Thiol-linked alkylation of RNA to assess expression dynamics.

Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (s4U) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM seq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase-II-dependent transcriptional output scaled with Oct4/Sox2/Nanog-defined enhancer activity, and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective and scalable manner.

SNPhood: investigate, quantify and visualise the epigenomic neighbourhood of SNPs using NGS data.

MOTIVATION: The vast majority of the many thousands of disease-associated single nucleotide polymorphisms (SNPs) lie in the non-coding part of the genome. They are likely to affect regulatory elements, such as enhancers and promoters, rather than the function of a protein. To understand the molecular mechanisms underlying genetic diseases, it is therefore increasingly important to study the effect of a SNP on nearby molecular traits such as chromatin or transcription factor binding. RESULTS: We developed SNPhood, a user-friendly Bioconductor R package to investigate, quantify and visualise the local epigenetic neighbourhood of a set of SNPs in terms of chromatin marks or TF binding sites using data from NGS experiments. AVAILABILITY AND IMPLEMENTATION: SNPhood is publicly available and maintained as an R Bioconductor package at http://bioconductor.org/packages/SNPhood/ CONTACT: judith.zaugg@embl.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SLAMseq resolves the kinetics of maternal and zygotic gene expression during early zebrafish embryogenesis.

The maternal-to-zygotic transition (MZT) is a key developmental process in metazoan embryos that involves the activation of zygotic transcription (ZGA) and degradation of maternal transcripts. We employed metabolic mRNA sequencing (SLAMseq) to deconvolute the compound embryonic transcriptome in zebrafish. While mitochondrial zygotic transcripts prevail prior to MZT, we uncover the spurious transcription of hundreds of short and intron-poor genes as early as the 2-cell stage. Upon ZGA, most zygotic transcripts originate from thousands of maternal-zygotic (MZ) genes that are transcribed at rates comparable to those of hundreds of purely zygotic genes and replenish maternal mRNAs at distinct timescales. Rapid replacement of MZ transcripts involves transcript decay features unrelated to major maternal degradation pathways and promotes de novo synthesis of the core gene expression machinery by increasing poly(A)-tail length and translation efficiency. SLAMseq hence provides insights into the timescales, molecular features, and regulation of MZT during zebrafish embryogenesis.

Multimodal imaging of an acute presentation of ocular histoplasmosis syndrome in an immunocompetent patient.

Purpose: Presumed ocular histoplasmosis syndrome (POHS) is a posterior segment disorder that is usually subclinical unless choroidal neovascular membrane (CNVM) develops. It is thought to be the sequela of a prior systemic infection with Histoplasma capsulatum, and evidence supporting this association is based on epidemiologic, animal, and few enucleation studies. Acute presentation of chorioretinal involvement during an initial histoplasmosis systemic infection in immunocompetent patients is rarely reported, presumably due to the usual lack of or minimal symptoms of both the systemic and ocular disease. We report on an immunocompetent male with choroidal lesions detected during disseminated histoplasmosis infection and characterize the lesions using multimodal imaging. Observations: A 17-year-old male presented when routine optometry screening detected two deep, yellowish-white lesions in the left fundus. Optical coherence tomography (OCT) imaging confirmed a choroidal mass with extension through Bruch's membrane into the subretinal space and a small amount of subretinal fluid. Fluorescein angiography was suggestive of CNVM. There were no clinical findings of intraocular inflammation, and the patient was initially lost to follow-up. Eight weeks after last follow-up, the patient presented to the emergency department with fatigue, mild respiratory symptoms, and abdominal pain for the last month. Imaging revealed a mediastinal mass with hilar extension and innumerable nodules throughout the lung and spleen. Serum Histoplasma IgM/IgG were positive, and biopsy of the mediastinal mass revealed Histoplasma organisms. The patient was treated with antifungals and discharged. The patient underwent an extensive immunologic evaluation while admitted, which did not reveal an underlying immunodeficiency. On last follow-up, the choroidal lesions were smaller and more consolidated, and the subretinal fluid had resolved. Conclusions and Importance: We present a patient with choroidal lesions in the setting of disseminated systemic histoplasmosis infection and characterize a lesion using multimodal imaging. The presentation of acute chorioretinal lesions in the setting of biopsy proven systemic Histoplasma infection supports H. capsulatum as the etiology of POHS.

