A trivalent nucleosome interaction by PHIP/BRWD2 is disrupted in neurodevelopmental disorders and cancer.

Mutations in the PHIP/BRWD2 chromatin regulator cause the human neurodevelopmental disorder Chung-Jansen syndrome, while alterations in PHIP expression are linked to cancer. Precisely how PHIP functions in these contexts is not fully understood. Here we demonstrate that PHIP is a chromatin-associated CRL4 ubiquitin ligase substrate receptor and is required for CRL4 recruitment to chromatin. PHIP binds to chromatin through a trivalent reader domain consisting of a H3K4-methyl binding Tudor domain and two bromodomains (BD1 and BD2). Using semisynthetic nucleosomes with defined histone post-translational modifications, we characterize PHIPs BD1 and BD2 as respective readers of H3K14ac and H4K12ac, and identify human disease-associated mutations in each domain and the intervening linker region that likely disrupt chromatin binding. These findings provide new insight into the biological function of this enigmatic chromatin protein and set the stage for the identification of both upstream chromatin modifiers and downstream targets of PHIP in human disease.

A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization.

Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.

Editor's Choice - Hybrid vs. Open Surgical Reconstruction for Iliofemoral Occlusive Disease: A Prospective Randomised Trial.

OBJECTIVE: The aim of this non-inferiority randomised trial was to compare the short and midterm safety and efficacy of hybrid repair (HR) and open reconstruction (OR) for patients with co-existing iliac and common femoral artery (CFA) occlusive disease. METHODS: The study was registered on the ClinicalTrials.gov register (identifier: NCT02580084). From 2015 to 2017, eligible patients presenting with combined iliac and CFA occlusive disease were randomised to either HR or OR. HR group patients underwent recanalisation and stenting of iliac arteries combined with CFA endarterectomy and patch angioplasty. The OR group underwent aortofemoral bypass with simultaneous CFA endarterectomy. Short (30 day) and midterm (36 month) outcomes including morbidity, mortality, and patency rates were compared between groups. RESULTS: Of 427 patients assessed, 202 were randomised (102 HR and 100 OR). The average hospital length of stay was shorter in the HR group (8.2 ± 4.2 days HR group vs. 15.7 ± 6.9 days OR group, p < .001); the 30 day peri-operative morbidity rate was 8.8% in the HR group vs. 21% in the OR group (p = .030). There was no significant difference in the 36 month mortality rate (p = .16). The cumulative primary patency rates were 93% (HR) vs. 93% (OR) at 12 months and 91% (HR) vs. 89% (OR) at 36 months (p = .38). The limb salvage rates were 99% (HR) vs. 99% (OR) at 12 months and 98% (HR) vs. 97% (OR) at 36 months (p = .49). CONCLUSION: The results of this first non-inferiority randomised study support the safety and midterm efficacy of hybrid procedures for patients with iliofemoral peripheral arterial disease. HR patients had a shorter length of stay with reduced peri-operative morbidity and similar medium term patency rates.

Hydrogen sulfide content in pregnant women with preeclampsia in late gestation and their newborns.

We examined 70 women who were 22-40 weeks pregnant and their newborns. Of these, 15 women with moderate PE made up group 1, 22 women with severe PE-group 2, and 55 women with uncomplicated pregnancy without hypertensive disorders - the control group. Blood was collected from women when they were admitted to the clinic, and blood was taken from newborns for 3-5 days of life. The concentration of hydrogen sulfide was determined by the method of K. Qu et al [17]. There was a decrease in the level of hydrogen sulfide in the blood serum of women whose pregnancy was complicated by severe preeclampsia. In newborns born to mothers with preeclampsia, an increase in the concentration of hydrogen sulfide was detected in the blood, which is probably a compensatory reaction aimed at restoring vascular homeostasis during early postnatal adaptation.

Selective hippocampal cell damage and mossy fiber sprouting induced by chronic intracerebral injections of 2-deoxy-D-glucose.

A reduction in glucose consumption has been shown in both patients with acquired epilepsy and in animal epilepsy models. However, the question remains whether the disturbance of glucose metabolism is the driving force of epileptogenesis. We have recently reported that a chronic partial inhibition of brain glycolysis by the non-metabolizable glucose analogue 2-deoxy-D-glucose (2-DG) triggers epileptogenesis in initially healthy rats. In this study, we further investigated whether chronic 2-DG treatment caused a cellular loss in the dorsal hippocampus and mossy fiber sprouting in the dentate gyrus. We found that prolonged (four weeks) treatment with 2-DG induced a neuronal loss in the CA1 field and the dentate hilus. We also found mossy fibers reorganization in the 2-DG group. In addition, we showed that pentylenetetrazole-induced convulsions were considerably strengthened and prolonged in 2-DG-treated rats. Our results demonstrate that the chronically impaired brain glucose metabolism likely leads to a structural remodeling resembling epileptogenesis and has a proconvulsive effect.

