Anthropometry of deep-set eyes with respect to difficulty in docking during femtosecond laser procedures.

Purpose: To identify the facial anthropometric parameters that predict the difficulty during femtosecond (FS) laser. Methods: This was a: single-center observational study was conducted on participants between the ages 18 and 30 years who were planned for FS-LASIK (femtosecond laser-assisted laser in situ keratomileusis) or SMILE (small incision lenticule extraction) at Dr. Rajendra Prasad Centre for Ophthalmic Sciences, AIIMS, New Delhi, India. The front and side-facing images of the participants were analyzed using Image J software to measure different anthropometric parameters. The nasal bridge index, facial convexity, and other parameters were measured. The difficulty faced by the surgeon during docking was recorded for each subject. The data were analyzed on Stata 14. Results: A total of 97 subjects were included. The mean age was 24 (±7) years. Twenty-three (23.71%) subjects were females while the rest were males. Difficulty in docking was seen in 1 (4.34%) female and 14 (19%) males. The mean nasal bridge index was 92.58 (±4.01) in subjects with deep-set eyes and 89.72 (±4.30) in normal subjects. The mean total facial convexity was 129.28 (±4.24) in deep-set eyes, and 140.23 (±4.74) in normal subjects. Conclusion: Total facial convexity appeared as the most important feature, with the value being less than 133° in most subjects with unfavorable facial anthropometry.

The expanding family of T follicular regulatory cells.

The coevolution of multiple specialized T follicular regulatory cell subsets has led to fine-tuning of human germinal center responses in providing optimal antibody production and preventing events leading to autoimmunity (see the related Research Article by Le Coz et al.).

Adult-onset Coats disease.

Coats disease is an idiopathic retinal vasculopathy characterized by telangiectasia and aneurysm of retinal vessels along with intra and subretinal exudation and fluid. While Coats disease is classically described in young male population, there is an adult variant of Coats disease presenting in adulthood. Adult onset Coats disease have a similar presentation but a slower progression, localised lipid deposition, both peripheral and juxta-macular involvement. In this review article, we have attempted to describe in detail the characteristic clinical features, pathogenesis, investigation modalities and treatment in adult-onset Coats disease.

Altered and allele-specific open chromatin landscape reveals epigenetic and genetic regulators of innate immunity in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes severe COVID-19 in some patients and mild COVID-19 in others. Dysfunctional innate immune responses have been identified to contribute to COVID-19 severity, but the key regulators are still unknown. Here, we present an integrative single-cell multi-omics analysis of peripheral blood mononuclear cells from hospitalized and convalescent COVID-19 patients. In classical monocytes, we identified genes that were potentially regulated by differential chromatin accessibility. Then, sub-clustering and motif-enrichment analyses revealed disease condition-specific regulation by transcription factors and their targets, including an interaction between C/EBPs and a long-noncoding RNA LUCAT1, which we validated through loss-of-function experiments. Finally, we investigated genetic risk variants that exhibit allele-specific open chromatin (ASoC) in COVID-19 patients and identified a SNP rs6800484-C, which is associated with lower expression of CCR2 and may contribute to higher viral loads and higher risk of COVID-19 hospitalization. Altogether, our study highlights the diverse genetic and epigenetic regulators that contribute to COVID-19.

Technique of re-enclavation of post-traumatic disenclaved haptic of posterior iris claw lens in a vitrectomised eye.

A 58-year-old woman presented to us with sudden onset diminution of vision for 10 days following trauma while using mobile phone. Patient had a history of posterior iris claw implantation 3 years ago. On examination, the patient was aphakic and intraocular lens (IOL) was seen enclaved on nasal side and disenclaved on temporal side on ultrasound biomicroscopy. Patient underwent surgery for re-enclavation of temporal haptic by lifting the IOL using 23-gauge pars plana trocar. Patient had a postoperative uncorrected visual acuity of 6/9 and best-corrected visual acuity of 6/6 with refraction. Re-enclavation of partially disenclaved posterior iris claw lens is a minimally invasive technique to restore visual acuity in such cases.

Developmental bifurcation of human T follicular regulatory cells.

Germinal centers (GCs) are anatomic structures where B cells undergo affinity maturation, leading to production of high-affinity antibodies. The balance between T follicular helper (TFH) and regulatory (TFR) cells is critical for adequate control of GC responses. The study of human TFH and TFR cell development has been hampered because of the lack of in vitro assays reproducing in vivo biology, along with difficult access to healthy human lymphoid tissues. We used a single-cell transcriptomics approach to study the maturation of TFH and TFR cells isolated from human blood, iliac lymph nodes (LNs), and tonsils. As independent tissues have distinct proportions of follicular T cells in different maturation states, we leveraged the heterogeneity to reconstruct the maturation trajectory for human TFH and TFR cells. We found that the dominant maturation of TFR cells follows a bifurcated trajectory from precursor Treg cells, with one arm of the bifurcation leading to blood TFR cells and the other leading to the most mature GC TFR cells. Overall, our data provide a comprehensive resource for the transcriptomics of different follicular T cell populations and their dynamic relationship across different tissues.