Publication bias, Open-Labeled Trials and Sky Rocketing Costs- Past, Present and Future of Repository Corticotropin Injection?

Repository corticotropin injection (RCI) has recently gained attention in the field of ocular inflammatory disease. Data supporting the use of RCI therapy in ocular inflammation are limited to case reports or small series subject to publication bias toward positive results. How this therapy differs significantly from oral corticosteroids, which are significantly cheaper, is unknown. Clinical trials to investigate the efficacy of RCI are currently limited to open-labeled non-comparative studies. Side effects of RCI are not insignificant, have been reported in other fields of medicine, and require further scrutiny. Finally, the price of RCI has skyrocketed with average yearly cost of therapy estimated to be between $480,000-$850,000 with allegations of the RCI manufacturing drug company providing remuneration to induce healthcare providers to prescribe RCIs but without any repercussions from a regulatory standpoint. The significant cost of RCI combined with lack of evidence-based guidance on efficacy, safety, and indications for use in ocular inflammation warrant caution in utilizing this therapy.

Dexamethasone Intravitreal Implant for the Treatment of Macular Edema and Uveitis: A Comprehensive Narrative Review.

Purpose: The purpose of this review article is to provide a comprehensive review of the current applications of intravitreal DEX implant (Ozurdex®, Allergan Inc, Irvine, CA) for a variety of ophthalmic conditions - ranging from FDA approved indications to off-label uses. We have attempted to provide relevant evidence from the literature to help a reader develop an understanding of the biological and pharmacokinetic properties of DEX implant, its uses, and potential side effects. Methods: PubMed searches were performed using the terms "Ozurdex", or "intravitreal DEX implant", AND "retinal vein occlusion", or "diabetic macular edema", or "uveitis". The search was performed in July of 2021, with an additional search in October 2021. All original English language articles were considered for this review. Results: DEX implant has evidence of efficacy in a variety of clinical situations including macular edema associated with retinal vein occlusion, diabetes, uveitis, and others. Safety concerns include cataract formation and progression, intraocular pressure elevation, complications related to intravitreal injection, and opportunistic infections secondary to steroid-induced immune suppression. Conclusion: DEX implant is a useful tool in the management of several retinal disorders. Further studies are needed for head-to-head comparison with other treatment modalities and to determine its precise place in clinical practice.

Patient Adherence to Immunosuppressive Therapy in Treatment of Chronic Inflammatory Eye Disease.

TITLE: Patient Adherence to Immunosuppressive Therapy for Chronic Inflammatory Eye Disease. PURPOSE: To investigate adherence rates to immunosuppressive therapy (IMT) for treatment of noninfectious inflammatory eye disease (IED), adherence and disease control, and factors associated with nonadherence. METHOD: Retrospective review of medical charts from 2015 to 2020 was conducted on patients with IED at 6 months, 1 and 2 years after initiation of IMT. RESULTS: Of 183 patients, adherence rates at 6 months and 1 year were 70% and 58% by 2 years. Eighty-two percent, 78%, and 65% of patients with disease quiescence were adherent at 6 months, 1 and 2 years, respectively. Adherent patients have 1.86 (95% CI 1.09, 3.20) times greater likelihood for disease control compared to nonadherent. Primary reason for nonadherence was patient self-discontinuation. No specific factors were associated with nonadherence. CONCLUSION: Patients on IMT for IED had steady adherence rates up to 1 year, with decreased adherence at 2 years. Adherence to IMT significantly correlates with disease quiescence.

Outcomes of Repository Corticotropin Gel for Ocular Sarcoidosis.

