Hepatocellular Carcinoma Surveillance Patterns and Outcomes in Patients With Cirrhosis.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) surveillance is associated with improved early detection and reduced mortality, although practice patterns and effectiveness vary in clinical practice. We aimed to characterize HCC surveillance patterns in a large, diverse cohort of patients with HCC. METHODS: We conducted a retrospective cohort study of patients diagnosed with HCC between January 2008 and December 2022 at 2 large US health systems. We recorded imaging receipt in the year before HCC diagnosis: ultrasound plus α-fetoprotein (AFP), ultrasound alone, multiphasic contrast-enhanced computed tomography (CT)/magnetic resonance imaging (MRI), and no liver imaging. We used multivariable logistic and Cox regression analysis to compare early tumor detection, curative treatment receipt, and overall survival between surveillance strategies. RESULTS: Among 2028 patients with HCC (46.7% Barcelona Clinic Liver Cancer stage A), 703 (34.7%) had ultrasound plus AFP, 293 (14.5%) had ultrasound alone, 326 (16.1%) had multiphasic CT/MRI, and 706 (34.8%) had no imaging in the year before HCC diagnosis. Over the study period, proportions without imaging were stable, whereas use of CT/MRI increased. Compared with no imaging, CT/MRI and ultrasound plus AFP, but not ultrasound alone, were associated with early stage HCC detection and curative treatment. Compared with ultrasound alone, CT/MRI and ultrasound plus AFP were associated with increased early stage detection. CONCLUSIONS: HCC surveillance patterns vary in clinical practice and are associated with differing clinical outcomes. While awaiting data to determine if CT or MRI surveillance can be performed in a cost-effective manner in selected patients, AFP has a complementary role to ultrasound-based surveillance, supporting its adoption in practice guidelines.

Single-cell chromatin accessibility reveals principles of regulatory variation.

Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'.

A Positive Cocaine Urine Toxicology Test and the Effect on Intraoperative Hemodynamics Under General Anesthesia.

BACKGROUND: Cocaine has a short biological half-life, but inactive urine metabolites may be detectable for a week following use. It is unclear if patients who test positive for cocaine but have a normal electrocardiogram and vital signs have a greater percentage of hemodynamic events intraoperatively. METHODS: A total of 328 patients with a history of cocaine use who were scheduled for elective noncardiac surgery under general anesthesia were enrolled. Patients were categorized into cocaine-positive versus cocaine-negative groups based on the results of their urine cocaine toxicology test. The primary aim of this study was to evaluate whether asymptomatic cocaine-positive patients had similar percentages of intraoperative hemodynamic events, defined as (1) a mean arterial blood pressure (MAP) of <65 or >105 mm Hg and (2) a heart rate (HR) of <50 or >100 beats per minute (bpm) compared to cocaine-negative patients. The study was powered to assess if the 2 groups had an equivalent mean percent of intraoperative hemodynamic events within specific limits using an equivalence test of means consisting of 2 one-sided tests. RESULTS: The cocaine-positive group had a blood pressure (BP) that was outside the set limits 19.4% (standard deviation [SD] 17.7%) of the time versus 23.1% (SD 17.7%) in the cocaine-negative group (95% confidence interval [CI], 0.5-7.0). The cocaine-positive group had a HR outside the set limits 9.6% (SD 16.2%) of the time versus 8.2% (SD 14.9%) in the cocaine-negative group (95% CI, 4.3-1.5). Adjusted for age, sex, body mass index (BMI), smoking status, and the presence of comorbid hypertension, renal disease, and psychiatric illness, the cocaine-positive and cocaine-negative patients were similar within a 7.5% margin of equivalence for MAP data (ß coefficient = 2%, P = .003, CI, 2-6) and within a 5% margin of equivalence for HR data (ß coefficient = 0.2%, P < .001, CI, 4-3). CONCLUSIONS: Asymptomatic cocaine-positive patients undergoing elective noncardiac surgery under general anesthesia have similar percentages of intraoperative hemodynamic events compared to cocaine-negative patients.

