Separation of small metabolites and lipids in spectra from biopsies by diffusion-weighted HR-MAS NMR: a feasibility study.

High Resolution Magic Angle Spinning (HR-MAS) NMR allows metabolic characterization of biopsies. HR-MAS spectra from tissues of most organs show strong lipid contributions that are overlapping metabolite regions, which hamper metabolite estimation. Metabolite quantification and analysis would benefit from a separation of lipids and small metabolites. Generally, a relaxation filter is used to reduce lipid contributions. However, the strong relaxation filter required to eliminate most of the lipids also reduces the signals for small metabolites. The aim of our study was therefore to investigate different diffusion editing techniques in order to employ diffusion differences for separating lipid and small metabolite contributions in the spectra from different organs for unbiased metabonomic analysis. Thus, 1D and 2D diffusion measurements were performed, and pure lipid spectra that were obtained at strong diffusion weighting (DW) were subtracted from those obtained at low DW, which include both small metabolites and lipids. This subtraction yielded almost lipid free small metabolite spectra from muscle tissue. Further improved separation was obtained by combining a 1D diffusion sequence with a T2-filter, with the subtraction method eliminating residual lipids from the spectra. Similar results obtained for biopsies of different organs suggest that this method is applicable in various tissue types. The elimination of lipids from HR-MAS spectra and the resulting less biased assessment of small metabolites have potential to remove ambiguities in the interpretation of metabonomic results. This is demonstrated in a reproducibility study on biopsies from human muscle.

Transglutaminase 1-deficient recessive lamellar ichthyosis associated with a LINE-1 insertion in Jack Russell terrier dogs.

BACKGROUND: Congenital, nonepidermolytic cornification disorders phenotypically resembling human autosomal recessive ichthyosis have been described in purebred dog breeds, including Jack Russell terrier (JRT) dogs. One cause of gene mutation important to humans and dogs is transposon insertions. OBJECTIVES: To describe an autosomal recessive, severe nonepidermolytic ichthyosis resembling lamellar ichthyosis (LI) in JRT dogs due to insertion of a long interspersed nucleotide element (LINE-1) in the transglutaminase 1 (TGM1) gene. METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Phenotypic information and genotyping with a canine microsatellite marker suggested TGM1 to be a candidate gene. Genomic DNA samples and cDNA generated from epidermal RNA were examined. Consequences of the mutation were evaluated by Western blotting, quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme activity from cultured keratinocytes. RESULTS: Affected dogs had generalized severe hyperkeratosis. Histological examination defined laminated to compact hyperkeratosis without epidermolysis; ultrastructurally, cornified envelopes were thin. Affected dogs were homozygous for a 1980-bp insertion within intron 9 of TGM1. The sequence of the insertion was that of a canine LINE-1 element. Quantitative RT-PCR indicated a significant decrease in TGM1 mRNA in affected dogs compared with wild-type. TGM1 protein was markedly decreased on immunoblotting, and membrane-associated enzyme activity was diminished in affected dogs. CONCLUSIONS: Based on morphological and molecular features, this disease is homologous with TGM1-deficient LI in humans, clinically models LI better than the genetically modified mouse and represents its first spontaneous animal model. This is the first reported form of LI due to transposon insertion.

Circadian clock-coordinated 12 Hr period rhythmic activation of the IRE1alpha pathway controls lipid metabolism in mouse liver.

The mammalian circadian clock plays a fundamental role in the liver by regulating fatty acid, glucose, and xenobiotic metabolism. Impairment of this rhythm has been shown to lead to diverse pathologies, including metabolic syndrome. Currently, it is supposed that the circadian clock regulates metabolism mostly by regulating expression of liver enzymes at the transcriptional level. Here, we show that the circadian clock also controls hepatic metabolism by synchronizing a secondary 12 hr period rhythm characterized by rhythmic activation of the IRE1alpha pathway in the endoplasmic reticulum. The absence of circadian clock perturbs this secondary clock and provokes deregulation of endoplasmic reticulum-localized enzymes. This leads to impaired lipid metabolism, resulting in aberrant activation of the sterol-regulated SREBP transcription factors. The resulting aberrant circadian lipid metabolism in mice devoid of the circadian clock could be involved in the appearance of the associated metabolic syndrome.