PURPOSE: To evaluate the utility and side effect profile of subcutaneous repository corticotropin gel (RCI) in ocular sarcoidosis. METHODS: Retrospective chart review. RESULTS: Among six identified patients on RCI therapy, 4 had uveitis, one had optic neuritis and one had uveitis and optic neuritis secondary to sarcoidosis. The average follow-up was 43.5 months. RCI therapy was continuous in 4 patients (average 7.7 months) and intermittent in 2 patients (24 and 12 months). Five of the 6 patients continued with local and/or systemic corticosteroids for ocular inflammation control while on RCI therapy.  Two-thirds of patients experienced adverse effects including hyperpigmentation, alopecia, and severe hypertension. RCI therapy was discontinued in 5 of the 6 patients due to continued inflammation and side/adverse effects (4 patients) and loss of follow-up (1 patient). CONCLUSION: In this small cohort, the majority of patients failed to achieve adequate steroid-sparing ocular inflammation control and experienced side effects while on RCI therapy. Additional studies are needed to elucidate the role of RCI in ocular inflammation.

Systematic refinement of gene annotations by parsing mRNA 3' end sequencing datasets.

Alternative cleavage and polyadenylation generates mRNA 3' isoforms in a cell type-specific manner. Due to finite available RNA sequencing data of organisms with vast cell type complexity, currently available gene annotation resources are incomplete, which poses significant challenges to the comprehensive interpretation and quantification of transcriptomes. In this chapter, we introduce 3'GAmES, a stand-alone computational pipeline for the identification and quantification of novel mRNA 3'end isoforms from 3'mRNA sequencing data. 3'GAmES expands available repositories and improves comprehensive gene-tag counting by cost-effective 3' mRNA sequencing, faithfully mirroring whole-transcriptome RNAseq measurements. By employing R and bash shell scripts (assembled in a Singularity container) 3'GAmES systematically augments cell type-specific 3' ends of RNA polymerase II transcripts and increases the sensitivity of quantitative gene expression profiling by 3' mRNA sequencing. Public access: https://github.com/AmeresLab/3-GAmES.git.

Paraneoplastic conjunctival cicatrization: two different pathogenic types.

PURPOSE: To describe the clinical and immunopathologic features of patients with 2 different types of paraneoplastic conjunctival cicatrization. DESIGN: Retrospective observational case analyses with a review of the literature. PARTICIPANTS: One patient with paraneoplastic ocular cicatricial pemphigoid (POCP) and 1 patient with paraneoplastic pemphigus (PNP) with ocular involvement. METHODS: Critical review of clinical history, diagnostic studies, and immunopathologic results of biopsies in the 2 cases, together with a review of the literature. MAIN OUTCOME MEASURES: Ability to recognize paraneoplastic conjunctival cicatrization and to diagnose the conditions accurately. RESULTS: The first patient, 46 years of age, presented with conjunctival scarring and symblephara, cough, oral lesions, and chest rash. Concurrently, a diagnosis of pulmonary squamous cell carcinoma was made. Conjunctival biopsy revealed a subepithelial bulla, an inflammatory infiltrate of T and B lymphocytes, and basement membrane zone deposition of immunoglobulin (Ig)-G and C3 consistent with POCP. The second patient, 54 years of age, had a recently diagnosed B-cell chronic lymphocytic leukemia, followed 1 month later with ocular irritation and bilateral extensive symblephara. Extensive oral lesions and skin involvement of the lower half of the body were seen. Skin biopsy disclosed subepidermal bullae and mostly T cells with virtually no B cells in the dermal infiltrate (the patient was being treated with rituximab). Linear subepithelial deposition of IgG and C3 and deposition within the epidermis were consistent with PNP. Further indirect immunofluorescence and immunoprecipitation studies with the patient's serum-derived antibodies established PNP as the definitive diagnosis. CONCLUSIONS: Underlying malignancy is an important consideration in younger patients with puzzling bilateral cicatrizing conjunctivitis, and a paraneoplastic condition can be established from either a conjunctival or a skin biopsy. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Palpebro-orbital apocrine cystadenoma: immunohistochemical verification of a unique variant with a critical differential diagnosis.