Evaluation of oxidative stress and antioxidant activity in women with moderate and severe preeclampsia in the third trimester of pregnancy and their newborns.

We examined 66 women who were 22-40 weeks pregnant and their newborns. Of these, 15 women with moderate PE were in group 1, 22 women with severe PE were in group 2, and 55 women with uncomplicated pregnancy without hypertensive disorders were in the control group. Blood was taken from women when they were admitted to the clinic, and newborns ' blood was taken for 3-5 days of life. Free radical oxidation and antioxidant activity were evaluated by induced chemiluminescence. It was found that in patients with severe and moderate preeclampsia, the development of oxidative stress is accompanied by a weakening of antioxidant activity. In newborns born to mothers with preeclampsia, oxidative stress is accompanied by a compensatory increase in antioxidant activity.

Seizure-induced reduction in glucose utilization promotes brain hypometabolism during epileptogenesis.

Brain glucose hypometabolism is an early symptom of acquired epilepsy, its causative mechanism yet unclear. We suggest that a bidirectional positive feedback linking seizures and hypometabolism (hypometabolism induces seizures while seizures disrupt glucose metabolism) may be a primary cause for acquired epileptogenesis. We reported recently that chronic partial inhibition of brain glycolysis triggers epileptogenesis in healthy rats. Here, by monitoring dynamic electrical and multiple metabolic parameters before and following seizure generation in mouse hippocampal slices using the 4-aminopyridine model of epileptiform activity, we show that in turn seizures are followed by a long-lasting glucose hypometabolism, indicating possible existence of a positive feedback in the mechanism of epileptogenesis. Seizures were associated with acute oxidative stress that may contribute to the subsequent glucose metabolism impairment, since exogenous application of H2O2 replicated the post-seizure metabolic effects. Exogenous pyruvate, the principal mitochondrial energy substrate with a broad spectrum of neuroprotective properties, effectively normalized the post-seizure glucose consumption. We have shown recently that pyruvate exhibited a strong antiepileptic action in three rodent chronic epilepsy models, while in the present study we find that pyruvate effectively normalizes impaired glucose metabolism following seizures. Together, our results provide the mechanistic basis for the metabolic concept of acquired epileptogenesis and an efficient treatment strategy.

Sensitivity analysis of consumption cycles.

We study the special case of a nonlinear stochastic consumption model taking the form of a 2-dimensional, non-invertible map with an additive stochastic component. Applying the concept of the stochastic sensitivity function and the related technique of confidence domains, we establish the conditions under which the system's complex consumption attractor is likely to become observable. It is shown that the level of noise intensities beyond which the complex consumption attractor is likely to be observed depends on the weight given to past consumption in an individual's preference adjustment.

The Behavior of VLF/LF Variations Associated with Geomagnetic Activity, Earthquakes, and the Quiet Condition Using a Neural Network Approach.

The neural network approach is proposed for studying very-low- and low-frequency (VLF and LF) subionospheric radio wave variations in the time vicinities of magnetic storms and earthquakes, with the purpose of recognizing anomalies of different types. We also examined the days with quiet geomagnetic conditions in the absence of seismic activity, in order to distinguish between the disturbed signals and the quiet ones. To this end, we trained the neural network (NN) on the examples of the representative database. The database included both the VLF/LF data that was measured during four-year monitoring at the station in Petropavlovsk-Kamchatsky, and the parameters of seismicity in the Kuril-Kamchatka and Japan regions. It was shown that the neural network can distinguish between the disturbed and undisturbed signals. Furthermore, the prognostic behavior of the VLF/LF variations indicative of magnetic and seismic activity has a different appearance in the time vicinity of the earthquakes and magnetic storms.

Chronic inhibition of brain glycolysis initiates epileptogenesis.

