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.

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.

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.

Influence of lamina vastoadductoria dissection on the outcomes of femoral artery extensive lesion stenting: A pilot randomised investigation.

OBJECTIVE: Aim of the study was to improve the immediate and long-term results of stenting of the superficial femoral artery in extended lesions with the changing of the biomechanics of superficial ffemoral artery and of the first portion of the popliteal artery. METHODS: Pilot randomized prospective single-center study were included 70 patients. Patients were randomized into two groups in 1 × 1 format for 35 people using the envelope method. Self-expanding bare metal stents were used in all cases. At the first group standard revascularization procedures with SFA stenting were performed; in the second group, the superficial femoral artery stenting was supplemented with fasciotomy in the Hunter's canal with the superficial femoral artery intersection. The total observation period was 2 years. During the observation period an assessment of the clinical symptoms of the lower extremities, measurement of the ankle-brachial index and ultrasound duplex scanning of the operated segment were performed. RESULTS: All procedures in both groups were successfully performed. Primary patency through 24 months was 28.5% (10 of 35) in group 1 and 60% (21 of 35) in group 2 (p = 0,015). CONCLUSIONS: Changing the biomechanical properties of the distal of the superficial femoral artery segment and of the first portion of the popliteal artery is safe and contributes to the primary patency improvement during the stenting of extended of the superficial femoral artery lesions compared to standard SFA stenting. Dissection of the lamina vastoadductoria with transection of the collateral branches of the knee joint network reduces frequent and severe damages of stents after the stenting of the superficial femoral artery extended lesion. According to the frequency of complications in the early and mid-term postoperative period, limb salvage, mortality and the secondary patency rates, the new method is comparable with standard of the superficial femoral artery stenting.

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.

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.

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.

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.

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.

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.

Randomized Controlled Trial of Conventional Versus MicroNet-Covered Stent in Carotid Artery Revascularization.

OBJECTIVES: The aim of this study was to compare procedure-related ipsilateral cerebral embolism with a conventional (Acculink, Abbott Vascular) versus a MicroNet-covered (CGuard, InspireMD) stent in carotid artery stenting (CAS). BACKGROUND: The MicroNet-covered stent may reduce periprocedural cerebral embolism in CAS, but level 1 evidence is lacking. METHODS: A total of 100 consecutive patients were randomized 1:1 to filter-protected CAS using the Acculink or the CGuard device. The study was powered for its primary endpoint of at least 50% reduction in ipsilateral diffusion-weighted magnetic resonance imaging lesion average volume 48 hours postprocedure (blinded external core laboratory analysis). RESULTS: The baseline characteristics of the study groups were similar. Eighty-two (total volume = 18,212 mm3) diffusion-weighted magnetic resonance imaging postprocedural cerebral lesions occurred in 26 Acculink-treated patients and 45 lesions (total volume = 3,930 mm3; 78.4% reduction) in 25 CGuard-treated patients. New cerebral lesion average volume was 171 mm3 vs 73 mm3 (P = 0.017) per affected patient and 222 mm3 vs 84 mm3 (P = 0.038) per lesion (Acculink vs CGuard). In lesion-affected patients, the average sum of lesion volumes was 701 mm3 vs 157 mm3 (P = 0.007). The Acculink significantly increased the risk for multiple (≥5) cerebral lesions (relative risk: 7.8; 95% CI: 1.3-14.9; P = 0.021). At 30 days, new permanent (fluid-attenuated inversion recovery) lesion prevalence was 3:1 (P < 0.001), with total permanent lesion volume 7,474 mm3 vs 574 mm3 (92.3% reduction with the CGuard). There were 6 vs 0 new ipsilateral lesions (P = 0.030) and 2 versus 0 strokes. CONCLUSIONS: The MicroNet-covered stent significantly reduced periprocedural and abolished postprocedural cerebral embolism in relation to a conventional carotid stent. This is consistent with the MicroNet-covered stent's sustained embolism prevention, translating into cerebral protection not only during but also after CAS. The present findings may influence decision making in carotid revascularization. (The SIBERIA Trial [Acculink™ Versus CGuard™]; NCT03488199).

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.

Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies.

Proteases are involved in the control of numerous physiological processes, and their dysregulation has been identified in a wide range of pathologies, including cancer. Protease activity is normally tightly regulated post-translationally and therefore cannot be accurately estimated based on mRNA or protein expression alone. While several types of zymography approaches to estimate protease activity exist, there remains a need for a robust and reliable technique to measure protease activity in biological tissues. We present a novel quantitative ex vivo zymography (QZ) technology based on Probody® therapeutics (Pb-Tx), a novel class of protease-activated cancer therapeutics that contain a substrate linker cleavable by tumor-associated proteases. This approach enables the measurement and comparison of protease activity in biological tissues via the detection of Pb-Tx activation. By exploiting substrate specificity and selectivity, cataloguing and differentiating protease activities is possible, with further refinement achieved using protease-specific inhibitors. Using the QZ assay and human tumor xenografts, patient tumor tissues, and patient plasma, we characterized protease activity in preclinical and clinical samples. The QZ assay offers the potential to increase our understanding of protease activity in tissues and inform diagnostic and therapeutic development for diseases, such as cancer, that are characterized by dysregulated proteolysis.

Food Intolerance: The Role of Histamine.

