Axenfeld-Rieger syndrome: more than meets the eye.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is characterised by typical anterior segment anomalies, with or without systemic features. The discovery of causative genes identified ARS subtypes with distinct phenotypes, but our understanding is incomplete, complicated by the rarity of the condition. METHODS: Genetic and phenotypic characterisation of the largest reported ARS cohort through comprehensive genetic and clinical data analyses. RESULTS: 128 individuals with causative variants in PITX2 or FOXC1, including 81 new cases, were investigated. Ocular anomalies showed significant overlap but with broader variability and earlier onset of glaucoma for FOXC1-related ARS. Systemic anomalies were seen in all individuals with PITX2-related ARS and the majority of those with FOXC1-related ARS. PITX2-related ARS demonstrated typical umbilical anomalies and dental microdontia/hypodontia/oligodontia, along with a novel high rate of Meckel diverticulum. FOXC1-related ARS exhibited characteristic hearing loss and congenital heart defects as well as previously unrecognised phenotypes of dental enamel hypoplasia and/or crowding, a range of skeletal and joint anomalies, hypotonia/early delay and feeding disorders with structural oesophageal anomalies in some. Brain imaging revealed highly penetrant white matter hyperintensities, colpocephaly/ventriculomegaly and frequent arachnoid cysts. The expanded phenotype of FOXC1-related ARS identified here was found to fully overlap features of De Hauwere syndrome. The results were used to generate gene-specific management plans for the two types of ARS. CONCLUSION: Since clinical features of ARS vary significantly based on the affected gene, it is critical that families are provided with a gene-specific diagnosis, PITX2-related ARS or FOXC1-related ARS. De Hauwere syndrome is proposed to be a FOXC1opathy.

Comprehensive phenotypic and functional analysis of dominant and recessive FOXE3 alleles in ocular developmental disorders.

The forkhead transcription factor FOXE3 is critical for vertebrate eye development. Recessive and dominant variants cause human ocular disease but the full range of phenotypes and mechanisms of action for the two classes of variants are unknown. We identified FOXE3 variants in individuals with congenital eye malformations and carried out in vitro functional analysis on selected alleles. Sixteen new recessive and dominant families, including six novel variants, were identified. Analysis of new and previously reported genetic and clinical data demonstrated a broad phenotypic range with an overlap between recessive and dominant disease. Most families with recessive alleles, composed of truncating and forkhead-domain missense variants, had severe corneal opacity (90%; sclerocornea in 47%), aphakia (83%) and microphthalmia (80%), but some had milder features including isolated cataract. The phenotype was most variable for recessive missense variants, suggesting that the functional consequences may be highly dependent on the type of amino acid substitution and its position. When assessed, aniridia or iris hypoplasia were noted in 89% and optic nerve anomalies in 60% of recessive cases, indicating that these defects are also common and may be underrecognized. In dominant pedigrees, caused by extension variants, normal eye size (96%), cataracts (99%) and variable anterior segment anomalies were seen in most, but some individuals had microphthalmia, aphakia or sclerocornea, more typical of recessive disease. Functional studies identified variable effects on the protein stability, DNA binding, nuclear localization and transcriptional activity for recessive FOXE3 variants, whereas dominant alleles showed severe impairment in all areas and dominant-negative characteristics.

CRISPR-Cas9-mediated functional dissection of the foxc1 genomic region in zebrafish identifies critical conserved cis-regulatory elements.

