Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment.

Cryptococcus neoformans is an opportunistic human fungal pathogen commonly associated with infection in immunocompromised individuals (e.g., patients with HIV/AIDS). Important virulence determinants include the production of a polysaccharide capsule, melanin, and extracellular enzymes, as well as the ability to grow at 37°C. C. neoformans controls a plethora of host defense and evasion mechanisms to survive during infection and to proliferate within the host, causing meningoencephalitis and death. Traditionally, characterization of C. neoformans under different environmental conditions and stresses has relied on genetic and phenotypic analyses, as well as biochemical assays. However, advances in mass spectrometry instrumentation, sample preparation protocols, and bioinformatic tools and databases promote comprehensive profiling of fungal cellular processes, secretion or protein release into the extracellular environment, and vesicle contents. Moreover, proteomics provides insight into regulatory mechanisms influencing signal transduction cascades and protein complexes or networks through profiling of post-translational modifications and protein-protein interactions. Given the medical impact of C. neoformans infections and the recent emergence of antifungal-resistant strains, defining proteins produced in response to unique environments provides an opportunity to uncover antivirulence strategies and alternative therapeutic options to combat infection. Here, we describe culturing and sample preparation of C. neoformans and outline protocols for comprehensively profiling changes in protein abundance within the cellular proteome and secretome. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Growth and sample preparation of Cryptococcus neoformans Basic Protocol 2: Protein extraction from supernatant Basic Protocol 3: Protein extraction from cell pellet Basic Protocol 4: Proteomic profiling and bioinformatics.

Amebic Keratitis in a Wearer of Disposable Contact Lenses due to a Mixed Vahlkampfia and Hartmannella Infection

Stiffness-related eye diseases such as keratoconus require comprehensive visualization of the complex morphological matrix changes. The aim of this study was to use three-dimensional (3D) light sheet fluorescence microscopy (LSFM) to analyze unlabeled corneal tissue samples, qualitatively visualizing changes in corneal stiffness. Isolated porcine corneal tissue samples were treated with either NaCl or 0.1% glutaraldehyde (GTA) prior to clearing with benzyl alcohol/benzyl benzoate (BABB) and subsequently scanned with LSFM. After analysis of the LSFM data sets, the samples were embedded in paraffin to validate the results by conventional planar microscopy. In the unlabeled corneal tissue samples the 2D/3D morphology of the entire tissue volume was identified by specific autofluorescence signals. An enhancement of collagen crosslinking was induced by applying GTA to the corneal tissue. Subsequent LSFM scans showed specific morphological changes due to altered autofluorescence signals of the corneal stroma, which were confirmed by conventional histology. Therefore, LSFM analysis of corneal tissue samples allowed label-free 3D autofluorescence assessment of the corneal morphology in its anatomical context. It provides the technical basis for the examination of the pathologically altered cornea and facilitates ophthalmologic examinations of corneal diseases based on the altered tissue stiffness.

PurposeTransportation of human corneal tissue for transplantation always needs to be conducted in a timely manner. For this reason, even single corneal tissue samples are frequently transported by cars. This...

The effectiveness of the fluoroquinolone ciprofloxacin is dependent on stromal drug concentrations which exceed the minimum inhibitory concentration90 (MIC90). The purpose of this study is to compare corneal tissue ciprofloxacin levels in patients exposed to three ciprofloxacin dosing regimens before undergoing penetrating keratoplasty. Thirty-one patients were assigned to one of three treatment groups. Group 1 followed a ciprofloxacin dosing regimen compatible with home use [two drops of 0.3% ciprofloxacin (Ciloxan; Alcon Laboratories, Fort Worth, TX, U.S.A.) every 4 h over a 24-h period]. Groups 2 and 3 followed a more tightly controlled dosing regimen designed for a health-care setting (two drops of Ciloxan applied by a trained professional every 15 min over a 4-h period). In groups 1 and 2, corneal epithelium was left intact, whereas in group 3 corneas were abraded. Corneal tissue samples were surgically obtained. Excised buttons were frozen and Ciloxan concentration determined by high-pressure liquid chromatography. Ciloxan corneal tissue concentrations (mean +/- SD) were 8.82 +/- 8.24 micrograms/g tissue in group 1, 166.20 +/- 336.94 micrograms/g tissue in group 2, and 938.30 +/- 1,081.51 micrograms/g tissue in group 3. Ciloxan penetration can be improved by administering the drug in a controlled setting at 15-min intervals over a 4-h period. Individual Ciloxan concentrations exceeded the MIC90 for most key ocular pathogens despite wide variability in all experimental groups.

