Mycotoxins can be found in food and feed storage as well as in several kinds of foodstuff and are capable of harming mammals and some of them even in small doses. This study investigated on the undifferentiated neuronal cell line SH-SY5Y the effects of two mycotoxins: patulin (PAT) and citrinin (CTN), which are predominantly produced by fungi species Penicillium and Aspergillus. Here, the individual and combined cytotoxicity of PAT and CTN was investigated using the cytotoxic assay MTT. Our findings indicate that after 24 h of treatment, the IC50 value for PAT is 2.01 μM, which decreases at 1.5 μM after 48 h. In contrast, CTN did not attain an IC50 value at the tested concentration. Therefore, we found PAT to be the more toxic compared to CTN. However, the combined treatment suggests an additive toxic effect. With 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) DCFH-DA assay, ROS generation was demonstrated after CTN treatment, but PAT showed only small changes. The mixture presented a very constant behavior over time. Finally, the median-effect/combination index (CI-) isobologram equation demonstrated an additive effect after 24 h, but an antagonistic effect after 48 h for the interaction of the two mycotoxins.

Canine non-infectious deep ulcerative keratitis is considered a severe ocular disorder that possibly can progress to perforation. Immediate treatment should be directed to stimulate corneal wound healing, control infection, and minimize self-trauma while eliminating the underlying causes.
This retrospective study was aimed to compare the difference in non-infectious deep corneal wound healing time between cases treated with medical therapy alone and those treated with medical therapy combined with a nictitating membrane flap.
The medical records at the Ophthalmology Clinic, Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University between January 2018 and March 2020 were retrospectively reviewed. Sixty-six eyes (from 65 dogs) diagnosed with non-infectious deep ulcerative keratitis from the medical treatment group (n = 34) and the combined treatment group (n = 32) were included. The combined treatment group was prescribed the same conservative medical administrations plus a surgical nictitating membrane flap for 14 days.
Healing time was defined as the duration of time from the day that the dog had been diagnosed with deep ulcerative keratitis by a fluorescein staining test to the day that the corneal fluorescein stain was negative. Overall, the mean age of dogs with deep ulcerative keratitis was 10.49 ± 4.7 years. The disease was commonly evident in females more than males. Shih Tzu was the most prevalent dog breed. The corneal healing time between dogs receiving medical therapy alone and those receiving combined treatment was not statistically significant (p = 0.386). Healing times were not significantly different between sex and breed (p = 0.41). The median corneal healing time for dogs older than 10 years in the combined treatments group (29.5 days; ranging from 20 to 46 days) was longer than for those receiving medical therapy alone (21 days; ranging from 9.5 to 30.5 days).
Supportive therapy including a nictitating membrane flap is suggested in dogs prone to deep corneal ulcers not involving infection. Even though the healing time is not statistically significant, a nictitating membrane flap acts as a tissue bandage to reduce friction over the cornea, and it also alleviates the healing process by moistening the ocular surface.

BACKGROUND: Solid-phase fluorescence spectroscopy (SPFS) is a very useful non-destructive technique for directly analyzing samples in solid form without the use of solvents. However, due to the so-called inner-filter effect, it is sometimes necessary to dilute solid samples using non-fluorescent solids as diluents.
OBJECTIVE: This study aimed to explore the potential of SPFS in the quantitative analysis of fluorescent species based on: (1) the type of solid diluent; and (2) the sampling method used in the SPFS analysis.
METHODS: Four different solids were used as solid diluents in the preparation of standard mixtures having different concentrations of rhodamine b and fluorescein as model compounds. Standard mixtures of model compounds were sampled by two different methods called: (1) the powder-cell method; and (2) the adhesive tape method. LOQ and calibration sensitivity calculated from the calibration graphs were used to assess the measurement performance. The usability of SPFS in real-sample analyses was also evaluated in detail.
RESULTS: Among the solid diluents studied, the best results were obtained with sodium carbonate. The powder-cell method yielded a significant advantage over the adhesive tape method. The lowest LOQs for rhodamine b and fluorescein were obtained by sodium carbonate and the powder-cell method as 0.06 and 0.11 mg/kg, respectively. The results of real-sample analyses were verified using conventional liquid-phase fluorescence spectroscopy (LPFS).
CONCLUSIONS: Solid-diluent type and sampling method were found to affect the performance of the SPFS technique. A combination of sodium carbonate and the powder-cell method gave the best results. According to the t-test, no difference was observed between the means obtained by SPFS and LPFS techniques in real-sample analyses.
CONCLUSIONS: In SPFS, toxic organic solvents and difficult sample preparation steps are not required. This makes the method advantageous over conventional fluorescence analyses performed in the liquid phase.

