Phospho-H1 Decorates the Inter-chromatid Axis and Is Evicted along with Shugoshin by SET during Mitosis.

Precise control of sister chromatid separation during mitosis is pivotal to maintaining genomic integrity. Yet, the regulatory mechanisms involved are not well understood. Remarkably, we discovered that linker histone H1 phosphorylated at S/T18 decorated the inter-chromatid axial DNA on mitotic chromosomes. Sister chromatid resolution during mitosis required the eviction of such H1S/T18ph by the chaperone SET, with this process being independent of and most likely downstream of arm-cohesin dissociation. SET also directed the disassembly of Shugoshins in a polo-like kinase 1-augmented manner, aiding centromere resolution. SET ablation compromised mitotic fidelity as evidenced by unresolved sister chromatids with marked accumulation of H1S/T18ph and centromeric Shugoshin. Thus, chaperone-assisted eviction of linker histones and Shugoshins is a fundamental step in mammalian mitotic progression. Our findings also elucidate the functional implications of the decades-old observation of mitotic linker histone phosphorylation, serving as a paradigm to explore the role of linker histones in bio-signaling processes.

Noninvasive biomarkers for the diagnosis and management of autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a rare disease of unclear etiology characterized by loss of self-tolerance that can lead to liver injury, cirrhosis, and acute liver failure. First-line treatment consists of systemic corticosteroids, or budesonide, and azathioprine, to which most patients are initially responsive, although predictors of response are lacking. Relapses are very common, correlate with histological activity despite normal serum transaminases, and increase hepatic fibrosis. Furthermore, current regimens lead to adverse effects and reduced quality of life, whereas medication titration is imprecise. Biomarkers that can predict the clinical course of disease, identify patients at elevated risk for relapse, and improve monitoring and medication dosing beyond current practice would have high clinical value. Herein, we review novel candidate biomarkers in adult and pediatric AIH based on prespecified criteria, including gene expression profiles, proteins, metabolites, and immune cell phenotypes in different stages of AIH. We also discuss biomarkers relevant to AIH from other immune diseases. We conclude with proposed future directions in which biomarker implementation into clinical practice could lead to advances in personalized therapeutic management of AIH.

Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption.

Dysferlin is a membrane-associated protein implicated in membrane resealing; loss of dysferlin leads to muscular dystrophy. We examined the same loss-of-function Dysf mutation in two different mouse strains, 129T2/SvEmsJ (Dysf(129)) and C57BL/6J (Dysf(B6)). Although there are many genetic differences between these two strains, we focused on polymorphisms in Anxa6 because these variants were previously associated with modifying a pathologically distinct form of muscular dystrophy and increased the production of a truncated annexin A6 protein. Dysferlin deficiency in the C57BL/6J background was associated with increased Evan's Blue dye uptake into muscle and increased serum creatine kinase compared to the 129T2/SvEmsJ background. In the C57BL/6J background, dysferlin loss was associated with enhanced pathologic severity, characterized by decreased mean fiber cross-sectional area, increased internalized nuclei, and increased fibrosis, compared to that in Dysf(129) mice. Macrophage infiltrate was also increased in Dysf(B6) muscle. High-resolution imaging of live myofibers demonstrated that fibers from Dysf(B6) mice displayed reduced translocation of full-length annexin A6 to the site of laser-induced sarcolemmal disruption compared to Dysf(129) myofibers, and impaired translocation of annexin A6 associated with impaired resealing of the sarcolemma. These results provide one mechanism by which the C57BL/6J background intensifies dysferlinopathy, giving rise to a more severe form of muscular dystrophy in the Dysf(B6) mouse model through increased membrane leak and inflammation.

EHD1 mediates vesicle trafficking required for normal muscle growth and transverse tubule development.

EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton.

Providing Oligonucleotides with Steric Selectivity by Brush-Polymer-Assisted Compaction.

