Identification of fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no Food and Drug Administration (FDA)-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 y of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo and inhibits activation of the NLRP3-ASC inflammasome and inflammatory cytokine release in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other antidepressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies identify fluoxetine as a potential drug-repurposing candidate for dry AMD.

Cytoplasmic synthesis of endogenous Alu complementary DNA via reverse transcription and implications in age-related macular degeneration.

Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1-reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493-0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

MicroRNA-874-mediated inhibition of the major G1/S phase cyclin, CCNE1, is lost in osteosarcomas.

The tumor microenvironment is characterized by nutrient-deprived conditions in which the cancer cells have to adapt for survival. Serum starvation resembles the growth factor deprivation characteristic of the poorly vascularized tumor microenvironment and has aided in the discovery of key growth regulatory genes and microRNAs (miRNAs) that have a role in the oncogenic transformation. We report here that miR-874 down-regulates the major G1/S phase cyclin, cyclin E1 (CCNE1), during serum starvation. Because the adaptation of cancer cells to the tumor microenvironment is vital for subsequent oncogenesis, we tested for miR-874 and CCNE1 interdependence in osteosarcoma cells. We observed that miR-874 inhibits CCNE1 expression in primary osteoblasts, but in aggressive osteosarcomas, miR-874 is down-regulated, leading to elevated CCNE1 expression and appearance of cancer-associated phenotypes. We established that loss of miR-874-mediated control of cyclin E1 is a general feature of osteosarcomas. The down-regulation of CCNE1 by miR-874 is independent of E2F transcription factors. Restoration of miR-874 expression impeded S phase progression, suppressing aggressive growth phenotypes, such as cell invasion, migration, and xenograft tumors, in nude mice. In summary, we report that miR-874 inhibits CCNE1 expression during growth factor deprivation and that miR-874 down-regulation in osteosarcomas leads to CCNE1 up-regulation and more aggressive growth phenotypes.

Identification of an RNA aptamer binding hTERT-derived peptide and inhibiting telomerase activity in MCF7 cells.

Human telomerase reverse transcriptase is an essential rate-limiting component of telomerase complex. hTERT protein in association with other proteins and the human telomerase RNA (hTR) shows telomerase activity, essential for maintaining genomic integrity in proliferating cells. hTERT binds hTR through a decapeptide located in the RID2 (RNA interactive domain 2) domain of N-terminal region. Since hTERT is essential for telomerase activity, inhibitors of hTERT are of great interest as potential anti-cancer agent. We have selected RNA aptamers against a synthetic peptide from the RID2 domain of hTERT by employing in vitro selection protocol (SELEX). The selected RNAs could bind the free peptide, as CD spectra suggested conformational change in aptamer upon RID2 binding. Extracts of cultured breast cancer cells (MCF7) expressing this aptamer showed lower telomerase activity as estimated by TRAP assay. hTERT-binding RNA aptamers hold promise as probable anti-cancer therapeutic agent.

RPA70 depletion induces hSSB1/2-INTS3 complex to initiate ATR signaling.

The primary eukaryotic single-stranded DNA-binding protein, Replication protein A (RPA), binds to single-stranded DNA at the sites of DNA damage and recruits the apical checkpoint kinase, ATR via its partner protein, ATRIP. It has been demonstrated that absence of RPA incapacitates the ATR-mediated checkpoint response. We report that in the absence of RPA, human single-stranded DNA-binding protein 1 (hSSB1) and its partner protein INTS3 form sub-nuclear foci, associate with the ATR-ATRIP complex and recruit it to the sites of genomic stress. The ATRIP foci formed after RPA depletion are abrogated in the absence of INTS3, establishing that hSSB-INTS3 complex recruits the ATR-ATRIP checkpoint complex to the sites of genomic stress. Depletion of homologs hSSB1/2 and INTS3 in RPA-deficient cells attenuates Chk1 phosphorylation, indicating that the cells are debilitated in responding to stress. We have identified that TopBP1 and the Rad9-Rad1-Hus1 complex are essential for the alternate mode of ATR activation. In summation, we report that the single-stranded DNA-binding protein complex, hSSB1/2-INTS3 can recruit the checkpoint complex to initiate ATR signaling.

Non-coding RNAs: biological functions and applications.

Analyses of the international human genome sequencing results in 2004 converged to a consensual number of ~20,000 protein-coding genes, spanning over <2% of the total genomic sequence. Therefore, the developmental and physiological complexity of human beings remains unaccounted if viewed only in terms of the number of protein-coding genes; the epigenetic influences involving chromatin remodelling and RNA interference and alternative precursor messenger RNA splicing of functional protein-coding transcripts as well as post-translational modifications of proteins increase the diversity and the functionality of the proteome and likely explain the increased complexity. In addition, there has been an explosion of research addressing possible functional roles for the other 98% of the human genome that does not encode proteins. In fact, >90% of the human genome is likely to be transcribed yielding a complex network of overlapping transcripts that include tens of thousands of long RNAs with little or no protein forming capacity; they are collectively called non-coding RNA. This review highlights the fundamental concepts of biological roles of non-coding RNA and their importance in regulation of cellular physiology under disease conditions like cancer.

