Delineating the phenotype of RNU4ATAC-related spliceosomopathy.

Biallelic pathogenic variants in RNU4ATAC cause microcephalic osteodysplastic primordial dwarfism type I (MOPD1), Roifman syndrome (RS) and Lowry-Wood syndrome (LWS). These conditions demonstrate significant phenotypic heterogeneity yet have overlapping features. Although historically described as discrete conditions they appear to represent a phenotypic spectrum with clinical features not always aligning with diagnostic categories. Clinical variability and ambiguity in diagnostic criteria exist among each disorder. Here we report an individual with a novel genotype and phenotype spanning all three disorders, expanding the phenotypic spectrum of RNU4ATAC-related spliceosomeopathies.

'Propped and prone' positioning reduces respiratory events in spontaneously breathing preterm infants: A randomised triple crossover study.

AIM: We compared effects of infant positioning and feed-rate interventions on respiratory events and oximetry parameters in spontaneously breathing preterm infants born <32 weeks gestation managed in a neonatal unit. METHODS: A randomised triple crossover design was employed. n = 68 infants underwent three test conditions A: control (supine/flat, gravity bolus feeds), B: position intervention (propped/prone) and C: feed-rate intervention (continuous pump feeds) in randomised sequence over three consecutive days. Primary outcomes were number of events (apnoea, bradycardia and desaturation) and percentage time SpO2 < 80% over 24 h. The secondary outcome was percentage time SpO2 ≥ 88%. Treatment effects were estimated using linear mixed-effects models. RESULTS: Propped/prone positioning significantly reduced events and improved percentage time SpO2 < 80% and ≥88% compared to both other conditions (all P < 0.001). Outcomes for the feed-rate intervention were not significantly different to control. CONCLUSIONS: Alternative infant positioning should be considered in preterm infants managed in the neonatal unit.

Delineating the CCDC22-related Ritscher-Schinzel syndrome phenotype in the original family.

Pathogenic variants in CCDC22 were initially described in 2012 in a large Australian family with intellectual disability and were subsequently noted to cause a phenotype consistent with the previously described Ritscher-Schinzel syndrome (RSS). The phenotypes of the original family were not described in detail and remains limited phenotypic data reported in medical literature. We detail the phenotypes of the original family, including newly diagnosed family members. With these eight phenotypic descriptions, more than triple the number of individuals for whom detailed clinical information is available. In addition to typical facies, common phenotypic features included intellectual disability, congenital heart disease and posterior fossa malformations, postnatal short stature, ectodermal abnormalities, and digital anomalies as previously described. Spinal curvature and genital anomalies were seen in most patients, while gastrointestinal features and disturbed sleep were also recurrently seen. We propose a possible mechanism linking the familial variant to a diagnosis of sarcoidosis in one individual. Given the clinical and genetic heterogeneity of RSS, we suggest a dyadic naming convention.

Clinical overlap of PHACE and LUMBAR syndromes.

Segmental infantile hemangiomas affecting the upper body are associated with PHACE(S) (Posterior fossa anomalies, Hemangioma, Arterial anomalies, Cardiac anomalies, Eye anomalies, and Sternal defects) syndrome, whereas segmental infantile hemangiomas affecting the lower body are the cutaneous hallmark of LUMBAR (Lower body hemangioma and other skin defects, Urogenital anomalies and Ulceration, Myelopathy, Bony deformities, Anorectal malformations and Arterial anomalies, and Renal anomalies) syndrome. We present two individuals with concurrent features of both PHACE and LUMBAR syndromes demonstrating an overlap phenotype. The overlapping features seen in our patients suggest that these syndromes occur on the same phenotypic spectrum and derive from a common embryonic pathophysiology.

Inherited variants in CHD3 show variable expressivity in Snijders Blok-Campeau syndrome.

