CRISPR screens in sister chromatid cohesion defective cells reveal PAXIP1-PAGR1 as regulator of chromatin association of cohesin.

The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.

Late Toxicity and Health-Related Quality of Life Following Definitive Chemoradiotherapy for Esophageal Cancer: A Systematic Review and Meta-analysis.

PURPOSE: Definitive chemoradiotherapy (dCRT) is a treatment option with curative intent for patients with esophageal cancer that could result in late toxicities and affect health-related quality of life (HRQoL). This study aimed to review the literature and perform a meta-analysis to investigate the effect of dCRT on late toxicities and HRQoL in esophageal cancer. METHODS AND MATERIALS: A systematic search was performed in MEDLINE, EMBASE, and PsychINFO. Prospective phase II and III clinical trials, population-based studies, and retrospective chart reviews investigating late toxicity or HRQoL after dCRT (≥50 Gy) were included. The HRQoL outcomes were analyzed using linear mixed-effect models with restricted cubic spline transformation. Any HRQoL changes of ≥10 points were considered clinically relevant. The risk of toxicities was calculated using the number of events and the total study population. RESULTS: Among 41 included studies, 10 assessed HRQoL and 31 late toxicity. Global health status remained stable over time and improved after 36 months compared with baseline (mean change, +11). Several tumor-specific symptoms, including dysphagia, eating restrictions, and pain, improved after 6 months compared with baseline. Compared with baseline, dyspnea worsened after 6 months (mean change, +16 points). The risk of any late toxicity was 48% (95% CI, 33%-64%). Late toxicity risk of any grade for the esophagus was 17% (95% CI, 12%-21%), pulmonary 21% (95% CI, 11%-31%), cardiac 12% (95% CI, 6%-17%), and any other organ 24% (95% CI, 2%-45%). CONCLUSIONS: Global health status remained stable over time, and tumor-specific symptoms improved within 6 months after dCRT compared with baseline, with the exception of dyspnea. In addition, substantial risks of late toxicity were observed.

Predictive Value of SLCO1B1 c.521T>C Polymorphism on Observed Changes in the Treatment of 1136 Statin-Users.

Pharmacogenomic testing is a method to prevent adverse drug reactions. Pharmacogenomics could be relevant to optimize statin treatment, by identifying patients at high risk for adverse drug reactions. We aim to investigate the clinical validity and utility of pre-emptive pharmacogenomics screening in primary care, with SLCO1B1 c.521T>C as a risk factor for statin-induced adverse drug reactions. The focus was on changes in therapy as a proxy for adverse drug reactions observed in statin-users in a population-based Dutch cohort. In total, 1136 statin users were retrospectively genotyped for the SLCO1B1 c.521T>C polymorphism (rs4149056) and information on their statin dispensing was evaluated as cross-sectional research. Approximately half of the included participants discontinued or switched their statin treatment within three years. In our analyses, we could not confirm an association between the SLCO1B1 c.521T>C genotype and any change in statin therapy or arriving at a stable dose sooner in primary care. To be able to evaluate the predictive values of SLCO1B1 c.521T>C genotype on adverse drug reactions from statins, prospective data collection of actual adverse drug reactions and reasons to change statin treatment should be facilitated.

The histone methyltransferase SETD2 negatively regulates cell size.

Cell size varies between cell types but is tightly regulated by cell intrinsic and extrinsic mechanisms. Cell size control is important for cell function, and changes in cell size are frequently observed in cancer. Here, we uncover a role for SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Using multiple independent perturbation strategies, we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that overexpression of the H3K36 demethylase KDM4A or the oncohistone H3.3K36M also increase cell size. In addition, ectopic overexpression of a decoy SRI domain increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development.

SETD2: from chromatin modifier to multipronged regulator of the genome and beyond.

Histone modifying enzymes play critical roles in many key cellular processes and are appealing proteins for targeting by small molecules in disease. However, while the functions of histone modifying enzymes are often linked to epigenetic regulation of the genome, an emerging theme is that these enzymes often also act by non-catalytic and/or non-epigenetic mechanisms. SETD2 (Set2 in yeast) is best known for associating with the transcription machinery and methylating histone H3 on lysine 36 (H3K36) during transcription. This well-characterized molecular function of SETD2 plays a role in fine-tuning transcription, maintaining chromatin integrity, and mRNA processing. Here we give an overview of the various molecular functions and mechanisms of regulation of H3K36 methylation by Set2/SETD2. These fundamental insights are important to understand SETD2's role in disease, most notably in cancer in which SETD2 is frequently inactivated. SETD2 also methylates non-histone substrates such as α-tubulin which may promote genome stability and contribute to the tumor-suppressor function of SETD2. Thus, to understand its role in disease, it is important to understand and dissect the multiple roles of SETD2 within the cell. In this review we discuss how histone methylation by Set2/SETD2 has led the way in connecting histone modifications in active regions of the genome to chromatin functions and how SETD2 is leading the way to showing that we also have to look beyond histones to truly understand the physiological role of an 'epigenetic' writer enzyme in normal cells and in disease.

Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L-dose dependency in HDAC1-deficient thymic lymphoma.

DOT1L methylates histone H3K79 and is aberrantly regulated in MLL-rearranged leukemia. Inhibitors have been developed to target DOT1L activity in leukemia, but cellular mechanisms that regulate DOT1L are still poorly understood. We have identified the histone deacetylase Rpd3 as a negative regulator of budding yeast Dot1. At its target genes, the transcriptional repressor Rpd3 restricts H3K79 methylation, explaining the absence of H3K79me3 at a subset of genes in the yeast genome. Similar to the crosstalk in yeast, inactivation of the murine Rpd3 homolog HDAC1 in thymocytes led to an increase in H3K79 methylation. Thymic lymphomas that arise upon genetic deletion of Hdac1 retained the increased H3K79 methylation and were sensitive to reduced DOT1L dosage. Furthermore, cell lines derived from Hdac1Δ/Δ thymic lymphomas were sensitive to a DOT1L inhibitor, which induced apoptosis. In summary, we identified an evolutionarily conserved crosstalk between HDAC1 and DOT1L with impact in murine thymic lymphoma development.

Review: Challenges in the histopathological classification of ganglioglioma and DNT: microscopic agreement studies and a preliminary genotype-phenotype analysis.

Low-grade epilepsy-associated brain tumours (LEAT) are the second most common cause for drug-resistant, focal epilepsy, that is ganglioglioma (GG) and dysembryoplastic neuroepithelial tumours (DNT). However, molecular pathogenesis, risk factors for malignant progression and their frequent association with drug-resistant focal seizures remain poorly understood. This contrasts recent progress in understanding the molecular-genetic basis and targeted treatment options in diffuse gliomas. The Neuropathology Task Force of the International League Against Epilepsy examined available literature to identify common obstacles in diagnosis and research of LEAT. Analysis of 10 published tumour series from epilepsy surgery pointed to poor inter-rater agreement for the histopathology diagnosis. The Task Force tested this hypothesis using a web-based microscopy agreement study. In a series of 30 LEAT, 25 raters from 18 countries agreed in only 40% of cases. Highest discordance in microscopic diagnosis occurred between GG and DNT variants, when oligodendroglial-like cell patterns prevail, or ganglion cells were difficult to discriminate from pre-existing neurons. Suggesting new terminology or major histopathological criteria did not satisfactorily increase the yield of histopathology agreement in four consecutive trials. To this end, the Task Force applied the WHO 2016 strategy of integrating phenotype analysis with molecular-genetic data obtained from panel sequencing and 450k methylation arrays. This strategy was helpful to distinguish DNT from GG variants in all cases. The Task Force recommends, therefore, to further develop diagnostic panels for the integration of phenotype-genotype analysis in order to reliably classify the spectrum of LEAT, carefully characterize clinically meaningful entities and make better use of published literature.

Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1.

Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features.

Expression of neurodegenerative disease-related proteins and caspase-3 in glioneuronal tumours.

AIMS: Recent evidence supports the activation of mechanisms underlying cellular ageing and neurodegeneration in developmental lesions associated with epilepsy. The present study examined the ongoing cell injury and vulnerability to neuronal degeneration in glioneuronal tumours (GNT). METHODS: We evaluated a series of GNT (n = 31 gangliogliomas, GG and n = 30 dysembryoplastic neuroepithelial tumours, DNT). Sections were processed for immunohistochemistry using markers for the evaluation of caspase-3 and neurodegeneration-related proteins/pathways and their expression was correlated with the tumour features and the clinical history of epilepsy. RESULTS: Both GG and DNT specimens contained caspase-3-positive cells. In GG, expression of activated caspase-3 was negatively correlated the with the BRAF V600E mutation status. We also observed an abnormal expression of death receptor-6 and ß-amyloid precursor protein (APP). Moreover, dysplastic neurones expressed p62, phosphorylated (p)TDP43 and pTau. Double labelling experiments showed colocalization of phosphorylated S6 (marker of mammalian target of rapamycin, mTOR, pathway activation) with pTau and p62. In GG, neuronal p62 expression was positively correlated with pS6. The immunoreactivity score (IRS) of caspase-3, APP, DR6, p62 and pTDP43 were found to be significantly higher in GG than in DNT. Expression of APP, DR6, pTau (in GG and DNT) and caspase-3 (in GG) positively correlated with duration of epilepsy. In GG, the expression of neuronal caspase-3, DR6 and glial p62 was associated with a worse postoperative seizure outcome. CONCLUSIONS: Our observations in GNT provide evidence of premature activation of mechanisms of neurodegeneration which are associated with the clinical course of epilepsy in patient with GG.

