Diet-dependent function of the extracellular matrix proteoglycan Lumican in obesity and glucose homeostasis.

OBJECTIVE: Extracellular matrix remodeling is required for adipose expansion under increased caloric intake. In turn, inhibited expandability due to aberrant collagen deposition promotes insulin resistance and progression towards the metabolic syndrome. An emerging role for the small leucine-rich proteoglycan Lumican in metabolically driven nonalcoholic fatty liver disease sparks an interest in further understanding its role in diet-induced obesity and metabolic complications. METHODS: Whole body ablation of Lumican (Lum-/-) gene and adeno-associated virus-mediated over-expression were used in combination with control or high fat diet to assess energy balance, glucose homeostasis as well as adipose tissue health and remodeling. RESULTS: Lumican was found to be particularly enriched in the stromal cells isolated from murine gonadal white adipose tissue. Likewise murine and human visceral fat showed a robust increase in Lumican as compared to fat from the subcutaneous depot. Lumican null female mice exhibited moderately increased fat mass, decreased insulin sensitivity and increased liver triglycerides in a diet-dependent manner. These changes coincided with inflammation in adipose tissue and no overt effects in adipose expandability, i.e. adipocyte formation and hypertrophy. Lumican over-expression in visceral fat and liver resulted in improved insulin sensitivity and glucose clearance. CONCLUSIONS: These data indicate that Lumican may represent a functional link between the extracellular matrix, glucose homeostasis, and features of the metabolic syndrome.

Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma.

Background: A major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients. Patients and methods: Matched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing. Results: For exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection. Conclusions: Combining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone. Clinical Trials: NCT01526928.

Interface Adhesion and Structural Characterization of Rolled-up GaAs/In0.2Ga0.8As Multilayer Tubes by Coherent Phonon Spectroscopy.

We present a detailed experimental and theoretical study of the acoustic phonon modes in rolled-up multilayers with thickness of the layers in the nanometre and diameters in the micrometre range. We compare our results to planar, unrolled multilayers grown by molecular beam epitaxy. For the planar multilayers the experimentally obtained acoustic modes exhibit properties of a superlattice and match well to calculations obtained by the Rytov model. The rolled-up superlattice tubes show intriguing differences compared to the planar structures which can be attributed to the imperfect adhesion between individual tube windings. A transfer matrix method including a massless spring accounting for the imperfect adhesion between the layers yields good agreement between experiment and calculations for up to five windings. Areas with sufficient mechanical coupling between all windings can be distinguished by their acoustic mode spectrum from areas where individual windings are only partially in contact. This allows the spatially resolved characterization of individual tubes with micrometre spatial resolution where areas with varying interface adhesion can be identified.

Progressing MoodSwings. The upgrade and evaluation of MoodSwings 2.0: An online intervention for bipolar disorder.

MoodSwings 2.0 is a self-guided online intervention for bipolar disorder. The intervention incorporates technological improvements on an earlier validated version of the intervention (MoodSwings 1.0). The previous MoodSwings trial provides this study with a unique opportunity to progress previous work, whilst being able to take into consideration lesson learnt, and technological enhancements. The structure and technology of MoodSwings 2.0 are described and the relevance to other online health interventions is highlighted. An international team from Australia and the US updated and improved the programs content pursuant to changes in DSM-5, added multimedia components and included larger numbers of participants in the group discussion boards. Greater methodological rigour in this trial includes an attention control condition, quarterly telephone assessments, and red flag alerts for significant clinical change. This paper outlines these improvements, including additional security and safety measures. A 3 arm RCT is currently evaluating the enhanced program to assess the efficacy of MS 2.0; the primary outcome is change in depressive and manic symptoms. To our knowledge this is the first randomized controlled online bipolar study with a discussion board attention control and meets the key methodological criteria for online interventions.

Death receptor-based enrichment of Cas9-expressing cells.

