Feasibility of wearable technology for 'real-world' gait analysis in children with Prader-Willi and Angelman syndromes.

BACKGROUND: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders in need of innovative 'real-world' outcome measures to evaluate treatment effects. Instrumented gait analysis (IGA) using wearable technology offers a potentially feasible solution to measure "real-world' neurological and motor dysfunction in these groups. METHODS: Children (50% female; 6-16 years) diagnosed with PWS (n = 9) and AS (n = 5) completed 'real-world' IGA assessments using the Physilog®5 wearable. PWS participants completed a laboratory assessment and a 'real-world' long walk. The AS group completed 'real-world' caregiver-assisted assessments. Mean and variability results for stride time, cadence, stance percentage (%) and stride length were extracted and compared across three different data reduction protocols. RESULTS: The wearables approach was found to be feasible, with all participants able to complete at least one assessment. This study also demonstrated significant agreement, using Lin's concordance correlation coefficient (CCC), between laboratory and 'real-world' assessments in the PWS group for mean stride length, mean stance % and stance % CV (n = 7, CCC: 0.782-0.847, P = 0.011-0.009). CONCLUSION: 'Real-world' gait analysis using the Physilog®5 wearable was feasible to efficiently assess neurological and motor dysfunction in children affected with PWS and AS.

Sleep in autism: A biomolecular approach to aetiology and treatment.

People with autism spectrum disorder (ASD) commonly experience other comorbidities. Studies indicate that between 50% and 83% of individuals with ASD have sleep problems or disorders. The most commonly reported sleep problems are: (a) insomnia symptoms including the inability to get to sleep or stay asleep; and (b) circadian rhythm sleep-wake disorders, defined as a misalignment between the timing of endogenous circadian rhythms and the external environment. The circadian system provides timing information for the sleep-wake cycle that is regulated by the interaction of an endogenous processes (circadian - Process C, and homeostatic - Process S) and synchronizing agents (neurohormones and neurotransmitters), which produce somnogenic activity. A clinical priority in ASD is understanding the cause of these sleep problems in order to improve treatment outcomes. This review approaches sleep in autism from several perspectives: Sleep-wake mechanisms and problems, and brain areas and molecules controlling sleep (e.g., GABA and melatonin) and wake maintenance (e.g., serotonin, acetylcholine and glutamate). Specifically, this review examines how altered sleep structure could be related to neurobiological alterations or genetic mutations and the implications this may have for potential pharmacological treatments in individuals with ASD.

Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells.

Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenic cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic ß-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/ß-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice.

Increased miR-155-5p and reduced miR-148a-3p contribute to the suppression of osteosarcoma cell death.

Osteosarcoma (OS) is the most common cancer of bone and the 5th leading cause of cancer-related death in young adults. Currently, 5-year survival rates have plateaued at ~70% for patients with localized disease. Those with disseminated disease have an ~20% 5-year survival. An improved understanding of the molecular genetics of OS may yield new approaches to improve outcomes for OS patients. To this end, we applied murine models that replicate human OS to identify and understand dysregulated microRNAs (miRNAs) in OS. miRNA expression patterns were profiled in murine primary osteoblasts, osteoblast cultures and primary OS cell cultures (from primary and paired metastatic locations) isolated from two genetically engineered murine models of OS. The differentially expressed miRNA were further assessed by a cross-species comparison with human osteoblasts and OS cultures. We identified miR-155-5p and miR-148a-3p as deregulated in OS. miR-155-5p suppression or miR-148a-3p overexpression potently reduced proliferation and induced apoptosis in OS cells, yet strikingly, did not impact normal osteoblasts. To define how these miRNAs regulated OS cell fate, we used an integrated computational approach to identify putative candidate targets and then correlated these with the cell biological impact. Although we could not resolve the mechanism through which miR-148a-3p impacts OS, we identified that miR-155-5p overexpression suppressed its target Ripk1 (receptor (TNFRSF)-interacting serine-threonine kinase 1) expression, and miR-155-5p inhibition elevated Ripk1 levels. Ripk1 is directly involved in apoptosis/necroptosis. In OS cells, small interfering RNA against Ripk1 prevented cell death induced by the sequestration of miR-155-5p. Collectively, we show that miR-148a-3p and miR-155-5p are species-conserved deregulated miRNA in OS. Modulation of these miRNAs was specifically toxic to tumor cells but not normal osteoblasts, raising the possibility that these may be tractable targets for miRNA-based therapies for OS.