PURPOSE: To describe a unique apocrine cystadenoma of the superonasal eyelid and anterior orbit. METHODS: Clinical evaluation with axial and coronal CT; histopathologic and immunohistochemical studies including sections stained with hematoxylin-eosin, periodic acid Schiff, alcian blue, mucicarmine, and Prussian blue for iron; and monoclonal antibodies against cytokeratin-7, epithelial membrane antigen, smooth muscle actin for myoepithelial cells, gross cystic disease fluid protein-15 for apocrine differentiation, and CD-68 and lysozyme for histiocytes. RESULTS: The cyst possessed a multilaminar lining composed of polygonal to low cuboidal cells. A stalk of solid tumor ingrowth from the wall was composed of adenomatous units of eosinophilic cells with apical snouts ("decapitation secretion"). No goblet cells were discovered. Both the cyst's lining cells and the adenoma expressed gross cystic disease fluid protein-15 indicative of apocrine differentiation. The adenoma displayed inner adlumenal cells that were CK-7 and epithelial membrane antigen positive, and outer myoepithelial cells that were smooth muscle actin positive. Simple local excision was curative. CONCLUSION: This unique lesion is the first example in the ophthalmic and dermatopathologic literatures of a solid adenoma encompassed by an apocrine cyst, which more typically features short or blunt papillae. It must be distinguished from other eyelid and/or anterior orbital cystic lesions including eccrine hydrocystomas; classical and extratarsal dermoid cysts; congenital and acquired conjunctival cysts and dermoids; dacryocystocoeles/mucoceles; canaliculops and lacrimal gland dacryops; and congenital cystic odontogenic choristomas.

MDM2-positive atypical lipomatous neoplasm/well-differentiated liposarcoma versus spindle cell lipoma of the orbit.

PURPOSE: To distinguish, in a 36-year-old man, an atypical lipomatous neoplasm/well-differentiated liposarcoma from a spindle cell lipoma in a recurrent orbital tumor. METHODS: Clinical, radiographic, and histopathologic evaluations coupled with immunohistochemical studies for CD34, factor XIII, bcl-2, S-100, muscle-specific actin, Ki-67, and MDM2. RESULTS: MDM2 gene amplification was discovered in the CD34 tumor cells. An average Ki-67 proliferation index of 28% was ascertained for the original and recurrent lesions. These findings established the diagnosis of a well-differentiated liposarcoma with lipoma-like and spindle cell features and ruled out a spindle cell lipoma. CONCLUSION: Well-differentiated liposarcoma is a slow growing, infiltrative, and nonmetastasizing neoplasm that is microscopically and diagnostically challenging. It can be reliably separated from a benign spindle cell or an atypical lipoma by using the markers MDM2 and Ki-67.

Intravenous daclizumab for recalcitrant ocular inflammatory disease.

BACKGROUND: To evaluate outcomes of patients with recalcitrant ocular inflammation treated with intravenous daclizumab. METHODS: Retrospective case-series. Seventeen patients (33 eyes) with ocular inflammation who had failed several previous systemic immunomodulatory therapies were included in this study. Daclizumab was infused intravenously at a dose of 1 mg per kilogram every 2 weeks. The dose and intervals were adjusted according to control of inflammation off systemic and/or topical corticosteroid therapy. Control of ocular inflammation without corticosteroid therapy was the primary efficacy end point. RESULTS: The mean patient age at the commencement of daclizumab therapy was 34.8(range 8-64 years). Three patients were less than 16 years of age at the initiation of therapy. The duration of drug use was 23.6 +/- 15.7 months (range 8-68 months), and the time to control inflammation was 9.8 +/- 11.3 weeks, which was achieved in 15 patients (88.2%). Flare-up rate was 44 per 100 person-years follow-up. Fifteen of 33 eyes (45%) had an improvement in the visual acuity, 13 eyes (39.3%) had stable acuities, and five eyes (15%) had a worsening of their acuities. Two patients (14%) exhibited worsening of ocular inflammation, and were declared as treatment failures. Side effects associated with daclizumab included nausea, fatigue and muscle aches. CONCLUSION: Daclizumab is effective in controlling inflammation in patients with stubborn non-infectious ocular inflammation. It has a corticosteroid-sparing effect, can be used as long-term therapy in children and adults, and is capable of inducing durable remission.

Winer's dilated pore of the eyelid.