BACKGROUND: The CRISPR/Cas9 genome editing system has greatly facilitated and expanded our capacity to engineer mammalian genomes, including targeted gene knock-outs. However, the phenotyping of the knock-out effect requires a high DNA editing efficiency. RESULTS: Here, we report a user-friendly strategy based on the extrinsic apoptosis pathway that allows enrichment of a polyclonal gene-edited cell population, by selecting Cas9-transfected cells that co-express dominant-negative mutants of death receptors. The extrinsic apoptosis pathway can be triggered in many mammalian cell types, and ligands are easy to produce, do not require purification and kill much faster than the state-of-the-art selection drug puromycin. Stringent assessment of our advanced selection strategy via Sanger sequencing, T7 endonuclease I (T7E1) assay and direct phenotyping confirmed a strong and rapid enrichment of Cas9-expressing cell populations, in some cases reaching up to 100 % within one hour. Notably, the efficiency of target DNA cleavage in these enriched cells reached high levels that exceeded the reliable range of the T7E1 assay, a conclusion that can be generalized for editing efficiencies above 30 %. Moreover, our data emphasize that the insertion and deletion pattern induced by a specific gRNA is reproducible across different cell lines. CONCLUSIONS: The workflow and the findings reported here should streamline a wide array of future low- or high-throughput gene knock-out screens, and should largely improve data interpretation from CRISPR experiments.

[Schooling of patients exhibiting Autism Spectrum Disorders without mental retardation]. / La scolarisation des patients atteints par des troubles du spectre autistique sans retard mental.

INTRODUCTION: Autism Spectrum Disorders belong to Pervasive Development Disorders. Although access to education is recommended by the French National High Authority for Health (HAS), the practice remains limited and the reasons for the low education rate of these children have still not been sufficiently explored in the literature. OBJECTIVE: The main objective of this study was to analyze the links between Autism Spectrum Disorder without mental retardation, psychiatric comorbidity and education. The secondary objective was to analyze the cognitive and contextual factors that could limit educational inclusion. METHOD: Eighty-three autistic patients (3-18years old; 73 males and 10 females) with childhood autism, atypical autism or Asperger's syndrome (criteria from the International Classification of Diseases-10) without mental retardation and in education were assessed at the Alpine Centre for Early Diagnosis of Autism. The sample included 45 subjects with childhood autism, 12 subjects with atypical autism and 26 subjects with Asperger's syndrome. The diagnosis was based on the Autism Diagnostic Interview Revised (ADI-R), in accordance with the recommendations of the HAS, the Autism Diagnostic Observation Schedule (ADOS) and the Wechsler Intelligence Scale for Children, 4th edition (WISC-IV). RESULTS: Our results showed that childhood autism and atypical autism were mainly found in nursery and primary school, whereas Asperger's syndrome was mainly found in secondary school (Chi(2)=18.23; df=6; P<.006). Individuals with childhood autism and atypical autism were more likely to receive the support of a special educational assistant (Chi(2)=15.61; df=2; P<.000) and underwent a higher number of consultations and treatment episodes than those with Asperger's syndrome (Chi(2)=27.83; df=14; P<.015). The cognitive profiles obtained with the WISC-IV also differed: the Verbal Comprehension Index (VCI) and Working Memory Index (WMI) were higher for Asperger's syndrome than for childhood autism and atypical autism (respectively, F=23.11, P<.000; df=2; partial η(2)=.576 and F=8.06, P<.001; df=2; partial η(2)=.357). Linear regression showed that the VCI and Processing Speed Index (PSI) were inversely correlated to the number of hours spent with a special educational assistant: the lower these indexes, the greater the amount of time spent with a special educational assistant. No link was found between psychiatric comorbidity, type of psychological and psychiatric treatment, and education. DISCUSSION: The use of special educational assistants seems to be linked to the diagnosis of Autism Spectrum Disorders and neuropsychological functioning, as assessed by WISC-IV, along a continuum that ranges from childhood autism (more needs and deficits) to atypical autism to Asperger's syndrome. The Verbal Comprehension Index (VCI) and the Processing Speed Index (PSI) could be used to evaluate the number of hours of support needed by children and to better target the deficits and specific needs of children without mental retardation who are in education. A study on a larger scale could help to more closely address the question of the cognitive abilities of children with Autism Spectrum Disorder without mental retardation, so as to better help them in their education.

New onset of diabetes after transplantation is associated with improved patient survival after liver transplantation due to confounding factor.

