Piezo1 channel causes lens sclerosis via transglutaminase 2 activation.

Presbyopia is caused by age-related lenticular hardening, resulting in near vision loss, and it occurs in almost every individual aged ≥50 years. The lens experiences mechanical pressure during for focal adjustment to change its thickness. As lenticular stiffening results in incomplete thickness changes, near vision is reduced, which is known as presbyopia. Piezo1 is a mechanosensitive channel that constantly senses pressure changes during the regulation of visual acuity, and changes in Piezo1 channel activity may contribute to presbyopia. However, no studies have reported on Piezo1 activation or the onset of presbyopia. To elucidate the relevance of Piezo1 activation and cross-linking in the development of presbyopia, we analysed the function of Piezo1 in the lens. The addition of Yoda1, a Piezo1 activator, induced an increase in transglutaminase 2 (TGM2) mRNA expression and activity through the extra-cellular signal-regulated kinase (ERK) 1/2 and c-Jun-NH2-terminal kinase1/2 pathways. In ex vivo lenses, Yoda1 treatment induced γ-crystallin cross-linking via TMG2 activation. Furthermore, Yoda1 eye-drops in mice led to lenticular hardening via TGM2 induction and activation in vivo, suggesting that Yoda1-treated animals could serve as a model for presbyopia. Our findings indicate that this presbyopia-animal model could be useful for screening drugs for lens-stiffening inhibition.

Effect of Ointment Base on the Skin Wound-Healing Deficits in Streptozotocin-Induced Diabetic Rat.

Wound-healing deficits of the skin, one of the most common complications in patients with diabetes, delay wound healing, significantly reducing the patient's QOL. Therefore, the topical treatment of wound areas with drug-containing ointments and dressings is important. In this study, we investigated the effect of various ointment bases on skin wound healing in normal and streptozotocin-induced diabetic rats (STZ rats). Three ointment bases were used: white ointment (oil-based), absorbent cream (emulsion-based, w/o), and macrogol ointment (water-based). Skin wound healing in STZ rats was delayed compared with that in normal rats. Each of the three ointment bases was applied to the skin wound area in normal rats, and there was no difference in the therapeutic effect. The therapeutic effect of both white ointment and absorbent cream was higher in the STZ rats group than that in the non-treated group, and delayed wound healing was observed in STZ rats treated with macrogol ointment. In conclusion, skin wound healing in STZ rats is affected by the properties of the ointment base, and it is important to use an ointment base that controls the drying of the wound area in STZ rats. These findings provide information for the selection of ointment bases useful for application to skin wounds in patients with diabetes.

Application of Granulated Nickel-Aluminum-Zirconium Complex Hydroxide in the Flow Method for Recovery of Chromium(VI) Ions.

A colloidal silicate granulated nickel-aluminum-zirconium (CSG-NAZ) was prepared, and the chromium(VI) (Cr(VI)) ions recovery capacity was evaluated using a sodium sulfate solution in a column experiment. The amount adsorbed and breakthrough time were enhanced by decreasing the flow rate (flow rate is in the order of 3.0 > 2.0 > 0.5 mL). The breakthrough curves and model parameters were estimated using the Thomas and Yoon-Nelson models. The obtained data confirmed to fit both the Yoon-Nelson model (0.858-0.906) and the Thomas model (0.813-0.906). Additionally, Cr(VI) ions that adsorbed onto CSG-NAZ could be desorbed using a sodium sulfate solution in a column experiment. The total recovery percentage of Cr(VI) ions was 80.9% after six repetitions of adsorption/desorption. Finally, the obtained results revealed that CSG-NAZ was a candidate adsorbent for the recovery of Cr(VI) ions owing to its applicability toward a continuous system.

5-ALA/SFC Ameliorates Endotoxin-Induced Ocular Inflammation in Rats by Inhibiting the NF-κB Signaling Pathway and Activating the HO-1/Nrf2 Signaling Pathway.

