Human Lens Research Articles

A De Novo Noncoding RARB Variant Associated with Complex Microphthalmia Alters a Putative Regulatory Element.

Retinoic acid receptor beta (RARB) is a transcriptional regulator crucial for coordinating retinoic acid- (RA-) mediated morphogenic movements, cell growth, and differentiation during eye development. Loss- or gain-of-function RARB coding variants have been associated with microphthalmia, coloboma, and anterior segment defects. We identified a de novo variant c.157+1895G>A located within a conserved region (CR1) in the first intron of RARB in an individual with complex microphthalmia and significant global developmental delay. Based on the phenotypic overlap, we further investigated the possible effects of the variant on mRNA splicing and/or transcriptional regulation through in silico and functional studies. In silico analysis identified the possibility of alternative splicing, suggested by one out of three (HSF, SpliceAI, and MaxEntScan) splicing prediction programs, and a strong indication of regulatory function based on publicly available DNase hypersensitivity, histone modification, chromatin folding, and ChIP-seq data sets. Consistent with the predictions of SpliceAI and MaxEntScan, in vitro minigene assays showed no effect on RARB mRNA splicing. Evaluation of CR1 for a regulatory role using luciferase reporter assays in human lens epithelial cells demonstrated a significant increase in the activity of the RARB promoter in the presence of wild-type CR1. This activity was further significantly increased in the presence of CR1 carrying the c.157+1895G>A variant, suggesting that the variant may promote RARB overexpression in human cells. Induction of RARB overexpression in human lens epithelial cells resulted in increased cell proliferation and elevated expression of FOXC1, a known downstream target of RA signaling and a transcription factor whose down- and upregulation is associated with ocular phenotypes overlapping the RARB spectrum. These results support a regulatory role for the CR1 element and suggest that the de novo c.157+1895G>A variant affecting this region may alter the proper regulation of RARB and, as a result, its downstream genes, possibly leading to abnormal development.

Exploring the Ocular Absorption Pathway of Fasudil Hydrochloride towards Developing a Nanoparticulate Formulation with Improved Performance.

Rho-kinase (ROCK) inhibitors represent a new category of anti-glaucoma medications. Among them, Fasudil hydrochloride, a selective ROCK inhibitor, has demonstrated promising outcomes in glaucoma treatment. It works by inhibiting the ROCK pathway, which plays a crucial role in regulating the trabecular meshwork and canal of Schlemm's aqueous humor outflow. This study aims to investigate the ocular absorption pathway of Fasudil hydrochloride and, subsequently, develop a nanoparticle-based delivery system for enhanced corneal absorption. Employing the ionic gelation method and statistical experimental design, the factors influencing chitosan nanoparticle (Cs NP) characteristics and performance were explored. Fasudil in vitro release and ex vivo permeation studies were performed, and Cs NP ocular tolerability and cytotoxicity on human lens epithelial cells were evaluated. Permeation studies on excised bovine eyes revealed significantly higher Fasudil permeation through the sclera compared to the cornea (370.0 μg/cm2 vs. 96.8 μg/cm2, respectively). The nanoparticle size (144.0 ± 15.6 nm to 835.9 ± 23.4 nm) and entrapment efficiency range achieved (17.2% to 41.4%) were predominantly influenced by chitosan quantity. Cs NPs showed a substantial improvement in the permeation of Fasudil via the cornea, along with slower release compared to the Fasudil aqueous solution. The results from the Hen's Egg Test Chorioallantoic Membrane (HET-CAM) and Bovine Corneal Opacity and Permeability (BCOP) tests indicated good conjunctival and corneal biocompatibility of the formulated chitosan nanoparticles, respectively. Lens epithelial cells displayed excellent tolerance to low concentrations of these nanoparticles (>94% cell viability). To the best of our knowledge, this is the first report on the ocular absorption pathway of topically applied Fasudil hydrochloride where the cornea has been identified as a potential barrier that could be overcome using Cs NPs.

