Intraluminal release of citrullinated histone 3 from various cellular origins coincides with microvascular thrombosis in burn wounds.

Loss of perfusion in the burn wound might cause wound deepening and impaired healing. We previously showed persistent microvascular thrombosis coinciding with intraluminal neutrophils extracellular traps in human burned skin. This study investigates the presence of intraluminal citrullinated histone 3 (H3cit) from different cellular origins (neutrophils, monocytes, and lymphocytes) in relation to microvascular thrombosis of burn wounds. Eschar was obtained from burn patients (n = 18) 6-40 days postburn with a mean total burned body surface area of 23%. Microvascular presence of tissue factor (TF), factor XII (FXII) and thrombi was assessed by immunohistochemistry. Intramicrovascular cell death was analyzed via immunofluorescent microscopy, combining antibodies for neutrophils (MPO), monocytes (CD14), and lymphocytes (CD45) with endothelial cell markers CD31 and H3cit. Significantly increased microvascular expression of TF, FXII, and thrombi (CD31+) was found in all eschar samples compared with control uninjured skin. Release of H3cit from different cellular origins was observed in the lumen of the dermal microvasculature in the eschar tissue 7-40 days postburn, with release from neutrophilic origin being 2.7 times more abundant. Intraluminal presence of extracellular H3cit colocalizing with either MPO, CD14, or CD45 is correlated to increased microvascular thrombosis in eschar of burn patients.

Nup98 regulation of histone methylation promotes normal gene expression and may drive leukemogenesis.

Nuclear pore proteins (Nups) interact with chromosomes to regulate gene expression and chromatin structure. A new study by Franks and colleagues (pp. 2222-2234) provides new mechanistic insight into the molecular basis by which Nup98 promotes gene activation in normal hematopoietic cells and how that process is altered by translocations to cause excess expression of developmental genes in leukemia.

Nup98 recruits the Wdr82-Set1A/COMPASS complex to promoters to regulate H3K4 trimethylation in hematopoietic progenitor cells.

Recent studies have shown that a subset of nucleoporins (Nups) can detach from the nuclear pore complex and move into the nuclear interior to regulate transcription. One such dynamic Nup, called Nup98, has been implicated in gene activation in healthy cells and has been shown to drive leukemogenesis when mutated in patients with acute myeloid leukemia (AML). Here we show that in hematopoietic cells, Nup98 binds predominantly to transcription start sites to recruit the Wdr82-Set1A/COMPASS (complex of proteins associated with Set1) complex, which is required for deposition of the histone 3 Lys4 trimethyl (H3K4me3)-activating mark. Depletion of Nup98 or Wdr82 abolishes Set1A recruitment to chromatin and subsequently ablates H3K4me3 at adjacent promoters. Furthermore, expression of a Nup98 fusion protein implicated in aggressive AML causes mislocalization of H3K4me3 at abnormal regions and up-regulation of associated genes. Our findings establish a function of Nup98 in hematopoietic gene activation and provide mechanistic insight into which Nup98 leukemic fusion proteins promote AML.

Novel quantitative immunohistochemistry method using histone H3, family 3B as the internal reference standard for measuring human epidermal growth factor receptor 2 expression in breast cancer.

BACKGROUND: Immunohistochemistry (IHC) is an essential technique in surgical and clinical pathology for detecting diagnostic, prognostic, and predictive biomarkers for personalized cancer therapy. However, the lack of standardization and reference controls results in poor reproducibility, and a reliable tool for IHC quantification is urgently required. The objective of this study was to describe a novel approach in which H3F3B (histone H3, family 3B) can be used as an internal reference standard to quantify protein expression levels using IHC. METHODS: The authors enrolled 89 patients who had human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC). They used a novel IHC-based assay to measure protein expression using H3F3B as the internal reference standard. H3F3B was uniformly expressed at the protein level in all tumor regions in cancer tissues. HER2 expression levels were measured with the H-score using HALO software. RESULTS: Kaplan-Meier analysis indicated that, among patients who had HER2-positive BC in The Cancer Genome Atlas data set and the authors' data set, the subgroup with low HER2 expression had a significantly better prognosis than the subgroup with high HER2 expression. Furthermore, the authors observed that HER2 expression levels were precisely evaluated using the proposed method, which can classify patients who are at higher risk of HER2-positive BC to receive trastuzumab-based adjuvant therapy. Dual-color IHC with H3F3B is an excellent tool for internal and external quality control of HER2 expression assays. CONCLUSIONS: The proposed IHC-based quantification method accurately assesses HER2 expression levels and provides insights for predicting clinical prognosis in patients with HER2-positive BC who receive trastuzumab-based adjuvant therapy.