We aimed to predict response to biologics in inflammatory bowel disease (IBD) using computerized image analysis of probe confocal laser endomicroscopy (pCLE) in vivo and assess the binding of fluorescent-labeled biologics ex vivo. Additionally, we investigated genes predictive of anti-tumor necrosis factor (TNF) response.
Twenty-nine patients (15 with Crohn\'s disease [CD], 14 with ulcerative colitis [UC]) underwent colonoscopy with pCLE before and 12 to 14 weeks after starting anti-TNF or anti-integrin α4β7 therapy. Biopsies were taken for fluorescein isothiocyanate-labeled infliximab and vedolizumab staining and gene expression analysis. Computer-aided quantitative image analysis of pCLE was performed. Differentially expressed genes predictive of response were determined and validated in a public cohort.
In vivo, vessel tortuosity, crypt morphology, and fluorescein leakage predicted response in UC (area under the receiver-operating characteristic curve [AUROC], 0.93; accuracy 85%, positive predictive value [PPV] 89%; negative predictive value [NPV] 75%) and CD (AUROC, 0.79; accuracy 80%; PPV 75%; NPV 83%) patients. Ex vivo, increased binding of labeled biologic at baseline predicted response in UC (UC) (AUROC, 83%; accuracy 77%; PPV 89%; NPV 50%) but not in Crohn\'s disease (AUROC 58%). A total of 325 differentially expressed genes distinguished responders from nonresponders, 86 of which fell within the most enriched pathways. A panel including ACTN1, CXCL6, LAMA4, EMILIN1, CRIP2, CXCL13, and MAPKAPK2 showed good prediction of anti-TNF response (AUROC >0.7).
Higher mucosal binding of the drug target is associated with response to therapy in UC. In vivo, mucosal and microvascular changes detected by pCLE are associated with response to biologics in inflammatory bowel disease. Anti-TNF-responsive UC patients have a less inflamed and fibrotic state pretreatment. Chemotactic pathways involving CXCL6 or CXCL13 may be novel targets for therapy in nonresponders.

Our purpose is to demonstrate the changes in cornea nerve parameters and symptoms and signs in dry eye disease (DED) patients after oral vitamin B1 and mecobalamin treatment. In this randomized double-blind controlled trial, DED patients were randomly assigned to either the treatment group (oral vitamin B1 and mecobalamin, artificial tears) or the control group (artificial tears). Corneal nerve parameters via in vivo confocal microscopy (IVCM), DED symptoms, and signs were assessed at baseline and 1 and 3 months post-treatment. In total, 398 eyes from 199 patients were included. In the treatment group, there were significant improvements in corneal nerve length, width, and neuromas, the sign of conjunctival congestion score (CCS), symptoms of dryness, pain, photophobia, blurred vision, total symptom score, and OSDI (OSDI) at 1/3 months post-treatment (all p < 0.05). Patients who received vitamin B1 and mecobalamin showed greater improvement in CCS, dryness scores at 1 month (p < 0.05), corneal fluorescein staining (CFS) (p = 0.012), photophobia (p = 0.032), total symptom scores (p = 0.041), and OSDI (p = 0.029) at 3 months. Greater continuous improvement in CFS (p = 0.045), dryness (p = 0.033), blurred vision (p = 0.031) and total symptom scores (p = 0.023) was demonstrated at 3 months than at 1 month post-treatment in the treatment group. We found that oral vitamin B1 and mecobalamin can improve corneal nerve length, width, reflectivity and the number of neuromas in IVCM, thereby repairing epithelial cells and alleviating some ocular symptoms. Thus, vitamin B1 and mecobalamin are potential treatment options for patients with DED.