FOXC1 encodes a forkhead-domain transcription factor associated with several ocular disorders. Correct FOXC1 dosage is critical to normal development, yet the mechanisms controlling its expression remain unknown. Together with FOXQ1 and FOXF2, FOXC1 is part of a cluster of FOX genes conserved in vertebrates. CRISPR-Cas9-mediated dissection of genomic sequences surrounding two zebrafish orthologs of FOXC1 was performed. This included five zebrafish-human conserved regions, three downstream of foxc1a and two remotely upstream of foxf2a/foxc1a or foxf2b/foxc1b clusters, as well as two intergenic regions between foxc1a/b and foxf2a/b lacking sequence conservation but positionally corresponding to the area encompassing a previously reported glaucoma-associated SNP in humans. Removal of downstream sequences altered foxc1a expression; moreover, zebrafish carrying deletions of two or three downstream elements demonstrated abnormal phenotypes including enlargement of the anterior chamber of the eye reminiscent of human congenital glaucoma. Deletions of distant upstream conserved elements influenced the expression of foxf2a/b or foxq1a/b but not foxc1a/b within each cluster. Removal of either intergenic sequence reduced foxc1a or foxc1b expression during late development, suggesting a role in transcriptional regulation despite the lack of conservation at the nucleotide level. Further studies of the identified regions in human patients may explain additional individuals with developmental ocular disorders.

Disruption of foxc1 genes in zebrafish results in dosage-dependent phenotypes overlapping Axenfeld-Rieger syndrome.

The Forkhead Box C1 (FOXC1) gene encodes a forkhead/winged helix transcription factor involved in embryonic development. Mutations in this gene cause dysgenesis of the anterior segment of the eye, most commonly Axenfeld-Rieger syndrome (ARS), often with other systemic features. The developmental mechanisms and pathways regulated by FOXC1 remain largely unknown. There are two conserved orthologs of FOXC1 in zebrafish, foxc1a and foxc1b. To further examine the role of FOXC1 in vertebrates, we generated foxc1a and foxc1b single knockout zebrafish lines and bred them to obtain various allelic combinations. Three genotypes demonstrated visible phenotypes: foxc1a-/- single homozygous and foxc1-/- double knockout homozygous embryos presented with similar characteristics comprised of severe global vascular defects and early lethality, as well as microphthalmia, periocular edema and absence of the anterior chamber of the eye; additionally, fish with heterozygous loss of foxc1a combined with homozygosity for foxc1b (foxc1a+/-;foxc1b-/-) demonstrated craniofacial defects, heart anomalies and scoliosis. All other single and combined genotypes appeared normal. Analysis of foxc1 expression detected a significant increase in foxc1a levels in homozygous and heterozygous mutant eyes, suggesting a mechanism for foxc1a upregulation when its function is compromised; interestingly, the expression of another ARS-associated gene, pitx2, was responsive to the estimated level of wild-type Foxc1a, indicating a possible role for this protein in the regulation of pitx2 expression. Altogether, our results support a conserved role for foxc1 in the formation of many organs, consistent with the features observed in human patients, and highlight the importance of correct FOXC1/foxc1 dosage for vertebrate development.

De Novo Missense Variants in WDR37 Cause a Severe Multisystemic Syndrome.

While genetic causes are known for many syndromes involving developmental anomalies, a large number of individuals with overlapping phenotypes remain undiagnosed. Using exome-sequencing analysis and review of matchmaker databases, we have discovered four de novo missense variants predicted to affect the N-terminal region of WDR37-p.Ser119Phe, p.Thr125Ile, p.Ser129Cys, and p.Thr130Ile-in unrelated individuals with a previously unrecognized syndrome. Features of WDR37 syndrome include the following: ocular anomalies such as corneal opacity/Peters anomaly, coloboma, and microcornea; dysmorphic facial features; significant neurological impairment with structural brain defects and seizures; poor feeding; poor post-natal growth; variable skeletal, cardiac, and genitourinary defects; and death in infancy in one individual. WDR37 encodes a protein of unknown function with seven predicted WD40 domains and no previously reported human pathogenic variants. Immunocytochemistry and western blot studies showed that wild-type WDR37 is localized predominantly in the cytoplasm and mutant proteins demonstrate similar protein levels and localization. CRISPR-Cas9-mediated genome editing generated zebrafish mutants with novel missense and frameshift alleles: p.Ser129Phe, p.Ser129Cys (which replicates one of the human variants), p.Ser129Tyr, p.Lys127Cysfs, and p.Gln95Argfs. Zebrafish carrying heterozygous missense variants demonstrated poor growth and larval lethality, while heterozygotes with frameshift alleles survived to adulthood, suggesting a potential dominant-negative mechanism for the missense variants. RNA-seq analysis of zebrafish embryos carrying a missense variant detected significant upregulation of cholesterol biosynthesis pathways. This study identifies variants in WDR37 associated with human disease and provides insight into its essential role in vertebrate development and possible molecular functions.