Myopia, Melatonin and Conjunctival Ultraviolet Autofluorescence: A Comparative Cross-sectional Study in Indian Myopes.

Purpose: To explore the role of outdoor light exposure by estimating ocular sun exposure measured by Conjunctival Ultraviolet Autofluorescence (CUVAF) imaging and serum melatonin levels in myopes and non-myopes.Materials & Methods: Age and sex matched emmetropes and myopes (60 each) aged 10-25 years participated. Those with a history of ocular surgery or any ocular or systemic co-morbidity were excluded. Socio-demographic parameters, sun exposure questionnaires, indoor and outdoor activity profile, morning serum melatonin levels, sleep pattern, degree of myopia, ocular biometry and area of CUVAF on ultraviolet photography were noted and analyzed.Results: Mean age of myopes (18 ± 4.5 years) and emmetropes (18.5 ± 4 years) was similar (P = .523). Serum melatonin levels were significantly higher (P = .001) among myopes (89.45 pg/ml) as compared to emmetropes (52.83 pg/ml). Lifetime sun exposure was significantly lower in myopes than emmetropes (P = .0003). Area of CUVAF was inversely related to degree of myopia (P < .0001). Day time sleepiness was greater in myopes (51.7%) than emmetropes (15%) (P < .0001). There was a positive correlation between serum melatonin levels and axial length among myopes (correlation coefficient = 0.27; P = .03). Age and gender had no association with serum melatonin levels.Conclusion: This study demonstrates an inverse relationship between serum melatonin levels and degree of CUVAF in myopes. A novel link between serum melatonin, axial length and outdoor sun exposure is highlighted in the current study.

Retinoblastoma in an adult: a diagnostic dilemma.

We report a case of a man aged 35 years who presented with the chief complaint of painless diminution of vision in the right eye for 4 months. Examination revealed a large inferior retinochoroidal mass along with retinal detachment. An anterior choroidal mass with moderate internal reflectivity was seen on B-scan ocular ultrasonography and MRI and CT scan were indicative of a mitotic aetiology. Fluorodeoxyglucose-positron emission tomography scan ruled out any other systemic foci of involvement. Based on the above findings, a provisional diagnosis of amelanotic choroidal melanoma was made and he was taken up for choroidal aspiration biopsy, wherein the cytopathology report revealed hypercellularity with no identifiable pigments. In view of the above, a diagnosticandtherapeutic enucleation was performed; however, the histopathology report of the enucleated specimen revealed poorly differentiated retinoblastoma. This case highlights that the diagnosis of retinoblastoma should be kept in mind even in adult patients.

The Relationship Between Body Mass Index in Children and Insurance Type, Parental Eating Concern, Asthma, and Allergies.

INTRODUCTION: Trust for America's Health reported rising levels of obesity contributed to increased disease rates and health care costs (Levi et al., 2013). Factors associated with overweight and obesity rates include lower socioeconomic status, public insurance, and increased chronic disease rates. METHODS: Body mass index percentile, health insurance, parental eating concern, asthma, and allergy information were evaluated from a dataset of 870 de-identified health assessment records. RESULTS: Among overweight/obese children, we found significant differences in children insured by Medicaid (26%) versus commercial insurance (15.9%), children with asthma (22.1%) versus children without asthma (14.8%), and children with known allergies (7.8%) versus children without known allergies (16.7%). The difference between children with parental eating concerns (3.7%) and children without (18.1%) was nearly significant. DISCUSSION: The associations depicted can assist pediatric providers in recognizing risk factors for overweight/obesity among their patients. Combating obesity in childhood can improve health outcomes.

Molecular Markers Distinguishing T Cell Subtypes With TSDR Strand-Bias Methylation.

Human regulatory CD4+CD25+FOXP3+ T cells (Treg) play important roles in the maintenance of self-tolerance and immune homeostasis in various disease settings and are also involved in the suppression of effective immune responses. These cells are heterogeneous in phenotype and function, and the ability to reliably distinguish between various FOXP3-expressing subpopulations can affect the development of successful therapies. This study demonstrates that hypomethylated CpG sites, present in four regions of the FOXP3 locus, CAMTA1 and FUT7 gene regions, can be used to distinguish several subsets of Treg from conventional CD4+ T lymphocytes (Tcon) in donors of both genders. We describe a previously unreported strand-bias hemimethylation pattern in FOXP3 promoter and TSDR in donors of both genders, with the coding strand being demethylated within promoter and methylated within TSDR in all CD4+ lymphocyte subtypes, whereas the template strand follows the previously described pattern of methylation with both regions being more demethylated in Treg subtypes and mostly methylated in Tcon. This strand-specific approach within the TSDR may prove to be instrumental in correctly defining Treg subsets in health and in disease.