Metabolic abnormalities found in epileptogenic tissue provide considerable evidence of brain hypometabolism, while major risk factors for acquired epilepsy all share brain hypometabolism as one common outcome, suggesting that a breakdown of brain energy homeostasis may actually precede epileptogenesis. However, a causal link between deficient brain energy metabolism and epilepsy initiation has not been yet established. To address this issue we developed an in vivo model of chronic energy hypometabolism by daily intracerebroventricular (i.c.v.) injection of the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG) and also investigated acute effects of 2-DG on the cellular level. In hippocampal slices, acute glycolysis inhibition by 2-DG (by about 35%) led to contrasting effects on the network: a downregulation of excitatory synaptic transmission together with a depolarization of neuronal resting potential and a decreased drive of inhibitory transmission. Therefore, the potential acute effect of 2-DG on network excitability depends on the balance between these opposing pre- and postsynaptic changes. In vivo, we found that chronic 2-DG i.c.v. application (estimated transient inhibition of brain glycolysis under 14%) for a period of 4 weeks induced epileptiform activity in initially healthy male rats. Our results suggest that chronic inhibition of brain energy metabolism, characteristics of the well-established risk factors of acquired epilepsy, and specifically a reduction in glucose utilization (typically observed in epileptic patients) can initiate epileptogenesis. © 2017 Wiley Periodicals, Inc.

Cancer-associated DNA hypermethylation of Polycomb targets requires DNMT3A dual recognition of histone H2AK119 ubiquitination and the nucleosome acidic patch.

During tumor development, promoter CpG islands that are normally silenced by Polycomb repressive complexes (PRCs) become DNA-hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) [DNMT(s)] catalyze CpG methylation at PRC-regulated regions remains unclear. Here, we report a cryo-electron microscopy structure of the DNMT3A long isoform (DNMT3A1) amino-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine-119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 amino terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Further, aberrant redistribution of DNMT3A1 to Polycomb target genes recapitulates the cancer-associated DNA hypermethylation signature and inhibits their transcriptional activation during cell differentiation. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for mediating promoter CpG island DNA hypermethylation, a major molecular hallmark of cancer.

Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch.

During tumor development, promoter CpG islands (CGIs) that are normally silenced by Polycomb repressive complexes (PRCs) become DNA hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) catalyze CpG methylation at PRC-regulated regions remains unclear. Here we report a cryo-EM structure of the DNMT3A long isoform (DNMT3A1) N-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine 119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 N-terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Furthermore, aberrant redistribution of DNMT3A1 to Polycomb target genes inhibits their transcriptional activation during cell differentiation and recapitulates the cancer-associated DNA hypermethylation signature. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for countering promoter CGI DNA hypermethylation, a major molecular hallmark of cancer.

Nucleosome conformation dictates the histone code.

Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has been proposed that these PTMs form localized 'codes' that are read by specialized regions (reader domains) in chromatin-associated proteins (CAPs) to regulate downstream function. Substantial effort has been made to define [CAP: histone PTM] specificities, and thus decipher the histone code and guide epigenetic therapies. However, this has largely been done using the reductive approach of isolated reader domains and histone peptides, which cannot account for any higher-order factors. Here, we show that the [BPTF PHD finger and bromodomain: histone PTM] interaction is dependent on nucleosome context. The tandem reader selectively associates with nucleosomal H3K4me3 and H3K14ac or H3K18ac, a combinatorial engagement that despite being in cis is not predicted by peptides. This in vitro specificity of the BPTF tandem reader for PTM-defined nucleosomes is recapitulated in a cellular context. We propose that regulatable histone tail accessibility and its impact on the binding potential of reader domains necessitates we refine the 'histone code' concept and interrogate it at the nucleosome level.

Immunochemical characterization of the capsular polysaccharide of Azospirillum irakense KBC1.

The repeating unit structure of Azospirillum irakense KBC1 capsular polysaccharide (CPS) was established and was found to be identical to that of the O polysaccharide of A. irakense KBC1 lipopolysaccharide (LPS). The antigenic heterogeneity of the LPS and the CPS was shown to be related to differences in the macromolecular organization of these glycopolymers. After an immune response activation, R-form CPS molecules were found to be predominant.

An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability.

In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g. K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has a broader extension. Here, we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation 'chromatin switch' on the H3 tail that modulates read-write accessibility in nucleosomes and resolves the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.

Unifying mechanism behind the onset of acquired epilepsy.

Acquired epilepsy (AE) can result from a number of brain insults and neurological diseases with wide etiological diversity sharing one common outcome of brain epileptiform activity. This implies that despite their disparity, all these initiating pathologies affect the same fundamental brain functions underlying network excitability. Identifying such mechanisms and their availability as therapeutic targets would help develop an effective strategy for epileptogenesis prevention. In this opinion article, we propose that the vicious cycle of NADPH oxidase (NOX)-mediated oxidative stress and glucose hypometabolism is the underlying cause of AE, as available data reveal a critical role for both pathologies in epileptogenesis and the process of seizure initiation. Altogether, here we present a novel view on the mechanisms behind the onset of AE and identify therapeutic targets for potential clinical applications.

