Change in Visual Acuity of Patients With Fuchs Endothelial Corneal Dystrophy Over 1 Year.

PURPOSE: To determine whether the clinical and paraclinical course of Fuchs endothelial corneal dystrophy (FECD) over 1 year is related to the extent of triplet repetition in the transcription factor 4 (TCF4) gene. METHODS: A prospective study with a 1-year follow-up was conducted. A total of 104 patients (160 eyes) with FECD and an equivalent number of age- and sex-matched control subjects without FECD were included. At inclusion, the corneas were graded using the modified Krachmer grade (KG) and patients were genotyped for the number of trinucleotide repeats (TNRs) in the TCF4 gene by the short tandem repeat assay. Visual acuity, Scheimpflug tomographic features, and the Visual Function and Corneal Health Status using a visual disability instrument were measured on 2 visits at 1-year intervals. RESULTS: KGs ranged from 1 to 6, and 46% of eyes had grades 1 to 4. 71% of the patients harbored TNR expansion (>40) versus 13% in control subjects (P < 0.001). Severity at inclusion was higher in the presence of TNR expansion when considering eyes independently (mean grade ±SD, 4.08 ± 1.42) without TNR expansion and 4.66 ± 1.27 with TNR expansion (P = 0.024). In 1 year, the ETDRS score significantly decreased by -2.97 (95% confidence interval -4.69 to -1.26, P = 0.001) and the ETDRS score with glare by -4.25 (95% confidence interval -6.22 to -2.27, P < 10-5). There was no relationship between the rate of decline and TNR expansion or KG. Central corneal thickness and Visual Function and Corneal Health Status scores did not significantly vary. CONCLUSIONS: It is possible to measure a subtle progression of FECD over a period as short as 1 year. We did not find a relationship between the presence of TNR expansion and the speed of deterioration over 1 year. This work should facilitate the design of future clinical trials on FECD.Trial Registration-URL: ClinicalTrials.gov. Identifier: French Fuchs Follow-up Study (F3S): NCT03974230.

Bioinformatics prediction and experimental verification identify a cuproptosis-related gene signature as prognosis biomarkers of hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) of which its prognostic prediction is still unclarified is a highly heterogeneous disease. Cuproptosis is a form of cell death that depends on copper regulation. Whether the cuproptosis-related genes can be the prognostic indicators of HCC is yet to be elucidated. The aim of this study is to investigate whether cuproptosis-related genes play a role in HCC and can be used as a diagnostic index to predict the occurrence of liver cancer. Methods: We downloaded HCC patients' gene expression profiles and their corresponding clinical data from a public database. To screen data, we used single factor Cox regression analysis, meanwhile, polymerase chain reaction (PCR) was used for the verification. After that, the risk score was calculated and the relationship between risk score and clinical factors was analyzed. Besides, a nomogram map was constructed for predicting the prognosis of HCC, and calibration map and decision curve analysis (DCA) map were used to test the model. Results: Compared to the high expression group of four cuproptosis-related genes, the low expression group showed better overall survival (OS) [hazard ratio (HR) =2.58; 95% confidence interval (CI): 1.72-3.89, P<0.01]. The expression of the four cuproptosis-relate genes increased in liver cancer cell lines compared to liver cell lines (P<0.05). Based on these four genes, we calculated the risk score and divided them into two groups as high-risk group and low-risk group. The risk factor map showed the high-risk group had shorter survival time and the four genes were highly expressed. The area under curve (AUC) of receiver operating characteristic (ROC) prediction curve for the first year was 0.726. Risk scores were closely related to clinical factors and immune cells. Finally, we constructed a nomogram for predicting the prognosis of HCC. Conclusions: The risk score for cuproptosis-related genes was established and involved in the construction of the nomogram, providing a new perspective on the prognosis and copper metabolism of HCC.

Femtosecond Laser Cutting of Human Crystalline Lens Capsule and Decellularization for Corneal Endothelial Bioengineering.