Reduction of ETV1 is Identified as a Prominent Feature of Age-Related Cataract

Purpose To identify the inactive genes in cataract lenses and explore their function in lens epithelial cells (LECs). Methods Lens epithelium samples obtained from both age-related cataract (ARC) patients and normal donors were subjected to two forms of histone H3 immunoprecipitation: H3K9ac and H3K27me3 chromatin immunoprecipitation (ChIP), followed by ChIP-seq. The intersection set of “active genes in normal controls” and “repressed genes in cataract lenses” was identified. To validate the role of a specific gene, ETV1, within this set, quantitative polymerase chain reaction (qPCR), western blot, and immunofluorescence were performed using clinical lens epithelium samples. Small interference RNA (siRNA) was utilized to reduce the mRNA level of ETV1 in cultured LECs. Following this, transwell assay and western blot was performed to examine the migration ability of the cells. Furthermore, RNA-seq analysis was conducted on both cell samples with ETV1 knockdown and control cells. Additionally, the expression level of ETV1 in LECs was examined using qPCR under H2O2 treatment. Results Six genes were identified in the intersection set of “active genes in normal controls” and “repressed genes in ARC lenses”. Among these genes, ETV1 showed the most significant fold-change decrease in the cataract samples compared to the control samples. After ETV1 knockdown by siRNA in cultured LECs, reduced cell migration was observed, along with a decrease in the expression of β-Catenin and Vimentin, two specific genes associated with cell migration. In addition, under the oxidative stress induced by H2O2 treatment, the expression level of ETV1 in LECs significantly decreased. Conclusions Based on the findings of this study, it can be concluded that ETV1 is significantly reduced in human ARC lenses. The repression of ETV1 in ARC lenses appears to contribute to the disrupted differentiation of lens epithelium, which is likely caused by the inhibition of both cell differentiation and migration processes.

Revamped mini-αA-crystallin showed improved skin permeation and therapeutic activity against melittin-induced toxicity

Melittin is honey bee venom's primary and most toxic pharmacologically active component. Melittin causes haemolysis, lymphocyte lysis, long-term pain, localised inflammation followed by rhabdomyolysis, and severe renal failure. Renal failure or cardiovascular complications could lead to the victim's death. Severe honey bee bites are treated with general medication involving antihistaminic, anti-inflammatory, and analgesic drugs, as a specific treatment option is unavailable. An earlier study showed the anti-hemolysis and anti-lymphocyte lysis activity of mini- αA-crystallin (MAC), a peptide derived from human eye lens alpha-crystallin. MAC's use has often been restricted despite its high therapeutic potential due to its poor skin permeability. This study compared the skin permeation, anti-inflammatory and analgesic activities of natural peptide MAC and its modified version (MAC-GRD) formed by attaching cell-penetrating peptide (CPP) and GRD amino residues into MAC. Gel formulations were prepared for MAC and MAC-GRD peptides using carbopol (1% w/w), Tween 80 (1%), and ethanol (10%). An ex-vivo skin permeation study was performed using a vertical-type Franz diffusion apparatus. Preclinical in-vivo experiments were conducted to compare the native and modified peptide formulations against melittin-induced toxicity in Wistar rats. MAC gel, MAC–GRD gel and 1% hydrocortisone cream significantly reduced the melittin-induced writhing (20.16 ± 0.792) response in rats with 15.16 ± 0.47, 11.16 ± 0.477 and 12.66 ± 0.66 wriths, respectively. There was a significant reduction in melittin-induced inflammation when MAC-GRD gel was applied immediately after melittin administration. At 0.5, 1, 3, and 5 h, the MAC-GRD-treated rat paws were 0.9 ± 0.043 mm, 0.750 ± 0.037 mm, 0.167 ± 0.0070 mm, and 0.133 ± 0.031 mm thick. Administration of melittin resulted in reduced GSH (antioxidant) levels (47.33 ± 0.760 μg/mg). However, treatment with MAC-GRD gel (71.167 ± 0.601 μg/mg), MAC gel (65.167 ± 1.138 μg/mg), and 1% hydrocortisone (68.33 ± 0.667 μg/mg) significantly increased the antioxidant enzyme levels. MAC-GRD gel significantly reduced the elevated MDA levels (6.933 ± 0.049 nmol/mg) compared to the melittin group (12.533 ± 0.126 nmol/mg), followed by the 1% hydrocortisone (7.367 ± 0.049 nmol/mg) and MAC gel (7.917 ± 0.048 nmol/mg). MAC-GRD demonstrated more skin permeability and superior anti-inflammatory, analgesic, and antioxidant activities when compared to MAC gel. When compared to standard 1% hydrocortisone cream, MAC-GRD had better anti-inflammatory, analgesic, antioxidant, and comparable action in anti-oxidant restoration against melittin. These findings suggest that the developed MAC-GRD gel formulation could help to treat severe cases of honey bee stings.

