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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 domain–containing protein 3, and apoptosis-associated speck-like protein–containing 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 RNA–driven diseases.

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.

Description, Endogenous Alu cDNA triggers inflammasome-mediated cell death in age-related macular degeneration., 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 cDNA–induced 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 RNA–induced 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.

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.

Nonfibrillar amyloid-β oligomers (AβOs) 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 AβOs induce RPE degeneration, a clinical hallmark of geographic atrophy (GA), a vision-threatening late stage of AMD that is currently untreatable. We demonstrate that AβOs 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 AβO-induced RPE degeneration. Two classes of small molecule inflammasome inhibitors—nucleoside reverse transcriptase inhibitors (NRTIs) and their antiretrovirally inert modified analog Kamuvudines—both inhibit AβOs-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.

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.

Significance Alu elements, comprising more than 10% of the human genome, propagate via retrotransposition. This genomic expansion requires enzymatic activity of L1 that reverse transcribes Alu RNA into Alu cDNA in the nucleus. We report Alu also undergoes L1-mediated reverse transcription via self-priming in the cytoplasm independent of retrotransposition, providing evidence of human DNA synthesis in this cellular compartment. This newly discovered shunt molecule in the Alu replication cycle also induces death of the retinal pigmented epithelium, a hallmark of atrophic age-related macular degeneration. A Big Data Archeology analysis of multiple health insurance databases reveals that use of FDA-approved nucleoside reverse transcriptase inhibitors is associated with protection against macular degeneration, identifying a repurposing candidate for this blinding disease.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no FDA-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 years 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 that it inhibits activation of the NLRP3-ASC inflammasome in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other anti-depressant 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 triangulate to link fluoxetine as a potential drug repurposing candidate for a major unmet medical need that causes blindness in millions of people in the United States and across the world.Significance StatementDry age-related macular degeneration (AMD) affects the vision of millions of people worldwide. There is currently no FDA-approved treatment for dry AMD. The inflammasome components NLRP3 and ASC have been implicated in the pathogenesis of dry AMD. We report that fluoxetine, which is approved for the treatment of clinical depression, directly binds the NLRP3 protein and prevents the assembly and activation of the NLRP3-ASC inflammasome. As a result, it also blocks the degeneration of retinal pigmented epithelium (RPE) cells in an animal model of dry AMD. Furthermore, we demonstrate through an analysis of health insurance databases that use of this FDA-approved anti-depressant drug is associated with reduced incidence of dry AMD. These studies identify that fluoxetine is a potential repurposing candidate for AMD, a prevalent cause of blindness.

The urokinase plasminogen activator system is a family of serine proteases which consists of uPA (urokinase plasminogen activator), uPAR (urokinase type plasminogen activator receptor) and PAI-1 (plasminogen activator inhibitor 1). In addition to their significant roles in activation, these proteases act as key regulators of the tumor microenvironment and are involved in the metastatic process in many cancers. High levels of uPA system proteases in many human cancer predicts poor patient prognosis and strongly indicated a key role of uPA system in cancer metastasis. Individual components of uPA system are found to be differentially expressed in cancer cells compared to normal cells and therefore are potential therapeutic targets. In this review, we present the molecular and cellular mechanisms underlying the role of uPA system in cancer progression. Epithelial to mesenchymal transitions (EMT) is the main cause of the cancer cell metastasis. We have also attempted to relate the role of uPA signaling in EMT of cancer cells.

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.

Geographic atrophy is a blinding form of age-related macular degeneration characterized by death of the retinal pigmented epithelium (RPE). In this disease, the RPE displays evidence of DICER1 deficiency, resultant accumulation of endogenous Alu retroelement RNA, and NLRP3 inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human cell culture and in mouse models is driven by a non-canonical inflammasome pathway that results in activation of caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-β (IFN-β) production and gasdermin D-dependent interleukin-18 (IL-18) secretion. Reduction of DICER1 levelsor accumulation of Alu RNA triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, IFN-β, and cGAS levels are elevated in the RPE of human eyes with geographic atrophy. Collectively, these data highlight an unexpected role for cGAS in responding to mobile element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial damage-induced inflammasome activation, expand the immune sensing repertoire of cGAS and caspase-4 to non-infectious human disease, and identify new potential targets for treatment of a major cause of blindness.

This study reports differentially expressed proteins in the middle silkglands of normal and cold stress treated 5th instar Philosamia ricini larvae by using high resolution two-dimensional polyacrylamide gel electrophoresis. About 300 protein spots were resolved in both type of the larvae. Most of the proteins were shown to be distributed in the area from 20 to 70 kDa ranging between pH 4 and 8. Six proteins showed considerably decreased expression in cold stress treated samples. Three proteins displayed increased level of expression after cold stress treatment, while two other proteins were expressed exclusively in cold stress treated silkgland tissues.