The bioengineering of corneal endothelial grafts consists of seeding in vitro cultured corneal endothelial cells onto a thin, transparent, biocompatible, and sufficiently robust carrier which can withstand surgical manipulations. This is one of the most realistic alternatives to donor corneas, which are in chronic global shortage. The anterior capsule of the crystalline lens has already been identified as one of the best possible carriers, but its challenging manual preparation has limited its use. In this study, we describe a femtosecond laser cutting process of the anterior capsule of whole lenses in order to obtain capsule discs of 8 mm diameter, similar to conventional endothelial grafts. Circular marks made on the periphery of the disc indicate its orientation. Immersion in water for 3 days is sufficient to completely remove the lens epithelial cells and to enable the seeding of corneal endothelial cells, which remain viable after 27 days of culture. Therefore, this method provides a transparent, decellularized disc ready to form viable tissue engineered endothelial grafts.

Design and validation of a custom-made system to measure transepithelial electrical impedance in human corneas preserved in active storage machine.

Corneal epithelial barrier represents one of the major limitations to ocular drug delivery and can be explored non-invasively through the evaluation of its electrical properties. Human corneas stored in active storage machine (ASM) could represent an interesting physiological model to explore transcorneal drug penetration. We designed a new system adapted to human corneas preserved in ASM to explore corneal epithelial barrier function ex-vivo. A bipolar set-up including Ag/AgCl electrodes adaptors to fit the corneal ASM and a dedicated software was designed and tested on freshly excised porcine corneas (n = 59) and human corneas stored 14 days in ASM (n = 6). Porcine corneas presented significant and proportional decrease in corneal impedance in response to increasing-size epithelial ulcerations and acute exposure to benzalkonium chloride (BAC) 0.01 and 0.05%. Human corneas stored 14 days in ASM presented a significant increase in corneal impedance associated with the restoration of a multi-layer epithelium and an enhanced expression of tight junctions markers zonula occludens 1, claudin 1 and occludin. These results support the relevance of the developed approach to pursue the exploration and development of human corneas stored in ASM as a physiological pharmacological model.

Effect of interaction between dissolved organic matter and iron/manganese (hydrogen) oxides on the degradation of organic pollutants by in-situ advanced oxidation techniques.

Iron and manganese (hydrogen) oxides (IMHOs) exhibit excellent redox capabilities for environmental pollutants and are commonly used in situ chemical oxidation (ISCO) technologies for the degradation of organic pollutants. However, the coexisting dissolved organic matter (DOMs) in surface environments would influence the degradation behavior and fate of organic pollutants in IMHOs-based ISCO. This review has summarized the interactions and mechanisms between DOMs and IMHOs, as well as the properties of DOM-IMHOs complexes. Importantly, the promotion or inhibition impact of DOM was discussed from three perspectives. First, the presence of DOMs may hinder the accessibility of active sites on IMHOs, thus reducing their efficiency in degrading organic pollutants. The formation of compounds between DOMs and IMHOs alters their stability and activity in the degradation process. Second, the presence of DOMs may also affect the generation and transport of active species, thereby influencing the oxidative degradation process of organic pollutants. Third, specific components within DOMs also participate and affect the degradation pathways and rates. A comprehensive understanding of the interaction between DOMs and IMHOs helps to better understand and predict the degradation process of organic pollutants mediated by IMHOs in real environmental conditions and contributes to the further development and application of IMHO-mediated ISCO technology.

Using solid phase adsorption toxin tracking and extended local similarity analysis to monitor lipophilic shellfish toxins in a mussel culture ranch in the Yangtze River Estuary.

