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Objective: To understand the etiological landscape and phenotypic differences between 2 developmental and epileptic encephalopathy (DEE) syndromes: DEE with spike-wave activation in sleep (DEE-SWAS) and epileptic encephalopathy with spike-wave activation in sleep (EE-SWAS)., Methods: All patients fulfilled International League Against Epilepsy (ILAE) DEE-SWAS or EE-SWAS criteria with a Core cohort (n = 91) drawn from our Epilepsy Genetics research program, together with 10 etiologically solved patients referred by collaborators in the Expanded cohort (n = 101). Detailed phenotyping and analysis of molecular genetic results were performed. We compared the phenotypic features of individuals with DEE-SWAS and EE-SWAS. Brain-specific gene co-expression analysis was performed for D/EE-SWAS genes., Results: We identified the etiology in 42/91 (46%) patients in our Core cohort, including 29/44 (66%) with DEE-SWAS and 13/47 (28%) with EE-SWAS. A genetic etiology was identified in 31/91 (34%). D/EE-SWAS genes were highly co-expressed in brain, highlighting the importance of channelopathies and transcriptional regulators. Structural etiologies were found in 12/91 (13%) individuals. We identified 10 novel D/EE-SWAS genes with a range of functions: ATP1A2, CACNA1A, FOXP1, GRIN1, KCNMA1, KCNQ3, PPFIA3, PUF60, SETD1B, and ZBTB18, and 2 novel copy number variants, 17p11.2 duplication and 5q22 deletion. Although developmental regression patterns were similar in both syndromes, DEE-SWAS was associated with a longer duration of epilepsy and poorer intellectual outcome than EE-SWAS., Interpretation: DEE-SWAS and EE-SWAS have highly heterogeneous genetic and structural etiologies. Phenotypic analysis highlights valuable clinical differences between DEE-SWAS and EE-SWAS which inform clinical care and prognostic counseling. Our etiological findings pave the way for the development of precision therapies. ANN NEUROL 2024;96:932-943., (© 2024 The Author(s). Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Ketogenic diet therapy (KDT) is an established treatment for people with epilepsy. As increasing evidence demonstrates effectiveness and safety of KDT on seizure reduction, cognition and behaviour, it is essential to evaluate factors hindering and supporting neurologists in prescribing KDT to strengthen quality, evidence-based, appropriate and equitable care. A study of Australian and New Zealand (ANZ) neurologists was undertaken via an online survey. Demographics, clinical role characteristics, perceptions of knowledge, use and experiences of KDT for epilepsy treatment were assessed. Responses were analysed using the Capability, Opportunity, Motivation and Behaviour (COM-B) model. 114 neurologists participated (18 % response rate). All were aware of KDT for epilepsy treatment, most (90 %) perceived it as acceptable and 85 % identified suitable patients in their practice. Poor knowledge of the KDT referral processes was a barrier for 64 %. Clinical role characteristics were significantly associated with perceived level of knowledge and use of KDT in practice, being more likely among paediatric neurologists), epileptologists and those in urban practices (p < 0.00001). Most neurologists (90 %) endorsed adoption of a KDT guideline to facilitate use of KDT in epilepsy management. This study established that KDT is accepted as a suitable treatment for epilepsy in ANZ. There is high variability in perceived knowledge and skills related to KDT, which impacts on utilization in clinical practice. Further education and resources for clinicians, allied health and community support agencies are needed to optimise the use of this valuable therapy. Additionally, a clear referral pathway would improve patient access., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024 Published by Elsevier Inc.)

Background: Kabuki syndrome (KS) is a genetic disorder caused by DNA mutations in KMT2D, a lysine methyltransferase that methylates histones and other proteins, and therefore modifies chromatin structure and subsequent gene expression. Ketones, derived from the ketogenic diet, are histone deacetylase inhibitors that can 'open' chromatin and encourage gene expression. Preclinical studies have shown that the ketogenic diet rescues hippocampal memory neurogenesis in mice with KS via the epigenetic effects of ketones., Methods: Single-cell RNA sequencing and mass spectrometry-based proteomics were used to explore molecular mechanisms of disease in individuals with KS (n = 4) versus controls (n = 4)., Findings: Pathway enrichment analysis indicated that loss of function mutations in KMT2D are associated with ribosomal protein dysregulation at an RNA and protein level in individuals with KS (FDR <0.05). Cellular proteomics also identified immune dysregulation and increased abundance of other lysine modification and histone binding proteins, representing a potential compensatory mechanism. A 12-year-old boy with KS, suffering from recurrent episodes of cognitive decline, exhibited improved cognitive function and neuropsychological assessment performance after 12 months on the ketogenic diet, with concomitant improvement in transcriptomic ribosomal protein dysregulation., Interpretation: Our data reveals that lysine methyltransferase deficiency is associated with ribosomal protein dysfunction, with secondary immune dysregulation. Diet and the production of bioactive molecules such as ketone bodies serve as a significant environmental factor that can induce epigenetic changes and improve clinical outcomes. Integrating transcriptomic, proteomic, and clinical data can define mechanisms of disease and treatment effects in individuals with neurodevelopmental disorders., Funding: This study was supported by the Dale NHMRC Investigator Grant (APP1193648) (R.D), Petre Foundation (R.D), and The Sydney Children's Hospital Foundation/Kids Research Early and Mid-Career Researcher Grant (E.T)., Competing Interests: Declaration of interests The authors report no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objectives: The ketogenic diet (KD) is a treatment for children with intractable epilepsy (IE), can cause gastrointestinal symptoms, and have an adverse effect on growth, nutrition and quality of life (QOL). This study investigated the extent of these side effects by comparing children with IE on KDs to their counterparts on normal diets., Methods: Patients with IE were categorized into patients with KD or control groups. Gastrointestinal side effects and QOL were assessed using the PedsQL Gastrointestinal Symptoms Module. Cross sectional growth, gut microbiome compositions, and inflammation levels were also analyzed., Results: Fourteen patients on the KD and 13 control patients were enrolled. Patients had been on KD for a median duration of 15 months (interquartile range: 9.8-60 months). The patients on the KD reported a trend to lower total gastrointestinal symptoms scores (more symptoms) compared to control patients, at 71.1 and 84.9, respectively ( P = 0.06, not significant). Patients on the KD had significantly lower QOL scores compared to control patients ( P = 0.01). Patients on the KD were found to have consistently lower median height/length, weight, and body mass index z scores compared to the controls although these were not statistically significant. Patients on the KD had a lower microbial diversity, Both groups had a normal level of S100A12, a marker of gut inflammation., Conclusions: Patients on the KD reported a trend to more gastrointestinal symptoms and more QOL concerns compared to controls. Although microbial differences were noted in patients on the KD, this did not result in detectable gut inflammation., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 by European Society for European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

