Human peritoneal fluid exerts ovulation- and nonovulation-sourced oncogenic activities on transforming fallopian tube epithelial cells.

Secretory cells in the fallopian tube fimbria epithelium (FTE) are regarded as the main cells of origin of ovarian high-grade serous carcinoma (HGSC). Ovulation is the main cause of FTE oncogenesis, which proceeds through a sequence of TP53 mutations, chromosomal instability due to Rb/cyclin E aberration, in situ carcinoma (STIC), and metastasis to the ovary and peritoneum (metastatic HGSC). Previously, we have identified multiple oncogenic activities of the ovulatory follicular fluid (FF), which exerts the full spectrum of transforming activity on FTE cells at different stages of transformation. After ovulation, the FF is transfused into the peritoneal fluid (PF), in which the FTE constantly bathes. We wondered whether PF exerts the same spectrum of oncogenic activities as done by FF and whether these activities are derived from FF. By using a panel of FTE cell lines with p53 mutation (FT282-V), p53/CCNE1 aberrations (FT282-CCNE1), and p53/Rb aberrations plus spontaneous transformation, and peritoneal metastasis (FEXT2), we analyzed the changes of different transformation phenotypes after treating with FF and PF collected before or after ovulation. Similar to effects exhibited by FF, we found that, to a lesser extent, PF promoted anchorage-independent growth (AIG), migration, anoikis resistance, and peritoneal attachment in transforming FTE cells. The more transformed cells were typically more affected. Among the transforming activities exhibited by PF treatment, AIG, Matrigel invasion, and peritoneal attachment growth were higher with luteal-phase PF treatment than with the proliferative-phase PF treatment, suggesting an ovulation source. In contrast, changes in anoikis resistance and migration activities were similar in response to treatment with PF collected before and after ovulation, suggesting an ovulation-independent source. The overall transforming activity of luteal-phase PF was verified in an i.p. co-injection xenograft mouse model. Co-injection of Luc-FEXT2 cells with either FF or luteal-phase PF supported early peritoneal implantation, whereas co-injection with follicular-phase PF did not. This study, for the first time, demonstrates that PF from ovulating women can promote different oncogenic phenotypes in FTE cells at different stages of malignant transformation. Most of these activities, other than anoikis resistance and cell migration, are sourced from ovulation.

Multispectral label-free in vivo cellular imaging of human retinal pigment epithelium using adaptive optics fluorescence lifetime ophthalmoscopy improves feasibility for low emission analysis and increases sensitivity for detecting changes with age and eccentricity.

Significance: Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE). Aim: To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO. Approach: AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( λ ex / λ em ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter. Results: A repeatability analysis revealed < 11.8 % change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters. Conclusions: Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.

Attenuated replication and damaging effects of SARS-CoV-2 Omicron variants in an intestinal epithelial barrier model.

Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function is thought to play a key role in Long COVID. Despite its importance, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin, and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir, the fusion inhibitor EK1 and the transmembrane serine protease 2 inhibitor Camostat inhibited SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.

Bioaccessibility and Caco-2 Cell Uptake of Iron Chlorophyllin Using A Biologically Relevant Digestion Model.

Iron deficiency remains a top nutrient deficiency worldwide. Iron chlorophyllin (IC), a compound structurally analogous to heme, utilizes the protoporphyrin ring of chlorophyll to bind iron. IC has previously been shown to deliver more iron to Caco-2 cells than FeSO4, the most common form prescribed for supplementation. However, previous test conditions used digestive conditions outside of those observed in humans. This study sought to assess IC bioaccessibility and Caco-2 cell uptake using physiologically relevant digestive solutions, pH, and incubation time, as compared to other iron sources (i.e. FeSO4, and hemoglobin (Hb)). Co-digestion with ascorbic acid (AA) and albumin was also investigated. Following gastric, duodenal, and jejunal digestion, IC-bound iron was less bioaccessible than iron delivered as FeSO4, and IC-bound iron was less bioaccessible than Hb-bound iron. IC-bound iron bioaccessibility was not affected by AA and was enhanced 2x with co-digested with a low dose of albumin. However, Caco-2 cell incubation with IC-containing digesta increased cell ferritin 2.5x more than FeSO4 alone, and less than Hb. IC with AA or with 400 mg albumin also increased cell ferritin more than IC alone, with the greatest increases observed following incubation of digesta containing IC + AA + 400 mg albumin. These results suggest IC can serve as an improved source of iron for supplementation as compared to FeSO4. These results also support further in vivo investigations of IC-based iron delivery in populations at risk of iron deficiency.

