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Messenger RNA (mRNA)-based therapies are a promising approach to medical treatment. Except for infectious diseases, no other disease has mRNA-based therapies available. The eye is an ideal model for mRNA therapeutic development because it requires limited dosing. Proliferative vitreoretinopathy (PVR) is a blinding condition caused by retinal detachment that now lacks available medical treatment, with surgery as the only treatment option. We previously implicated runt-related transcription factor-1 (RUNX1) as a driver of epithelial-to-mesenchymal transition (EMT) in PVR and as a critical mediator of aberrant ocular angiogenesis when up-regulated. On the basis of these findings, an mRNA was designed to express a dominant-negative inhibitor of RUNX1 (RUNX1-Trap). We show that RUNX1-Trap delivered in polymer-lipidoid complexes or lipid nanoparticles sequestered RUNX1 in the cytosol and strongly reduced proliferation in primary cell cultures established from fibrotic membranes derived from patients with PVR. We assessed the preclinical efficacy of intraocular delivery of mRNA-encoded RUNX1-Trap in a rabbit model of PVR and in a laser-induced mouse model of aberrant angiogenesis often used to study wet age-related macular degeneration. mRNA-encoded RUNX1-Trap suppressed ocular pathology, measured as pathological scores in the rabbit PVR model and leakage and lesion size in the laser-induced choroidal neovascularization mouse model. mRNA-encoded RUNX1-Trap also strongly reduced proliferation in a human ex vivo explant model of PVR. These data demonstrate the therapeutic potential of mRNA-encoded therapeutic molecules with dominant-negative properties, highlighting the potential of mRNA-based therapies beyond standard gene supplementation approaches.

Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements. Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results. We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.

The energy transition is a multinational challenge to mitigate climate change, with a joint reduction target for greenhouse gas emissions. Simultaneously, each country is interested in minimizing its own energy supply cost. Still, most energy system models neglect national interests when identifying cost-optimal transition pathways. We design the European energy system transition until 2050, considering competition between countries in a shared electricity and carbon market using bilevel optimization. We find that national objectives substantially impact the transition pathway: Compared to the model solved using the common centralized optimization, the overall installed capacity increases by just 3% when including national interests. However, the distribution of the installed capacity changes dramatically by more than 40% in most countries. Our results underline the risk of miscalculating the need for national capacity expansion when neglecting stakeholder representation in energy system models and demonstrate the need for cooperation for an efficient energy transition., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Background: Increased age is a risk factor for the development and progression of retinal diseases including age-related macular degeneration (AMD). Understanding the changes that occur in the eye due to aging is important in enhancing our understanding of AMD pathogenesis and the development of novel AMD therapies. Microglia, the resident brain and retinal immune cells are associated with both maintaining homeostasis and protection of neurons and loss of microglia homeostasis could be a significant player in age related neurodegeneration. One important characteristic of retinal aging is the migration of microglia from the inner to outer retina where they reside in the subretinal space (SRS) in contact with the retinal pigment epithelial (RPE) cells. The role of aged subretinal microglia is unknown. Here, we depleted microglia in aged C57/BL6 mice fed for 6 weeks with a chow containing PLX5622, a small molecule inhibitor of colony-stimulating factor-1 receptor (Csf1r) required for microglial survival., Results: The subretinal P2RY12 + microglia in aged mice displayed a highly amoeboid and activated morphology and were filled with autofluorescence droplets reminiscent of lipofuscin. TEM indicates that subretinal microglia actively phagocytize shed photoreceptor outer segments, one of the main functions of retinal pigmented epithelial cells. PLX5622 treatment depleted up to 90% of the retinal microglia and was associated with significant loss in visual function. Mice on the microglia depletion diet showed reduced contrast sensitivity and significantly lower electroretinogram for the c-wave, a measurement of RPE functionality, compared to age-matched controls. The loss of c-wave coincided with a loss of RPE cells and increased RPE swelling in the absence of microglia., Conclusions: We conclude that microglia preserve visual function in aged mice and support RPE cell function, by phagocytosing shed photoreceptor outer segments and lipids, therefore compensating for the known age-related decline of RPE phagocytosis., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

