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Background Studies have shown conflicting results regarding associations of preoperative comorbidities with outcomes after total ankle arthroplasty (TAA). Our aim was to analyze preoperative risk factors for complications, longer hospital stay, and readmission within 30 days after TAA. Methods We conducted a retrospective study using the American College of Surgeons National Surgical Quality Improvement Program database. We included 294 patients who underwent TAA from 2009 through 2012. We used multivariate logistic regression to identify risk factors for complications, longer hospital stay, and hospital readmission. Results Surgical site infection was the most common complication. Diabetes was associated with greater odds of complications as was current smoker status. Notably, obesity was not associated with greater odds of complications. Age, chronic obstructive pulmonary disease, and diabetes mellitus were associated with longer hospital stays. Surgical site infection was the most common reason for hospital readmission. Conclusions TAA has a low complication rate, with surgical site infection being the most common complication and the most common reason for hospital readmission. Patients with diabetes have greater odds of poor outcomes and prolonged hospital stays after TAA than patients without diabetes. Obesity was not associated with poor outcomes after TAA. Levels of Evidence: Level III

There is a need for methods that can image chromosomes with genome-wide coverage, as well as greater genomic and optical resolution. We introduce OligoFISSEQ, a suite of three methods that leverage fluorescence in situ sequencing (FISSEQ) of barcoded Oligopaint probes to enable the rapid visualization of many targeted genomic regions. Applying OligoFISSEQ to human diploid fibroblast cells, we show how four rounds of sequencing are sufficient to produce 3D maps of 36 genomic targets across six chromosomes in hundreds to thousands of cells, implying a potential to image thousands of targets in only five to eight rounds of sequencing. We also use OligoFISSEQ to trace chromosomes at finer resolution, following the path of the X chromosome through 46 regions, with separate studies showing compatibility of OligoFISSEQ with immunocytochemistry. Finally, we combined OligoFISSEQ with OligoSTORM, laying the foundation for accelerated single-molecule super-resolution imaging of large swaths of, if not entire, human genomes. OligoFISSEQ combines Oligopaints with fluorescence in situ sequencing to enable the 3D mapping of many regions across the genome in human cells to interrogate genome organization at improved genomic resolution. OligoFISSEQ is compatible with immunochemistry and OligoSTORM for super-resolution imaging.

The human genome can be segmented into topologically associating domains (TADs), which have been proposed to spatially sequester genes and regulatory elements through chromatin looping. Interactions between TADs have also been suggested, presumably because of variable boundary positions across individual cells. However, the nature, extent and consequence of these dynamic boundaries remain unclear. Here, we combine high-resolution imaging with Oligopaint technology to quantify the interaction frequencies across both weak and strong boundaries. We find that chromatin intermingling across population-defined boundaries is widespread but that the extent of permissibility is locus-specific. Cohesin depletion, which abolishes domain formation at the population level, does not induce ectopic interactions but instead reduces interactions across all boundaries tested. In contrast, WAPL or CTCF depletion increases inter-domain contacts in a cohesin-dependent manner. Reduced chromatin intermingling due to cohesin loss affects the topology and transcriptional bursting frequencies of genes near boundaries. We propose that cohesin occasionally bypasses boundaries to promote incorporation of boundary-proximal genes into neighboring domains.

Higher-order chromatin structure regulates gene expression, and mutations in proteins mediating genome folding underlie developmental disorders known as cohesinopathies. However, the relationship between three-dimensional genome organization and embryonic development remains unclear. Here we define a role for bromodomain-containing protein 4 (BRD4) in genome folding, and leverage it to understand the importance of genome folding in neural crest progenitor differentiation. Brd4 deletion in neural crest results in cohesinopathy-like phenotypes. BRD4 interacts with NIPBL, a cohesin agonist, and BRD4 depletion or loss of the BRD4-NIPBL interaction reduces NIPBL occupancy, suggesting that BRD4 stabilizes NIPBL on chromatin. Chromatin interaction mapping and imaging experiments demonstrate that BRD4 depletion results in compromised genome folding and loop extrusion. Finally, mutation of individual BRD4 amino acids that mediate an interaction with NIPBL impedes neural crest differentiation into smooth muscle. Remarkably, loss of WAPL, a cohesin antagonist, rescues attenuated smooth muscle differentiation resulting from BRD4 loss. Collectively, our data reveal that BRD4 choreographs genome folding and illustrates the relevance of balancing cohesin activity for progenitor differentiation.

The calcium-activated chloride channel (CaCC) TMEM16A plays crucial roles in regulating neuronal excitability, smooth muscle contraction, fluid secretion and gut motility. While opening of TMEM16A requires binding of intracellular Ca2+, prolonged Ca2+-dependent activation results in channel desensitization or rundown, the mechanism of which is unclear. Here we show that phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates TMEM16A channel activation and desensitization via binding to a putative binding site at the cytosolic interface of transmembrane segments (TMs) 3–5. We further demonstrate that the ion-conducting pore of TMEM16A is constituted of two functionally distinct modules: a Ca2+-binding module formed by TMs 6–8 and a PIP2-binding regulatory module formed by TMs 3–5, which mediate channel activation and desensitization, respectively. PIP2 dissociation from the regulatory module results in ion-conducting pore collapse and subsequent channel desensitization. Our findings thus provide key insights into the mechanistic understanding of TMEM16 channel gating and lipid-dependent regulation., The calcium-activated chloride channel (CaCC) TMEM16A plays crucial roles in regulating neuronal excitability and muscle contraction. Here authors show that phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates TMEM16A channel activation and desensitization via binding to a putative binding site.

In virtually all eukaryotes, telomerase counteracts chromosome erosion by adding repetitive sequence to terminal ends.Drosophila melanogasterinstead relies on specialized retrotransposons that insert preferentially at telomeres. This exchange of goods between host and mobile element—wherein the mobile element provides an essential genome service and the host provides a hospitable niche for mobile element propagation—has been called a ‘genomic symbiosis’. However, these telomere-specialized, ‘jockey’ family elements may actually evolve to selfishly over-replicate in the genomes that they ostensibly serve. Under this intra-genomic conflict model, we expect rapid diversification of telomere-specialized retrotransposon lineages and possibly, the breakdown of this tenuous relationship. Here we report data consistent with both predictions. Searching the raw reads of the 15-million-year-old ‘melanogaster species group’, we generatedde novojockey retrotransposon consensus sequences and used phylogenetic tree-building to delineate four distinct telomere-associated lineages. Recurrent gains, losses, and replacements account for this striking retrotransposon lineage diversity. Moreover, an ancestrally telomere-specialized element has ‘escaped,’ residing now throughout the genome ofD. rhopaloa.InD. biarmipes,telomere-specialized elements have disappeared completely.De novoassembly of long-reads and cytogenetics confirmed this species-specific collapse of retrotransposon-dependent telomere elongation. Instead, telomere-restricted satellite DNA and DNA transposon fragments occupy its terminal ends. We infer thatD. biarmipesrelies instead on a recombination-based mechanism conserved from yeast to flies to humans. Combined with previous reports of adaptive evolution at host proteins that regulate telomere length, telomere-associated retrotransposon diversification and disappearance offer compelling evidence that intra-genomic conflict shapes Drosophila telomere evolution.