Harmful algal blooms (HABs) and their associated phycotoxins are increasing globally, posing great threats to local coastal ecosystems and human health. Nutrients have been carried by the freshwater Yangtze River and have entered the estuary, which was reported to be a biodiversity-rich but HAB-frequent region. Here, in situ solid phase adsorption toxin tracking (SPATT) was used to monitor lipophilic shellfish toxins (LSTs) in seawaters, and extended local similarity analysis (eLSA) was conducted to trace the temporal and special regions of those LSTs in a one-year trail in a mussel culture ranch in the Yangtze River Estuary. Nine analogs of LSTs, including okadaic acid (OA), dinophysistoxin-1 (DTX1), yessotoxin (YTX), homoyessotoxin (homoYTX), 45-OH-homoYTX, pectenotoxin-2 (PTX2), 7-epi-PTX2 seco acid (7-epi-PTX2sa), gymnodimine (GYM) and azaspiracids-3 (AZA3), were detected in seawater (SPATT) or rope farmed mussels. The concentrations of OA + DTX1 and homoYTX in mussels were positively correlated with those in SPATT samplers (Pearson test, p < 0.05), indicating that SPATT (with resin HP20) would be a good monitoring tool and potential indicator for OA + DTX1 and homoYTX in mussel Mytilus coruscus. The eLSA results indicated that late summer and early autumn were the most phycotoxin-contaminated seasons in the Yangtze River Estuary. OA + DTX1, homoYTX, PTX2 and GYM were most likely driven by the local growing HAB species in spring and summer, while Yangtze River diluted water may impact the accumulation of HAB species, causing potential phycotoxin contamination in the Yangtze River Estuary in autumn and winter. Together, the results showed that the mussel harvesting season, late summer and early autumn, would be the season with the greatest phycotoxin risk and would be the most contaminated by local growing toxic algae. Routine monitoring sites should be set up close to the local seawaters.

Lightweight Pneumatically Elastic Backbone Structure with Modular Construction and Nonlinear Interaction for Soft Actuators.

There has been a growing need for soft robots operating various force-sensitive tasks due to their environmental adaptability, satisfactory controllability, and nonlinear mobility unique from rigid robots. It is of desire to further study the system instability and strongly nonlinear interaction phenomenon that are the main influence factors to the actuations of lightweight soft actuators. In this study, we present a design principle on lightweight pneumatically elastic backbone structure (PEBS) with the modular construction for soft actuators, which contains a backbone printed as one piece and a common strip balloon. We build a prototype of a lightweight (<80 g) soft actuator, which can perform bending motions with satisfactory output forces (∼20 times self-weight). Experiments are conducted on the bending effects generated by interactions between the hyperelastic inner balloon and the elastic backbone. We investigated the nonlinear interaction and system instability experimentally, numerically, and parametrically. To overcome them, we further derived a theoretical nonlinear model and a numerical model. Satisfactory agreements are obtained between the numerical, theoretical, and experimental results. The accuracy of the numerical model is fully validated. Parametric studies are conducted on the backbone geometry and stiffness, balloon stiffness, thickness, and diameter. The accurate controllability, operation safety, modularization ability, and collaborative ability of the PEBS are validated by designing PEBS into a soft laryngoscope, a modularized PEBS library for a robotic arm, and a PEBS system that can operate remote surgery. The reported work provides a further applicability potential of soft robotics studies.

NIR-II Excitable Water-Dispersible Two-Dimensional Conjugated Polymer Nanoplates for In Vivo Two-Photon Luminescence Bioimaging.

While two-dimensional conjugated polymers (2DCPs) have shown great promise in two-photon luminescence (TPL) bioimaging, 2DCP-based TPL imaging agents that can be excited in the second near-infrared window (NIR-II) have rarely been reported so far. Herein, we report two 2DCPs including 2DCP1 and 2DCP2, with octupolar olefin-linked structures for NIR-II-excited bioimaging. The 2DCPs are customized with the fully conjugated donor-acceptor (D-A) linkage and aggregation-induced emission (AIE) active building blocks, leading to good two-photon absorption into the NIR-II window with a 2PACS of ∼64.0 GM per choromophore for both 2DCPs. Moreover, 2DCP1 powders can be exfoliated into water-dispersible nanoplates with a Pluronic F-127 surfactant-assisted temperature-swing method, accompanied by both a drastic reduction of 2PACS throughout the range of 780-1080 nm and a sharp increase of photoluminescence quantum yield to 33.3%. The 2DCP1 nanoplates are subsequently proven to be capable of assisting in visualizing mouse brain vasculatures with a penetration depth of 421 µm and good contrast in vivo, albeit that only 19% of previous 2PACS at 1040 nm is preserved. This work not only provides important insights on how to construct NIR-II excitable 2DCPs for TPL bioimaging but also how to investigate the exfoliation-photophysical property correlation of 2DCPs, which should aid in future research on developing highly efficient TPL bioimaging agents.

