Technical Brief: A novel strategy for enrichment of trabecular meshwork protease proteome

We present a novel and simple enrichment strategy to capture trabecular meshwork (TM) protease proteome. The method relies on fractionation of TM tissue into cytosolic and nuclear extracts and subsequent affinity enrichment of proteases on peptide inhibitors. A large repertoire of available protease substrate analog peptides enables an improved capture of TM protease proteome compared to SDS–PAGE fractionation alone. Peptide analog inhibitors of protease substrates are immobilized on a protein A or G column using 254 nm intense ultraviolet (UV) light. The TM cytosolic protein extract incubated on the column is eluted with salt or a buffer with a low hydrogen ion concentration. The resultant protein solution is precipitated with acetone, fractionated on SDS–PAGE, in situ trypsin digested, and subjected to mass spectrometry. This relatively simple protocol enables improved capture of cytosolic proteases. We identified 20 previously reported TM proteins from a single donor tissue using affinity enrichment. The majority of identified proteins were either intracellular proteases or known protease inhibitors. Both serine and cysteine proteases were captured using this strategy with improved coverage compared to our previous identification without affinity enrichment

A Health-Related Quality of Life Measure for Patients Who Undergo Minimally Invasive Glaucoma Surgery

PurposeTo develop a patient-reported outcome measure to assess the impact of glaucoma and treatment, including minimally invasive glaucoma surgery (MIGS).DesignObservational study before and after concomitant cataract and Food and Drug Administration-approved implantable MIGS device surgery.SettingSurvey administration was on a computer, iPad, or similar device.Patient population184 adults completed the baseline survey, 124 a survey 3 months after surgery, and 106 the 1-month test-retest reliability survey. The age range was 37 to 89 (average age = 72). Most were female (57%), non-Hispanic White (81%), and had a college degree (56%).Main outcome measuresThe Glaucoma Outcomes Survey (GOS) assesses functional limitations (27 items), vision-related symptoms (7 items), psychosocial issues (7 items), and satisfaction with microinvasive glaucoma surgery (1 item). These multiple-item scales were scored on a 0 to 100 range, with a higher score indicating worse health.ResultsInternal consistency reliability estimates ranged from 0.75 to 0.93, and 1-month test-retest intraclass correlations ranged from 0.83 to 0.92 for the GOS scales. Product-moment correlations among the scales ranged from 0.56 to 0.60. Improvement in visual acuity in the study eye from baseline to the 3-month follow-up was significantly related to improvements in GOS functional limitations (r = 0.18, P = .0485), vision-related symptoms (r = 0.19, P = .0386), and psychosocial concerns (r = 0.18, P = .0503). Responders to treatment ranged from 17% for vision-related symptoms to 48% for functional limitations.ConclusionsThis study supports using the GOS for ophthalmic procedures such as MIGS. Further evaluation of the GOS in different patient subgroups and clinical settings is needed

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure

Lokale Anwendung von Metronidazol-Gel als wirksame Therapiealternative in der Behandlung der chronischen Demodex-Blepharitis - ein Fallbericht

