RLSuite: An Integrative R-Loop Bioinformatics Framework.

We recently described the development of a database of 810 R-loop mapping datasets and used this data to conduct a meta-analysis of R-loops. R-loops are three-stranded nucleic acid structures containing RNA:DNA hybrids and we were able to verify that 30% of expressed genes have an associated R-loop in a location conserved manner.. Moreover, intergenic R-loops map to enhancers, super enhancers and with TAD domain boundaries. This work demonstrated that R-loop mapping via high-throughput sequencing can reveal novel insight into R-loop biology, however the analysis and quality control of these data is a non-trivial task for which few bioinformatic tools exist. Herein we describe RLSuite, an integrative R-loop bioinformatics framework for pre-processing, quality control, and downstream analysis of R-loop mapping data. RLSuite enables users to compare their data to hundreds of public datasets and generate a user-friendly analysis report for sharing with non-bioinformatician colleagues. Taken together, RLSuite is a novel analysis framework that should greatly benefit the emerging R-loop bioinformatics community in a rapidly expanding aspect of epigenetic control that is still poorly understood.

Quantifying biomarkers of axonal degeneration in early glaucoma to find the disc at risk.

To evaluate regional axonal-related parameters as a function of disease stage in primary open angle glaucoma (POAG) and visual field (VF) sensitivity. Spectral domain optical coherence tomography was used to acquire 20° scans of POAG (n = 117) or healthy control (n = 52) human optic nerve heads (ONHs). Region specific and mean nerve fibre layer (NFL) thicknesses, border NFL and peripapillary NFL, minimum rim width (MRW)/ area (MRA) and prelamina thickness; and volume were compared across POAG disease stages and with visual field sensitivity. Differences identified between early glaucoma (EG), preperimetric glaucoma (PG) and control (C) ONHs included thinner PG prelamina regions than in controls (p < 0.05). Mean border NFL was thinner in EG (p < 0.001) and PG (p = 0.049) compared to control eyes; and EG mean, and inferior and ST, border NFL was thinner than in PG (p < 0.01). Mean, superior and inferior PG peripapillary NFL were thinner than in controls (p < 0.05), and EG ST peripapillary NFL was thinner than in PG (p = 0.023). MRW differences included: PG SN and inferior less than in controls (p < 0.05); thinner EG mean regional, inferior, nasal, and ST MRW versus PG MRW (p < 0.05). Regional border NFL, peripapillary NFL, MRW, MRA, prelamina thickness (except centre, p = 0.127) and prelamina volume (p < 0.05) were significantly associated with VF mean deviation (MD). Novel axon-derived indices hold potential as biomarkers to detect early glaucoma and identify ONHs at risk.

A randomized controlled trial adding fluvastatin to peginterferon and ribavirin for naïve genotype 1 hepatitis C patients.

Fluvastatin or simvastatin has demonstrable antiviral activity against hepatitis C virus (HCV) as monotherapy. The safety and efficacy of adding fluvastatin or simvastatin to peginterferon/ribavirin for 48 weeks was tested in HCV genotype 1 naïve-to-treatment veterans. Thirty-seven naïve-to-treatment genotype 1 HCV patients were randomized to either a control group (n = 20) to receive peginterferon alfa plus ribavirin or an experimental group (n = 18) to similarly receive peginterferon alfa plus ribavirin as well as fluvastatin 20 mg/day. In addition, seven patients who presented for HCV treatment already were on simvastatin and could not be withdrawn. These simvastatin users were not randomized but were entered into a concurrent prospective pilot arm. There were no unique safety issues with fluvastatin or simvastatin when these drugs were given with peginterferon/ribavirin for 48 weeks. Thirteen of 25 statin patients achieved sustained viral response (SVR), while 5 of 20 control patients achieved SVR. Analysis of SVR by intention-to-treat showed P = 0.078. In this phase 2 study, there were no safety issues with the addition of fluvastatin or simvastatin to peginterferon and ribavirin for 48 weeks. There was a trend towards improvement in SVR when fluvastatin or simvastatin was administered with peginterferon/ribavirin. The size of the groups did not reach the prestudy size thought needed to show significant difference (type II error). These results support the significant results of two other larger randomized controlled trials reported using the same dose of fluvastatin in naïve-to-treatment genotype 1 HCV patients.

Myth: necrotizing enterocolitis: probiotics will end the disease, and surgical intervention improves the outcome.