PURPOSE: Common diagnostic next-generation sequencing strategies are not optimized to identify inherited variants in genes associated with dominant neurodevelopmental disorders as causal when the transmitting parent is clinically unaffected, leaving a significant number of cases with neurodevelopmental disorders undiagnosed. METHODS: We characterized 21 families with inherited heterozygous missense or protein-truncating variants in CHD3, a gene in which de novo variants cause Snijders Blok-Campeau syndrome. RESULTS: Computational facial and Human Phenotype Ontology-based comparisons showed that the phenotype of probands with inherited CHD3 variants overlaps with the phenotype previously associated with de novo CHD3 variants, whereas heterozygote parents are mildly or not affected, suggesting variable expressivity. In addition, similarly reduced expression levels of CHD3 protein in cells of an affected proband and of healthy family members with a CHD3 protein-truncating variant suggested that compensation of expression from the wild-type allele is unlikely to be an underlying mechanism. Notably, most inherited CHD3 variants were maternally transmitted. CONCLUSION: Our results point to a significant role of inherited variation in Snijders Blok-Campeau syndrome, a finding that is critical for correct variant interpretation and genetic counseling and warrants further investigation toward understanding the broader contributions of such variation to the landscape of human disease.

Arginase-1 deficiency in neural cells does not contribute to neurodevelopment or functional outcomes after sciatic nerve injury.

Arginase-1 (Arg1) is an enzyme controlling the final step of the urea cycle, with highest expression in the liver and lower expression in the lungs, pancreas, kidney, and some blood cells. Arg1 deficiency is an inherited urea cycle disorder presenting with neurological dysfunction including spastic diplegia, intellectual and growth retardation, and encephalopathy. The contribution of Arg1 expression in the central and peripheral nervous system to the development of neurological phenotypes remains largely unknown. Previous studies have shown prominent arginase-1 expression in the nervous system and post-peripheral nerve injury in mice, but very low levels in the naïve state. To investigate neurobiological roles of Arg1, we created a conditional neural (n)Arg1 knockout (KO) mouse strain, with expression eliminated in neuronal and glial precursors, and compared them to littermate controls. Long-term analysis did not reveal any major differences in blood amino acid levels, body weight, or stride gait cycle from 8 to 26-weeks of age. Brain structure measured by magnetic resonance imaging at 16-weeks of age observed only a significant decrease in the volume of the mammillary bodies. We also assessed whether nArg1, which is expressed by sensory neurons after injury, may play a role in regeneration following sciatic nerve crush. Only subtle differences were observed in locomotor and sensory recovery between nArg1 KO and control mice. These results suggest that arginase-1 expression in central and peripheral neural cells does not contribute substantially to the phenotypes of this urea cycle disorder, nor is it likely crucial for post-injury regeneration in this mouse model.

Clinical and laboratory reporting impact of ACMG-AMP and modified ClinGen variant classification frameworks in MYH7-related cardiomyopathy.

PURPOSE: ClinGen provides gene-specific guidance for interpretation of sequence variants in MYH7. We assessed laboratory and clinical impact of reclassification by the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) and ClinGen recommendations in 43 MYH7 variants reported by a diagnostic laboratory between 2013 and 2017. METHODS: Fifty-two proband reports containing MYH7 variants were reinterpreted by original ACMG-AMP and ClinGen guidelines. Evidence items were compared across schemes and reasons for classification differences recorded. Laboratory impact was assessed by number of recommended report reissues, and reclassifications coded as clinically "actionable" or "equivalent." Available pedigrees were reviewed to describe projected cascade impact. RESULTS: ClinGen produced a higher proportion of diagnostic classifications (65% of variants) compared with ACMG-AMP (54%) and fewer variants of uncertain significance (30% versus 42%). ClinGen classification resulted in actionable changes in 18% of variants with equal upgrades and downgrades from original report. ClinGen's revisions to PM1 and PS4 contributed to classification differences in 21% and 19% of variants respectively. Each classification change per proband report impacted, on average, 3.1 cascade reports with a further 6.3 first- and second-degree relatives potentially available for genotyping per family. CONCLUSION: ClinGen's gene-specific criteria provide expert-informed guidance for interpretation of MYH7 sequence variants. Periodic re-evaluation improves diagnostic confidence and should be considered by clinical and laboratory teams.

JARID2 haploinsufficiency is associated with a clinically distinct neurodevelopmental syndrome.

