 3-week period of discussion, all comments were collated and group-generated critical appraisal summaries were fed back to participants. In addition, letters to the journal editors based on the group appraisal were submitted. A questionnaire survey to evaluate the VJC concept was also conducted., Findings: After eight cycles of the VJC, the mean trainee participation rate was 29.6 per cent (range 21.1-42.1%). Senior trainees (≥4 years of postgraduate experience) were more likely to participate than more junior trainees (75.0 versus 21.1%; p = 0.005). The majority of participants thought that the VJC was educationally valuable, easy to participate in, helpful in keeping up to date with recent papers and useful in developing critical appraisal skills. Barriers to participation were lack of time, motivation and lack of experience in critical appraisal. In addition, the group-generated critical appraisal summaries derived from VJC discussions led to eight published 'letters to the editor'. Traditional journal club models based on didactic presentation sessions followed by group discussion have many limitations, Conclusion: This novel VJC model is a feasible and popular method of delivering a journal club in the postgraduate setting., (© 2015 John Wiley & Sons Ltd.)

The cation-independent mannose 6-phosphate receptor (CI-MPR) plays a critical role in intracellular transport of lysosomal enzymes as well as the uptake of recombinant proteins. To define the minimal glycan structure determinants necessary for receptor binding and cellular uptake, we synthesized a series of glycans containing mono-, di-, tri-, tetra-, and hexamannoses terminated with either one or two phosphates for conjugating to a model protein, recombinant human acid α-glucosidase. A high affinity interaction with the CI-MPR can be achieved for the enzyme conjugated to a dimannose glycan with a single phosphate. However, tightest binding to a CI-MPR affinity column was observed with a hexamannose structure containing two phosphates. Moreover, maximal cellular uptake and a 5-fold improvement in in vivo potency were achieved when the bisphosphorylated hexamannose glycan is conjugated to the protein by a β linker. Nevertheless, even a monophosphorylated dimannose glycan conjugate showed stronger binding to the receptor affinity column, higher cellular uptake, and significantly greater in vivo efficacy compared to the unconjugated protein which contains a low level of high affinity glycan structure. These results demonstrate that the phosphorylated dimannose moiety appears to be the minimal structure determinant for enhanced CI-MPR binding and that the orientation of the glycan is critical for maximum receptor interaction. In summary, we have improved the understanding of the mechanism of CI-MPR binding and developed a simple alternative for CI-MPR targeting.

Engineering proteins for selective tissue targeting can improve therapeutic efficacy and reduce undesired side effects. The relatively high dose of recombinant human acid α-glucosidase (rhGAA) required for enzyme replacement therapy of Pompe disease may be attributed to less than optimal muscle uptake via the cation-independent mannose 6-phosphate receptor (CI-MPR). To improve muscle targeting, Zhu et al. (1) conjugated periodate oxidized rhGAA with bis mannose 6-phosphate bearing synthetic glycans and achieved 5-fold greater potency in a murine Pompe efficacy model. In the current study, we systematically evaluated multiple strategies for conjugation based on a structural homology model of GAA. Glycan derivatives containing succinimide, hydrazide, and aminooxy linkers targeting free cysteine, lysines, and N-linked glycosylation sites on rhGAA were prepared and evaluated in vitro and in vivo. A novel conjugation method using enzymatic oxidation was developed to eliminate side oxidation of methionine. Conjugates derived from periodate oxidized rhGAA still displayed the greatest potency in the murine Pompe model. The efficiency of conjugation and its effect on catalytic activity were consistent with predictions based on the structural model and supported its use in guiding selection of appropriate chemistries.

Purpose: I respond to Kamhi's (2011) conclusion in his article "Balancing Certainty and Uncertainty in Clinical Practice" that rational or critical thinking is an essential complement to evidence-based practice (EBP)., Method: I expand on Kamhi's conclusion and briefly describe what clinicians might need to know to think critically within an EBP profession. Specifically, I suggest how critical thinking is relevant to EBP, broadly summarize the relevant skills, indicate the importance of thinking dispositions, and outline the various ways our thinking can go wrong., Conclusion: I finish the commentary by suggesting that critical thinking skills should be considered a required outcome of our professional training programs.