Objective: WWOX is an autosomal recessive cause of early infantile developmental and epileptic encephalopathy (WWOX-DEE), also known as WOREE (WWOX-related epileptic encephalopathy). We analyzed the epileptology and imaging features of WWOX-DEE, and investigated genotype-phenotype correlations, particularly with regard to survival., Methods: We studied 13 patients from 12 families with WWOX-DEE. Information regarding seizure semiology, comorbidities, facial dysmorphisms, and disease outcome were collected. Electroencephalographic (EEG) and brain magnetic resonance imaging (MRI) data were analyzed. Pathogenic WWOX variants from our cohort and the literature were coded as either null or missense, allowing individuals to be classified into one of three genotype classes: (1) null/null, (2) null/missense, (3) missense/missense. Differences in survival outcome were estimated using the Kaplan-Meier method., Results: All patients experienced multiple seizure types (median onset = 5 weeks, range = 1 day-10 months), the most frequent being focal (85%), epileptic spasms (77%), and tonic seizures (69%). Ictal EEG recordings in six of 13 patients showed tonic (n = 5), myoclonic (n = 2), epileptic spasms (n = 2), focal (n = 1), and migrating focal (n = 1) seizures. Interictal EEGs demonstrated slow background activity with multifocal discharges, predominantly over frontal or temporo-occipital regions. Eleven of 13 patients had a movement disorder, most frequently dystonia. Brain MRIs revealed severe frontotemporal, hippocampal, and optic atrophy, thin corpus callosum, and white matter signal abnormalities. Pathogenic variants were located throughout WWOX and comprised both missense and null changes including five copy number variants (four deletions, one duplication). Survival analyses showed that patients with two null variants are at higher mortality risk (p-value = .0085, log-rank test)., Significance: Biallelic WWOX pathogenic variants cause an early infantile developmental and epileptic encephalopathy syndrome. The most common seizure types are focal seizures and epileptic spasms. Mortality risk is associated with mutation type; patients with biallelic null WWOX pathogenic variants have significantly lower survival probability compared to those carrying at least one presumed hypomorphic missense pathogenic variant., (© 2023 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

In this work, we present a new experimental setup for the assessment of the anisotropic properties of Bovine Pericardium (BP) membranes. The chemically fixed BP samples have been subjected to a bulge test with in situ confocal laser scanning at increasing applied pressure. The high resolution topography provided by the confocal laser scanning has allowed to obtain a quantitative measure of the bulge displacement; after polynomial fitting, principal curvatures have been obtained and a degree of anisotropy (DA) has been defined as the normalized difference between the maximum and minimum principal curvatures. The experiments performed on the BP membranes have allowed us to obtain pressure-displacement data which clearly exhibit distinct principal curvatures indicating an anisotropic response. A comparison with curvatures data obtained on isotropic Nitrile Buthadiene Rubber (NBR) samples has confirmed the effectiveness of the experimental setup for this specific purpose. Numerical simulations of the bulge tests have been performed with the purpose of identifying a range of constitutive parameters which well describes the obtained range of DA on the BP membranes. The DA values have been partially validated with biaxial tests available in literature and with suitably performed uni-axial tensile tests., (Copyright © 2023 by ASME.)

Aim: To improve delivery of acute therapies for acute ischaemic stroke (AIS)., Method: We identified factors influencing the speed of diagnosis and delivery of acute therapies in a prospective cohort of 21 children with suspected AIS (eight with AIS, 13 stroke mimics) and explored them in a retrospective cohort with confirmed AIS., Results: Approximately half of the prospective and total AIS cohorts presented with acute, sustained hemiparesis, and were diagnosed relatively quickly. AIS was suspected and diagnosed more slowly in the half presenting with symptoms other than sustained hemiparesis. Thirty-one out of 51 patients with AIS (19 females, 32 males, mean age 8 years 6 months, SD 5 years 4 months) had arterial abnormalities identified by computed tomography angiography (CTA) or magnetic resonance angiography (MRA): 11 with large vessel occlusion, six with dissection, five with moyamoya disease, nine with other arteriopathies. Among these patients, those initially imaged with CTA were diagnosed more quickly than those with initial magnetic resonance imaging/angiography, which facilitated thrombectomy and thrombolytic therapy. Twenty out of 51 had AIS without arterial abnormalities on CTA or MRA: eight with lenticulostriate vasculopathy and 12 with other small-vessel AIS. Among these patients, 80% were ineligible for thrombolysis for reasons beyond delay to diagnosis, and all showed good outcomes with supportive treatments alone., Interpretation: Clinical features at presentation influence rapidity with which childhood AIS is suspected and diagnosed. Readily available CTA can direct thrombectomy in patients with large vessel occlusion and thrombolysis in most, but not all, eligible patients., What This Paper Adds: Children with acute ischaemic stroke (AIS) commonly present with symptoms other than sustained hemiparesis. Stroke is more slowly recognized in these patients, which limits potential therapies. Computed tomography angiography (CTA) accurately identifies AIS with large vessel occlusion, enabling timely endovascular thrombectomy. CTA is sufficient to direct thrombolytic therapy in most eligible children. Most childhood AIS without arterial abnormalities identified by CTA had good outcomes., (© 2022 The Authors. Developmental Medicine & Child Neurology published by John Wiley & Sons Ltd on behalf of Mac Keith Press.)

Aim: To determine if the management of paediatric status epilepticus (SE) follows accepted clinical practice guidelines., Methods: Retrospective, consecutive series of patients with SE who attended the emergency departments from two NSW sites over a 12-month period. SE was defined as a convulsive seizure, 5 min or more in duration. Time to presentation to the ED, time to first- and second-line treatment, number of benzodiazepine (BZD) doses given prior to intubation and adherence to guidelines were evaluated. The outcomes included seizure duration, need for respiratory support, admission to intensive care, morbidity and mortality., Results: The time from onset of seizure to ED presentation was a median (p25-p75) time of 22 (15-40) min. Forty-eight of 59 presentations received pre-hospital midazolam. The median (p25-p75) time to first-line treatment was 15 (8-25) min and to second-line treatment was 43.5 (35-59) min. There was no significant difference in the results in the two hospitals. The total number of BZD doses ranged from 1 to 7 (median 3). There was non-adherence to the clinical practice guidelines in 55 (93.2%) of 59 presentations., Conclusions: We found excessive benzodiazepine use and delay in both definitive treatment of status epilepticus and in escalation from first- to second-line anticonvulsant treatment. This raises the need for rapid escalation of treatment. We propose a 'status epilepticus code' for emergency departments., (© 2021 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).)

Objective: To assess the benefits and limitations of whole genome sequencing (WGS) compared to exome sequencing (ES) or multigene panel (MGP) in the molecular diagnosis of developmental and epileptic encephalopathies (DEE)., Methods: We performed WGS of 30 comprehensively phenotyped DEE patient trios that were undiagnosed after first-tier testing, including chromosomal microarray and either research ES (n = 15) or diagnostic MGP (n = 15)., Results: Eight diagnoses were made in the 15 individuals who received prior ES (53%): 3 individuals had complex structural variants; 5 had ES-detectable variants, which now had additional evidence for pathogenicity. Eleven diagnoses were made in the 15 MGP-negative individuals (68%); the majority (n = 10) involved genes not included in the panel, particularly in individuals with postneonatal onset of seizures and those with more complex presentations including movement disorders, dysmorphic features, or multiorgan involvement. A total of 42% of diagnoses were autosomal recessive or X-chromosome linked., Conclusion: WGS was able to improve diagnostic yield over ES primarily through the detection of complex structural variants (n = 3). The higher diagnostic yield was otherwise better attributed to the power of re-analysis rather than inherent advantages of the WGS platform. Additional research is required to assist in the assessment of pathogenicity of novel noncoding and complex structural variants and further improve diagnostic yield for patients with DEE and other neurogenetic disorders., (© 2021 American Academy of Neurology.)