Sodium ferrous citrate (SFC) is involved in the metabolism of 5-aminolevulinic acid (5-ALA) and enhances its anti-inflammatory effects. The effects of 5-ALA/SFC on inflammation in rats with endotoxin-induced uveitis (EIU) have yet to be elucidated. In this study, during lipopolysaccharide injection, 5-ALA/SFC (10 mg/kg 5-ALA plus 15.7 mg/kg SFC) or 5-ALA (10 or 100 mg/kg) was administered via gastric gavage, wherein we saw that 5-ALA/SFC ameliorated ocular inflammation in EIU rats by suppressing clinical scores; by infiltrating cell counts, aqueous humor protein, and inflammatory cytokine levels; and by improving histopathological scores to the same extent as 100 mg/kg 5-ALA. Immunohistochemistry showed that 5-ALA/SFC suppressed iNOS and COX-2 expression, NF-κB activation, IκB-α degradation, and p-IKKα/ß expression, and activated HO-1 and Nrf2 expression. Therefore, this study has investigated how 5-ALA/SFC reduces inflammation and revealed the pathways involved in EIU rats. 5-ALA/SFC is shown to inhibit ocular inflammation in EIU rats by inhibiting NF-κB and activating the HO-1/Nrf2 pathways.

Sodium Ferrous Citrate and 5-Aminolevulinic Acid Exert a Therapeutic Effect on Endotoxin-Induced Uveitis in Rats.

The metabolism of 5-aminolevulinic acid (ALA) is more efficient when combined with sodium ferrous citrate (SFC). Our previous study revealed that oral administration of ALA, which has anti-inflammatory properties, and SFC (ALA/SFC) immediately before lipopolysaccharide (LPS) inoculation suppressed endotoxin-induced uveitis (EIU) in rats. However, the therapeutic effect of ALA/SFC post-administration remains unexplored. Hence, this study aimed to evaluate the therapeutic efficacy of ALA/SFC on EIU in rats, which were administered with a gastric gavage of ALA/SFC (100/157 mg/kg) or prednisolone (Pred, 10 mg/kg) after 4 h of LPS inoculation. The treatment groups showed ameliorated clinical scores, inflammatory cells, protein levels in the aqueous humor (AqH), and histopathologic evaluation 24 h after LPS inoculation. Furthermore, the treatment groups had reduced tumor necrosis factor-α, nitric oxide, prostaglandin E2, and interleukin-6 levels in the AqH. ALA/SFC demonstrated an anti-inflammatory effect equivalent to that demonstrated by Pred. These findings indicate that ALA/SFC exerts a therapeutic effect on EIU in rats, indicating its clinical usefulness in uveitis treatment.

Aquaporins contribute to vacuoles formation in Nile grass type II diabetic rats.

Regulation of ion and water microcirculation within the lens is tightly controlled through aquaporin channels and connexin junctions. However, cataracts can occur when the lens becomes cloudy. Various factors can induce cataracts, including diabetes which is a well-known cause. The most common phenotype of diabetic cataracts is a cortical and/or posterior subcapsular opacity. In addition to the three main types and two subtypes of cataracts, a vacuole formation is frequently observed; however, their origin remains unclear. In this study, we focused on the aquaporins and connexins involved in diabetes-induced cataracts and vacuoles in Nile grass type II diabetes. The results showed that the expression of aquaporin 0 and aquaporin 5 increased, and that of connexin 43 decreased in diabetic rat lenses. Additionally, aquaporin 0 and 5 were strongly localized in peripheral of vacuoles, suggesting that aquaporins are involved in vacuoles formation. Transillumination photography revealed large vacuoles at the tip of the Y-suture in the anterior capsule of the diabetic lens, and several small vacuoles were observed in the posterior capsule. Within the vacuoles, cytoplasmic degradation and aggregation of fibrous material were observed. Our findings suggest that aquaporins are potential candidate proteins for preventing vacuole formation.

Role of mucoadhesive agent in ocular delivery of ganciclovir microemulsion: cytotoxicity evaluation in vitro and ex vivo.