Clinical features as predictors of histologically confirmed inflammation in patients with lumbar disc herniation with associated radiculopathy.

BACKGROUND: An understanding of the clinical features of inflammation in low back pain with or without leg symptoms may allow targeted evaluations of anti-inflammatory treatment in randomised-controlled-trials and clinical practice. PURPOSE: This study evaluated the diagnostic accuracy of clinical features to predict the presence/absence of histologically confirmed inflammation in herniated disc specimens removed at surgery in patients with lumbar disc herniation and associated radiculopathy (DHR). STUDY DESIGN: Cohort Study. METHODS: Disc material from patients with DHR undergoing lumbar discectomy was sampled and underwent histological/immunohistochemistry analyses. Control discs were sampled from patients undergoing surgical correction for scoliosis. Baseline assessment comprising sociodemographic factors, subjective examination, physical examination and psychosocial screening was conducted and a range of potential clinical predictors of inflammation developed based on the existing literature. Multi-variate analysis was undertaken to determine diagnostic accuracy. RESULTS: Forty patients with DHR and three control patients were recruited. None of the control discs had evidence of inflammation compared to 28% of patients with DHR. Predictors of the presence of histologically confirmed inflammation included back pain < 5/10, symptoms worse the next day after injury, lumbar flexion range between 0 and 30° and a positive clinical inflammation score (at least 3 of: constant symptoms, morning pain/stiffness greater than 60-min, short walking not easing symptoms and significant night symptoms). The model achieved a sensitivity of 90.9%, a specificity of 92.9%, and a predictive accuracy of 92.3%. CONCLUSION: In a sample of patients with lumbar DHR a combination of clinical features predicted the presence or absence of histologically confirmed inflammation. CLINICAL RELEVANCE: These clinical features may enable targeted anti-inflammatory treatment in future RCTs and in clinical practice.

Rituximab in patients with primary CNS lymphoma (HOVON 105/ALLG NHL 24): a randomised, open-label, phase 3 intergroup study.

