Fluorescent protein tags affect the condensation properties of a phase-separating viral protein.

Fluorescent protein (FP) tags are extensively used to visualize and characterize the properties of biomolecular condensates despite a lack of investigation into the effects of these tags on phase separation. Here, we characterized the dynamic properties of µNS, a viral protein hypothesized to undergo phase separation and the main component of mammalian orthoreovirus viral factories. Our interest in the sequence determinants and nucleation process of µNS phase separation led us to compare the size and density of condensates formed by FP::µNS to the untagged protein. We found an FP-dependent increase in droplet size and density, which suggests that FP tags can promote µNS condensation. To further assess the effect of FP tags on µNS droplet formation, we fused FP tags to µNS mutants to show that the tags could variably induce phase separation of otherwise noncondensing proteins. By comparing fluorescent constructs with untagged µNS, we identified mNeonGreen as the least artifactual FP tag that minimally perturbed µNS condensation. These results show that FP tags can promote phase separation and that some tags are more suitable for visualizing and characterizing biomolecular condensates with minimal experimental artifacts.

Transcriptomic signatures of feline chronic gingivostomatitis are influenced by upregulated IL6.

Feline chronic gingivostomatitis (FCGS) is a relatively common and debilitating disease characterized by bilateral inflammation and ulceration of the caudal oral mucosa, alveolar and buccal mucosa, and varying degrees of periodontal disease. The etiopathogenesis of FCGS remains unresolved. In this study, we performed bulk RNA-seq molecular profiling of affected tissues derived from a cohort of client-owned cats with FCGS compared to tissues from unaffected animals, to identify candidate genes and pathways that can help guide future exploration of novel clinical solutions. We complemented transcriptomic findings with immunohistochemistry and in situ hybridization assays to better understand the biological significance of the results and performed RNA-seq validation of biologically relevant differentially expressed genes using qPCR assays to demonstrate technical reproducibility. Transcriptomic profiles of oral mucosal tissues in cats with FCGS are enriched with immune- and inflammation-related genes and pathways that appear to be largely influenced by IL6, and include NFKB, JAK/STAT, IL-17 and IFN type I and II signaling, offering new opportunities to develop novel clinical applications based on a more rational understanding of the disease.

Transcriptomic signatures of feline chronic gingivostomatitis are influenced by upregulated IL6.

Feline chronic gingivostomatitis (FCGS) is a relatively common and debilitating disease characterized by bilateral inflammation and ulceration of the caudal oral mucosa, alveolar and buccal mucosa, and varying degrees of periodontal disease. The etiopathogenesis of FCGS remains unresolved. In this study, we performed bulk RNA-seq molecular profiling of affected tissues derived from a cohort of client-owned cats with FCGS compared to tissues from unaffected animals, to identify candidate genes and pathways that can help guide future exploration of novel clinical solutions. We complemented transcriptomic findings with immunohistochemistry and in situ hybridization assays to better understand the biological significance of the results and performed RNA-seq validation of selected differentially expressed genes using qPCR assays to demonstrate technical reproducibility. Transcriptomic profiles of oral mucosal tissues in cats with FCGS are enriched with immune- and inflammation-related genes and pathways that appear to be largely influenced by IL6 , and include NFKB, JAK/STAT, IL-17 and IFN type I and II signaling, offering new opportunities to develop novel clinical applications based on a more rational understanding of the disease.

Intracorneal ring segment implantation in advanced Keratoconus.

PURPOSE: To compare the clinical outcomes of intracorneal ring segment (ICRS) implantation in eyes with advanced vs. mild/moderate keratoconus (KCN). METHODS: A retrospective analysis of 141 eyes of 111 patients with KCN who underwent ICRS implantation. Preoperative maximum keratometry (Kmax) was <57 diopters (D) in 70 eyes and >57 D in 71 eyes. Postoperatively, corrected distance visual acuity (CDVA), Kmax, and intraoperative and postoperative complications were assessed at 1 day, 1 month, and 1 year. RESULTS: Corneas with a preoperative Kmax >57 D experienced greater reduction in axial curvature after ICRS implantation than corneas with a preoperative Kmax <57 D (7.0 D vs. 5.5 D, p=0.005) and gained more Snellen lines of CDVA (3 vs. 1, p<0.001) by 1 year postoperatively. The incidences of the most prevalent complications (explantation, extrusion, and infectious keratitis) did not differ significantly between the two groups (p=0.29, p=0.99, p=0.98). CONCLUSIONS: The visual and topographic effects of ICRS implantation are greater in eyes with more advanced KCN, with no increase in the incidence of the most common complications.

