Mediator kinase inhibition suppresses hyperactive interferon signaling in Down syndrome.

Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A, and this occurred through suppression of IFN-responsive transcription factor activity. Moreover, we discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. Kinase inhibition altered splicing in pathway-specific ways and selectively affected IFN-responsive gene splicing in T21 cells. To further probe Mediator kinase function, we completed cytokine screens and untargeted metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways, including broad up-regulation of anti-inflammatory lipid mediators. Elevated levels of lipid mediators persisted at least 24hr after Mediator kinase inhibition, and many identified lipids serve as ligands for nuclear receptors (e.g. PPAR, LXR) or G-protein coupled receptors (GPCRs; e.g. FFAR4). Notably, ligand-dependent activation of these GPCRs or nuclear receptors will propagate anti-inflammatory signaling pathways and gene expression programs, and this mechanistic link suggests that metabolic changes caused by CDK8/CDK19 inhibition can durably and independently suppress pro-inflammatory IFN responses. Collectively, our results establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.

Endoscopic enucleation and clinicopathologic correlation of a small choroidal melanoma hiding massive extrascleral extension.

Purpose: To report the unusual case of a previously stable choroidal nevus, closely followed for over 15 years, which underwent malignant transformation into small choroidal melanoma with massive extrascleral extension. Observations: A 67-year-old Caucasian female was referred to the Stanford Ocular Oncology Service with concern for malignant transformation of a previously stable choroidal nevus in her left eye. Her funduscopic examination demonstrated a dome-shaped choroidal lesion with overlying associated lipofuscin and subretinal fluid, consistent with a diagnosis of small choroidal melanoma. By B-scan ultrasonography, the lesion measured 8.0 × 6.0 mm in base and 2.1 mm in thickness. B-scan ultrasonography also disclosed an associated retroscleral mass, which appeared contiguous with the intraocular melanoma and was confirmed on subsequent orbital magnetic resonance imaging. A decision was made to proceed with enucleation. Under direct endoscopic visualization, the globe and extrascleral mass were fully isolated, mobilized, and removed in toto. At 24 months post-enucleation, the patient remains disease-free without evidence of systemic metastasis or local recurrence. Conclusions/importance: This case describes a small choroidal melanoma hiding massive extrascleral extension, underscoring the value of B-scan ultrasonography. This case also describes the unique management of choroidal melanoma with extrascleral extension using endoscopic enucleation. Performing enucleation under direct endoscopic visualization ensures complete resection and prevents inadvertent transection of the extrascleral component.

Xrn2 substrate mapping identifies torpedo loading sites and extensive premature termination of RNA pol II transcription.

The exonuclease torpedo Xrn2 loads onto nascent RNA 5'-PO4 ends and chases down pol II to promote termination downstream from polyA sites. We report that Xrn2 is recruited to preinitiation complexes and "travels" to 3' ends of genes. Mapping of 5'-PO4 ends in nascent RNA identified Xrn2 loading sites stabilized by an active site mutant, Xrn2(D235A). Xrn2 loading sites are approximately two to 20 bases downstream from where CPSF73 cleaves at polyA sites and histone 3' ends. We propose that processing of all mRNA 3' ends comprises cleavage and limited 5'-3' trimming by CPSF73, followed by handoff to Xrn2. A similar handoff occurs at tRNA 3' ends, where cotranscriptional RNase Z cleavage generates novel Xrn2 substrates. Exonuclease-dead Xrn2 increased transcription in 3' flanking regions by inhibiting polyA site-dependent termination. Surprisingly, the mutant Xrn2 also rescued transcription in promoter-proximal regions to the same extent as in 3' flanking regions. eNET-seq revealed Xrn2-mediated degradation of sense and antisense nascent RNA within a few bases of the TSS, where 5'-PO4 ends may be generated by decapping or endonucleolytic cleavage. These results suggest that a major fraction of pol II complexes terminates prematurely close to the start site under normal conditions by an Xrn2-mediated torpedo mechanism.