A framework for Frizzled-G protein coupling and implications to the PCP signaling pathways

The ten Frizzled receptors (FZDs) are essential in Wnt signaling and play important roles in embryonic development and tumorigenesis. Among these, FZD6 is closely associated with lens development. Understanding FZD activation mechanism is key to unlock these emerging targets. Here we present the cryo-EM structures of FZD6 and FZD3 which are known to relay non-canonical planar cell polarity (PCP) signaling pathways as well as FZD1 in their G protein-coupled states and in the apo inactive states, respectively. Comparison of the three inactive/active pairs unveiled a shared activation framework among all ten FZDs. Mutagenesis along with imaging and functional analysis on the human lens epithelial tissues suggested potential crosstalk between the G-protein coupling of FZD6 and the PCP signaling pathways. Together, this study provides an integrated understanding of FZD structure and function, and lays the foundation for developing therapeutic modulators to activate or inhibit FZD signaling for a range of disorders including cancers and cataracts.

Mapping a research-advocacy-policy agenda on human rights and albinism: a mixed methods project.

Persons with albinism face challenges to their wellbeing, safety, and security, ranging from vision impairment and skin cancer to stigma and discrimination. In some regions, they also face human rights atrocities including mutilation and murder. Research on human rights and albinism is a relatively new field that has gained momentum since the United Nations appointment of an Independent Expert on the enjoyment of human rights by persons with albinism. In this paper, we present the results of a mixed methods study undertaken to identify priorities for research, advocacy, and policy on albinism and human rights. The first component was a synthesis of peer-reviewed and grey literatures at the nexus of albinism, spiritual/cultural beliefs and practices, and human rights. We then conducted a priority-setting survey, informed by Delphi methods, on extant knowledge-practice gaps and research, advocacy, and policy priorities. Inclusion criteria included demonstrated expertise in the field (e.g., peer-reviewed publications, funded research), membership on national or international associations, or advocacy (civil society organizations) of more than 2years in albinism and human rights. Thereafter, we gathered leading researchers, policy-makers, and civil society stakeholders for a Roundtable to gain consensus on these priorities. Access to skin and vision care, and education were not deemed high priority for research, likely because the evidence supporting the need for these is well established. However, they were priorities for advocacy and policy: what is needed is mobilization of this evidence through advocacy and implementation of such services (policy). Other social determinants of health (rurality, poverty, and gender equality) are present as subtext in the findings, more so than priorities for research, advocacy, or policy, despite their preponderance in the lives of persons with albinism. Research was prioritized on stigma and discrimination; advocacy; and witchcraft, but with some differentiation between Global North and Global South priorities. Priorities for research, advocacy, and policy vary in keeping with the explanatory frameworks at play, including how harmful practices and witchcraft are viewed. The lived experience of albinism is profoundly shaped by the social determinants of health (SDOH). Threats to the security and well-being of persons with albinism should be viewed through a human rights lens that encompasses the explanatory frameworks at play.

Fabrication of a 3D bioprinting model for posterior capsule opacification using GelMA and PLMA hydrogel-coated resin.