TMEM16 Ca(2+)-activated phospholipid scramblases (CaPLSases) mediate rapid transmembrane phospholipid flip-flop and as such play essential roles in various physiological and pathological processes such as blood coagulation, skeletal development, viral infection, cell-cell fusion, and ataxia. Pharmacological tools specifically targeting TMEM16 CaPLSases are urgently needed to understand these novel membrane transporters and their contributions to health and disease. Tannic acid (TA) and epigallocatechin gallate (EGCG) were recently reported as promising TMEM16F CaPLSase inhibitors. However, our present study shows that TA and EGCG do not inhibit the phospholipid-scrambling or ion conduction activities of the dual-functional TMEM16F. Instead, we found that TA and EGCG mainly acted as fluorescence quenchers that rapidly suppress the fluorophores conjugated to annexin V, a phosphatidylserine-binding probe commonly used to report on TMEM16 CaPLSase activity. These data demonstrate the false positive effects of TA and EGCG on inhibiting TMEM16F phospholipid scrambling and discourage the use of these polyphenols as CaPLSase inhibitors. Appropriate controls as well as a combination of both fluorescence imaging and electrophysiological validation are necessary in future endeavors to develop TMEM16 CaPLSase inhibitors.

The aim of this study was to investigate genetic structures and expression of blaOXA-58 gene in five Acinetobacter baumannii clinical isolates recovered from two hospitals in southern Vietnam during 2012-2014. A. baumannii isolates were identified by automated microbiology systems and confirmed by PCR. All isolates were characterized as multidrug resistant by antimicrobial testing using the disk diffusion method. Four imipenem susceptible and one nonsusceptible isolates (MIC > 32 μg·ml−1) were identified by E-test. PCR amplification of blaOXA-58 gene upstream and downstream sequences revealed the presence of ISAba3 at both locations in one multidrug-resistant isolate. Semiquantitation of blaOXA-51 and blaOXA-58 gene expression was performed by the 2-ΔΔCt method. The blaOXA-51 gene expression of five isolates showed little difference, but the isolate bearing ISAba3-blaOXA-58-ISAba3 exhibited significantly higher blaOXA-58 mRNA level. Higher β-lactamases activity in periplasmic than cytoplasmic fraction was found in most isolates. The isolate overexpressing blaOXA-58 gene possessed very high periplasmic enzyme activity. In conclusion, the A. baumannii isolate bearing ISAba3-blaOXA-58 gene exhibited high resistance to imipenem, corresponding to an overexpression of blaOXA-58 gene and very high periplasmic β-lactamase activity.

Background Micronutrient malnutrition has been associated with maternal depressive symptoms (MDS), but little is known about the effects of preconceptional micronutrient supplementation. This paper examined the effects of preconceptional micronutrient supplementation on MDS during pregnancy and postpartum. Methods We used data from a double-blind controlled trial (PRECONCEPT) in which 5011 Vietnamese women were randomized to receive weekly supplements containing either a) multiple micronutrients (MM) b) iron and folic acid (IFA) or c) folic acid (FA) until conception (n = 1813). Maternal mental health was assessed using the Center for Epidemiologic Studies Depression Scale (CES-D) at baseline (preconception), and the Edinburgh Postnatal Depression Scale (EPDS) during pregnancy and 3 months postpartum. Elevated MDS was defined as EPDS score ≥ 4. All group comparisons were done using ANOVA or chi-square tests of proportions intention to treat and per protocol analyses (women consumed supplements ≥26 weeks before conception). We also conducted stratified analyses by preconception CES-D scores, underweight, or anemia status using generalized linear models. Results Baseline CES-D scores were similar across treatment groups. The proportion of women experiencing elevated MDS was 11.3, 8.1 and 4.9% at first, second and third trimesters of pregnancy, respectively, and 3.6% at 3 mo postpartum. Mean EPDS scores at first (1.5 ± 2.7), second (1.1 ± 2.4), and third trimester of pregnancy (0.7 ± 2.0) and early postpartum (0.6 ± 1.8) were low and did not differ by treatment group. However, among women in the highest tertile of CES-D scores at preconception, mean EPDS scores in the first and second trimesters of pregnancy were lower in the MM and IFA groups compared to FA only (P

Cell-type-specific 3D organization of the genome is unrecognizable during mitosis. It remains unclear how essential positional information is transmitted through cell division such that a daughter cell recapitulates the spatial genome organization of the parent. Lamina-associated domains (LADs) are regions of repressive heterochromatin positioned at the nuclear periphery that vary by cell type and contribute to cell-specific gene expression and identity. Here we show that histone 3 lysine 9 dimethylation (H3K9me2) is an evolutionarily conserved, specific mark of nuclear peripheral heterochromatin and that it is retained through mitosis. During mitosis, phosphorylation of histone 3 serine 10 temporarily shields the H3K9me2 mark allowing for dissociation of chromatin from the nuclear lamina. Using high-resolution 3D immuno-oligoFISH, we demonstrate that H3K9me2-enriched genomic regions, which are positioned at the nuclear lamina in interphase cells prior to mitosis, re-associate with the forming nuclear lamina before mitotic exit. The H3K9me2 modification of peripheral heterochromatin ensures that positional information is safeguarded through cell division such that individual LADs are re-established at the nuclear periphery in daughter nuclei. Thus, H3K9me2 acts as a 3D architectural mitotic guidepost. Our data establish a mechanism for epigenetic memory and inheritance of spatial organization of the genome.

Current methods for chromosome painting via fluorescence in situ hybridization (FISH) are costly, time consuming, and are limited in complexity. In contrast to conventional sources of probe, Oligopaints are computationally designed, synthesized on microarrays, and amplified by PCR. This approach allows for precise control over the sequences they target, which can range from a few kilobases to entire chromosomes with the same basic protocol. We have utilized the flexibility and scalability of Oligopaints to generate low-cost and renewable chromosome paints for Drosophila, mouse, and human chromosomes. These Oligopaint libraries can be customized to label any genomic feature(s) in a chromosome-wide manner. Additionally, this method is compatible with sequential FISH to label entire genomes with a single denaturation step. Here, we outline a protocol and considerations to scale the Oligopaint technology for fluorescent labeling of whole chromosomes.

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. While links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions, but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared to non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism utilized by a transcription factor to boost gene expression.

SUMMARY Calcium (Ca2+) is the primary stimulus for transmembrane protein 16 (TMEM16) Ca2+-activated chloride channels and phospholipid scramblases, which regulate important physiological processes ranging from smooth muscle contraction to blood coagulation and tumor progression. Binding of intracellular Ca2+ to two highly conserved orthosteric binding sites in transmembrane helices (TMs) 6–8 efficiently opens the permeation pathway formed by TMs 3–7. Recent structures of TMEM16K and TMEM16F scramblases revealed an additional Ca2+ binding site between TM2 and TM10, whose functional relevance remains unknown. Here, we report that Ca2+ binds with high affinity to the equivalent third Ca2+ site in TMEM16A to enhance channel activation. Our cadmium (Cd2+) metal bridging experiments reveal that the third Ca2+ site’s conformational states can profoundly influence TMEM16A’s opening. Our study thus confirms the existence of a third Ca2+ site in TMEM16A, defines its functional importance in channel gating, and provides insight into a long-range allosteric gating mechanism of TMEM16 channels and scramblases., Graphical Abstract, In Brief Le and Yang present electrophysiological evidence for a third Ca2+ binding site in the Ca2+-activated chloride channel TMEM16A. They show that Ca2+ binds with high affinity to this site to facilitate channel opening, providing insight into an allosteric gating mechanism in TMEM16 ion channels and scramblases.

