Chaperoning heterochromatin: new roles of FACT in chromatin silencing.

The multitasking histone chaperone FACT (FAcilitates Chromatin Transcription) contributes to actively transcribed euchromatin and repressed heterochromatin. However, its precise role in gene silencing has remained obscure. Here, we discuss new insights into the silent chromatin functions and recruitment mechanisms of FACT, and their possible implications in cell identity and cancer.

The histone chaperone FACT facilitates heterochromatin spreading by regulating histone turnover and H3K9 methylation states.

Heterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT (facilitates chromatin transcription) has been implicated in heterochromatin silencing, but the mechanisms by which FACT engages in this process remain opaque. Here, we pinpoint its function to the heterochromatin spreading process in fission yeast. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1. Together, our study identifies FACT as a histone chaperone that promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of repressive methylation marks.

Essential histone chaperones collaborate to regulate transcription and chromatin integrity.

Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions.

Bromodomain AAA+ ATPases get into shape.

Bromodomain AAA+ ATPases (ATPases associated with diverse cellular activities) are emerging as oncogenic proteins and compelling targets for anticancer therapies. However, structural and biochemical insight into these machines is missing. A recent study by Cho et al. reports the first cryo-EM structure of a bromodomain AAA+ ATPase and provides first insights into the functions of this putative histone chaperone.

The Chaperone FACT and Histone H2B Ubiquitination Maintain S. pombe Genome Architecture through Genic and Subtelomeric Functions.

The histone chaperone FACT and histone H2B ubiquitination (H2Bub) facilitate RNA polymerase II (Pol II) passage through chromatin, yet it is not clear how they cooperate mechanistically. We used genomics, genetic, biochemical, and microscopic approaches to dissect their interplay in Schizosaccharomyces pombe. We show that FACT and H2Bub globally repress antisense transcripts near the 5' end of genes and inside gene bodies, respectively. The accumulation of these transcripts is accompanied by changes at genic nucleosomes and Pol II redistribution. H2Bub is required for FACT activity in genic regions. In the H2Bub mutant, FACT binding to chromatin is altered and its association with histones is stabilized, which leads to the reduction of genic nucleosomes. Interestingly, FACT depletion globally restores nucleosomes in the H2Bub mutant. Moreover, in the absence of Pob3, the FACT Spt16 subunit controls the 3' end of genes. Furthermore, FACT maintains nucleosomes in subtelomeric regions, which is crucial for their compaction.

Spt6 Is Required for the Fidelity of Promoter Selection.

Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome.

Neoatherosclerosis development following bioresorbable vascular scaffold implantation in diabetic and non-diabetic swine.

BACKGROUND: DM remains a risk factor for poor outcome after stent-implantation, but little is known if and how DM affects the vascular response to BVS. AIM: The aim of our study was to examine coronary responses to bioresorbable vascular scaffolds (BVS) in swine with and without diabetes mellitus fed a 'fast-food' diet (FF-DM and FF-NDM, respectively) by sequential optical coherence tomography (OCT)-imaging and histology. METHODS: Fifteen male swine were evaluated. Eight received streptozotocin-injection to induce DM. After 9 months (M), 32 single BVS were implanted in epicardial arteries with a stent to artery (S/A)-ratio of 1.1:1 under quantitative coronary angiography (QCA) and OCT guidance. Lumen, scaffold, neointimal coverage and composition were assessed by QCA, OCT and near-infrared spectroscopy (NIRS) pre- and/or post-procedure, at 3M and 6M. Additionally, polarization-sensitive (PS)-OCT was performed in 7 swine at 6M. After sacrifice at 3M and 6M, histology and polymer degradation analysis were performed. RESULTS: Late lumen loss was high (~60%) within the first 3M after BVS-implantation (P<0.01 FF-DM vs. FF-NDM) and stabilized between 3M and 6M (<5% change in FF-DM, ~10% in FF-NDM; P>0.20). Neointimal coverage was highly heterogeneous in all swine (DM vs. NDM P>0.05), with focal lipid accumulation, irregular collagen distribution and neointimal calcification. Likewise, polymer mass loss was low (~2% at 3M, ~5% at 6M;P>0.20) and not associated with DM or inflammation. CONCLUSION: Scaffold coverage showed signs of neo-atherosclerosis in all FF-DM and FF-NDM swine, scaffold polymer was preserved and the vascular response to BVS was not influenced by diabetes.