Endovascular clot retrieval (ECR) is an emerging therapy for treatment of acute ischaemic stroke (AIS) in adults, including basilar artery occlusion (BAO). Its role in children is not well established. We report four consecutive children with AIS due to BAO treated with ECR in Sydney, Australia. We reviewed the literature to characterize the 'natural course' of AIS due to BAO in children not treated with thrombolysis or ECR, and compared their outcome with our patients and reported children with BAO treated with ECR. Despite delays in diagnosis, ECR achieved recanalization in our four children. Three children had a good outcome (Paediatric Modified Rankin Score [PedmRS] 0-2). One child with acute leukaemia suffered recurrent basilar occlusion and died of brainstem dysfunction. Literature review identified 111 children exhibiting the natural course of AIS due to BAO, among whom 42% had good outcomes (PedmRS 0-2), 48% had significant residual disability (PedmRS 3-5), and 10% died. Of 34 children treated with ECR, 28 (82%) had good outcomes (PedmRS 0-2), five (15%) had significant residual disability (PedmRS 3-5), and one (3%) died. Complications of ECR were uncommon. These observations suggest ECR may be beneficial for children with AIS due to BAO. WHAT THIS PAPER ADDS: Children with acute ischaemic stroke (AIS) due to basilar artery occlusion (BAO) experience significant morbidity and mortality. Endovascular clot retrieval may be beneficial in children with AIS due to BAO., (© 2020 Mac Keith Press.)

Aim: To evaluate the influence of adherence to treatment guidelines on outcomes in children presenting with status epilepticus (SE) using the Newborn and Paediatric Emergency Transport Service, New South Wales prospective registry., Methods: We reviewed the treatment of children with SE, transported by the Newborn And Paediatric Emergency Transport Service to a tertiary paediatric hospital, over 1 year. We evaluated variation in management from the New South Wales clinical practice guideline., Results: There was excessive administration of benzodiazepines (BZD) and a delay in administration of appropriate second-line treatment of status (median 45 min). There was excessive use of BZD, with five or more doses of BZD associated with significantly higher odds for intubation., Conclusion: There is considerable scope to improve clinician compliance with the SE clinical practice guidelines., (© 2019 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).)

Objective: Myotonic dystrophy type 1 (DM1) is an autosomal-dominant neuromuscular disease with variable severity affecting all ages; however, current care guidelines are adult-focused. The objective of the present study was to profile DM1 in childhood and propose a framework to guide paediatric-focused management., Design, Setting and Patients: 40 children with DM1 (mean age 12.8 years; range 2-19) were studied retrospectively for a total of 513 follow-up years at Sydney Children's Hospital. 143 clinical parameters were recorded., Results: The clinical spectrum of disease in childhood differs from adults, with congenital myotonic dystrophy (CDM1) having more severe health issues than childhood-onset/juvenile patients (JDM1). Substantial difficulties with intellectual (CDM1 25/26 96.2%; JDM1 9/10, 90.0%), fine motor (CDM1 23/30, 76.6%; JDM1 6/10, 60.0%), gastrointestinal (CDM1 17/30, 70.0%; JDM1 3/10, 30.0%) and neuromuscular function (CDM1 30/30, 100.0%; JDM1 25/30, 83.3%) were evident., Conclusion: The health consequences of DM1 in childhood are diverse, highlighting the need for paediatric multidisciplinary management approaches that encompass key areas of cognition, musculoskeletal, gastrointestinal, respiratory, cardiac and sleep issues., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Objective: To evaluate the tolerability and safety of cannabidiol for treating drug-resistant epilepsy in children, and to describe adverse events associated with such treatment., Study Design: Prospective, open label cohort study., Setting: Three tertiary NSW referral centres with paediatric neurology services., Participants: First 40 children enrolled in the NSW Compassionate Access Scheme for children with drug-resistant epilepsy and uncountable daily seizures., Intervention: Children received cannabidiol as an adjunct anti-epileptic drug, titrated to a maximum of 25 mg/kg/day, for up to 12 weeks., Outcome Measures: Adverse events, withdrawals, and caregiver and physician Global Impression of Change assessments were recorded at 4, 8 and 12 weeks. Seizure frequency could not be reliably recorded because of disease severity., Results: Thirty-nine patients reported at least one adverse event; many were deemed unrelated to cannabidiol treatment. The most frequent treatment-related adverse event was somnolence (15 participants), which resolved spontaneously in ten patients; it was particularly frequent in patients taking higher clobazam doses. Gastrointestinal effects (nausea, vomiting, diarrhoea) were each reported by seven to nine participants. Four children were withdrawn from treatment, including one with elevated transaminase levels. The caregivers of 12 children felt the overall health of their children had much or very much improved; clinicians assessed seven children as being much or very much improved., Conclusion: Cannabidiol as an adjunct treatment had some subjective benefit for overall health, with a manageable adverse event profile. Monitoring changes in liver function and awareness of potential drug interactions is essential. Whether the reported benefit is attributable to cannabidiol cannot be established in an open label study of participants with severe intractable epilepsy.

Background: Multiple guidelines seek to alter rates of prostate-specific antigen (PSA)-based prostate cancer screening. The costs borne by payers associated with PSA-based screening for men of different age groups-including the costs of screening and subsequent diagnosis, treatment, and adverse events-remain uncertain. We sought to develop a model of PSA costs that could be used by payers and health care systems to inform cost considerations under a range of different scenarios., Methods: We determined the prevalence of PSA screening among men aged 50 and higher using 2013-2014 data from a large, multispecialty group, obtained reimbursed costs associated with screening, diagnosis, and treatment from a commercial health plan, and identified transition probabilities for biopsy, diagnosis, treatment, and complications from the literature to generate a cost model. We estimated annual total costs for groups of men ages 50-54, 55-69, and 70+ years, and varied annual prostate cancer screening prevalence in each group from 5 to 50% and tested hypothetical examples of different test characteristics (e.g., true/false positive rate)., Results: Under the baseline screening patterns, costs of the PSA screening represented 10.1% of the total costs; costs of biopsies and associated complications were 23.3% of total costs; and, although only 0.3% of all screen eligible patients were treated, they accounted for 66.7% of total costs. For each 5-percentage point decrease in PSA screening among men aged 70 and older for a single calendar year, total costs associated with prostate cancer screening decreased by 13.8%. For each 5-percentage point decrease in PSA screening among men 50-54 and 55-69 years old, costs were 2.3% and 7.3% lower respectively., Conclusions: With constrained financial resources and with national pressure to decrease use of clinically unnecessary PSA-based prostate cancer screening, there is an opportunity for cost savings, especially by focusing on the downstream costs disproportionately associated with screening men 70 and older.

Objective: Due to a gap between published clinical guidelines on status epilepticus SE and clinician management of SE, a systematic review was performed to investigate treatment adherence to SE guidelines and its impact on patient outcomes., Methods: Medline and Embase searches were conducted for studies appraising adherence to SE guidelines (from 1970 and 1st April 2018). The quality of eligible studies was assessed by QUADAS- 2 criteria. Comparison was made between patients where guidelines were followed and not followed. Various patient outcomes including intubation, ICU admission, morbidity and mortality were compared. A Forest plot was used to investigate the effect of adherence on outcome., Results: A total of 3424 titles and abstracts were screened from the initial search after removal of duplicates. A total of 441 full text articles were reviewed in detail, and 22 articles were included in this study. The proportion of deviations ranged from 10.7% to 66.1%. Four studies were descriptive. Eighteen studies looked at the adverse effects of non-adherence. Eight studies showed respiratory depression and intubation were associated with excessive benzodiazepine use. A subset analysis showed 5.79 times higher odds of respiratory depression and intubation], if excessive benzodiazepines were given. The next most common variations were delayed management and insufficient treatment. These variations from the guidelines were associated with prolonged seizures., Conclusions: This review provides preliminary evidence that non-adherence to SE guidelines negatively impacts on patient outcomes. Appropriate and timely treatment is imperative for rapid seizure termination and improving outcomes., (Copyright © 2018 British Epilepsy Association. All rights reserved.)