Gartland Type IIa Supracondylar Humerus Fractures: Outcomes of Attempted Nonoperative Management.

BACKGROUND: The optimal treatment of Gartland type IIa supracondylar humerus fractures remains controversial. We report the results of a series of patients with type IIa fractures who underwent closed reduction and immobilization using conscious sedation in the emergency department. Our goal was to identify variables associated with fractures that were successfully managed nonoperatively. METHODS: This was a retrospective cohort study of pediatric patients who underwent closed reduction of Gartland type IIa supracondylar humerus fractures with the use of conscious sedation in the emergency department. Prereduction and postreduction radiographs were reviewed to determine the degree of fracture extension, anterior humeral line index, Baumann angle, and splint flexion angle. The success of closed reduction was defined as a reduction that was maintained without the need for surgical intervention. RESULTS: A total of 54 patients (54 elbows) were included in this study. The mean overall age was 5.2±2.5 years. Following the closed reduction in the emergency department, 38 (70%) patients were successfully managed nonoperatively with casting, and 16 (30%) patients required operative intervention. The degree of fracture extension on the injury radiograph was 13.2±8.4 degrees in the nonoperative group compared with 19.8±7.5 degrees in the operative group (P=0.008). The postreduction degree of fracture extension was 3.0±3.4 degrees in the nonoperative group and 10.0±7.2 degrees in the operative group (P<0.0001). The mean anterior humeral line index on the injury radiograph was 0.34 in the nonoperative group and 0.13 in the operative group (P=0.104). The mean anterior humeral line index on the postreduction radiograph was 1.2 in the nonoperative group and 0.38 in the operative group (P=0.0002). Patient age, prereduction and postreduction Baumann angle, and the postreduction splint flexion angle did not differ significantly between groups. CONCLUSIONS: Closed reduction under conscious sedation in the emergency department is a viable treatment option for Gartland type IIa supracondylar humerus fractures. Increasing fracture extension on injury radiographs can help predict failure of nonoperative management following closed reduction. LEVEL OF EVIDENCE: Level III-retrospective comparative study.

Information system use antecedents of nursing employee turnover in a hospital setting.

BACKGROUND: Voluntary turnover (VTO) of nursing employees is expensive for hospital systems and is often associated with lower levels of patient satisfaction, as well as adverse patient outcomes such as falls and medication errors. PURPOSE: The aim of this study was to establish nurses' electronic medical record (EMR) use patterns and test if they can be used to predict VTO. METHODOLOGY/APPROACH: The study followed 1,836 hospital nurses via the collection of EMR metadata through two 1-month time periods that were 1 year apart. Machine learning algorithms were then used to derive patterns of EMR utilization using VTO as a key variable for classification. Post hoc analysis of the most predictive variables was conducted. RESULTS: The predictive model was effective in identifying which nurses would turnover 73.4% of the time and which nurses would not turnover 84.1% of the time. PRACTICE APPLICATIONS: The ability to accurately predict nurses' intentions to leave is critical to reducing turnover. Early identification can lead to specific interventions to mitigate factors that are adversely impacting the nursing experience. Post hoc analysis and the key informant interviews indicated that many nurses do not appear to have good EMR navigation skills and spend significant effort in search of patient information.

WDR37 syndrome: identification of a distinct new cluster of disease-associated variants and functional analyses of mutant proteins.