Histamine is a natural amine derived from L-histidine. Although it seems that our knowledge about this molecule is wide and diverse, the importance of histamine in many regulatory processes is still enigmatic. The interplay between different types of histamine receptors and the compound may cause ample effects, including histamine intoxication and so-called histamine intolerance or non-allergic food intolerance, leading to disturbances in immune regulation, manifestation of gastroenterological symptoms, and neurological diseases. Most cases of clinical manifestations of histamine intolerance are non-specific due to tissue-specific distribution of different histamine receptors and the lack of reproducible and reliable diagnostic markers. The diagnosis of histamine intolerance is fraught with difficulties, in addition to challenges related to the selection of a proper treatment strategy, the regular course of recovery, and reduced amelioration of chronic symptoms due to inappropriate treatment prescription. Here, we reviewed a history of histamine uptake starting from the current knowledge about its degradation and the prevalence of histamine precursors in daily food, and continuing with the receptor interactions after entering and the impacts on the immune, central nervous, and gastrointestinal systems. The purpose of this review is to build an extraordinarily specific method of histamine cycle assessment in regard to non-allergic intolerance and its possible dire consequences that can be suffered.

Aß initiates brain hypometabolism, network dysfunction and behavioral abnormalities via NOX2-induced oxidative stress in mice.

A predominant trigger and driver of sporadic Alzheimer's disease (AD) is the synergy of brain oxidative stress and glucose hypometabolism starting at early preclinical stages. Oxidative stress damages macromolecules, while glucose hypometabolism impairs cellular energy supply and antioxidant defense. However, the exact cause of AD-associated glucose hypometabolism and its network consequences have remained unknown. Here we report NADPH oxidase 2 (NOX2) activation as the main initiating mechanism behind Aß1-42-related glucose hypometabolism and network dysfunction. We utilize a combination of electrophysiology with real-time recordings of metabolic transients both ex- and in-vivo to show that Aß1-42 induces oxidative stress and acutely reduces cellular glucose consumption followed by long-lasting network hyperactivity and abnormalities in the animal behavioral profile. Critically, all of these pathological changes were prevented by the novel bioavailable NOX2 antagonist GSK2795039. Our data provide direct experimental evidence for causes and consequences of AD-related brain glucose hypometabolism, and suggest that targeting NOX2-mediated oxidative stress is a promising approach to both the prevention and treatment of AD.

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.

Two competing mechanisms of DNMT3A recruitment regulate the dynamics of de novo DNA methylation at PRC1-targeted CpG islands.

Precise deposition of CpG methylation is critical for mammalian development and tissue homeostasis and is often dysregulated in human diseases. The localization of de novo DNA methyltransferase DNMT3A is facilitated by its PWWP domain recognizing histone H3 lysine 36 (H3K36) methylation1,2 and is normally depleted at CpG islands (CGIs)3. However, methylation of CGIs regulated by Polycomb repressive complexes (PRCs) has also been observed4-8. Here, we report that DNMT3A PWWP domain mutations identified in paragangliomas9 and microcephalic dwarfism10 promote aberrant localization of DNMT3A to CGIs in a PRC1-dependent manner. DNMT3A PWWP mutants accumulate at regions containing PRC1-mediated formation of monoubiquitylated histone H2A lysine 119 (H2AK119ub), irrespective of the amounts of PRC2-catalyzed formation of trimethylated histone H3 lysine 27 (H3K27me3). DNMT3A interacts with H2AK119ub-modified nucleosomes through a putative amino-terminal ubiquitin-dependent recruitment region, providing an alternative form of DNMT3A genomic targeting that is augmented by the loss of PWWP reader function. Ablation of PRC1 abrogates localization of DNMT3A PWWP mutants to CGIs and prevents aberrant DNA hypermethylation. Our study implies that a balance between DNMT3A recruitment by distinct reader domains guides de novo CpG methylation and may underlie the abnormal DNA methylation landscapes observed in select human cancer subtypes and developmental disorders.

Overall survival in patients with metastatic renal cell carcinoma in Russia, Kazakhstan, and Belarus: a report from the RENSUR3 registry.

BACKGROUND: Real-world data describing outcomes of treatment among metastatic renal cell carcinoma (mRCC) patients are limited and heterogeneous. AIM: RENSUR3 registry study assessed real-world data on the use of therapies in mRCC and overall survival (OS) in Russia, Kazakhstan, and Belarus. METHODS: Patients were included in the retrospective multicenter registry study. To be eligible, patients were required to have mRCC diagnosed from January 2015 to January 2016. Anonymized data were collected through an online registry. The outcomes of interest were patient characteristics, treatment patterns, and OS. RESULTS: 1094 mRCC patients were identified. Mean age was 62.3 (SD, 11.2) years. Four hundred and forty-four (41%) patients were 65 years and older. Primary tumor has not been removed in 503 (46%) patients. Subtype of RCC based on WHO classification (clear-cell or other) has been reported in 402 (37%) patients. In total, 595 (54.4%) patients received systemic therapy for metastatic disease. 58% of elderly patients (≥65) were not treated compared to 37% of younger patients. Cytokines and targeted therapy were used in 298 (50.1%) and 297 (49.9%) of 595 treated patients, respectively. Median OS was 11.9 months (95% CI 10.9-12.9). The 1- and 3-year OS rates were 49.6% and 19.3%. CONCLUSIONS: Half of patients received no systemic therapy or had only cytokines for mRCC in Russia, Kazakhstan, and Belarus, which doubtless negatively affected OS in this population. Novel therapies should be considered as life prolonging and a priority.