Background: Epileptic encephalopathies are a devastating group of neurological conditions in which etiological diagnosis can alter management and clinical outcome. Exome sequencing and gene panel testing can improve diagnostic yield but there is no cost-effectiveness analysis of their use or consensus on how to best integrate these tests into clinical diagnostic pathways., Methods: We conducted a retrospective cost-effectiveness study comparing trio exome sequencing with a standard diagnostic approach, for a well-phenotyped cohort of 32 patients with epileptic encephalopathy, who remained undiagnosed after "first-tier" testing. Sensitivity analysis was included with a range of commercial exome and multigene panels., Results: The diagnostic yield was higher for the exome sequencing (16/32; 50%) than the standard arm (2/32; 6.2%). The trio exome sequencing pathway was cost-effective compared to the standard diagnostic pathway with a cost saving of AU$5,236 (95% confidence intervals $2,482; $9,784) per additional diagnosis; the standard pathway cost approximately 10 times more per diagnosis. Sensitivity analysis demonstrated that the majority of commercial exome sequencing and multigene panels studied were also cost-effective. The clinical utility of all diagnoses was reported., Conclusion: Our study supports the integration of exome sequencing and gene panel testing into the diagnostic pathway for epileptic encephalopathy, both in terms of cost effectiveness and clinical utility. We propose a diagnostic pathway that integrates initial rapid screening for treatable causes and comprehensive genomic screening. This study has important implications for health policy and public funding for epileptic encephalopathy and other neurological conditions., (© 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Certain mutations can cause proteins to accumulate in neurons, leading to neurodegeneration. We recently showed, however, that upregulation of a wild-type protein, Ataxin1, caused by haploinsufficiency of its repressor, the RNA-binding protein Pumilio1 (PUM1), also causes neurodegeneration in mice. We therefore searched for human patients with PUM1 mutations. We identified eleven individuals with either PUM1 deletions or de novo missense variants who suffer a developmental syndrome (Pumilio1-associated developmental disability, ataxia, and seizure; PADDAS). We also identified a milder missense mutation in a family with adult-onset ataxia with incomplete penetrance (Pumilio1-related cerebellar ataxia, PRCA). Studies in patient-derived cells revealed that the missense mutations reduced PUM1 protein levels by ∼25% in the adult-onset cases and by ∼50% in the infantile-onset cases; levels of known PUM1 targets increased accordingly. Changes in protein levels thus track with phenotypic severity, and identifying posttranscriptional modulators of protein expression should identify new candidate disease genes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Research is expanding for the use of cannabidiol as an anticonvulsant drug. The mechanism of cannabidiol in paediatric epilepsy is unclear but is thought to play a role in modulation of synaptic transmission. Evidence for its efficacy in treating epilepsy is limited but growing, with a single pharmaceutical company-funded randomised double-blind controlled trial in children with Dravet syndrome. Progress towards the use of medicinal cannabinoids incorporates a complex interplay of social influences and political and legal reform. Access to unregistered but available cannabidiol in Australia outside of clinical trials and compassionate access schemes is state dependent and will require Therapeutic Goods Administration approval, although the cost may be prohibitive. Further clinical trials are needed to clearly define efficacy and safety, particularly long term.

Improved survival of children with congenital heart disease has led to increasing focus on neurodevelopmental outcome, as close to half of the infants undergoing cardiac surgery are affected by neurodevelopmental disability. Stroke is particularly important as it frequently results in permanent neurologic sequelae. The aim of this study was to investigate risk factors for peri-procedural arterial ischaemic stroke (AIS) in children with cardiac disease. A retrospective case-control analysis of children aged <18 years with radiologically confirmed AIS following a cardiac procedure admitted to the Royal Children's Hospital Melbourne between 1993 and 2010. Each case was matched with two controls with similar cardiac diagnosis, procedure type, age and date of procedure. Demographics and peri-procedural data were collected from medical records and departmental database. Fifty-two cases were identified. Multivariable analysis identified post-procedural infection (OR 6.1, CI 1.3-27, p = 0.017) and length of ICU stay (OR 4.0, CI 1.4-11, p = 0.009) as risk factors for AIS. Although the study is limited to a single-centre cohort, length of ICU stay and post-procedural infection were identified as risk factors for AIS. These findings demonstrate these factors to be important areas to focus attention for stroke prevention in children with cardiac disease.

BAT-controlled thermogenic activity is thought to be required for its capacity to prevent the development of insulin resistance. This hypothesis predicts that mediators of thermogenesis may help prevent diet-induced insulin resistance. We report that the mitochondrial fusion protein Mitofusin 2 (Mfn2) in BAT is essential for cold-stimulated thermogenesis, but promotes insulin resistance in obese mice. Mfn2 deletion in mice through Ucp1-cre (BAT-Mfn2-KO) causes BAT lipohypertrophy and cold intolerance. Surprisingly however, deletion of Mfn2 in mice fed a high fat diet (HFD) results in improved insulin sensitivity and resistance to obesity, while impaired cold-stimulated thermogenesis is maintained. Improvement in insulin sensitivity is associated with a gender-specific remodeling of BAT mitochondrial function. In females, BAT mitochondria increase their efficiency for ATP-synthesizing fat oxidation, whereas in BAT from males, complex I-driven respiration is decreased and glycolytic capacity is increased. Thus, BAT adaptation to obesity is regulated by Mfn2 and with BAT-Mfn2 absent, BAT contribution to prevention of insulin resistance is independent and inversely correlated to whole-body cold-stimulated thermogenesis., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Purpose: Palliative care inpatient units (PCUs) can improve symptoms, family perception of care, and lower per-diem costs compared with usual care. In March 2013, Johns Hopkins Medical Institutions (JHMI) added a PCU to the palliative care (PC) program. We studied the financial impact of the PC program on JHMI from March 2013 to March 2014., Methods: This study considered three components of the PC program: PCU, PC consultations, and professional fees. Using 13 months of admissions data, the team calculated the per-day variable cost pre-PCU (ie, in another hospital unit) and after transfer to the PCU. These fees were multiplied by the number of patients transferred to the PCU and by the average length of stay in the PCU. Consultation savings were estimated using established methods. Professional fees assumed a collection rate of 50%., Results: The total positive financial impact of the PC program was $3,488,863.17. There were 153 transfers to the PCU, 60% with cancer, and an average length of stay of 5.11 days. The daily loss pretransfer to the PCU of $1,797.67 was reduced to $1,345.34 in the PCU (-25%). The PCU saved JHMI $353,645.17 in variable costs, or $452.33 per transfer. Cost savings for PC consultations in the hospital, 60% with cancer, were estimated at $2,765,218. $370,000 was collected in professional fees savings., Conclusion: The PCU and PC program had a favorable impact on JHMI while providing expert patient-centered care. As JHMI moves to an accountable care organization model, value-based patient-centered care and increased intensive care unit availability are desirable.