Complement-Mediated Two-Step NETosis: Serum-Induced Complement Activation and Calcium Influx Generate NADPH Oxidase-Dependent NETs in Serum-Free Conditions.

The complement system and neutrophils play crucial roles in innate immunity. Neutrophils release neutrophil extracellular traps (NETs), which are composed of decondensed DNA entangled with granular contents, as part of their innate immune function. Mechanisms governing complement-mediated NET formation remain unclear. In this study, we tested a two-step NETosis mechanism, as follows: classical complement-mediated neutrophil activation in serum and subsequent NET formation in serum-free conditions, using neutrophils from healthy donors, endothelial cells, and various assays (Fluo-4AM, DHR123, and SYTOX), along with flow cytometry and confocal microscopy. Our findings reveal that classical complement activation on neutrophils upregulated the membrane-anchored complement regulators CD46, CD55, and CD59. Additionally, complement activation increased CD11b on neutrophils, signifying activation and promoting their attachment to endothelial cells. Complement activation induced calcium influx and citrullination of histone 3 (CitH3) in neutrophils. However, CitH3 formation alone was insufficient for NET generation. Importantly, NET formation occurred only when neutrophils were in serum-free conditions. In such environments, neutrophils induced NADPH oxidase-dependent reactive oxygen species (ROS) production, leading to NET formation. Hence, we propose that complement-mediated NET formation involves a two-step process, as follows: complement deposition, neutrophil priming, calcium influx, CitH3 formation, and attachment to endothelial cells in serum. This is followed by NADPH-dependent ROS production and NET completion in serum-free conditions. Understanding this process may unveil treatment targets for pathologies involving complement activation and NET formation.

Molecular and Pathological Features of Paediatric High-Grade Gliomas.

Paediatric high-grade gliomas are among the most common malignancies found in children. Despite morphological similarities to their adult counterparts, there are profound biological and molecular differences. Furthermore, and thanks to molecular biology, the diagnostic pathology of paediatric high-grade gliomas has experimented a dramatic shift towards molecular classification, with important prognostic implications, as is appropriately reflected in both the current WHO Classification of Tumours of the Central Nervous System and the WHO Classification of Paediatric Tumours. Emphasis is placed on histone 3, IDH1, and IDH2 alterations, and on Receptor of Tyrosine Kinase fusions. In this review we present the current diagnostic categories from the diagnostic pathology perspective including molecular features.

Neutrophil Extracellular Trap Formation in Advanced Heart Failure Patients-Preliminary Report.

In end-stage heart failure, which is characterized by persistent or progressive ventricular dysfunction despite optimal medical therapy, a left ventricular assist device (LVAD) can be beneficial. Congestive heart failure provokes inflammatory and prothrombotic activation. The aim of this study was to evaluate the serum concentration of citrullinated histone 3 (CH3) representing neutrophil extracellular trap (NET) formation in patients referred for LVAD implantation. There were 10 patients with a median age of 61 (57-65) years enrolled in a prospective single-center analysis who underwent LVAD implantation. The CH3 plasma concentration was measured preoperatively and on the 1st and 7th postoperative days, followed by control measurements on the median (Q1-3) 88th (49-143) day. The preoperative CH3 concentration strongly correlated with brain natriuretic peptide (r = 0.879, p < 0.001). Significant differences in CH3 serum concentration were observed between pre- and postoperative measurements, including an increase on the first postoperative day (p < 0.001), as well as a decrease on the seventh day (p = 0.016) and in follow-up (p < 0.001). CH3 concentration, as a marker of NET formation, decreases after LVAD implantation.