The dCypher Approach to Interrogate Chromatin Reader Activity Against Posttranslational Modification-Defined Histone Peptides and Nucleosomes.

Bulk chromatin encompasses complex sets of histone posttranslational modifications (PTMs) that recruit (or repel) the diverse reader domains of Chromatin-Associated Proteins (CAPs) to regulate genome processes (e.g., gene expression, DNA repair, mitotic transmission). The binding preference of reader domains for their PTMs mediates localization and functional output, and are often dysregulated in disease. As such, understanding chromatin interactions may lead to novel therapeutic strategies, However the immense chemical diversity of histone PTMs, combined with low-throughput, variable, and nonquantitative methods, has defied accurate CAP characterization. This chapter provides a detailed protocol for dCypher, a novel approach for the rapid, quantitative interrogation of CAPs (as mono- or multivalent Queries) against large panels (10s to 100s) of PTM-defined histone peptide and semisynthetic nucleosomes (the potential Targets). We describe key optimization steps and controls to generate robust binding data. Further, we compare the utility of histone peptide and nucleosome substrates in CAP studies, outlining important considerations in experimental design and data interpretation.

Heat Release by Isolated Mouse Brain Mitochondria Detected with Diamond Thermometer.

The production of heat by mitochondria is critical for maintaining body temperature, regulating metabolic rate, and preventing oxidative damage to mitochondria and cells. Until the present, mitochondrial heat production has been characterized only by methods based on fluorescent probes, which are sensitive to environmental variations (viscosity, pH, ionic strength, quenching, etc.). Here, for the first time, the heat release of isolated mitochondria was unambiguously measured by a diamond thermometer (DT), which is absolutely indifferent to external non-thermal parameters. We show that during total uncoupling of transmembrane potential by CCCP application, the temperature near the mitochondria rises by 4-22 °C above the ambient temperature with an absolute maximum of 45 °C. Such a broad variation in the temperature response is associated with the heterogeneity of the mitochondria themselves as well as their aggregations in the isolated suspension. Spontaneous temperature bursts with comparable amplitude were also detected prior to CCCP application, which may reflect involvement of some mitochondria to ATP synthesis or membrane potential leaking to avoid hyperproduction of reactive oxygen species. The results obtained with the diamond temperature sensor shed light on the "hot mitochondria" paradox.

Fecal Microbiota Analysis in Cats with Intestinal Dysbiosis of Varying Severity.

Recent studies have shown that the gut microbiota plays an important role in the pathogenesis of gastrointestinal diseases in various animal species. There are only limited data on the microbiome in cats with varying grades of dysbiosis. The purpose of the study was a detailed analysis of the quantitative and qualitative fecal microbiota spectrum in cats with intestinal dysbiosis of varying severity. The data obtained indicate that, depending on the dysbiosis severity in cats, the intestinal microbiome landscape changes significantly. It has been established that, depending on the dysbiosis severity, there is a shift in the balance between the Gram-positive and Gram-negative bacterial pools and in the nature of the isolation of specific bacteria forms, in the amount of obligate microbiota isolation, as well as individual facultative strains. When analyzing the serotyping of E. coli cultures isolated at various grades of intestinal dysbiosis severity, differences were found both in the isolation amount of various serotypes from one animal and in the prevalence of certain serotypes for each disease severity. A retrospective analysis of the fecal microbiota sensitivity in cats with dysbiosis to antibacterial drugs showed that, depending on the disease severity, the number of isolates sensitive to antibiotics increases significantly.

Structure and flexibility of the yeast NuA4 histone acetyltransferase complex.

The NuA4 protein complex acetylates histones H4 and H2A to activate both transcription and DNA repair. We report the 3.1-Å resolution cryo-electron microscopy structure of the central hub of NuA4, which flexibly tethers the histone acetyltransferase (HAT) and Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) modules. The hub contains the large Tra1 subunit and a core that includes Swc4, Arp4, Act1, Eaf1, and the C-terminal region of Epl1. Eaf1 stands out as the primary scaffolding factor that interacts with the Tra1, Swc4, and Epl1 subunits and contributes the conserved HSA helix to the Arp module. Using nucleosome-binding assays, we find that the HAT module, which is anchored to the core through Epl1, recognizes H3K4me3 nucleosomes with hyperacetylated H3 tails, while the TINTIN module, anchored to the core via Eaf1, recognizes nucleosomes that have hyperacetylated H2A and H4 tails. Together with the known interaction of Tra1 with site-specific transcription factors, our data suggest a model in which Tra1 recruits NuA4 to specific genomic sites then allowing the flexible HAT and TINTIN modules to select nearby nucleosomes for acetylation.