Structural and Biochemical Insights into Methylation Site and State Specificity of JMJD2 Lysine Demethylases.

The human JMJD2/KDM4 family of histone lysine demethylases comprises four homologs: JMJD2A, JMJD2B, JMJD2C and JMJD2D. These enzymes have been implicated in a number of biological processes such as transcriptional activation, development and cell cycle control. The biological functions of these enzymes are defined by their distinct methylation site and state specificities. JMJD2A, JMJD2B and JMJD2C display dual specificity for trimethylated histone H3 Lys9 and Lys36 (H3K9me3 and H3K36me3), whereas JMJD2D is specific for H3K9me3. Furthermore, while most JMJD2 homologs are predominantly trimethyllysine-specific, JMJD2D can demethylate both tri- and dimethyllysines. To enable quantitative kinetic studies of JMJD2 demethylases, we developed and applied a new affinity purification protocol that minimizes contamination by transition state metals. In order to delineate the molecular basis of site and state specific demethylation by the JMJD2 enzymes, we determined the first crystal structure of JMJD2D in the apoenzyme form and in a ternary complex with 2-OG and an H3K9me3 peptide. Our site specificity studies with JMJD2A and JMJD2D revealed surprising differences in H3K9me3 recognition by these enzymes despite the overall similarity in the substrate binding conformation. In addition, our docking studies with H3K36me3 and biochemical analysis with histone H3 hybrid peptides underscored the role of steric clashes, electrostatic clashes and loss of productive hydrogen bonds in occluding recognition of the H3K36me3 site by JMJD2D. Our structural and biochemical analysis of the active site also revealed the basis for differential state specificity in the JMJD2 enzymes and highlighted the role of CH---O hydrogen bonds in di- and trimethyllysine substrate recognition. Together, these structural and biochemical studies elucidate the molecular basis of the different substrate specificities within the JMJD2 family, which is not only key to understanding their distinct biological functions but will also aid in the structure-based design of selective inhibitors of JMJD2 enzymes implicated in disease.PHDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/96120/1/swathik_1.pd

Structure and Function of Histone H3 Lysine 9 Methyltransferases and Demethylases

Histone lysine methylation is a dynamic chromatin modification that plays key regulatory roles in gene expression and other genomic functions. Methylation of Lys9 in histone H3 (H3K9) is a prominent modification that has been implicated in diverse processes, including transcriptional silencing, heterochromatin formation, and DNA methylation. In this review, we summarize recent advances in understanding the structure and substrate specificity of the H3K9-specific methyltransferases G9A and GLP and explore current efforts to develop inhibitors of these enzymes. In addition, we discuss the structure and specificity of the recently discovered PHF8 family of histone demethylases that target H3K9 as well as other methylation sites in histones H3 and H4. Finally, we conclude by comparing the H3K9 binding modes displayed by these enzymes and examine the relevance of these studies to their biological functions and to structure-based inhibitor design.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79431/1/254_ftp.pd

Crosslinguistic Generalization of Semantic Treatment in Aphasia: Evidence from the Indian Context

The last two decades witnessed several novel treatment approaches to aphasia therapy. Semantic feature-based therapy is one of such treatment approaches that gained considerable research attention (Boyle & Coelho, 1995). More importantly, this treatment approach has been found effective in bilingual persons with aphasia. For instance, Edmonds and Kiran (2006) administered semantic feature based therapy in Spanish-English bilingual persons with aphasia and reported of crosslinguistic generalization of treatment effect to untreated language. This promising research, however, needs to be replicated and extended to novel language pairs. Research on crosslinguistic generalization of treatment effects is of paramount importance to multilingual countries like India. For instance, with several hundreds of languages and dialects spoken across India and with the pervasive use of English as second language, speech language pathologists (SLPs) in the country are often baffled on the selection of language for treatment in bilingual persons with aphasia. Empirical evidence from Indian languages would add confidence to the SLPs while selecting language for treatment in person with aphasia. In this context, the current study aimed to replicate and extend the earlier findings on crosslinguistic generalization of treatment effects in bilingual persons with aphasia to the Indian context

Decitabine, a DNA-demethylating agent, promotes differentiation via NOTCH1 signaling and alters immune-related pathways in muscle-invasive bladder cancer