Decoding cellular plasticity and niche regulation of limbal stem cells during corneal wound healing.

BACKGROUND: Dysfunction or deficiency of corneal epithelium results in vision impairment or blindness in severe cases. The rapid and effective regeneration of corneal epithelial cells relies on the limbal stem cells (LSCs). However, the molecular and functional responses of LSCs and their niche cells to injury remain elusive. METHODS: Single-cell RNA sequencing was performed on corneal tissues from normal mice and corneal epithelium defect models. Bioinformatics analysis was performed to confirm the distinct characteristics and cell fates of LSCs. Knockdown of Creb5 and OSM treatment experiment were performed to determine their roles of in corneal epithelial wound healing. RESULTS: Our data defined the molecular signatures of LSCs and reconstructed the pseudotime trajectory of corneal epithelial cells. Gene network analyses characterized transcriptional landmarks that potentially regulate LSC dynamics, and identified a transcription factor Creb5, that was expressed in LSCs and significantly upregulated after injury. Loss-of-function experiments revealed that silencing Creb5 delayed the corneal epithelial healing and LSC mobilization. Through cell-cell communication analysis, we identified 609 candidate regeneration-associated ligand-receptor interaction pairs between LSCs and distinct niche cells, and discovered a unique subset of Arg1+ macrophages infiltrated after injury, which were present as the source of Oncostatin M (OSM), an IL-6 family cytokine, that were demonstrated to effectively accelerate the corneal epithelial wound healing. CONCLUSIONS: This research provides a valuable single-cell resource and reference for the discovery of mechanisms and potential clinical interventions aimed at ocular surface reconstruction.

Inhibition of hypoxia-inducible factors suppresses subretinal fibrosis.

Age-related macular degeneration (AMD) is a common cause of vision loss. The aggressive form of AMD is associated with ocular neovascularization and subretinal fibrosis, representing a responsive outcome against neovascularization mediated by epithelial-mesenchymal transition of retinal pigment epithelium (RPE) cells. A failure of the current treatment (anti-vascular endothelial growth factor therapy) has also been attributed to the progression of subretinal fibrosis. Hypoxia-inducible factors (HIFs) increase gene expressions to promote fibrosis and neovascularization. HIFs act as a central pathway in the pathogenesis of AMD. HIF inhibitors may suppress ocular neovascularization. Nonetheless, further investigation is required to unravel the aspects of subretinal fibrosis. In this study, we used RPE-specific HIFs or von Hippel-Lindau (VHL, a regulator of HIFs) conditional knockout (cKO) mice, along with pharmacological HIF inhibitors, to demonstrate the suppression of subretinal fibrosis. Fibrosis was suppressed by treatments of HIF inhibitors, and similar suppressive effects were detected in RPE-specific Hif1a/Hif2a- and Hif1a-cKO mice. Promotive effects were observed in RPE-specific Vhl-cKO mice, where fibrosis-mediated pathologic processes were evident. Marine products' extracts and their component taurine suppressed fibrosis as HIF inhibitors. Our study shows critical roles of HIFs in the progression of fibrosis, linking them to the potential development of therapeutics for AMD.

Mucoepidermoid Carcinoma of the Thyroglossal Duct: Two Rare Entities in a Unique Case Report.

BACKGROUND: Mucoepidermoid Carcinoma (MEC) of the thyroid represents less than 0.5% of all thyroid neoplasms. Thyroglossal duct cyst carcinoma is a rare condition with only approximately 300 cases reported. CASE REPORT: A 34-year-old pregnant woman at 37 weeks gestation presented to an endocrinological center for primary autoimmune hypothyroidism. The thyroid ultrasound revealed a pseudonodular pattern. The patient was followed up after two years. She reported a full-term delivery without complications. A new thyroid ultrasound was performed, showing a cystic lesion in the median suprathyroid area, measuring 6 x 9 x 10 mm, not previously reported. After 4 months, the suprathyroid cystic lesion was confirmed by thyroid ultrasound, measuring 6 x 11 x 12 mm. The patient was referred for fine-needle aspiration cytology. Cytological examination showed lymphocytes, red blood cells, and some epithelial aggregates with large cytoplasm and nuclear polymetrism with oxyphilic aspects. The patient underwent the Sistrunk procedure for the suprathyroid lesion. The histological examination revealed lymphocytic thyroiditis in heterotopic thyroid tissue with solid cell nest, epidermoid epithelium, and mucus-secreting cells suggestive of low-grade mucoepidermoid carcinoma. The immunohistochemistry study was positive, exhibiting thyroid transcription factor 1 and cytokeratin-19. No positivity was observed for thyroglobulin, calcitonin, galectin-3, and Hector Battifora mesothelial antigen 1. The recent follow-up examination, 13 months after the surgery, has been found negative for disease recurrence. CONCLUSION: This is the first case of an MEC occurring within a thyroglossal duct. Considering the age of the patient, the histological diagnosis, and the absence of thyroid nodules and metastasis, we decided on the Sistrunk procedure without total thyroidectomy.