BACKGROUND: The influence of NODAT on survival of liver transplant recipients has not been clarified. Therefore, we evaluated the effect of NODAT on survival in LT recipients. METHODS: Data from 352 LT patients were totally analyzed. 97 patients with pretransplant diabetes mellitus were excluded, and 255 patients without diabetes mellitus at time of transplantation were included. RESULTS: NODAT was diagnosed in 41 patients (16.1%). There was no difference in frequency of NODAT according to the etiology of liver cirrhosis. NODAT was associated with a higher body weight (p=0.004) and BMI (p=0.002) 5years after LT, but not with weight gain (p=0.201) or increase in BMI (p=0.335) 5years after LT. HbA1c 5years after LT was significantly higher in patients with NODAT (p=0.001), but mean HbA1c still remained lower than 6.5% (6.4(±1.2) %). Patients with NODAT showed better survival rates (log rank: p=0.002) compared to LT recipients without diabetes. According to all existing knowledge of diabetes mellitus (DM) better survival cannot be a direct effect of this disease. Our results are rather influenced by an not known confounding factor (possibly recovery from cachexia) associated with better survival and NODAT, while complications of NODAT will not appear during the relatively short postoperative time and observation period (mean follow up 6.08 (±2.67) years). CONCLUSION: NODAT is frequently diagnosed in LT recipients and is associated with an improved 5year survival after LT due to a not exactly known confounding factor.

Preoperative left ventricular hypertrophy is associated with reduced patient survival after liver transplantation.

UNLABELLED: Left ventricular hypertrophy (LVH) has been described in the context of cirrhotic cardiomyopathy. The influence of LVH on survival of liver transplant (LT) recipients has not been clarified. Therefore, we evaluated the effect of LVH on survival in LT recipients. In total, data from 352 LT patients were analyzed. LVH was diagnosed by echocardiographic measurement of left ventricular wall thickness before LT. Patients were followed up for a mean of 4.2 yr. LVH was diagnosed in 135 (38.4%) patients. Patients with LVH had significantly more frequently male gender (p = 0.046), diastolic dysfunction (p < 0.001), and hepatocellular carcinoma (HCC; p = 0.004). Furthermore, LVH patients were older (p < 0.001) and had a higher body mass index (BMI; p = 0.001). There was no difference in frequency of arterial hypertension, pre-transplant diabetes mellitus, or etiology of liver cirrhosis. Patients without LVH had a better survival (log rank: p = 0.05) compared with LVH patients. In a multivariate Cox regression LVH (p = 0.031), end-stage renal disease (ESRD; p = 0.003) and lack of arterial hypertension (p = 0.004) but not MELD score (p = 0.885) were associated with poorer survival. CONCLUSION: LVH is frequently diagnosed in patients on the waiting list and influences survival after LT.

The effects of weightlessness on the human organism and mammalian cells.

It has always been a desire of mankind to conquest Space. A major step in realizing this dream was the completion of the International Space Station (ISS). Living there for several months confirmed early observations of short-term spaceflights that a loss of gravity affects the health of astronauts. Space medicine tries to understand the mechanism of microgravity-induced health problems and to conceive potent countermeasures. There are four different aspects which make space medicine appealing: i) finding better strategies for adapting astronauts to weightlessness; ii) identification of microgravity-induced diseases (e.g. osteoporosis, muscle atrophy, cardiac problems and others); iii) defining new therapies to conquer these diseases which will benefit astronauts as well as people on Earth in the end; and iv) on top of that, unveiling the mechanisms of weightlessness-dependent molecular and cellular changes is a requirement for improving space medicine. In mammalian cells, microgravity induces apoptosis and alters the cytoskeleton and affects signal transduction pathways, cell differentiation, growth, proliferation, migration and adhesion. This review focused on gravi-sensitive signal transduction elements and pathways as well as molecular mechanisms in human cells, aiming to understand the cellular changes in altered gravity. Moreover, the latest information on how these changes lead to clinically relevant health problems and current strategies of countermeasures are reviewed.

[Genome-wide association studies]. / Genomweite Assoziationsstudien.

Genome-wide association studies (GWAS) are aimed to identify genetic markers of complex human diseases and individual traits. In this context more than 150 gene loci have been found to be associated with about 60 different diseases and personal characteristics. A recent example is the relevance of a polymorphism in the interleukin 28B gene for the natural course of hepatitis C virus infection and the efficacy of antiviral therapy. It is to be expected that GWAS will increasingly contribute to the understanding of the pathogenesis and consquently improve prediction, prevention, diagnosis and therapy of human diseases and eventually will become part of clinical practice.

Diagnostic and therapeutic use of membrane proteins in cancer cells.

As proteomics technologies develop, increasing number of membrane-associated proteins specific for cancer cells are being discovered. These proteins are of great interest, particularly because they are rich in targets for antibodies. Amongst them candidate biomarkers for early tumor diagnosis, prognosis and treatment have been detected. The suitability of several membrane-associated proteins as targets for drugs or antibodies has already been tested in preclinical and clinical studies. The results were encouraging in some cases, but not in all. They demonstrate that each type of tumor has its specific "Achilles heel", and that suitable targets of cancer diagnosis and therapy must be found for each kind of neoplasm. This implies that membrane-associated proteins for each type of tumor cell need to be investigated. This review describes the current technologies of membrane protein characterization in a first part and subsequently summarizes the membrane associated proteins currently being tested as targets for diagnosis and treatment in breast, prostate, thyroid, and colon cancer. Their function will be explained and their role in tumor biology will be discussed.