BACKGROUND: The prognosis for primary CNS lymphoma has improved with the use of high-dose methotrexate-based chemotherapy, but patient outcomes remain poor. Rituximab, a chimeric monoclonal antibody that targets the CD20 cell surface protein, has substantial activity in systemic CD20-positive diffuse large B-cell lymphoma, but its efficacy in primary CNS lymphoma is unknown and low penetration of the large rituximab molecule through the blood-brain barrier could limit its effect. We aimed to investigate the addition of rituximab to a high-dose methotrexate-based chemotherapy regimen in patients with newly diagnosed primary CNS lymphoma. METHODS: This intergroup, multicentre, open-label, randomised phase 3 study was done at 23 hospitals in the Netherlands, Australia, and New Zealand. Non-immunocompromised patients aged 18-70 years with newly diagnosed primary CNS lymphoma were randomly assigned (1:1) to receive methotrexate-based chemotherapy with or without intravenous rituximab. We used a web-based randomisation system with stratification by centre, age, and Eastern Cooperative Oncology Group-WHO performance status, and a minimisation procedure. All group assignment was open label and neither investigators nor patients were masked to allocation. All patients were treated with two 28-day cycles of induction chemotherapy, consisting of intravenous methotrexate 3 g per m2 on days 1 and 15, intravenous carmustine 100 mg per m2 on day 4, intravenous teniposide 100 mg per m2 on days 2 and 3, and oral prednisone 60 mg per m2 on days 1-5, with (R-MBVP) or without (MBVP) intravenous rituximab 375 mg per m2 on days 0, 7, 14, and 21 in cycle one and days 0 and 14 in cycle two. Patients with response at the end of induction subsequently received high-dose cytarabine and, in patients aged 60 years or younger, low-dose whole-brain radiotherapy. The primary endpoint was event-free survival, with events defined as not reaching complete response or complete response unconfirmed at the end of treatment, or progression or death after response; analysis was adjusted for age and performance score. Patients were analysed on a modified intention-to-treat basis. This trial is registered with the Nederlands Trial Register, number NTR2427, and the Australian New Zealand Clinical Trials Registry, number ACTRN12610000908033. The trial was closed on May 27, 2016, after achieving complete accrual, and follow-up is ongoing. FINDINGS: Between Aug 3, 2010, and May 27, 2016, we recruited 200 patients (109 men and 91 women; median age was 61 years [IQR 55-67]). We randomly assigned 100 patients to MBVP and 99 patients to R-MBVP. One patient was randomly assigned to the R-MBVP group but found to be ineligible because of an incorrect diagnosis and was excluded from all analyses. After a median follow-up of 32·9 months (IQR 23·9-51·5), 98 patients had had an event (51 in the MBVP group and 47 in the R-MBVP group), of whom 79 had died (41 in the MBVP group and 38 in the R-MBVP group). Event-free survival at 1 year was 49% (95% CI 39-58) in the MBVP group (no rituximab) and 52% (42-61) in the R-MBVP group (with rituximab; hazard ratio 1·00, 95% CI 0·70-1·43, p=0·99). Grade 3 or 4 adverse events occurred in 58 (58%) patients in the MBVP group and 63 (64%) patients in the R-MBVP group, with infections (24 [24%] patients receiving MBVP vs 21 [21%] patients receiving R-MBVP), haematological toxicity (15 [15%] vs 12 [12%]), and nervous system disorders (ten [10%] vs 15 [15%]) being the most common. Life-threatening or fatal serious adverse events occurred in 12 (12%) patients in the MBVP group and ten (10%) patients in the R-MBVP group, and five (5%) patients in the MBVP group and three (3%) in the R-MBVP group died from treatment-related causes. INTERPRETATION: We found no clear benefit of addition of rituximab to methotrexate, carmustine, teniposide, and prednisone chemotherapy in primary CNS lymphoma. Therefore, the results of this study do not support the use of rituximab as a component of standard treatment in primary CNS lymphoma. FUNDING: Roche, the Dutch Cancer Society, and Stichting STOPhersentumoren.

The influence of morbid obesity on difficult intubation and difficult mask ventilation.

PURPOSE: To determine the influence of morbid obesity on the incidence of difficult mask ventilation and difficult intubation. METHODS: Over a 6-year period, all tracheal intubations in the operating room of a large tertiary teaching hospital were analyzed. A modified version of the intubation difficulty scale (mIDS) was used to define easy versus difficult intubation, where a score of two or greater was defined as difficult intubation. Difficult mask ventilation was defined as the use of one or more adjuncts to achieve successful mask ventilation. RESULTS: Of 45,447 analyzed cases, 1893 (4.2%) were classified as difficult intubations. Morbidly obese patients were not more likely to have difficult intubation [Odds Ratio (OR) = 1.131, 95% confidence interval (CI): 0.958, 1.334, p = 0.146]. Factors that were associated with difficult intubation included patient age > 46 years, male sex, Mallampati 3-4, thyromental distance < 6 cm, and the presence of intact dentition. Of 37,016 cases in which mask ventilation was attempted, 1069 (2.9%) were difficult. Morbidly obese patients were more likely to have difficult mask ventilation (OR = 3.785, 95% CI: 3.188, 4.493, p < 0.0001). Other factors associated with difficult mask ventilation included patient age > 46 years, male sex, Mallampati 3-4, and a history of obstructive sleep apnea. Having intact dentition decreased the likelihood of difficult mask ventilation. CONCLUSION: Morbidly obese patients do not have a higher incidence of difficult intubation compared to non-morbidly obese patients. However, they have a significantly higher incidence of difficult mask ventilation. Other factors that are predictive of both difficult mask ventilation and difficult intubation include age > 46 years, male sex, and Mallampati 3-4.