Difficult biopharmaceutical characteristics of oligonucleotides, such as poor enzymatic stability, rapid clearance by reticuloendothelial organs, immunostimulation, and coagulopathies, limit their application as therapeutics. Many of these side effects are initiated via sequence-specific or nonsequence-specific interactions with proteins. Herein, we report a novel form of brush-polymer/DNA conjugate that provides the DNA with nanoscale steric selectivity: Hybridization kinetics with complementary DNA remains nearly unaffected, but interactions with proteins are significantly retarded. The relative lengths of the brush side chain and the DNA strand are found to play a critical role in the degree of selectivity. Being able to evade protein adhesion also improves in vivo biodistribution, thus making these molecular nanostructures promising materials for oligonucleotide-based therapies.

Purification and assay protocols for obtaining highly active Jumonji C demethylases.

Jumonji C (JmjC) lysine demethylases (KDMs) are Fe(II)-dependent hydroxylases that catalyze the oxidative demethylation of methyllysine residues in histones and nonhistone proteins. These enzymes play vital roles in regulating cellular processes such as gene expression, cell cycle progression, and stem cell self-renewal and differentiation. Despite their biological importance, recombinant forms of JmjC KDMs generally display low enzymatic activity and have remained challenging to isolate in a highly active form. Here we present a simple affinity purification scheme for Strep(II)-tagged JmjC KDMs that minimizes contamination by transition state metal ions, yielding highly active and pure enzyme. We also describe an optimized continuous fluorescent assay for KDMs that detects formaldehyde production during demethylation via a coupled reaction using formaldehyde dehydrogenase. Purification and kinetic analysis of the human KDMs JMJD2A and JMJD2D using these methods yielded activities substantially higher than those previously reported for these enzymes, which are comparable to that of the flavin-dependent KDM LSD1. In addition, we show that JMJD2A exhibited a lower catalytic efficiency toward a histone peptide bearing a chemically installed trimethyllysine analog compared with a bona fide trimethylated substrate. The methodology described here is broadly applicable to other JmjC KDMs, facilitating their biochemical characterization and high-throughput screening applications.

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.

eIF2B activator prevents neurological defects caused by a chronic integrated stress response.

The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts.

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-1ß 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.

Neutrophil elastase levels in the gingival crevicular fluid following hyaluronan gel application in the treatment of chronic periodontitis: A randomized split-mouth study.

BACKGROUND: Neutrophils are the predominant leukocytes in the periodontium, which prevent infection from periodontal pathogens and subsequent tissue destruction. A potentially destructive role has been elucidated, especially due to elastase enzyme. Controlling its levels might be crucial in minimizing the tissue destruction. Hyaluronan, known to inhibit the release of this enzyme from neutrophils, might be a viable option to treat chronic periodontitis. AIMS: The aim of this study is to evaluate and compare the effects of 0.2% hyaluronan gel adjunctive to scaling and root planing on the levels of elastase in gingival crevicular fluid (GCF). SETTINGS AND DESIGN: This split-mouth study included eighty (forty experimental and forty control) sites from twenty patients representing both sexes. MATERIALS AND METHODS: GCF samples were collected from all the eighty sites; simultaneously, clinical periodontal parameters were recorded. Enzyme-linked immunosorbent assay was used to determine the levels of elastase at baseline and 6 weeks after therapy, following mechanical debridement and subsequent subgingival placement of the experimental drug. STATISTICAL ANALYSIS USED: With the aid of statistical software (SPSS Version 13), Student's t-test and Pearson's correlation test were performed. RESULTS: There was a mean reduction in the elastase levels from baseline to 6 weeks after therapy in the experimental group. However, the difference between the groups was not statistically significant. CONCLUSIONS: Adjunctive use of hyaluronan following mechanical debridement resulted in comparable reduction in the elastase levels, suggesting that this substance has an inhibitory effect on elastase, and subsequent tissue destruction. Further long-term studies are mandatory to validate the results of this study.

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.

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

BACKGROUND: 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. METHOD: 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. RESULTS: 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. CONCLUSION: 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.

Structural and functional analysis of JMJD2D reveals molecular basis for site-specific demethylation among JMJD2 demethylases.