Expression of targeted ribozyme against telomerase RNA causes altered expression of several other genes in tumor cells.

Telomeres are tandem repeat sequences present at chromosome end that are synthesized by RNA-protein enzyme called telomerase. The RNA component (TR) serves as template for telomerase reverse transcriptase (TERT) for generating telomere repeats. TERT is overexpressed in actively dividing cells including cancerous cells, absent in differentiated somatic cells whereas human telomerase RNA (hTR) is present in normal as well as in cancer cells. Telomerase overexpression in cancer cells ensures telomere length maintenance that actually provides proliferative advantage to cells. Stable expression of ribozyme against hTR in HeLa cells results in reduction of hTR levels, telomerase activity, and telomere length which is accompanied by altered cell morphology and expression of several specific cellular genes. The altered genes deduced from differentially display PCR and 2D gel electrophoresis upon hTR knockdown have function in ribosome biogenesis, chromatin modulation, cell cycle control, and p63-dependant pathways. Our observations shows hTR participates in diverse cellular functions other than telomere maintenance, validates as a possible drug targets in p53- and pRB-negative status, and indicated possible cross-talks between telomerase and other cellular pathways.

A goat eye, wet lab model for training in Descemet membrane endothelial keratoplasty.

Here we describe a new, non-human, ex-vivo model (goat eye model) for training surgeons in DMEK surgeons. In a wet lab setting, goat eyes were used to obtain a pseudo-DMEK graft of 8 mm from the goat lens capsule that was injected into another goat eye with the same maneuvers described for human DMEK. The DMEK pseudo-graft can be easily prepared, stained, loaded, injected, and unfolded into the goat eye model reproducing the similar maneuvers used for DMEK in a human eye, except for the descemetorhexis, which cannot be performed. The pseudo-DMEK graft behaves similar to human DMEK graft and useful for surgeons to experience and understand steps of DMEK early in learning curve. The concept of a non-human ex-vivo eye model is simple and reproducible and obviates the need for human tissue and the issues of poor visibility in stored corneal tissue.

Efficient reduction of the scrolling of Descemet membrane endothelial keratoplasty grafts by engineering the medium.

The Descemet Membrane Endothelial Keratoplasty (DMEK) procedure for corneal transplantation is challenging due to the need to unscroll the donor graft within the recipient's eye. This process of unscrolling is complex, time-consuming, leads to a loss of endothelial cells and, most importantly, can negatively impact the graft's adhesion and integration with the host tissue after surgery. This problem is particularly evident when the graft is young. However, the physics behind this scrolling is not well understood, and therefore no sustainable solution is attained. Here, we propose that the concentration gradient of the medium used during transplant leads to a displacement gradient across the graft thickness, resulting in an out-of-plane folding or scrolling of the graft tissue. Using chitosan bilayer-based experimental models, it is experimentally demonstrated that this diffusion-coupled-deformation phenomenon can successfully explain why younger donor grafts tend to scroll tighter than older ones. Most importantly, we illustrate here through experiments that the medium can be engineered to reduce the scroll tightness and thus reduce the surgical inconveniences and improve post-transplant recovery. STATEMENT OF SIGNIFICANCE: This paper addresses a major issue that surgeons face while doing Descemet Membrane Endothelial Keratoplasty (DMEK) in unscrolling grafts during the graft insertion procedure. The currently used tapping method to unscroll the graft inside the patient's eye significantly reduces endothelial cell count, thus affecting its lifetime. Surprisingly, the physics behind graft scrolling is not well understood, so no sustainable solutions are proposed by the medical community. In this work, we present the underlying mechanism of DMEK graft scroll and illustrate experimentally the reason for scroll tightness through a chitosan bilayer based experiment model. Most importantly, we have successfully demonstrated that the preserving medium of the grafts can be engineered to reduce scroll tightness.

Retinoblastoma: A review of the molecular basis of tumor development and its clinical correlation in shaping future targeted treatment strategies.

Retinoblastoma is a retinal cancer that affects children and is the most prevalent intraocular tumor worldwide. Despite tremendous breakthroughs in our understanding of the fundamental mechanisms that regulate progression of retinoblastoma, the development of targeted therapeutics for retinoblastoma has lagged. Our review highlights the current developments in the genetic, epigenetic, transcriptomic, and proteomic landscapes of retinoblastoma. We also discuss their clinical relevance and potential implications for future therapeutic development, with the aim to create a frontline multimodal therapy for retinoblastoma.