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, Inflammasome activation may contribute to type 2 diabetes, but whether targeting inflammasome is beneficial is unclear. Here the authors show that repurposing nucleoside reverse transcriptase inhibitors for inhibiting inflammasome activation is associated with reduced diabetes development in people and improves insulin sensitivity in experimental settings.

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-1β 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-1β 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.

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.

The migration of chromosomes during mitosis is mediated primarily by kinesins that bind to the chromosomes and move along the microtubules, exerting pulling and pushing forces on the centrosomes. We report that a DNA replication protein, Sld5, localizes to the centrosomes, resisting the microtubular pulling forces experienced during chromosome congression. In the absence of Sld5, centriolar satellites, which normally cluster around the centrosomes, are dissipated throughout the cytoplasm, resulting in the loss of their known function of recruiting the centrosomal protein, pericentrin. We observed that Sld5-deficient centrosomes lacking pericentrin were unable to endure the CENP-E- and Kid-mediated microtubular forces that converge on the centrosomes during chromosome congression, resulting in monocentriolar and acentriolar spindle poles. The minus-end-directed kinesin-14 motor protein, HSET, sustains the traction forces that mediate centrosomal fragmentation in Sld5-depleted cells. Thus, we report that a DNA replication protein has an as yet unknown function of ensuring spindle pole resistance to traction forces exerted during chromosome congression.

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.

Analyses of the international human genome sequencing results in 2004 converged to a consensual number of ~20,000 protein-coding genes, spanning over 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.

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.

Telomerase is a specialized nucleoprotein enzyme complex that maintains the telomere length. The telomerase reverse transcriptase (TERT) is the catalytically active component of the telomerase complex. In humans, the protein component (hTERT) and RNA component (hTR) are found to differentially express in cancer cells. In contrast to differentiated cells, most of the cancer cells overexpress hTERT, which is needed to maintain the proliferative potential of cells. The overexpression of telomerase is not proportionate to telomere length in cancer cells, suggesting that the immortalizing phenotype can be mediated through other factors in addition to telomere length. To investigate the role of hTERT in immortalizing process, loss of gene function studies were carried out. Short interfering RNA (siRNA) and short hairpin RNA (shRNA) against hTERT showed the reduction of hTERT transcript, reduction of telomerase activity and alteration of gene expression in HeLa cells. The molecular basis of proliferative capacity of hTERT was investigated by gene expression microarray. Analysis of microarray data for HeLa cells following siRNA and shRNA mediated knockdown of hTERT showed that 80 genes were upregulated and 73 genes downregulated. Out of these, 37 genes are known to be involved in cancer. Further analyses of previously known genes involved in cancer like KLF4, FGF2, IRF-9 and PLAU by Real Time PCR showed their upregulation. We are documenting for the first time the effect of knocking down hTERT on expression of KLF4 and FGF2. Interestingly, it has been earlier reported that KLF4 and FGF2 up-regulate the expression of hTERT in cancer cells. This suggests that hTERT may be subject to its own auto-regulatory effects.

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.

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.

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.

Background Measles virus nucleoprotein (N) encapsidates the viral RNA, protects it from endonucleases and forms a virus specific template for transcription and replication. It is the most abundant protein during viral infection. Its C-terminal domain is intrinsically disordered imparting it the flexibility to interact with several cellular and viral partners. Principal Findings In this study, we demonstrate that expression of N within mammalian cells resulted in morphological transitions, nuclear condensation, DNA fragmentation and activation of Caspase 3 eventuating into apoptosis. The rapid generation of intracellular reactive oxygen species (ROS) was involved in the mechanism of cell death. Addition of ascorbic acid (AA) or inhibitor of caspase-3 in the extracellular medium partially reversed N induced apoptosis. We also studied the protein profile of cells expressing N protein. MS analysis revealed the differential expression of 25 proteins out of which 11 proteins were up regulated while 14 show signs of down regulation upon N expression. 2DE results were validated by real time and semi quantitative RT-PCR analysis. Conclusion These results show the pro-apoptotic effects of N indicating its possible development as an apoptogenic tool. Our 2DE results present prima facie evidence that the MV nucleoprotein interacts with or causes differential expression of a wide range of cellular factors. At this stage it is not clear as to what the adaptive response of the host cell is and what reflects a strategic modulation exerted by the virus.