Aberrant DNA methylation observed in cancer can provide survival benefits to cells by silencing genes essential for anti-tumor activity. DNA-demethylating agents such as Decitabine (DAC)/Azacitidine (AZA) activate otherwise silenced tumor suppressor genes, alter immune response and epigenetically reprogram tumor cells. In this study, we show that non-cytotoxic nanomolar DAC concentrations modify the bladder cancer transcriptome to activate NOTCH1 at the mRNA and protein level, increase double-stranded RNA sensors and CK5-dependent differentiation. Importantly, DAC treatment increases ICN1 expression (the active intracellular domain of NOTCH1) significantly inhibiting cell proliferation and causing changes in cell size inducing morphological alterations reminiscent of senescence. These changes were not associated with β-galactosidase activity or increased p16 levels, but instead were associated with substantial IL-6 release. Increased IL-6 release was observed in both DAC-treated and ICN1 overexpressing cells as compared to control cells. Exogenous IL-6 expression was associated with a similar enlarged cell morphology that was rescued by the addition of a monoclonal antibody against IL-6. Treatment with DAC, overexpression with ICN1 or addition of exogenous IL-6 showed CK5 reduction, a surrogate marker of differentiation. Overall this study suggests that in MIBC cells, DNA hypomethylation increases NOTCH1 expression and IL-6 release to induce CK5-related differentiation.Fil: Ramakrishnan, Swathi. Roswell Park Cancer Institute; Estados UnidosFil: Hu, Qiang. Roswell Park Cancer Institute; Estados UnidosFil: Krishnan, Nithya. Roswell Park Cancer Institute; Estados UnidosFil: Wang, Dan. Roswell Park Cancer Institute; Estados UnidosFil: Smit, Evelyn. Roswell Park Cancer Institute; Estados UnidosFil: Granger, Victoria. Roswell Park Cancer Institute; Estados UnidosFil: Rak, Monika. Jagiellonian University; PoloniaFil: Attwood, Kristopher. Roswell Park Cancer Institute; Estados UnidosFil: Johnson, Candace. Roswell Park Cancer Institute; Estados UnidosFil: Morrison, Carl. Roswell Park Cancer Institute; Estados UnidosFil: Pili, Roberto. Indiana University; Estados UnidosFil: Chatta, Gurkamal. Roswell Park Cancer Institute; Estados UnidosFil: Guru, Khurshid. Roswell Park Cancer Institute; Estados UnidosFil: Gueron, Geraldine. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: McNally, Lacey. University of Louisville; Estados UnidosFil: Wang, Jianmin. Roswell Park Cancer Institute; Estados UnidosFil: Woloszynska-Read, Anna. Roswell Park Cancer Institute; Estados Unido

Inhibition of EZH2 induces NK cell-mediated differentiation and death in muscle-invasive bladder cancer

Lysine-specific demethylase 6A (KDM6A) and members of the Switch/Sucrose Non-Fermentable (SWI/SNF) family are known to counteract the activity of Enhancer of Zeste Homolog 2 (EZH2), which is often overexpressed and is associated with poor prognosis in muscle-invasive bladder cancer. Here we provide evidence that alterations in chromatin modifying enzymes, including KDM6A and members of the SWI/SNF complex, are frequent in muscle-invasive bladder cancer. We exploit the loss of function mutations in KDM6A and SWI/SNF complex to make bladder cancer cells susceptible to EZH2-based epigenetic therapy that activates an immune response to drive tumor cell differentiation and death. We reveal a novel mechanism of action of EZH2 inhibition, alone and in combination with cisplatin, which induces immune signaling with the largest changes observed in interferon gamma (IFN-γ). This upregulation is a result of activated natural killer (NK) signaling as demonstrated by the increase in NK cell-associated genes MIP-1α, ICAM1, ICAM2, and CD86 in xenografts treated with EZH2 inhibitors. Conversely, EZH2 inhibition results in decreased expression of pluripotency markers, ALDH2 and CK5, and increased cell death. Our results reveal a novel sensitivity of muscle-invasive bladder cancer cells with KMD6A and SWI/SNF mutations to EZH2 inhibition alone and in combination with cisplatin. This sensitivity is mediated through increased NK cell-related signaling resulting in tumor cell differentiation and cell death.Fil: Ramakrishnan, Swathi. Roswell Park Comprehensive Cancer Center; Estados UnidosFil: Granger, Victoria. Roswell Park Comprehensive Cancer Center; Estados UnidosFil: Rak, Monica. Jagiellonian University; PoloniaFil: Hu, Qiang. Roswell Park Comprehensive Cancer Center; Estados UnidosFil: Attwood, Kristopher. Roswell Park Comprehensive Cancer Center; Estados UnidosFil: Aquila, Lanni. Roswell Park Comprehensive Cancer Center; Estados UnidosFil: Krishnan, Nithya. Roswell Park Comprehensive Cancer Center; Estados UnidosFil: Osiecki, Rafal. Medical University Of Warsaw; PoloniaFil: Azabdaftari, Gissou. Roswell Park Comprehensive Cancer Center; Estados UnidosFil: Guru, Khurshid. Roswell Park Comprehensive Cancer Center; Estados UnidosFil: Chatta, Gurkamal. Roswell Park Comprehensive Cancer Center; Estados UnidosFil: Gueron, Geraldine. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: McNally, Lacey. Wake Forest Comprehensive Cancer Center; Estados UnidosFil: Ohm, Joyce. Roswell Park Comprehensive Cancer Center; Estados UnidosFil: Wang, Jianmin. Roswell Park Comprehensive Cancer Center; Estados UnidosFil: Woloszynska-Read, Anna. Roswell Park Comprehensive Cancer Center; Estados Unido