Role of AS-OCT in Managing Corneal Disorders.

Optical coherence tomography (OCT) is analogous to ultrasound biometry in the cross sectional imaging of ocular tissues. Development of current devices with deeper penetration and higher resolution has made it popular tool in clinics for visualization of anterior segment structures. In this review, the authors discussed the application of AS-OCT for diagnosis and management of various corneal and ocular surface disorders. Further, recent developments in the application of the device for pediatric corneal disorders and extending the application of OCT angiography for anterior segment are introduced.

In vitro selected RNA aptamer recognizing glutathione induces ROS mediated apoptosis in the human breast cancer cell line MCF 7.

Glutathione (GSH) is an abundant natural tripeptide with antioxidant properties. Under different conditions, it can play protective as well as pathogenic roles. The redox state of the cell has an important role in the induction of apoptosis. Elevated level of glutathione in cancer cells provides resistance to a number of chemotherapeutic drugs. Inhibition of glutathione synthesis sensitizes the cells for apoptosis and enhances the activity of chemotherapeutic drugs. We have selected GSH-binding RNA aptamers by employing in vitro selection protocol SELEX. The Kd value of these aptamers with respect to GSH were determined by surface plasmon resonance (SPR) analysis and isocratic affinity chromatography. Two aptamers GSHapt 8.17 (class-III) and GSHapt 5.39 (class-IV) had Kd values of 4.18 and 4.89 x 10(-8) M, respectively and GSHapt class-I had a Kd value of 1.2 x 10(-6) M. CD spectra suggested conformational change in aptamers upon GSH binding. Cultured breast cancer cells (MCF7) responded to expression of GSH aptamers by accumulating ROS and undergoing morphological transition, nuclear condensation, and DNA fragmentation, with concurrent depletion of cellular GSH and activation of caspase 3 eventually leading to apoptosis. DTT and caspase-3 inhibitor partially rescued aptamer induced apoptosis. These aptamers exhibit high specificity to GSH over non specific competitor. The same aptamers did not induce apoptosis in 293T cells. The kinetic properties and pro-apoptotic effects suggest that glutathione-binding RNA aptamer could be developed into an effective anti-cancer chemotherapeutic agent.

Infectious Keratitis: An Update on Role of Epigenetics.

Epigenetic mechanisms modulate gene expression and function without altering the base sequence of DNA. These reversible, heritable, and environment-influenced mechanisms generate various cell types during development and orchestrate the cellular responses to external stimuli by regulating the expression of genome. Also, the epigenetic modifications influence common pathological and physiological responses including inflammation, ischemia, neoplasia, aging and neurodegeneration etc. In recent past, the field of epigenetics has gained momentum and become an increasingly important area of biomedical research As far as eye is concerned, epigenetic mechanisms may play an important role in many complex diseases such as corneal dystrophy, cataract, glaucoma, diabetic retinopathy, ocular neoplasia, uveitis, and age-related macular degeneration. Focusing on the epigenetic mechanisms in ocular diseases may provide new understanding and insights into the pathogenesis of complex eye diseases and thus can aid in the development of novel treatments for these diseases. In the present review, we summarize the clinical perspective of infectious keratitis, role of epigenetics in infectious keratitis, therapeutic potential of epigenetic modifiers and the future perspective.

Nucleoside reverse transcriptase inhibitors and Kamuvudines inhibit amyloid-ß induced retinal pigmented epithelium degeneration.

Nonfibrillar amyloid-ß oligomers (AßOs) are a major component of drusen, the sub-retinal pigmented epithelium (RPE) extracellular deposits characteristic of age-related macular degeneration (AMD), a common cause of global blindness. We report that AßOs induce RPE degeneration, a clinical hallmark of geographic atrophy (GA), a vision-threatening late stage of AMD that is currently untreatable. We demonstrate that AßOs induce activation of the NLRP3 inflammasome in the mouse RPE in vivo and that RPE expression of the purinergic ATP receptor P2RX7, an upstream mediator of NLRP3 inflammasome activation, is required for AßO-induced RPE degeneration. Two classes of small molecule inflammasome inhibitors-nucleoside reverse transcriptase inhibitors (NRTIs) and their antiretrovirally inert modified analog Kamuvudines-both inhibit AßOs-induced RPE degeneration. These findings crystallize the importance of P2RX7 and NLRP3 in a disease-relevant model of AMD and identify inflammasome inhibitors as potential treatments for GA.

DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retrotransposon RNAs.

Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domain­containing protein 3, and apoptosis-associated speck-like protein­containing CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNA­driven diseases.