JMJD2 lysine demethylases (KDMs) participate in diverse genomic processes. Most JMJD2 homologs display dual selectivity toward H3K9me3 and H3K36me3, with the exception of JMJD2D, which is specific for H3K9me3. Here, we report the crystal structures of the JMJD2D⋅2-oxoglutarate⋅H3K9me3 ternary complex and JMJD2D apoenzyme. Utilizing structural alignments with JMJD2A, molecular docking, and kinetic analysis with an array of histone peptide substrates, we elucidate the specific signatures that permit efficient recognition of H3K9me3 by JMJD2A and JMJD2D, and the residues in JMJD2D that occlude H3K36me3 demethylation. Surprisingly, these results reveal that JMJD2A and JMJD2D exhibit subtle yet important differences in H3K9me3 recognition, despite the overall similarity in the substrate-binding conformation. Further, we show that H3T11 phosphorylation abrogates demethylation by JMJD2 KDMs. Together, these studies reveal the molecular basis for JMJD2 site specificity and provide a framework for structure-based design of selective inhibitors of JMJD2 KDMs implicated in disease.

Crystal structure and functional analysis of JMJD5 indicate an alternate specificity and function.

JMJD5 is a Jumonji C (JmjC) protein that has been implicated in breast cancer tumorigenesis, circadian rhythm regulation, embryological development, and osteoclastogenesis. Recently, JMJD5 (also called KDM8) has been reported to demethylate dimethylated Lys-36 in histone H3 (H3K36me2), regulating genes that control cell cycle progression. Here, we report high-resolution crystal structures of the human JMJD5 catalytic domain in complex with the substrate 2-oxoglutarate (2-OG) and the inhibitor N-oxalylglycine (NOG). The structures reveal a ß-barrel fold that is conserved in the JmjC family and a long shallow cleft that opens into the enzyme's active site. A comparison with other JmjC enzymes illustrates that JMJD5 shares sequence and structural homology with the asparaginyl and histidinyl hydroxylase FIH-1 (factor inhibiting hypoxia-inducible factor 1 [HIF-1]), the lysyl hydroxylase JMJD6, and the RNA hydroxylase TYW5 but displays limited homology to JmjC lysine demethylases (KDMs). Contrary to previous findings, biochemical assays indicate that JMJD5 does not display demethylase activity toward methylated H3K36 nor toward the other methyllysines in the N-terminal tails of histones H3 and H4. Together, these results imply that JMJD5 participates in roles independent of histone demethylation and may function as a protein hydroxylase given its structural homology with FIH-1 and JMJD6.

A model of malaria epidemiology involving weather, exposure and transmission applied to north East India.

BACKGROUND: Quantitative relations between weather variables and malaria vector can enable pro-active control through meteorological monitoring. Such relations are also critical for reliable projections in a changing climate, especially since the vector abundance depends on a combination of weather variables, each in a given range. Further, such models need to be region-specific as vector population and exposure depend on regional characteristics. METHODS: We consider days of genesis based on daily temperature, rainfall and humidity in given ranges. We define a single model parameter based on estimates of exposure and transmission to calibrate the model; the model is applied to 12 districts of Arunachal Pradesh, a region endemic to malaria. The epidemiological data is taken as blood samples that test positive. The meteorological data is adopted from NCEP daily Reanalysis on a global grid; population data is used to estimate exposure and transmission coefficients. RESULTS: The observed annual cycles (2006-2010) and the interannual variability (2002-2010) of epidemiology are well simulated for each of the 12 districts by the model. While no single weather variable like temperature can reproduce the observed epidemiology, a combination of temperature, rainfall and humidity provides an accurate description of the annual cycle as well as the inter annual variability over all the 12 districts. CONCLUSION: Inclusion of the three meteorological variables, along with the expressions for exposure and transmission, can quite accurately represent observed epidemiology over multiple locations and different years. The model is potentially useful for outbreak forecasts at short time scales through high resolution weather monitoring; however, validation with longer and independent epidemiological data is required for more robust estimation of realizable skill. While the model has been examined over a specific region, the basic algorithm is easily applicable to other regions; the model can account for shifting vulnerability due to regional climate change.