The role of hydrodynamics for the spatial distribution of high-temperature hydrothermal vent-endemic fauna in the deep ocean environment.

Active hydrothermal vents provide the surrounding submarine environment with substantial amounts of matter and energy, thus serving as important habitats for diverse megabenthic communities in the deep ocean and constituting a unique, highly productive chemosynthetic ecosystem on Earth. Vent-endemic biological communities gather near the venting site and are usually not found beyond a distance of the order of 100 m from the vent. This is surprising because one would actually expect matter ejected from high-temperature vents, which generate highly turbulent buoyancy plumes, to be suspended and carried far away by the plume flows and deep-sea currents. Here, we study this problem from a fluid dynamics perspective by simulating the vent hydrodynamics using a numerical model that couples the plume flow with induced matter and energy transport. We find that both low- and high-temperature vents deposit most vent matter relatively close to the plume. In particular, the tendency of turbulent buoyancy plumes to carry matter far away is strongly counteracted by generated entrainment flows back into the plume stem. The deposition ranges of organic and inorganic hydrothermal particles obtained from the simulations for various natural high-temperature vents are consistent with the observed maximum spatial extent of biological communities, evidencing that plume hydrodynamics exercises strong control over the spatial distribution of vent-endemic fauna. While other factors affecting the spatial distribution of vent-endemic fauna, such as geology and geochemistry, are site-specific, the main physical features of plume hydrodynamics unraveled in this study are largely site-unspecific and therefore universal across vent sites on Earth.

Nanotopography by chromatic confocal microscopy of the endothelium in Fuchs endothelial corneal dystrophy, pseudophakic bullous keratopathy and healthy corneas.

AIM: To investigate the interest of chromatic confocal microscopy (CCM) to characterise guttae in Fuchs endothelial corneal dystrophy (FECD). METHODS: Descemet's membranes (DM) were obtained during endothelial keratoplasty in patients with FECD and pseudophakic bullous keratopathy (PBK). They were compared with healthy samples obtained from body donation to science. Samples were fixed in 0.5% paraformaldehyde and flat mounted. Surface roughness of DMs was quantified using CCM and the AltiMap software that provided the maximum peak (Sp) and valley (Sv) heights, the mean square roughness (Rq) and the asymmetry coefficient (Ssk). RESULTS: The physiological roughness of healthy samples was characterised by an Rq of 0.12±0.05 µm, which was two times rougher than in PBK (Rq=0.06±0.03 µm), but both were still flat with a symmetrical distribution between peaks and valleys (Ssk close to 0, npeaks=nvalleys), smaller than 1 µm. In FECD, the maximum peak height was 5.10±2.40 µm, up to 5.8 and 8.3 times higher than the control and PBK, respectively. The maximum valley depth was half than the peak (2.28±0.89 µm). The surface with guttae was very rough (Rq=0.45±0.14 µm) and the Ssk=1.84± 0.43 µm, greater than 0, confirms an asymmetric surface with high peaks and low valleys (npeaks>nvalleys). Moreover, the CCM provided quantitative parameters allowing to distinguish different types of guttae from different patients. CONCLUSIONS: CCM is an innovative approach to describe and quantify different morphologies of guttae. It could be useful to analyse the different stages of FECD and define subgroups of patients.

P02-A121†Precut DSAEK stored in an active storage machine: feasibility study.