PURPOSE: JARID2, located on chromosome 6p22.3, is a regulator of histone methyltransferase complexes that is expressed in human neurons. So far, 13 individuals sharing clinical features including intellectual disability (ID) were reported with de novo heterozygous deletions in 6p22-p24 encompassing the full length JARID2 gene (OMIM 601594). However, all published individuals to date have a deletion of at least one other adjoining gene, making it difficult to determine if JARID2 is the critical gene responsible for the shared features. We aim to confirm JARID2 as a human disease gene and further elucidate the associated clinical phenotype. METHODS: Chromosome microarray analysis, exome sequencing, and an online matching platform (GeneMatcher) were used to identify individuals with single-nucleotide variants or deletions involving JARID2. RESULTS: We report 16 individuals in 15 families with a deletion or single-nucleotide variant in JARID2. Several of these variants are likely to result in haploinsufficiency due to nonsense-mediated messenger RNA (mRNA) decay. All individuals have developmental delay and/or ID and share some overlapping clinical characteristics such as facial features with those who have larger deletions involving JARID2. CONCLUSION: We report that JARID2 haploinsufficiency leads to a clinically distinct neurodevelopmental syndrome, thus establishing gene-disease validity for the purpose of diagnostic reporting.

Sleep Disturbance in Older Adults With or Without Mild Cognitive Impairment and Its Associated Factors Residing in Rural Area, China.

AIMS: To evaluate the prevalence of sleep disturbance in older adults with or without mild cognitive impairment (MCI) and associated factors among residents in rural central China. METHODS: A cross-sectional survey was conducted in adults in rural areas of the Hunan province aged≥60 years. Study participants (N = 1213) included 479 individuals meeting the criteria for MCI and 734 with normal cognitive abilities. The participants completed the Athens Insomnia Scale, Stress Resilience Quotient Scale, Affect Balance Scale and Activities of Daily Living (ADL) Scale. Chi-square test, Wilcoxon rank sum analyses and multiple logistic regression were used in this study. RESULTS: A total of 60.33% of participants with MCI demonstrated sleep disturbance (60.33%, 95% CI: 0.559-0.649), which was significantly higher than in the non-MCI group (43.73%, 95% CI: 0.759-0.838). Multiple logistic regression conducted separately in the populations of older adults with or without MCI showed that age, drinking habits, affect balance and activities of daily life were correlates of self-reported sleep disturbance in rural older adults with MCI (B = -5.469), whereas age, ADL, living arrangement and resilience were the main influencing factors in older adults without MCI (B = 2.991). CONCLUSION: Sleep disturbance is more common in older adults with MCI than without MCI in rural areas of China. The factors influencing sleep disturbances vary between older adults with or without MCI, with age and ADL representing common factors influencing sleep disturbance in both groups. Interventions focusing on the age, drinking habits, affect balance and ADL may improve sleep quality in MCI older adults.

Feasibility of Ultra-Rapid Exome Sequencing in Critically Ill Infants and Children With Suspected Monogenic Conditions in the Australian Public Health Care System.

Importance: Widespread adoption of rapid genomic testing in pediatric critical care requires robust clinical and laboratory pathways that provide equitable and consistent service across health care systems. Objective: To prospectively evaluate the performance of a multicenter network for ultra-rapid genomic diagnosis in a public health care system. Design, Setting, and Participants: Descriptive feasibility study of critically ill pediatric patients with suspected monogenic conditions treated at 12 Australian hospitals between March 2018 and February 2019, with data collected to May 2019. A formal implementation strategy emphasizing communication and feedback, standardized processes, coordination, distributed leadership, and collective learning was used to facilitate adoption. Exposures: Ultra-rapid exome sequencing. Main Outcomes and Measures: The primary outcome was time from sample receipt to ultra-rapid exome sequencing report. The secondary outcomes were the molecular diagnostic yield, the change in clinical management after the ultra-rapid exome sequencing report, the time from hospital admission to the laboratory report, and the proportion of laboratory reports returned prior to death or hospital discharge. Results: The study population included 108 patients with a median age of 28 days (range, 0 days to 17 years); 34% were female; and 57% were from neonatal intensive care units, 33% were from pediatric intensive care units, and 9% were from other hospital wards. The mean time from sample receipt to ultra-rapid exome sequencing report was 3.3 days (95% CI, 3.2-3.5 days) and the median time was 3 days (range, 2-7 days). The mean time from hospital admission to ultra-rapid exome sequencing report was 17.5 days (95% CI, 14.6-21.1 days) and 93 reports (86%) were issued prior to death or hospital discharge. A molecular diagnosis was established in 55 patients (51%). Eleven diagnoses (20%) resulted from using the following approaches to augment standard exome sequencing analysis: mitochondrial genome sequencing analysis, exome sequencing-based copy number analysis, use of international databases to identify novel gene-disease associations, and additional phenotyping and RNA analysis. In 42 of 55 patients (76%) with a molecular diagnosis and 6 of 53 patients (11%) without a molecular diagnosis, the ultra-rapid exome sequencing result was considered as having influenced clinical management. Targeted treatments were initiated in 12 patients (11%), treatment was redirected toward palliative care in 14 patients (13%), and surveillance for specific complications was initiated in 19 patients (18%). Conclusions and Relevance: This study suggests feasibility of ultra-rapid genomic testing in critically ill pediatric patients with suspected monogenic conditions in the Australian public health care system. However, further research is needed to understand the clinical value of such testing, and the generalizability of the findings to other health care settings.