Evaluation of glutathione level in gingival crevicular fluid in periodontal health, in chronic periodontitis and after nonsurgical periodontal therapy: A clinicobiochemical study

Context: Periodontitis is predominantly due to exaggerated host response to pathogenic microorganisms and their products which causes an imbalance between the reactive oxygen species-antioxidant in gingival crevicular fluid (GCF). Glutathione is an important redox regulator in GCF and maintenance of stable reduced glutathione (GSH):oxidized glutathione (GSSG) ratio is essential for periodontal health. Aims: The present study was undertaken to evaluate and compare the level of glutathione and redox balance (GSH: GSSG ratio) in GCF of chronic periodontitis patients, periodontally healthy controls and also to evaluate the effect of nonsurgical periodontal therapy on the level of glutathione and redox balance during 3 months postoperative visit. Study Design: Baseline GCF samples were collected from 20 chronic periodontitis patients and 20 periodontally healthy subjects for GSH and GSSG levels estimation. Periodontitis patients were recalled 3 months postnonsurgical periodontal therapy to re-sample GCF. Materials and Methods: GSH and GSSG levels were measured by high-performance liquid chromatography. The values were statistically analyzed by Paired t-test. Results: The mean GSH and GSSG values in GCF were found to be significantly lower in periodontitis patients pre- and 3 months post-nonsurgical periodontal therapy, compared with those in the control group subjects. In addition, the successful nonsurgical therapy even though leading to a significant improvement in the GSH and GSSG levels, does not restore glutathione concentration to the levels seen in healthy subjects. Conclusion: Successful nonsurgical periodontal therapy leads to significant improvement in the redox balance (GSH: GSSG ratio) in chronic periodontitis patients

MicroRNA-223 Induced Repolarization of Peritoneal Macrophages Using CD44 Targeting Hyaluronic Acid Nanoparticles for Anti-Inflammatory Effects.

The aim of this study was to evaluate macrophages repolarization from pro-inflammatory M1 to anti-inflammatory M2 phenotype upon transfection with microRNA-223 (miR-223) duplexes and miR-223 expressing plasmid DNA encapsulated in CD44-targeting hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles (NPs). The HA-PEI/miR-223 NPs with spherical shape and an average diameter of 200 nm were efficiently internalized by J774A.1 alveolar and primary peritoneal macrophages and non-cytotoxic at HA-PEI concentration less than 200 μg/mL. Transfection of HA-PEI/miR-223 NPs in J774A.1 macrophages showed significantly higher miR-223 expression than that with HA-PEI/plasmid DNA expressing miR-223 (pDNA-miR-223). HA-PEI/miR-223 NPs mediated transfection increased miR-223 expression to 90 fold in primary peritoneal macrophages compared to untreated cells. The overexpression of miR-223 in both J774A.1 and peritoneal macrophages induced a phenotypic change from M1 to M2 state as indicated by a decrease in iNOS-2 (M1 marker) and an increase in Arg-1 (M2 marker) levels compared to those in lipopolysaccharide (LPS) and interferon-gamma (IFN-γ)-stimulated macrophages (M1). The change in macrophage phenotype by HA-PEI/miR-223 NPs could suppress the inflammation in peritoneal macrophages induced by LPS as evidenced by a significant decrease in pro-inflammatory cytokine levels TNF-α, IL-1β and IL-6, compared to LPS-stimulated peritoneal macrophages without treatment. The results demonstrated that miR-223-encapsulated HA-PEI NPs modulated macrophage polarity toward an anti-inflammatory M2 phenotype, which has potential for the treatment of inflammatory diseases

Relative roles of weather variables and change in human population in malaria: comparison over different states of India.

Pro-active and effective control as well as quantitative assessment of impact of climate change on malaria requires identification of the major drivers of the epidemic. Malaria depends on vector abundance which, in turn, depends on a combination of weather variables. However, there remain several gaps in our understanding and assessment of malaria in a changing climate. Most of the studies have considered weekly or even monthly mean values of weather variables, while the malaria vector is sensitive to daily variations. Secondly, rarely all the relevant meteorological variables have been considered together. An important question is the relative roles of weather variables (vector abundance) and change in host (human) population, in the change in disease load.We consider the 28 states of India, characterized by diverse climatic zones and changing population as well as complex variability in malaria, as a natural test bed. An annual vector load for each of the 28 states is defined based on the number of vector genesis days computed using daily values of temperature, rainfall and humidity from NCEP daily Reanalysis; a prediction of potential malaria load is defined by taking into consideration changes in the human population and compared with the reported number of malaria cases.For most states, the number of malaria cases is very well correlated with the vector load calculated with the combined conditions of daily values of temperature, rainfall and humidity; no single weather variable has any significant association with the observed disease prevalence.The association between vector-load and daily values of weather variables is robust and holds for different climatic regions (states of India). Thus use of all the three weather variables provides a reliable means of pro-active and efficient vector sanitation and control as well as assessment of impact of climate change on malaria