Due to the lack of acid alpha-glucosidase (GAA) activity, Pompe mice develop glycogen storage pathology and progressive skeletal muscle dysfunction with age. Applying either gene or enzyme therapy to reconstitute GAA levels in older, symptomatic Pompe mice effectively reduces glycogen storage in skeletal muscle but provides only modest improvements in motor function. As strategies to stimulate muscle hypertrophy, such as by myostatin inhibition, have been shown to improve muscle pathology and strength in mouse models of muscular dystrophy, we sought to determine whether these benefits might be similarly realized in Pompe mice. Administration of a recombinant adeno-associated virus serotype 8 vector encoding follistatin, an inhibitor of myostatin, increased muscle mass and strength but only in Pompe mice that were treated before 10 months of age. Younger Pompe mice showed significant muscle fiber hypertrophy in response to treatment with follistatin, but maximal gains in muscle strength were achieved only when concomitant GAA administration reduced glycogen storage in the affected muscles. Despite increased grip strength, follistatin treatment failed to improve rotarod performance. These findings highlight the importance of treating Pompe skeletal muscle before pathology becomes irreversible, and suggest that adjunctive therapies may not be effective without first clearing skeletal muscle glycogen storage with GAA.

Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1-2years of age to a more slowly progressive course that causes significant morbidity and early mortality in children and adults. Recombinant human GAA (rhGAA) improves clinical outcomes with variable results. Adjunct therapy that increases the effectiveness of rhGAA may benefit some Pompe patients. Co-administration of the mTORC1 inhibitor rapamycin with rhGAA in a GAA knockout mouse reduced muscle glycogen content more than rhGAA or rapamycin alone. These results suggest mTORC1 inhibition may benefit GSDs that involve glycogen accumulation in muscle., (Copyright 2010 Elsevier Inc. All rights reserved.)

Tissue turnover during wound healing, regeneration or integration of biomedical materials depends on the rate and extent of materials trafficking into and out of cells involved in extracellular matrix (ECM) remodeling. To exploit these processes, we report the first model for matrix trafficking in which these issues are quantitatively assessed for cells grown on both native collagen (normal tissue) and denatured collagen (wound state) substrates. Human fibroblasts more rapidly remodeled denatured versus normal collagen type I to form new ECM. Fluxes to and from the cells from the collagen substrates and the formation of new ECM were quantified using radioactively labeled substrates. The model can be employed for the systematic and quantitative study of the impact of a broad range of physiological factors and disease states on tissue remodeling, integrating extracellular matrix structures and cell biology.

EGF suppresses proteolysis via class 1 phosphatidylinositol 3-kinase (PI3K) in renal tubular cells. EGF also increases the abundance of glycolytic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and transcription factors (e.g., pax2) that are degraded by the lysosomal pathway of chaperone-mediated autophagy. To determine if EGF regulates chaperone-mediated autophagy through PI3K signaling, this study examined the effect of inhibiting PI3K and its downstream mediators Akt and the mammalian target of rapamycin (mTOR). Inhibition of PI3K with LY294002 prevented EGF-induced increases in GAPDH and pax2 abundance in NRK-52E renal tubular cells. Similar results were seen with an adenovirus encoding a dominant negative Akt (DN Akt). Expression of a constitutively active Akt increased GAPDH and pax2 abundance. An mTOR inhibitor, rapamycin, did not prevent EGF-induced increases in these proteins. Neither DN Akt nor rapamycin alone had an effect on total cell protein degradation, but both partially reversed EGF-induced suppression of proteolysis. DN Akt no longer affected proteolysis after treatment with a lysosomal inhibitor, methylamine. In contrast, methylamine or the inhibitor of macroautophagy, 3-methyladenine, did not prevent rapamycin from partially reversing the effect of EGF on proteolysis. Notably, rapamycin did not increase autophagasomes detected by monodansylcadaverine staining. Blocking the proteasomal pathway with either MG132 or lactacystin prevented rapamycin from partially reversing the effect of EGF on proteolysis. It is concluded that EGF regulates pax2 and GAPDH abundance and proteolysis through a PI3K/Akt-sensitive pathway that does not involve mTOR. Rapamycin has a novel effect of regulating proteasomal proteolysis in cells that are stimulated with EGF.