Lysine 27 of histone H3.3 is a fine modulator of developmental gene expression and stands as an epigenetic checkpoint for lignin biosynthesis in Arabidopsis.

Chromatin is a dynamic platform within which gene expression is controlled by epigenetic modifications, notably targeting amino acid residues of histone H3. Among them is lysine 27 of H3 (H3K27), the trimethylation of which by the Polycomb Repressive Complex 2 (PRC2) is instrumental in regulating spatiotemporal patterns of key developmental genes. H3K27 is also subjected to acetylation and is found at sites of active transcription. Most information on the function of histone residues and their associated modifications in plants was obtained from studies of loss-of-function mutants for the complexes that modify them. To decrypt the genuine function of H3K27, we expressed a non-modifiable variant of H3 at residue K27 (H3.3K27A ) in Arabidopsis, and developed a multi-scale approach combining in-depth phenotypical and cytological analyses, with transcriptomics and metabolomics. We uncovered that the H3.3K27A variant causes severe developmental defects, part of them are reminiscent of PRC2 mutants, part of them are new. They include early flowering, increased callus formation and short stems with thicker xylem cell layer. This latest phenotype correlates with mis-regulation of phenylpropanoid biosynthesis. Overall, our results reveal novel roles of H3K27 in plant cell fates and metabolic pathways, and highlight an epigenetic control point for elongation and lignin composition of the stem.

Heparanase-mediated histone 3 acetylation regulates VEGF gene transcription in the hyperglycemia and hypoxia human retinal endothelial cells.

Heparanase (HPA) is believed that might mediate histone 3 lysine 9 acetylation (H3K9ac) to regulate vascular endothelial growth factor (VEGF) gene expressions in the hyperglycemia and hypoxia human retinal endothelial cells (HRECs). Cultured human retinal endothelial cells (HRECs) in hyperglycemia, hypoxia, siRNA, and normal medium, respectively. Distributions of H3K9ac and HPA in HRECs were analyzed by immunofluorescence. Western blot and real-time PCR were respectively used to evaluate the expression of HPA, H3K9ac, and VEGF. The differences in occupancies of H3K9ac and RNA polymerase II at VEGF gene promoter among three groups were studied by Chromatin immunoprecipitation (ChIP) combined with real-time PCR. Co-immunoprecipitation (Co-IP) was used to measure the status of HPA and H3K9ac. Re-ChIP was used to verify whether HPA and H3K9ac associate to the transcription of VEGF gene. HPA was consistent with that of H3K9ac in the hyperglycemia and hypoxia groups. And the fluorescent lights of H3K9ac and HPA in siRNA groups were similar to the control group, fainter than that of hyperglycemia, hypoxia, and non-silencing groups. Western blot results showed that the expressions of HPA, H3K9ac, and VEGF in hyperglycemia and hypoxia HRECs were statistically higher than that of the control. HPA, H3K9ac, and VEGF expressions in siRNA groups were statistically lower than hyperglycemia and hypoxia HRECs. The same trends also were found in real-time PCR. ChIP exhibited the occupancies of H3K9ac and RNA Pol II at VEGF gene promoter in hyperglycemia and hypoxia groups were significantly more increased than in the control group. Co-IP revealed that HPA combined with H3K9ac in hyperglycemia and hypoxia groups; while it was not discovered in the control group. Re-ChIP showed that HPA combined with H3K9ac at VEGF gene promoter in the hyperglycemia and hypoxia HRECs nuclear. In our study HPA can influence expressions of H3K9ac and VEGF in the hyperglycemia and hypoxia HRECs. HPA can probably combine with H3K9ac and regulate the transcription of the VEGF gene in the hyperglycemia and hypoxia HRECs.

Phase II study of tazemetostat for relapsed or refractory B-cell non-Hodgkin lymphoma with EZH2 mutation in Japan.