Epithelial-mesenchymal transition (EMT) is a dedifferentiation program in which polarized, differentiated epithelial cells lose their cell-cell adhesions and transform into matrix-producing mesenchymal cells. EMT of retinal pigment epithelial (RPE) cells plays a crucial role in many retinal diseases, including age-related macular degeneration, proliferative vitreoretinopathy, and diabetic retinopathy. This dynamic process requires complex metabolic reprogramming to accommodate the demands of this dramatic cellular transformation. Both transforming growth factor-beta 2 (TGFβ2) and tumor necrosis factor-alpha (TNFα) have the capacity to induce EMT in RPE cells; however, little is known about their impact on the RPE metabolome. Untargeted metabolomics using high-resolution mass spectrometry was performed to reveal the metabolomic signatures of cellular and secreted metabolites of primary human fetal RPE cells treated with either TGFβ2 or TNFα for 5 days. A total of 638 metabolites were detected in both samples; 188 were annotated as primary metabolites. Metabolomics profiling showed distinct metabolomic signatures associated with TGFβ2 and TNFα treatment. Enrichment pathway network analysis revealed alterations in the pentose phosphate pathway, galactose metabolism, nucleotide and pyrimidine metabolism, purine metabolism, and arginine and proline metabolism in TNFα-treated cells compared to untreated control cells, whereas TGFβ2 treatment induced perturbations in fatty acid biosynthesis metabolism, the linoleic acid pathway, and the Notch signaling pathway. These results provide a broad metabolic understanding of the bioenergetic rewiring processes governing TGFβ2- and TNFα-dependent induction of EMT. Elucidating the contributions of TGFβ2 and TNFα and their mechanistic differences in promoting EMT of RPE will enable the identification of novel biomarkers for diagnosis, management, and tailored drug development for retinal fibrotic diseases.

Redox homeostasis is a delicate balancing act of maintaining appropriate levels of antioxidant defense mechanisms and reactive oxidizing oxygen and nitrogen species. Any disruption of this balance leads to oxidative stress, which is a key pathogenic factor in several ocular diseases. In this review, we present the current evidence for oxidative stress and mitochondrial dysfunction in conditions affecting both the anterior segment (e.g., dry eye disease, keratoconus, cataract) and posterior segment (age-related macular degeneration, proliferative vitreoretinopathy, diabetic retinopathy, glaucoma) of the human eye. We posit that further development of therapeutic interventions to promote pro-regenerative responses and maintenance of the redox balance may delay or prevent the progression of these major ocular pathologies. Continued efforts in this field will not only yield a better understanding of the molecular mechanisms underlying the pathogenesis of ocular diseases but also enable the identification of novel druggable redox targets and antioxidant therapies.

The retinal pigment epithelium (RPE) acts as a metabolic gatekeeper between photoreceptors and the choroidal vasculature to maintain retinal function. RPE dysfunction is a key feature of age-related macular degeneration (AMD), the leading cause of blindness in developed countries. Inflammation is a key pathogenic mechanism in AMD and tumor necrosis factor-alpha (TNFα) has been implicated as a pro-inflammatory cytokine involved in AMD. While mitochondrial dysfunction has been implicated in AMD pathogenesis, the interplay between inflammation and cellular metabolism remains elusive. The present study explores how the pro-inflammatory cytokine, TNFα, impacts mitochondrial morphology and metabolic function in RPE. Matured human primary RPE (H-RPE) were treated with TNFα (10 ng/ml) for up to 5 days. TNFα-induced upregulation of IL-6 secretion and inflammatory genes (IL-6, IL-8, MCP-1) was accompanied by increased oxidative phosphorylation (OXPHOS) and reduced glycolysis, leading to an increase in cellular adenosine triphosphate (ATP) content. Transmission electron microscopy (TEM) revealed defects in mitochondrial morphology with engorged mitochondria and loss of cristae integrity following TNFα treatment. Pre-treatment with the anti-inflammatory drug, 80 μM dimethyl fumarate (DMFu), blocked TNFα-induced inflammatory activation of RPE (IL-6, IL-8, MCP-1, CFH, CFB, C3) and normalized their bioenergetic profile to control levels by regulating PFKFB3 and PKM2 gene expression. Furthermore, DMFu prevented TNFα-induced mitochondrial dysfunction and morphological anomalies. Thus, our results indicate that DMFu serves as a novel therapeutic avenue for combating inflammatory activation and metabolic dysfunction of RPE in AMD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shu, Frank, Fitch, Karg, Butcher, Nnuji-John, Kim and Saint-Geniez.)