Interictal psychosis following temporal lobe surgery: dentate gyrus pathology.

BACKGROUND: De novo interictal psychosis, albeit uncommon, can develop in patients following temporal lobe surgery for epilepsy. Pathological alterations of the dentate gyrus, including cytoarchitectural changes, immaturity and axonal reorganization that occur in epilepsy, may also underpin co-morbid psychiatric disorders. Our aim was to study candidate pathways that may be associated with the development of interictal psychosis post-operatively in patients with hippocampal sclerosis (HS). METHOD: A total of 11 patients with HS who developed interictal psychosis (HS-P) post-operatively were compared with a matched surgical HS group without psychosis (HS-NP). Resected tissues were investigated for the extent of granule cell dispersion, mossy fibre sprouting and calbindin expression in the granule cells. We quantified doublecortin, mini-chromosome maintenance protein 2 (MCM2) and reelin-expressing neuronal populations in the dentate gyrus as well as the distribution of cannabinoid type 1 receptor (CBR1). RESULTS: The patterns of neuronal loss and gliosis were similar in both groups. HS-P patients demonstrated less mossy fibre sprouting and granule cell dispersion (p < 0.01) and more frequent reduction in calbindin expression in granule cells. There were no group differences in the densities of immature MCM2, doublecortin and reelin-positive cells. CBR1 labelling was significantly lower in Cornu ammonis area CA4 relative to other subfields (p < 0.01); although reduced staining in all hippocampal regions was noted in HS-P compared with HS-NP patients, the differences were not statistically significant. CONCLUSIONS: The alterations in dentate gyrus pathology found in HS-P patients could indicate underlying differences in the cellular response to seizures. These mechanisms may predispose to the development of psychosis in epilepsy and warrant further investigation.

Antecolic versus retrocolic route of the gastroenteric anastomosis after pancreatoduodenectomy: a randomized controlled trial.

OBJECTIVE: To investigate the relationship between the route of gastroenteric (GE) reconstruction after pancreatoduodenectomy (PD) and the postoperative incidence of delayed gastric emptying (DGE). BACKGROUND: DGE is one of the most common complications after PD. Recent studies suggest that an antecolic route of the GE reconstruction leads to a lower incidence of DGE, compared to a retrocolic route. In a nonrandomized comparison within our trial center, we found no difference in DGE after antecolic or retrocolic GE reconstruction. METHODS: Ten middle- to high-volume centers participated in the patient inclusion. Patients scheduled for PD who gave written informed consent were included and randomized during surgery after resection. Standard operation was a pylorus-preserving PD. Primary endpoint was DGE. Secondary endpoints included other complications and length of hospital stay. RESULTS: There were 125 patients in the retrocolic group, and 121 patients in the antecolic group. Baseline and treatment characteristics did not differ between the study groups. In the retrocolic group, 45 patients (36%) developed clinically relevant DGE compared with 41 (34%) in the antecolic group (absolute risk difference: 2.1%; 95% confidence interval: -9.8% to 14.0%). There were no differences in need for postoperative (par)enteral nutritional support, other complications, hospital mortality, and median length of hospital stay. CONCLUSIONS: The route of GE reconstruction after PD does not influence the postoperative incidence of DGE or other complications. The etiology and treatment of DGE, which occurs frequently after both procedures, need further investigation. The GE reconstruction after PD should be routed according to the surgeon's preference.

Production and characterization of two monoclonal antibodies to bovine tumour necrosis factor alpha (TNF-alpha) and their cross-reactivity with ovine TNF-alpha.