Spatial-temporal comparison of Eph/Ephrin gene expression in ocular lenses from aging and knockout mice.

Cataracts, defined as any opacity in the transparent ocular lens, remain the leading cause of blindness and visual impairment in the world; however, the etiology of this pathology is not fully understood. Studies in mice and humans have found that the EphA2 receptor and the ephrin-A5 ligand play important roles in maintaining lens homeostasis and transparency. However, due to the diversity of the family of Eph receptors and ephrin ligands and their promiscuous binding, identifying functional interacting partners remains a challenge. Previously, 12 of the 14 Ephs and 8 of 8 ephrins in mice were characterized to be expressed in the mouse lens. To further narrow down possible genes of interest in life-long lens homeostasis, we collected and separated the lens epithelium from the fiber cell mass and isolated RNA from each compartment in samples from young adult and middle-aged mice that were either wild-type, EphA2-/- (knockout), or ephrin-A5 -/- . Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was implemented to compare transcript levels of 33 Eph and ephrin gene variants in each tissue compartment. Our results show that, of the Eph and ephrin variants screened, 5 of 33 showed age-related changes, and 2 of 33 showed genotype-related changes in lens epithelium. In the isolated fibers, more dynamic gene expression changes were observed, in which 12 of 33 variants showed age-related changes, and 6 of 33 showed genotype-related changes. These data allow for a more informed decision in determining mechanistic leads in Eph-ephrin-mediated signaling in the lens.

The Drosophila TNF Eiger promotes Myc super-competition independent of canonical JNK signaling.

Numerous factors have been implicated in the cell-cell interactions that lead to elimination of cells via cell competition, a context-dependent process of cell selection in somatic tissues that is based on comparisons of cellular fitness. Here we use a series of genetic tests in Drosophila to explore the relative contribution of the pleiotropic cytokine Tumor Necrosis Factor ⍺ (TNF⍺) in Myc-mediated cell competition (also known as Myc super-competition or Myc cell competition). We find that the sole Drosophila TNF, Eiger (Egr), its receptor Grindelwald (Grnd/TNFR), and the adaptor proteins Traf4 and Traf6 are required to eliminate wild-type "loser" cells during Myc cell competition. Although typically the interaction between Egr and Grnd leads to cell death by activating the intracellular Jun N-terminal Kinase (JNK) stress signaling pathway, our experiments reveal that many components of canonical JNK signaling are dispensable for cell death in Myc cell competition, including the JNKKK Tak1, the JNKK Hemipterous (Hep) and the JNK Basket (Bsk). Our results suggest that Egr/Grnd signaling participates in Myc cell competition, but functions in a role that is largely independent of the JNK signaling pathway.

Daily Light Onset and Plasma Membrane Tethers Regulate Mitochondria Redistribution within the Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) is an essential component of the retina that plays multiple roles required to support visual function. These include light onset- and circadian rhythm-dependent tasks, such as daily phagocytosis of photoreceptor outer segments. Mitochondria provide energy to the highly specialized and energy-dependent RPE. In this study, we examined the positioning of mitochondria and how this is influenced by the onset of light. We identified a population of mitochondria that are tethered to the basal plasma membrane pre- and post-light onset. Following light onset, mitochondria redistributed apically and interacted with melanosomes and phagosomes. In a choroideremia mouse model that has regions of the RPE with disrupted or lost infolding of the plasma membrane, the positionings of only the non-tethered mitochondria were affected. This provides evidence that the tethering of mitochondria to the plasma membrane plays an important role that is maintained under these disease conditions. Our work shows that there are subpopulations of RPE mitochondria based on their positioning after light onset. It is likely they play distinct roles in the RPE that are needed to fulfil the changing cellular demands throughout the day.

Activation of fetal-like molecular programs during regeneration in the intestine and beyond.