Objectives: Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disease with variable severity that affects all ages. Sleepiness is an important co-morbidity affecting a large proportion of paediatric DM1 patients. The current study examined the relationship between sleepiness and quality of life in a paediatric DM1 cohort., Methods: A cross-sectional study was conducted in children and adolescents with DM1 attending a multi-disciplinary neuromuscular clinic in a tertiary paediatric centre. The modified Epworth sleepiness scale (ESS), the PedsQL™ quality of life (version 4.0) and neuromuscular modules (version 3.0) were used to measure sleepiness, generic quality of life and neuromuscular-specific quality of life, respectively., Results: Seventeen current patients with DM1 completed all questionnaires and assessments. Of them, 35.5% had abnormal scores on the modified ESS, which is indicative of excessive daytime sleepiness (EDS). Higher ESS scores were highly significantly related to reduced quality of life in neuromuscular-specific (r = 0.77, p < 0.001) and generic measures (r = 0.78, p < 0.001). EDS was not significantly related to intellectual function or sleep disorders as detected on polysomnography., Conclusions: EDS is common in children and adolescents with DM1. It is associated with reduced quality of life and should be routinely assessed. Further studies to develop treatments of EDS in this population are required and may improve overall outcomes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Non-proteolytic ubiquitin signaling mediated by Lys 63 ubiquitin chains plays a critical role in multiple pathways that are key to the development and activation of immune cells. Our previous work indicates that GPS2 (G-protein Pathway Suppressor 2) is a multifunctional protein regulating TNFα signaling and lipid metabolism in the adipose tissue through modulation of Lys 63 ubiquitination events. However, the full extent of GPS2-mediated regulation of ubiquitination and the underlying molecular mechanisms are unknown. Here, we report that GPS2 is required for restricting the activation of TLR and BCR signaling pathways and the AKT/FOXO1 pathway in immune cells based on direct inhibition of Ubc13 enzymatic activity. Relevance of this regulatory strategy is confirmed in vivo by B cell-targeted deletion of GPS2, resulting in developmental defects at multiple stages of B cell differentiation. Together, these findings reveal that GPS2 genomic and non-genomic functions are critical for the development and cellular homeostasis of B cells., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: Insulin signaling plays a unique role in the regulation of energy homeostasis and the impairment of insulin action is associated with altered lipid metabolism, obesity, and Type 2 Diabetes. The main aim of this study was to provide further insight into the regulatory mechanisms governing the insulin signaling pathway by investigating the role of non-proteolytic ubiquitination in insulin-mediated activation of AKT., Methods: The molecular mechanism of AKT regulation through ubiquitination is first dissected in vitro in 3T3-L1 preadipocytes and then validated in vivo using mice with adipo-specific deletion of GPS2, an endogenous inhibitor of Ubc13 activity (GPS2-AKO mice)., Results: Our results indicate that K63 ubiquitination is a critical component of AKT activation in the insulin signaling pathway and that counter-regulation of this step is provided by GPS2 preventing AKT ubiquitination through inhibition of Ubc13 enzymatic activity. Removal of this negative checkpoint, through GPS2 downregulation or genetic deletion, results in sustained activation of insulin signaling both in vitro and in vivo . As a result, the balance between lipid accumulation and utilization is shifted toward storage in the adipose tissue and GPS2-AKO mice become obese under normal laboratory chow diet. However, the adipose tissue of GPS2-AKO mice is not inflamed, the levels of circulating adiponectin are elevated, and systemic insulin sensitivity is overall improved., Conclusions: Our findings characterize a novel layer of regulation of the insulin signaling pathway based on non-proteolytic ubiquitination of AKT and define GPS2 as a previously unrecognized component of the insulin signaling cascade. In accordance with this role, we have shown that GPS2 presence in adipocytes modulates systemic metabolism by restricting the activation of insulin signaling during the fasted state, whereas in absence of GPS2, the adipose tissue is more efficient at lipid storage, and obesity becomes uncoupled from inflammation and insulin resistance.

Aim: To develop and evaluate an online educational package instructing paediatricians and trainees in the diagnosis and management of a first unprovoked seizure in children., Methods: The E-learning content was created following a comprehensive literature review that referenced current international guidelines. Rigorous consultation with local paediatric neurologists, paediatricians and epilepsy nurses was undertaken. A series of learning modules was created and sequenced to reflect steps needed to achieve optimal diagnosis and management in a real-life situation of a child presenting with a paroxysmal event. Paediatric registrars and advanced trainees from the Sydney Children's Hospitals Network were assessed before and after using the E-learning Resource. Measures included general epilepsy knowledge, case-based scenario knowledge; self-rated measures of satisfaction with instruction and confidence regarding clinical approach to the child with first unprovoked seizure; and open ended questions evaluating the usefulness of the E-learning resource., Results: Performance on measures of general epilepsy knowledge and on the seizure-related case scenarios improved significantly following completion of the E-learning as did self-rated satisfaction with instruction and confidence across all aspects of managing first seizure., Conclusions: The E-learning resource has been validated as a useful educational resource regarding the first afebrile unprovoked seizure for paediatricians., (© 2016 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).)

Objective: To describe the spectrum of cardiac disorders, timing in relation to interventional procedures, and outcome in children with cardiac disease and arterial ischemic stroke (AIS)., Methods: Children younger than 18 years with cardiac disease and radiologically confirmed AIS admitted to the Royal Children's Hospital Melbourne between 1993 and 2010 were retrospectively identified using ICD-9 and ICD-10 searches., Results: Seventy-six children with cardiac disease and radiologically confirmed AIS were identified with the median age at diagnosis of 5 months (interquartile range 0-58). Cardiac lesions included cyanotic congenital heart disease (CHD) in 42 (55%), acyanotic heart disease in 24 (29%), cardiomyopathies/myocarditis in 6 (8%), infective endocarditis in 3 (4%), and primary arrhythmias in 3 (4%). Stroke occurred following cardiac procedures in 52 patients (68%): 41 post cardiac surgery (4.6 strokes per 1,000 surgical procedures) and 11 post cardiac catheterization (1.7 strokes per 1,000 catheterizations). The median time from procedure to diagnosis of stroke was 3 days (interquartile range 2-7), with 68% (95% confidence interval 58%-79%) of strokes estimated to occur within the periprocedural period. Prevalence of periprocedural stroke varied by diagnostic category, but was most common in patients with cyanotic CHD undergoing palliative surgery (22/2,256, 1%) (p < 0.005). There were 3 AIS-related deaths, and 54 survivors (84%) had persisting neurologic deficits., Conclusions: Infants with cyanotic CHD were most frequently affected by AIS during the periprocedural period. Prospective cohort studies are required to determine effective primary and secondary prevention strategies., (© 2015 American Academy of Neurology.)

Myotonic dystrophy type 1 (DM1) is multisystem disease arising from mutant CTG expansion in the non-translating region of the dystrophia myotonica protein kinase gene. While DM1 is the most common adult muscular dystrophy, with a worldwide prevalence of one in eight thousand, age of onset varies from before birth to adulthood. There is a broad spectrum of clinical severity, ranging from mild to severe, which correlates with number of DNA repeats. Importantly, the early clinical manifestations and management in congenital and childhood DM1 differ from classic adult DM1. In neonates and children, DM1 predominantly affects muscle strength, cognition, respiratory, central nervous and gastrointestinal systems. Sleep disorders are often under recognised yet a significant morbidity. No effective disease modifying treatment is currently available and neonates and children with DM1 may experience severe physical and intellectual disability, which may be life limiting in the most severe forms. Management is currently supportive, incorporating regular surveillance and treatment of manifestations. Novel therapies, which target the gene and the pathogenic mechanism of abnormal splicing are emerging. Genetic counselling is critical in this autosomal dominant genetic disease with variable penetrance and potential maternal anticipation, as is assisting with family planning and undertaking cascade testing to instigate health surveillance in affected family members. This review incorporates discussion of the clinical manifestations and management of congenital and childhood DM1, with a particular focus on hypersomnolence and sleep disorders. In addition, the molecular genetics, mechanisms of disease pathogenesis and development of novel treatment strategies in DM1 will be summarised.