The 2016 revision of the WHO Classification of Central Nervous System Tumours: retrospective application to a cohort of diffuse gliomas.

The classification of central nervous system tumours has more recently been shaped by a focus on molecular pathology rather than histopathology. We re-classified 82 glial tumours according to the molecular-genetic criteria of the 2016 revision of the World Health Organization (WHO) Classification of Tumours of the Central Nervous System. Initial diagnoses and grading were based on the morphological criteria of the 2007 WHO scheme. Because of the impression of an oligodendroglial component on initial histological assessment, each tumour was tested for co-deletion of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH-1 and 2) genes. Additionally, expression of proteins encoded by alpha-thalassemia X-linked mental retardation (ATRX) and TP53 genes was assessed by immunohistochemistry. We found that all but two tumours could be assigned to a specific category in the 2016 revision. The most common change in diagnosis was from oligoastrocytoma to specifically astrocytoma or oligodendroglioma. Analysis of progression free survival (PFS) for WHO grade II and III tumours showed that the objective criteria of the 2016 revision separated diffuse gliomas into three distinct molecular categories: chromosome 1p/19q co-deleted/IDH mutant, intact 1p/19q/IDH mutant and IDH wild type. No significant difference in PFS was found when comparing IDH mutant grade II and III tumours suggesting that IDH status is more informative than tumour grade. The segregation into distinct molecular sub-types that is achieved by the 2016 revision provides an objective evidence base for managing patients with grade II and III diffuse gliomas based on prognosis.

IDH1 and IDH2 mutations in postoperative diffuse glioma-associated epilepsy.

OBJECTIVE: Isocitrate dehydrogenase 1 and 2 mutations (IDH1/2) have an established association with preoperative seizures in patients with grades II-IV diffuse gliomas. Here, we examined if IDH1/2 mutations are a biomarker of postoperative seizure frequency. METHODS: This was a retrospective study. Patients with grades II-IV supratentorial diffuse glioma, immunohistochemistry results of IDH1-R132H, and antiepileptic drug (AED) prescribed postoperatively were included. The primary outcome was seizure frequency over the first 12 postoperative months: Group A - postoperative seizure freedom; Group B - 1-11 seizures over 12months (less than one seizure per month); and Group C - greater than one seizure per month. Rates of IDH1-R132H mutation were compared between the three outcome groups in univariate and multivariate analyses. Subgroup analysis was performed in 64 patients with IDH1/2 pyrosequencing data. RESULTS: One hundred cases were included in the analysis: 30.0% grade II, 20.0% grade III, and 50.0% grade IV gliomas. Group B patients averaged 1 seizure over 12months, compared with 2 seizures per month in Group C. Isocitrate dehydrogense 1-R132H mutation was present in 29.3% (17/58) of Group A, 18.2% (14/22) of Group B, and 70.0% (14/20) of Group C patients (p=0.001). On multivariate analysis, after controlling for preoperative seizure, grade, and temporal tumor location, IDH1-R132H was associated with Group C when compared with both Group A (RR 4.75, p=0.032) and Group B (RR 9.70, p=0.012). In the subgroup with IDH1/2 molecular data, an IDH1/2 mutation was present in 64.7% (22/34) of Group A, 28.6% (4/14) of Group C, and 87.5% (14/16) of Group C patients (p=0.004). SIGNIFICANCE: In patients with supratentorial diffuse gliomas, IDH1-R132H mutations are associated with a more severe phenotype of postoperative epilepsy. These findings support further research into IDH mutations, and the potential for an antiepileptic therapeutic effect of their inhibitors, in patients with glioma-associated epilepsy.

Primary orbital melanoma: a case series and literature review.