Bone morphogenetic proteins (BMPs) are a diverse class of growth factors that belong to the transforming growth factor-beta (TGFβ) superfamily. Although originally discovered to possess osteogenic properties, BMPs have since been identified as critical regulators of many biological processes, including cell-fate determination, cell proliferation, differentiation and morphogenesis, throughout the body. In the ocular lens, BMPs are important in orchestrating fundamental developmental processes such as induction of lens morphogenesis, and specialized differentiation of its fiber cells. Moreover, BMPs have been reported to facilitate regeneration of the lens, as well as abrogate pathological processes such as TGFβ-induced epithelial-mesenchymal transition (EMT) and apoptosis. In this review, we summarize recent insights in this topic and discuss the complexities of BMP-signaling including the role of individual BMP ligands, receptors, extracellular antagonists and cross-talk between canonical and non-canonical BMP-signaling cascades in the lens. By understanding the molecular mechanisms underlying BMP activity, we can advance their potential therapeutic role in cataract prevention and lens regeneration.

Lens homeostasis and transparency are dependent on the function and intercellular communication of its epithelia. While the lens epithelium is uniquely equipped with functional repair systems to withstand reactive oxygen species (ROS)-mediated oxidative insult, ROS are not necessarily detrimental to lens cells. Lens aging, and the onset of pathogenesis leading to cataract share an underlying theme; a progressive breakdown of oxidative stress repair systems driving a pro-oxidant shift in the intracellular environment, with cumulative ROS-induced damage to lens cell biomolecules leading to cellular dysfunction and pathology. Here we provide an overview of our current understanding of the sources and essential functions of lens ROS, antioxidative defenses, and changes in the major regulatory systems that serve to maintain the finely tuned balance of oxidative signaling vs. oxidative stress in lens cells. Age-related breakdown of these redox homeostasis systems in the lens leads to the onset of cataractogenesis. We propose eight candidate hallmarks that represent common denominators of aging and cataractogenesis in the mammalian lens: oxidative stress, altered cell signaling, loss of proteostasis, mitochondrial dysfunction, dysregulated ion homeostasis, cell senescence, genomic instability and intrinsic apoptotic cell death., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Transforming growth factor beta (TGFβ) and bone morphogenetic protein (BMP) signaling play opposing roles in epithelial-mesenchymal transition (EMT) of lens epithelial cells, a cellular process integral to the pathogenesis of fibrotic cataract. We previously showed that BMP-7-induced Smad1/5 signaling blocks TGFβ-induced Smad2/3-signaling and EMT in rat lens epithelial cell explants. To further explore the antagonistic role of BMPs on TGFβ-signaling, we tested the capability of BMP-4 or newly described BMP agonists, ventromorphins, in blocking TGFβ-induced lens EMT. Primary rat lens epithelial explants were treated with exogenous TGFβ2 alone, or in combination with BMP-4 or ventromorphins. Treatment with TGFβ2 induced lens epithelial cells to undergo EMT and transdifferentiate into myofibroblastic cells with upregulated α-SMA and nuclear translocation of Smad2/3 immunofluorescence. BMP-4 was able to suppress this EMT without blocking TGFβ2-nuclear translocation of Smad2/3. In contrast, the BMP agonists, ventromorphins, were unable to block TGFβ2-induced EMT, despite a transient and early ability to significantly reduce TGFβ2-induced nuclear translocation of Smad2/3. This intriguing disparity highlights new complexities in the responsiveness of the lens to differing BMP-related signaling. Further research is required to better understand the antagonistic relationship between TGFβ and BMPs in lens EMT leading to cataract., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

PGC-1α, a key orchestrator of mitochondrial metabolism, plays a crucial role in governing the energetically demanding needs of retinal pigment epithelial cells (RPE). We previously showed that silencing PGC-1α induced RPE to undergo an epithelial-mesenchymal-transition (EMT). Here, we show that induction of EMT in RPE using transforming growth factor-beta 2 (TGFβ2) suppressed PGC-1α expression. Correspondingly, TGFβ2 induced defects in mitochondrial network integrity with increased sphericity and fragmentation. TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial OXPHOS levels consistent with reduced expression of NDUFB5 . The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic reserve, glucose uptake and gene expression of glycolytic enzymes ( PFKFB3 , PKM2 , LDHA ). Treatment with ZLN005, a selective small molecule activator of PGC-1α, blocked TGFβ2-induced upregulation of mesenchymal genes ( αSMA , Snai1 , CTGF , COL1A1 ) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. ZLN005 effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment of subretinal fibrosis.