PURPOSE: The aim of the present study was to investigate increase in delivery of drug upon formulation as mucoadhesive microemulsion system and further to investigate possibility of any cytotoxic effects using such formulation. MATERIAL AND METHODS: Considering hydrophilic and small molecular nature of the drug, it was attempted to be formulated as microemulsion, by using pseudo ternary phase diagram method. Thus, three types of microemulsions were prepared; oil in water, water in oil type and chitosan-coated microemulsion. These microemulsions were characterized for several physicochemical properties like size, zeta potential, Polydispersity index, and compared for in vitro cell viability and ex vivo corneal irritation study. RESULTS: All three microemulsions were quite stable, transparent and homogenous systems. They showed similar drug release pattern, but highest ex vivo goat corneal permeation was observed with Chitosan coated microemulsion when compared with ganciclovir solution. CONCLUSION: All microemulsions were found to be non-irritant in in vitro cell viability assay and ex vivo corneal irritation study, indicating the potential of using such systems for delivery of drug to eye.

Optimization of the Hydrothermal Activation Treatment with Sodium Hydroxide Solution for the Conversion of Coal Fly Ash to Zeolite and Its Adsorption Capability of Lead (II) Ions from the Liquid Phase.

Coal fly ash (FA) was treated by hydrothermal activation with sodium hydroxide solution at different concentrations to optimize the conversion method. Zeolite of the sodium type is prepared from coal FA by 1, 1.5, and 3 mol/L sodium hydroxide solutions (ZE1, ZE1.5, and ZE3). These adsorbents' morphology, crystal structure, scanning electron microscopy, Fourier transform (FT)-IR spectra, cation exchange capacity (CEC), specific surface area and pore volumes, and pHpzc were determined. An adsorption experiment was performed to evaluate the effects of contact time, pH, temperature, and coexistence. From the results, the values of CEC and specific surface area of prepared samples was in the order ZE3 < ZE1.5 < ZE1. The similar trends were observed in lead ions adsorption. In addition, our obtained data elucidate that the ion exchange with sodium ions in the interlayer ZE1 is one of the adsorption mechanisms of Pb2+ from water layer. Finally, lead ions adsorbed on ZE1 could be desorbed using a hydrochloric acid solution, showing that ZE1 could be reused as a water purification agent.

N,N-Diethyl-3-toluamide Formulation Based on Ethanol Containing 0.1% 2-Hydroxypropyl-ß-cyclodextrin Attenuates the Drug's Skin Penetration and Prolongs the Repellent Effect without Stickiness.

N,N-diethyl-3-toluamide (DEET) is one of the most widely used insect repellents in the world. It was reported that a solution containing 6-30% cyclodextrin (CD) as a solvent instead of ethanol (EtOH) provided an enhancement of the repellent action time duration of the DEET formulation, although the high-dose CD caused stickiness. In order to overcome this shortcoming, we attempted to prepare a 10% DEET formulation using EtOH containing low-dose CDs (ß-CD, 2-hydroxypropyl-ß-CD (HPßCD), methyl-ß-CD, and sulfobutylether-ß-CD) as solvents (DEET/EtOH/CD formulations). We determined the CD concentration to be 0.1% in the DEET/EtOH/CD formulations, since the stickiness of 0.1% CDs was not felt (approximately 8 × 10-3 N). The DEET residue on the skin superficial layers was prolonged, and the drug penetration into the skin tissue was decreased by the addition of 0.1% CD. In particular, the retention time and attenuated penetration of DEET on the rat skin treated with the DEET/EtOH/HPßCD formulation was significantly higher in comparison with that of the DEET/EtOH formulation without CD. Moreover, the repellent effect of DEET was more sustained by the addition of 0.1% HPßCD in the study using Aedes albopictus. In conclusion, we found that the DEET/EtOH/HPßCD formulations reduced the skin penetration of DEET and prolonged the repellent action without stickiness.

Iris-derived induced pluripotent stem cells that express GFP in all somatic cells of mice and differentiate into functional retinal neurons.

When regenerated tissue is generated from induced pluripotent stem cells (iPSCs), it is necessary to track and identify the transplanted cells. Fluorescently-labeled iPSCs synthesize a fluorescent substance that is easily tracked. However, the expressed protein should not affect the original genome sequence or pluripotency. To solve this problem, we created a cell tool for basic research on iPSCs. Iris tissue-derived cells from GFP fluorescence-expressing mice (GFP-DBA/2 mice) were reprogrammed to generate GFP mouse iris-derived iPSCs (M-iris GFP iPSCs). M-iris GFP iPSCs expressed cell markers characteristic of iPSCs and showed pluripotency in differentiating into the three germ layers. In addition, when expressing GFP, the cells differentiated into functional recoverin- and calbindin-positive cells. Thus, this cell line will facilitate future studies on iPSCs.