Tissue regeneration after damage is generally thought to involve the mobilization of adult stem cells that divide and differentiate into progressively specialized progeny. However, recent studies indicate that tissue regeneration can be accompanied by reversion to a fetal-like state. During this process, cells at the injury site reactivate programs that operate during fetal development but are typically absent in adult homeostasis. Here, we summarize our current understanding of the molecular signals and epigenetic mediators that orchestrate "fetal-like reversion" during intestinal regeneration. We also explore evidence for this phenomenon in other organs and species and highlight open questions that merit future examination.

Aging of the eye: Lessons from cataracts and age-related macular degeneration.

Aging is the greatest risk factor for chronic human diseases, including many eye diseases. Geroscience aims to understand the effects of the aging process on these diseases, including the genetic, molecular, and cellular mechanisms that underlie the increased risk of disease over the lifetime. Understanding of the aging eye increases general knowledge of the cellular physiology impacted by aging processes at various biological extremes. Two major diseases, age-related cataract and age-related macular degeneration (AMD) are caused by dysfunction of the lens and retina, respectively. Lens transparency and light refraction are mediated by lens fiber cells lacking nuclei and other organelles, which provides a unique opportunity to study a single aging hallmark, i.e., loss of proteostasis, within an environment of limited metabolism. In AMD, local dysfunction of the photoreceptors/retinal pigmented epithelium/Bruch's membrane/choriocapillaris complex in the macula leads to the loss of photoreceptors and eventually loss of central vision, and is driven by nearly all the hallmarks of aging and shares features with Alzheimer's disease, Parkinson's disease, cardiovascular disease, and diabetes. The aging eye can function as a model for studying basic mechanisms of aging and, vice versa, well-defined hallmarks of aging can be used as tools to understand age-related eye disease.

Lattice light sheet microscopy reveals 4D force propagation dynamics and leading-edge behaviors in an embryonic epithelium in Drosophila.

How pulsed contractile dynamics drive the remodeling of cell and tissue topologies in epithelial sheets has been a key question in development and disease. Due to constraints in imaging and analysis technologies, studies that have described the in vivo mechanisms underlying changes in cell and neighbor relationships have largely been confined to analyses of planar apical regions. Thus, how the volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface in three dimensions, including especially the apical-basal axis, is unclear. Here, we perform lattice light sheet microscopy (LLSM)-based analysis to determine how far and fast forces propagate across different apical-basal layers, as well as where topological changes initiate from in a columnar epithelium. These datasets are highly time- and depth-resolved and reveal that topology-changing forces are spatially entangled, with contractile force generation occurring across the observed apical-basal axis in a pulsed fashion, while the conservation of cell volumes constrains instantaneous cell deformations. Leading layer behaviors occur opportunistically in response to favorable phasic conditions, with lagging layers "zippering" to catch up as new contractile pulses propel further changes in cell topologies. These results argue against specific zones of topological initiation and demonstrate the importance of systematic 4D-based analysis in understanding how forces and deformations in cell dimensions propagate in a three-dimensional environment.

Deciphering the M-cell niche: insights from mouse models on how microfold cells "know" where they are needed.

Known for their distinct antigen-sampling abilities, microfold cells, or M cells, have been well characterized in the gut and other mucosa including the lungs and nasal-associated lymphoid tissue (NALT). More recently, however, they have been identified in tissues where they were not initially suspected to reside, which raises the following question: what external and internal factors dictate differentiation toward this specific role? In this discussion, we will focus on murine studies to determine how these cells are identified (e.g., markers and function) and ask the broader question of factors triggering M-cell localization and patterning. Then, through the consideration of unconventional M cells, which include villous M cells, Type II taste cells, and medullary thymic epithelial M cells (microfold mTECs), we will establish the M cell as not just a player in mucosal immunity but as a versatile niche cell that adapts to its home tissue. To this end, we will consider the lymphoid structure relationship and apical stimuli to better discuss how the differing cellular programming and the physical environment within each tissue yield these cells and their unique organization. Thus, by exploring this constellation of M cells, we hope to better understand the multifaceted nature of this cell in its different anatomical locales.

Reassessing retinal pigment epithelial ketogenesis: Enzymatic assays for ketone body levels provide inaccurate results.

