Reactive Metabolite-induced Protein Glutathionylation: A Potentially Novel Mechanism Underlying Acetaminophen Hepatotoxicity.

Although covalent protein binding is established as the pivotal event underpinning acetaminophen (APAP) toxicity, its mechanistic details remain unclear. In this study, we demonstrated that APAP induces widespread protein glutathionylation in a time-, dose- and bioactivation-dependent manner in HepaRG cells. Proteo-metabonomic mapping provided evidence that APAP-induced glutathionylation resulted in functional deficits in energy metabolism, elevations in oxidative stress and cytosolic calcium, as well as mitochondrial dysfunction that correlate strongly with the well-established toxicity features of APAP. We also provide novel evidence that APAP-induced glutathionylation of carnitine O-palmitoyltransferase 1 (CPT1) and voltage-dependent anion-selective channel protein 1 are respectively involved in inhibition of fatty acid ß-oxidation and opening of the mitochondrial permeability transition pore. Importantly, we show that the inhibitory effect of CPT1 glutathionylation can be mitigated by PPARα induction, which provides a mechanistic explanation for the prophylactic effect of fibrates, which are PPARα ligands, against APAP toxicity. Finally, we propose that APAP-induced protein glutathionylation likely occurs secondary to covalent binding, which is a previously unknown mechanism of glutathionylation, suggesting that this post-translational modification could be functionally implicated in drug-induced toxicity.

Combating Microbial Contamination with Robust Polymeric Nanofibers: Elemental Effect on the Mussel-Inspired Cross-Linking of Electrospun Gelatin.

Designing biocompatible nanofibrous mats capable of preventing microbial colonization from resident and nosocomial bacteria for an extended period remains an unmet clinical need. In the present work, we designed antibiotic free durable antimicrobial nanofiber mats by taking advantage of synergistic interactions between polydopamine (pDA) and metal ions with varying degree of antimicrobial properties (Ag+, Mg2+, Ca2+, and Zn2+). Microscopic analysis showed successful pDA-mediated cross-linking of the gelatin nanofibers, which further improved by the inclusion of Ag+, Mg2+, and Ca2+ ions as supported by mechanical and thermal studies. Spectroscopic results reinforce the presence of strong interactions between pDA and metal ions in the composite nanofibers, leading to generation of robust polymeric nanofibers. We further showed that strong pDA-Ag interactions attenuated the cell cytotoxicity and anticell proliferative properties of silver ions for immortalized keratinocytes and primary human dermal fibroblasts. pDA-Ca2+/Zn2+ interactions rendered the composite structure sterile against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium strains, whereas the silver ion-incorporated composite mats displayed broad spectrum antibacterial activity against both Gram-positive/-negative bacteria and yeast strains. We showed that the strong pDA-Ag interactions help retaining long-term antimicrobial activity of the mats for at least 40 days while attenuating mammalian cell cytotoxicity of silver ions for skin cells. Overall, the results suggest the potential of pDA-metal ion interactions for engineering sterile nanofibrous mats and expanding the antibiotic armamentarium against drug-resistant pathogens.

Tear Proteins Calcium binding protein A4 (S100A4) and Prolactin Induced Protein (PIP) are Potential Biomarkers for Thyroid Eye Disease.

There are no reliable biomarkers to predict thyroid eye disease (TED) in patients with autoimmune thyroid disease (AITD) currently. Several evidences support the involvement of the lacrimal gland in TED. The aim of our study was to quantitatively correlate the changes in tear protein profile with increasing severity of TED. Tear samples were collected from four groups of patients; AITD without TED (AITD), AITD with mild TED (mild TED), AITD with severe TED (severe TED) and normal controls. A total of 72 patients were recruited for the study. In discovery phase, isobaric tags for relative and absolute quantification (iTRAQ) 4-plex was used for quantitative proteomics analysis. For verification of results from discovery phase, sequential window acquisition of all theoretical fragment ion spectra (SWATH) was used to analyze an independent cohort from normal controls, AITD, mild TED and severe TED. Two proteins, S100A4 and PIP showed consistent dysregulation trends in the discovery and validation phase experiments. Our study demonstrated the differences in tear proteome across the spectrum of different severity and activity of TED in patients with AITD. Two tear proteins, S100A4 and PIP may serve as potential biomarkers to predict progression to severe TED in patients with AITD.