Necrotizing enterocolitis remains an important contributor to neonatal morbidity and mortality. Recent studies suggest that probiotic supplementation may reduce the risk of the disease in premature infants, and some authors recommend that this approach is ready to be utilized as standard-of-care. Once necrotizing enterocolitis is diagnosed and progresses toward peritonitis or perforation, surgical intervention is thought to improve the outcome, and investigators have suggested that peritoneal drainage is as effective as an exploratory laparotomy. In this chapter, we review the current state of knowledge, and suggest that additional studies are necessary to confirm that probiotics will end this disease, and that surgical intervention may not significantly improve the outcome after diagnosis in these compromised patients.

Use of intrathecal chemoprophylaxis in children after SCT and the risk of central nervous system relapse.

Conflicting conclusions can be drawn from the available data concerning antileukemic efficacy and risks of intrathecal (i.t.) chemoprophylaxis to children after hematopoietic SCT (HSCT). To address this, we enrolled six transplantation centers with similar treatment and patient material. Of the 397 children included, 136 patients had received post-HSCT i.t. treatment (i.t. group) and 261 had not (non-i.t. group). The two groups were, apart from the i.t. therapy given or not given, at equal risk of post-HSCT central nervous system (CNS) relapse, which was the primary endpoint studied. Isolated CNS relapses were observed in 2 (1.5%) patients from the i.t. group and 2 (1%) from the non-i.t. group. Combined relapses, including CNS, involved 4 (3%) patients from the i.t. group and 6 (2%) from the non-i.t. group. Overall survival and the occurrence of neurological side effects did not differ significantly between the groups. There was no statistically significant difference in the incidence of isolated or mixed CNS relapses between the two groups, suggesting little or no benefit from i.t. therapy post-HSCT in children.

Prosthetic repair of prevascular femoral herniation.

INTRODUCTION: In prevascular femoral hernias the sac is situated anterior to the femoral vessels in the groin. These hernias are rare and a preoperative diagnosis is seldom considered. Traditionally the surgeon had to do the best with the situation that confronted him/her. The aim of this study was to evaluate the use of a prosthetic mesh-plug via a low anterior groin approach. METHODS AND RESULTS: Over a nine-year period, six patients (all men, aged 46-82 year) were treated for prevascular herniation using a mesh-plug. Five patients had previous groin surgery. Follow-up ranged from 15 months to nine years, during which no recurrence, infection or vascular compromise was detected. DISCUSSION: Prevascular herniation is rare and the diagnosis may be difficult. Our results suggest that mesh-plug repair via a low approach is feasible and safe and may provide another option for a surgeon encountering a prevascular hernia.

Temporal artery biopsy...who needs one?

OBJECTIVE: To review a 10 year period of temporal artery biopsies, using the American College of Rheumatology (ACR) 1990 criteria: a five point scoring system for the diagnosis of giant cell arteritis (GCA). DESIGN: Population based, retrospective cohort analysis. SETTING: One district general hospital in the United Kingdom, over one decade. PARTICIPANTS: All patients who underwent temporal artery biopsy from July 1994 to June 2004. MAIN OUTCOME MEASURES: ACR score and temporal artery biopsy result. RESULTS: During the 10 year period 111 patients were identified. The median (range) age at presentation was 71 (29-85) years. Seventy five patients had an initial ACR score of three or four at presentation. There were 20 positive biopsy specimens. In 19 of these cases at least three of the other criteria were positive so there was already sufficient clinical information for a confident diagnosis. In only one case did the positive result influence the diagnosis by changing the ACR score from two to three. In our series, corticosteroid treatment before biopsy did not significantly reduce the yield of the biopsy. CONCLUSIONS: The ACR score of three or more has a sensitivity of 93.5% and specificity of 91.2% for the diagnosis of GCA. Using these criteria, 68% of patients had sufficient clinical features when referred to make a confident diagnosis of GCA. Temporal artery biopsy was therefore unnecessary in this group. In the remaining group (ACR score < or =2) there was one positive biopsy. The biopsy only changed the diagnosis in this one case-less than 3% of the uncertain cases and less than 1% of the total cases. Using the ACR criteria and restricting biopsy to those cases in which it might change the diagnosis will reduce the number of biopsies by two thirds without jeopardising diagnostic accuracy.

Cellular drug sensitivity in MLL-rearranged childhood acute leukaemia is correlated to partner genes and cell lineage.