The retinal pigment epithelium (RPE) is omnivorous and can utilize a wide range of substrates for oxidative phosphorylation. Certain tissues with high mitochondrial metabolic load are capable of ketogenesis, a biochemical pathway that consolidates acetyl-CoA into ketone bodies. Earlier work demonstrated that the RPE expresses the rate-limiting enzyme for ketogenesis, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), and that the RPE indeed produces ketone bodies, including beta-hydroxybutyrate (ß-HB). Prior work, based on detecting ß-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export ß-HB across the apical membrane. Here, we compare the accuracy of measuring ß-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of ß-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete ß-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming ß-HB as levels rise. Using isotopically labeled glucose, amino acid, and fatty acid tracers, we found that carbons from both fatty acids and ketogenic amino acids, but not from glucose, produce ß-HB. Altogether, we substantiate ß-HB secretion in RPE but find that the secretion is equal apically and basally, RPE ß-HB can derive from ketogenic amino acids or fatty acids, and accurate ß-HB assessment requires mass spectrometric analysis.

Effective intravitreal gene delivery to retinal pigment epithelium with hyaluronic acid nanospheres.

Inherited retinal degeneration (IRD) can cause a wide range of different forms of vision loss and blindness, and in spite of extensive advancements in gene therapy research, therapeutic approaches for targeting IRDs are still lacking. We have recently developed an approach for the intravitreal co-delivery of hyaluronic-acid nanospheres (HA-NSs) with sulfotyrosine (ST), effectively reaching the outer retina from the vitreal cavity. Here, our goal was to understand whether DNA-filled HA-NSs could generate gene expression in the outer retina. TxRed-labeled HA-NSs were compacted with plasmid DNA carrying a GFP reporter gene and intravitreally injected into the mouse retina. Follow-up at 4 weeks showed widespread gene expression in the outer retina and reduced, albeit present, expression at 8 weeks post-injection. Further analysis revealed this expression to be largely localized to the retinal pigment epithelium (RPE). These data show that intravitreal delivery of HA-NSs is a promising non-viral platform for the delivery of therapeutic genes and can generate pan-tissue, persistent gene expression in the RPE.

Establishment of a human nasal epithelium model of histamine-induced inflammation to assess the activity of fexofenadine as an inverse agonist and its link to clinical benefit.

Background: Fexofenadine (FEX) is an antihistamine that acts as an inverse agonist against histamine (HIS) receptor 1 (H1R), which mediates the allergic reaction. Inverse agonists may be more potent than neutral antagonists, as they bind the same receptor as the agonist (HIS) but stabilize the inactive form and induce an opposite pharmacological response, suppressing the basal activity of H1R and preventing HIS from binding. This study aims to establish and validate a model of HIS-induced inflammation based on fully reconstituted human nasal epithelial tissue to assess the activity of FEX as an inverse agonist in this model and explore its link to clinical benefit. Methods: The model was developed using nasal MucilAir™ (Epithelix) in vitro epithelium challenged by HIS. Two conditions were assessed in a side-by-side comparison: tissue was exposed to HIS + FEX with or without FEX pre-treatment (one-hour prior to HIS challenge). Tissue functionality, cytotoxicity, H1R gene expression, and inflammatory cytokines were assessed. Results: HIS at 100 µM induced significant 3.1-fold and 2.2-fold increases for inflammatory biomarkers interleukin (IL)-8 and IL-6, respectively (p < 0.0001), as well as rapid upregulation of H1R mRNA. Inflammatory biomarkers were inhibited by FEX and H1R expression was significantly reduced (p < 0.0001). FEX alone decreased H1R expression at all doses tested. With one-hour FEX pre-treatment, there was significantly higher downregulation of IL-8 (p < 0.05) and further downregulation of H1R expression and IL-6 versus without FEX pre-treatment; the effects of FEX were improved from 22% to 40%. Conclusion: A model of HIS-induced airway inflammation was established based on IL-8, IL-6 and H1R gene expression and was validated with FEX. FEX works as an inverse agonist, with a higher effect when used before+during versus only during the HIS challenge. Taking FEX before+during allergen exposure, or when symptoms first occur, may reduce basal activity and H1R gene expression, providing stronger protection against the worsening of symptoms upon allergen exposure.

Intraoperative variability of corneal epithelium thickness in photorefractive keratectomy.