Improvement in adsorption of Hg2+ from aqueous media using sodium-type fine zeolite grains.

To increase the adsorption capability of Hg2+ from aqueous media, we prepared sodium-type fine zeolite grains with various particle sizes (denoted as ZE1, ZE2 and ZE3). The particle sizes of ZE1, ZE2 and ZE3 were 16.363 ± 0.365, 1.454 ± 0.357 and 0.607 ± 0.377 µm, respectively. Moreover, the CEC, specific surface area and pore volume were in the order ZE1 (42 mmol/g and 23.5 m2/g) < ZE2 (72 mmol/g and 67.1 m2/g) < ZE3 (135 mmol/g and 176.6 m2/g). Subsequently, the Hg2+ adsorption capability was investigated. The performance of tested agents on Hg2+ adsorbed was in the order ZE1 (5.0 mg/g) < ZE2 (9.4 mg/g) < ZE3 (20.2 mg/g). It was concluded that fine crystalline zeolite was important in enhancing the adsorption capability of Hg2+. In addition, the mechanism of adsorption of Hg2+ on the ZE samples was evaluated. Our results suggested that Hg2+ was exchanged with sodium ions in the interlayers of ZE samples with correlation coefficients of 0.966-0.979. Our findings revealed that these ZE samples constitute potential agents for the adsorption of Hg2+ from aqueous media.

Capsaicin attenuates TGFß2-induced epithelial-mesenchymal-transition in lens epithelial cells in vivo and in vitro.

Posterior capsule opacification (PCO), the most common complication of cataract surgery occurring in 20-50% of patients after 2-5 years of cataract surgery, is a major problem in the aging society. The epithelial-mesenchymal transition (EMT) of lens epithelial cells after cataract surgery has been proposed as a major cause of PCO. Capsaicin, widely used as a food additive and analgesic agent, is a major pungent ingredient in red pepper. Although the effect of capsaicin on EMT has been reported in cancer cells, the biological reaction of capsaicin was unique in each cell type, and there have been no reports describing its effects on EMT earlier. In this study, we demonstrated that treatment with capsaicin inhibited TGFß2-induced EMT in vitro lens epithelial cells and ex vivo explant lens epithelial cells. Furthermore, eye drops of capsaicin inhibited the PCO model mice in vivo. Finally, we showed that capsaicin inhibited non-canonically induced Smad2/3 activation via suppression of EGFR activation and ERK phosphorylation. Our findings indicate that capsaicin and its derivatives are good candidate compounds for preventing PCO after cataract surgery.

Exploiting the Different Parameters on the Adsorption of Phosphate Ions and Its Subsequent Recovery Using Complex Nickel-Aluminum-Zirconium Hydroxide.

In this study, the effect of contact time, temperature, pH, and coexistences on the adsorption of phosphate ions using the complex nickel-aluminum-zirconium hydroxide (NAZ) was evaluated. Moreover, the recovery of adsorbed phosphate ions from NAZ using desorption solution with different concentrations was demonstrated. The results showed that the quantity of phosphate ions adsorbed gradually increased with time, and the adsorption equilibrium was achieved within 24 h after adsorption. This kinetic data could be well described by the pseudo-second-order model with the correlation coefficient in the value of 0.997. Additionally, the quantity of phosphate which was adsorbed increased as temperature increased, and these results corresponded well with both the Langmuir, the correlation coefficient ranged from 0.920-0.949, and Freundlich models, the correlation coefficient ranged from 0.863-0.995. These results showed that the adsorption of phosphate ion was monolayer adsorption onto the NAZ surface. The optimal pH for removal of phosphate ions from aqueous media was during 4-8. In addition, chloride, nitrate, and sulfate ions did not significantly affect to the adsorption capability of phosphate ions in the complex solution system. Finally, the phosphate ions which were adsorbed onto NAZ could be recovered using sodium sulfate solution (recovery percentage: approx. 50% using sodium sulfate solution at 1000 mmol/L). These results highlight the potential of using NAZ as the cost-effectiveness adsorbent for phosphate ions removal from aqueous media.