PURPOSE: The number of endothelial grafts precut by eye banks increases. Their shelf life is limited to a few days. We previously demonstrated the superiority of an active storage machine (ASM) over organ culture (passive) for whole corneas. AIMS: to measure endothelial viability of precut DSAEK after 3 or 10 days of storage in our ASM in a preclinical study. METHODS: Human pairs of corneas were included. The endothelial cell density (ECD in cells/mm2), and central corneal thickness (CCT in µm) were measured to ensure their initial intra pair comparability. After deswelling (CorneaJet, Eurobio) grafts preparation was performed by cutting the anterior stroma with a Moria linear microkeratome and keeping the anterior lamellae attached during storage. After randomization, one cornea was kept in the corneajet bottle (CJ) and the other was inserted into the ASM allowing a renewal or storage medium (CorneaMax, Eurobio) at 2.6 µL/min with 21 mmHg of pressure in the endothelial chamber. Both group of corneas were stored for 3 or 10 days at 31°C. The final viable ECD (vECD) was determined using the triple staining with Hoechst-Ethidium-Calcein-AM by an independent experimenter in a masked fashion. RESULTS: Initial ECDs were comparable: 2595±878 in ASM versus 2654±954 cells/mm2 in CJ for the 3-days period (n=5 pairs) and 2416±712 in ASM versus 2492±764 cells/mm2 in CJ for the 10-period (n=5 pairs). CCTs were also comparable. The anterior lamellae stayed attached in either the ASM or CJ. vECD was significantly higher in ASM than in CJ with respectively 2062±695 cells/mm2 versus 1632±633 cells/mm2 after 3 days either a cell loss of 20.5% and 38.5% respectively (p=0.0062) and 1082±649 versus 935±691 cells/mm2 for the 10-day period either a cell loss of 132% and 164% respectively (p=0.005). Grafts thickness did not differ after 3 days 219±25 µm in ASM versus 182±39 µm (p=0.063) or 10 days respectively 221±58 µm versus 189±48 µm (p=0.06). CONCLUSION: The storage of precut DSAEKs into the ASM allows a better preservation of grafts without use on deswelling storage medium. Nevertheless, the cell loss remains high after 10 days, suggesting a significant cell stress.

P01-A120†Pre-dissected dmek stored in an active storage machine: feasibility study.

PURPOSE: The number of endothelial grafts precut by eye banks increases. Their shelf life is limited to a few days. We previously demonstrated the superiority of an active storage machine (ASM) over organ culture (passive) for whole corneas. AIMS: To measure the endothelial viability of pre-dissected DMEK after 3 and 10 days of storage in our ASM in a preclinical study. METHODS: Pairs of human corneas were included. The endothelial cell density (ECD in cells/mm2), thickness and transparency of corneas were measured before graft preparation. Descemet's membrane (DM) was peeled using the no-touch technique leaving the graft attached to the center of the cornea (on approx. 1mm2). After randomization, one cornea was kept in organ culture (OC) and the other in the ASM (21 mmHg, 2.6 µL/min) in the same medium (CorneaMax, Eurobio). The final viable ECD was determined using the triple staining with Hoechst-Ethidium-Calcein-AM. In addition, the expression of CD166 and NCAM (lateral membranes), ZO-1 (apical junctions), Na+/K+ ATPase (endothelial pump function) and COX-IV (mitochondrial content) was studied by immunostaining to characterize endothelial cells after the storage. RESULTS: Initial ECDs were comparable: 2185±232 cells/mm2 in the ASM versus 2276±328 in OC for the 3-day period and 2680±416 cells/mm2 in the ASM versus 2644±420 in OC for the 10-day period. The DMs did not fold back in either BR or OC. The viable ECD did not differ significantly between the ASM and OC for either storage period: 2378±501 (ASM) versus 2342±503 (OC) for the 3-day period (n=8 pairs and p=0.624) and 2482±288 (ASM) versus 2579±315 (OC) for the 10-day period (n=5 pairs and p=0.176). Corneas were more transparent and thinner in the ASM than in OC after 3 days (916±86 versus 1193±136µm, p=0.0001) and 10 days (957±128 versus 1220±105µm, p=0.0625). The functional and structural markers studied were expressed in both groups after 3 and 10 days, some better preserved in the ASM. CONCLUSION: The storage of precut DMEKs is possible in ASM and OC for at least 10 days. Interestingly, a pre-dissected endothelium continues to partially exert its pump function into the ASM. In practice, this could allow the stroma to be used for DALK without further deswelling. In addition to improving the storage of whole grafts, the ASM allows the storage of precut DMEKs for up to 10 days with excellent endothelial survival.