Mammals survive starvation by activating proteolysis and lipolysis in many different tissues. These responses are triggered, at least in part, by changing hormonal and neural statuses during starvation. Pathways of proteolysis that are activated during starvation are surprisingly diverse, depending on tissue type and duration of starvation. The ubiquitin-proteasome system is primarily responsible for increased skeletal muscle protein breakdown during starvation. However, in most other tissues, lysosomal pathways of proteolysis are stimulated during fasting. Short-term starvation activates macroautophagy, whereas long-term starvation activates chaperone-mediated autophagy. Lipolysis also increases in response to starvation, and the breakdown of triacylglycerols provides free fatty acids to be used as an energy source by skeletal muscle and other tissues. In addition, glycerol released from triacylglycerols can be converted to glucose by hepatic gluconeogenesis. During long-term starvation, oxidation of free fatty acids by the liver leads to the production of ketone bodies that can be used for energy by skeletal muscle and brain. Tissues that cannot use ketone bodies for energy respond to these small molecules by activating chaperone-mediated autophagy. This is one form of interaction between proteolytic and lipolytic responses to starvation.

Autophagy, including macroautophagy (MA), chaperone-mediated autophagy (CMA), crinophagy, pexophagy and microautophagy, are processes by which cells select internal components such as proteins, secretory vesicles, organelles, or foreign bodies, and deliver them to lysosomes for degradation. MA and CMA are activated during conditions of serum withdrawal in cell culture and during short-term and prolonged starvation in organisms, respectively. Although MA and CMA are activated under similar conditions, they are regulated by different mechanisms. We used pulse/chase analysis under conditions in which most intracellular proteolysis is due to CMA to test a variety of compounds for effects on this process. We show that inhibitors of MA such as 3-methyladenine, wortmannin, and LY294002 have no effect on CMA. Protein degradation by MA is sensitive to microtubule inhibitors such as colcemide and vinblastine, but protein degradation by CMA is not. Activators of MA such as rapamycin also have no effect on CMA. We demonstrate that CMA, like MA, is inhibited by protein synthesis inhibitors anisomycin and cycloheximide. CMA is also partially inhibited when the p38 mitogen activated protein kinase is blocked. Finally we demonstrate that the glucose-6-phophate dehydrogenase inhibitor, 6-aminonicotinamide, and heat shock protein of 90 kilodaltons inhibitor, geldanamycin, have the ability to activate CMA.

Purpose: The purpose of this tutorial is to describe 10 criteria that may help clinicians distinguish between scientific and pseudoscientific treatment claims. The criteria are illustrated, first for considering whether to use a newly developed treatment and second for attempting to understand arguments about controversial treatments., Method: Pseudoscience refers to claims that appear to be based on the scientific method but are not. Ten criteria for distinguishing between scientific and pseudoscientific treatment claims are described. These criteria are illustrated by using them to assess a current treatment for stuttering, the SpeechEasy device. The authors read the available literature about the device and developed a consensus set of decisions about the 10 criteria. To minimize any bias, a second set of independent judges evaluated a sample of the same literature. The criteria are also illustrated by using them to assess controversies surrounding 2 treatment approaches: Fast ForWord and facilitated communication., Conclusions: Clinicians are increasingly being held responsible for the evidence base that supports their practice. The power of these 10 criteria lies in their ability to help clinicians focus their attention on the credibility of that base and to guide their decisions for recommending or using a treatment.

Chaperone-mediated autophagy (CMA) is a selective lysosomal protein degradative process that is activated in higher organisms under conditions of prolonged starvation and in cell culture by the removal of serum. Ketone bodies are comprised of three compounds (beta-hydroxybutyrate, acetoacetate, and acetone) that circulate during starvation, especially during prolonged starvation. Here we have investigated the hypothesis that ketone bodies induce CMA. We found that physiological concentrations of beta-hydroxybutyrate (BOH) induced proteolysis in cells maintained in media with serum and without serum; however, acetoacetate only induced proteolysis in cells maintained in media with serum. Lysosomes isolated from BOH-treated cells displayed an increased ability to degrade both glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A, substrates for CMA. Isolated lysosomes from cells maintained in media without serum also demonstrated an increased ability to degrade glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A when the reaction was supplemented with BOH. Such treatment did not affect the levels of lysosome-associated membrane protein 2a or lysosomal heat shock cognate protein of 70 kDa, two rate-limiting proteins in CMA. However, pretreatment of glyceraldehyde-3-phosphate and ribonuclease A with BOH increased their rate of degradation by isolated lysosomes. Lysosomes pretreated with BOH showed no increase in proteolysis, suggesting that BOH acts on the substrates to increase their rates of proteolysis. Using OxyBlot analysis to detect carbonyl formation on proteins, one common marker of protein oxidation, we showed that treatment of substrates with BOH increased their oxidation. Neither glycerol, another compound that increases in circulation during prolonged starvation, nor butanol or butanone, compounds closely related to BOH, had an effect on CMA. The induction of CMA by ketone bodies may provide an important physiological mechanism for the activation of CMA during prolonged starvation.