EcR recruits dMi-2 and increases efficiency of dMi-2-mediated remodelling to constrain transcription of hormone-regulated genes.

Gene regulation by steroid hormones plays important roles in health and disease. In Drosophila, the hormone ecdysone governs transitions between key developmental stages. Ecdysone-regulated genes are bound by a heterodimer of ecdysone receptor (EcR) and Ultraspiracle. According to the bimodal switch model, steroid hormone receptors recruit corepressors in the absence of hormone and coactivators in its presence. Here we show that the nucleosome remodeller dMi-2 is recruited to ecdysone-regulated genes to limit transcription. Contrary to the prevalent model, recruitment of the dMi-2 corepressor increases upon hormone addition to constrain gene activation through chromatin remodelling. Furthermore, EcR and dMi-2 form a complex that is devoid of Ultraspiracle. Unexpectedly, EcR contacts the dMi-2 ATPase domain and increases the efficiency of dMi-2-mediated nucleosome remodelling. This study identifies a non-canonical EcR-corepressor complex with the potential for a direct regulation of ATP-dependent nucleosome remodelling by a nuclear hormone receptor.

Low-Molecular-Weight Hydrogels as New Supramolecular Materials for Bioelectrochemical Interfaces.

Controlling the interface between biological tissues and electrodes remains an important challenge for the development of implantable devices in terms of electroactivity, biocompatibility, and long-term stability. To engineer such a biocompatible interface a low molecular weight gel (LMWG) based on a glycosylated nucleoside fluorocarbon amphiphile (GNF) was employed for the first time to wrap gold electrodes via a noncovalent anchoring strategy, that is, self-assembly of GNF at the electrode surface. Scanning electron microscopy (SEM) studies indicate that the gold surface is coated with the GNF hydrogels. Electrochemical measurements using cyclic voltammetry (CV) clearly show that the electrode properties are not affected by the presence of the hydrogel. This coating layer of 1 to 2 µm does not significantly slow down the mass transport through the hydrogel. Voltammetry experiments with gel coated macroporous enzyme electrodes reveal that during continuous use their current is improved by 100% compared to the noncoated electrode. This demonstrates that the supramolecular hydrogel dramatically increases the stability of the bioelectrochemical interface. Therefore, such hybrid electrodes are promising candidates that will both offer the biocompatibility and stability needed for the development of more efficient biosensors and biofuel cells.

CENPs and Sweet Nucleosomes Face the FACT.

Chaperones mediate vital interactions between histones and DNA during chromatin assembly and reorganization. Two recent studies reveal novel substrates for the essential and conserved histone chaperone FAcilitates Chromatin Transcription (FACT). Prendergast et al. show that FACT helps deposit important histone-fold proteins on centromeres. Raj et al. find that FACT preferentially binds O-GlcNAcylated nucleosomes, suggesting that FACT may contribute to nutrient-regulated cellular programs.

Breast cancer leptomeningeal metastasis: the results of combined treatment and the comparison of methotrexate and liposomal cytarabine as intra-cerebrospinal fluid chemotherapy.

BACKGROUND: This was a prospective observational study to assess the results of the treatment of patients with breast cancer leptomeningeal metastasis (LM) and to compare the efficacy of methotrexate and liposomal cytarabine in patients treated intrathecally by lumbar puncture. PATIENTS AND METHODS: In this prospective observational study, 149 consecutive patients with breast cancer and LM treated between the years 1999 and 2011 were assessed. Multimodality treatment methods were used: systemic therapy in 77 patients, radiotherapy in 92 patients, intrathecal methotrexate in 81 patients, and intrathecal liposomal cytarabine in 15 patients. RESULTS: The median survival of all patients was 4.2 months. The median survival of patients in whom systemic intravenous/oral treatment was used was 6 months, in those who did not have systemic treatment, the median survival was 2 months (P < .001). The median survival of patients treated with intrathecal methotrexate was 4.2 months; in patients treated with intrathecal liposomal cytarabine, the median survival was 4.6 months, and in patients who did not receive intrathecal treatment, the median survival was 3.7 months (P = .717). Median survival after whole-brain radiotherapy was 4.6 months and with no radiotherapy, it was 3.2 months (P = .028). Multivariate analysis revealed a Karnofsky performance status (KPS) of > 70. Systemic intravenous/oral treatment and bone as a site of metastasis were factors prolonging survival from LM. CONCLUSION: Among treatment methods, only systemic therapy prolonged survival in patients with LM. Neither radiotherapy nor lumbar intrathecal therapy influenced survival in those patients; however, both methods alleviated signs and symptoms of LM. No difference in survival was observed in patients treated intrathecally with methotrexate and those treated with liposomal cytarabine. Treatment with both drugs was comparable.