Rearrangements in the 11q23 region, the site of the mixed lineage leukaemia (MLL) gene, are found in both childhood acute myeloid (AML) and lymphoblastic (ALL) leukaemia. We studied the in vitro drug resistance by the fluorometric microculture cytotoxicity assay (FMCA) in 132 children with AML and 178 children with ALL (aged 0-17 years). In AML, children with t(9;11) (n = 10) were significantly more sensitive to cytarabine (P < 0.001) and doxorubicin (P = 0.005) than non-11q23 rearranged patients (n = 108). Children with other 11q23 rearrangements (n = 14) differed less from non-rearranged children. The 'AML-profile' common to all three groups included relative resistance to glucocorticoids and vincristine. In ALL, children with 11q23 rearrangement (n = 22) were significantly more sensitive to cytarabine (P = 0.026) than children without 11q23 rearrangement (n = 156), also after stratification for white blood cell count. In conclusion, the findings indicate that the cellular drug resistance is correlated to both the cell lineage and the type of 11q23 rearrangement. High cellular sensitivity to cytarabine and doxorubicin might explain the excellent treatment results in children with AML and t(9;11). The present study supports the strategy of contemporary protocols to include high-dose cytarabine in the treatment of 11q23-positive patients both in AML and ALL.

Study of the field around magnetic force microscopy probes using electron holography.

We have used electron holography to perform quantitative investigations of the leakage flux of thin film tips used as probes in magnetic force microscopy. A method to deduce an arrangement of magnetic domains in a thin magnetic whisker from the knowledge of the stray flux is also described. A simple analytical model of the magnetic properties of the probes allows the extraction of computer images, which simulate the experimental results satisfactory. The reliability of the recorded experimental maps of the magnetic flux arising from these kinds of sensors allows an evaluation of the total flux affecting the sample and the calculation of the magnetic field profile along the tip axis.

Vincristine in childhood leukaemia: no pharmacokinetic rationale for dose reduction in adolescents.

AIM: To investigate whether there is any pharmacokinetic rationale for the common practice of administering vincristine to adolescents at relatively lower doses than those to younger children. METHODS: A total of 98 children, aged 1.3-17.3 y, with acute lymphoblastic leukaemia (ALL) were studied on day 1 of induction therapy. Plasma samples were drawn before and 10, 30, 360 and 1380 min after injection of vincristine 2.0 mg/m2 (maximum dose 2.0 mg) and analysed by high-performance liquid chromatography. RESULTS: The median value (and range) for distribution half-life was 6.4 min (0.8-11.8), elimination half-life 1014 min (258-2570), volume of distribution 445 L/m2 (137-1241) and total body clearance 362 ml/min/m2 (134-2553). No correlation was found between age and any of these pharmacokinetic parameters. The area under the concentration time curve (AUC) was significantly correlated to age (p = 0.002; p - 0.31), as expected from the dosage of vincristine. The lower AUC in children with a body surface area > 1 m2, which is reached at 8-9 y of age, indicates that they received a less intense treatment because of the capping of the vincristine dose at 2.0 mg. CONCLUSIONS: Vincristine pharmacokinetics were not age dependent in this paediatric population. Thus, we found no pharmacokinetic rationale for dose reduction in adolescents. The common practice of limiting the vincristine dose to 2.0 mg should be carefully reconsidered.

Deletion of the Ink4-locus (the p16ink4a, p14ARF and p15ink4b genes) predicts relapse in children with ALL treated according to the Nordic protocols NOPHO-86 and NOPHO-92.

Inactivation of the Ink4 gene locus locus on 9p comprising the tumour suppressor gene p16ink4a and its neighbours p14ARF and p15ink4b is common in childhood acute lymphoblastic leukaemia (ALL), but the prognostic significance is controversial. DNA from 230 patients was retrospectively analysed by Southern blotting, single strand conformation polymorphism (SSCP) and sequencing techniques. The results were correlated with clinical characteristics and outcome. One hundred and ninety-four fully analysed patients, similarly treated using the Nordic NOPHO-86 or the current NOPHO-92 protocols, were included in the outcome analysis. Deletions approached a minimally deleted region between the p16ink4a and p15ink4b genes, making the p14ARF gene the most commonly deleted coding sequence. Bi-allelic deletion was associated with high white blood cell count (WBC) (P < 0.001), T cell phenotype (P < 0.001) and mediastinal mass (P < 0.001). Patients with Ink4 locus bi-allelic deletions had an inferior pEFS (P < 0.01) and multivariate analysis indicated that bi-allelic deletion of the p16ink4a and the p14ARF genes was an independent prognostic risk factor (P < 0.05). Sub-group analysis revealed a pronounced impact of deletion status for high-risk patients, ie with high WBC. Deletion-status and clinical risk criteria (WBC) could thus be combined to further differentiate risk within the high-risk group. The analysis of the Ink4 locus adds independent prognostic information in childhood ALL treated by Nordic protocols and may help in selection of patients for alternative treatment.

In vitro activity of the novel cytotoxic agent CHS 828 in childhood acute leukemia.