Knockdown of PTHR1 in osteosarcoma cells decreases invasion and growth and increases tumor differentiation in vivo.

Osteosarcoma (OS) is the most common cancer of bone. Parathyroid hormone (PTH) regulates calcium homeostasis and bone development, while the paracrine/autocrine PTH-related protein (PTHrP) has central roles in endochondral bone formation and bone remodeling. Using a murine OS model, we found that OS cells express PTHrP and the common PTH/PTHrP receptor (PTHR1). To investigate the role of PTHR1 signaling in OS cell behavior, we used shRNA to reduce PTHR1 expression. This only mildly inhibited proliferation in vitro, but markedly reduced invasion through collagen and reduced expression of RANK ligand (RANKL). Administration of PTH(1-34) did not stimulate OS proliferation in vivo but, strikingly, PTHR1 knockdown resulted in a profound growth inhibition and increased differentiation/mineralization of the tumors. Treatment with neutralizing antibody to PTHrP did not recapitulate the knockdown of PTHR1. Consistent with this lack of activity, PTHrP was predominantly intracellular in OS cells. Knockdown of PTHR1 resulted in increased expression of late osteoblast differentiation genes and upregulation of Wnt antagonists. RANKL production was reduced in knockdown tumors, providing for reduced homotypic signaling through the receptor, RANK. Loss of PTHR1 resulted in the coordinated loss of gene signatures associated with the polycomb repressive complex 2 (PRC2). Using Ezh2 inhibitors, we demonstrate that the increased expression of osteoblast maturation markers is in part mediated by the loss of PRC2 activity. Collectively these results demonstrate that PTHR1 signaling is important in maintaining OS proliferation and undifferentiated state. This is in part mediated by intracellular PTHrP and through regulation of the OS epigenome.

Platelet activation by a relapsing fever spirochaete results in enhanced bacterium-platelet interaction via integrin alphaIIbbeta3 activation.

Borrelia hermsii, a spirochaete responsible for relapsing fever in humans, grows to high density in the bloodstream and causes thrombocytopenia. We show here that B. hermsii binds to human platelets. Extended culture in bacteriological medium resulted in both diminished infectivity in vivo and diminished platelet binding in vitro. Platelet binding was promoted by the platelet integrin alphaIIbbeta3: the bacterium bound to purified integrin alphaIIbbeta3, and bacterial binding to platelets was diminished by alphaIIbbeta3 antagonists or by a genetic defect in this integrin. Integrin alphaIIbbeta3 undergoes a conformational change upon platelet activation, and bacteria bound more efficiently to activated rather than resting platelets. Nevertheless, B. hermsii bound at detectable levels to preparations of resting platelets. The bacterium did not recognize a point mutant of alphaIIbbeta3 that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin alphaIIbbeta3 activation, as indicated by the expression of the platelet activation marker P-selectin and integrin alphaIIbbeta3 in its active conformation. The degree of platelet activation varied depending upon bacterial strain and growth conditions. Prostacyclin I2, an inhibitor of platelet activation, diminished bacterial attachment, indicating that activation enhanced bacterial binding. Thus, B. hermsii signals the host cell to activate a critical receptor for the bacterium, thereby promoting high-level bacterial attachment.

Profiling methyl-CpG specific determinants on transcriptionally silent chromatin.