Rapid Identification of Biallelic SPTB Mutation in a Neonate with Severe Congenital Hemolytic Anemia and Liver Failure.

Heterozygous pathogenic variants in SPTB cause autosomal dominant hereditary spherocytosis, an important cause of neonatal nonimmune hemolytic anemia. Biallelic mutations are rarely reported, all with severe neonatal presentation. We describe rapid (68 h) genomic diagnosis of homozygous ß-spectrin deficiency in a newborn with severe transfusion-dependent hemolytic anemia, conjugated hyperbilirubinemia, and progressive liver failure. Trio whole-exome sequencing identified a novel biallelic SPTB variant (c.6119C>T; p.Thr2040Ile) located in the critical spectrin repeat region. Pretransfusion blood film showed marked spherocytosis including microspherocytes and nucleated erythrocytes, and eosin-5-maleimide (E5M) staining was markedly reduced, supporting pathogenicity. Both asymptomatic heterozygous parents demonstrated mildly reduced E5M staining, with occasional spherocytes and elliptocytes. Early molecular diagnosis facilitated hypertransfusion to suppress ineffective erythropoiesis and reverse hepatic dysfunction. This report broadens the genotypic and phenotypic spectrum of spectrin deficiency and highlights the utility of rapid genomic testing in facilitating early diagnosis and informing targeted therapy in critically ill patients.

LASIK flap stability after severe ocular injury.

PURPOSE: To report two cases of LASIK flap stability after severe ocular trauma.Observations: Two patients suffered open globe injuries several years after undergoing uneventful LASIK with femtosecond laser corneal flap. Both underwent primary ruptured globe repair, during which no LASIK flap dislocation was identified. Histopathologic examination of one of the cornea specimens confirmed an intact LASIK flap. CONCLUSION AND IMPORTANCE: In these cases, the femtosecond LASIK flap remained in place despite significant injury to the cornea. The presence of a femtosecond LASIK flap did not complicate surgical management of the injury, and did not contribute to the patient's loss of BCVA.

Military Teleconsultation Services Facilitate Prompt Recognition and Treatment of a Case of Syphilitic Uveitis Aboard a United States Navy Aircraft Carrier at Sea During Combat Operations Without Evacuation Capability.

Background: In the United States Armed Forces, telemedicine can bring specialist care into the field, augmenting the capabilities of health care providers in remote, austere environments to nearly the point of injury or illness. The early intervention enabled by telemedical consultation can not only guide crucial temporizing measures to safeguard life, limb, and eyesight, but can also facilitate care in resource limited environments, potentially avoiding need for patient evacuation. In circumstances when a higher level of care is needed, but unavailable or operationally not possible, telemedicine can guide management in the field until transport can be achieved. Methods: In the present case, a young male patient presented to medical company aboard a U.S. Navy aircraft carrier while deployed at sea for evaluation of an acute red eye. Despite initial therapeutic measures, his vision subsequently deteriorated. Uveitis was suspected, but transport off the ship to an eye care specialist was not possible during combat operations. Results: Telemedical consultation with shore-based ophthalmologists guided initial diagnostic and therapeutic efforts, resulting in the presumed diagnosis of syphilitic uveitis. With remote support of ophthalmology and infectious disease specialist care, the patient was treated at sea for this vision-threatening condition. As operational conditions allowed, the patient was later evacuated to definitive care. Conclusions: At follow up after treatment, the patient had an excellent visual outcome, and was promptly returned to duty.