Tumour necrosis factor alpha (TNF-alpha) is an innate pro-inflammatory cytokine involved in protection against intracellular pathogens. Existing methods for measuring TNF-alpha production and function in ruminants are limited to ELISA and many rely on polyclonal antisera. With a view to developing improved detection methods for bovine (bov) TNF-alpha, monoclonal antibodies (mAb) were produced by immunising mice with a plasmid encoding bov TNF-alpha. Two of the resulting mAb, termed CC327 and CC328, were used to develop a sandwich ELISA capable of detecting both native and recombinant bov TNF-alpha. This ELISA did not detect recombinant ovine (ov) TNF-alpha. A luminometric method was applied to the ELISA to improve sensitivity for detection of native bov TNF-alpha in culture supernatants derived from bovine monocyte-derived dendritic cells (DC) infected with Mycobacterium bovis. Both CC327 and CC328 detected intracytoplasmic expression of TNF-alpha in mitogen-activated bovine T lymphocytes. However, only CC328 detected intracytoplasmic ovine TNF-alpha in transfected cells, explaining the failure of the sandwich ELISA to detect recombinant ov TNF-alpha. These mAbs have generated the capability to study the role of TNF-alpha in host immune protection and disease pathogenesis in ruminants.

Bilateral reorganization of the dentate gyrus in hippocampal sclerosis: a postmortem study.

BACKGROUND: Hippocampal sclerosis (HS) is the most common surgical pathology associated with mesial temporal lobe epilepsy (MTLE). HS is typically characterized by mossy fiber sprouting (MFS) and reorganization of neuropeptide Y (NPY) fiber networks in the dentate gyrus. One potential cause of postoperative seizure recurrence following temporal lobe surgery may be the presence of seizure-associated bilateral hippocampal damage. We aimed to investigate patterns of hippocampal abnormalities in a postmortem series as identified by NPY and dynorphin immunohistochemistry. METHODS: Analysis of dentate gyrus fiber reorganization, using dynorphin (to demonstrate MFS) and NPY immunohistochemistry, was carried out in a postmortem epilepsy series of 25 cases (age range 21-96 years). In 9 patients, previously refractory seizures had become well controlled for up to 34 years prior to death. RESULTS: Bilateral MFS or abnormal NPY patterns were seen in 15 patients including those with bilateral symmetric, asymmetric, and unilateral HS by conventional histologic criteria. MFS and NPY reorganization was present in all classical HS cases, more variably in atypical HS, present in both MTLE and non-MTLE syndromes and with seizure histories of up to 92 years, despite seizure remission in some patients. CONCLUSION: Synaptic reorganization in the dentate gyrus may be a bilateral, persistent process in epilepsy. It is unlikely to be sufficient to generate seizures and more likely to represent a seizure-induced phenomenon.

Expression patterns of glial fibrillary acidic protein (GFAP)-delta in epilepsy-associated lesional pathologies.

AIMS: Glial fibrillary acidic protein (GFAP)-delta is a novel isoform that differs in its C-terminal sequence from other GFAP isoforms. Previous studies suggest restriction of expression to the subpial layer, subventricular zone and the subgranular zone astrocytes, with an absence in pathological conditions causing reactive gliosis. GFAP-delta is speculated to have roles in regulation of astrocyte size and motility and a subpopulation of GFAP-delta-positive glia may be multipotent stem cells. The aim of this study was to investigate its expression in common causes of lesion-related refractory epilepsy. METHODS: Hippocampal sclerosis (HS), focal cortical dysplasia (FCD) type IIB, cortical tuberous sclerosis (TSC) lesions, gangliogliomas, grey matter heterotopias and hemimegalencephaly from a wide age range of patients using both surgical and post mortem tissue specimens were studied. RESULTS: GFAP-delta expression was observed in CA4 and CA1 astrocytes in HS with less frequent labelling in the granule cell layer, even where granule cell dispersion was present. No significant labelling was noted in the subiculum in HS cases or in any subfields in non-HS epilepsy cases. Balloon cells in FCDIIB and hemimegalencephaly, giant cells in TSC and the astrocytic component of gangliogliomas showed immunoreactivity, colocalizing with conventional GFAP. No neuronal expression for GFAP-delta was seen in any of the pathologies. Quantitative analysis in 10 FCDIIB and five TSC cases revealed greater numbers of GFAP-delta-positive balloon cells than conventional GFAP. There was no GFAP-delta expression within nodular heterotopia. CONCLUSIONS: GFAP-delta expression patterns in HS overall appears to mirror regional reactive gliosis. It is a useful marker for the demonstration of balloon cells in FCD and TSC, which may be relevant to their abnormal size and localization. The lack of GFAP-delta within heterotopia supports their composition from cells destined for deeper cortical layers.