Epithelial-mesenchymal transition (EMT) and endothelial-mesenchymal transition (EndMT) are physiological processes required for normal embryogenesis. However, these processes can be hijacked in pathological conditions to facilitate tissue fibrosis and cancer metastasis. In the eye, EMT and EndMT play key roles in the pathogenesis of subretinal fibrosis, the end-stage of age-related macular degeneration (AMD) that leads to profound and permanent vision loss. Predominant in subretinal fibrotic lesions are matrix-producing mesenchymal cells believed to originate from the retinal pigment epithelium (RPE) and/or choroidal endothelial cells (CECs) through EMT and EndMT, respectively. Recent evidence suggests that EMT of RPE may also be implicated during the early stages of AMD. Transforming growth factor-beta (TGFβ) is a key cytokine orchestrating both EMT and EndMT. Investigations in the molecular mechanisms underpinning EMT and EndMT in AMD have implicated a myriad of contributing factors including signaling pathways, extracellular matrix remodelling, oxidative stress, inflammation, autophagy, metabolism and mitochondrial dysfunction. Questions arise as to differences in the mesenchymal cells derived from these two processes and their distinct mechanistic contributions to the pathogenesis of AMD. Detailed discussion on the AMD microenvironment highlights the synergistic interactions between RPE and CECs that may augment the EMT and EndMT processes in vivo. Understanding the differential regulatory networks of EMT and EndMT and their contributions to both the dry and wet forms of AMD can aid the development of therapeutic strategies targeting both RPE and CECs to potentially reverse the aberrant cellular transdifferentiation processes, regenerate the retina and thus restore vision.

The ocular lens is exposed to numerous growth factors that influence its behavior in diverse ways. While many of these, such as FGF and EGF promote normal cell behavior, TGFβ is unique in that it can also induce lens cell pathology, namely, the epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) leading to fibrotic cataract formation. The present study explores how EGF impacts on TGFβ-induced EMT in the lens. LECs in explants prepared from 21-day-old Wistar rats were treated with either 200 pg/ml TGFβ2, 5 ng/ml EGF, or a combination of these, with or without a 2-h pre-treatment of an EGFR inhibitor (PD153035), MEK inhibitor (U0126) or Smad3 inhibitor (SIS3). Co-treatment of LECs with TGFβ2 and EGF, compared with TGFβ2 alone, resulted in a more pronounced elongation and transdifferentiation of the LECs into myofibroblastic cells, with higher protein levels of mesenchymal cell markers (α-SMA and tropomyosin). Combining EGF with a less potent lower dose of TGFβ2 (50 pg/ml) induced LECs to undergo EMT equivalent to treatment with a higher dose of TGFβ2 (200 pg/ml) within 5 days of culture. EGF alone, nor the lower dose of TGFβ2, were able to induce EMT in LECs within 5 days. Co-treatment of LECs with EGF and TGFβ2 induced a temporal shift in the phosphorylation levels of Smad2/3, ERK1/2 and EGFR and changed the expression patterns of downstream EMT target genes, compared to treatment of LECs with either growth factor alone. Inhibition of EGFR-signaling with PD153035 blocked the EMT response induced by co-treatment with EGF and TGFβ2. Taken together, our data demonstrate that EGF can potentiate TGFβ2 activity to enhance EMT in LECs, further highlighting the importance of EGFR-signaling in cataract formation. By directly blocking EGFR signaling, the activity of both EGF and TGFβ2 can be simultaneously reduced, thereby serving as a potential target for cataract prevention., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We previously demonstrated that inhibition of epidermal growth factor receptor (EGFR) slowed corneal epithelial migration. Here we examine the effect of EGF on transforming growth factor-beta receptor II (TGF-βRII) in a corneal wound-healing model and primary human corneal epithelial cells (pHCE). Corneal debridement wounds were made and allowed to heal ± Tyrphostin AG1478 (EGFR inhibitor), and assayed for EGFR activation and EGFR and TGF-βRII localization. Primary HCE were treated with EGF ± U0126 (MEK inhibitor) and assayed for TGF-βRII expression. EGFR activation was maximal 15 minutes after wounding and localized in the migrating epithelial cells. TGF-βRII localization was also observed in the migrating epithelium and was reduced when EGFR was blocked. When pHCE were treated with EGF for 6 hours, the cells produced enhanced levels of TGF-βRII, which was blocked by U0126. Downstream signaling pathways of MEK (p38 MAPK and ERK1/2 MAPK ) were then examined, and TGF-β1 and EGF were found to have differential effects on the phosphorylation of p38 and ERK1/2, with TGF-β1 upregulating p-p38 but not pERK1/2 and EGF upregulating pERK1/2 but not p-p38. Taken together, these data indicate that EGF stimulates TGF-βRII through ERK1/2 and EGFR signaling, suggesting interplay between EGF- and TGF-β-signaling pathways during corneal wound repair.