Nucleotide-level linkage of transcriptional elongation and polyadenylation.

Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' untranslated regions. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et al., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters - from the last isoform of one cluster to the first isoform of the next - is much less pronounced, even over large distances. GC content in a region 13-30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at single nucleotide resolution within clusters but not between them. Pol II occupancy increases just downstream of poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that (1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, (2) poly(A) site clusters are linked to the local elongation rate, and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, (3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and (4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.

Evaluation of Interventions Targeting Follow-up Appointment Scheduling After Emergency Department Referral to Ophthalmology Clinics Using A3 Problem Solving.

Importance: Many patients seen for eye-related issues in the emergency department do not receive recommended follow-up care. Prior evidence supports that scheduling appointments is a barrier to accomplishing the transition to outpatient ophthalmology care. Objective: To evaluate time until appointment scheduling following emergency department discharge with urgent outpatient ophthalmology referral. Design, Setting, and Participants: The A3 problem solving process was implemented by a multidisciplinary team as part of a structured quality improvement program with the goal of reducing the mean time between urgent referral placement in the emergency department and outpatient ophthalmology appointment scheduling. The study was conducted at Stanford Health Care, an academic medical center in Palo Alto, California, affiliated with Stanford University School of Medicine. Using medical center administrative records, all patients discharged from the adult emergency department with an urgent outpatient referral to the Stanford Department of Ophthalmology from August 9 to September 19, 2020 (baseline; n = 43), and from October 26 to November 29, 2020 (after implementation of all interventions; n = 21), were included. Interventions: Interventions developed to target the workflow of the ophthalmology resident, emergency department, ophthalmology clinic, and health system schedulers to address key drivers of the referral-scheduling process included medical record documentation guidelines, identification of responsible parties, preidentified appointment slots, patient education materials, and education of stakeholders, and were implemented by October 25, 2020. Main Outcomes and Measures: Mean time between urgent referral placement (ie, emergency department discharge) and appointment scheduling with outpatient ophthalmology at baseline vs postintervention. Results: At baseline, appointments were scheduled a mean (range) 2.8 (0-7) days after referral placement. In the 5 weeks following implementation of interventions, the mean (range) decreased to 1.3 (0-4) days, a difference of 1.5 days (95% CI, 0.20-2.74; P = .02). This corresponds to 642 (95% CI, 86-1173) days of reduced patient wait time annually. In addition, there was less variability in the number of days between referral and appointment scheduling after intervention compared with baseline. Conclusions and Relevance: The results suggest improvement in efficiency of outpatient ophthalmology appointment scheduling of urgent emergency department referrals could be achieved through application of a quality improvement methodology by a multidisciplinary team representing key stakeholders in the process.

Alternative RNA structures formed during transcription depend on elongation rate and modify RNA processing.

Most RNA processing occurs co-transcriptionally. We interrogated nascent pol II transcripts by chemical and enzymatic probing and determined how the "nascent RNA structureome" relates to splicing, A-I editing and transcription speed. RNA folding within introns and steep structural transitions at splice sites are associated with efficient co-transcriptional splicing. A slow pol II mutant elicits extensive remodeling into more folded conformations with increased A-I editing. Introns that become more structured at their 3' splice sites get co-transcriptionally excised more efficiently. Slow pol II altered folding of intronic Alu elements where cryptic splicing and intron retention are stimulated, an outcome mimicked by UV, which decelerates transcription. Slow transcription also remodeled RNA folding around alternative exons in distinct ways that predict whether skipping or inclusion is favored, even though it occurs post-transcriptionally. Hence, co-transcriptional RNA folding modulates post-transcriptional alternative splicing. In summary, the plasticity of nascent transcripts has widespread effects on RNA processing.

Histopathologic Observations of Eyes in Exenterated Orbits After Neoadjuvant Intra-Arterial Cytoreductive Chemotherapy for Adenoid Cystic Carcinoma of the Lacrimal Gland.