Posterior capsule opacification (PCO) remains the predominant complication following cataract surgery, significantly impairing visual function restoration. In this study, we developed a PCO model that closely mimics the anatomical structure of the crystalline lens capsule post-surgery. The model incorporated a threaded structure for accurate positioning and observation, allowing for opening and closing. Utilizing 3D printing technology, a stable external support system was created using resin material consisting of a rigid, hollow base and cover. To replicate the lens capsule structure, a thin hydrogel coating was applied to the resin scaffold. The biocompatibility and impact on cellular functionality of various hydrogel compositions were assessed through an array of staining techniques, including calcein-AM/PI staining, rhodamine staining, BODIPY-C11 staining and EdU staining in conjunction with transwell assays. Additionally, the PCO model was utilized to investigate the effects of eight drugs with anti-inflammatory and anti-proliferative properties, including 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), THZ1, sorbinil, 4-octyl itaconate (4-OI), xanthohumol, zebularine, rapamycin and caffeic acid phenethyl ester, on human lens epithelial cells (HLECs). Confocal microscopy facilitated comprehensive imaging of the PCO model. The results demonstrated that the GelMA 60 5% + PLMA 2% composite hydrogel exhibited superior biocompatibility and minimal lipid peroxidation levels among the tested hydrogels. Moreover, compared to using hydrogel as the material for 3D printing the entire model, applying surface hydrogel spin coating with parameters of 2000 rpm × 2 on the resin-based 3D printed base yielded a more uniform cell distribution and reduced apoptosis. Furthermore, rapamycin, 4-OI and AICAR demonstrated potent antiproliferative effects in the drug intervention study. Confocal microscopy imaging revealed a uniform distribution of HLECs along the anatomical structure of the crystalline lens capsule within the PCO model, showcasing robust cell viability and regular morphology. In conclusion, the PCO model provides a valuable experimental platform for studying PCO pathogenesis and exploring potential therapeutic interventions.

3D visualization of cellular and molecular distributions in human crystalline lenses at different ages.

The human lens is a highly organized tissue, and it is constructed of delicate inner architectures that ensure its transparency. However, the pattern of cell distribution in the intact lens has rarely been observed or traced in a three-dimensional (3D) perspective. Here, we modified and compared three different kinds of tissue transparency methods to investigate the cellular and molecular changes in the human lens at different ages from a 3D perspective. First, we analyzed the general 3D parameters of cleared human lenses from 6 months to 72 years of age and found that the equator proportion remained constant with age (23.05% ± 0.36). Next, we visualized the cellular distribution patterns in the anterior capsule and equator, as well as the distribution of cortical fiber cells. Interestingly, we observed the accumulation of equatorial epithelium in adolescents and the asymmetrical denucleation of cortical fiber cells in the elderly. Zonula occludens-1 and tropomyosin receptor kinase A were also identified in the pre-equatorial germinative zone, and its presence decreased when comparing lenses of a 17-year-old to those of a 49-year-old. We present a 3D cellular and molecular reconstruction of the human lens, illustrating the observed alterations in human lens epithelial cells across different ages.

Chemical Castration in Indonesian Criminal Law: Human Rights Perspective

This study delves into the controversial practice of chemical castration for sexual offenders against children within Indonesian criminal law, examining it through a human rights lens. Adopting a normative legal method, the research incorporates legislative and conceptual analysis with data sourced from primary and secondary legal materials. The study reveals a stark incongruity between chemical castration and rehabilitation goals of the penal system, highlighting its inhumane nature and violation of human dignity. Furthermore, the research discusses the additional sanction of electronic detection devices, scrutinizing its infringement on privacy rights and inconsistency with the International Covenant on Civil and Political Rights (ICCPR). This study underscores the critical need for a penal system that respects human rights while addressing sexual violence against children, calling for a shift from punitive to rehabilitative approaches.Highlights: Chemical castration in Indonesia's penal system conflicts with rehabilitation goals and human rights principles, being inhumane and degrading. The additional use of electronic detection devices infringes on perpetrators' privacy rights and contradicts international human rights conventions. The study advocates for a penal system that balances the need for justice in cases of sexual violence against children with respect for human rights and dignity. Keywords: Human Rights, Chemical Castration, Indonesian Law, Sexual Violence, Penal System

Protecting migrant children's well-being in Ecuador's public schools

Education plays a crucial role in the adaptation and broader societal inclusion of immigrant students and their families. The current study explored the inclusion of migrants in Ecuador's school system as a fundamental tenet of protecting their health and well-being. We conducted a thematic analysis of 13 policy documents, 12 national laws and international agreements, and 31 key informant interviews, applying a human rights lens. We find that, although the 2008 Ecuadorian Constitution and migration laws protect the right of migrants to health and education, key informants argue that these laws are poorly implemented. Furthermore, they agree that the health sector generally does not consider education as a relevant setting for health. In contrast, the public education sector and non-profit organizations at the national and local levels appear to recognize the importance of coordination across the health and education sectors, including school-based initiatives for migrant inclusion.