The retinal pigment epithelium (RPE) supports visual processing and photoreceptor homeostasis via energetically demanding cellular functions. Here, we describe the consequences of repressing peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α), a master regulator of mitochondrial function and biogenesis, on RPE epithelial integrity. The sustained silencing of PGC-1α in differentiating human RPE cells affected mitochondria/autophagy function, redox state, and impaired energy sensor activity ultimately inducing epithelial to mesenchymal transition (EMT). Adult conditional knockout of PGC-1 coactivators in mice resulted in rapid RPE dysfunction and transdifferentiation associated with severe photoreceptor degeneration. RPE anomalies were characteristic of autophagic defect and mesenchymal transition comparable with the ones observed in age-related macular degeneration. These findings demonstrate that PGC-1α is required to maintain the functional and phenotypic status of RPE by supporting the cells' oxidative metabolism and autophagy-mediated repression of EMT., (© 2019 Rosales et al.)

Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) plays a critical role in the pathogenesis of fibrotic cataract. Transforming growth factor-beta (TGFβ) is a potent inducer of this fibrotic process in lens. Recent studies in cancer progression have shown that in addition to activating the canonical Smad signaling pathway, TGFβ can also transactivate the epidermal growth factor receptor (EGFR) to enhance invasive cell migration. The present study aims to elucidate the involvement of EGFR-signaling in TGFβ-induced EMT in LECs. Treatment with TGFβ2 induced transdifferentiation of LECs into myofibroblastic cells, typical of an EMT. TGFβ2 induced the phosphorylation of the EGFR and upregulation of Egfr and Hb-egf gene expression. Pharmacologic inhibition of EGFR-signaling using PD153035 inhibited TGFβ-induced EMT, including the upregulation of mesenchymal markers and downregulation of epithelial markers. Crosstalk between TGFβ2-induced EGFR and ERK1/2 was evident, with both pathways impacting on Smad2/3-signaling. Our finding that TGFβ2 transactivates downstream EGFR-signaling reveals a previously unknown mechanism in the pathogenesis of cataract. Understanding the complex interplay between divergent canonical and non-canonical signaling pathways, as well as downstream target genes involved in TGFβ-induced EMT, will enable the development of more effective targeted therapies in the pharmacological treatment of cataract., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Purpose: This study aims to highlight some of the genes that are differentially regulated by ERK1/2 signaling in TGFβ-induced EMT in lens, and their potential contribution to this pathological process., Materials and Methods: Rat lens epithelial explants were cultured with or without TGFβ over a 3-day-culture period to induce EMT, in the presence or absence of UO126 (ERK1/2 signaling inhibitor), both prior to TGFβ-treatment, or 24 or 48 hours after TGFβ treatment. Smad2/3-nuclear immunolabeling was used to indicate active TGFβ signaling, and quantitative RT-PCR was used to analyze changes in the different treatment groups in expression of the following representative genes: TGFβ signaling (Smad7, Smurf1, and Rnf111), epithelial markers (Pax6, Cdh1, Zeb1, and Zeb2), cell survival/death regulators (Bcl2, Bax, and Bad) and lens mesenchymal markers (Mmp9, Fn1, and Col1a1), over the 3 days of culture., Results: ERK1/2 was found to regulate the expression of Smurf1, Smad7, Rnf11, Cdh1, Pax6, Zeb1, Bcl2, Bax, and Bad genes in lens cells. TGFβ signaling was evident by nuclear localization of Smad2/3 and this was effectively blocked by pre-treatment with UO126, but not by post-treatment with this ERK1/2 signaling inhibitor. TGFβ induced the expression of its signaling partners (Smad7, Smurf1, and Rnf111), as well as lens mesenchymal genes (Mmp9, Fn1, and Col1a1), consistent with its role in inducing an EMT. These TGFβ-responsive signaling genes, as well as the mesenchymal markers, were all positively regulated by ERK1/2-activity. The expression levels of the lens epithelial genes we examined, and genes that were associated with cell death/survival, were not directly impacted by TGFβ., Conclusions: TGFβ-mediated ERK1/2 signaling positively modulates the expression of mesenchymal genes in lens epithelial explants undergoing EMT, in addition to regulating TGFβ-mediated regulatory genes. Independent of TGFβ, ERK1/2 activity can also regulate the expression of endogenous lens epithelial genes, highlighting its potential key role in regulation of both normal and pathological lens cellular processes.