Transcriptional activity is closely associated with DNA methylation and chromatin remodelling. Evidence is emerging that a family of methylation specific (methyl-CpG binding domain, MBD) proteins have the capacity to bind to methylated sequences and repress transcription. Recent advances in this area reveal that many of the MBD proteins are associated with histone deacetylase (HDAC) dependant repression. The capacity of MBD association to repress transcription would largely be defined by promoter structure and this is best explained by the position and density of DNA methylation. The mechanism of specific targeting of MBD family members to methylated sequences remains largely unknown. In order to understand the mechanistic details of silencing the current challenge is to identify and map these molecular determinants assembled on native chromatin in model systems of human development and disease. Downstream targets such as the methylated Fragile X Mental Retardation gene 1 (FMR1) gene and tumour suppressor genes are likely candidates. In this article, we describe a powerful strategy that involves the immunoprecipitation of in vivo formaldehyde fixed chromatin to identify MBD binding complexes directly isolated from the natural chromosomal environment. We demonstrate the methylated human Multidrug Resistance gene 1 (MDR1) is enriched with transcriptional repressors that belong to the MBD family and this would account for transcriptional silencing.

A mutation in the alpha subunit of the platelet integrin alphaIIbbeta3 identifies a novel region important for ligand binding.

An unbiased genetic approach was used to identify a specific amino acid residue in the alphaIIb subunit important for the ligand binding function of the integrin alphaIIbbeta. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in alphaIIb at position 224 (D-->V) was identified. Although well expressed on the surface of transfected cells, alphaIIbD224Vbeta3 as well as alphaIIbD224Abeta3 did not bind alphaIIbbeta3-specific ligands or a RGD peptide, a ligand shared in common with alphavbeta3. Insertion of exon 5 of alphaIIb, residues G193-W235, into the backbone of the alphav subunit did not enable the chimeric receptor to bind alphaIIbbeta3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. alphaIIbD224 is not well conserved among other integrin alpha subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed beta-propeller model for integrin alpha subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the alphaIIbbeta3 receptor.

A genetic analysis of integrin function: Glanzmann thrombasthenia in vitro.

Glanzmann thrombasthenia, an inherited bleeding disorder, can be caused by a defect or deficiency in platelet integrin alphaIIb beta3 (GPIIb-IIIa). Studies of thrombasthenia variants have facilitated identification of sites involved in the functions of alphaIIb beta3 and other integrins. Such sites include those that bind ligand and those that participate in the "activation" of alphaIIb beta3 required for high affinity binding of ligands such as fibrinogen or PAC1, a monoclonal antibody. Here we describe the isolation of such variants, created in vitro with Chinese hamster ovary cells that express an activated form of alphaIIb beta3. These cells were exposed to a mutagen, ethyl methane sulfonate, and variants that lost the capacity to bind PAC1 were isolated by fluorescence-activated cell sorting. These variants were grouped into three phenotypic classes. One comprised integrin mutations that disrupt ligand binding function; a second comprised mutations that interfere with the capacity of cells to activate the integrin. Most of these activation-defective mutations were in the integrin cytoplasmic domain, but surprisingly, several were caused by mutations affecting three closely spaced residues in the beta3 extracellular domain. A third class of mutants exhibited a defect in integrin activation not ascribable to changes in the integrin sequence. Thus, these may represent mutated signaling molecules required for integrin activation. This unbiased genetic approach provides new insights into the structural basis of integrin function and may assist in identifying the cellular events that regulate integrin function.

Continuously nebulized albuterol in severe exacerbations of asthma in adults: a case-controlled study.

A retrospective, case-controlled analysis comparing patients admitted to a medical intensive care unit with severe exacerbations of asthma who received continuously nebulized albuterol (CNA) versus intermittent albuterol (INA) treatments is reported. Forty matched pairs of patients with asthma are compared. CNA was administered for a mean of 11 +/- 10 hr. The incidence of cardiac dysrhythmias was similar between groups. Symptomatic hypokalemia did not occur. CNA patients had higher heart rates during treatment, which may reflect severity of illness. The incidence of intubation was similar. We conclude that CNA and INA demonstrated similar profiles with regard to safety, morbidity, and mortality.

Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration.

Retinitis pigmentosa (RP) is a group of hereditary human diseases that cause retinal degeneration and lead to eventual blindness. More than 25% of all RP cases in humans appear to be caused by dominant mutations in the gene encoding the visual pigment rhodopsin. The mechanism by which the mutant rhodopsin proteins cause dominant retinal degeneration is still unclear. Interestingly, the great majority of these mutants appear to produce misfolded rhodopsin. We now report the isolation and characterization of 13 rhodopsin mutations that act dominantly to cause retinal degeneration in Drosophila; four of these correspond to identical substitutions in human autosomal dominant RP patients. We demonstrate that retinal degeneration results from interference in the maturation of wild-type rhodopsin by the mutant proteins.

The cyclophilin homolog NinaA functions as a chaperone, forming a stable complex in vivo with its protein target rhodopsin.

In Drosophila, biogenesis of the major rhodopsin, Rh1, is dependent on the presence of a photoreceptor cell-specific cyclophilin, NinaA. In ninaA mutants, Rh1 is retained within the endoplasmic reticulum and rhodopsin levels are reduced > 100-fold. Cyclophilins have been shown to be peptidyl-prolyl cis-trans isomerases and have been implicated in catalyzing protein folding. We have generated transgenic animals expressing different functional rhodopsins containing a histidine tag. We isolated these molecules from wild-type and ninaA mutant retinas, and have demonstrated that in vivo NinaA forms a specific stable protein complex with its target Rh1. We also expressed ninaA under an inducible promoter and showed that NinaA is required quantitatively for Rh1 biogenesis. These results provide the first evidence for a biologically relevant physical interaction between a cyclophilin and its cellular target, and suggest that the normal cellular role of this class of cyclophilins is to function as chaperones, possibly escorting their protein substrates through the secretory pathway.

Genetic dissection of cyclophilin function. Saturation mutagenesis of the Drosophila cyclophilin homolog ninaA.

Cyclophilins, the intracellular receptors for the widely used immunosuppressant cyclosporin A have been found to be peptidyl-prolyl cis/trans isomerases and have been implicated in intracellular protein folding and trafficking. The Drosophila ninaA gene encodes a photoreceptor-specific cyclophilin homolog involved in rhodopsin biogenesis. ninaA mutants have a 90% reduction in the levels of Rh1 rhodopsin. To gain insight into the role of cyclophilins in vivo, we carried out a genetic screen designed to identify functionally important regions in the ninaA protein. Over 700,000 mutagenized flies were screened for a visible ninaA phenotype and 70 independent mutations in ninaA were isolated and characterized. These mutations provide a detailed dissection of the structure/function relationships in cyclophilin. We also show that mammalian cyclophilins engineered to contain missense mutations found in two temperature-sensitive ninaA alleles display temperature-sensitive prolyl cis/trans isomerase activity.

The cyclophilin homolog ninaA is required in the secretory pathway.

In Drosophila, the major rhodopsin Rh1 is synthesized in endoplasmic reticulum (ER)-bound ribosomes of the R1-R6 photoreceptor cells and is then transported to the rhabdomeres where it functions in phototransduction. Mutations in the cyclophilin homolog ninaA lead to a 90% reduction in Rh1 opsin. Cyclophilins have been shown to be peptidyl-prolyl cis-trans isomerases and have been implicated in catalyzing protein folding. We now show that mutations in the ninaA gene severely inhibit opsin transport from the ER, leading to dramatic accumulations of ER cisternae in the photoreceptor cells. These results demonstrate that ninaA functions in the ER. Interestingly, ninaA and Rh1 also colocalize to secretory vesicles, suggesting that Rh1 may require ninaA as it travels through the distal compartments of the secretory pathway. These results are discussed in relation to the possible role of cyclophilins in protein folding and intracellular protein trafficking.