Plasma Metabonomic Profiling of Diabetic Retinopathy.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and the leading cause of visual impairment in working-age adults. Patients with diabetes often develop DR despite appropriate control of systemic risk factors, suggesting the involvement of other pathogenic factors. We hypothesize that the plasma metabolic signature of DR is distinct and resolvable from that of diabetes alone. A nested population-based case-control metabonomic study was first performed on 40 DR cases and 40 control subjects with diabetes using gas chromatography-mass spectrometry. Eleven metabolites were found to be correlated with DR, and the majority were robust when adjusted for metabolic risk factors and confounding kidney disease. The metabolite markers 2-deoxyribonic acid; 3,4-dihydroxybutyric acid; erythritol; gluconic acid; and ribose were validated in an independent sample set with 40 DR cases, 40 control subjects with diabetes, and 40 individuals without diabetes. DR cases and control subjects with diabetes were matched by HbA1c in the validation set. Activation of the pentose phosphate pathway was identified from the list of DR metabolite markers. The identification of novel metabolite markers for DR provides insights into potential new pathogenic pathways for this microvascular complication and holds translational value in DR risk stratification and the development of new therapeutic measures.

Interaction of gelatin with polyenes modulates antifungal activity and biocompatibility of electrospun fiber mats.

Topical application of antifungals does not have predictable or well-controlled release characteristics and requires reapplication to achieve therapeutic local concentration in a reasonable time period. In this article, the efficacy of five different US Food and Drug Administration-approved antifungal-loaded (amphotericin B, natamycin, terbinafine, fluconazole, and itraconazole) electrospun gelatin fiber mats were compared. Morphological studies show that incorporation of polyenes resulted in a two-fold increase in fiber diameter and the mats inhibit the growth of yeasts and filamentous fungal pathogens. Terbinafine-loaded mats were effective against three filamentous fungal species. Among the two azole antifungals compared, the itraconazole-loaded mat was potent against Aspergillus strains. However, activity loss was observed for fluconazole-loaded mats against all of the test organisms. The polyene-loaded mats displayed rapid candidacidal activities as well. Biophysical and rheological measurements indicate strong interactions between polyene antifungals and gelatin matrix. As a result, the polyenes stabilized the triple helical conformation of gelatin and the presence of gelatin decreased the hemolytic activity of polyenes. The polyene-loaded fiber mats were noncytotoxic to primary human corneal and sclera fibroblasts. The reduction of toxicity with complete retention of activity of the polyene antifungal-loaded gelatin fiber mats can provide new opportunities in the management of superficial skin infections.

Femtosecond laser-assisted endothelial keratoplasty: a laboratory model.

PURPOSE: To evaluate the ability of a 40-kHz femtosecond laser in performing posterior stromal ablation for endothelial keratoplasty. METHODS: Human corneoscleral rims were mounted on an artificial anterior chamber. After corneal pachymetry, the femtosecond laser was used to create a donor lenticule by using a variety of diameter ablations, 150 microm from the Descemet membrane. After ablation, the donor lenticule was peeled from the posterior surface and examined under light and scanning electron microscopy. Grading of ease of peeling and removing of the donor lenticule was assessed. RESULTS: The 40-kHz laser was able to produce effective dissection at low power in the posterior stroma. Modification of laser parameter settings was needed to improve the quality of stromal bed ablation. Double pass ablation of the bed significantly improved ease of peeling and removing of the donor lenticule. This was corroborated by smoother ablations on light and scanning electron microscopy. However, multiple pass ablations did not improve vertical rim dissections, which were satisfactory when single passes were used. CONCLUSIONS: Femtosecond laser-assisted endothelial keratoplasty is a viable alternative to microkeratome-assisted endothelial keratoplasty. Customized nomograms are needed for deep stromal ablation.

Primary graft failure after Descemet-stripping automated endothelial keratoplasty: clinico-pathological study.

PURPOSE: Descemet-stripping automated endothelial keratoplasty (DSAEK) is a selective tissue corneal transplantation procedure in which only the diseased endothelium and the Descemet membrane is replaced. Refractive and visual results have been encouraging with this procedure, but higher rates of primary graft failure have been noted. We herein report histopathologic and ultrastructural changes in 2 cases of primary graft failure after DSAEK. We are not aware of these features being reported previously. METHODS: Two cases of primary graft failure after DSAEK. One patient underwent DSAEK for Fuchs endothelial dystrophy and the other for pseudophakic bullous keratopathy. Both DSAEK procedures were uneventful. Postoperatively, there was no graft dislocation, but 1 patient had a nasal Descemet detachment that was reapposed with intracameral air. One month postoperatively, there was no improvement in the vision, and both patients had pronounced swelling of the recipient and donor corneas. Both patients underwent graft exchange. Both the recipient and donor corneal edema resolved. RESULTS: Histopathologic evaluation showed marked corneal edema and loss of endothelial cells. Ultrastructural evaluation showed only remnant endothelial cell membranes present in 1 sample devoid of any intracellular contents. Transmission electron microscopy showed the presence of extensively damaged keratocytes in the deep posterior stroma and also in the interface at the graft-host junction. CONCLUSIONS: These cases show widespread and irrecoverable endothelial and keratocyte damage in patients with primary graft failure after DSAEK.