“Like Home”: Gerrymandering the Physical Public Sphere in Female Journalist Narratives

AbstractThe cultural figure of the female journalist most clearly embodies the opportunities given to, and the anxieties caused by, the period’s working women. As writers, they fought for rhetorical space in the pages of newspapers and periodicals, and as women, they faced social pressure to avoid the male-dominated physical public sphere or move within it under specific conditions. Even the increasing number of female journalists at the turn of the century could not guarantee their place within the newsroom itself, let alone the world beyond its walls. Instead, their struggle to stake a claim in physical public spaces manifested in fiction and nonfiction narratives as a search for “home,” or a place of belonging. This article explores the exclusively white-woman fiction and nonfiction narratives by and about nonwhite and nonmainstream women through a human geography lens, arguing that their shared central issue is the social “gerrymandering” of women and other overlapping marginalized groups out of physical public spaces, as well as the efforts of women to “redistrict” the social spheres into a comfortable public place in which everyone could thrive.

Противодействие живых систем развитию возрастной катаракты. Физико-химическое объяснение

Simultaneous processes occur in living systems that lead to both an increase in entropy (chaos) and its decrease. A decrease in entropy is possible only in open systems, associated with a contin-uous flow of energy-intensive compounds into them, such as glucose, the oxidation of which is energetically associated with the synthesis of ATP, the universal carrier of chemical energy to en-sure functioning of living systems. When energy-intensive compounds are oxidized, entropy in-creases, and when ATP is synthesized, it decreases. In living systems, not only reactions associated with the synthesis of ATP and their functioning occur, but also aging does, namely, the oxidation of hydrophobic formations in cells, in particular, the membrane matrix, following the type of chain reactions with degenerate branching. The development of such chain reactions of oxidation in cells can be prevented by the penetration into them of various antioxidants, for example, resvera-trol, which is contained in grapes and other plants, and quenchers of molecular oxygen in the sin-glet state, for example, iodides, as well as by creating cyclic processes involving desaturases that prevent an increase in viscosity in the matrix of membranes up to certain limiting values, after which cell death becomes inevitable. In this regard, the processes leading to cataracts (clouding of the eye lenses) are indicative. In the absence of desaturases in the lens fibers of rats, exposure to UV light leads to cataracts in 2–4 months, while the presence of desaturases in human eye lens fi-bers makes it possible to avoid the occurrence of cataracts for many decades. The reaction of con-version of lipids with saturated fatty acids into unsaturated ones, catalyzed by desaturases, leads to counteracting the aging of the membrane matrix. The vital feature of this reaction is that it oc-curs only when the membrane matrix is cured. This reaction first leads to liquefaction of the ma-trix up to a certain state, after which it stops. The reasons leading either to revival of desaturase or to decrease in its activity are discussed in this article.

Sestrin 2 protects human lens epithelial cells from oxidative stress and apoptosis induced by hydrogen peroxide by regulating the mTOR/Nrf2 pathway.

We aimed to explore the effect and potential mechanism of Sestrin 2 (SESN2) in human lens epithelial cells (HLECs). To mimic the oxidative stress environment, SAR01/04 cells were treated with 200μM hydrogen peroxide (H2O2) for 24h. Cell viability and apoptosis were checked by cell counting kit-8 and flow cytometry. Western blot was taken to check the protein changes of SESN2, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), mechanistic target of rapamycin (mTOR), phosphorylated (p)-mTOR, ribosomal protein S6 kinase B1 (p70S6K), p-p70S6K, and nuclear factor erythroid 2-related factor 2 (Nrf2). Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and reactive oxygen species (ROS) were detected via the corresponding reagent kit. The levels of interleukin (IL)-1β, IL-18, and tumor necrosis factor (TNF)-α were measured using enzyme-linked immunosorbent assay. SESN2 was down-regulated in cataract lens tissue and up-regulated in SAR01/04 cells treated with H2O2. Under treatment of H2O2, up-regulation of SESN2 improved cell viability, enhanced the activity of SOD and CAT, inhibited cell apoptosis, and reduced the levels of MDA, ROS, IL-1β, IL-18, and TNF-α, while down-regulation of SESN2 caused the contrary effects. Further bioinformatics analysis suggested that SESN2 regulated the mTOR signaling pathway. Treatment of H2O2 inhibited p-mTOR and p-p70S6K protein expression, while overexpression of SESN2 increased p-mTOR and p-p70S6K protein expression in the H2O2 group and down-regulation of SESN2 further decreased p-mTOR and p-p70S6K protein expression in the H2O2 group. Additionally, H2O2 increased Nrf2 protein expression, and overexpression of SESN2 further increased Nrf2 protein expression in the H2O2 group. Importantly, rapamycin (an inhibitor of mTOR signaling pathway) and knockdown of Nrf2 reversed the promotive effects of SESN2 on cell viability and the inhibitive effects of SESN2 on cell apoptosis, oxidative stress, and inflammatory reaction. SESN2 protected HLECs damage induced by H2O2, which was related to the activation of mTOR/Nrf2 pathway.