Asparagine Synthetase Deficiency is a recently described cause of profound intellectual disability, marked progressive cerebral atrophy and variable seizure disorder. To date there has been limited functional data explaining the underlying pathophysiology. We report a new case with compound heterozygous mutations in the ASNS gene (NM&#95;183356.3:c. [866G>C]; [1010C>T]). Both variants alter evolutionarily conserved amino acids and were predicted to be pathogenic based on in silico protein modelling that suggests disruption of the critical ATP binding site of the ASNS enzyme. In patient fibroblasts, ASNS expression as well as protein and mRNA stability are not affected by these variants. However, there is markedly reduced proliferation of patient fibroblasts when cultured in asparagine-limited growth medium, compared to parental and wild type fibroblasts. Restricting asparagine replicates the physiology within the blood-brain-barrier, with limited transfer of dietary derived asparagine, resulting in reliance of neuronal cells on intracellular asparagine synthesis by the ASNS enzyme. These functional studies offer insight into the underlying pathophysiology of the dramatic progressive cerebral atrophy associated with Asparagine Synthetase Deficiency., (Copyright © 2015 Elsevier Inc. All rights reserved.)

G protein pathway suppressor 2 (GPS2) is a multifunctional protein involved in the regulation of a number of metabolic organs. First identified as part of the NCoR-SMRT corepressor complex, GPS2 is known to play an important role in the nucleus in the regulation of gene transcription and meiotic recombination. In addition, we recently reported a non-transcriptional role of GPS2 as an inhibitor of the proinflammatory TNFα pathway in the cytosol. Although this suggests that the control of GPS2 localization may be an important determinant of its molecular functions, a clear understanding of GPS2 differential targeting to specific cellular locations is still lacking. Here we show that a fine balance between protein stabilization and degradation tightly regulates GPS2 nuclear function. Our findings indicate that GPS2 is degraded upon polyubiquitination by the E3 ubiquitin ligase Siah2. Unexpectedly, interaction with the exchange factor TBL1 is required to protect GPS2 from degradation, with methylation of GPS2 by arginine methyltransferase PRMT6 regulating the interaction with TBL1 and inhibiting proteasome-dependent degradation. Overall, our findings indicate that regulation of GPS2 by posttranslational modifications provides an effective strategy for modulating its molecular function within the nuclear compartment., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objectives: To evaluate the efficacy, tolerability, and compliance of 3 ketogenic diets, the classical ketogenic diet, medium-chain triglyceride (MCT), and modified Atkins diet., Study Design: A single-center, retrospective study of 48 children with intractable epilepsy receiving ketogenic diets from 2003 to 2012. Patient demographics, epilepsy history, nutritional management, and side effects were collated. Compliance and tolerability were assessed by recording reasons for diet modification and cessation. The value of potassium citrate supplementation for preventing nephrolithiasis was reviewed., Results: Median age at ketogenic diet initiation was 3.8 years (IQR: 2.3-7 years). The majority had intractable epilepsy, and 33 of the 48 children (69%) had epileptic encephalopathies. Three (6%) patients became seizure free, 35 (73%) reported <50%-90% reduction, and 10 (21%) had 0%-50% reduction during a 2-year period. Diet duration or ketogenic diet type did not predict reduction in seizures (P = .381; P = .272). Constipation (n = 31, 65%) was very common. Food refusal (n = 3, 6%) and poor parental compliance (n = 5, 10%) were common reasons cited for cessation. There were lower rates of side effects for modified Atkins diet. Diet cessation was greatest for MCT; however, 3 patients on MCT ceased therapy because adequate seizure control was achieved. Nephrolithiasis was reported in 1 patient before potassium citrate was used and 2 patients noncompliant with potassium citrate supplementation developed hypercalciuria., Conclusion: The 3 ketogenic diets were comparably effective in seizure control and generally well-tolerated. Potassium citrate supplementation is an effective prophylactic supplement for the prevention of nephrolithiasis., (Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.)

Timely and selective recruitment of transcription factors to their appropriate DNA-binding sites represents a critical step in regulating gene activation; however, the regulatory strategies underlying each factor's effective recruitment to specific promoter and/or enhancer regions are not fully understood. Here, we identify an unexpected regulatory mechanism by which promoter-specific binding, and therefore function, of peroxisome proliferator-activator receptor γ (PPARγ) in adipocytes requires G protein suppressor 2 (GPS2) to prime the local chromatin environment via inhibition of the ubiquitin ligase RNF8 and stabilization of the H3K9 histone demethylase KDM4A/JMJD2. Integration of genome-wide profiling data indicates that the pioneering activity of GPS2/KDM4A is required for PPARγ-mediated regulation of a specific transcriptional program, including the lipolytic enzymes adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). Hence, our findings reveal that GPS2 exerts a biologically important function in adipose tissue lipid mobilization by directly regulating ubiquitin signaling and indirectly modulating chromatin remodeling to prime selected genes for activation., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: To evaluate the efficacy and side effects of oral mammalian target of rapamycin (mTOR) inhibitors in children and adolescents with tuberous sclerosis complex (TSC) and intractable epilepsy or subependymal giant cell astrocytoma (SEGA)., Study Design: Single-center series of 13 children and adolescents with TSC who received sirolimus or everolimus (mTOR inhibitors). The anticonvulsant response was evaluated in 7 patients with TSC and refractory seizures. Six patients with SEGAs were treated with either sirolimus or everolimus for nonsurgical management. SEGA volumes were assessed longitudinally using 1.5-T magnetic resonance imaging., Results: Of the intractable seizure group (7 patients), 1 patient had >90% reduction, 4 had 50%-90% reduction, and 2 had <50% reduction. Three reported subjective improvements in learning. By 12 months of treatment, there were statistically significant reductions in the SEGA volumes in 4 patients who received mTOR inhibitors (P < .04). The mean SEGA volume after 6 months of treatment was 2.18 cm(3), which represents 33% reduction in the mean baseline volume of 3.26 cm(3). The mTOR inhibitors were well tolerated. Adverse effects include dyslipidaemia (3 of 13), gingivitis (1 of 13), anorexia (1 of 13), and mild gastrointestinal side effects (1 of 13)., Conclusion: This case series suggests that mTOR inhibitors can improve seizures in those with TSC and refractory epilepsy. They are also an effective treatment for reducing the volume of SEGAs in patients with TSC not amenable to surgery with an acceptable side effect profile., (Crown Copyright © 2014. Published by Mosby, Inc. All rights reserved.)