P33-A110†Ex vivo models of corneal epithelial regeneration in bioreactor: respective roles of limbal, conjunctival and corneal epithelia.

PURPOSE: Human corneas preserved in bioreactor (BR) are characterized by not only a better endothelial viability, but also a more differentiated and stratified epithelium compared to corneas preserved in organoculture. By using corneal preservation in BR, we aimed to analyze the respective contribution of corneal (C), limbal (L), and conjunctival (Conj) epithelia in corneal epithelial regeneration. METHODS: Five pairs of corneas from body donation to Science were used with a death-to-collection time <20 hours. A 3- to 5-mm-wide conjunctival flange was kept intact. Five patterns were set up by complete mechanical removal of 1, 2, or 3 epithelia (-): C-L+Conj+, C-L-Conj+, C-L+Conj-, C+L-Conj-, C-L-Conj- (control) (n=2 for each pattern). The L epithelia was destroyed by scraping and thermocoagulation. Corneas were then kept in BR (21mmHg, 2.5µl/min of Corneamax Eurobio, 31°C) for 3 weeks to allow epithelial regeneration. The epithelium was then analyzed using immunofluorescence (IF) on flat mounted cornea by targeting CK12 (corneal epithelium) and CK15 (limbal epithelium). Cell nuclei were counterstained with DAPI. Corneal transparency was quantified using a transparometer. RESULTS: No epithelium was reconstituted in the C-L-Conj- control group. In the other 4 models including the C-L-Conj+ group, the cornea was transparent and covered by a pluristratified corneal epithelium, characterized by CK12 expression. CONCLUSION: In this BR model, conjunctival epithelial cells alone allowed the regeneration of a typical corneal epithelium whereas corneal epithelium was able to migrate to the limbus and conjunctiva. We hypothesize that all 3 ocular surface epithelia contain stem cells or progenitors able to migrate throughout the cornea and restore the corneal epithelium independently of each other. The main difference between our ex vivo model and in vivo situation is the absence of neovascularization. This suggests that the main cause of limbic insufficiency is due to the loss of the anti-angiogenic barrier rather than the loss of limbic stem cells.

P38-A108†Optimization of the triple HEC staining for laboratory assessment of DMEK graft quality.

INTRODUCTION: The quality of the endothelial graft is critical to the success of DMEK and to the survival time of the graft. The peeling technique, preservation method, and skill level of graft preparers need to be evaluated and validated. The most reliable method of evaluation is the viability test based on a triple staining of Hoechst- Ethidium-Calcein AM (H-E-C) which allows the determination of the total number of viable cells on the graft. However, this test has some shortcomings for DMEK grafts: 1) The undesirable fluorescence of the Calcein AM stain prevents accurate viability analysis, especially in cases where the graft is attached to the cornea for preservation; 2) Incompatibility with immunofluorescence (IF) that could provide additional information. The objective of this study is to develop technical tricks to overcome these drawbacks. METHODS: Two strategies were employed to improve Calcein AM staining: 1. Increase the specific fluorescence intensity by changing the diluent and the concentration of Calcein AM; 2. Decrease undesired fluorescence from keratocytes by adding Trypan Blue (BT). In order to combine the IF after the HEC test, an extension wash in PBS was performed. RESULTS: Calcein AM at 4µM diluted in OptiMEM increased fluorescence intensity 3-fold (p=0.0017, n=5) compared with conventional staining at 2µM in PBS. BT decreased the undesired fluorescence of Calcein and thus optimized count variability between different operators by 42% (p=0.0027, n=10) and saved 40% (p=0.0002, n=10) of count time. To perform IF after HEC, prolonged washing in PBS is an effective method to remove residual Calcein fluorescence and allows release of the FITC/Alexa 488 filter. CONCLUSION: This study provides effective technical tips for optimizing the endothelial viability assay using Calcein AM and for performing IF after the viability assay.