PtIr-WO3 nanostructured alloy for electrocatalytic oxidation of ethylene glycol and ethanol.

In this article, we characterized tungsten oxide-decorated carbon-supported PtIr nanoparticles and tested it for the electrooxidation reactions of ethylene glycol and ethanol. Phase and morphological evaluation of the proposed electrocatalytic materials are investigated employing various characterization techniques including X-ray diffraction (XRD) and transmission electron microscopy (TEM). Electrochemical diagnostic measurements such as cyclic voltammetry, chronoamperometry, and linear sweep voltammetry revealed that the tungsten oxide-modified PtIr/Vulcan nanoparticles have higher catalytic activity for ethylene glycol and ethanol electrooxidation than that of PtIr/Vulcan. A significant enhancement for electrooxidation of CO-adsorbate monolayers occurred in the presence of a transition metal oxide relative to that of pure PtIr/Vulcan electrocatalyst. The likely reasons for this are modification on the Pt center electronic structure and/or increasing the population of reactive oxo groups at the PtIr/Vulcan electrocatalytic interface in different potential regions.

Acute phase proteins in drain fluid: a new screening tool for colorectal anastomotic leakage? The APPEAL study: analysis of parameters predictive for evident anastomotic leakage.

BACKGROUND: We aim to determine if C-reactive protein (CRP), lipopolysaccharide-binding protein (LBP), and procalcitonin (PCT) in drain fluid can serve as screening tools for colorectal anastomotic leakage (CAL). METHODS: Patients included in this multicenter prospective observational study underwent left hemicolectomy, sigmoid resection, high anterior resection, low anterior resection, or subtotal colectomy. During the first 5 postoperative days, CRP, LBP, and PCT were determined on drain fluid. RESULTS: In total 243 patients were included, of whom 19 (8%) developed CAL. CRP levels were higher in patients with leakage on day 3 and day 5, levels of LBP were higher on days 2, 3, and 4, and PCT levels were higher on day 5. Multivariate analysis showed LBP to be significantly related to CAL. An increase in the average initial value at the first postoperative day with 1 standard deviation increased the risk of leakage by 1.6 times. CONCLUSION: Increased concentrations of LBP in drain fluid are significantly associated to a higher chance of CAL and could contribute in a future prognostic model for CAL.

Relationships among manual body functions, manual capacity, and bimanual performance using the prosthetic upper extremity functional index in children with congenital hand differences.

BACKGROUND: Most surgical techniques intervene at the level of body functions of the upper limb, aiming to improve manual capacity and activity performance. However, the nature of the relationships among these levels of functioning and evidence for hand function variables predicting performance have scarcely been investigated. OBJECTIVE: The primary aim of this study was to assess aspects of hand function and manual capacity that influence bimanual performance in children with congenital hand differences (CHDs), ranging from surgically corrected polydactyly or syndactyly to radial dysplasia. A secondary aim was to assess whether the number of items on the Prosthetic Upper Extremity Functional Index (PUFI) can be reduced without losing information on bimanual performance in this population. DESIGN: A cross-sectional design was used. METHODS: One hundred six 10- to 14-year-old children with CHD participated in the study, which was conducted in a university hospital's outpatient clinic. Bimanual performance was evaluated with child self-reports on an adapted version of the PUFI, calculating ease of performance and actual use of the affected hand. Additionally, hand function and manual capacity were assessed. RESULTS: The median score on ease of performance was high, and, on average, the children used their affected hand actively in 97% of all activities. Manual capacity of the nondominant hand and lateral pinch strength of the dominant hand predicted attainment of maximum PUFI scores. Nonmaximum PUFI scores were predicted by opposition strength of the nondominant hand and lateral pinch strength of the dominant hand. In addition, in this patient group, only 6 items of the PUFI explained all variance in PUFI scores. LIMITATIONS: The generalizability of the results is limited by the carefully selected age range. Second, the cross-sectional design of the study limits statements on causality on the relationships found. CONCLUSION: Children with a CHD generally have good bimanual performance and, on average, perform activities with active use of the affected hand. Therapy directed toward increasing manual capacity and finger muscle strength might assist in improving bimanual performance in children with CHD. Furthermore, the number of items on the PUFI could be reduced from 38 to 6 items in children with CHD.