Busulphan-melphalan is the superior myeloablative therapy (mat) for high risk neuroblastoma: results from the hr-nbl1/siopen trial

Purpose: The HR-NBL1 trial of the European SIOP Neuroblastoma Group randomised 2 MAT regimens to demonstrate superiority based on event free survival (EFS).Method: Response eligibility criteria prior to randomisation after Rapid COJEC Induction (J Clin Oncol, 2010) 3 4 2 courses of TVD (Cancer, 2003) included complete bone marrow remission andA ^ 3, but improved, mIBG positive spots. The MAT regimens were BuMel (oral busulfan till 2006, 4_150 mg/m2 in 4 equal doses, or after 2006 intravenous use according to body weight and melphalan 140 mg/m__/day) and CEM (carboplatin ctn. infusion (4xAUC 4.1 mg/ml.min/day), etoposide ctn. infusion (4_338 mg/m__day or 4_200 mg/m__/ day*), melphalan (3_70 mg/m__/day or 3_60 mg/m__/day*. *reduced if GFR<100 ml/ min/1.73m__)). A minimum of 3_10E6 CD34/kgBW PBSC were requested. VOD prophylaxis included ursadiol, but not prophylactic defibrotide. Local control included surgery and radiotherapy of 21 Gy. A total of 598 high risk neuroblastoma patients were randomised (296 BuMel, 302 CEM). The median age at randomisation was 3 years (1-17.2).Results: A significant difference in EFS in favour of BuMel (3-years EFS 49% vs. 33%) was observed as well as for overall survival (3-years OS 60% vs. 48%, p¼0.004) with a median follow up of 3 years. This difference was mainly related to the relapse and progression incidence, which was significantly (p<0.001) lower with BuMel (48% vs. 60%). The severe toxicity rate up to day 100 (ICU and toxic deaths) was below 10%, but was significantly higher for CEM (p¼0.014). The acute toxic death rate was 3% for BuMel and 5% for CEM (NS). The acute MAT toxicity profile favours the BuMel regimen in spite of a total VOD incidence of 18% (grade 3:5%). Based on these results and following advice from the DMC, the randomisation was closed early.

No evidence for mutations of CTCFL/BORIS in Silver-Russell syndrome patients with IGF2/H19 imprinting control region 1 hypomethylation.

BACKGROUND: Silver-Russell syndrome (SRS) is a genetically and clinically heterogeneous disease. Although no protein coding gene defects have been reported in SRS patients, approximately 50% of SRS patients carry epimutations (hypomethylation) at the IGF2/H19 imprinting control region 1 (ICR1). Proper methylation at ICR1 is crucial for the imprinted expression of IGF2, a fetal growth factor. CTCFL, a testis-specific protein, has recently been proposed to play a role in the establishment of DNA methylation at the murine equivalent of ICR1. A screen was undertaken to assess whether CTCFL is mutated in SRS patients with hypomethylation, to explore a link between the observed epimutations and a genetic cause of the disease. METHODOLOGY/PRINCIPAL FINDINGS: DNA was obtained from 36 SRS patients with hypomethylation at ICR1. All CTCFL coding exons were sequenced and analyzed for duplications/deletions using both multiplex ligation-dependent probe amplification, with a custom CTCFL probe set, and genomic qPCR. Novel SNP alleles were analyzed for potential differential splicing in vitro utilizing a splicing assay. Neither mutations of CTCFL nor duplications/deletions were observed. Five novel SNPs were identified and have been submitted to dbSNP. In silico splice prediction suggested one novel SNP, IVS2-66A>C, activated a cryptic splice site, resulting in aberrant splicing and premature termination. In vitro splicing assays did not confirm predicted aberrant splicing. CONCLUSIONS/SIGNIFICANCE: As no mutations were detected at CTCFL in the patients examined, we conclude that genetic alterations of CTCFL are not responsible for the SRS hypomethylation. We suggest that analysis of other genes involved in the establishment of DNA methylation at imprinted genes, such as DNMT3A and DNMT3L, may provide insight into the genetic cause of hypomethylation in SRS patients.

Randomized Trial of prophylactic granulocyte colony-stimulating factor during rapid COJEC induction in pediatric patients with high-risk neuroblastoma: the European HR-NBL1/SIOPEN study.