The EU Water Framework Directive requires the monitoring and evaluation of surface water sediment quality based on the assessment of risk posed by contamination on the biotic receptors. Floodplain sediments are important receptors of potentially toxic element (PTE) contamination from the upstream catchment areas, and floodplains host climate-sensitive riverine ecosystems and fertile agricultural areas at the same time. This study investigates the effect of PTE contamination on microbial communities in floodplain sediments and soils using the fast, inexpensive and reliable fluorescein diacetate (FDA) method in order to estimate its applicability for sediment quality monitoring and preliminary toxicity-based risk assessment. Sediment and soil samples were collected from the actively flooded alluvial plain and the river terrace areas along a 130-km stretch of the large Drava River floodplain known to be widely contaminated by historical mining, smelting and the associated industry in the upstream Alpine region. Results of detailed data analysis show that the total microbial activity represented by the measured FDA values is related to PTE (As, Cu, Zn, Cd, Pb) concentrations, but this relationship shows significant heterogeneity and depends on the spatial location and on the soil properties such as organic matter content, dissolved salt and nutrient content, and it is specific to the toxic elements. Results show that some microbe species appear to be able to adapt to the elevated PTE concentrations in toxic soil micro-environments, over time. Despite the observed heterogeneity of microbial activity, the results revealed a breakpoint in the FDA dataset around the FDA = 3 FC (fluorescein concentration) value suggesting that microbial activity is controlled by thresholds.

Purpose: To assess the safety and technical feasibility of in-vivo needle-based forward-looking confocal laser endomicroscopy in prostate tissue. Methods: For this feasibility study, 2 patients with a suspicion of prostate cancer underwent transperineal needle-based confocal laser endomicroscopy during ultrasound-guided transperineal template mapping biopsies. After intravenous administration of fluorescein, needle-based confocal laser endomicroscopy imaging was performed with a forward-looking probe (outer diameter 0.9 mm) in 2 trajectories during a manual push-forward and pullback motion. A biopsy was taken in a coregistered parallel adjacent trajectory to the confocal laser endomicroscopy trajectory for histopathologic comparison. Peri- and postprocedural adverse events, confocal laser endomicroscopy device malfunction and procedural failures were recorded. Needle-based confocal laser endomicroscopy image quality assessment, image interpretation, and histology were performed by an experienced confocal laser endomicroscopy rater and uro-pathologist, blinded to any additional information. Results: In both patients, no peri- and post-procedural adverse events were reported following needle-based confocal laser endomicroscopy. No confocal laser endomicroscopy device malfunction nor procedural failures were reported. Within 1.5 min after intravenous administration of fluorescein, needle-based confocal laser endomicroscopy image quality was sufficient for interpretation for at least 14 min, yielding more than 5000 confocal laser endomicroscopy frames per patient. The pullback confocal laser endomicroscopy recordings and most of the push-forward recordings almost only visualized erythrocytes, being classified as non-representative. During the push-forward recordings, prostate tissue was occasionally visualized in single frames, insufficient for histopathologic comparison. Prostate carcinoma was identified by biopsy in one patient (Gleason score 4 + 3 = 7, >50%), while the biopsy from the other patient showed no malignancy. Conclusion: Needle-based confocal laser endomicroscopy imaging of in-vivo prostate tissue with a forward-looking confocal laser endomicroscopy probe is safe without device malfunctions or procedural failures. Needle-based confocal laser endomicroscopy is technically feasible, but the acquired confocal laser endomicroscopy datasets are non-representative. The confocal laser endomicroscopy images\' non-representative nature is possibly caused by bleeding artifacts, movement artifacts and a lack of contact time with the tissue of interest. A different confocal laser endomicroscopy probe or procedure might yield representative images of prostatic tissue.