Background Small for gestational age (SGA) is a global health problem. Identifying the timing of fetal growth faltering is critical for developing preventive interventions. We aim to describe patterns of fetal growth and to predict SGA at birth using fetal ultrasound measurements. Methods We studied 1412 pregnant women enrolled in a randomised-controlled trial evaluating maternal micronutrient supplementation in Thai Nguyen province, Vietnam. Ultrasound examinations included biparietal diameter (BPD), head circumference (HC) and abdominal circumference (AC), and femur length (FL). Measures were assessed using the new international fetal growth standards (INTERGROWTH-21st Project). Generalised linear mixed logit regression models were used to examine the association between ultrasound measures and SGA at birth. Results Overall fetal growth restriction began in early pregnancy and continued through delivery, but the timing of growth faltering varied by measure: it began by 20 weeks for HC, BPD and AC, earlier as compared to FL growth that started >30 weeks. SGA infants had significantly lower mean fetal growth parameters as early as 14 weeks. Ultrasound measures below the 10th percentile were associated with a two to four times higher risk of SGA at birth compared to fetuses greater than the 50th percentile, with the largest odds ratios for AC (OR 3.9, 95% confidence interval (CI) 2.7, 5.7). Conclusions Fetal growth faltering by ultrasound begins in early gestation among rural Vietnamese populations; these patterns clearly identified those to be born SGA. Efforts to prevent fetal growth faltering must begin early in pregnancy and perhaps even before pregnancy.

Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.

The loss of insulin-secreting β cells is characteristic among type I and type II diabetes. Stimulating proliferation to expand sources of β cells for transplantation remains a challenge because adult β cells do not proliferate readily. The cell cycle inhibitor p57 has been shown to control cell division in human β cells. Expression of p57 is regulated by the DNA methylation status of the imprinting control region 2 (ICR2), which is commonly hypomethylated in Beckwith-Wiedemann syndrome patients who exhibit massive β cell proliferation. We hypothesized that targeted demethylation of the ICR2 using a transcription activator-like effector protein fused to the catalytic domain of TET1 (ICR2-TET1) would repress p57 expression and promote cell proliferation. We report here that overexpression of ICR2-TET1 in human fibroblasts reduces p57 expression levels and increases proliferation. Furthermore, human islets overexpressing ICR2-TET1 exhibit repression of p57 with concomitant upregulation of Ki-67 while maintaining glucose-sensing functionality. When transplanted into diabetic, immunodeficient mice, the epigenetically edited islets show increased β cell replication compared with control islets. These findings demonstrate that epigenetic editing is a promising tool for inducing β cell proliferation, which may one day alleviate the scarcity of transplantable β cells for the treatment of diabetes.

Transmembrane protein 16F (TMEM16F) is an enigmatic Ca2+-activated phospholipid scramblase (CaPLSase) that passively transports phospholipids down their chemical gradients and mediates blood coagulation, bone development and viral infection. Despite recent advances in the structure and function understanding of TMEM16 proteins, how mammalian TMEM16 CaPLSases open and close, or gate their phospholipid permeation pathways remains unclear. Here we identify an inner activation gate, which is established by three hydrophobic residues, F518, Y563 and I612, in the middle of the phospholipid permeation pathway of TMEM16F-CaPLSase. Disrupting the inner gate profoundly alters TMEM16F phospholipid permeation. Lysine substitutions of F518 and Y563 even lead to constitutively active CaPLSases that bypass Ca2+-dependent activation. Strikingly, an analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to the Ca2+-activated Cl− channel (CaCC). The identification of an inner activation gate can help elucidate the gating and permeation mechanism of TMEM16 CaPLSases and channels., TMEM16F is an enigmatic Ca2 + -activated phospholipid scramblase (CaPLSase) that passively transports phospholipids. Here authors identify an inner activation gate and its disruption profoundly alters TMEM16F phospholipid permeation.

Chromosome organization is crucial for genome function. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method–integrative modeling of genomic regions (IMGR)–to increase the genomic resolution of our traces to 10 kb., Author summary Questions regarding the impact of chromosome structure on genome function are focusing increasingly on the manner in which chromosomes are organized within the nucleus. In fact, studies of processes as diverse as gene activation and repression as well as genome repair and stability are all querying how the 3D organization of chromosomal DNA may be a major player. Here, we apply our strategy for tracing chromosomes at super-resolution, traversing over 8 megabases of human chromosome 19 while visualizing genomic features ranging in size from kilobases to megabases. This technology has enabled exploration of the physical nature of a genomic feature called the compartment; compartments are widely hypothesized to reflect the partitioning of genomes into relatively more and less active regions. Excitingly, we find that compartments are, indeed, physical structures, that they are sometimes distinct and other times entangled. We also find that the maternally-derived and the paternally-derived homologous regions can differ more than would be expected by chance. Finally, by integrating image data with information regarding the frequency with which genomic segments contact each other, we produce a 3D model of how the 8 megabases we have imaged may be organized within a single nucleus, achieving 10 kb genomic resolution.

Chromatin loops enable transcription factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors, such as active forms of Notch, can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3-dimensional (3D) organization of the cancer genome is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B-cell lymphoma, we show that far beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes through establishing new long-range regulatory interactions. Moreover, a large fraction of Notch-promoted regulatory loops forms highly interacting enhancer and promoter spatial clusters, termed “3D cliques”. Loss-and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

Significance FISH enables researchers to visualize the subcellular distribution of RNA and DNA molecules in individual cells. The recent development of FISH methods employing probes composed of synthetic DNA oligonucleotides (oligos) allows researchers to tightly control aspects of probe design such as binding energy and genomic specificity. Although oligo FISH probes are central to many recently developed massively multiplexed and superresolution imaging methods, no dedicated computational utility exists to facilitate the design of such probes on the genome-wide scale. Here, we introduce a streamlined pipeline for the rapid, genome-scale design of oligo FISH probes and validate our approach by using conventional and superresolution imaging. Our method provides a framework with which to design oligo-based hybridization experiments., Oligonucleotide (oligo)-based FISH has emerged as an important tool for the study of chromosome organization and gene expression and has been empowered by the commercial availability of highly complex pools of oligos. However, a dedicated bioinformatic design utility has yet to be created specifically for the purpose of identifying optimal oligo FISH probe sequences on the genome-wide scale. Here, we introduce OligoMiner, a rapid and robust computational pipeline for the genome-scale design of oligo FISH probes that affords the scientist exact control over the parameters of each probe. Our streamlined method uses standard bioinformatic file formats, allowing users to seamlessly integrate new and existing utilities into the pipeline as desired, and introduces a method for evaluating the specificity of each probe molecule that connects simulated hybridization energetics to rapidly generated sequence alignments using supervised machine learning. We demonstrate the scalability of our approach by performing genome-scale probe discovery in numerous model organism genomes and showcase the performance of the resulting probes with diffraction-limited and single-molecule superresolution imaging of chromosomal and RNA targets. We anticipate that this pipeline will make the FISH probe design process much more accessible and will more broadly facilitate the design of pools of hybridization probes for a variety of applications.

BackgroundMid-upper-arm circumference (MUAC) and calf circumference (CC) are correlated with body mass index (BMI) in adults and may be useful for screening women with underweight (BMI < 18.5 kg/m2). However, there is no consensus on appropriate MUAC and CC cutoff points in diverse populations, especially in women of reproductive age.ObjectiveTo assess the accuracy of different MUAC and CC cutoff points to screen for underweight and to identify the most appropriate cutoff points in a sample of women of reproductive age from rural northern Vietnam.MethodsAnthropometric measurements (weight, height, MUAC, CC, and triceps and subscapular skinfold thicknesses) were obtained for 4,981 women of reproductive age who participated in a micronutrient intervention trial (PRECONCEPT) in Thái Nguyên Province, Viet- nam. Receiver operating characteristic (ROC) analysis was used to evaluate different cutoff values of MUAC and CC and identify the most appropriate cutoff values to predict underweight.ResultsThe overall prevalence of underweight was 32%. The MUAC value of 23.5 cm and the CC value of 31 cm were identified as the best cutoffs based on low misclassification (16% for MUAC and 21% for CC) and good balance of sensitivity (89% and 85%, respectively) and specificity (71% and 67%, respectively. The ROC curves were similar across different ethnic groups, with the area under the curve (AUC) values reaching 0.89 to 0.93 for MUAC and 0.83 to 0.89 for CC.ConclusionsMUAC and CC perform adequately in screening for underweight in women. The utility of these measurements in predicting functional outcomes should be examined.