Unlabelled: In light of emerging findings concerning untreated recovery and neural plasticity, this paper re-examines the viability of an NIH conference recommendation [Cooper, J. A. (1990). Research directions in stuttering: Consensus and conflict. In Cooper, J. A. (Ed.), Research needs in stuttering: Roadblocks and future directions (pp. 98-100). Rockville, MD: American Speech-Language-Hearing Association.] that adults who have recovered from stuttering might inform our understanding of the nature and treatment of persistent stuttering. It is suggested that those who have recovered could constitute a behavioral, cognitive, and neurophysiologic benchmark for evaluating stuttering treatment for adolescents and adults, while helping to identify the limits of recovery from a persistent disorder. This possibility seems especially promising because of findings from recent studies investigating untreated recovery during childhood and adulthood, the emerging evidence concerning neural plasticity and reorganization, and reports of neural system changes during stuttering treatment. Potential obstacles to applying findings from unassisted recovery to treatment do exist, but the benefits of attempts to fully understand stuttering certainly outweigh the difficulties., Educational Objectives: After completing this activity, the learner will be able to: (1) describe two complexities involved in determining whether recovery from stuttering was assisted or unassisted; (2) discuss the implications for stuttering research of two neural plasticity research findings from areas other than stuttering; and (3) evaluate the possible implications for stuttering treatment of a coordinated research program that addresses behavioral, cognitive, and neurological characteristics of assisted and unassisted recovery from stuttering.

Unlabelled: The purpose of this study was to investigate the nature of recovery from stuttering based on the experiences of adults who recovered without treatment. Using a semi-structured, open-ended interview format, 15 speakers verified as persons who recovered without treatment were asked to describe their status as everyday speakers. Seven speakers reported that they no longer stuttered and eight reported that they still stuttered on occasion. Interview material was coded and analyzed by the investigators and checked by independent judges. Results suggested that complete recovery was possible for speakers who reported that they no longer stuttered; whereas, those who still stuttered occasionally appeared to no longer be handicapped by stuttering, but required some vigilance to maintain their relatively fluent speech., Educational Objectives: After completing this activity, the learner will be able to: (1) describe the relevance of self-report data for evaluating the nature of recovery from stuttering without treatment; (2) describe the differences in self-perception concerning the nature of recovery for those who no longer have any tendency to stutter compared to those who still have an occasional tendency to stutter; and (3) suggest the possible implications for understanding the nature of recovery from persistent stuttering based on investigations of late recovery without treatment.

A number of terminal phosphate-labeled nucleotides with three or more phosphates and with varied length linkers attached between the terminal phosphate and the dye have been synthesized. These nucleotides have been tested as substrates for different DNA and RNA polymerases. We have also explored their utility in DNA sequencing, SNP analysis, nucleic acid amplification, quantitative PCR, and other biochemical assays.

Unlabelled: This paper overviews recent developments in an ongoing program of brain imaging research on developmental stuttering that is being conducted at the University of Texas Health Science Center, San Antonio. This program has primarily used H(2)15O PET imaging of different speaking tasks by right-handed adult male and female persistent stutterers, recovered stutterers and controls in order to isolate the neural regions that are functionally associated with stuttered speech. The principal findings have emerged from studies using condition contrasts and performance correlation techniques. The emerging findings from these studies are reviewed and referenced to a neural model of normal speech production recently proposed by Jürgens [Neurosci. Biobehav. Rev. 26 (2002) 235]. This paper will report (1) the reconfiguration of previous findings within the Jürgens Model; (2) preliminary findings of an investigation with late recovered stutterers; (3) an investigation of neural activations during a treatment procedure designed to produce a sustained improvement in fluency; and (4) an across-studies comparison that seeks to isolate neural regions within the Jürgens Model that are consistently associated with stuttering. Two regions appear to meet this criterion: right anterior insula (activated) and anterior middle and superior temporal gyri (deactivated) mainly in right hemisphere. The implications of these findings and the direction of future imaging investigations are discussed., Educational Objectives: The reader will learn about (1) recent uses of H(2)15O PET imaging in stuttering research; (2) the use of a new neurological model of speech production in imaging research on stuttering; and (3) initial findings from PET imaging investigations of treated and recovered stutterers.