PURPOSE: To assess whether exenteration specimens obtained after neoadjuvant intra-arterial cytoreductive chemotherapy (IACC) for adenoid cystic carcinoma of the lacrimal gland demonstrate significant ocular histopathologic alterations that might preclude future pursuit of globe-preserving therapy. METHODS: Retrospective histopathologic analysis of globes in IACC-treated exenteration specimens among the same cohort of patients whose survival outcomes have been reported. RESULTS: Twenty patients had specimens available. Nineteen globes revealed no abnormalities of the iris, ciliary body, lens, retinal pigment epithelium, choroid, or chorioretinal vasculature. Eighteen globes showed no optic nerve abnormalities. One globe from a patient who refused exenteration until adenoid cystic carcinoma recurrence supervened demonstrated optic nerve edema with a peripapillary hemorrhage and cotton wool spot, as well as hemorrhage and necrosis within an extraocular muscle. Eighteen globes showed no retinal abnormalities attributable to intra-arterial chemotherapy. Three globes showed incidental retinal findings: 2 globes contained 1 to 2 small peripheral retinal hemorrhages and 1 had a pigmented retinal hole. Seven demonstrated mild, chronic extraocular muscle inflammation, and 13 had unremarkable musculature. The single patient who received IACC via the internal carotid rather than the external carotid artery developed ophthalmic artery occlusion with orbital apex syndrome prior to exenteration, and diffuse necrosis and hemorrhage were evident histopathologically. CONCLUSIONS: Neoadjuvant IACC does not cause significant histopathologic damage to key ocular structures or compromise visual function in patients receiving intra-arterial chemotherapy through the external carotid artery. However, delivering chemotherapy through the internal carotid artery may result in visually significant thrombotic vascular events. The generally benign histopathological findings in these exenteration specimens support the concept of IACC delivery through the external carotid system as the cornerstone of a future globe-preserving strategy for lacrimal gland adenoid cystic carcinoma.

Metastasis of Lung Adenocarcinoma to the Lacrimal Sac.

The authors report an unusual case of lung adenocarcinoma metastasis to the lacrimal sac. A 61-year-old woman with stage IV non-small cell lung cancer presented with left facial pain and epiphora. She was found to have an elevated tear meniscus associated with a firm, fixed medial canthal mass. Orbital imaging demonstrated nodular enlargement of the lacrimal drainage apparatus. Biopsy of the lacrimal sac was performed, and it revealed a metastatic lung adenocarcinoma. The patient received targeted radiation therapy to the lacrimal sac, and her dose of maintenance chemotherapy was increased. The patient's symptoms have since improved. This case of lung cancer involving the lacrimal sac highlights the importance of thorough oncologic surveillance, even with respect to locations atypical for metastatic spread.

Orbital Bony Reconstruction With Presized and Precontoured Porous Polyethylene-Titanium Implants.

PURPOSE: Complex bony orbital defects are reconstructively challenging due to loss of intraoperative anatomical landmarks and adjacent support. Presized and precontoured porous polyethylene-titanium implants (Medpor Titan 3D Orbital Floor Implant) are designed to reestablish normal orbital floor and medial wall anatomy and are modeled after anatomically averaged orbits. This is the first study to report clinical outcomes with this implant. METHODS: This retrospective case series reviewed clinical data and outcomes for patients undergoing orbital reconstruction with a presized and precontoured porous polyethylene-titanium orbital implant from January 2016 to June 2018. RESULTS: A total of 34 orbits of 33 patients were identified (mean age: 43 ± 16 years, 70% men). Most bony defects were a result of trauma and included large orbital floor deformities (100%), medial wall defects (74%), disrupted inferomedial struts (68%), and broken posterior ledges (82%). Symptomatic diplopia (73%) and enophthalmos (89%, mean: 3.7 ± 2.1 mm) were common preoperatively. Many cases were revisions (44%). Mean follow up was 7.8 ± 6.7 months. All patients had improved globe positioning, enophthalmos, and hypoglobus. Seven patients had persistent postoperative diplopia: 6 responded to prism therapy and 1 required strabismus surgery. One patient required retrobulbar hematoma drainage and 1 patient required implant explantation due to chronic infection. CONCLUSIONS: Commercially available presized and precon toured porous polyethylene-titanium implants are useful for complex orbital bony defects and can achieve functional improve ments in diplopia, enophthalmos, and extraocular motility with a low incidence of postoperative complications or revisional surgery.

Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription.

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

Punctal agenesis and delayed-onset dacryocystocele in CHARGE syndrome.

Punctal agenesis and other nasolacrimal abnormalities have been infrequently reported in CHARGE syndrome-a constellation of findings affecting the eyes, heart, choana, and ears-which generally presents at birth. We present a rare case of punctal agenesis with delayed-onset dacryocystocele/lacrimal sac mucocele in a teenager with CHARGE syndrome.

Periorbital Subcutaneous Perfluoron Is Well Tolerated Following Intravitreal Surgery.

Purpose: Perfluorocarbon liquids (PFCLs) are well tolerated in intraocular surgery, but chronic exposure can cause inflammation. PFCL leakage into the orbit without significant sequelae has been reported, but PFCL leakage into the preseptal subcutaneous tissues has not been described. Methods: A case report is presented. Results: A 46-year-old man presented with hand motion vision from a ruptured globe and retained intraocular foreign body. Intraoperatively, the foreign body could not be removed, and PFCL extravasated from the posterior globe rupture. Postoperative imaging revealed hyperdense material in the orbit, lids, and superficial adnexal tissues. The patient tolerated the retained PFCL, and imaging 10 months later demonstrated interval resorption. The patient eventually developed ocular siderosis and underwent transconjunctival orbitotomy with foreign body extraction. Two years following the initial injury, his vision remained stable at 20/40 without further sequelae. Conclusions: PFCL is well tolerated in the extraocular space and may resorb with conservative management.

Gun-related eye injuries: A primer.

Gun-related eye injuries are relatively common in the context of gunshot wounds to the head and neck. Many of the fundamental principles of gunshot wound management apply to the care of these patients, but the complex anatomy and functional relationships of the periocular region do pose special challenges. We provide a focused primer for physicians seeking a more in-depth understanding of gun-related eye injuries and present 3 representative cases outlining the spectrum of pathology, provide a focused review of the relevant ballistics concepts, and discuss the management of injuries to the periocular soft tissues, orbital structures, and globe. We found that good cosmetic and functional results can often be achieved with appropriate early intervention, but visual prognosis may remain guarded despite optimal treatment.

Control of RNA Pol II Speed by PNUTS-PP1 and Spt5 Dephosphorylation Facilitates Termination by a "Sitting Duck Torpedo" Mechanism.

Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA polymerase II (Pol II) elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, Pol II decelerates from >2 kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a model for termination, the "sitting duck torpedo" mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert Pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homolog NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.

Orbital cholesterol granuloma: A report and discussion of orbital findings.

PURPOSE: To report a case of orbital cholesterol granuloma and discuss the orbital findings seen in this entity. OBSERVATION: A 38-year-old male presented with an 8-month history of progressive left upper lid ptosis and hypoglobus. Clinical examination was significant for 3 mm of hypoglobus and restricted supraduction in the left eye. Contrasted computed tomography imaging revealed a well-circumscribed lesion in the superotemporal orbit causing extensive bone erosion that appeared to arise from the lacrimal gland. An incisional biopsy was performed, and histopathological evaluation demonstrated fibrovascular tissue surrounding a mixture of histiocytes and cholesterol clefts, consistent with a cholesterol granuloma. CONCLUSIONS AND IMPORTANCE: Orbital cholesterol granulomas are rare lesions that are predominantly found in the superotemporal orbit. These lesions can be associated with marked bony changes in the superotemporal fossa that can be mistaken for a lacrimal gland neoplasm; however, bony erosion is a hallmark of this lesion and should be considered on the differential diagnosis of any lacrimal gland mass with extensive bony erosion.