DNA double-strand breaks (DSBs) are most often repaired by two pathways in mammalian cells, homologous recombination or non-homologous end joining. Biochemical and genetic studies showed that DSBs can also be joined via microhomology-mediated end joining (MHEJ), which is always mutagenic and may result in diseases, such as cancer. In this study we established a human cell-based reporter system to determine the prevalence of MHEJ events and factors that modulate MHEJ. A nonfunctional puromycin acetyltransferase (Pac) gene, disrupted by an insertion flanked by two microhomologous repeats, was integrated into chromosomes of human HT1080 cells. Repair of DSBs via MHEJ using the repeats resulted in deletion of the insertion and restoration of the Pac gene function, thus rendering the cells puromycin resistant. Our results showed that MHEJ spontaneously occurs at the reporter locus (loci), manifested by formation of puromycin resistant (puro(r)) colonies after culturing reporter cells in medium containing puromycin. The frequency of puro(r) cells can be greatly increased by site-directed DSB inside the insertion. Our results also demonstrated that the frequency of puro(r) cells is affected by the length of the repeat and by the size of the intervening sequence. Thus, this cell-based assay provides a platform for evaluating factors modulating in vivo MHEJ.

To examine ethnic differences in optic nerve head and retinal nerve fibre layer (RNFL) parameters between European Caucasian and East Asian children aged 6-12 years.Of 4118 children examined in the Sydney Childhood Eye Study (incorporating the Sydney Myopia Study) from 34 randomly selected primary and 21 secondary schools during 2003-5, 3382 (82.1%) had optical coherence tomography (OCT; Zeiss Stratus) data suitable for analysis. 'Fast' optic disc and RNFL scans were used. Ethnicity was defined only when both parents were of the same ethnicity.East Asian children tended to have a lower birth weight, were shorter with a smaller body mass index and were less hyperopic than European Caucasian children of the same age. After adjusting for age, gender, axial length, birth weight and optic-disc area, East Asian children had similar mean vertical disc diameters to European Caucasians (p=0.38, p=0.64 for 6-12 years, respectively) but 30-43% larger mean vertical cup diameters (p0.0001 for both), resulting in larger mean cup/disc ratios (p0.0001 for both). Compared with European Caucasians (101.95 microm and 104.57 microm, respectively), East Asian children had thicker mean average RNFL (105.45 microm and 107.92 microm, respectively; p=0.0006 and 0.0001) and thicker non-nasal RNFL quadrants in both ages.Compared with European Caucasian children, East Asian children generally had thicker RNFL and larger mean cup/disc ratios. Given the relatively lower prevalence of open angle glaucoma in Asians, these anatomical variations could contribute to better understanding of apparent racial differences in glaucoma susceptibility.

To compare the measurements of optic nerve head parameters from digital photographic images and optical coherence tomography (OCT) in normal children.The Sydney Childhood Eye Study assessed 1765 children aged 6 years from 34 randomly selected primary schools during 2003 to 2005. Optic nerve head parameters were measured from digital photographs captured using a Canon fundus camera (CF-60Uvi)/EOS 10D and OCT3 (Zeiss Stratus) using the "fast" optic disc protocol. Retinal images of 333 sequential child participants were graded using both methods and are included in analyses. Optic disc and cup area, vertical and horizontal disc and cup diameters, vertical and horizontal cup/disc diameter ratios, and cup/disc area ratios were calculated using both modalities. Magnification of the planimetric images was corrected using the Bengtsson formula.Mean vertical and horizontal disc and cup diameter and mean disc and cup area, as measured using OCT (1.76, 1.50, 0.71, and 0.68 mm and 2.15 and 0.47 mm, respectively) were significantly (P0.0001; cup area P=0.0014) smaller than when measured using digital photography (1.85, 1.66, 0.76, and 0.74 mm and 2.40 and 0.51 mm, respectively). All 3 cup/disc ratio measures did not vary significantly (P0.05) between the 2 methods (0.41, 0.45, and 0.22 vs. 0.41, 0.44, and 0.21, respectively).Linear and area measures by Stratus-OCT, compared with digital planimetry measurements, are around 10% smaller; however, all 3 cup/disc ratios were preserved. Where OCT produces unexpectedly small cup/disc ratios, manual viewing is advisable. However, OCT can be considered moderately reliable in measuring and monitoring cup/disc ratio in clinical settings.

DNA nonhomologous end-joining (NHEJ) and homologous recombination are two distinct pathways of DNA double-strand break repair in mammalian cells. Biochemical and genetic studies showed that DNA ends can also be joined via microhomology-mediated end joining (MHEJ), especially when proteins responsible for NHEJ, such as Ku, are reduced or absent. While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we used a cell-free assay to determine the roles of DNA ligases I, III and IV in MHEJ and NHEJ. We found that siRNA mediated down-regulation of DNA ligase I or ligase III in human HTD114 cells led to impaired end joining that was mediated by 2-, 3- or 10-bp microhomology. In addition, nuclear extract from human fibroblasts harboring a mutation in DNA ligase I displayed reduced MHEJ activity. Furthermore, treatment of HTD114 nuclear extracts with an antibody against DNA ligase I or III also significantly reduced MHEJ. These data indicate that DNA ligases I and III are required in MHEJ. DNA ligase IV, on the contrary, is not required in MHEJ but facilitates Ku-dependent NHEJ. Therefore, MHEJ and NHEJ require different DNA ligases.

To examine the prevalence of refractive error and distribution of ocular biometric parameters among major ethnic groups in a population-based sample of 11-15-year-old Australian children.The Sydney Myopia Study examined 2353 students (75.3% response) from a random cluster-sample of 21 secondary schools across Sydney. Examinations included cycloplegic autorefraction, and measures of corneal radius of curvature, anterior chamber depth, and axial length.Participants mean age was 12.7 years (range 11.1-14.4); 49.4% were female. Overall, 60.0% of children had European Caucasian ethnicity, 15.0% East Asian, 7.1% Middle Eastern, and 5.5% South Asian. The most frequent refractive error was mild hyperopia (59.4%, 95% confidence interval (CI), 53.2-65.6), defined as spherical equivalent (SE) +0.50 to +1.99 D. Myopia (SE-0.50 D or less) was found in 11.9%, 95% (CI 6.6-17.2), and moderate hyperopia (SEor =+2.00 D) in 3.5%, 95% (CI 2.8-4.1). Myopia prevalence was lower among European Caucasian children (4.6%, 95% CI 3.1-6.1) and Middle Eastern children (6.1%, 95% CI 1.3-11.0) than among East Asian (39.5%, 95%, CI 25.6-53.5) and South Asian (31.5%, 95%, CI 21.6-41.4) children. European Caucasian children had the most hyperopic mean SE (+0.82 D) and shortest mean axial length (23.23 mm). East Asian children had the most myopic mean SE (-0.69 D) and greatest mean axial length (23.86 mm).The overall myopia prevalence in this sample was lower than in recent similar-aged European Caucasian population samples. East Asian children in our sample had both a higher prevalence of myopia and longer mean axial length.