Missense variants located in the N-terminal region of WDR37 were recently identified to cause a multisystemic syndrome affecting neurological, ocular, gastrointestinal, genitourinary, and cardiac development. WDR37 encodes a WD40 repeat-containing protein of unknown function. We identified three novel WDR37 variants, two likely pathogenic de novo alleles and one inherited variant of uncertain significance, in individuals with phenotypes overlapping those previously reported but clustering in a different region of the protein. The novel alleles are C-terminal to the prior variants and located either within the second WD40 motif (c.659A>G p.(Asp220Gly)) or in a disordered protein region connecting the second and third WD40 motifs (c.778G>A p.(Asp260Asn) and c.770C>A p.(Pro257His)). The three novel mutants showed normal cellular localization but lower expression levels in comparison to wild-type WDR37. To investigate the normal interactions of WDR37, we performed co-immunoprecipitation and yeast two-hybrid assays. This revealed the ability of WDR37 to form homodimers and to strongly bind PACS1 and PACS2 phosphofurin acidic cluster sorting proteins; immunocytochemistry confirmed colocalization of WDR37 with PACS1 and PACS2 in human cells. Next, we analyzed previously reported and novel mutants for their ability to dimerize with wild-type WDR37 and bind PACS proteins. Interaction with wild-type WDR37 was not affected for any variant; however, one novel mutant, p.(Asp220Gly), lost its ability to bind PACS1 and PACS2. In summary, this study presents a novel region of WDR37 involved in human disease, identifies PACS1 and PACS2 as major binding partners of WDR37 and provides insight into the functional effects of various WDR37 variants.

Effect of Side Struts on the Strength of Long Arm Plaster Splints: A Biomechanical Study.

BACKGROUND: There are multiple methods of achieving upper extremity immobilization after pediatric elbow injuries; however, no biomechanical study has established an optimal construct. The goal of this study was to compare the strength of commonly used long arm splints and to evaluate the effect of reinforcing plaster splints with side struts. METHODS: Five categories of long arm posterior slab splints were tested: 4-inch plaster without side struts, 4-inch plaster with a medial side strut, 4-inch plaster with medial and lateral side struts, 5-inch plaster without side struts, and 4-inch fiberglass splint material without side struts. There were 4 splints in each group. As a control, 4 half fiberglass long arm casts were also tested. Each splint or cast was mounted on a single-column tensile tester and a 3-point bending load was applied to simulate an extension moment at the elbow. The maximum load before failure was measured and an ANOVA model was used to analyze the differences between groups. Additionally, a retrospective chart review was performed of pediatric patients who were immobilized postoperatively in a long arm plaster splint with side struts. We collected data on patient age, type of fracture, time from splint application in the operating room to removal in clinic, length of follow-up, and any complications. RESULTS: The 4-inch plaster splints reinforced with 2 struts had the highest average maximum load to failure (731±143 N), which was significantly higher than the 4-inch plaster splints with one strut (505±48 N) (P=0.01) and the 4-inch plaster splints without struts (100±10 N) (P<0.001). The half fiberglass casts failed at an average maximum load of 655±96 N, however there was no statistically significant difference compared with 4-inch plaster splints with 2 struts (P=0.10). The 5-inch plaster splints without side struts failed at a greater average maximum load (341±110 N) compared with the splints constructed with fiberglass material without side struts (233±61 N) (P=0.03). A total of 140 patients were identified in the retrospective review. Splint-related complications occurred in 2 patients. CONCLUSIONS: The addition of both 1 and 2 side struts to a 4-inch long arm plaster splint significantly increased the load to failure. The strength of 4-inch plaster splints with 2 side struts was comparable to that of half fiberglass casts. LEVEL OF EVIDENCE: NA (biomechanical study).

Complication Rates in Total Knee Arthroplasty Performed for Osteoarthritis and Post-Traumatic Arthritis: A Comparison Study.