Purpose To reduce the incidence of febrile neutropenia during rapid COJEC (cisplatin, vincristine, carboplatin, etoposide, and cyclophosphamide given in a rapid delivery schedule) induction. In the High-Risk Neuroblastoma-1 (HR-NBL1) trial, the International Society of Paediatric Oncology European Neuroblastoma Group (SIOPEN) randomly assigned patients to primary prophylactic (PP) versus symptom-triggered granulocyte colony-stimulating factor (GCSF; filgrastim). Patients and Methods From May 2002 to November 2005, 239 patients in 16 countries were randomly assigned to receive or not receive PPGCSF. There were 144 boys with a median age of 3.1 years (range, 1 to 17 years) of whom 217 had International Neuroblastoma Staging System (INSS) stage 4 and 22 had stage 2 or 3 MYCN-amplified disease. The prophylactic arm received a single daily dose of 5 μg/kg GCSF, starting after each of the eight COJEC chemotherapy cycles and stopping 24 hours before the next cycle. Chemotherapy was administered every 10 days regardless of hematologic recovery, provided that infection was controlled. Results The PPGCSF arm had significantly fewer febrile neutropenic episodes (P = .002), days with fever (P = .004), hospital days (P = .017), and antibiotic days (P = .001). Reported Common Toxicity Criteria (CTC) graded toxicity was also significantly reduced: infections per cycle (P = .002), fever (P < .001), severe leucopenia (P < .001), neutropenia (P < .001), mucositis (P = .002), nausea/vomiting (P = .045), and constipation (P = .008). Severe weight loss was reduced significantly by 50% (P = .013). Protocol compliance with the rapid induction schedule was also significantly better in the PPGCSF arm shown by shorter time to completion (P = .005). PPGCSF did not adversely affect response rates or success of peripheral-blood stem-cell harvest. Following these results, PPG-GSF was advised for all patients on rapid COJEC induction.

Inter- and intraobserver variability of measurements with Pentacam HR® in patients with mild to moderate keratoconus

Purpose: To evaluate inter- and intraobserver variability of indices crucial for detection of keratoconus progression derived from the Pentacam HR® (high-resolution) tomographer (OCULUS Optikgeräte GmbH, Wetzlar, Germany) in patients with mild to moderate keratoconus. Methods: Three repeated corneal topography measurements in the 25-picture mode by two independent observers were performed. The extent of variability across a large range of measurement parameters was analyzed including anterior and posterior corneal surface measurements, pachymetry values, corneal volume, anterior chamber volume and depth, and iridocorneal angle. The intraclass correlation coefficient (ICC) between and within each investigator was calculated to assess reproducibility and repeatability, respectively. Results: 31 eyes of 20 patients (mean age 31.6, SD ± 8.6) were included. Overall, the repeatability and reproducibility were excellent. The range of variability was reported by calculating the standard deviation of measurements. The detailed results are shown in Table 1. Conclusions: This study shows that the Pentacam HR® tomographer provides reliable measurements in patients with mild to moderate keratoconus. However, all parameters showed a certain range of variability. This should be taken into account when assessing keratoconus progression in order to distinguish true progression from variability in measurements. In addition, the excellent reproducibility suggests that the measurements can be reliably performed by different individuals from one visit to another.

Bone structure assessed by HR-pQCT, TBS and DXL in adult patients with different types of osteogenesis imperfecta.

UNLABELLED: Bone microarchitecture by high-resolution peripheral quantitative computed tomography (HR-pQCT) was assessed in adult patients with mild, moderate, and severe osteogenesis imperfecta (OI). The trabecular bone score (TBS), bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA), and dual X-ray and laser (DXL) at the calcaneus were likewise assessed in patients with OI. Trabecular microstructure and BMD in particular were severely altered in patients with OI. INTRODUCTION: OI is characterized by high fracture risk but not necessarily by low BMD. The main purpose of this study was to assess bone microarchitecture and BMD at different skeletal sites in different types of OI. METHODS: HR-pQCT was performed in 30 patients with OI (mild OI-I, n = 18 (41.8 [34.7, 55.7] years) and moderate to severe OI-III-IV, n = 12 (47.6 [35.3, 58.4] years)) and 30 healthy age-matched controls. TBS, BMD by DXA at the lumbar spine and hip, as well as BMD by DXL at the calcaneus were likewise assessed in patients with OI only. RESULTS: At the radius, significantly lower trabecular parameters including BV/TV (p = 0.01 and p < 0.0001, respectively) and trabecular number (p < 0.0001 and p < 0.0001, respectively) as well as an increased inhomogeneity of the trabecular network (p < 0.0001 and p < 0.0001, respectively) were observed in OI-I and OI-III-IV in comparison to the control group. Similar results for trabecular parameters were found at the tibia. Microstructural parameters were worse in OI-III-IV than in OI-I. No significant differences were found in cortical thickness and cortical porosity between the three subgroups at the radius. The cortical thickness of the tibia was thinner in OI-I (p < 0.001), but not OI-III-IV, when compared to controls. CONCLUSIONS: Trabecular BMD and trabecular bone microstructure in particular are severely altered in patients with clinical OI-I and OI-III-IV. Low TBS and DXL and their significant associations to HR-pQCT parameters of trabecular bone support this conclusion.