The Flip-Back Myocutaneous Advancement Flap for Periocular Reconstruction.

PURPOSE: To present a novel myocutaneous flap for anterior lamellar reconstruction. METHODS: Retrospective interventional case series of consecutive patients who underwent Mohs reconstruction using the flip-back flap. Operations were performed by a single surgeon (DTT) between January 2012 and May 2016. For lower eyelid defects, an extended subciliary incision was made and a skin-muscle flap developed and suspended in the manner of lower eyelid blepharoplasty. A back-cut was used to develop a pedicle from the overlapping tissue, which was then rotated 180 degrees into the defect. A similar method was employed in an inverted manner for upper eyelid defects. Postoperative eyelid function, cosmesis, complications, and need for further interventions were assessed. RESULTS: Ten patients-8 with lower and 2 with upper eyelid defects-were reconstructed using this method. Mean follow up was 18.3 ± 15.5 months with a minimum interval of 4 months. Despite the 180-degree rotation of a relatively narrow pedicle, none of the patients experienced flap necrosis. Postoperative function and cosmesis was satisfactory, with no tissue puckering, notching, or symptomatic retraction. No antimetabolite/steroid injection or surgical revision was required. CONCLUSIONS: The flip-back flap expands the armamentarium of the periocular reconstructive surgeon. Its particular forte is in addressing broad and relatively shallow anterior lamellar defects where sufficient tissues are not available for transposition via a uni- or bipedicle flap. By leveraging the robust periocular vascular plexus and defying traditional guidelines governing pedicle formation and rotation, it permits creation of a local flap in cases where skin grafts or extensive Mustarde-style flaps might otherwise be required.The flip-back myocutaneous flap offers a novel alternative to skin grafting or more extensive cheek rotational flaps for reconstruction of challenging anterior lamellar defects involving the eyelids and adjacent periocular tissues.

Dog bite injuries of the eye and ocular adnexa.

Dog bites result in a diverse range of injuries and complications in the periocular region, particularly in school aged children. It is therefore incumbent on the oculoplastic surgeon to be well versed in both acute and long-term management. The intent of this review is to provide a systematic evaluation of the epidemiology, principles of dog bite wound care, and specific considerations related to common patterns of ophthalmic injury. Review of clinical literature from 1976 to 2014. The majority of periocular injuries result from seemingly benign interactions between young children and familiar dogs. Aggressive saline lavage combined with selective debridement of devitalized tissue is essential. High-risk wounds and vulnerable patient groups may benefit from preventive antibiotic coverage as well as appropriate rabies and tetanus prophylaxis. While the nuances of surgical repair are variable given the heterogeneity of presentation, systematic examination and an algorithm-driven approach underlie the optimal management of these complex injuries.

Ocular and cerebral infarction from periocular filler injection.

A 20-year-old woman presented with loss of vision in her right eye and a "black nose" after receiving hyaluronic acid filler injections in her right glabella 1 month prior. Her vision was no light perception, and external examination revealed resolving skin necrosis at the nasal tip. A dilated fundus exam showed a fibrotic membrane emanating from a pale optic nerve and a diffusely atrophic retina with sclerotic vessels. An MRI demonstrated scattered right-sided parietal lobe infarcts. These findings were consistent with inadvertent cannulation of the supraorbital artery, followed by injection of filler into the internal carotid circulation. The product traveled in a retrograde fashion, occluding the right ophthalmic artery, right dorsal nasal artery, and arterial segments to the Circle of Willis. This case highlights the importance of understanding the complex vascular architecture of the periorbita and the mechanism by which such occlusions occur.