Differences in career paths of alumni of the Cornell Leadership Program from North American universities compared with those alumni from universities in other countries.

OBJECTIVE: To characterize and compare the careers of alumni of the Cornell Leadership Program for Veterinary Students according to the countries where they studied and obtained their veterinary qualification. The Cornell Leadership Program is a 10-week residential research experience program for veterinary students from around the world who have ambitions for research-related careers. SAMPLE: Data on the career development of all 672 alumni were collected each year over the period of 1990 to 2019. PROCEDURES: The annual career profile of each alumnus was recorded and coded in 1 of 28 different categories. The careers and postveterinary qualifications of alumni from universities in the US and Canada (referred to as North American universities) were compared with those alumni who graduated from universities in other countries. RESULTS: Analysis of this 30-year database revealed that a considerable proportion (45.7% [307/672]) of the total 672 alumni are following the traditional career path of veterinary clinical practice rather than the research-related careers they aspired to as students during the Leadership Program. Furthermore, a higher proportion of the 325 North American alumni (56% [182/325]) were in clinical practice compared with 33.6% (112/333) of the 333 alumni from other countries. CLINICAL RELEVANCE: Many veterinary schools now provide research experience programs to encourage highly talented students who have ambitions for careers in which they can advance knowledge about animal disease and contribute to solving the health problems of animals through hypothesis-based research. Comparison of the careers of the Leadership Program alumni indicates that research experience alone is not sufficient to maintain the career goals of alumni. Follow-up mentoring of alumni of such programs is recommended while they complete their veterinary studies to reinforce their career aspirations and provide advice on how to achieve research-related careers.

DMEK Without Postoperative Supine Posturing.

PURPOSE: The purpose of this study was to describe the incidence of graft detachment after Descemet membrane endothelial keratoplasty (DMEK) without postoperative supine posturing. METHODS: A total of 106 eyes of 84 patients with Fuchs endothelial corneal dystrophy or bullous keratopathy (BK) were operated by a single experienced surgeon with DMEK with a 99% anterior chamber air bubble fill, recovered in an upright (seated) position, and then discharged without instructions to remain supine. Postoperatively, all eyes were evaluated for graft detachment through anterior segment optical coherence tomography at predetermined intervals (1 d, 1 wk, and 1 mo). Detachments were regarded as clinically significant if they subtended 30% of the total graft surface area or involved the visual axis. RESULTS: Clinically significant graft detachments were observed in 23 of 106 eyes (22%) in the no-supine posturing cohort, including 22 of 85 eyes (26%) operated for Fuchs endothelial corneal dystrophy and 1 of 21 eyes (5%) operated for BK. Compared with a historical comparison group of eyes undergoing DMEK with 48 hours of postoperative supine posturing, the risk of graft detachment was not increased. In both cohorts, 6% of operated eyes required regrafting for either persistent detachment or primary graft failure. No additional intraoperative or postoperative complications were experienced. CONCLUSIONS: Particularly in eyes operated for BK, the supine posturing requirement after DMEK may be eliminated without increasing the absolute risk for clinically significant graft detachment.

Clinical outcomes of Descemet membrane endothelial keratoplasty performed in eyes with keratoconus and corneal endothelial dysfunction.