Epileptic encephalopathies are characterized by frequent severe seizures, and/or prominent interictal epileptiform discharges on the electroencephalogram, developmental delay or deterioration, and usually a poor prognosis. The epileptiform abnormalities themselves are believed to contribute to the progressive disturbance in cerebral function. Determining the underlying aetiology responsible for infantile epileptic encephalopathy is a clinical challenge worth undertaking to facilitate advice on the recurrence risk and to allow for the option of prenatal testing, as often this category of epilepsy is associated with devastating hardship for families. This review takes advantage of recently published studies that have identified new genes associated with epilepsy and focuses on known monogenic causes where detection is useful for the process of genetic counselling. Based on the review, we present a diagnostic work-up in order to triage specific genetic testing for infants presenting with an epileptic encephalopathy., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

The association between hyperinflammatory states and numerous diseases is widely recognized, but our understanding of the molecular strategies that have evolved to prevent uncontrolled activation of inflammatory responses remains incomplete. Here, we report a critical, nontranscriptional role of GPS2 as a guardian against hyperstimulation of the TNF-α-induced gene program. GPS2 cytoplasmic actions are required to specifically modulate RIP1 ubiquitylation and JNK activation by inhibiting TRAF2/Ubc13 enzymatic activity. In vivo relevance of GPS2 anti-inflammatory role is confirmed by inhibition of TNF-α target genes in macrophages and by improved insulin signaling in the adipose tissue of aP2-GPS2 transgenic mice. As the nontranscriptional role is complemented by GPS2 functioning as positive and negative cofactor for nuclear receptors, in vivo overexpression also results in elevated circulating level of Resistin and development of hepatic steatosis. Together, these studies define GPS2 as a molecular guardian required for precise control of inflammatory responses involved in immunity and homeostasis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aim: This article describes the association of severe iron-deficiency anaemia with Helicobacter pylori gastritis., Results: We report three children who had symptomatic iron-deficiency anaemia with no obvious clinical cause and refractory to iron replacement therapy. All three underwent a diagnostic endoscopy and were found to have H. pylori gastritis. Histopathology confirmed inflammatory changes consisting of dense bands of clusters of plasma cells within the lamina propria and two of the three adolescents were noted to have numerous H. pylori in gastric crypts and glands. Two of the three cases had a urease positive test. Iron deficiency was successfully corrected following antibiotic eradication of H. pylori infection., Conclusions: This case series highlights the importance of considering H. pylori infection as a cause of refractory iron-deficiency anaemia in adolescents, even in the absence of gastrointestinal symptoms.

The inherited myopathies and muscular dystrophies are a diverse group of muscle diseases presenting with common complaints and physical signs: weakness, motor delay, and respiratory and bulbar dysfunction. The myopathies are caused by genetic defects in the contractile apparatus of muscle, and defined by distinctive histochemical or ultrastructural changes on muscle biopsy. The muscular dystrophies, in contrast, are diseases of muscle membrane or supporting proteins, which are generally characterized by pathological evidence of ongoing muscle degeneration and regeneration. Diagnosis of these disorders is contingent on a targeted history and examination, biochemical and neurophysiological assessment, muscle biopsy, and genetic testing. Treatment is focused on symptomatic management and rehabilitation, and monitoring for disease complications.

A solid formulation of a potent anthelmintic macrocyclic lactone, moxidectin, was administered using a non-degradable delivery device to discharge the agent into the subcutaneous tissues of sheep. In vivo release was monitored in sheep indirectly using faecal egg counts. Using a dose of 0.2 mg moxidectin/kg body weight when applied in the form of a solid pellet, protection of sheep against Haemonchus contortus challenge was conferred to a level greater than that of sheep which received Cydectin, the commercial liquid injectable form delivered at the same dosage. The anthelmintic efficacy of the solid formulation was assessed at four dosage levels in sheep and it was demonstrated that the dosage of anthelmintic agent could be reduced to 1/6 of the present recommended injectable dose. When two pellets containing the recommended dose of moxidectin were loaded into a non-degradable delivery device, the period of H. contortus control was extended from 42 to 183 days. Antibody levels of sheep receiving repeated infections of H. contortus L3 larvae and treated with moxidectin-loaded devices were reduced significantly compared to the levels observed in sheep treated with Cydectin (p < 0.0005). This implies that the group treated with the moxidectin-loaded devices was exposed to a reduced antigenic load compared to sheep treated with placebo devices, and sheep treated with Cydectin. The antibody levels generated in the sheep treated with placebo devices were no different to those treated with Cydectin. Application of this sustained release device may allow the control of nematode diseases in livestock throughout an entire season with a single administration.

Overexpression of cloned genes in bacteria often leads to insoluble refractile body formation requiring solubilization and refolding to obtain biologically active proteins. A refolding pathway was established for a model protein, porcine growth hormone (PGH), yielding an appreciably high recovery of 85%. The conditions include the dilution of a urea, beta-mercaptoethanol (beta-ME) denatured PGH solution in a refolding environment containing 3.5 M urea and 10 mM beta-ME/HED at a 10:1 ratio at pH 9.1 and 0.5 mg/mL PGH. The intrinsic fluorescence-detected transition of PGH in urea gives 3.8 kcal/mol for the free energy of denaturation (delta GH2O) of PGH. The native-like conformation of PGH is dependent on disulfide bonds because reduced and carboxymethylated PGH is devoid of tertiary structure as assessed by intrinsic tryptophan fluorescence. Physical analysis of C-terminally truncated recombinant PGH indicated no significant difference in the free energy of denaturation of P-band in urea as full-length PGH. This suggests that the first disulfide, forming the large loop domain of PGH, provides a significantly greater contribution to the conformational stability of PGH than the second disulfide, which forms the carboxy-terminal small loop domain. The rate of formation of native structure during refolding was biphasic, with native structure identified by intrinsic fluorescence and hydrophobicity spectroscopy prior to disulfide bond formation. Thus "framework" intermediates are prerequisites for correct disulfide formation and tertiary folding of PGH. This study shows how a protein refolds, forms disulfides, and self-associates, which may be useful for examining the refolding of other recombinant proteins.

Overexpression of cloned eukaryote genes in bacteria often leads to the formation of insoluble refractile bodies which require solubilization by harsh denaturants or detergents. We describe the conformational changes associated with the binding of a surfactant, cetyltrimethylammonium chloride (CTAC) to recombinant porcine growth hormone (PGH). The stoichiometry of binding by CTAC to the soluble and insoluble forms of recombinant PGH was also assessed. Optimum CTAC binding and protein solubilisation were obtained at 50 degrees C and at extreme pH. Increased ionic strength and changes in pH towards the isoelectric point of PGH (pH 6) decreased both the binding of CTAC and the efficiency of solubilising PGH from inclusion bodies. The positive charge on the quaternary ammonium head group of CTAC was found to be critical in the binding of CTAC to PGH and for the subsequent solubilisation of inclusion bodies. The binding of CTAC to the soluble form of PGH caused appreciable changes to the tertiary structure of the protein but did not significantly alter secondary structure, or cause complete unfolding. These observations help to explain earlier results which demonstrate that urea, guanidine hydrochloride and CTAC solubilized recombinant PGH molecules behave differently during in vitro refolding (Puri, N.K., Crivelli, E.C., Cardamone, M., Fiddes, R., Bertolini, J., Ninham, B. and Brandon, M.R. (1992) Biochem. J. 285, 871-879.).