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity.

Long interspersed nuclear element-1 (L1)­mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA­induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA­induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

A Clinical Metabolite of Azidothymidine Inhibits Experimental Choroidal Neovascularization and Retinal Pigmented Epithelium Degeneration.

Purpose: Azidothymidine (AZT), a nucleoside reverse transcriptase inhibitor, possesses anti-inflammatory and anti-angiogenic activity independent of its ability to inhibit reverse transcriptase. The aim of this study was to evaluate the efficacy of 5'-glucuronyl azidothymidine (GAZT), an antiretrovirally inert hepatic clinical metabolite of AZT, in mouse models of retinal pigment epithelium (RPE) degeneration and choroidal neovascularization (CNV), hallmark features of dry and wet age-related macular degeneration (AMD), respectively. Methods: RPE degeneration was induced in wild-type (WT) C57BL/6J mice by subretinal injection of Alu RNA. RPE degeneration was assessed by fundus photography and confocal microscopy of zonula occludens-1-stained RPE flat mounts. Choroidal neovascularization was induced by laser injury in WT mice, and CNV volume was measured by confocal microscopy. AZT and GAZT were delivered by intravitreous injections. Inflammasome activation was monitored by western blotting for caspase-1 and by ELISA for IL-1ß in Alu RNA-treated bone marrow-derived macrophages (BMDMs). Results: GAZT inhibited Alu RNA-induced RPE degeneration and laser-induced CNV. GAZT also reduced Alu RNA-induced caspase-1 activation and IL-1ß release in BMDMs. Conclusions: GAZT possesses dual anti-inflammatory and anti-angiogenic properties and could be a viable treatment option for both forms of AMD.

Repurposing anti-inflammasome NRTIs for improving insulin sensitivity and reducing type 2 diabetes development.

Innate immune signaling through the NLRP3 inflammasome is activated by multiple diabetes-related stressors, but whether targeting the inflammasome is beneficial for diabetes is still unclear. Nucleoside reverse-transcriptase inhibitors (NRTI), drugs approved to treat HIV-1 and hepatitis B infections, also block inflammasome activation. Here, we show, by analyzing five health insurance databases, that the adjusted risk of incident diabetes is 33% lower in patients with NRTI exposure among 128,861 patients with HIV-1 or hepatitis B (adjusted hazard ratio for NRTI exposure, 0.673; 95% confidence interval, 0.638 to 0.710; P < 0.0001; 95% prediction interval, 0.618 to 0.734). Meanwhile, an NRTI, lamivudine, improves insulin sensitivity and reduces inflammasome activation in diabetic and insulin resistance-induced human cells, as well as in mice fed with high-fat chow; mechanistically, inflammasome-activating short interspersed nuclear element (SINE) transcripts are elevated, whereas SINE-catabolizing DICER1 is reduced, in diabetic cells and mice. These data suggest the possibility of repurposing an approved class of drugs for prevention of diabetes.

KLF4 sensitizes the colon cancer cell HCT-15 to cisplatin by altering the expression of HMGB1 and hTERT.

AIMS: Insensitivity of cancer cells to therapeutic drugs is the most daunting challenge in cancer treatment. The mechanism of developing chemo-resistance is only partly understood to date. In continuation of some earlier reports, we hypothesize that KLF4, a key transcription factors that also has a crucial role in maintaining the stemness in cancer cells, may offer a basis for chemo-resistance. MAIN METHODS: Sensitivity of cells to cisplatin was analyzed by cell proliferation, colony formation, and cell growth assay. Cell cycle analysis and immunophenotyping were used to measure cell cycle arrest and level of reactive oxygen species respectively. Immunoblotting was used to analyze the change in expression hTERT and HMGB1 involved in KLF4 mediated cisplatin resistance. KEY FINDINGS: We found that KLF4 expression sensitizes cancer cell to cisplatin cytotoxicity. Further, KLF4 promotes the cisplatin-mediated G2/M cell cycle arrest while KLF4 knocked down induces cisplatin-mediated S-phase arrest compared to control. Decreased level of reactive oxygen species (ROS) in cisplatin-treated and KLF4 knocked down HCT-15 cells compared to vector control, accounting for increased cell survival. Immuno-blotting showed that KLF4 positively regulates expression of the survival proteins hTERT and HMGB1 while in presence of cisplatin, expression of HMGB1 and hTERT is negatively regulated by KLF4. SIGNIFICANCE: This study suggests the involvement of KLF4-HMGB1/hTERT signaling in offering the basis for chemo-resistance in colon cancer cells and KLF4 overexpression as a probable strategy for sensitizing drug-resistant cancer cells to chemotherapy. The present study opens up new avenues for cancer research and therapeutics.