PURPOSE: To evaluate the clinical outcome of Descemet membrane endothelial keratoplasty (DMEK) performed in eyes with comorbid keratoconus (KCN) and corneal endothelial dysfunction. METHODS: Twenty-five consecutive eyes of 14 patients with comorbid stable KCN underwent DMEK for corneal endothelial dysfunction; best spectacle corrected visual acuity (BSCVA), maximum corneal curvature (Kmax), maximum corneal power (Pmax), central corneal thickness (CCT), and intra- and postoperative complications were assessed. RESULTS: Excluding eyes requiring re-transplantation for primary graft failure (n = 3), all eyes showed improvement in BSCVA, reaching ≥ 20/40 (0.5) in 86%, ≥ 20/25 (0.8) in 55%, and ≥ 20/20 (1.0) in 27% by one month postoperatively; 90%, 76%, and 48% by 6 months postoperatively; and 88%, 76%, and 47% by 12 months postoperatively. CCT decreased from 571µm preoperatively to 485µm at 1 month (p < 0.001) and 481µm at 12 months (p < 0.001). Kmax decreased by a median of 1.4 diopters (D) at 1 month (p = 0.003) and 3.1 D at 12 months (p = 0.021), and every eye with a preoperative Kmax ≥ 46 D demonstrated flattening. Pmax decreased by 2.1 D at 1 month (p = 0.001) and 4.0 D at 12 months (p = 0.016). CONCLUSION: DMEK is technically feasible in eyes with comorbid KCN and may give excellent outcomes visual and refractive outcomes, including significant corneal flattening, which may potentially create a visually significant hyperopic shift in patients with severely ectatic corneas.

Spatial mapping of the total transcriptome by in situ polyadenylation.

Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNA enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We demonstrate that our spatial total RNA-sequencing (STRS) approach captures coding RNAs, noncoding RNAs and viral RNAs. We apply STRS to study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify spatially defined expression of noncoding transcripts in skeletal muscle regeneration and highlight host transcriptional responses associated with local viral RNA abundance. STRS requires adding only one step to the widely used Visium spatial total RNA-sequencing protocol from 10x Genomics, and thus could be easily adopted to enable new insights into spatial gene regulation and biology.

Curriculum Guidelines for Graduate and Undergraduate Virology Courses.

Curriculum guidelines for virology are needed to best guide student learning due to the continuous and ever-increasing volume of virology information, the need to ensure that undergraduate and graduate students have a foundational understanding of key virology concepts, and the importance in being able to communicate that understanding to both other virologists and nonvirologists. Such guidelines, developed by virology educators and the American Society for Virology Education and Career Development Committee, are described herein.

DMEK with endophotocoagulation and cyst wall removal for corneal endothelial decompensation due to iris cyst.

PURPOSE: Iris cysts may arise secondary to surgical or nonsurgical trauma, potentially leading to corneal decompensation via mechanical injury to the adjacent endothelium. However, no well-established protocol exists for the treatment for corneal edema arising therefrom. OBSERVATIONS: A 58-year-old white male presented with an iris mass of his left eye; it occupied 1/3rd the anterior chamber volume and directly contacted the corneal endothelium. The cornea was diffusely edematous, and best corrected visual acuity (BCVA) measured 20/70 (0.3). Corneal endothelial decompensation secondary to iris cyst was diagnosed. Treatment consisted of endophotocoagulation and vitrectomy probe removal of the cyst wall, with Descemet membrane endothelial keratoplasty (DMEK) also performed as a single, combined procedure. The patient subsequently experienced a resolution of his corneal edema and disappearance of his iris cyst, without recurrence of either condition. BCVA improved to 20/25 (0.8). CONCLUSIONS AND IMPORTANCE: Iris cyst may be a rare cause of corneal decompensation. Viable treatment may entail a single-stage procedure involving endophotocoagulation and vitrectomy probe application to the cyst wall combined with DMEK.

Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis.

A significant fraction of sudden death in children and young adults is due to viral myocarditis, an inflammatory disease of the heart. In this study, by using integrated single-cell and spatial transcriptomics, we created a high-resolution, spatially resolved transcriptome map of reovirus-induced myocarditis in neonatal mouse hearts. We assayed hearts collected at three timepoints after infection and studied the temporal, spatial and cellular heterogeneity of host-virus interactions. We further assayed the intestine, the primary site of reovirus infection, to establish a full chronology of molecular events that ultimately lead to myocarditis. We found that inflamed endothelial cells recruit cytotoxic T cells and undergo pyroptosis in the myocarditic tissue. Analyses of spatially restricted gene expression in myocarditic regions and the border zone identified immune-mediated cell-type-specific injury and stress responses. Overall, we observed a complex network of cellular phenotypes and spatially restricted cell-cell interactions associated with reovirus-induced myocarditis in neonatal mice.

Preimplantation dehydration for corneal allogenic intrastromal ring segment implantation.