Energy-Dependent Endocytosis Is Responsible for Skin Penetration of Formulations Based on a Combination of Indomethacin Nanoparticles and l-Menthol in Rat and Göttingen Minipig.

We previously designed a Carbopol gel formulation (N-IND/MEN) based on a combination of indomethacin solid nanoparticles (IND-NPs) and l-menthol, and we reported that the N-IND/MEN showed high transdermal penetration. However, the detailed mechanism for transdermal penetration of IND-NPs was not clearly defined. In this study, we investigated whether endocytosis in the skin tissue of rat and Göttingen minipig is related to the transdermal penetration of IND-NPs using pharmacological inhibitors of endocytosis. The pharmacological inhibitors used in this study are as follows: 54 µM nystatin, a caveolae-mediated endocytosis (CavME) inhibitor; 40 µM dynasore, a clathrin-mediated endocytosis (CME) inhibitor; and 2 µM rottlerin, a micropinocytosis (MP) inhibitor. The N-IND/MEN was prepared by a bead mill method, and the particle size of solid indomethacin was 79-216 nm. In both rat and Göttingen minipig skin, skin penetration of approximately 80% IND-NPs was limited by the stratum corneum (SC), although the penetration of SC was improved by the combination of l-menthol. On the other hand, the treatment of nystatin and dynasore decreased the transdermal penetration of indomethacin in rats and Göttingen minipigs treated with N-IND/MEN. Moreover, in addition to nystatin and dynasore, rottlerin attenuated the transdermal penetration of IND-NPs in the Göttingen minipigs' skin. In conclusion, we found that l-menthol enhanced the SC penetration of IND-NPs. In addition, this study suggests that the SC-passed IND-NPs are absorbed into the skin tissue by energy-dependent endocytosis (CavME, CME, and/or MP pathways) on the epidermis under the SC, resulting in an enhancement in transdermal penetration of IND-NPs. These findings provide significant information for the design of nanomedicines in transdermal formulations.

Regulation of the Membrane Trafficking of the Mechanosensitive Ion Channels TRPV1 and TRPV4 by Zonular Tension, Osmotic Stress and Activators in the Mouse Lens.

Lens water transport generates a hydrostatic pressure gradient that is regulated by a dual-feedback system that utilizes the mechanosensitive transient receptor potential vanilloid (TRPV) channels, TRPV1 and TRPV4, to sense changes in mechanical tension and extracellular osmolarity. Here, we investigate whether the modulation of TRPV1 or TRPV4 activity dynamically affects their membrane trafficking. Mouse lenses were incubated in either pilocarpine or tropicamide to alter zonular tension, exposed to osmotic stress, or the TRPV1 and TRPV4 activators capsaicin andGSK1016790A (GSK101), and the effect on the TRPV1 and TRPV4 membrane trafficking in peripheral fiber cells visualized using confocal microscopy. Decreases in zonular tension caused the removal of TRPV4 from the membrane of peripheral fiber cells. Hypotonic challenge had no effect on TRPV1, but increased the membrane localization of TRPV4. Hypertonic challenge caused the insertion of TRPV1 and the removal of TRPV4 from the membranes of peripheral fiber cells. Capsaicin caused an increase in TRPV4 membrane localization, but had no effect on TRPV1; while GSK101 decreased the membrane localization of TRPV4 and increased the membrane localization of TRPV1. These reciprocal changes in TRPV1/4 membrane localization are consistent with the channels acting as mechanosensitive transducers of a dual-feedback pathway that regulates lens water transport.

Morphological comparison between three-dimensional structure of immortalized human lens epithelial cells and Soemmering's ring.

The incidence rate of post-cataract surgery posterior capsule opacification (PCO) and lens turbidity is about 20% in 5 years. Soemmering's ring, which is a type of PCO also called a regenerated lens with similar tissue structure to that of a human lens, is an important proxy for elucidating the mechanism of lens regeneration and maintenance of transparency. The authors created new human immortalized crystalline lens epithelial cells (iHLEC-NY1s) with excellent differentiation potential, and as a result of culturing the cells by static and rotation-floating methods, succeeded in producing a three-dimensional cell structure model (3D-iHLEC-NY1s) which is similar to Soemmering's ring in tissue structure and expression characteristics of αA-crystalline, ßB2-crystalline, vimentin proteins. 3D-iHLEC-NY1s is expected to be a proxy in vitro experimental model of Soemmering's ring to enable evaluation of drug effects on suppression of cell aggregate formation and transparency. By further improving the culture conditions, we aim to control the cell sequence and elucidate the mechanism underlying the maintenance of lens transparency.