Primary orbital melanoma (POM) is a very rare condition. We report further four cases and review all previously reported cases. We present a multicentre retrospective review of patients with POM. Clinical, radiological, surgical, histological, and follow-up data is presented. Four patients with POM were identified between 2000 and 2013. All presented with proptosis and diplopia without reduced vision. Two had known pre-existing blue cell naevi. All were stage T1N0M0. All underwent exenteration with adjuvant radiotherapy. All are disease free at follow-up durations of 24-151 months. The present three cases and review of all cases in the literature suggest a higher likelihood of disease-free survival from primary exenteration (7/8 disease-free survival, 1/8 death from metastatic disease) than wide local excision (7/16 disease-free survival, 9 recurrence or metastasis of whom 4 died). Adjuvant radiotherapy may additionally improve outcomes.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

BACKGROUND: Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS: We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. CONCLUSION: As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

Synaptotagmin 2 mutations cause an autosomal-dominant form of lambert-eaton myasthenic syndrome and nonprogressive motor neuropathy.

Synaptotagmin 2 is a synaptic vesicle protein that functions as a calcium sensor for neurotransmission but has not been previously associated with human disease. Via whole-exome sequencing, we identified heterozygous missense mutations in the C2B calcium-binding domain of the gene encoding Synaptotagmin 2 in two multigenerational families presenting with peripheral motor neuron syndromes. An essential calcium-binding aspartate residue, Asp307Ala, was disrupted by a c.920A>C change in one family that presented with an autosomal-dominant presynaptic neuromuscular junction disorder resembling Lambert-Eaton myasthenic syndrome. A c.923C>T variant affecting an adjacent residue (p.Pro308Leu) produced a presynaptic neuromuscular junction defect and a dominant hereditary motor neuropathy in a second family. Characterization of the mutation homologous to the human c.920A>C variant in Drosophila Synaptotagmin revealed a dominant disruption of synaptic vesicle exocytosis using this transgenic model. These findings indicate that Synaptotagmin 2 regulates neurotransmitter release at human peripheral motor nerve terminals. In addition, mutations in the Synaptotagmin 2 C2B domain represent an important cause of presynaptic congenital myasthenic syndromes and link them with hereditary motor axonopathies.

Motor neuron degeneration in spastic paraplegia 11 mimics amyotrophic lateral sclerosis lesions.

The most common form of autosomal recessive hereditary spastic paraplegia is caused by mutations in the SPG11/KIAA1840 gene on chromosome 15q. The nature of the vast majority of SPG11 mutations found to date suggests a loss-of-function mechanism of the encoded protein, spatacsin. The SPG11 phenotype is, in most cases, characterized by a progressive spasticity with neuropathy, cognitive impairment and a thin corpus callosum on brain MRI. Full neuropathological characterization has not been reported to date despite the description of >100 SPG11 mutations. We describe here the clinical and pathological features observed in two unrelated females, members of genetically ascertained SPG11 families originating from Belgium and Italy, respectively. We confirm the presence of lesions of motor tracts in medulla oblongata and spinal cord associated with other lesions of the central nervous system. Interestingly, we report for the first time pathological hallmarks of SPG11 in neurons that include intracytoplasmic granular lysosome-like structures mainly in supratentorial areas, and others in subtentorial areas that are partially reminiscent of those observed in amyotrophic lateral sclerosis, such as ubiquitin and p62 aggregates, except that they are never labelled with anti-TDP-43 or anti-cystatin C. The neuropathological overlap with amyotrophic lateral sclerosis, associated with some shared clinical manifestations, opens up new fields of investigation in the physiopathological continuum of motor neuron degeneration.

Mice with an isoform-ablating Mecp2 exon 1 mutation recapitulate the neurologic deficits of Rett syndrome.

Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT OMIM 312750). Alternative inclusion of MECP2/Mecp2 exon 1 with exons 3 and 4 encodes MeCP2-e1 or MeCP2-e2 protein isoforms with unique amino termini. While most MECP2 mutations are located in exons 3 and 4 thus affecting both isoforms, MECP2 exon 1 mutations but not exon 2 mutations have been identified in RTT patients, suggesting that MeCP2-e1 deficiency is sufficient to cause RTT. As expected, genetic deletion of Mecp2 exons 3 and/or 4 recapitulates RTT-like neurologic defects in mice. However, Mecp2 exon 2 knockout mice have normal neurologic function. Here, a naturally occurring MECP2 exon 1 mutation is recapitulated in a mouse model by genetic engineering. A point mutation in the translational start codon of Mecp2 exon 1, transmitted through the germline, ablates MeCP2-e1 translation while preserving MeCP2-e2 production in mouse brain. The resulting MeCP2-e1 deficient mice developed forelimb stereotypy, hindlimb clasping, excessive grooming and hypo-activity prior to death between 7 and 31 weeks. MeCP2-e1 deficient mice also exhibited abnormal anxiety, sociability and ambulation. Despite MeCP2-e1 and MeCP2-e2 sharing, 96% amino acid identity, differences were identified. A fraction of phosphorylated MeCP2-e1 differed from the bulk of MeCP2 in subnuclear localization and co-factor interaction. Furthermore, MeCP2-e1 exhibited enhanced stability compared with MeCP2-e2 in neurons. Therefore, MeCP2-e1 deficient mice implicate MeCP2-e1 as the sole contributor to RTT with non-redundant functions.

A Comparative Epigenomic Analysis of Polyploidy-Derived Genes in Soybean and Common Bean.

Soybean (Glycine max) and common bean (Phaseolus vulgaris) share a paleopolyploidy (whole-genome duplication [WGD]) event, approximately 56.5 million years ago, followed by a genus Glycine-specific polyploidy, approximately 10 million years ago. Cytosine methylation is an epigenetic mark that plays an important role in the regulation of genes and transposable elements (TEs); however, the role of DNA methylation in the fate/evolution of genes following polyploidy and speciation has not been fully explored. Whole-genome bisulfite sequencing was used to produce nucleotide resolution methylomes for soybean and common bean. We found that, in soybean, CG body-methylated genes were abundant in WGD genes, which were, on average, more highly expressed than single-copy genes and had slower evolutionary rates than unmethylated genes, suggesting that WGD genes evolve more slowly than single-copy genes. CG body-methylated genes were also enriched in shared single-copy genes (single copy in both species) that may be responsible for the broad and high expression patterns of this class of genes. In addition, diverged methylation patterns in non-CG contexts between paralogs were due mostly to TEs in or near genes, suggesting a role for TEs and non-CG methylation in regulating gene expression post polyploidy. Reference methylomes for both soybean and common bean were constructed, providing resources for investigating epigenetic variation in legume crops. Also, the analysis of methylation patterns of duplicated and single-copy genes has provided insights into the functional consequences of polyploidy and epigenetic regulation in plant genomes.

R-loop formation at Snord116 mediates topotecan inhibition of Ube3a-antisense and allele-specific chromatin decondensation.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted autism-spectrum disorders with known genetic bases, but complex epigenetic mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11-q13 is regulated by an imprinting control region that is maternally methylated and silenced. The PWS imprinting control region is the promoter for a one megabase paternal transcript encoding the ubiquitous protein-coding Snrpn gene and multiple neuron-specific noncoding RNAs, including the PWS-related Snord116 repetitive locus of small nucleolar RNAs and host genes, and the antisense transcript to AS-causing ubiquitin ligase encoding Ube3a (Ube3a-ATS). Neuron-specific transcriptional progression through Ube3a-ATS correlates with paternal Ube3a silencing and chromatin decondensation. Interestingly, topoisomerase inhibitors, including topotecan, were recently identified in an unbiased drug screen for compounds that could reverse the silent paternal allele of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus is unknown. Here, we demonstrate that topotecan treatment stabilizes the formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within paternal Snord116, corresponding to increased chromatin decondensation and inhibition of Ube3a-ATS expression. Neural precursor cells from paternal Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated neurons and show a reduced effect of topotecan compared with wild-type neurons. These results demonstrate that the AS candidate drug topotecan acts predominantly through stabilizing R loops and chromatin decondensation at the paternally expressed PWS Snord116 locus. Our study holds promise for targeted therapies to the Snord116 locus for both AS and PWS.