Aqueous Extract Analysis of Lens Epithelial Cells: Unraveling the Link between Oxidative DNA Damage and Malondialdehyde in Senile Cataract

Background and Aims: The purpose of this study was to examine the level of oxidative stress in people with cataracts, based on previous studies that indicated its role in causing DNA damage and cataract formation. The study also sought to determine the correlation between the degree of DNA damage and the concentration of malondialdehyde, a marker of lipid peroxidation, in patients with senile cataracts. Methods: The study involved 30 senile cataract patients aged 50-80 years and 12 cadaveric donors of corneal tissue who died in road accidents. They were selected from the ophthalmology ward of Acharya Vinoba Bhave Hospital. The aim was to measure DNA damage in human lens epithelial cells (HLECs) by Comet assay and malondialdehyde levels in the aqueous extract of HLECs. Results: The study found that the oxidative DNA damage level of human lens epithelial cells is not correlated with the malondialdehyde levels in the aqueous extract of HLECs in senile cataract patients. Conclusions: The study did not support the hypothesis that oxidative stress is a major factor in the development of cataracts. The results suggest that other mechanisms, such as genetic factors, environmental factors may be more relevant in the pathogenesis of cataracts.

The human capsular bag model as a pre‐clinical evaluation tool for IOLs

Cataract affects the vision and well‐being of millions. At present the only means of treating cataract is through surgical intervention. Modern cataract surgery typically retains a ring of anterior lens capsule and the entire posterior capsule. This is known as a capsular bag, which can house an intraocular lens (IOL) implant. Initial surgical outcomes are excellent, but with time lens cells that remain following surgery undergo a wound‐healing response. In essence the trauma of surgery provokes cells to proliferate and migrate across the acellular capsular surfaces and in some case the IOL surface. Most importantly, cells ultimately grow within the visual axis, on the central posterior capsule, and undergo fibrotic events that are associated with transdifferentiation to myofibroblasts, matrix deposition and matrix contraction that can lead to a secondary loss of vision termed posterior capsule opacification (PCO). PCO affects a large proportion of patients and is one of the most common fibrotic conditions in the world. It is therefore of great importance to better understand this condition and thus strong experimental systems are required for this purpose. To this end cataract surgery can be performed on human donor eyes within a laboratory setting to provide a biological product that is configured in the same manner as in the patient. This is known as the human capsular bag model. Using this clinically relevant model it has been possible to tease our key regulatory molecules driving human lens growth and fibrosis. While the human capsular bag model has been a useful tool to identify therapeutic targets or test putative therapeutics no pharmacological treatment is currently in clinical use. As a result, the intraocular lens is currently the major tool employed to manage PCO progression. Characteristics required for an IOL include strong optical performance, stability within the lens capsular bag and anti‐PCO properties. To meet international standards for IOL manufacture a number of tests are performed to predict IOL behaviour when implanted. However, ISO guided tests take place in a non‐biological setting and are limited to assessing purely physical characteristics. Therefore, understanding how the IOL interacts with the capsular bag and how wound‐healing responses could influence IOL position and stability is an area of great importance. The human capsular bag model can address this shortfall in the IOL development pipeline to relate design features to IOL stability and anti‐PCO performance in a clinically relevant setting. The presentation will highlight the evolution of the model as a tool for IOL testing and development.