Corneal allogenic intrastromal ring segments (CAIRS) are semicircular pieces of donor corneal stroma, which may be surgically implanted to flatten keratoconic corneas. These segments can be trimmed to different thicknesses; whereas thicker segments confer greater flattening, their bulk renders them more technically challenging to insert. Consequently, thinner segments are often preferred, especially for starting surgeons. Here, we describe a technique for transiently thinning CAIRS to facilitate easy insertion, thereby permitting the use of thicker segments to achieve the maximal flattening effect.

Reovirus Nonstructural Protein σNS Recruits Viral RNA to Replication Organelles.

The function of the mammalian orthoreovirus (reovirus) σNS nonstructural protein is enigmatic. σNS is an RNA-binding protein that forms oligomers and enhances the stability of bound RNAs, but the mechanisms by which it contributes to reovirus replication are unknown. To determine the function of σNS-RNA binding in reovirus replication, we engineered σNS mutants deficient in RNA-binding capacity. We found that alanine substitutions of positively charged residues in a predicted RNA-binding domain decrease RNA-dependent oligomerization. To define steps in reovirus replication facilitated by the RNA-binding property of σNS, we established a complementation system in which wild-type or mutant forms of σNS could be tested for the capacity to overcome inhibition of σNS expression. Mutations in σNS that disrupt RNA binding also diminish viral replication and σNS distribution to viral factories. Moreover, viral mRNAs only incorporate into viral factories or factory-like structures (formed following expression of nonstructural protein µNS) when σNS is present and capable of binding RNA. Collectively, these findings indicate that σNS requires positively charged residues in a putative RNA-binding domain to recruit viral mRNAs to sites of viral replication and establish a function for σNS in reovirus replication. IMPORTANCE Viral replication requires the formation of neoorganelles in infected cells to concentrate essential viral and host components. However, for many viruses, it is unclear how these components coalesce into neoorganelles to form factories for viral replication. We discovered that two mammalian reovirus nonstructural proteins act in concert to form functioning viral factories. Reovirus µNS proteins assemble into exclusive factory scaffolds that require reovirus σNS proteins for efficient viral mRNA incorporation. Our results demonstrate a role for σNS in RNA recruitment to reovirus factories and, more broadly, show how a cytoplasmic non-membrane-enclosed factory is formed by an RNA virus. Understanding the mechanisms of viral factory formation will help identify new targets for antiviral therapeutics that disrupt assembly of these structures and inform the use of nonpathogenic viruses for biotechnological applications.

The multi-functional reovirus σ3 protein is a virulence factor that suppresses stress granule formation and is associated with myocardial injury.

The mammalian orthoreovirus double-stranded (ds) RNA-binding protein σ3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection is largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus infection, we engineered a panel of thirteen σ3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are formed at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not mediate formation of SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.

The Paradoxes of Viral mRNA Translation during Mammalian Orthoreovirus Infection.

De novo viral protein synthesis following entry into host cells is essential for viral replication. As a consequence, viruses have evolved mechanisms to engage the host translational machinery while at the same time avoiding or counteracting host defenses that act to repress translation. Mammalian orthoreoviruses are dsRNA-containing viruses whose mRNAs were used as models for early investigations into the mechanisms that underpin the recognition and engagement of eukaryotic mRNAs by host cell ribosomes. However, there remain many unanswered questions and paradoxes regarding translation of reoviral mRNAs in the context of infection. This review summarizes the current state of knowledge about reovirus translation, identifies key unanswered questions, and proposes possible pathways toward a better understanding of reovirus translation.

Flattening the curve: manual method for corneal allogenic intrastromal ring segment implantation.

Corneal allogenic ring segments are semicircular pieces of donor corneal stroma that may be surgically implanted to flatten keratoconic corneas. Conventionally, these donor segments are inserted into channels created using femtosecond laser dissection. However, access to femtosecond technology is not universal. In this study, an alternate, manual technique for channel creation, which is femtosecond laser independent, is described.

Reovirus σ3 Protein Limits Interferon Expression and Cell Death Induction.