This paper was aimed to explore the physics of Cherenkov radiation and its potential application in boron neutron capture therapy (BNCT). The Monte Carlo toolkit Geant4 was used to simulate the interaction between the epithermal neutron beam and the phantom containing boron-10. Results showed that Cherenkov photons can only be generated from secondary charged particles of gamma rays in BNCT, in which the 2.223 MeV prompt gamma rays are the main contributor. The number of Cherenkov photons per unit mass generated in the measurement region decreases linearly with the increase of boron concentration in both water and tissue phantom. The work presented the fundamental basis for applications of Cherenkov radiation in BNCT., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Wound healing is one of the most complex biological processes to occur in life. Repair of tissue following injury involves dynamic interactions between multiple cell types, growth factors, inflammatory mediators and components of the extracellular matrix (ECM). Aberrant and uncontrolled wound healing leads to a non-functional mass of fibrotic tissue. In the eye, fibrotic disease disrupts the normally transparent ocular tissues resulting in irreversible loss of vision. A common feature in fibrotic eye disease is the transdifferentiation of cells into myofibroblasts that can occur through a process known as epithelial-mesenchymal transition (EMT). Myofibroblasts rapidly produce excessive amounts of ECM and exert tractional forces across the ECM, resulting in the distortion of tissue architecture. Transforming growth factor-beta (TGFβ) plays a major role in myofibroblast transdifferentiation and has been implicated in numerous fibrotic eye diseases including corneal opacification, pterygium, anterior subcapsular cataract, posterior capsular opacification, proliferative vitreoretinopathy, fibrovascular membrane formation associated with proliferative diabetic retinopathy, submacular fibrosis, glaucoma and orbital fibrosis. This review serves to introduce the pathological functions of the myofibroblast in fibrotic eye disease. We also highlight recent developments in elucidating the multiple signaling pathways involved in fibrogenesis that may be exploited in the development of novel anti-fibrotic therapies to reduce ocular morbidity due to scarring., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Transforming Growth Factor Beta (TGFβ) potently induces lens epithelial to mesenchymal transition (EMT). The resultant mesenchymal cells resemble those found in plaques of human forms of subcapsular cataract. Smad signaling has long been implicated as the sole driving force of TGFβ-mediated activity. Rat lens epithelial explants were used to examine the role of the Smad-independent signaling, namely the MAPK/ERK1/2 signaling pathway, in the initiation and progression of TGFβ-induced EMT. Phase contrast microscopy was used to observe the morphological changes associated with TGFβ-induced EMT in this model, including cell elongation, cell membrane blebbing, cell loss as indicated by the area of bare capsule and capsular wrinkling. The levels of Smad2, Smad2/3 and ERK1/2 phosphorylation measured using western blotting confirmed that the addition of UO126 was sufficient in blocking all TGFβ-induced ERK1/2 activation, as well as reducing Smad signaling at 18 h. Immunofluorescent labeling and further western blotting confirmed that TGFβ-induced EMT was associated with an increase in α-smooth muscle actin (α-SMA) and a reduction of E-cadherin at cell borders. Pre-treatment with UO126 was effective at blocking the TGFβ-induced EMT, as evidenced by a reduction of α-SMA expression and protein labeling, E-cadherin labeling at cell borders, and a reduction of cell loss, cell elongation and capsular wrinkling. Post-treatment with UO126 at 2 and 6 h after TGFβ addition was also effective at blocking EMT while post-treatment with UO126 at 24 and 48 h was not sufficient in hampering TGFβ-induced EMT. Our data implicates ERK1/2 signaling in the initiation but not the progression of TGFβ-induced EMT in rat lens epithelial cells. The tight regulation of intracellular signaling pathways such as ERK1/2 are required for the maintenance of lens epithelial cell integrity and hence tissue transparency. A greater understanding of the molecular mechanisms that drive the induction and progression of EMT in the lens will provide the basis for potential therapeutics for human cataract., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Optimization of the Compton camera for measuring prompt gamma rays (0.478MeV) emitted during boron neutron capture therapy (BNCT) was performed with Geant4. The parameters of the Compton camera were determined as follows: 3cm thick - 10cm wide scatter detector (Silicon), 10cm thick - 10cm wide absorber detector (Germanium), and 1cm distance between the scatter and absorber detectors. For a typical brain tumor treatment, the overall detection efficiency of the optimized Compton camera was approximately 0.1425% using the Snyder's head phantom with a sphere tumor (4cm diameter and ~1cm depth)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: To report the histopathological features of anterior subcapsular cataract associated with atopic dermatitis., Case Report: A 29-year-old man with atopic dermatitis presented with bilateral anterior subcapsular cataract. After routine cataract surgery, the anterior subcapsular cataractous tissue was obtained as an anterior capsulorhexis flap and prepared as a wholemount for histological analysis. The wholemount consisted of a well-demarcated central grayish-white plaque surrounded by transparent capsule, corroborating the slit-lamp biomicroscopic appearance. Higher magnification of the plaque revealed a fibrous and amorphous mass, most likely extracellular matrix owing to the presence of irregularly arranged bundled strands of fibrils, typical of collagen. Lens epithelial cells at the plaque were densely packed and myofibroblast-like and immunoreactive for alpha-smooth muscle actin. In contrast, lens epithelial cells more distant from the plaque retained their regular cuboidal arrangement and regular spacing, and were not labeled for alpha-smooth muscle actin, similar to lens epithelial cells obtained from a non-cataractous case., Conclusions: The presence of alpha-smooth muscle actin-reactive elongated cells at the plaque suggests that the cuboidal lens epithelial cells making up the anterior subcapsular cataract have transdifferentiated into spindle-shaped myofibroblastic cells that produce and deposit aberrant extracellular matrix. This transdifferentiation process, more commonly known as an epithelial-mesenchymal transition, contributes to a fibrotic response leading to the development of human anterior subcapsular cataract.