The significance of growth shells in development of symmetry, transparency, and refraction of the human lens.

Human visual function depends on the biological lens, a biconvex optical element formed by coordinated, synchronous generation of growth shells produced from ordered cells at the lens equator, the distal edge of the epithelium. Growth shells are comprised of straight (St) and S-shaped (SSh) lens fibers organized in highly symmetric, sinusoidal pattern which optimizes both the refractile, transparent structure and the unique microcirculation that regulates hydration and nutrition over the lifetime of an individual. The fiber cells are characterized by diversity in composition and age. All fiber cells remain interconnected in their growth shells throughout the life of the adult lens. As an optical element, cellular differentiation is constrained by the physical properties of light and its special development accounts for its characteristic symmetry, gradient of refractive index (GRIN), short range transparent order (SRO), and functional longevity. The complex sinusoidal structure is the basis for the lens microcirculation required for the establishment and maintenance of image formation.

Synchrotron‐based Fourier transform infrared microspectroscopy examined differences between the cultured and post‐operative human lens epithelial cells important for understanding posterior capsular opacification and lens regeneration

Aims/Purpose: To compare the bio‐macromolecular composition and the differences between the human cultured and post‐operative lens epithelial cells (LECs) on lens capsules (LCs) for a better understanding of posterior capsular opacification (PCO)—the most common complication of cataract surgery, and lens regeneration.Methods: The explants of the anterior portion of the LC containing the LECs, obtained from cataract surgery and cultivated under adherent conditions, as well as the post‐operative LC with LECs, were analysed by using synchrotron radiation‐based Fourier transform infrared (SR‐FTIR) microspectroscopy, a vibrational spectroscopic technique that allows monitoring of the entire biochemical status of the biological processes. The SR‐FTIR microspectroscopy setup installed on the beamline MIRAS at the Spanish synchrotron light source ALBA was used, where measurements were set to achieve single‐cell resolution, with high spectral stability and high photon flux.Results: We found that the differences exist between the composites of cultured and post‐operative LECs on LCs on the level of all chemical constituents: proteins, lipids, nucleic acids and carbohydrates as well as oxidative stress. The strongest differences are found in protein secondary structure contribution where the composite of primary LECs cultures on LC have more α‐helix (1652 cm−1) and post‐operative LC have more β‐sheet (1624 cm−1 and 1697 cm−1) secondary structures. The oxidative stress is more expressed in the composite of primary LECs cultures on LC and relatively less in the composite of post‐operative lens epithelium LECs on LC.Conclusions: Our results obtained by SR‐FTIR increase the knowledge about the total proteins, lipids, and nucleic acids in human primary cultures LECs while offering also evidence that SR‐FTIR is sensitive to the pathologic processes of LECs transdifferentiation. We showed that the composite of primary cultures LECs on LC have a distinct bio‐macromolecular composition compared to the composite of post‐operative LECs on LC, which gives additional information for a better understanding of PCO and lens regeneration.

CircMAP3K4 Suppresses H2O2-Induced Human Lens Epithelial Cell Injury by miR-630/ERCC6 Axis in Age-Related Cataract

Background Dysregulated circular RNAs (circRNAs) is involved in the pathogenesis of age-related cataract (ARC). Here, this study aimed to explore the function and mechanism of circMAP3K4 in ARC. Methods Human lens epithelial cells were exposed to hydrogen peroxide (H2O2) for functional experiments. qRT-PCR and western blotting analyses were used for the expression detection of genes and proteins. Cell proliferation was tested using cell counting kit-8 and EdU. Flow cytometry was applied to analyze cell apoptosis and cell cycle. The oxidative stress was evaluated by detecting the production of malondialdehyde (MDA), reactive oxygen species (ROS), and superoxide dismutase (SOD). The target relationship between miR-630 and circMAP3K4 or Excision repair cross-complementing group 6 (ERCC6) was analyzed by dual-luciferase reporter assay and RIP assay. Results CircMAP3K4 was lowly expressed in ARC patients and H2O2-induced HLECs. Functionally, forced expression of circMAP3K4 protected HLECs against H2O2-evoked proliferation inhibition, cell cycle arrest and the promotion of cell apoptosis and oxidative stress. Mechanistically, circMAP3K4 acted as a sponge for miR-630 to regulate the expression of its target ERCC6. MiR-630 was highly expressed while ERCC6 was lowly expressed in ARC patients and H2O2-induced HLECs. Up-regulation of miR-630 could reverse the protective effects of circMAP3K4 on HLECs under H2O2 treatment. In addition, inhibition of miR-630 suppressed H2O2-induced HLEC injury, which was abolished by ERCC6 silencing. Conclusion Forced expression of circMAP3K4 protected HLECs against H2O2-evoked apoptotic and oxidative injury via miR-630/ERCC6 axis, suggesting that circMAP3K4 may function as a potential therapeutic target for ARC.