Optimization and Production of Exopolysaccharides (EPS) and Indole-3-Acetic Acid (IAA) Under Chromium by Halophilic Bacteria Oceanobacillus oncorhynchi W4.

The current study assessed the levels of exopolysaccharides (EPS) and indole-3-acetic acid (IAA) and the impacts of halophilic bacteria Oceanobacillus oncorhynchi W4 under Cr (VI). The effects of W4 were tested for its ability to remove Cr (VI) at several concentrations, the removal rate was reached after 48 h at 58.4%, 53.3%, 49.2%, and 43.1%). After 12-48 h, the maximum removal rate of 29-58% was found at an initial concentration of 50 mg/L (Cr (VI)). The Box-Behnken design based on response surface methodology was utilized to optimize the EPS, including pH, sucrose concentration, and incubation period. The highest EPS yield (314.5 mg/L) was obtained under 96 h at pH 7.0, with 5% sucrose concentration. The strain Oceanobacillus oncorhynchi W4 was tested for its ability to create EPS at various concentrations of Cr (VI). After 96 h, it generated the maximum amount of EPS (216.3 mg/L) at a concentration of 50 mg/L. By using FT-IR spectrum measurements, it was confirmed that hexavalent chromium and EPS had surface chemical interactions. At various Cr (VI) concentrations, the isolate W4 was tested for its ability to secrete Indole-3 acetic acid. IAA secretion (control) without Cr (VI) achieved a maximum of 1.45 mg/ml at 120 h. At 200 mg/L Cr (VI) concentration, 1.65 mg/ml of IAA was also produced after 48 h. According to the findings, Oceanobacillus oncorhynchi W4 was a promising isolate in a stressful environment.

Shotgun proteomics identification of proteins expressed in the Descemet's membrane of patients with Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a slowly evolving, bilateral disease of the corneal endothelium, characterized by an abnormal accumulation of extracellular matrix (ECM) in the basement membrane (Descemet's membrane, DM). This results in the formation of small round excrescences, called guttae, and a progressive disappearance of endothelial cells. In the intermediate stage, the numerous guttae create significant optical aberrations, and in the late stage, the loss of endothelial function leads to permanent corneal edema. The molecular components of guttae have not been fully elucidated. In the current study, we conducted shotgun proteomics of the DMs, including guttae, obtained from patients with FECD and revealed that 32 proteins were expressed only in the FECD-DMs but not in the DMs of control subjects. Subsequent enrichment analyses identified associations with multiple ECM-related pathways. Immunostaining of flat-mounted DMs confirmed that 4 of the top 5 identified proteins (hemoglobin α, SRPX2, tenascin-C, and hemoglobin γδεß) were expressed in FECD-DMs but not in non-FECD-DMs. Fibrinogen α was strongly expressed in FECD-DMs, but weakly expressed in non-FECD-DMs. We also demonstrated that matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) can display the in situ spatial distribution of biomolecules expressed in the DM, including the guttae.

Investigating the Role of TGF-ß Signaling Pathways in Human Corneal Endothelial Cell Primary Culture.

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial-mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-ßs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-ß signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-ß1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-ß-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-ß1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-ß receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-ß1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD.

NIR-IIb fluorescence-image guided synergistic surgery/starvation/chemodynamic therapy: an innovative treatment paradigm for malignant non-small cell lung cancers.