Purpose: Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a key pathologic mechanism underlying cataract. Two members of the transforming growth factor-β (TGFβ) superfamily, TGFβ and bone morphogenetic protein-7 (BMP-7) have functionally distinct roles in EMT. While TGFβ is a potent inducer of EMT, BMP-7 counteracts the fibrogenic activity of TGFβ. We examine the modulating effect of BMP-7 on TGFβ-induced EMT in LECs., Methods: Rat lens epithelial explants were treated exogenously with TGFβ2 alone or in combination with BMP-7 for up to 5 days. Expression levels of E-cadherin, β-catenin, α-smooth muscle actin (α-SMA), and phosphorylated downstream Smads were determined using immunofluorescence and Western blotting. Reverse transcriptase quantitative PCR (RT-qPCR) was used to study gene expression levels of EMT markers and downstream BMP target genes, including the Inhibitors of differentiation (Id)., Results: Transforming growth factor-β2 induced LECs to transdifferentiate into myofibroblastic cells. Addition of BMP-7 suppressed TGFβ2-induced α-SMA protein levels and mesenchymal gene expression, with retention of E-cadherin and β-catenin expression to the cell membrane. Addition of BMP-7 prevented lens capsular wrinkling and cellular loss associated with TGFβ2-induced EMT over the 5-day treatment period. The inhibitory effect of BMP-7 was accompanied by an early induction of pSmad1/5 and suppression of TGFβ2-induced pSmad2/3. Treatment with TGFβ2 alone suppressed gene expression of Id2/3 and addition of BMP-7 restored Id2/3 expression., Conclusions: Exogenous administration of BMP-7 abrogated TGFβ2-induced EMT in rat lens epithelial explants. Understanding the complex interplay between the TGFβ- and BMP-7-associated Smad signaling pathways and their downstream target genes holds therapeutic promise in cataract prevention.