Balloon cells associated with granule cell dispersion in the dentate gyrus in hippocampal sclerosis.

Granule cell dispersion (GCD) is a common finding in hippocampal sclerosis in patients with intractable focal epilepsy. It is considered to be an acquired, post-developmental rather than a pre-existing abnormality, involving dispersion of either mature or newborn neurones, but the precise factors regulating it and its relationship to seizures are unknown. We present two cases of GCD with associated CD34-immunopositive balloon cells, a cell phenotype associated with focal cortical dysplasia type IIB, considered to be a developmental cortical lesion promoting epilepsy. This observation opens up the debate regarding the origin of balloon cells and CD34 expression and their temporal relationship to seizures.

Mixed glioneuronal tumour of the fourth ventricle with prominent rosette formation.

We describe three unusual tumours characterized by a mixture of glial and neuronal differentiation, involvement of the posterior fossa and formation of rosettes. Mixed glial-neuronal tumours of the posterior fossa are rare and poorly described neoplasms. However, several distinctive entities have appeared in the literature over recent years under a variety of different names. Our cases demonstrate the morphological features of the 'rosette-forming glioneuronal tumour of the fourth ventricle', a recently identified tumour characterised by its unique location, neurocytic pseudo-rosette formation and the presence of a low grade astrocytoma component. The long term prognosis of these tumours remains unclear. However, the clinical data available including the cases presented here, along with the histological features, suggest that these are low grade tumours with a good prognosis after surgical resection.

Vascular colocalization of P-glycoprotein, multidrug-resistance associated protein 1, breast cancer resistance protein and major vault protein in human epileptogenic pathologies.

Multidrug transporters, such as P-glycoprotein (P-gp), multidrug-resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP), are associated with multidrug resistance in cancers; other molecules, such as major vault protein (MVP), have a similar association with drug-resistant cancer. These proteins are postulated to generate drug resistance in epilepsy. They have been shown individually to be up-regulated in epileptogenic brain tissue. In any consideration of the function, inhibition or evasion of the activity of such proteins, the colocalization of such proteins needs to be understood. We systematically determined the presence of such colocalization, focusing on microvascular endothelium from epileptogenic human brain tissue. Double labelling immunofluorescence and confocal laser scanning microscopy were used to determine colocalization of P-gp, MRP1, BCRP and MVP in one case of hippocampal sclerosis and two cases of focal cortical dysplasia type IIb. Endothelial colocalization was examined with double labelling using antibodies to CD34 and Factor VIII. The presence of P-gp, BCRP and MVP in microvascular endothelium was confirmed. P-gp, BCRP and MVP colocalized in microvascular endothelium, though not all proteins appeared to be identically distributed within this tissue. MRP1 did not colocalize to endothelium. These findings were not unexpected but required formal confirmation. The demonstrated colocalization of P-gp, BCRP and MVP in microvascular endothelium in epileptogenic human brain tissue has important implications for functional experiments (including single knock-out mice studies), work with specific and broad-spectrum inhibitors of transport function, and any eventual trials of treatment of refractory epilepsy involving modulation of the function of these proteins.

Mcm2 labelling of balloon cells in focal cortical dysplasia.

Balloon cells (BC) are the prominent and defining cellular component of type IIB Focal Cortical Dysplasia (FCD), a common cause of focal epilepsy in patients undergoing surgical treatment. BC are considered immature cells of uncommitted cellular differentiation having immunophenotypical characteristics of both neurones and glia. They are often located in the lower cortical layers and white matter underlying the dysplastic cortex, suggesting migratory arrest during development. We investigated the proliferative potential of BC in 15 cases of FCD from patients with a wide range of ages using immunohistochemistry for Mcm2 (mini chromosome maintenance protein) and Ki67. In the majority of cases, BC showed Mcm2 nuclear positivity. In addition, cells with intermediate neuronal-glial characteristics were labelled whilst the dysmorphic or hypertrophic pyramidal neuronal components of FCD were not. Ki67 labelled only occasional BC. These findings support the view that BC cells represent a pool of less differentiated glial cells with proliferative capacity which may have potential for delayed neuronal differentiation. Furthermore, as Mcm2 specifically identifies BC populations, this marker may be of diagnostic value in the subtyping of FCD lesions in patients with epilepsy.