Impact of keratocyte differentiation on corneal opacity resolution and visual function recovery in male rats.

Intrastromal cell therapy utilizing quiescent corneal stromal keratocytes (qCSKs) from human donor corneas emerges as a promising treatment for corneal opacities, aiming to overcome limitations of traditional surgeries by reducing procedural complexity and donor dependency. This investigation demonstrates the therapeutic efficacy of qCSKs in a male rat model of corneal stromal opacity, underscoring the significance of cell-delivery quality and keratocyte differentiation in mediating corneal opacity resolution and visual function recovery. Quiescent CSKs-treated rats display improvements in escape latency and efficiency compared to wounded, non-treated rats in a Morris water maze, demonstrating improved visual acuity, while stromal fibroblasts-treated rats do not. Advanced imaging, including multiphoton microscopy, small-angle X-ray scattering, and transmission electron microscopy, revealed that qCSK therapy replicates the native cornea's collagen fibril morphometry, matrix order, and ultrastructural architecture. These findings, supported by the expression of keratan sulfate proteoglycans, validate qCSKs as a potential therapeutic solution for corneal opacities.

Mesenchymal Stem Cell Exosomes as Immunomodulatory Therapy for Corneal Scarring.

Corneal scarring is a leading cause of worldwide blindness. Human mesenchymal stem cells (MSC) have been reported to promote corneal wound healing through secreted exosomes. This study investigated the wound healing and immunomodulatory effects of MSC-derived exosomes (MSC-exo) in corneal injury through an established rat model of corneal scarring. After induction of corneal scarring by irregular phototherapeutic keratectomy (irrPTK), MSC exosome preparations (MSC-exo) or PBS vehicle as controls were applied to the injured rat corneas for five days. The animals were assessed for corneal clarity using a validated slit-lamp haze grading score. Stromal haze intensity was quantified using in-vivo confocal microscopy imaging. Corneal vascularization, fibrosis, variations in macrophage phenotypes, and inflammatory cytokines were evaluated using immunohistochemistry techniques and enzyme-linked immunosorbent assays (ELISA) of the excised corneas. Compared to the PBS control group, MSC-exo treatment group had faster epithelial wound closure (0.041), lower corneal haze score (p = 0.002), and reduced haze intensity (p = 0.004) throughout the follow-up period. Attenuation of corneal vascularisation based on CD31 and LYVE-1 staining and reduced fibrosis as measured by fibronectin and collagen 3A1 staining was also observed in the MSC-exo group. MSC-exo treated corneas also displayed a regenerative immune phenotype characterized by a higher infiltration of CD163+, CD206+ M2 macrophages over CD80+, CD86+ M1 macrophages (p = 0.023), reduced levels of pro-inflammatory IL-1ß, IL-8, and TNF-α, and increased levels of anti-inflammatory IL-10. In conclusion, topical MSC-exo could alleviate corneal insults by promoting wound closure and reducing scar development, possibly through anti-angiogenesis and immunomodulation towards a regenerative and anti-inflammatory phenotype.

Parameter Selection for a Microvolume Electrochemical Escherichia coli Detector for Pairing with a Concentration Device.

Waterborne infections are responsible for health problems worldwide and their prompt and sensitive detection in recreational and potable water is of great importance. Bacterial identification and enumeration in water samples ensures water is safe for its intended use. Culture-based methods can be time consuming and are usually performed offsite. There is a need to for automated and distributed at-source detectors for water quality monitoring. Herein we demonstrate a microvolume Escherichia coli (E. coli) detector based on a screen printed electrode (SPE) bioelectroanalytical system and explore to what extent performance can be improved by coupling it with a filtration device. To confidently benchmark detector performance, we applied a statistical assessment method to target optimal detection of a simulated concentrated sample. Our aim was to arrive at a holistic understanding of device performance and to demonstrate system improvements based on these insights. The best achievable detection time for a simulated 1 CFU mL-1 sample was 4.3 (±0.6) h assuming no loss of performance in the filtration step. The real filtered samples fell short of this, extending detection time to 16-18 h. The loss in performance is likely to arise from stress imposed by the filtration step which inhibited microbial growth rates.

Naphthoquinone glycosides for bioelectroanalytical enumeration of the faecal indicator Escherichia coli.

Microbial water quality monitoring for the presence of faecal indicator bacteria (FIB) is a mandatory activity in many countries and is key in public health protection. Despite technological advances and a need for methodological improvements, chromogenic and fluorogenic enzymatic techniques remain the mainstays of water quality monitoring for both public health agencies and regulated utilities. We demonstrated that bioelectroanalytical approaches to FIB enumeration are possible and can be achieved using commercially available enzyme-specific resorufin glycosides, although these are expensive, not widely available or designed for purpose. Following this, we designed two naphthoquinone glycosides which performed better, achieving Escherichia coli detection in the range 5.0 × 102 to 5.0 × 105 CFU ml-1 22-54% quicker than commercially available resorufin glycosides. The molecular design of the naphthoquinone glycosides requires fewer synthetic steps allowing them to be produced for as little as US$50 per kg. Tests with environmental samples demonstrated the low tendency for abiotic interference and that, despite specificity being maintained between ß-glucuronidase and ß-galactosidase, accurate enumeration of E. coli in environmental samples necessitates development of a selective medium. In comparison to a commercially available detection method, which has U.S. Environmental Protection Agency (EPA) approval, our approach performed better at high organism concentrations, detecting 500 organisms in 9 h compared with 13.5 h for the commercial method. Bioelectroanalytical detection is comparable to current approved methods and with further development could result in improved detection times. A recent trend for low-cost open-source hardware means that automated, potentiostatically controlled E. coli detection systems could be constructed for less than US$100 per channel.