A 71-year-old woman had a whitish mass of the outer right lower eyelid with an eccentric pore plugged with keratin. Histopathologic evaluation disclosed an epidermoid cyst that opened widely to the surface and was filled with flaky keratin. The wall displayed a proliferation of rete ridge-type structures that projected in the surrounding dermis. The lesion was diagnosed as a dilated pore of Winer and has not recurred after simple excision.

Perioperative Management of Uveitic Cataracts.

Uveitis patients represent a unique subset of the population undergoing cataract surgery and pose several challenges that require special consideration and strategy. Maintenance of disease quiescence for at least three months prior to surgery maximizes postoperative outcomes, though these patients remain at increased risk for pseudophakic cystoid macular edema, which can be refractory to the traditional steroid treatments. In this review, we detail the pillars of preoperative optimization, intraoperative considerations, and postoperative management of uveitic cataracts, with special attention on the evidence surrounding prevention and treatment of refractory postoperative cystoid macular edema.

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis.

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.

Retinoschisis in Pars Planitis.

PURPOSE: To describe the course of retinoschisis in patients with pars planitis (PP). METHODS: Chart review of PP patients seen July 2012-September 2014 at a single institution. RESULTS: Included were 34 patients (68 eyes). Uveitis was bilateral in all cases. Thirteen eyes (19%) developed retinoschisis. In six patients (86%), the schisis was bilateral. The average follow-up of patients with schisis was 7 years; the average best-corrected visual acuity (BCVA) was 20/22 at last follow-up. Schisis was noted to develop or progress in patients with both active and inactive inflammation. Five eyes of five patients underwent vitrectomy; three for disease control, with scleral buckle to reduce residual traction. Two eyes required vitrectomy for retinal detachment with progressive schisis, despite inactive uveitis. Seven eyes remained stable without intervention. CONCLUSIONS: Retinoschisis is a common complication in patients with PP. It is typically bilateral, and may develop or progress, despite control of uveitis.

Changes in Scleral Architecture in Chronic Vogt-Koyanagi-Harada Disease.

PURPOSE: To describe scleral changes in chronic VKH. METHODS: Medical records of patients with chronic VKH were retrospectively reviewed. Change of scleral architecture was defined as progressive posterior bowing on OCT, axial length elongation, and/or increased myopia more than -1.0 D, not explicable by other etiologies. RESULTS: In total, 28 eyes (16 patients) with mean age of disease onset 32.5 ± 14.0 years were included in the study. Disease duration was 15.1 ± 10.2 years. Eight eyes (28.6%) showed progressive scleral architectural changes. Five eyes (18%) developed scleral changes on OCT, not seen on prior imaging (2-12 years earlier). One eye had posterior bowing on OCT with increased axial length, both eyes of a bilateral pseudophake developed increased myopia with increased axial length. Well-circumscribed chorioretinal atrophy within the arcade was associated with progressive scleral change. CONCLUSIONS: Progressive scleral change may develop as a late complication of VKH. The association with well-circumscribed chorioretinal atrophy suggests that chronic choroidal inflammation may be responsible.

CTCF-Mediated Chromatin Loops between Promoter and Gene Body Regulate Alternative Splicing across Individuals.

The CCCTC-binding factor (CTCF) is known to establish long-range DNA contacts that alter the three-dimensional architecture of chromatin, but how the presence of CTCF influences nearby gene expression is still poorly understood. Here, we analyze CTCF chromatin immunoprecipitation sequencing, RNA sequencing, and Hi-C data, together with genotypes from a healthy human cohort, and measure statistical associations between inter-individual variability in CTCF binding and alternative exon usage. We demonstrate that CTCF-mediated chromatin loops between promoters and intragenic regions are prevalent and that when exons are in physical proximity with their promoters, CTCF binding correlates with exon inclusion in spliced mRNA. Genome-wide, CTCF-bound exons are enriched for genes involved in signaling and cellular stress-response pathways. Structural analysis of three specific examples, checkpoint kinase 2 (CHK2), CDC-like kinase 3 (CLK3), and euchromatic histone-lysine N-methyltransferase (EHMT1), suggests that CTCF-mediated exon inclusion is likely to downregulate enzyme activity by disrupting annotated protein domains. In total, our study suggests that alternative exon usage is regulated by CTCF-dependent chromatin structure.