MPO-derived oxidants including HOCl contribute to tissue damage and the initiation and propagation of inflammatory diseases. The search for small molecule inhibitors of myeloperoxidase, as molecular tools and potential drugs, requires the application of high throughput screening assays based on monitoring the activity of myeloperoxidase. In this study, we have compared three classes of fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid, including boronate-, aminophenyl- and thiol-based fluorogenic probes and we show that all three classes of probes are suitable for this purpose. However, probes based on the coumarin fluorophore turned out to be not reliable indicators of the inhibitors\' potency. We have also determined the rate constants of the reaction between HOCl and the probes and they are equal to 1.8 × 104 M-1s-1 for coumarin boronic acid (CBA), 1.1 × 104 M-1s-1 for fluorescein based boronic acid (FLBA), 3.1 × 104 M-1s-1 for 7-(p-aminophenyl)-coumarin (APC), 1.6 × 104 M-1s-1 for 3\'-(p-aminophenyl)-fluorescein (APF), and 1 × 107 M-1s-1 for 4-thiomorpholino-7-nitrobenz-2-oxa-1,3-diazole (NBD-TM). The high reaction rate constant of NBD-TM with HOCl makes this probe the most reliable tool to monitor HOCl formation in the presence of compounds showing HOCl-scavenging activity.

UNASSIGNED: To assess the effects of systemic isotretinoin therapy (SIT) on the ocular surface, meibomian glands (MG) and cornea microstructure in acne vulgaris (AV) patients.
UNASSIGNED: Patients with AV (n = 20) and healthy controls (n = 20) were enrolled in the study. All participants underwent ocular surface tests in the order of ocular surface disease index (OSDI) questionnaire, corneal sensitivity, tear break-up time (BUT), fluorescein and lissamine green (LG) staining and Schirmer II test with anaesthesia. MG alterations were evaluated with meibography for upper (UE) and lower eyelids (LE) separately. Corneal basal epithelium and subbasal nerve plexus (SNP) were evaluated using In Vivo Confocal Microscopy (IVCM).
UNASSIGNED: Schirmer II test with anaesthesia, BUT, corneal sensitivity, fluorescein and LG staining grades and OSDI score results showed no difference between the control group and the baseline of the patient group. Whereas the meibomian gland dysfunction (MGD) grades, UE and LE meiboscores were higher in the patient group at the baseline (p = 0.013, p = 0.004, p = 0.008 respectively). The Control group possessed higher numbers of total and long nerve fibres compared with patients at the baseline (p ≤ 0.001 for both two values). Compared to the baseline and the third month, BUT decreased and fluorescein staining grades increased (p = 0.017 and p = 0.043, respectively). MGD grades, UE and LE meiboscores increased in the third month compared to the baseline (p < 0.001, p < 0.001, p = 0.008 respectively). Basal epithelial cell density (BECD) decreased in the third month of SIT (p = 0.043).
UNASSIGNED: This prospective study showed that systemic Isotretinoin treatment effects not only ocular surface parameters but also corneal and Meibomian glands structure. Considering early alterations in the course of treatment, ophthalmological assessment and follow-up during SIT are mandatory.

Exosomes represent an important group of extracellular vesicles. They are formed in endosomal compartments and are actively secreted to extracellular spaces. Several membrane proteins, including integrins, are present on the surface of exosomes. As exosomal integrins are competent for binding to ligand, they can play important roles in directing the tissue distribution of exosomes. Integrin-directed exosomal trafficking in vivo is involved in regulating the remodeling of cell homing niches for metastatic cancers and migrating lymphocytes. This chapter describes the methods used to study integrin functions on exosomes including: isolation and biophysical characterization of exosomes, exosomal integrin-ligand binding assays, and in vivo competitive exosome homing assays.