BACKGROUND: The number of total knee arthroplasty (TKA) procedures performed in the United States has been increasing. Increased complication rates have been demonstrated in patients with post-traumatic arthritis (PTA) undergoing TKA. However, there remains limited data directly comparing outcomes of TKA performed for osteoarthritis (OA) and PTA. METHODS: The National Inpatient Sample was utilized to identify patients undergoing elective TKA between 2006 and 2015 for OA and PTA. The prevalence of preoperative comorbidities and the incidence of postoperative complications including superficial wound infection, deep joint infection, acute deep venous thrombosis, and pulmonary embolus were analyzed. RESULTS: Between 2006 and 2015, the National Inpatient Sample database accounted for 1,301,394 patients diagnosed with either PTA (14,206) or OA (1,287,188) undergoing TKA. The incidence of superficial wound infection, deep joint infection, and acute deep venous thrombosis was found to occur at a higher rate in patients with a diagnosis of PTA compared to OA. The incidence of pulmonary embolus was not found to be statistically different between the 2 groups. Patients with PTA had a higher prevalence of drug and alcohol abuse, psychosis, and liver disease, whereas patients with OA had a higher prevalence of obesity, diabetes, heart disease, and lung disease. CONCLUSION: This study demonstrates an increased risk of complications in patients undergoing TKA for PTA compared to OA. Surgeons can use this information to help aid in counseling patients preoperatively. Furthermore, these data provide objective evidence that could have implications with regards to establishing bundled payment reimbursement in this patient population.

Characterising extinction debt following habitat fragmentation using neutral theory.

Habitat loss leads to species extinctions, both immediately and over the long term as 'extinction debt' is repaid. The same quantity of habitat can be lost in different spatial patterns with varying habitat fragmentation. How this translates to species loss remains an open problem requiring an understanding of the interplay between community dynamics and habitat structure across temporal and spatial scales. Here we develop formulas that characterise extinction debt in a spatial neutral model after habitat loss and fragmentation. Central to our formulas are two new metrics, which depend on properties of the taxa and landscape: 'effective area', measuring the remaining number of individuals and 'effective connectivity', measuring individuals' ability to disperse through fragmented habitat. This formalises the conventional wisdom that habitat area and habitat connectivity are the two critical requirements for long-term preservation of biodiversity. Our approach suggests that mechanistic fragmentation metrics help resolve debates about fragmentation and species loss.

Reconciling the contribution of environmental and stochastic structuring of tropical forest diversity through the lens of imaging spectroscopy.

Both niche and stochastic dispersal processes structure the extraordinary diversity of tropical plants, but determining their relative contributions has proven challenging. We address this question using airborne imaging spectroscopy to estimate canopy ß-diversity for an extensive region of a Bornean rainforest and challenge these data with models incorporating niches and dispersal. We show that remotely sensed and field-derived estimates of pairwise dissimilarity in community composition are closely matched, proving the applicability of imaging spectroscopy to provide ß-diversity data for entire landscapes of over 1000 ha containing contrasting forest types. Our model reproduces the empirical data well and shows that the ecological processes maintaining tropical forest diversity are scale dependent. Patterns of ß-diversity are shaped by stochastic dispersal processes acting locally whilst environmental processes act over a wider range of scales.

Transition-Metal-Catalyzed C-H Alkylation Using Alkenes.

Alkylation reactions represent an important organic transformation to form C-C bonds. In addition to conventional approaches with alkyl halides or sulfonates as alkylating agents, the use of unactivated olefins for alkylations has become attractive from both cost and sustainability viewpoints. This Review summarizes transition-metal-catalyzed alkylations of various carbon-hydrogen bonds (addition of C-H bonds across olefins) using regular olefins or 1,3-dienes up to May 2016. According to the mode of activation, the Review is divided into two sections: alkylation via C-H activation and alkylation via olefin activation.

Synthesis and applications of rhodium porphyrin complexes.

Organotransition metal complexes capable of forming metalloradicals have been an intriguing subject of study for the past fifty years. Of these, rhodium porphyrin complexes have proven particularly interesting due to their straightforward synthesis and unique reactivity; indeed, these complexes are responsible for some highly influential transformations of organic compounds, including rare C-H and C-C bond activations. The complexity and selectivity of rhodium porphyrins has been attractive for catalytic transformations, with specific interest in their usage for selective carbon monoxide reduction for fuel cell applications. This review will highlight historical and modern syntheses of rhodium porphyrins, as well as their respective reactions with small molecules and applications therein. The discussion will be limited to rhodium porphyrins consisting of four pyrrolic rings bridged with four methine units.