Background: The L25 mouse line was generated by random genomic insertion of a lens-specific transgene. Inbreeding of L25 hemizygotes revealed an unanticipated spastic phenotype in the hind limbs., Objective: The goals were to characterize the motor phenotype in the L25 mice and to map the transgene insert site within the mouse genome., Methods: Six pairs of L25+/- mice were repeatedly mated. Beginning at weaning, all progeny were inspected for body weight and motor signs twice weekly until they displayed predefined ethical criteria for termination. The transgene insert site was determined by whole genome sequencing. Western blotting was used to compare the expression levels of beta-IV spectrin protein in the brain., Results: Matings of hemizygous L25+/- × L25+/- mice yielded 20% (29/148) affected weanlings, identified by an abnormal retraction of the hind limbs when lifted by the tail, and a fine tremor. Affected mice were less mobile and grew more slowly than wild-type littermates. All affected mice required termination due to >15% loss of body weight (50% survival age 92 days). At the endpoint, mice showed varying degrees of spastic paresis or spastic paralysis localised to the hind limbs. Motor endplates remained fully innervated. Genome sequencing confirmed that the transgene was inserted in the locus of βIV spectrin of L25 mice. Western blotting indicated that this random insertion had greatly reduced the expression of βIV spectrin protein in the affected L25 mice., Conclusions: The results confirm the importance of βIV spectrin for maintaining central motor pathway control of the hind limbs, and provide a developmental time course for the phenotype.

To investigate the efficacy of interferon alpha 2 b plus ribavirin combination therapy in sixty-two patients with chronic hepatitis c (CHC) infection originating from a single blood donor. The 62 patients who developed CHC following blood transfusion from a known single infected donor were treated with interferon and ribavirin combination therapy for 48 weeks and followed-up for 96 weeks. The therapy regimen consisted of subcutaneous administration of 3-500 MIU interferon alpha 2 b every other day and daily oral administration of 0.6-1.0 g of ribavirin. Patients were monitored during treatment and in follow-up for sustained virological response (SVR), early virology response (EVR), treatment end virology response (ETVR), biochemical response of withdrawals, and side effects. The SVR rate was 83.9% (52/62). The EVR rate was 95.2% (59/62). The ETVR rate was 87.1% (54/62). The biochemical response rate after withdrawal of treatment was 100.0%. Eight patients developed mildly abnormal thyroid function as a result of the interferon therapy, but all were able to complete the antiviral treatment regimen under the care of endocrinologists. Younger age, relatively short course of disease, low viral load, and better compliance, but not sex, were correlated to curative effect of the combination therapy. Interferon alpha 2 b plus ribavirin combination therapy had a significant curative effect on a group of 62 CHC patients originating from a single case, with 52 of the patients showing SVR out to 96 weeks after therapy. Antiviral treatment is recommended for hepatitis C virus-positive patients to eradicate the virus and prevent disease progression.

Supercritical CO(2)-based fluid is not only being considered as environmentally benign medium for photoresist (PR) removal in electronic device manufacture, but also capable of challenging feature dimensions. Despite many attractive properties, clear supercritical CO(2) has little solvating power for PR. Here, two acetate modifiers were selective to add in the CO(2) and evaluated individual contribution to the overall stripping rate by factorial experiment design, which included four other factors with four level ranges for each one and concluded the best 90% extraction efficiency would be obtained under the optimized parameters: 2.5min static time, 35min dynamic time, 1.25ml ethylacetate spiked, 125 degrees C oven temperature and 480atm CO(2) pressure. As analyzing the variances of these contributors to this system, it disclosed that dynamics controlled the stripping mechanism before near 35min purging but thermodynamics took over after then; and that increasing pressure was more competent for removing PR than increasing temperature. All supercritical extracts were from two commercial PR coated on two substrates and analyzed by UV absorption spectrometry. Removing PR coated on silicon oxide layer was easier than that on Al-Cu alloy one.