PURPOSE: To evaluate the intraoperative central corneal epithelial thickness (ET) as measured by optical coherence pachymetry (OCP) in myopic eyes undergoing alcohol-assisted photorefractive keratectomy (PRK). METHODS: A retrospective review of patients who underwent alcohol-assisted PRK was performed. Data were abstracted on age, gender, contact lens (CL) wear, preoperative refractive errors, keratometry, topographic and ultrasonic pachymetry, and intraoperative OCP measurements before and after epithelium removal. The central ET was calculated by subtracting OCP measurement after epithelium removal from the OCP measurement prior to epithelium removal. RESULTS: The study comprised of 162 consecutive eyes from 81 patients. Mean age was 26.73 ± 6.47 years, 50.6% were males. CL was used in 92 eyes (56.8%). The mean sphere and spherical equivalent were -3.60 ± 1.84 D and -3.26 ± 1.85D, respectively. The mean intraoperative ET was 58.22 ± 17.53 µm (range, 15-121µm). Fifty-five percent of the eyes had an ET measurement above or below the range of 40-60µm. ET was significantly higher in the second operated eye compared to the first operated eye (p = 0.006), and an association was found to CL-wear (p = 0.03). There was no significant difference in thickness between genders (p = 0.62), and no correlation to patient age (p = 0.45, rp = 0.06), refractive errors (p > 0.30,rp=-0.07-0.08), nor keratometry(p > 0.80, rp=-0.01- (-0.02)). CONCLUSION: The intraoperative assessment of ET in alcohol-assisted PRK showed a high variability of the central corneal epithelium, with a significant difference between the first and second operated eyes. This difference may have implications when the epithelium is not included in the surgical planning in surface ablation.

Rho-Associated Protein Kinase Activity Is Required for Tissue Homeostasis in the Xenopus laevis Ciliated Epithelium.

Lung epithelial development relies on the proper balance of cell proliferation and differentiation to maintain homeostasis. When this balance is disturbed, it can lead to diseases like cancer, where cells undergo hyperproliferation and then can undergo migration and metastasis. Lung cancer is one of the deadliest cancers, and even though there are a variety of therapeutic approaches, there are cases where treatment remains elusive. The rho-associated protein kinase (ROCK) has been thought to be an ideal molecular target due to its role in activating oncogenic signaling pathways. However, in a variety of cases, inhibition of ROCK has been shown to have the opposite outcome. Here, we show that ROCK inhibition with y-27632 causes abnormal epithelial tissue development in Xenopus laevis embryonic skin, which is an ideal model for studying lung cancer development. We found that treatment with y-27632 caused an increase in proliferation and the formation of ciliated epithelial outgrowths along the tail edge. Our results suggest that, in certain cases, ROCK inhibition can disturb tissue homeostasis. We anticipate that these findings could provide insight into possible mechanisms to overcome instances when ROCK inhibition results in heightened proliferation. Also, these findings are significant because y-27632 is a common pharmacological inhibitor used to study ROCK signaling, so it is important to know that in certain in vivo developmental models and conditions, this treatment can enhance proliferation rather than lead to cell cycle suppression.

Extensive Contact Lens Wear Modulates Expression of miRNA-320 and miRNA-423-5p in the Human Corneal Epithelium: Possible Biomarkers of Corneal Health and Environmental Impact.

The identification of new biomarkers of ocular diseases is nowadays of outmost importance both for early diagnosis and treatment. Epigenetics is a rapidly growing emerging area of research and its involvement in the pathophysiology of ocular disease and regulatory mechanisms is of undisputable importance for diagnostic purposes. Environmental changes may impact the ocular surface, and the knowledge of induced epigenetic changes might help to elucidate the mechanisms of ocular surface disorders. In this pilot study, we investigated the impact of extensive contact lens (CL) wearing on human corneal epithelium epigenetics. We performed ex vivo analysis of the expression of the miR-320 and miR-423-5p involved in the processes of cellular apoptosis and chronic inflammation. The human corneal epithelium was harvested from healthy patients before the photorefractive keratectomy (PRK). The patients were divided into two age- and sex-matched groups accordingly to CL wearing history with no CL wearers used as a control. The epithelium was stored frozen in dry ice at -80 °C and forwarded for miRNA extraction; afterwards, miRNA levels were detected using real-time PCR. Both miRNAs were highly expressed in CL wearers (p < 0.001), suggesting epigenetic modifications occurring in chronic ocular surface stress. These preliminary results show the relationships between selected miRNA expression and the chronic ocular surface stress associated with extensive CL use. MicroRNAs might be considered as biomarkers for the diagnosis of ocular surface conditions and the impact of environmental factors on ocular surface epigenetic. Furthermore, they might be considered as new therapeutic targets in ocular surface diseases.