Cerebellar ataxia with normal intellect associated with a homozygous truncating variant in CA8.

Biallelic pathogenic variants in CA8 cause cerebellar ataxia, mental retardation and dysequilibrium syndrome 3 (CAMRQ3), a rare form of hereditary ataxia characterised by cerebellar hypoplasia/atrophy, variable intellectual disability and often quadrupedal gait. The few cases reported in the medical literature are all caused by pathogenic homozygous or compound heterozygous missense variants in CA8. We report a 9 year-old boy with marked gross motor delay, ataxia and progressive cerebellar atrophy with limited bipedal gait, but without intellectual disability. Singleton whole exome sequencing was performed. A novel homozygous truncating variant in CA8 (c.232C>T) with a predicted premature termination codon at position 78 (p.Arg78*) was identified. Both parents and the proband's healthy sister are heterozygous for the variant. This variant is likely pathogenic and the cause of the condition in this child. Functional evidence in the form of a spontaneous mouse model involving homozygous intragenic deletion of the mouse analogue of CA8 with nonsense-mediated decay and similar clinical features to the proband support pathogenicity. Identification of this truncating variant broadens the genotypic and phenotypic spectrum of CA8-related cerebellar ataxia.

Early diagnosis of Pearson syndrome in neonatal intensive care following rapid mitochondrial genome sequencing in tandem with exome sequencing.

Rapid genomic testing is a valuable new diagnostic tool for acutely unwell infants, however exome sequencing does not deliver clinical-grade mitochondrial genome sequencing and may fail to diagnose mitochondrial disorders caused by mitochondrial DNA (mtDNA) variants. Rapid mitochondrial genome sequencing and analysis are not routinely available in rapid genomic diagnosis programmes. We present two critically ill neonates with transfusion-dependent anaemia and persistent lactic acidosis who underwent rapid mitochondrial genome sequencing in tandem with exome sequencing as part of an exome sequencing-based rapid genomic diagnosis programme. No diagnostic variants were identified on examination of the nuclear exome data for either infant. Mitochondrial genome sequencing identified a large mtDNA deletion in both infants, diagnosing Pearson syndrome within 74 and 55 h, respectively. Early diagnosis in the third week of life allowed the avoidance of a range of other investigations and appropriate treatment planning. Rapid mitochondrial genome analysis provides additional diagnostic and clinical utility and should be considered as an adjunct to exome sequencing in rapid genomic diagnosis programmes.

Resilience and affect balance of empty-nest older adults with mild cognitive impairment in poor rural areas of Hunan province, China.

AIM: To evaluate the prevalence of mild cognitive impairment (MCI) within the empty-nest older adults population in poor rural areas of the Hunan province of China, and to explore the effects of resilience and affective balance on cognitive functioning within this specific population. METHODS: A cross-sectional, multistage, random cluster survey was administered to participants from March 2013 to December 2014 in the Hunan province. There were a total of 1164 participants. These participants were empty-nest older adults who were residing in poor rural areas of the Hunan province. The data was collected in two stages. In stage 1, the participants were administered the Montreal Cognitive Assessment for screening cognitive impairment. In stage 2, the participants were screened for any potential cognitive impairment, were administered a series of neuropsychological tests and received a definitive diagnosis for MCI, if the criteria were met. Resilience and affect balance were assessed by the Chinese modified version of the Stress Resilience Quotient and the Affect Balance Scale. RESULTS: The prevalence of MCI was 38.40% within this empty-nest older adult population. Significant differences were found between MCI and non-MCI empty-nest older adults specific to resilience and affect balance. Path analysis showed that resilience mediated the relationship between MCI and affect balance. CONCLUSIONS: Resilience and affect balance were less prominent within the MCI empty-nest older adults than those in the non-MCI group. The results suggest that resilience is a mediating variable between MCI and affect balance. Geriatr Gerontol Int 2019; 19: 222-227.

Efficacy of a Bicistronic Vector for Correction of Sandhoff Disease in a Mouse Model.