Tazemetostat is a selective, reversible, small-molecule inhibitor of the histone methyltransferase enzyme, enhancer of zest homolog 2 (EZH2). In this multicenter, open-label, phase II study, we assessed the efficacy and safety of tazemetostat in Japanese patients with relapsed or refractory (R/R) B-cell non-Hodgkin lymphoma harboring the EZH2 mutation. Tazemetostat (800 mg twice daily) was given orally (28-day cycle) until disease progression or unacceptable toxicity. Among the 20 eligible patients, 17 were enrolled in cohort 1 (follicular lymphoma [FL]), and three were enrolled in cohort 2 (diffuse large B-cell lymphoma). At data cut-off, the objective response rate in cohort 1 was 76.5%, including six patients (35.3%) with complete response and seven patients (41.2%) with partial response (PR). All three patients in cohort 2 achieved PR. In cohort 1, median progression-free survival (PFS) was not reached at the median follow-up of 12.9 months. The estimated PFS rate at 12 and 15 months was 94.1% and 73.2%, respectively. The most common grade 3 treatment-emergent adverse event (TEAE) was lymphopenia (n = 2). Grade 4 TEAEs included hypertriglyceridemia and pneumonia aspiration (n = 1 each), which were not related to tazemetostat. Treatment-emergent adverse events leading to study drug discontinuation were reported in four of the 20 patients, indicating that the safety profile of tazemetostat was acceptable and manageable. Tazemetostat 800 mg twice daily showed encouraging efficacy in patients with R/R EZH2 mutation-positive FL with a manageable safety profile in the overall population. Thus, tazemetostat could be a potential treatment for R/R EZH2 mutation-positive FL.

Characterizing circulating nucleosomes in the plasma of dogs with lymphoma.

BACKGROUND: Nucleosomes consist of DNA wrapped around a histone octamer core like beads on a string so that DNA can be condensed as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death where chromatin is fragmentated and released as mononucleosomes into the blood. The Nu.Q™ H3.1 assay measures total nucleosome concentration in plasma of humans and has been used to detect and identify cancer even at early stages. The objectives of this study were to determine if nucleosome levels could be used to distinguish between healthy dogs and dogs with various stages of lymphoma (LSA) using the Nu.Q™ H3.1 assay. A total of 126 dogs diagnosed with LSA and 134 healthy controls were recruited for this study. Plasma was collected from each dog and stored in K2-EDTA tubes. The LSA patient samples were recruited from TAMU or purchased from various biobanks. All control cases were recruited from TAMU. RESULTS: Dogs with LSA had an approximately 7-fold increase in their plasma nucleosome concentrations compared to controls (AUC 87.8%). Nucleosome concentrations increased with cancer stage and dogs with B cell lymphomas had significantly higher nucleosome concentrations than dogs with T cell lymphomas. CONCLUSIONS: The Nu.Q™ H3.1 assay was able to reliably detect elevated nucleosome concentrations in the plasma of dogs with LSA. Furthermore, it appears that nucleosomes are useful for differentiating cancer from healthy individuals in canines.

Insulin-Like Growth Factor Binding Protein-3 Binds to Histone 3.

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is an essential protein that regulates cellular processes such as cell proliferation, apoptosis, and differentiation. It is known to bind with several proteins to carry out various cellular functions. In this study, we report for the first time that IGFBP-3 is a histone 3 (H3) binding protein. Sub-cellular fractionation was performed to separate into cytosolic fraction, nucleic acid binding protein fraction and insoluble nuclear fraction. Using ligand blot analysis, we identified a ~15 kDa protein that can interact with IGFBP-3 in the insoluble nuclear fraction. The 15 kDa protein was confirmed as histone 3 by far-Western blot analysis and co-immunoprecipitation experiments. A dot-blot experiment further validated the binding of IGFBP-3 with H3. The intensity of IGFBP-3 on dot-blot showed a proportional increase with H3 concentrations between 2.33 pmol-37.42 pmol. Our results support the presence of protein-protein interaction between IGFBP-3 and H3. The physical binding between IGFBP-3 and H3 could indicate its yet another cellular role in regulating the chromatin remodeling for gene transcription.

