The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

A proximity proteomics pipeline with improved reproducibility and throughput.

Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.

To compare central visual field progression using mean deviation and pointwise linear regression analysis.

Purpose: To compare central visual field progression using mean deviation and pointwise linear regression (PLR) analysis. Methods: We analyzed the 10-2 Humphrey visual field (HVF) tests for moderate and advanced primary glaucoma who had undergone at least five reliable 10-2 visual field tests with a minimum follow-up of at least two years and best-corrected visual acuity better than 6/12. Regression slope less than -1 dB/year at P < 0.01 at a point was defined as an individual threshold point progression. Results: Ninety-six eyes of 74 patients were included. The median follow-up duration was of 4 years (±1.97). Median 10-2 mean deviation (MD) at inclusion was -19.01 dB (interquartile range [IQR] -13.2, -24.14) and -21.90 (IQR - 13.4, -27.8) on 24-2 HVF. The median rate of MD change was -0.13 dB/year (IQR - 0.46, 0.08) for 10-2. The median rate for visual field index (VFI) change was 0.9% per year (IQR - 1.5, 0.4). Twenty-eight percent of eyes (27 eyes) showed progression. Twelve percent (12 eyes) showed progression of two or more points in the same hemifield on pointwise linear regression (PLR) analysis, and 16% of eyes (15 eyes) showed progression of one point. The median rate of MD change was significantly more in progressing eyes based on PLR analysis than eyes with no progression (-0.5 vs. -0.06 dB/year P < 0.001). One patient had likely and the second had possible progression on 24-2. In 24 eyes, event analysis did not show any change; the rest mean deviation was out of range. Conclusion: Central visual field PLR analysis is useful in detecting progression in advanced glaucomatous damage.

An automated proximity proteomics pipeline for subcellular proteome and protein interaction mapping.

Proximity labeling (PL) coupled with mass spectrometry has emerged as a powerful technique to map proximal protein interactions in living cells. Large-scale sample processing for proximity proteomics necessitates a high-throughput workflow to reduce hands-on time and increase quantitative reproducibility. To address this issue, we developed a scalable and automated PL pipeline, including generation and characterization of monoclonal cell lines, automated enrichment of biotinylated proteins in a 96-well format, and optimization of the quantitative mass spectrometry (MS) acquisition method. Combined with data-independent acquisition (DIA) MS, our pipeline outperforms manual enrichment and data-dependent acquisition (DDA) MS regarding reproducibility of protein identification and quantification. We apply the pipeline to map subcellular proteomes for endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, using serotonin receptor (5HT2A) as a model, we investigated agonist-induced dynamics in protein-protein interactions. Importantly, the approach presented here is universally applicable for PL proteomics using all biotinylation-based PL enzymes, increasing both throughput and reproducibility of standard protocols.

Effect of immediate shallow anterior chamber after Ahmed glaucoma valve implantation on intermediate-term intraocular pressure control.

Purpose: To evaluate the incidence of shallow anterior chamber in the early postoperative period following Ahmed glaucoma valve (AGV) implantation and its effect on the hypertensive phase (HP), intermediate-term intraocular pressure (IOP) control, and success rate. Methods: A retrospective analysis of 369 eyes of 360 patients who underwent AGV implantation between January 2005 and January 2020 with a minimum follow-up of 2 months was performed. Twenty-six patients developed shallow anterior chamber (AC) within 8 weeks following surgery (cases). They were compared with 39 randomly selected controls (no shallow AC post AGV). HP (IOP spike >21 mmHg), use of ocular hypotensive medications, and other associations were compared. Results: Incidence of shallow AC post AGV was 7% (95% confidence interval [CI] 4, 9). The onset of shallow AC was 3 ± 2.1 days and resolved within 6 ± 4.7 days. Hypotony (12 [47%] vs. 1 [2.5%], P 0.0001) and choroidal detachment (CD; 7 [27%] vs. 3 [8%], P 0.03) were more common in cases compared to controls. The HP occurred in 11 (43%) cases versus 13 (34%) controls (P 0.4). Cases required more ocular hypotensive medications than controls at the end of 8 weeks (1.1 ± 1 vs. 0.5 ± 0.5, P 0.01). There was no significant difference in the qualified success between the groups at 1 year. Conclusion: The development of postoperative shallow AC post AGV implantation was not detrimental to IOP control at 1 year. However, there is a need to monitor the occurrence of HP in these eyes.

Mycobacterium porcinum peritonitis in a patient on continuous ambulatory peritoneal dialysis.

Mycobacterium porcinum has been reported to cause a variety of illnesses including wound infections, respiratory tract infections, osteomyelitis and catheter-related bacteremias. We report the first case of M. porcinum peritonitis in a patient on continuous ambulatory peritoneal dialysis (CAPD). A 67-year-old woman on CAPD presented with three weeks of constitutional symptoms and abdominal pain. Peritoneal fluid cultures on day three grew acid-fast rods. Nocardiosis was suspected and the patient was empirically treated with amikacin and trimethoprim-sulfamethoxazole. The dialysis catheter was removed. Two weeks later final culture results revealed M. porcinum. Ciprofloxacin and trimethoprim-sulfamethoxazole were initiated with good clinical response.

Staphylococcus lugdunensis native tricuspid valve endocarditis: a case report and review of literature.

Coagulase negative staphylococci are skin commensals and are generally disregarded as contaminants in clinical specimens. Repeated isolation of coagulase negative staphylococci in blood cultures should warrant a species identification to recognize unusually virulent organisms that demand aggressive treatment, such as Staphylococcus lugdunensis. Staphylococcus lugdunensis is known to cause a wide variety of infections, including a predominant left-sided endocarditis. We report a rare case of native tricuspid valve Staphylococcus lugdunensis endocarditis in a non-intravenous drug user and include a brief literature review.