GM2 gangliosidoses are a family of severe neurodegenerative disorders resulting from a deficiency in the ß-hexosaminidase A enzyme. These disorders include Tay-Sachs disease and Sandhoff disease, caused by mutations in the HEXA gene and HEXB gene, respectively. The HEXA and HEXB genes are required to produce the α and ß subunits of the ß-hexosaminidase A enzyme, respectively. Using a Sandhoff disease mouse model, we tested for the first time the potential of a comparatively lower dose (2.04 × 1013 vg/kg) of systemically delivered single-stranded adeno-associated virus 9 expressing both human HEXB and human HEXA cDNA under the control of a single promoter with a P2A-linked bicistronic vector design to correct the neurological phenotype. A bicistronic design allows maximal overexpression and secretion of the Hex A enzyme. Neonatal mice were injected with either this ssAAV9-HexB-P2A-HexA vector or a vehicle solution via the superficial temporal vein. An increase in survival of 56% compared with vehicle-injected controls and biochemical analysis of the brain tissue and serum revealed an increase in enzyme activity and a decrease in brain GM2 ganglioside buildup. This is a proof-of-concept study showing the "correction efficacy" of a bicistronic AAV9 vector delivered intravenously for GM2 gangliosidoses. Further studies with higher doses are warranted.

Biallelic loss of function variants in PPP1R21 cause a neurodevelopmental syndrome with impaired endocytic function.

Next-generation sequencing (NGS) has been instrumental in solving the genetic basis of rare inherited diseases, especially neurodevelopmental syndromes. However, functional workup is essential for precise phenotype definition and to understand the underlying disease mechanisms. Using whole exome (WES) and whole genome sequencing (WGS) in four independent families with hypotonia, neurodevelopmental delay, facial dysmorphism, loss of white matter, and thinning of the corpus callosum, we identified four previously unreported homozygous truncating PPP1R21 alleles: c.347delT p.(Ile116Lysfs*25), c.2170_2171insGGTA p.(Ile724Argfs*8), c.1607dupT p.(Leu536Phefs*7), c.2063delA p.(Lys688Serfs*26) and found that PPP1R21 was absent in fibroblasts of an affected individual, supporting the allele's loss of function effect. PPP1R21 function had not been studied except that a large scale affinity proteomics approach suggested an interaction with PIBF1 defective in Joubert syndrome. Our co-immunoprecipitation studies did not confirm this but in contrast defined the localization of PPP1R21 to the early endosome. Consistent with the subcellular expression pattern and the clinical phenotype exhibiting features of storage diseases, we found patient fibroblasts exhibited a delay in clearance of transferrin-488 while uptake was normal. In summary, we delineate a novel neurodevelopmental syndrome caused by biallelic PPP1R21 loss of function variants, and suggest a role of PPP1R21 within the endosomal sorting process or endosome maturation pathway.

Transplantation of Gene-Edited Hepatocyte-like Cells Modestly Improves Survival of Arginase-1-Deficient Mice.

Progress in gene editing research has been accelerated by utilizing engineered nucleases in combination with induced pluripotent stem cell (iPSC) technology. Here, we report transcription activator-like effector nuclease (TALEN)-mediated reincorporation of Arg1 exons 7 and 8 in iPSCs derived from arginase-1-deficient mice possessing Arg1Δ alleles lacking these terminal exons. The edited cells could be induced to differentiate into hepatocyte-like cells (iHLCs) in vitro and were subsequently used for transplantation into our previously described (Sin et al., PLoS ONE 2013) tamoxifen-inducible Arg1-Cre arginase-1-deficient mouse model. While successful gene-targeted repair was achieved in iPSCs containing Arg1Δ alleles, only minimal restoration of urea cycle function could be observed in the iHLC-transplanted mice compared to control mice, and survival in this lethal model was extended by up to a week in some mice. The partially rescued phenotype may be due to inadequate regenerative capacity of arginase-1-expressing cells in the correct metabolic zones. Technical hurdles exist and will need to be overcome for gene-edited iPSC to iHLC rescue of arginase-1 deficiency, a rare urea cycle disorder.

Laser in situ keratomileusis flap stability in an aviator following aircraft ejection.

We present the case of a 28-year-old male F/A-18F Super Hornet naval flight officer who ejected from an aircraft at 13 000 feet at a speed in excess of 350 knots 7 years after uneventful laser in situ keratomileusis (LASIK). The patient was evaluated the day after the ejection. No LASIK flap complications or epithelial defects were found, and the corrected distance visual acuity was 20/15 in both eyes. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.