Prognostic impact of distinct genetic entities in pediatric diffuse glioma WHO-grade II-Report from the German/Swiss SIOP-LGG 2004 cohort.

Reports on pediatric low-grade diffuse glioma WHO-grade II (DG2) suggest an impaired survival rate, but lack conclusive results for genetically defined DG2-entities. We analyzed the natural history, treatment and prognosis of DG2 and investigated which genetically defined sub-entities proved unfavorable for survival. Within the prospectively registered, population-based German/Swiss SIOP-LGG 2004 cohort 100 patients (age 0.8-17.8 years, 4% neurofibromatosis [NF1]) were diagnosed with a DG2. Following biopsy (41%) or variable extent of resection (59%), 65 patients received no adjuvant treatment. Radiologic progression or severe neurologic symptoms prompted chemotherapy (n = 18) or radiotherapy (n = 17). Multiple lines of salvage treatment were necessary for 19/35 patients. Five years event-free survival dropped to 0.44, while 5 years overall survival was 0.90 (median observation time 8.3 years). Extensive genetic profiling of 65/100 DG2 identified Histone3-K27M-mutation in 4, IDH1-mutation in 11, BRAF-V600-mutation in 12, KIAA1549-BRAF-fusions in 6 patients, while the remaining 32 tumor tissues did not show alterations of these genes. Progression to malignant glioma occurred in 12 cases of all genetically defined subgroups within a range of 0.5 to 10.8 years, except for tumors carrying KIAA1549-BRAF-fusions. Histone3-K27M-mutant tumors proved uniformly fatal within 0.6 to 2.4 years. The current LGG treatment strategy seems appropriate for all DG2-entities, with the exemption of Histone3-K27M-mutant tumors that require a HGG-related treatment strategy. Our data confirm the importance to genetically define pediatric low-grade diffuse gliomas for proper treatment decisions and risk assessment.

A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct from Kabuki syndrome.

PURPOSE: To investigate if specific exon 38 or 39 KMT2D missense variants (MVs) cause a condition distinct from Kabuki syndrome type 1 (KS1). METHODS: Multiple individuals, with MVs in exons 38 or 39 of KMT2D that encode a highly conserved region of 54 amino acids flanked by Val3527 and Lys3583, were identified and phenotyped. Functional tests were performed to study their pathogenicity and understand the disease mechanism. RESULTS: The consistent clinical features of the affected individuals, from seven unrelated families, included choanal atresia, athelia or hypoplastic nipples, branchial sinus abnormalities, neck pits, lacrimal duct anomalies, hearing loss, external ear malformations, and thyroid abnormalities. None of the individuals had intellectual disability. The frequency of clinical features, objective software-based facial analysis metrics, and genome-wide peripheral blood DNA methylation patterns in these patients were significantly different from that of KS1. Circular dichroism spectroscopy indicated that these MVs perturb KMT2D secondary structure through an increased disordered to ɑ-helical transition. CONCLUSION: KMT2D MVs located in a specific region spanning exons 38 and 39 and affecting highly conserved residues cause a novel multiple malformations syndrome distinct from KS1. Unlike KMT2D haploinsufficiency in KS1, these MVs likely result in disease through a dominant negative mechanism.

Nitric oxide signaling inhibits microglia proliferation by activation of protein kinase-G.