Confirmation of a protein's cysteine content and of its location within the amino acid sequence is crucial in investigating the structural integrity of recombinant proteins. A combination of thiol-specific chemistry and peptide mapping by reversed-phase microbore HPLC was used to confirm the presence and map the location of cysteine residues in the primary sequences of recombinant porcine growth hormone and human tumor necrosis factor alpha. Recombinant proteins were conjugated with a hydrophobic iodoacetamide derivative, dimethylaminoazo-benzene iodoacetamide, and digested with trypsin. The peptide fragments were separated on a C8 microbore reversed-phase column using a linear acetonitrile gradient. The peptides containing the cysteine residues were selectively identified by monitoring with a diode-array detector at 215 nm with the reference wavelength set at 450 nm. Cysteine-containing peptides could be readily distinguished as inverted "negative" peaks relative to the baseline and noncysteine-containing peptides. Isolated peptide fragments were then sequenced in order to confirm the location of the cysteines in the proteins. This approach offers the benefits of selectively and rapidly identifying, from a single chromatrophic step, the cysteine-containing peptides of proteins. Furthermore, the use of the labeling reagent renders the cysteine-containing peptides more hydrophobic, thereby making them easier to separate from noncysteine-containing peptides.

During the development of a novel solubilization procedure (1) for bacterial inclusion bodies (IB's) using the cationic surfactant cetyltrimethylammonium chloride (CTAC; (CH3)3-N(+)-C16H33Cl) significant proportions of an apparently truncated, lower molecular weight (MW) variant form of recombinant pig growth hormone (rPGH) were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis relative to pig pituitary derived GH. The formation of this rPGH-like species, designated P-band, was found to occur in vitro during solubilization of IB's by CTAC and was dependent on pH and temperature of solubilization, but was not due directly to the use of CTAC, as purified soluble rPGH of the correct MW could not be converted to P-band by exposure to CTAC alone. The bacterial proteolysis suspected as being responsible for the in vitro formation of P-band could not be inhibited by the use of a "cocktail" of defined antiproteolytic agents but was inhibited by pH and temperature, and by solubilization of IB's in 5% SDS, 6 M gnHCl or 7.5 M urea. Detailed characterization of the structure of P-band by N-terminal amino acid sequencing, electrospray mass spectrometry, radioreceptor binding assay, peptide mapping, and C-terminal peptide sequencing confirmed that P-band was approximately 950 mass units smaller than normal rPGH and lacked eight C-terminal amino acids. A significant finding was that P-band is unable to bind to the pig liver-membrane GH receptor in a competitive radioreceptor assay. Analysis of the relative secondary and tertiary structure of P-band by circular dichroism spectra, intrinsic tryptophan-dependent fluorescence, and average surface hydrophobicity (2) suggested small but measurable changes to the overall structure of P-band relative to normal rPGH. Consequently, our results also suggest that the C-terminal portion of rPGH, including in particular the last eight amino acids, is of major importance in the binding of rPGH to the pig liver membrane GH receptor.

Recombinant pig growth hormone (rPGH) was solubilized from inclusion bodies by using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The solubilizing action of CTAC appeared to be dependent on the presence of a positively charged head group, as a non-charged variant was inactive. Relatively low concentrations of CTAC were required for rapid solubilization, and protein-bound CTAC was easily removed by ion-exchange chromatography. Compared with solubilization and recovery of rPGH from inclusion bodies with 7.5 M-urea and 6 M-guanidinium chloride, the relative efficiency of solubilization was lower with CTAC. However, superior refolding efficiency resulted in final yields of purified rPGH being in the order of CTAC greater than urea greater than or equal to guanidinium chloride. Detailed comparison of the different rPGH preparations as well as pituitary-derived growth hormone by h.p.l.c., native PAGE, c.d. spectral analysis and radioreceptor-binding assay showed that the CTAC-derived rPGH was essentially indistinguishable from the urea and guanidinium chloride preparations. The CTAC-derived rPGH was of greater biopotency than pituitary-derived growth hormone. The advantages of CTAC over urea and guanidinium chloride for increasing recovery of monomeric rPGH by minimizing aggregation during refolding in vitro were also found with recombinant sheep interleukin-I beta and a sheep insulin-like growth factor II fusion protein. In addition, the bioactivity of the CTAC-derived recombinant interleukin-1 beta was approximately ten-fold greater than that of an equivalent amount obtained from urea and guanidinium chloride preparations. It is concluded that CTAC represents, in general, an excellent additional approach or a superior alternative to urea and in particular guanidinium chloride for solubilization and recovery of bioactive recombinant proteins from inclusion bodies.

Recombinant porcine growth hormone (rPGH) was solubilised from inclusion bodies (IB's) using either 6 M guanidinium hydrochloride (GnHCl), 7.5 M urea or by a novel method using a cationic surfactant, cetyltrimethylammonium chloride (CTAC). Circular dichroism (CD) analysis of the secondary (2 degrees) structure of the urea- and GnHCl-solubilised rPGH showed the absence of alpha-helical content with the majority of the molecule existing in a 'random coil' structure. In contrast, the CTAC-solubilised rPGH displayed significant starting 2 degrees structure (10-15% alpha helix; 30-40% beta structure). The three rPGH preparations were refolded in vitro against weak urea. GnHCl or aqueous buffers, resulting in an average refolding efficiency of 50% native (monomeric) rPGH for CTAC solubilised IB's and only 20% for urea or GnHCl solubilised IB's. We conclude that the method of solubilisation of IB's and the resultant difference in the starting 2 degrees structure of rPGH, particularly alpha-helical content, is a major in vitro factor that apparently predetermines the aggregation/refolding behaviour rPGH irrespective of refolding environment.

The equilibrium binding of the apolar fluorescent dye 1-anilinonaphthalene-8-sulphonate (ANS) to bacteriorhodopsin, BSA, chicken egg lysozyme, ovalbumin, porcine somatotrophin (PST) and bovine pancreatic ribonuclease (RNAase) was quantitatively evaluated using Scatchard- and Klotz-plot analyses. On the basis of the average association constant for ANS binding sites (Ka), the proteins could be ranked in order of surface hydrophobicity as: Bacteriorhodopsin greater than BSA greater than ovalbumin greater than PST greater than lysozyme greater than RNAase. The number of protein-ANS binding sites was determined as 54, 10, 3, 1, 2 and 1 respectively. The ANS-based assessment of the surface hydrophobicity of these proteins was generally in agreement with the average hydrophobicity based on amino acid sequence [Bigelow (1967) J. Theor. Biol. 16, 187-211], except for results with PST and ovalbumin. The proteins were also analysed by reversed-phase h.p.l.c. using C1 and C8 columns. There was no significant correlation between ANS and reversed-phase-h.p.l.c. assessment of hydrophobicity, with the results obtained by h.p.l.c. being dependent upon the column used. ANS-based measurement of surface hydrophobicity appears to be the most appropriate means for assessing proteins such as to reflect their overall three-dimensional structure in solution.

Expression of recombinant porcine growth hormone (rpGH) in E. coli cells resulted in the accumulation of the rpGH within inclusion bodies (IBs). The IBs were solubilized and the rpGH refolded in a single step using a cationic surfactant, N-cetyl pyridinium chloride (CPC; C21H38ClN) in the absence of reducing agents. No additional dialysis or rapid dilution steps of the solubilizing agent were required to obtain a 30% yield of refolded and oxidized rpGH monomer at protein concentrations of up to 15-20 mg/mL. In contrast, the refolding in vitro of rpGH in the absence of CPC resulted in the formation of significant amounts of higher molecular weight aggregate at the expense of the biologically active monomer. The fluorescence spectrum of the purified refolded rpGH was indistinguishable from that of biologically active, pituitary derived porcine GH and reverse-phase HPLC analysis of the purified rpGH showed similar retention times to that of pituitary GH. It is likely that the use of cetylpyridinium chloride is generally applicable to the simplified high yield recovery of biologically active recombinant proteins from IBs.