Maternal nutritional status before and during early pregnancy plays a critical role in fetal growth and development. The benefits of periconception folic acid (FA) supplementation in the prevention of neural tube defects is well recognized, but the evidence for preconception micronutrient interventions for improving pregnancy outcomes is limited.This study aimed to evaluate whether preconception supplementation with weekly iron and folic acid (IFA) or multiple micronutrients (MMs) improves birth outcomes compared with FA alone.We recruited 5011 women of reproductive age in a double-blind, randomized controlled trial in Vietnam and provided weekly supplements containing either 2800 μg FA, 60 mg Fe and 2800 μg FA (IFA), or the same amount of FA and iron plus other MMs until they conceived (n = 1813). All pregnant women received daily IFA through delivery, and were followed up for birth outcomes, including birth weight, gestational age, preterm delivery and small for gestational age (SGA). Group comparisons were done with the use of ANOVA or chi-square tests for both intention-to-treat (n = 1599) and per-protocol analyses (women consumed supplements ≥26 wk before conception; n = 824). Effect modification by baseline underweight or anemia status was tested with the use of generalized linear models.The mean age of the women was 26 y, 30% were underweight, and10% were nulliparous. The groups were similar for most baseline characteristics. The mean ± SD duration of the preconception intervention was 33 ± 25 wk and compliance was high (90%). Infants born to the 3 groups of women did not differ (P ≥ 0.05) on mean ± SD birth weight (3076.8 ± 444.5 g) or gestational age (39.2 ± 2.0 wk), or prevalence of SGA (12%), low birth weight (5%) and preterm delivery (10%). There were no significant differences in women who consumed supplements ≥26 wk before conception or by baseline underweight or anemia.Weekly supplementation with MMs or IFA before conception did not affect birth outcomes compared with FA in rural Vietnamese women. The trial was registered at clinicaltrials.gov as NCT01665378.

PURPOSE The purpose of this study was to evaluate and compare the Complete Ophthalmic Analysis System (COAS) G200 Aberrometer (Wavefront Sciences Inc., Albuquerque, NM) and Canon RK-F1 Autorefractor (Canon Inc., Tokyo, Japan) for measuring refractive errors in young children. METHODS The Sydney Myopia Study is a population-based study of refractive error and eye health in young Australian children. Cycloplegic refractions were performed on 1504 school year 1 students (mostly 6 years old) and 890 school year 7 (mostly 12 years old) students using both the COAS G200 Aberrometer and Canon RK-F1 autorefractor. Refractive data were analyzed using power vectors. Mean differences and 95% limits of agreement were determined for refractive components between the two instruments. RESULTS The mean age +/- standard deviation was 6.7 +/- 0.4 years (range, 5.5-9.1 years) and 12.6 +/- 0.5 years (range, 11.1-14.4 years) for the year 1 and year 7 students, respectively. Mean paired differences for the M component (spherical equivalent) between the COAS G200 and Canon RK-F1 were

To study the distribution of retinal nerve fiber layer (RNFL) thickness by ocular and demographic variables in a population-based study of young children.Population-based cross-sectional study.One thousand seven hundred sixty-five of 2238 (78.9%) eligible 6-year-old children participated in the Sydney Childhood Eye Study between 2003 and 2004. Mean age was 6.7 years (50.9% boys).Detailed examination included cycloplegic autorefraction and measurement of axial length. Retinal nerve fiber layer scans using an optical coherence tomographer were performed with a circular scan pattern of 3.4-mm diameter. Multivariate analyses were performed to examine the distribution of RNFL parameters with gender, ethnicity, axial length, and refraction.Peripapillary RNFL thickness and RNFL(estimated integral) (RNFL(EI)), which measures the total cross-sectional area of ganglion cell axons converging onto the optic nerve head.Peripapillary RNFL thickness and RNFL(EI) were normally distributed. The mean+/-standard deviation RNFL average thickness was 103.7+/-11.4 microm and RNFL(EI) was 1.05+/-0.12 mm2. Retinal nerve fiber layer thickness was least for the temporal quadrant (75.7+/-14.7 microm), followed by the nasal (81.7+/-19.6 microm), inferior (127.8+/-20.5 microm), and superior (129.5+/-20.6 microm) quadrants. Multivariate adjusted RNFL average thickness was marginally greater in boys than in girls (104.7 microm vs. 103.2 microm; P = 0.007) and in East Asian than in white children (107.7 microm vs. 102.7 microm; P0.0001). The RNFL was thinner with greater axial length (P(trend)0.0001) and less positive spherical equivalent refractions (P(trend) = 0.004).Retinal nerve fiber layer average thickness and RNFL(EI) followed a normal distribution. Retinal nerve fiber layer thickness varied marginally with gender, but differences were more marked between white and East Asian children. Retinal nerve fiber layer thinning was associated with increasing axial length and less positive refractions.

To assess the effects of refraction and axial length on optical coherence tomography (OCT) measures of childhood optic disk parameters.Population-based cross-sectional study.Of 4,118 children examined in the Sydney Myopia Study (Sydney Childhood Eye Study) from 34 randomly selected primary schools and 21 secondary schools from 2003 through 2005, 3,529 (85.7%) were included in the analysis (1,395 6-year-old children [year 1 students] and 2,134 12-year-old children [year 7 students]). Comprehensive standardized eye examinations included best-corrected visual acuity, cycloplegic autorefraction, biometry measurements, and fast optic disk scans using OCT.After adjusting for magnification, the mean optic disk area was positively associated with axial length (P(trend).0001, both age groups) but was not associated consistently with spherical equivalent refraction (SER).Optic disk parameters in childhood are influenced by axial length, but not by refractive error itself.

To examine symmetry of macular, peripapillary nerve fiber layer (NFL), and optic disk measurements in healthy children.Cross-sectional study.We examined a population-based sample of six-year-old children (n = 1,765) in the Sydney Childhood Eye Study. Optical coherence tomography (OCT) scan data for right and left eyes were compared.High interocular correlations (0.8) were found for foveal minimum thickness and cup-to-disk ratios. Average NFL thickness was moderately correlated (0.7). Other macular, NFL, and optic disk parameters showed negligible or small mean interocular differences. In 95% of children, interocular difference in macular thickness was22 microm for foveal minimum and40 microm for other areas, and 16 to 17 microm for average NFL thickness. Cup-to-disk ratio was highly symmetric, varying by0.25 in 95% of children.Interocular asymmetry of retinal/optic disk parameters should be interpreted in the context of other clinical measures because of the potential for large degrees of asymmetry among individuals.

Purpose To assess the accuracy of the Lang II stereotest in screening for strabismus, amblyopia, and anisometropia in 6-year-old children. Design Cross-sectional population-based study. Methods The Sydney Myopia Study examined 1765 6-year-old children (78.9% of eligible) who were identified by random cluster sampling of 34 schools in Sydney, Australia. Sensitivity and specificity of the Lang II stereotest was determined by best stereoacuity. Cycloplegic autorefraction, assessment of visual acuity, and ocular motility were conducted. Results Test sensitivity ranged from 21.4% for anisometropia (≥1.0 diopter) to 31.3% for amblyopia. The detection rate for new cases of amblyopia ranged from 20% to 40%; the detection rate for new cases of strabismus was 30%. Specificity was >98% in all three conditions. Children with false-negative results included newly diagnosed cases of strabismus (14 of 25 children) or amblyopia (5 of 12 children). Conclusion The Lang II stereotest, when used alone, has very limited value as a screening test of binocular dysfunction.

The study aims were to explore the relationships between perceived barriers to research use and the implementation of evidence-based practice among hospital nurses and to investigate the barriers as predictors of implementation of evidence-based practice.Evidence-based practice is critical in improving healthcare quality. Although barriers to research use have been extensively studied, little is known about the relationships between the barriers and the implementation of evidence-based practice in nursing. Cross-sectional study.Data were collected between December 2006-January 2007 for this cross-sectional study using computerised Evidence-Based Practice Questionnaire and BARRIERS surveys. A convenience sample (n=1301) of nurses from four hospitals in southern California, USA, participated. Hierarchical multiple regression analyses were performed for each of the three dependent variables: practice, attitude and knowledge/skills associated with evidence-based practice. BARRIERS subscales were used as predictor variables.The perceived barriers to research use predicted only 2·7, 2·4 and 4·5% of practice, attitude and knowledge/skills associated with evidence-based practice. Conclusions. It was unexpected that the barriers to research use predicted such small fractions of practice, attitude and knowledge/skills associated with evidence-based practice. The barriers appear to have minimal influence over the implementation of evidence-based practice for most hospital nurses.In implementing evidence-based practice, the focus on barriers to research use among general nursing staff may be misplaced. Further studies are needed to identify the predictors of evidence-based practice and to identify the subset of nurses who are most amenable to adopting evidence-based practice.