Microglia population is primarily determined by a finely-regulated proliferation process during early development of the central nervous system (CNS). Nitric oxide (NO) is known to inhibit proliferation in numerous cell types. However, how NO signaling regulates microglia proliferation remains elusive. Using wildtype (WT) and inducible nitric oxide synthase knockout (iNOS-/-) mice, this study investigated the role and underlying mechanisms of iNOS/NO signaling in microglia proliferation. Here we reported that iNOS-/- mice displayed significantly more BrdU-labeled proliferating microglia in the cortex than that in WT mice at postnatal day 10. Compared to microglia isolated from WT mouse cortex, significantly more iNOS-/- microglia displayed the specific cell-cycle markers Ki67 and phospho-histone H3 (pH3) in their nuclei. In addition, treating WT microglia with the NOS inhibitor LNAME drastically increased the percentage of cells expressing Ki67 and pH3, whereas treating iNOS-/- microglia with NOC18, a slow-release NO-donor, significantly decreased the percentage of microglia expressing the two cell-cycle markers. Moreover, inhibition of protein kinase-G (PKG) in WT microglia increased the proportion of microglia expressing Ki67 and pH3, whereas activation of PKG signaling using 8Br-cGMP in iNOS-/- microglia significantly decreased the fraction of microglia displaying Ki67 and pH3. Interestingly, in the presence of a PKG inhibitor, NOC18 increased the quantity of iNOS-/- microglia expressing Ki67 and pH3. Together, these results indicate that basal activity of iNOS/NO signaling impedes microglial cell-cycle progression and attenuates proliferation through activation of the cGMP-PKG pathway. However, NO increases microglia cell-cycle progression in the absence of cGMP-PKG signaling.

H3K4me3 Is a Potential Mediator for Antiproliferative Effects of Calcitriol (1α,25(OH)2D3) in Ovarian Cancer Biology.

Posttranslational histone modification plays an important role in tumorigenesis. Histone modification is a dynamic response of chromatin to various signals, such as the exposure to calcitriol (1α,25(OH)2D3). Recent studies suggested that histone modification levels could be used to predict patient outcomes in various cancers. Our study evaluated the expression level of histone 3 lysine 4 trimethylation (H3K4me3) in a cohort of 156 epithelial ovarian cancer (EOC) cases by immunohistochemical staining and analyzed its correlation to patient prognosis. The influence of 1α,25(OH)2D3 on the proliferation of ovarian cancer cells was measured by BrdU proliferation assay in vitro. We could show that higher levels of H3K4me3 were correlated with improved overall survival (median overall survival (OS) not reached vs. 37.0 months, p = 0.047) and identified H3K4me3 as a potential prognostic factor for the present cohort. Ovarian cancer cell 1α,25(OH)2D3 treatment induced H3K4me3 protein expression and exhibited antiproliferative effects. By this, the study suggests a possible impact of H3K4me3 expression on EOC progression as well as its relation to calcitriol (1α,25(OH)2D3) treatment. These results may serve as an explanation on how 1α,25(OH)2D3 mediates its known antiproliferative effects. In addition, they further underline the potential benefit of 1α,25(OH)2D3 supplementation in context of ovarian cancer care.

Epigenetic fates of gene silencing established by heterochromatin spreading in cell identity and genome stability.

Heterochromatin spreading, the propagation of repressive chromatin along the chromosome, is a reaction critical to genome stability and defense, as well as maintenance of unique cell fates. Here, we discuss the intrinsic properties of the spreading reaction and circumstances under which its products, formed distal to DNA-encoded nucleation sites, can be epigenetically maintained. Finally, we speculate that the epigenetic properties of heterochromatin evolved together with the need to stabilize cellular identity.

Genetic polymorphisms of histone methyltransferase SETD2 predicts prognosis and chemotherapy response in Chinese acute myeloid leukemia patients.