Thrombus composition in acute ischemic stroke: a histopathological study of thrombus extracted by endovascular retrieval.

BACKGROUND AND PURPOSE: The composition of occlusive thrombus in acute ischemic stroke may affect treatment success. Neuroimaging characteristics may correlate with thrombus composition. In this study we aimed to investigate the relationship between the hyperdense artery sign (HAS) on imaging and thrombus composition. MATERIALS AND METHODS: Acute ischemic stroke patients who underwent endovascular thrombus retrieval from 2010-2012 were prospectively recruited. One blinded pathologist prepared the histology sections of retrieved thrombi whereby staining with haematoxylin and eosin and CD34 immunostain were performed. Histology sections were categorised into 4 phases of thrombus formation: red blood cell (RBC) dominant, RBC proportion equal to fibrin, fibrin dominant and organised fibrin. Computed tomography (CT) brain scans were assessed for HAS. Fisher's exact test was performed to identify an association between HAS and thrombus composition. RESULTS: Forty patients were included. The mean age was 65.6±12.9 years and 67.5% were male. Atrial fibrillation was detected in 19 (47.5%) patients, diabetes mellitus in 6 (15.0%), hypercholesterolaemia in 11 (27.5%), hypertension in 20 (50.0%) and previous stroke or transient ischemic attack in 8 (20.0%) patients. Of the retrieved thrombi, 11 (27.5%) were RBC dominant, 11 (27.5%) RBC proportion was equal to fibrin, 7 (17.5%) fibrin dominant and 11 (27.5%) organised fibrin pathology. HAS was present in 29 (72.5%) patients and was significantly associated with thrombus early phase pathology (P<0.05). CONCLUSION: HAS was significantly associated with early phase thrombus composition. This may enable the prediction of thrombus composition and allow for targeted selection of therapeutic modality.

IDH1 mutation is associated with seizures and protoplasmic subtype in patients with low-grade gliomas.

OBJECTIVE: The isocitrate dehydrogenase 1 (IDH1) R132H mutation is the most common mutation in World Health Organization (WHO) grade II gliomas, reported to be expressed in 70-80%, but only 5-10% of high grade gliomas. Low grade tumors, especially the protoplasmic subtype, have the highest incidence of tumor associated epilepsy (TAE). The IDH1 mutation leads to the accumulation of 2-hydroxyglutarate (2HG), a metabolite that bears a close structural similarity to glutamate, an excitatory neurotransmitter that has been implicated in the pathogenesis of TAE. We hypothesized that expression of mutated IDH1 may play a role in the pathogenesis of TAE in low grade gliomas. METHODS: Thirty consecutive patients with WHO grade II gliomas were analyzed for the presence of the IDH1-R132H mutation using immunohistochemistry. The expression of IDH1 mutation was semiquantified using open-source biologic-imaging analysis software. RESULTS: The percentage of cells positive for the IDH1-R132H mutation was found to be higher in patients with TAE compared to those without TAE (median and interquartile range (IQR) 25.3% [8.6-53.5] vs. 5.2% [0.6-13.4], p = 0.03). In addition, we found a significantly higher median IDH1 mutation expression level in the protoplasmic subtype of low grade glioma (52.2% [IQR 19.9-58.6] vs. 13.8% [IQR 3.9-29.4], p = 0.04). SIGNIFICANCE: Increased expression of the IDH1-R132H mutation is associated with seizures in low grade gliomas and also with the protoplasmic subtype. This supports the hypothesis that this mutation may play a role in the pathogenesis of both TAE and low grade gliomas.