PURPOSE. To examine the influence of different signal strengths on measurements made with optical coherence tomography (OCT) of macular, optic nerve head, and retinal nerve fiber layer (RNFL) parameters. METHODS. From 2003 to 2005, 2092 children, mostly aged 12 years, were examined, and macular, optic nerve head, and RNFL parameters were measured by OCT. Multiple fast scans were acquired, and only right eyes were included in the analyses. Signal strength category was determined after averaging individual signal strengths from each scan and classifying scans as providing moderate (average signal strength, 5‐7.49), good (average signal strength, 7.5‐9.49), and excellent (average signal strength, 9.5) image quality. General linear models were used after adjustment for covariates. RESULTS. Significant differences were observed between measurements obtained at excellent signal strengths compared with those obtained at moderate and good signal strengths for both macular and optic nerve parameters. However, although statistically significant, the magnitude of the differences in macular parameters was very small (5 m, or a 2% difference). Differences in optic nerve head parameters were much greater (up to a 32% difference), with larger measurements recorded for most parameters with increasing signal strength category. Significant differences in RNFL parameters with increasing signal strength were not demonstrated. CONCLUSIONS. Significantly larger macular and optic nerve head OCT measurements were obtained with increasing signal strength measurements, although absolute differences in macular measurements were small and are of questionable clinical importance. The results support the robustness of OCT in providing precise macular imaging. (Invest Ophthalmol Vis Sci. 2010;51:4471‐4475) DOI:10.1167/iovs.09-3892

To examine macular and peripapillary retinal nerve fiber layer (RNFL) thickness in amblyopia.Population-based cross-sectional study.Of 4118 children examined in the Sydney Childhood Eye Study (incorporating the Sydney Myopia Study) from 34 randomly selected primary schools and 21 secondary schools from 2003 to 2005, 3529 (85.7%) were included in this analysis. The median age of the 2 samples was 6 years (n = 1395) and 12 years (n = 2134), respectively.A detailed eye examination was conducted on all children, including determination of best-corrected visual acuity (logarithm of the minimum angle of resolution [logMAR]), autorefraction (RK-F1 autorefractor, Canon, Tokyo, Japan) after cyclopentolate (1%), cover testing to identify strabismus, and optical coherence tomography (StratusOCT, Carl Zeiss Meditec, Dublin, CA) through dilated pupils to obtain macula and peripapillary RNFL thickness. Amblyopia was defined as best visual acuity0.3 logMAR units not explained by any obvious underlying eye or visual pathway abnormalities. Anisometropia was defined as an interocular difference of at least 1.0 diopter of the spherical equivalent refraction.Macular and peripapillary RNFL thickness.Amblyopic eyes had slightly greater foveal minimum thickness than the normal fellow eye (by 5.0 microm; 95% confidence interval 0.1-9.9) and right eyes of non-amblyopic children (by approximately 10 microm), both P0.05. This was more pronounced in 6-year-old children (6.9 microm) than 12-year-old children (4.2 microm). Amblyopic eyes also had slightly thicker central macula (1 mm diameter region) in both comparisons, although these differences were not statistically significant. The inner macular ring (outer radius 1.5 mm) was thinner in amblyopic than normal fellow eyes. Peripapillary RNFL thickness was not significantly different between amblyopic and normal fellow eyes or normal eyes of non-amblyopic children.In children aged predominantly 6 and 12 years, central macular thickness may be increased in eyes with amblyopia, although it is uncertain if this precedes or follows the development of amblyopia. No differences in peripapillary RNFL thickness were found when compared with normal eyes.The author(s) have no proprietary or commercial interest in any materials discussed in this article.

To assess the relationship of birth weight, birth length, and head circumference as proxy markers of intrauterine growth, cup/disc ratio, and other optic disc parameters measured using optical coherence tomography (OCT).Population-based cross sectional analysis.The Sydney Childhood Eye Study examined 2353 primarily 12-year-old children from 21 randomly selected secondary schools during 2003 to 2005.Of 2353 children examined, 2134 (90.7%) had OCT scans (Zeiss Stratus OCT, Carl Zeiss Meditec, Dublin, CA) and are included in this study. The "fast" optic disc scan protocol was used. Birth weight, birth length, and head circumference were ascertained from health records. Height and weight were measured using standardized protocols, body mass index (BMI) was defined as weight (kilograms)/ height squared (meters), and sociodemographic information was collected in a questionnaire completed by parents. Low birth weight was defined as birth weightor=2499 g, and prematurity was defined as gestation less than 37 weeks. Logistic and mixed model analyses were performed.Vertical optic disc and optic cup diameters, and cup/disc ratio.Children of low birth weight had decreased vertical disc diameter, increased cup diameter, and increased cup/disc ratio by 30 microm (P = 0.009), 44 microm (P = 0.004), and 0.03 (P0.0001), respectively. After adjusting for age, gender, ethnicity, height, axial length, and BMI, birth weight remained positively associated with vertical optic disc diameter and inversely associated with both vertical optic cup diameter and vertical cup/disc ratio (each kilogram increase was associated with a 0.0133-mm larger mean disc diameter; P = 0.04; a 0.0203-mm smaller mean cup diameter; P = 0.02; and a 0.0136 reduction in mean cup/disc ratio; P = 0.002). These associations were not present in children with gestational age less than 33 weeks. Smaller birth length and head circumference were similarly associated with larger cup/disc ratio.Low birth weight, short birth length, and small head circumference at birth were associated with larger cup/disc ratio in children aged 12 years. Our findings suggest that fetal growth restriction could adversely influence optic nerve head parameters. This may have implications for future risk of glaucomatous optic neuropathy.The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Purpose To determine whether differences exist in morphological structures of dominant and nondominant eyes in children ages 6 or 12 years. The following structural features were assessed: axial length, refraction, corneal radius of curvature, and retinal features—measured with the use of optical coherence tomography (OCT)—including macular and retinal nerve fiber layer thickness and optic disk parameters. Methods Of 4,118 children examined from 2003 to 2005 in the Sydney Myopia Study, 3,382 (82.1%) had OCT data for analysis. Comprehensive standardized eye examinations were performed, including best-corrected visual acuity. Ocular dominance was determined using the hole-in-card test, repeated on 3 occasions. Dominance was assigned only if the 3 measurements were identical. “Fast” scans of the optic disk and retina were performed using OCT. Results Dominance was observed in 85% of subjects, with boys having a tendency toward right eye dominance. The dominant eye was longer ( p = 0.001 and p = 0.0003 for 6- and 12-year-old patients, respectively) and more myopic ( p = 0.002 and p = 0.0001, respectively) than its counterpart. No retinal features consistently identified dominant from nondominant eyes in both age groups. Conclusions Although dominant eyes tended to be longer and slightly more myopic than nondominant eyes, we found no consistent ocular structural differences between dominant and nondominant eyes with the use of OCT.