BACKGROUND: SETD2, the single mediator of trimethylation of histone 3 at position lysine 36, has been reported associated with initiation progression and chemotherapy resistance in acute myeloid leukemia (AML). Whether polymorphisms of SETD2 affect prognosis and chemotherapy response of AML remains elusive. METHODS: Three tag single-nucleotide polymorphisms (tagSNPs) of SETD2 were genotyped in 579 AML patients by using Sequenom Massarray system. Association of the SNPs with complete remission (CR) rate after Ara-C based induction therapy, overall survival (OS) and relapse-free survival (RFS) were analyzed. RESULT: Survival analysis indicated that SETD2 rs76208147 TT genotype was significantly associated with poor prognosis of AML (TT vs. CC + CT hazard ratio: HR = 1.838, 95% confidence interval (CI) 1.005-3.360, p = 0.048). After adjusting for the known prognostic factors including risk stratification, age, allo-SCT, WBC count and LDH count, rs76208147 TT genotype was still associated with OS in the multivariate analysis (TT vs. CC + CT HR = 1.923, 95% CI 1.007-3.675, p = 0.048). In addition, after adjusting by other clinical features, patients with rs4082155 allele G carries showed higher rate of complete remission which indicated by CR rate (AG + GG vs. AA odd ratio (OR) = 0.544, 95% CI 0.338-0.876, p = 0.012). CONCLUSIONS: SETD2 genetic polymorphism is associated with AML prognosis and chemotherapy outcome, suggesting the possibility for development in AML diagnostics and therapeutics towards SETD2.

Resolving the basal divisions in the stylommatophoran land snails and slugs with special emphasis on the position of the Scolodontidae.

The deep divisions at the base of the stylommatophoran land snails and slugs have proved to be controversial, with the phylogenetic position of the Scolodontidae remaining unresolved. Here we present a phylogenetic analysis of 34 stylommatophoran genera based on a combined dataset (5782 sites) of four loci with the aim of resolving the position of the Scolodontidae and their relationship to the 'achatinoid' and 'non-achatinoid' clades. We also evaluate the phylogenetic utility of different genes and gene partitions. The deep phylogenetic relationships within the Stylommatophora are now clearly resolved. The Scolodontidae are shown categorically to be the sister group to all other stylommatophoran groups with robust support and with all phylogeny reconstruction methods. The 'achatinoid' and 'non-achatinoid' clades are also strongly supported in the tree. The original LSU 1-5 fragment used extensively in studies of the Stylommatophora was found to be the most informative gene fragment and works well at resolving relationships at most levels in the Stylommatophora but does not reliably resolve the deep level relationships at the very base of the clade. The concatenated dataset of four genes employed in this study is not only informative at lower levels but also resolves the deep level relationships at the base of the Stylommatophora with robust support.

Epigenetic Mechanisms Within the Cingulate Cortex Regulate Innate Anxiety-Like Behavior.

BACKGROUND: Pathological anxiety originates from a complex interplay of genetic predisposition and environmental factors, acting via epigenetic mechanisms. Epigenetic processes that can counteract detrimental genetic risk towards innate high anxiety are not well characterized. METHODS: We used female mouse lines of selectively bred high (HAB)- vs low (LAB)-innate anxiety-related behavior and performed select environmental and pharmacological manipulations to alter anxiety levels as well as brain-specific manipulations and immunohistochemistry to investigate neuronal mechanisms associated with alterations in anxiety-related behavior. RESULTS: Inborn hyperanxiety of high anxiety-like phenotypes was effectively reduced by environmental enrichment exposure. c-Fos mapping revealed that hyperanxiety in high anxiety-like phenotypes was associated with blunted challenge-induced neuronal activation in the cingulate-cortex, which was normalized by environmental enrichment. Relating this finding with epigenetic modifications, we found that high anxiety-like phenotypes (compared with low-innate anxiety phenotypes) showed reduced acetylation in the hypoactivated cingulate-cortex neurons following a mild emotional challenge, which again was normalized by environmental enrichment. Paralleling the findings using environmental enrichment, systemic administration of histone-deacetylase-inhibitor MS-275 elicited an anxiolytic-like effect, which was correlated with increased acetylated-histone-3 levels within cingulate-cortex. Finally, as a proof-of-principle, local MS-275 injection into cingulate-cortex rescued enhanced innate anxiety and increased acetylated-histone-3 within the cingulate-cortex, suggesting this epigenetic mark as a biomarker for treatment success. CONCLUSIONS: Taken together, the present findings provide the first causal evidence that the attenuation of high innate anxiety-like behavior via environmental/pharmacological manipulations is epigenetically mediated via acetylation changes within the cingulate-cortex. Finally, histone-3 specific histone-deacetylase-inhibitor could be of therapeutic importance in anxiety disorders.