Induction of necroptosis by mammalian reovirus requires both type I interferon (IFN)-signaling and viral replication events that lead to production of progeny genomic double-stranded RNA (dsRNA). The reovirus outer capsid protein µ1 negatively regulates reovirus-induced necroptosis by limiting RNA synthesis. To determine if the outer capsid protein σ3, which interacts with µ1, also functions in regulating necroptosis, we used small interfering RNA (siRNA)-mediated knockdown. Similarly to what was observed in diminishment of µ1 expression, knockdown of newly synthesized σ3 enhances necroptosis. Knockdown of σ3 does not impact reovirus RNA synthesis. Instead, this increase in necroptosis following σ3 knockdown is accompanied by an increase in IFN production. Furthermore, ectopic expression of σ3 is sufficient to block IFN expression following infection. Surprisingly, the capacity of σ3 protein to bind dsRNA does not impact its capacity to diminish production of IFN. Consistent with this, infection with a virus harboring a mutation in the dsRNA binding domain of σ3 does not result in enhanced production of IFN or necroptosis. Together, these data suggest that σ3 limits the production of IFN to control innate immune signaling and necroptosis following infection through a mechanism that is independent of its dsRNA binding capacity.IMPORTANCE We use mammalian reovirus as a model to study how virus infection modulates innate immune signaling and cell death induction. Here, we sought to determine how viral factors regulate these processes. Our work highlights a previously unknown role for the reovirus outer capsid protein σ3 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires production of interferon. The σ3 protein limits the induction of necroptosis by preventing excessive production of interferon following infection.

Bowman Layer Onlay Graft for Reducing Fluctuation in Visual Acuity After Previous Radial Keratotomy.

PURPOSE: To describe the clinical outcome of a first patient undergoing Bowman layer (BL) transplantation with an onlay graft to reduce fluctuation in visual acuity and refractive error after previous radial keratotomy (RK) surgery. METHODS: In 2018, a 66-year-old woman presented with complaints of long-standing diurnal fluctuation in best-spectacle corrected visual acuity (BSCVA) after RK in 1983. After the removal of host epithelium, a BL graft was positioned onto the host cornea. BSCVA, Scheimpflug-based corneal tomography, and anterior segment optical coherence tomography were evaluated up to 12 months postoperatively. RESULTS: The surgery and postoperative course were uneventful. After surgery, the subjective complaints of visual fluctuation were reduced from 10 to 3 on a scale from 1 to 10. BSCVA (20/40; 0.5) did not change from preoperative to postoperative. Corneal tomography showed an overall central corneal steepening of 5.9 diopters. Biomicroscopy, Scheimpflug imaging, and anterior segment optical coherence tomography showed a completely epithelialized and well-integrated graft, with some minor epithelial remnants located in the preexisting keratotomy incisions. CONCLUSIONS: BL onlay grafting may have the potential to manage patients with subjective complaints of diurnal fluctuation in visual acuity after previous RK.

DMEK in Super-Seniors: Clinical Outcomes of Descemet Membrane Endothelial Keratoplasty Performed in Patients ≥ 90 Years Old.

PURPOSE/AIMS OF THE STUDY: To evaluate the clinical outcomes of Descemet membrane endothelial keratoplasty (DMEK) performed in the "oldest old" patients, i.e. ≥ 90 years. MATERIALS AND METHODS: Between the years of 2009 and 2019, 20 consecutive eyes of 17 patients aged ≥ 90 underwent DMEK for endothelial dysfunction. Best corrected visual acuity (BCVA), central corneal thickness (CCT), endothelial cell density (ECD), graft survival, and intra- and postoperative complications were assessed. RESULTS: Except in one case in which the DMEK surgery could not be completed, all operated eyes experienced an improvement in BCVA, although only 50% achieved ≥ 20/40 (0.5) by 1 year postoperatively. One year after surgery, median CCT had declined from 641(±161) µm to 480 (±34) µm, and median endothelial cell density was reduced by 53%, from 2574 (±286) to 1226 (±404) cells/mm2. Six of 19 eyes receiving DMEK grafts (32%) developed partial graft detachments requiring re-bubbling. One eye experienced a secondary graft failure at 6 months and underwent repeat endothelial keratoplasty. CONCLUSION: DMEK is technically feasible in the oldest old patients and may yield significant visual improvements, although an elevated risk of some postoperative complications including graft detachment with corresponding need for re-bubbling may be anticipated.