Objective To assess the relationship of near, midworking distance, and outdoor activities with prevalence of myopia in school-aged children. Design Cross-sectional study of 2 age samples from 51 Sydney schools, selected using a random cluster design. Participants One thousand seven hundred sixty-five 6-year-olds (year 1) and 2367 12-year-olds (year 7) participated in the Sydney Myopia Study from 2003 to 2005. Methods Children had a comprehensive eye examination, including cycloplegic refraction. Parents and children completed detailed questionnaires on activity. Main Outcome Measures Myopia prevalence and mean spherical equivalent (SE) in relation to patterns of near, midworking distance, and outdoor activities. Myopia was defined as SE refraction ≤−0.5 diopters (D). Results Higher levels of outdoor activity (sport and leisure activities) were associated with more hyperopic refractions and lower myopia prevalence in the 12-year-old students. Students who combined high levels of near work with low levels of outdoor activity had the least hyperopic mean refraction (+0.27 D; 95% confidence interval [CI], 0.02–0.52), whereas students who combined low levels of near work with high levels of outdoor activity had the most hyperopic mean refraction (+0.56 D; 95% CI, 0.38–0.75). Significant protective associations with increased outdoor activity were seen for the lowest ( P = 0.04) and middle ( P = 0.02) tertiles of near-work activity. The lowest odds ratios for myopia, after adjusting for confounders, were found in groups reporting the highest levels of outdoor activity. There were no associations between indoor sport and myopia. No consistent associations between refraction and measures of activity were seen in the 6-year-old sample. Conclusions Higher levels of total time spent outdoors, rather than sport per se, were associated with less myopia and a more hyperopic mean refraction, after adjusting for near work, parental myopia, and ethnicity.

Purpose To study the distributions of refractive (RA), corneal (CA), and internal astigmatism (IA) in 12-year-old Australian children and to explore differences from previous findings in 6-year-old children. Methods Eligible year 7 students (2353/3144 [75.3%], median age, 12 years) from a random cluster sample of 21 high schools in Sydney, Australia, were examined by keratometry, cycloplegic autorefraction, and review of questionnaire data. Results Prevalence rates of RA, CA, and IA > or =1.0 D in right eyes were 6.7% (95% confidence interval [CI], 5.0-8.4), 26.6% (CI, 22.1-31.1), and 26.5% (CI, 22.9-30.0), respectively. RA was predominantly with-the-rule (WTR; 40.4%, CI, 32.6 to 48.2) and against-the-rule (ATR; 43.6%, CI, 35.7-51.5), CA was WTR (88.8%, CI, 86.3-91.3), and IA was ATR (90.2%, CI, 87.8-92.6). The girls had significantly greater CA and IA prevalence, with greater ATR astigmatism and lower oblique IA than did the boys. The European white-Australian children had lower CA prevalence than did the East Asian-Australian children and higher IA prevalence than did the South Asian-Australian children. Ethnic differences in RA prevalence were not significant, when adjusted for confounders. RA was more frequently ATR in European white than in other ethnic groups. Compensation between CA and IA reduced the magnitude of RA. Comparison with the data on 6-year-old children revealed minimal differences for all astigmatic components. Conclusions There was a relatively low prevalence of RA, due to compensation between CA and IA. The minimal differences in all components of astigmatism between the two age cohorts suggest that astigmatism is stable between ages 6 and 12 years, although this conclusion needs to be confirmed in longitudinal studies.

PURPOSE To study the distribution of optic disc, cup, and neural rim size by ocular and demographic variables in a population-based sample of 6-year-old children. METHODS The Sydney Childhood Eye Study examined 1765 of 2238 eligible 6-year-old children (78.9%) from 34 randomly selected Sydney schools during 2003 to 2004. Comprehensive standardized eye examination included cycloplegic autorefraction, optical biometry and "fast optic disc" scans performed using optical coherence tomography. RESULTS Scans of adequate quality were available for 1309 children (75% of participants), with 70% aged 6 years; 50.9% were boys. Mean (+/- SD) horizontal and vertical disc diameter and disc area was 1.53 +/- 0.21 mm, 1.79 +/- 0.28 mm, and 2.20 +/- 0.39 mm(2), respectively. Corresponding cup dimensions were 0.70 +/- 0.28 mm, 0.73 +/- 0.27 mm, and 0.48 +/- 0.32 mm(2). A definable optic cup was absent in 7.4%, 87% of whom were European white. Cup-to-disc diameter ratios were 0.46 +/- 0.16 horizontally and 0.42 +/- 0.15 vertically, whereas cup-to-disc area ratio was 0.22 +/- 0.13. Mean +/- SD neural rim area was 1.76 +/- 0.44 mm(2) and increased with disc size (Pearson correlation = 0.68, P < 0.0001). Horizontal and vertical average nerve widths were 0.36 +/- 0.05 and 0.28 +/- 0.05 mm, respectively. In analyses adjusting for potential confounders, disc area increased significantly with axial length (P(trend) < 0.0001) and refraction (P(trend) = 0.02). Rim area increased only with axial length (P(trend) = 0.01). There were no gender differences, except for average nerve width, marginally greater in boys. Most disc and cup dimensions were significantly larger in East-Asian than European white and Middle Eastern children. CONCLUSIONS Disc, cup, and neural rim parameters were generally normally distributed in this young population. Axial length appeared to be a stronger determinant of disc and rim size than refraction. Some ethnic but not gender differences were demonstrated for most parameters.

To determine the reproducibility of optical coherence tomography (OCT) measurements of macular thickness, peripapillary nerve fiber layer (NFL) thickness, and optic disk parameters and to investigate the effect of axial length and refractive error on these measurements in children with healthy eyes.Cross-sectional study.The Sydney Childhood Eye Study examined 2,353 year 7 students (75.3% response) from a random cluster sample of 21 secondary schools across Sydney. A consecutive subsample of 120 children had OCT (StratusOCT, Carl Zeiss, Dublin, California, USA) performed by a single operator, which was repeated with a brief rest between the two sessions. Scans of the NFL, macula, and optic disk were performed.Intersubject variability of measurements of macular thickness, NFL thickness, and optic disk parameters assessed using intraclass correlation coefficients accounted for85%,62%, and38% of total variability of measurements, respectively. Corresponding coefficients of variability were5%,8%, and13%. Magnification effects attributable to axial length and refractive error on the measurement of these parameters were statistically not significant.The StratusOCT demonstrated reproducible measurements of macular and NFL thickness. Measurement of most optic disk parameters were also reproducible. Magnification attributable to axial length and refractive error had minimal impact on measurements of macular and NFL thickness.

To report the distribution of stereoacuity thresholds and ocular characteristics associated with reduced stereoacuity in a representative sample of 12-year-old Australian children.Stereoacuity thresholds were determined using the three quantitative plates of the TNO test in 2343 children, either unaided or with spectacles, if worn. Logarithm of minimum angle of resolution (logMAR) visual acuity was measured. Cycloplegic autorefraction (using cyclopentolate), cover testing, and dilated fundus examination were performed. Reduced stereoacuity was defined as120 arcsec. Myopia was defined as spherical equivalent refraction (SER)or = -0.50 D hyperopia as spherical equivalent refractionor = +2.0 D, anisometropia as spherical equivalent refraction difference between eyesor =1.00 D, and astigmatism as cylinderor = 1.0 D.Stereoacuity was based on unaided visual acuity in 1975 children (84.3%) and on spectacle-corrected visual acuity in 368 children (15.7%); 87 children (3.7%) had reduced stereoacuity. Amblyopia was the most common identifiable cause, accounting for 32%, followed by strabismus (15%) and anisometropia (14%). Presence of anisometropia was significantly associated with reduced stereoacuity; 78.6% of anisometropic children achieved normal stereoacuity versus 98.9% without anisometropia (p0.0001).Reduced stereoacuity was relatively uncommon in a population of 12-year-old Australian children. Its functional and psychosocial impact on individuals and on the whole population remains uncertain.