Changes in Percutaneous Absorption of Fentanyl Patches in Rats Treated with a Sebum-Like Secretion.

The percutaneous absorption of a fentanyl (FEN)-patch is affected by various external factors including the volume of sebum secretion, which causes changes in the skin surface environment. In this study, we prepared a lard-based sebum-like secretion (SLS), and applied it to investigate the effect of different skin surface conditions on the drug penetration of a FEN-patch. In vitro work to test drug release using the Franz diffusion cell indicated that drug release was significantly suppressed by treatment with 5% SLS, which is equivalent to the amount of daily human sebum secretion. Conversely, in ex vivo experiments using rat skin, the amount of FEN that accumulated in the skin tissue of the 5% SLS-treated rats was higher in comparison with the non-SLS treated group. Furthermore, in vivo experiments indicated that the plasma FEN concentration in rats treated with the FEN-patch was significantly increased by treatment with 5% SLS. These results suggest that the sebum affected the release, accumulation, and absorption of FEN from the FEN-patch, and the FEN concentration in the blood was reflected by the balance of the suppression of drug release and the enhancement of drug accumulation in the skin with SLS.

Effects of the Ophthalmic Additive Mannitol on Antimicrobial Activity and Corneal Toxicity of Various Preservatives.

Ophthalmic preservatives are indispensable in eye drop formulations, but may be toxic to corneal structures. Corneal damage necessitates the discontinuation of treatment with ophthalmic solutions. Therefore, the development of a new and safe preservative system without corneal toxicity is needed. The present study investigated the effects of mannitol on the antimicrobial activities and corneal toxicities of various preservatives using Escherichia coli and a human corneal epithelial cell line (HCE-T cells). The following preservatives were examined: boric acid (BA), benzalkonium chloride (BAC), methyl parahydroxybenzoate (MP), propyl parahydroxybenzoate (PP), sodium chlorite (SC), and zinc chloride (ZC). The antimicrobial activities and HCE-T-cell toxicities of 50 µg/mL BA, MP, PP, SC, and ZC were reduced by a co-treatment with mannitol (0-300 µg/mL). The suppressed antimicrobial activities of BA, MP, PP, and SC by the co-treatment with mannitol were restored by the application of a mannitol content higher than 500 µg/mL. In contrast to these 5 preservatives, the addition of mannitol did not affect the antimicrobial activity of BAC and attenuated its HCE-T cell toxicity. Therefore, the balance between the contents of mannitol and preservatives is important in co-treatments. The present results will serve as a guide for the future development of eye drop formulations without corneal toxicity.

A Combination of Cryopreservation and Kneading Maintains the Usability of Mohs Paste.

Mohs paste is useful for controlling exudates from wounds and infections and is used to treat patients with inoperable skin tumors. Unfortunately, Mohs paste is difficult to preserve because its viscosity and stickiness increase dramatically immediately after preparation, resulting in decreased usability. In this study, the combined use of cryopreservation and kneading was shown to improve long-term storage of Mohs paste. At 25°C, Mohs pastes solidified rapidly, and viscosity reached approximately 700 Pa·s 5 h after preparation. In contrast, cryopreservation at -20°C attenuated hardening of Mohs pastes, and kneading also decreased viscosity. The viscosity of Mohs pastes cotreated with cryopreservation and kneading after 7 months of storage was <70 Pa·s. In addition, tissue invasion with these stored pastes was similar to freshly prepared Mohs paste. Results suggest that the combination of cryopreservation and kneading permits Mohs paste to be stored over extended periods, which may increase the utility of the paste for clinical use.

Correction: Sato, K., et al. Identification of a Novel Oligosaccharide in Maple Syrup as a Potential Alternative Saccharide for Diabetes Mellitus Patients. Int. J. Mol. Sci. 2019, 20, 5041.

The authors wish to make the following corrections to this paper [...].