Background: Few studies have systematically investigated pregnancy-induced changes in protein abundance of drug transporters in organs important for drug/xenobiotic disposition.Objective: The goal of this study was to compare protein abundance of important drug/xenobiotic trans-porters including Abcb1a, Abcg2, Abcc2, and Slco1b2 in the liver, kidney and brain of pregnant mice on gestation day 15 to that of non-pregnant mice.Methods: The mass spectrometry-based proteomics was used to quantify changes in protein abundance of transporters in tissues from pregnant and non-pregnant mice.Results: The protein levels of hepatic Abcc2, Abcc3, and Slco1a4 per µg of total membrane proteins were significantly decreased by pregnancy by 24%, 72%, and 70%, respectively. The protein levels of Abcg2, Abcc2, and Slco2b1 per µg of total membrane proteins in the kidney were significantly decreased by pregnancy by 43%, 50%, and 46%, respectively. After scaling to the whole liver with consideration of increase in liver weight in pregnant mice, the protein abundance of Abcb1a, Abcg2, Abcc2, Abcb11, Abcc4, Slco1a1, and Slco1b2 in the liver was ~50-100% higher in pregnant mice, while those of Abcc3 and Slco1a4 were ~40% lower. After scaling to the whole kidney, none of the transporters examined were significantly changed by pregnancy. Only Abcg2 and Abcb1a were quantifiable in the brain and their abundance in the brain was not influenced by pregnancy.Conclusion: Protein abundance of drug transporters can be significantly changed particularly in the liver by pregnancy. These results will be helpful to understand pregnancy-induced changes in drug/xenobiotic disposition in the mouse model.

To determine herpes simplex virus (HSV) DNA prevalence and mean cycle threshold of polymerase chain reaction (PCR) in corneal tissue of patients with penetrating keratoplasty (PKP), with (HSK+) and without (HSK-) previous clinical herpetic keratitis history. Retrospective review of recipient corneal buttons which were explanted through PKP between March 2010 and September 2018 at the Department of Ophthalmology, Saarland University Medical Center in Homburg/Saar, Germany. Corneal tissue samples were analysed by real-time PCR for the presence of HSV DNA. For each subject, clinical data, including patients' demographics and clinical diagnoses, were collected. In total, 2230 corneal samples (age at the time of the surgery 57.3 ± 19.2 years) of 1860 patients were analysed. HSV PCR was positive in 137 (6.1%) corneal samples, with a 30.57 ± 6.01 (range 14-39) mean cycle threshold (Ct) value. Two hundred ninety-eight (13.4%) corneas of 266 patients were clinically HSK+, and 1932 (86.6%) corneas of 1600 patients were clinically HSK-. HSV DNA was detected significantly more frequently (p < 0.0001) in HSK+ corneal samples (108 corneal samples; 36.2%), than in HSK- corneal samples (29 corneal samples; 1.5%). Ct value was significantly lower in HSK+ than in HSK- corneal samples (29.8 ± 5.8 versus 32.6 ± 5.9; p = 0.008). Our data demonstrate that a positive clinical history of HSK is related to HSV PCR positivity in about every 2.8th patient. In addition, about every 66th explanted corneal tissue is HSV PCR-positive despite the lack of clinical suspicion. These patients may need additional local/systemic antiviral treatment to avoid newly acquired HSK following penetrating keratoplasty.

The objective of this study was to assess the clinical diagnostic value of metagenomic next-generation sequencing (mNGS) in cases of challenging corneal infections using corneal tissue samples. This retrospective study involved 42 patients with corneal infections, where conventional diagnostic techniques failed to identify the causative pathogen. Corneal tissue specimens underwent mNGS, followed by microbial culture for validation. Sensitivity-guided antimicrobial therapy was administered upon identification of the pathogen. The diagnostic and therapeutic efficacy of mNGS was analyzed to evaluate its clinical utility. A total of 42 patients were included in this study, with mNGS detection results obtained for 38 cases (90.48%). Among them, 30 cases (71.43%) were clinically significant, eight cases (19.05%) had low clinical relevance, and four cases (9.52%) showed no detection. Following corresponding antimicrobial treatment, 30 patients exhibited significant improvement, resulting in a treatment effectiveness of 71.43%. The prognosis of mNGS-positive patients was superior to that of mNGS-negative patients, with statistically significant differences observed (P < 0.001). Corneal tissue mNGS facilitated the rapid identification of causative agents in challenging corneal infections with unclear clinical diagnoses. It could be seamlessly integrated with traditional diagnostic methods to guide the diagnosis and treatment of corneal diseases.

Proteomics Characterization of Extracellular Space Components in the Human Aorta

Moso bamboo is an important forest species with a variety of ecological, economic, and cultural values. However, the gene annotation information of moso bamboo is only based on the transcriptome sequencing, lacking the evidence of proteome. The lignification and fiber in moso bamboo leads to a difficulty in the extraction of protein using conventional methods, which seriously hinders research on the proteomics of moso bamboo. The purpose of this study is to establish efficient methods for extracting the total proteins from moso bamboo for following mass spectrometry-based quantitative proteome identification. Here, we have successfully established a set of efficient methods for extracting total proteins of moso bamboo followed by mass spectrometry-based label-free quantitative proteome identification, which further improved the protein annotation of moso bamboo genes. In this study, 10,376 predicted coding genes were confirmed by quantitative proteomics, accounting for 35.8% of all annotated protein-coding genes. Proteome analysis also revealed the protein-coding potential of 1015 predicted long noncoding RNA (lncRNA), accounting for 51.03% of annotated lncRNAs. Thus, mass spectrometry-based proteomics provides a reliable method for gene annotation. Especially, quantitative proteomics revealed the translation patterns of proteins in moso bamboo. In addition, the 3284 transcript isoforms from 2663 genes identified by Pacific BioSciences (PacBio) single-molecule real-time long-read isoform sequencing (Iso-Seq) was confirmed on the protein level by mass spectrometry. Furthermore, domain analysis of mass spectrometry-identified proteins encoded in the same genomic locus revealed variations in domain composition pointing towards a functional diversification of protein isoform. Finally, we found that part transcripts targeted by nonsense-mediated mRNA decay (NMD) could also be translated into proteins. In summary, proteomic analysis in this study improves the proteomics-assisted genome annotation of moso bamboo and is valuable to the large-scale research of functional genomics in moso bamboo. In summary, this study provided a theoretical basis and technical support for directional gene function analysis at the proteomics level in moso bamboo.