CHS 828, a pyridyl cyanoguanidine, is a new drug candidate now in phase I/II trials, that has shown promising anticancer activity in experimental tumor models and primary cultures of cancer cells from patients. In this study the fluorometric microculture cytotoxicity assay was used for evaluation of CHS 828 in primary cell cultures from children with acute leukemia. The activity of and interaction with the standard drugs, doxorubicin, melphalan, etoposide and cytosine arabinoside (Ara-C), were also assessed. Samples from 65 patients, 42 with acute lymphocytic leukemia (ALL) and 23 with acute myelocytic leukemia (AML) were tested with 72-h continuous drug exposure. There was 50% cell kill at very low CHS 828 concentrations; median IC50 was 0.01 microM in ALL and 0.03 in AML samples (NS) with large interindividual variability in both groups. ALL samples were significantly more sensitive than AML samples to melphalan, doxorubicin and etoposide, but not to Ara-C. In AML samples, combinations between CHS 828 and each of the four standard drugs resulted in significantly lower cell survival than either drug alone. This was also observed in ALL samples, except for Ara-C. Using the additive interaction model, CHS 828 showed a synergistic effect with melphalan in 67%, doxorubicin in 47%, etoposide in 38% and Ara-C in 14% of AML samples. In most ALL samples subadditive effects were found. Further exploration of CHS 828 in childhood leukemia is warranted, especially in AML.

Pharmacokinetics of doxorubicin in children with acute lymphoblastic leukemia: multi-institutional collaborative study.

BACKGROUND: In adults, it has been shown that the pharmacokinetics of doxorubicin are highly variable, despite standardization of the dose based on body surface area (BSA). The purpose of this study was to determine the plasma concentrations of doxorubicin and its active metabolite doxorubicinol in children treated for acute lymphoblastic leukemia (ALL). PROCEDURE: Children, 107 in number, aged 1.3-17.3 years, were studied at Day 1 of induction therapy according to the current Nordic protocol. Five infants, 3-9 months old, were also included. Plasma samples were drawn 23 hr after the start of a 24-hr infusion of doxorubicin 40 mg/m(2), and analyzed by reversed-phase liquid chromatography. RESULTS: There was a more than 10-fold difference between patients in dose normalized plasma concentration of doxorubicin, median 62.8 ng/ml, range 22.6-334 ng/ml. The doxorubicin concentrations differed significantly between age groups (P = 0.003). Children aged 4-6 years had the highest doxorubicin concentrations, median 77.9 ng/ml, followed by 2-4-year-old children, median 64.3 ng/ml. Both younger and older children had median values of about 50 ng/ml. Patients with white blood cell (WBC) count > 50 x 10(9)/L at diagnosis had significantly lower doxorubicin concentrations, median 55.3 ng/ml, than those with WBC count < 10 x 10(9)/L, median 64.4 ng/ml (P = 0.015). There was no difference in doxorubicin concentration between boys and girls. No correlation was found between doxorubicin levels and serum aminotransferases or serum creatinine. The concentration of doxorubicinol was 13% (median value) of that of doxorubicin. Four infants, 7-9 months old, had plasma clearance between 350-431 ml/min/m(2), which is in the same range as in older children. A 3-month-old infant had a clearance of 181 ml/min/m(2). CONCLUSIONS: The age groups who had the highest doxorubicin concentrations, (2-) 4-6-year-old children, are known to make up a large proportion of standard risk ALL cases with good prognosis. The correlation between doxorubicin plasma levels and clinical effect needs further study. The influence of age, body composition, and tumor burden on the pharmacokinetics of antineoplastic drugs should also be further explored, aiming at improvements in the current dosing regimen based on BSA.

Variations in the estimation of the contribution of environmental tobacco smoke (ETS) to respirable (< or = 5 microns) indoor air particulates obtained by the use of different analytical methods.

Several methods are in use for the identification of the contribution of particulate associated environmental tobacco smoke (ETS) and sidestream smoke to an atmosphere. These include the measurement of respirable suspended particulates (RSP), measurements of the total UV absorption and total fluorescence emission of a methanol extract of collected particulates and the use of specific marker compounds such as solanesol and scopoletin. Use of these methods gave values for the contribution of particulate ETS to total respirable (< or = 5 microns) particulates in the ranges 8.3-124.7% for smokers' houses and 9.6-121.2% for smokers' offices, respectively. However, using what we consider to be the most reliable methodology, based on the measurement of solanesol, the average contribution of particulate ETS to total respirable (< or = 5 microns) particulates for smokers' houses was 21.7% and for smokers' offices was 23.3%.