Zusammenfassung HINTERGRUND Blepharokonjunktivitis ist die häufigste Manifestation der okulären Rosazea. Die Epilation von Zilien ermöglicht den Nachweis von DEMODEX FOLLICULORUM, einer Haarbalgmilbe deren ätiologische Beteiligung an Rosazea diskutiert wird. Wichtige Differentialdiagnosen und Komplikationen sind trockene Augen, seborrhoische, bakterielle und allergische Blepharokonjunktivitiden. Die Therapie basiert auf Lidkantenpflege und Quecksilber-Präzipitat-Salbe, sie erfordert engmaschige ärztliche Kontrollen, ihre Dauer sollte wegen der Quecksilber-Toxizität 6 Wochen nicht überschriten. ANAMNESE UND BEFUND Eine 30jährige Patientin stellte sich wegen seit 21 Jahren geröteten, gereizten Augen vor. Antibiotika und Antiallergika brachten keinen therapeutischen Erfolg. Allgemeinanamnese unauffällig, Quecksilberallergie. Sehschärfe: R/L 1,0/1,0. Spaltlampenuntersuchung: R/L hyperämische, verdickte Lidkanten, verkrustete Ablagerungen an rarefizierten, teilweise gebrochenen Wimpern, Telangiektasien der Bindehaut, niedriger Tränenmeniskus, sonst regelrechter ophthalmologischer Befund. Diagnose: chronische Demodex Blepharokonjunktivitis. THERAPIE UND VERLAUF Die konventionelle Therapie war kontraindiziert aufgrund der Quecksilber-Allergie. Die alternative Behandlung mit Minocyclin oral, 100 mg täglich, brachte weder subjektive noch objektive Besserung. Eine Kombination von Lidkantenpflege mit 2% Metronidazol-Gel halbierte die Zahl der Haarbalgmilben nach einem Monat und bewirkte ein erneutes Wachstum der Wimpern nach 2 Monaten. Die Therapie wurde nach 6 Monaten bei negativem Demodex Nachweis abgeschlossen. Die Patientin ist rezidivfrei nach 1 Jahr. SCHLUßFOLGERUNGEN LJnser Fall demonstriert die Wirksamkeit der lokalen Applikation von 2% Metronidazol Gel in der chronischen Demodex-Blepharokonjunktivitis und bietet eine neue und sichere Therapiealternative. Weder okuläre noch systemische Nebenwirkungen wurden beobachtet. Kontroverse Hypothesen zur Ätiologie der Blepharitis werden diskutiert

Glaucomatous aqueous humor vesicles are smaller and differ in composition compared to controls

Cells communicate with each other using vesicles of varying sizes, including a specific repertoire known as exosomes. We isolated aqueous humor (AH)-derived vesicles using two different methods: ultracentrifugation and an exosome isolation kit. We confirmed a unique vesicle size distribution in the AH derived from control and primary open-angle glaucoma (POAG) patients using various techniques, including Nanotracker, dynamic light scattering, atomic force imaging, and electron microscopy. Bonafide vesicle and/or exosome markers were present by dot blot in both control and POAG AH-derived vesicles. Marker levels differed between POAG and control samples, while non-vesicle negative markers were absent in both. Quantitative labeled (iTRAQ) proteomics showed a reduced presence of a specific protein, STT3B, in POAG compared to controls, which was further confirmed using dot blot, Western blot, and ELISA assays. Along the lines of previous findings with AH profiles, we found vast differences in the total phospholipid composition of AH vesicles in POAG compared to controls. Electron microscopy further showed that the addition of mixed phospholipids alters the average size of vesicles in POAG. We found that the cumulative particle size of type I collagen decreased in the presence of Cathepsin D, which normal AH vesicles were able to protect against, but POAG AH vesicles did not. AH alone had no effect on collagen particles. We observed a protective effect on collagen particles with an increase in artificial vesicle sizes, consistent with the protective effects observed with larger control AH vesicles but not with the smaller-sized POAG AH vesicles. Our experiments suggest that AH vesicles in the control group provide greater protection for collagen beams compared to POAG, and their increased vesicle sizes are likely contributing factors to this protection.•Compared to control, aqueous humor (AH) derived vesicles/exosomes from primary open-angle glaucoma (POAG) patients contain smaller size vesicles, as demonstrated by electron microscopy, dynamic light scattering, atomic force microscopy, and Nanotracker analyses.•POAG vesicle composition differs from controls, with decreased levels of STT3B—an enzyme involved in protein and lipid N-glycosylation, and phospholipids, particularly phosphatidylserine (PS) lipids.•Vesicles derived from AH, whether POAG or control, have a potential protective role against collagen type I degradation by Cathepsin D. However, the protection conferred by POAG vesicles is minimal compared to control. Adding an ensemble of PS lipids to vesicles results in a size-dependent increase in protection against degradation