Stronger relation between impairment and manual capacity in the non-dominant hand than the dominant hand in congenital hand differences; implications for surgical and therapeutic interventions.

OBJECTIVES: To evaluate manual activity capacity (i.e. activity capacity to perform hand activities) and its relation with body functions of the hand and forearm in children with congenital hand differences (CHD) METHODS: We assessed 10-14 year-old children with CHD (N = 106) using a functional handgrips test. Measurements of body functions included joint mobility and muscle strength. Patient characteristics were hand dominance and severity. RESULTS: We found a stronger relation between body functions and manual activity capacity in non-dominant hands than dominant hands. Dominant hands scored significantly higher on manual activity capacity than nondominant hands that were similarly impaired at body functions level. Severity of the CHD and body functions had only small effects on manual activity capacity. CONCLUSION: The relation between body functions and manual activity capacity is stronger in non-dominant hands than dominant hands, indicating that improvement in body functions lead to larger changes in manual activity capacity in the non-dominant hand. This may suggest that in bilaterally-affected children surgery should be done at the non-dominant hand first since this hand would benefit most from surgery-induced body functions improvement.

Measuring upper airway volume: accuracy and reliability of Dolphin 3D software compared to manual segmentation in craniosynostosis patients.

PURPOSE: To test the accuracy and reliability of Dolphin 3-dimensional (3D) software airway analysis compared with manual segmentation in patients who underwent a Le Fort III osteotomy. MATERIALS AND METHODS: Computed tomographic scans of 20 patients with syndromic craniosynostosis at Sophia's Children's Hospital (Rotterdam, The Netherlands) were used for airway volume measurements using Dolphin 3D. The same scans had been used for measurement using a manual segmentation method. The results of this previous study were reported in 2010. The manual segmentation measuring result was used as a gold standard. The airway was subdivided into the oropharynx and the nasal passage. A linear mixed effects statistical model was applied. RESULTS: Dolphin 3D measurements differed from manual segmentation by 9 to 43%, depending on the observer, the time at which the measured scan was acquired (pre- or postoperative), and the airway compartment being measured. The highest accuracy for Dolphin 3D was found for measurements from postoperative scans of the nasal passage. CONCLUSION: The airway analysis tool of Dolphin 3D is not accurate or reliable enough to use in a Le Fort III osteotomy evaluation. When scanning properties are conditioned and measurements are standardized, accuracy and reliability may increase.

Breast cancer leptomeningeal metastasis: propensity of breast cancer subtypes for leptomeninges and the analysis of factors influencing survival.

The aim of the study was to define biological subtypes of breast cancer that have the propensity to metastasize to the leptomeninges and to assess factors influencing survival from detection of leptomeningeal metastatis (LM). One hundred and eighteen consecutive breast cancer patients with LM were treated in one institution, between the years 1999 and 2009; 40.5 % of patients had triple-negative subtype, 37.5 % had luminal A subtype and 22 % had HER2-positive subtypes (luminal B and HER2). Of patients with LM, 35 % had lobular cancer. Median survival from the detection of LM was 18 weeks, and 1-year survival was 16 %. Cox multivariate analysis revealed that performance status and systemic treatment statistically significantly influenced survival of patients with LM. Triple-negative biological subtype and lobular histological type of breast cancer had the propensity to metastasize to the leptomeninges. Performance status and systemic treatment ordered after detection of LM statistically significantly influenced survival.