Regulation of the Germinal Center Response.

The germinal center (GC) is a specialized microstructure that forms in secondary lymphoid tissues, producing long-lived antibody secreting plasma cells and memory B cells, which can provide protection against reinfection. Within the GC, B cells undergo somatic mutation of the genes encoding their B cell receptors which, following successful selection, can lead to the emergence of B cell clones that bind antigen with high affinity. However, this mutation process can also be dangerous, as it can create autoreactive clones that can cause autoimmunity. Because of this, regulation of GC reactions is critical to ensure high affinity antibody production and to enforce self-tolerance by avoiding emergence of autoreactive B cell clones. A productive GC response requires the collaboration of multiple cell types. The stromal cell network orchestrates GC cell dynamics by controlling antigen delivery and cell trafficking. T follicular helper (Tfh) cells provide specialized help to GC B cells through cognate T-B cell interactions while Foxp3+ T follicular regulatory (Tfr) cells are key mediators of GC regulation. However, regulation of GC responses is not a simple outcome of Tfh/Tfr balance, but also involves the contribution of other cell types to modulate the GC microenvironment and to avoid autoimmunity. Thus, the regulation of the GC is complex, and occurs at multiple levels. In this review we outline recent developments in the biology of cell subsets involved in the regulation of GC reactions, in both secondary lymphoid tissues, and Peyer's patches (PPs). We discuss the mechanisms which enable the generation of potent protective humoral immunity whilst GC-derived autoimmunity is avoided.

An N-Ethyl-N-Nitrosourea (ENU) Mutagenized Mouse Model for Autosomal Dominant Nonsyndromic Kyphoscoliosis Due to Vertebral Fusion.

Kyphosis and scoliosis are common spinal disorders that occur as part of complex syndromes or as nonsyndromic, idiopathic diseases. Familial and twin studies implicate genetic involvement, although the causative genes for idiopathic kyphoscoliosis remain to be identified. To facilitate these studies, we investigated progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and assessed them for morphological and radiographic abnormalities. This identified a mouse with kyphoscoliosis due to fused lumbar vertebrae, which was inherited as an autosomal dominant trait; the phenotype was designated as hereditary vertebral fusion (HVF) and the locus as Hvf. Micro-computed tomography (µCT) analysis confirmed the occurrence of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae in HVF mice, consistent with a pattern of blocked vertebrae due to failure of segmentation. µCT scans also showed the lumbar vertebral column of HVF mice to have generalized disc narrowing, displacement with compression of the neural spine, and distorted transverse processes. Histology of lumbar vertebrae revealed HVF mice to have irregularly shaped vertebral bodies and displacement of intervertebral discs and ossification centers. Genetic mapping using a panel of single nucleotide polymorphic (SNP) loci arranged in chromosome sets and DNA samples from 23 HVF (eight males and 15 females) mice, localized Hvf to chromosome 4A3 and within a 5-megabase (Mb) region containing nine protein coding genes, two processed transcripts, three microRNAs, five small nuclear RNAs, three large intergenic noncoding RNAs, and 24 pseudogenes. However, genome sequence analysis in this interval did not identify any abnormalities in the coding exons, or exon-intron boundaries of any of these genes. Thus, our studies have established a mouse model for a monogenic form of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae, and further identification of the underlying genetic defect will help elucidate the molecular mechanisms involved in kyphoscoliosis. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants.

BACKGROUND: Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method. RESULTS: We generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations. We observed that the use of lssDNA molecules as donors efficiently yielded founders bearing the conditional allele, with seven out of nine projects giving rise to modified alleles. However, rearranged alleles including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated by this method. Specifically, we found that alleles containing unexpected point mutations were found in three of the nine projects analyzed. Alleles originating from illegitimate repairs or partial integration of the donor were detected in eight projects. Furthermore, additional integrations of donor molecules were identified in four out of the seven projects analyzed by copy counting. This highlighted the requirement for a thorough allele validation by polymerase chain reaction, sequencing and copy counting of the mice generated through this method. We also demonstrated the feasibility of using lssDNA donors to generate thus far problematic point mutations distant from active CRISPR cutting sites by targeting two distinct genes (Gckr and Rims1). We propose a strategy to perform extensive quality control and validation of both types of mouse models generated using lssDNA donors. CONCLUSION: lssDNA donors reproducibly generate conditional alleles and can be used to introduce point mutations away from CRISPR/Cas9 cutting sites in mice. However, our work demonstrates that thorough quality control of new models is essential prior to reliably experimenting with mice generated by this method. These advances in genome editing techniques shift the challenge of mutagenesis from generation to the validation of new mutant models.