Background: Currently, the prognosis and survival rate for patients bearing non-small cell lung cancer (NSCLC) is still quite poor, mainly due to lack of efficient theranostic paradigms to exert in time diagnostics and therapeutics. Methods: Herein, for NSCLC treatment, we offer a customized theranostic paradigm, termed NIR-IIb fluorescence diagnosis and synergistic surgery/starvation/chemodynamic therapeutics, with a newly designed theranostic nanoplatform PEG/MnCuDCNPs@GOx. The nanoplatform is composed of brightly NIR-II emissive downconversion nanoparticles (DCNPs)-core and Mn/Cu-silica shell loaded with glucose oxidase (GOx) to achieve synergistic starvation and chemodynamic therapy (CDT). Results: It is found that 10% Ce3+ doped in the core and 100% Yb3+ doped in the middle shell greatly improves the NIR-IIb emission up to even 20.3 times as compared to the core-shell DCNPs without Ce3+ doping and middle shell. The bright NIR-IIb emission of the nanoplatform contributes to sensitive margin delineation of early-stage NSCLC (diameter < 1 mm) with a signal-to-background ratio (SBR) of 2.18, and further assists in visualizing drug distribution and guiding surgery/starvation/chemodynamic therapy. Notably, the starvation therapy mediated by GOx-driven oxidation reaction efficiently depletes intratumoral glucose, and supplies H2O2 to boost the CDT mediated by the Mn2+ and Cu2+, which consequently realized a highly effective synergistic treatment for NSCLC. Conclusion: This research demonstrates an efficient treatment paradigm for NSCLC with NIR-IIb fluorescence diganosis and image-guided synergistic surgery/starvation/chemodynamic therapeutics.

Melanophages give rise to hyperreflective foci in AMD, a disease-progression marker.

Retinal melanosome/melanolipofuscin-containing cells (MCCs), clinically visible as hyperreflective foci (HRF) and a highly predictive imaging biomarker for the progression of age-related macular degeneration (AMD), are widely believed to be migrating retinal pigment epithelial (RPE) cells. Using human donor tissue, we identify the vast majority of MCCs as melanophages, melanosome/melanolipofuscin-laden mononuclear phagocytes (MPs). Using serial block-face scanning electron microscopy, RPE flatmounts, bone marrow transplantation and in vitro experiments, we show how retinal melanophages form by the transfer of melanosomes from the RPE to subretinal MPs when the "don't eat me" signal CD47 is blocked. These melanophages give rise to hyperreflective foci in Cd47-/--mice in vivo, and are associated with RPE dysmorphia similar to intermediate AMD. Finally, we show that Cd47 expression in human RPE declines with age and in AMD, which likely participates in melanophage formation and RPE decline. Boosting CD47 expression in AMD might protect RPE cells and delay AMD progression.

Comprehensive mass spectrometry lipidomics of human biofluids and ocular tissues.

Evaluating lipid profiles in human tissues and biofluids is critical in identifying lipid metabolites in dysregulated metabolic pathways. Due to various chemical characteristics, single-run lipid analysis has not yet been documented. Such approach is essential for analyzing pathology-related lipid metabolites. Age-related macular degeneration, the leading cause of vision loss in western countries, is emblematic of this limitation. Several studies have identified alterations in individual lipids but the majority are based on targeted approaches. In this study, we analyzed and identified approximately 500 lipid species in human biofluids (plasma and erythrocytes) and ocular tissues (retina and retinal pigment epithelium) using the complementarity of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase chromatography (RPC), coupled to high-resolution mass spectrometry. For that, lipids were extracted from human eye globes and blood from 10 subjects and lipidomic analysis was carried out through analysis in HILIC and RPC, alternately. Furthermore, we illustrate the advantages and disadvantages of both techniques for lipid characterization. RPC showed greater sensitivity in hydrophobicity-based lipid separation, detecting diglycerides, triglycerides, cholesterol, and cholesteryl esters, whereas no signal of these molecules was obtained in HILIC. However, due to coelution, RPC was less effective in separating polar lipids like phospholipids, which were separated effectively in HILIC in both ionization modes. The complementary nature of these analytical approaches was essential for the detection and identification of lipid classes/subclasses, which can then provide distinct insights into lipid metabolism, a determinant of the pathophysiology of several diseases involving lipids, notably age-related macular degeneration.