Mass spectrometry-based proteomics enables the unbiased and sensitive profiling of cellular proteomes and extracellular environments. Recent technological and bioinformatic advances permit identifying dual biological systems in a single experiment, supporting investigation of infection from both the host and pathogen perspectives. At the ocular surface, Pseudomonas aeruginosa is commonly associated with biofilm formation and inflammation of the ocular tissues, causing damage to the eye. The interaction between P. aeruginosa and the immune system at the site of infection describes limitations in clearance of infection and enhanced pathogenesis. Here, the extracellular environment (eye wash) of murine ocular surfaces infected with a clinical isolate of P. aeruginosa is profiled and neutrophil marker proteins are detected, indicating neutrophil recruitment to the site of infection. The first potential diagnostic markers of P. aeruginosa-associated keratitis are also identified. In addition, the deepest murine corneal proteome to date is defined and proteins, categories, and networks critical to the host response are detected. Moreover, the first identification of bacterial proteins attached to the ocular surface is reported. The findings are validated through in silico comparisons and enzymatic profiling. Overall, the work provides comprehensive profiling of the host-pathogen interface and uncovers differences between general and site-specific host responses to infection.

Docosahexaenoic acid eliminates endoplasmic reticulum stress and inflammatory pathways in diabetic rat keratopathy

Conjunctival and corneal tissue samples from nine patients with severe changes after acid and alkali burns were examined immunohistochemically. Epithelial HLA Class II antigen expression was present in three of five conjunctival samples, and all of the antigen markers examined in the conjunctival stroma could be stained. HLA Class II expression was demonstrated in four out of five corneal tissue samples. The results show that in Grade III or IV chemical burns, with the diverse changes of the external eye that they produce, immunological and possibly also autoimmune mechanisms can influence the clinical course.

Small open reading frames (smORFs) encode previously unannotated polypeptides or short proteins that regulate translation in cis (eukaryotes) and/or are independently functional (prokaryotes and eukaryotes). Ongoing efforts for complete annotation and functional characterization of smORF-encoded proteins have yielded novel regulators and therapeutic targets. However, because they are excluded from protein databases, initiate at non-AUG start codons, and produce few unique tryptic peptides, unannotated small proteins cannot be detected with standard proteomic methods. Here,, we outline a procedure for mass spectrometry-based detection of translated smORFs in cultured human cells from protein extraction, digestion, and LC-MS/MS, to database preparation and data analysis. Following proteomic detection, translation from a unique smORF may be validated via siRNA-based silencing or overexpression and epitope tagging. This is necessary to unambiguously assign a peptide to a smORF within a specific transcript isoform or genomic locus. Provided that sufficient starting material is available, this workflow can be applied to any cell type/organism and adjusted to study specific (patho)physiological contexts including, but not limited to, development, stress, and disease. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Protein extraction, size selection, and trypsin digestion Alternate Protocol 1: In-solution C8 column size selection Support Protocol 1: Chloroform/methanol precipitation Support Protocol 2: Reduction, alkylation, and in-solution protease digestion Support Protocol 3: Peptide de-salting Basic Protocol 2: Two-dimensional LC-MS/MS with ERLIC fractionation Basic Protocol 3: Transcriptomic database construction Alternate Protocol 2: Transcriptomics database generation with gffread Basic Protocol 4: Non-annotated peptide identification from LC-MS/MS data Basic Protocol 5: Validation using isotopically labeled synthetic peptide standards and siRNA Basic Protocol 6: Transcript validation using transient overexpression.

Candida albicans biofilm formation in the presence of drugs can be examined through time-lapse microscopy. In many cases, the images are used qualitatively, which limits their utility for hypothesis testing. We employed a machine-learning algorithm implemented in the Orbit Image Analysis program to detect the percent area covered by cells from each image. This is combined with custom R scripts to determine the growth rate, growth asymptote, and time to reach the asymptote as quantitative proxies for biofilm formation. We describe step-by-step protocols that go from sample preparation for time-lapse microscopy through image analysis parameterization and visualization of the model fit. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation Basic Protocol 2: Time-lapse microscopy: Evos protocol Basic Protocol 3: Batch file renaming Basic Protocol 4: Machine learning analysis of Evos images with Orbit Basic Protocol 5: Parametrization of Orbit output in R Basic Protocol 6: Visualization of logistic fits in R.