A randomized controlled trial of manual therapy and pneumatic compression for recovery from prolonged running - an extended study.

Manual therapy (MT) and intermittent pneumatic compression (IPC) are recovery methods used by endurance athletes with little evidence supporting effectiveness. This randomized controlled trial evaluated effectiveness of four daily post-race treatments of a specific MT protocol and IPC compared with supine rest on recovery following an ultramarathon among 56 ultramarathoners. Groups were comparable across all characteristics examined, including post-race plasma creatine kinase concentration. Subject completed timed 400 m runs before the race and on days three, five, seven and 14 post- race, and also provided muscle pain and soreness ratings and fatigue scores immediately before and after treatments, and during the 14 days post- race. Daily subjective measures and 400 m run times were not improved by either treatment, but both treatments reduced (p < .05) muscular fatigue scores acutely after treatment following the race and on post-race day 1, and MT improved (p < .05) muscle pain and soreness acutely following the race.

Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer.

Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (ß) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α6) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α6 is assembled from dimers (α2) without a previously proposed tetramer intermediate (α4). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α6 can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α6 were further examined by SAXS in the presence of the ß subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α6 is more stable than the ATP-induced α6 and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the ß subunit to the active site of α.

Cyclic Ether Synthesis via Palladium-Catalyzed Directed Dehydrogenative Annulation at Unactivated Terminal Positions.

Here, a palladium-catalyzed functionalization of unactivated sp(3) C-H bonds with internal alcohol nucleophiles is described. Directed by an oxime-masked alcohol, annulation chemoselectively occurs at the ß position, leading to a range of aliphatic cyclic ethers with four- to seven-membered rings. Tethered primary, secondary, and tertiary free hydroxyl groups can all react to give the corresponding cyclized products. In addition, benzyl and silyl protected alcohols can also be directly coupled. An sp(3) C-H activation/intramolecular SN2 pathway was proposed.

Assistive technology as a predictor of general or alternate assessment among elementary-aged students with autism spectrum disorders.

The No Child Left Behind Act of 2001 specifically mandates that all students participate in the general assessment process or some form of alternate assessment as a measure of school accountability for student academic progress. Although levels of communication difficulties, intellectual impairment, and specific diagnoses such as autism spectrum disorders (ASDs) are correlated with increased probability of participating in alternate assessment methods, very little empirical research has focused on identifying predictors for students' assessment modality. Archival data from the Special Education Elementary Longitudinal Study (SEELS; 2005) were used to examine variables that predict whether elementary school students with ASD participated in the general or alternate assessment. Results indicated that receptive and expressive communication abilities appear to influence participation in the general vs. alternate assessment in tandem with access to assistive technology. Students with ASDs were approximately 2.71 times more likely to participate in the general assessment when they had access to assistive technology. Next, we performed a second, follow-up analysis for only ASD students with communication problems. The odds ratio value increased to 14.9 indicating that ASD students with communication problems that had access to assistive technology were almost 15 times more likely to participate in the general assessment than students with communication problems without access to assistive technology.

The genetic landscape of a metabolic interaction.

While much prior work has explored the constraints on protein sequence and evolution induced by physical protein-protein interactions, the sequence-level constraints emerging from non-binding functional interactions in metabolism remain unclear. To quantify how variation in the activity of one enzyme constrains the biochemical parameters and sequence of another, we focus on dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), a pair of enzymes catalyzing consecutive reactions in folate metabolism. We use deep mutational scanning to quantify the growth rate effect of 2696 DHFR single mutations in 3 TYMS backgrounds under conditions selected to emphasize biochemical epistasis. Our data are well-described by a relatively simple enzyme velocity to growth rate model that quantifies how metabolic context tunes enzyme mutational tolerance. Together our results reveal the structural distribution of epistasis in a metabolic enzyme and establish a foundation for the design of multi-enzyme systems.