A genetic modifier suggests that endurance exercise exacerbates Huntington's disease.

Polyglutamine expansions in the huntingtin gene cause Huntington's disease (HD). Huntingtin is ubiquitously expressed, leading to pathological alterations also in peripheral organs. Variations in the length of the polyglutamine tract explain up to 70% of the age-at-onset variance, with the rest of the variance attributed to genetic and environmental modifiers. To identify novel disease modifiers, we performed an unbiased mutagenesis screen on an HD mouse model, identifying a mutation in the skeletal muscle voltage-gated sodium channel (Scn4a, termed 'draggen' mutation) as a novel disease enhancer. Double mutant mice (HD; Scn4aDgn/+) had decreased survival, weight loss and muscle atrophy. Expression patterns show that the main tissue affected is skeletal muscle. Intriguingly, muscles from HD; Scn4aDgn/+ mice showed adaptive changes similar to those found in endurance exercise, including AMPK activation, fibre type switching and upregulation of mitochondrial biogenesis. Therefore, we evaluated the effects of endurance training on HD mice. Crucially, this training regime also led to detrimental effects on HD mice. Overall, these results reveal a novel role for skeletal muscle in modulating systemic HD pathogenesis, suggesting that some forms of physical exercise could be deleterious in neurodegeneration.

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair.

BACKGROUND: Nuclease-based technologies have been developed that enable targeting of specific DNA sequences directly in the zygote. These approaches provide an opportunity to modify the genomes of inbred mice, and allow the removal of strain-specific mutations that confound phenotypic assessment. One such mutation is the Cdh23 (ahl) allele, present in several commonly used inbred mouse strains, which predisposes to age-related progressive hearing loss. RESULTS: We have used targeted CRISPR/Cas9-mediated homology directed repair (HDR) to correct the Cdh23 (ahl) allele directly in C57BL/6NTac zygotes. Employing offset-nicking Cas9 (D10A) nickase with paired RNA guides and a single-stranded oligonucleotide donor template we show that allele repair was successfully achieved. To investigate potential Cas9-mediated 'off-target' mutations in our corrected mouse, we undertook whole-genome sequencing and assessed the 'off-target' sites predicted for the guide RNAs (≤4 nucleotide mis-matches). No induced sequence changes were identified at any of these sites. Correction of the progressive hearing loss phenotype was demonstrated using auditory-evoked brainstem response testing of mice at 24 and 36 weeks of age, and rescue of the progressive loss of sensory hair cell stereocilia bundles was confirmed using scanning electron microscopy of dissected cochleae from 36-week-old mice. CONCLUSIONS: CRISPR/Cas9-mediated HDR has been successfully utilised to efficiently correct the Cdh23 (ahl) allele in C57BL/6NTac mice, and rescue the associated auditory phenotype. The corrected mice described in this report will allow age-related auditory phenotyping studies to be undertaken using C57BL/6NTac-derived models, such as those generated by the International Mouse Phenotyping Consortium (IMPC) programme.

Current strategies for mutation detection in phenotype-driven screens utilising next generation sequencing.

Mutagenesis-based screens in mice are a powerful discovery platform to identify novel genes or gene functions associated with disease phenotypes. An N-ethyl-N-nitrosourea (ENU) mutagenesis screen induces single nucleotide variants randomly in the mouse genome. Subsequent phenotyping of mutant and wildtype mice enables the identification of mutated pathways resulting in phenotypes associated with a particular ENU lesion. This unbiased approach to gene discovery conducts the phenotyping with no prior knowledge of the functional mutations. Before the advent of affordable next generation sequencing (NGS), ENU variant identification was a limiting step in gene characterization, akin to 'finding a needle in a haystack'. The emergence of a reliable reference genome alongside advances in NGS has propelled ENU mutation discovery from an arduous, time-consuming exercise to an effective and rapid form of mutation discovery. This has permitted large mouse facilities worldwide to use ENU for novel mutation discovery in a high-throughput manner, helping to accelerate basic science at the mechanistic level. Here, we describe three different strategies used to identify ENU variants from NGS data and some of the subsequent steps for mutation characterisation.

A novel SOD1-ALS mutation separates central and peripheral effects of mutant SOD1 toxicity.

Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1(D83G/D83G) mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1(D83G/D83G) mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1(-/-)). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.