Unlabelled: An evidence-based framework can be described as an empirically-driven, measurement-based, client-sensitive approach for selecting treatments. It is believed that using such a framework is more likely to result in a clinically significant outcome. For this paper, a clinically significant outcome was defined as a meaningful treatment change. It was suggested that there are at least three groups for whom a treatment's outcome is meaningful. These groups include clinicians/clinical researchers, the clients, and relevant others who have some interest in the outcome (e.g., parents of a child who stutters). The meaning and measurement of clinical significance was discussed for each of these three groups, based on research from the behavioral stuttering treatment literature., Educational Objectives: The reader will learn about and be able to (1) broadly define a clinically significant outcome and identify some of the groups who are interested in such an outcome and (2) describe how clinical significance has been evaluated in stuttering treatment within an evidence-based framework.

A series of charge-modified, dye-labeled 2', 3'-dideoxynucleoside-5'-triphosphates have been synthesized and evaluated as reagents for dye-terminator DNA sequencing. Unlike the commonly used dye-labeled terminators, these terminators possess a net positive charge and migrate in the opposite direction to dye-labeled Sanger fragments during electrophoresis. Post-sequencing reaction purification is not required to remove unreacted nucleotide or associated breakdown products prior to electrophoresis. Thus, DNA sequencing reaction mixtures can be loaded directly onto a separating medium such as a sequencing gel. The charge-modified nucleotides have also been shown to be more efficiently incorporated by a number of DNA polymerases than regular dye-labeled dideoxynucleotide terminators or indeed normal dideoxynucleoside-5'-triphosphates.

A four-color set of negatively charged, single dye as well as energy transfer dye labeled-ddNTPs were synthesized and evaluated in combination with a novel polymerase in a "direct-load" DNA sequencing, obviating the laborious and time consuming post-reaction work-up.

A number of different energy transfer dye labeled-cassettes were synthesized using aminoacid based trifunctional linkers and coupled to the propargylamino-substituted dideoxynucleoside-5'-triphosphates (ddNTPs). These terminators were evaluated for their energy transfer efficiency and DNA sequencing potential using thermostable DNA polymerase.

Unlabelled: Generalization and maintenance are a widely recognized challenge for stuttering treatment. There are many reasons why this is the case. First, there is no accepted model of recovery, though self-efficacy may be a helpful construct and, secondly, the client's age, stuttering severity, and negative attitudes may be complicating factors. Three strategies for promoting generalization and maintenance in school settings are suggested based on current research evidence. They include probing and training for generalization, incorporating real-life elements into therapy, and training clients to self-regulate their behavior. These strategies appear to be viable and practical, but further research is needed to fully evaluate their effectiveness for school settings., Learning Outcomes: Readers will learn about (1) typical approaches to managing stuttering in the school setting, (2) barriers to generalization and maintenance of stuttering treatment gains, and (3) three strategies for promoting generalization and maintenance.

Self-regulation refers to a process by which individuals learn to direct and control their own behavior, thoughts, and feelings to manage or eliminate their stuttering. As a concept, it has been a vital and enduring component of most approaches to managing persistent and chronic stuttering. The theoretical basis for self-regulation is briefly reviewed and the main principles of self-regulation--goal setting, cue management, self-monitoring, and self-evaluation--are described and illustrated with clinical examples. Factors related to self-regulatory failure are also discussed.

A novel series of charge-modified, dye-labeled 2',3'-dideoxynucleoside-triphosphate terminators were synthesized and evaluated as reagents for DNA sequencing. These terminators possess an advantage over existing reagents in that no purification is required to remove unreacted nucleotide or associated breakdown products prior to electrophoretic separation of the sequencing fragments. This obviates the need for a time consuming post-reaction work up, allowing direct loading of DNA sequencing reaction mixtures onto a slab gel. Thermo Sequenase II DNA polymerase poorly incorporates the charge-modified terminators compared with regular dye-labeled terminators. However, extending the linker arm between dye and nucleotide and using a mutant form of a related DNA polymerase can in part mitigate the decrease in substrate efficiency. We also present evidence that these charge-modified terminators can relieve gel compression artefacts when used with dGTP in sequencing reactions.