A PtdIns4,5P2-regulated nuclear poly(A) polymerase controls expression of select mRNAs.

Phosphoinositides are a family of lipid signalling molecules that regulate many cellular functions in eukaryotes. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2), the central component in the phosphoinositide signalling circuitry, is generated primarily by type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIalpha, PIPKIbeta and PIPKIgamma). In addition to functions in the cytosol, phosphoinositides are present in the nucleus, where they modulate several functions; however, the mechanism by which they directly regulate nuclear functions remains unknown. PIPKIs regulate cellular functions through interactions with protein partners, often PtdIns4,5P2 effectors, that target PIPKIs to discrete subcellular compartments, resulting in the spatial and temporal generation of PtdIns4,5P2 required for the regulation of specific signalling pathways. Therefore, to determine roles for nuclear PtdIns4,5P2 we set out to identify proteins that interacted with the nuclear PIPK, PIPKIalpha. Here we show that PIPKIalpha co-localizes at nuclear speckles and interacts with a newly identified non-canonical poly(A) polymerase, which we have termed Star-PAP (nuclear speckle targeted PIPKIalpha regulated-poly(A) polymerase) and that the activity of Star-PAP can be specifically regulated by PtdIns4,5P2. Star-PAP and PIPKIalpha function together in a complex to control the expression of select mRNAs, including the transcript encoding the key cytoprotective enzyme haem oxygenase-1 (refs 8, 9) and other oxidative stress response genes by regulating the 3'-end formation of their mRNAs. Taken together, the data demonstrate a model by which phosphoinositide signalling works in tandem with complement pathways to regulate the activity of Star-PAP and the subsequent biosynthesis of its target mRNA. The results reveal a mechanism for the integration of nuclear phosphoinositide signals and a method for regulating gene expression.

Bronchoalveolar Lavage Fluid Cytology of Deployed Military Personnel With Chronic Respiratory Symptoms From the STAMPEDE III Study.

INTRODUCTION: Deployed military personnel may be at risk for developing acute and chronic lung disease. Prior studies of this patient population have revealed that unexplained exertional dyspnea is the most common diagnosis despite an extensive evaluation. There is a concern that an occult disorder may be affecting this population. This study evaluated the role for bronchoalveolar lavage (BAL) fluid analysis in the evaluation of chronic deployment-associated dyspnea. MATERIALS AND METHODS: Military personnel who reported chronic respiratory symptoms were evaluated as part of the Study of Active Duty Military for Pulmonary Disease Related to Environmental Deployment Exposures III study. Participants underwent bronchoscopy with BAL as part of a standardized evaluation. RESULTS: A total of 308 patients with a mean age of 38 ± 8.6 years underwent bronchoscopy with BAL. BAL cell-count percentages of macrophages, lymphocytes, neutrophils, and eosinophils were: 76.2 ± 17.0%, 16.3 ± 13.4%, 6.6 ± 8.9%, and 0.9 ± 3.2%, respectively. There was no clear differentiation between groups based on increases in lymphocyte counts (P = .640), although lymphocyte values were more elevated (21.4 ± 12.1%) in the interstitial lung disease category. Neutrophil counts (6.6 ± 8.9%) were elevated compared to the reported normal reference values and were increased in the isolated pulmonary function test abnormality (9.4 ± 11.6%), large airway disorder (10.0 ± 7.5%), miscellaneous (10.9 ± 20.2%), and obstructive lung disease (11.0 ± 15.6%) groups. Eosinophil counts were within normal limits (0.9 ± 3.2%) and showed no differences between groups (P = .545); asthma patients trended higher (1.6 ± 5.7%). BAL counts for the exertional dyspnea group were within normal reference values and showed no differences from the entire cohort. CONCLUSIONS: The addition of BAL cytology did not help differentiate those patients with unexplained dyspnea from other etiologies.