To evaluate the repeatability and comparability of keratometry measured by both the IOLMaster and RK-F1 AutoRef-Keratometer in children.Keratometry results from a sample (n = 447) of 6-year-old children who were examined in the Sydney Myopia Study were analyzed. Corneal power was analyzed along the flattest and steepest meridians to determine if there were any systematic differences between repeat measurements or between the two instruments. The 95% limits of repeatability (LR) and 95% limits of agreement (LA) (mean difference +/- 1.96 x standard deviation of differences) were calculated.There were no systematic differences in repeat measurements for each instrument. For the IOLMaster, mean difference of the flattest corneal meridian was -0.01 (D) (P = 0.3, 95% LR, -0.22, 0.21 D) and of the steepest corneal meridian, 0.01 D (P = 0.3, 95% LR, -0.35, 0.38 D). For the RK-F1, mean difference of the flattest corneal meridian was -0.02 D (P = 0.3, 95% LR, -0.25, 0.21 D); and of the steepest corneal meridian, 0.00 D (P = 0.9, 95% LR, -0.39, 0.39 D). Systematic differences, however, were found between the two instruments. The IOLMaster gave significantly (P0.0001) steeper readings than the RK-F1 for both the flattest corneal meridian, 0.29 D (95% LA, -0.08, 0.66 D), and the steepest corneal meridian, 0.18 D (95% LA, -0.29, 0.65 D).Keratometry was highly repeatable for both the IOLMaster and RK-F1 instruments when used in young children. These instruments would be suitable for use in monitoring changes of corneal curvature over time. Small significant systematic differences in keratometry between the two instruments were also found.

To study the distribution of anisometropia and aniso-astigmatism in young Australian children, together with clinical and ocular biometry relations.The Sydney Myopia Study examined 1765 predominantly 6 year old children from 34 randomly selected Sydney schools during 2003-4. Keratometry, cycloplegic autorefraction, and questionnaire data were collected.Spherical equivalent (SE) anisometropia (or =1 dioptre) prevalence was 1.6% (95% confidence interval (CI) 1.1% to 2.4%). Aniso-astigmatism (or =1D) prevalence was 1.0% (CI: 0.6% to 1.6%). Both conditions were significantly more prevalent among moderately hyperopic (SEor =2.0D) than mildly hyperopic (SE 0.5-1.9D) children. Myopic children (SEor =-0.5D) had higher anisometropia prevalence. Neither condition varied by age, sex, or ethnicity. In multivariate analyses, anisometropia was significantly associated with amblyopia, odds ratio (OR) 29, (CI: 8.7 to 99), exotropia (OR 7.7, CI: 1.2 to 50), and neonatal intensive care unit (NICU) admission (OR 3.6, CI: 1.1 to 12.6). Aniso-astigmatism was significantly associated with amblyopia (OR 8.2, CI: 1.4 to 47), maternal age35 years (OR 4.0, CI: 1.3 to 11.9), and NICU admission (OR 4.6, CI: 1.2 to 17.2). Anisometropia resulted from relatively large interocular differences in axial length (p0.0001) and anterior chamber depth (p = 0.0009). Aniso-astigmatism resulted from differences in corneal astigmatism (p0.0001).In this predominantly 6 year old population, anisometropia and aniso-astigmatism were uncommon, had important birth and biometry associations, and were strongly related to amblyopia and strabismus.

To document the prevalence of correctable and non-correctable visual impairment in a representative sample of Australian children, predominantly age 12 years.Population-based cross-sectional study.Logarithm of the minimum angle of resolution (logMAR) visual acuity was measured in both eyes unaided, with spectacles if worn, and after subjective refraction if required, in 2353 children, examined during 2004 to 2005. Cycloplegic autorefraction (using cyclopentolate) and dilated fundus examination were performed. Using a cut-off of 0.3 logMAR units (20/40), presenting visual impairment was defined using unaided visual acuity if spectacles were not worn or with usual correction if spectacles were worn. Impairment not eliminated by refraction was considered non-correctable; any difference between this and presenting impairment was defined as correctable impairment. Myopia was defined as spherical equivalent refraction (SER)or =-0.50 diopters (D), hyperopia as SERor =+2.0 diopters, anisometropia as SER differenceor =1.00 diopters, and astigmatism as cylinderor =1.0 diopters. Amblyopia was defined as corrected visual acuity0.3 logMAR not attributable to an underlying structural eye or visual pathway abnormality.Visual impairment was found in the worse eye of 117 children (5.0%) and comprised correctable (82%) and non-correctable impairment (18%). Correctable impairment was due to myopia in 67 (69.8%), hyperopia in 11 (11.5%) and astigmatism in 32 subjects (33.3%). Causes of non-correctable impairment were: amblyopia 66.7%, congenital glaucoma 9.5%, optic nerve hypoplasia 9.5%, congenital nystagmus 4.8%, and cortical blindness 4.8%.Visual impairment had a relatively low prevalence in this older childhood population, a large proportion of which was correctable by refraction alone.

PURPOSE: The purpose of the present study was to report the prevalence of refractive (RA), corneal (CA), and internal astigmatism (IA) in a population of 6-year-old children; examine their variation with gender, ethnicity, and refraction; and examine the effects of gender, ethnicity, and spherical equivalent refraction on the relationship between CA and RA in this population. METHODS: The Sydney Myopia Study is a population-based survey of refraction and eye health in 6-year-old children. A random cluster design was used to recruit children from schools across Sydney, Australia, during 2003 to 2004. Data collection used a detailed questionnaire and comprehensive eye examination. Keratometric and cycloplegic autorefraction data from right eyes were analyzed. RESULTS: Of 2238 eligible children, 1765 (78.9%; 50.7% boys) had parental consent to participate. Overall prevalence of RA (> or =1.0 diopter [D]) was 4.8% (95% confidence interval [CI] 3.8%-6.1%), CA (> or =1.0 D) 27.7% (CI 23.8%-32.3%), and IA (> or =1.0 D) 21.1% (CI 19.0%-23.5%). The RA axis was fairly evenly distributed, with predominance of oblique axis (39.1%; CI 35.9%-42.6%). CA axis was mainly with the rule (75.1%; CI 72.6%-77.8%), while IA axis was mainly against the rule (76.7%; CI 74.2%-79.3%). After adjustment for multiple variables, girls had significant, marginally greater mean CA and IA than boys. East Asian and South Asian children had significantly greater prevalence and mean RA and CA than European Caucasian children. There were no significant ethnic differences of mean IA. Compared to reference (spherical equivalent [SEq] 1.01-1.50 D), mean RA and CA increased significantly with more hyperopic and more myopic refractions. Mean IA was significantly greater only for hyperopic refractions (SEq > 2.00 D). CONCLUSIONS: The prevalence of astigmatism found in this population of 6-year-old children was relatively low, and showed significant variation with ethnicity. The data suggest that emmetropization for RA occurs by a compensatory process between CA and IA.

To examine influences of gestational age and birth parameters on peripapillary nerve fiber layer (NFL) and macular thickness at age six.Cross-sectional study.The Sydney Childhood Eye Study examined a random-cluster sample of 1765 six-year-old Sydney school children. Peripapillary NFL and macular thickness were measured (StratusOCT, Zeiss, Dublin, California, USA). Birth parameters were extracted from health records. Multivariate analyses were performed.Higher birth weight children had greater peripapillary NFL, inner and outer (all P(trend)0.03), but not central or average macular thickness (both P(trend)0.1). Peripapillary NFL (P(trend) = 0.001), inner (P(trend) = 0.01), outer (P(trend) = 0.002), and average macular thickness (P(trend) = 0.02), but not central macular thickness (P(trend) = 0.5) was greater in children with larger head circumference at birth. The central macula was thicker in prematurely (37 weeks) born children (195.0 microm) than those born at term (191.2 microm), P = 0.04.Birth weight and head circumference predicted peripapillary NFL and macular thickness. Prematurity was weakly associated with central macular thickness.