Human Cx50 Isoleucine177 prevents heterotypic docking and formation of functional gap junction channels with Cx43.

The human lens is an avascular organ, and its transparency is dependent on gap junction (GJ)-mediated microcirculation. Lens GJs are composed of three connexins with Cx46 and Cx50 being expressed in lens fiber cells and Cx43 and Cx50 in the epithelial cells. Impairment of GJ communication by either Cx46 or Cx50 mutations has been shown to be one of the main molecular mechanisms of congenital cataracts in mutant carrier families. The docking compatibility and formation of functional heterotypic GJs for human lens connexins have not been studied. Previous study on rodent lens connexins revealed that Cx46 can form functional heterotypic GJs with Cx50 and Cx43, but Cx50 cannot form heterotypic GJ with Cx43 due to its second extracellular (EL2) domain. To study human lens connexin docking and formation of functional heterotypic GJs, we developed a genetically engineered HEK293 cell line with endogenously expressed Cx43 and Cx45 ablated. The human lens connexins showed docking compatibility identical to those found in the rodent connexins. To reveal the structural mechanisms of the docking incompatibility between Cx50 and Cx43, we designed eight variants based on the differences between the EL2 of Cx50 and Cx46. We found that Cx50I177L is sufficient to establish heterotypic docking with Cx43 with some interesting gating properties. Our structure models indicate this residue is important for interdomain interactions within a single connexin, Cx50 I177L showed an increased interdomain interaction which might alter the docking interface structure to be compatible with Cx43.NEW & NOTEWORTHY The human lens is an avascular organ, and its transparency is partially dependent on gap junction (GJ) network composed of Cx46, Cx50, and Cx43. We found that human Cx46 can dock and form functional heterotypic GJs with Cx50 and Cx43, but Cx50 is unable to form functional heterotypic GJs with Cx43. Through mutagenesis and patch-clamp study of several designed variants, we found that Cx50 I177L was sufficient to form functional heterotypic GJs with Cx43.

Role of reactive oxygen species in epithelial-mesenchymal transition and apoptosis of human lens epithelial cells.

To investigate the role of reactive oxygen species (ROS) in epithelial-mesenchymal transition (EMT) and apoptosis of human lens epithelial cells (HLECs). Flow cytometry was used to assess ROS production after transforming growth factor β2 (TGF-β2) induction. Apoptosis of HLECs after H2O2 and TGF-β2 interference with or without ROS scavenger N-acetylcysteine (NAC) were assessed by flow cytometry. The corresponding protein expression levels of the EMT marker α-smooth muscle actin (α-SMA), the extracellular matrix (ECM), marker fibronectin (Fn), and apoptosis-associated proteins were detected by using Western blotting in the presence of an ROS scavenger (NAC). Wound-healing and Transwell assays were used to assess the migration capability of HLECs. TGF-β2 stimulates ROS production within 8h in HLECs. Additionally, TGF-β2 induced HLECs cell apoptosis, EMT/ECM synthesis protein markers expression, and pro-apoptotic proteins production; nonetheless, NAC treatment prevented these responses. Similarly, TGF-β2 promoted HLECs cell migration, whereas NAC inhibited cell migration. We further determined that although ROS initiated apoptosis, it only induced the accumulation of the EMT marker α-SMA protein, but not COL-1 or Fn. ROS contribute to TGF-β2-induced EMT/ECM synthesis and cell apoptosis of HLECs; however, ROS alone are not sufficient for EMT/ECM synthesis.