Multiple hemangiomas and hemangiomatosis--risk factors and outcome over an eight year period.

INTRODUCTION: Hemangiomas are the most common tumors of infancy. Multiple cutaneous hemangiomas may be associated with the presence of hemangiomas in inner organs. However, there is little data on the risk factors for organ involvement and the outcome of a large sample of patients. PATIENTS AND METHODS: patients with 3 or more cutaneous hemangiomas were evaluated with regard to patient characteristics, distribution of hemangiomas, results of radiological abdominal/cerebral imaging, clinical course, and therapeutic approach. We analyzed the risk factors for organ involvement and complications/outcome. RESULTS: The average gestational week at birth was 32.8; radiological imaging showed liver hemangiomas in 13.5% and mesenteric lesions in 1 (1.9%) but no cerebral lesions. Preterm infants (p=0.02) and patients with high numbers of cutaneous hemangiomas (p=0.02) were at higher risk of organ involvement. A life-threatening event occurred in 1 patient (1.9%). None of the patients died. CONCLUSIONS: Organ manifestation is relatively common in patients with multiple hemangiomas, complications are rare, but potentially life-threatening. We recommend abdominal imaging for patients with 3 or more hemangiomas, especially in preterm infants.

Frequency of calpain-3 c.550delA mutation in limb girdle muscular dystrophy type 2 and isolated hyperCKemia in German patients.

OBJECTIVE: Calpain-3 deficiency is the most common cause of autosomal-recessive limb girdle muscular dystrophy (LGMD2). The c.550delA mutation in the CAPN3 gene was frequently identified in LGMD2A patients from Eastern Europe and is considered a Slavic founder mutation. METHODS: We screened for the c.550delA mutation in unrelated German patients with LGMD2 (n = 98) and in patients with asymptomatic or minimally symptomatic (myalgia or fatigue) hyperCKemia of unknown origin (n = 102). Results of Western blot analysis were available in 75 patients with LGMD2 and 65 patients with hyperCKemia. In samples that were heterozygous for the c.550delA mutation, the whole CAPN3 gene was analyzed by sequencing in order to detect the second mutation. RESULTS: The c.550delA mutation was found in 8.1% of LGMD2 (n = 1 homozygous, n = 7 heterozygous) and 1.9% of hyperCKemia patients (n = 2 heterozygous). In 8 of the 9 hetrozygous patients, a second CAPN3 mutation was identified by direct sequencing. Two mutations (Val509Phe and Gln565Stop) have not been reported before. Absent or deficient calpain-3 protein in Western blot analysis was found in 22.5% of the LGMD2 patients and 11% of the patients with hyperCKemia. Western blot results were available in 9 out of the 10 patients with genetically confirmed LGMD2A and were clearly abnormal in 6 patients, suspicious in 2 and entirely normal in 1. Two LGMD2 patients with the c.550delA mutation and onset within the first 2 decades had joint contractures. Muscle biopsy revealed inflammatory changes in three patients. CONCLUSION: The CAPN3 gene mutation c.550delA is rather frequently observed in German patients with LGMD2, but also occasionally in cases with isolated hyperCKemia.

Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity: impact of vascular endothelial growth factor.

Endothelial cells play a crucial role in the pathogenesis of many diseases and are highly sensitive to low gravity conditions. Using a three-dimensional random positioning machine (clinostat) we investigated effects of simulated weightlessness on the human EA.hy926 cell line (4, 12, 24, 48 and 72 h) and addressed the impact of exposure to VEGF (10 ng/ml). Simulated microgravity resulted in an increase in extracellular matrix proteins (ECMP) and altered cytoskeletal components such as microtubules (alpha-tubulin) and intermediate filaments (cytokeratin). Within the initial 4 h, both simulated microgravity and VEGF, alone, enhanced the expression of ECMP (collagen type I, fibronectin, osteopontin, laminin) and flk-1 protein. Synergistic effects between microgravity and VEGF were not seen. After 12 h, microgravity further enhanced all proteins mentioned above. Moreover, clinorotated endothelial cells showed morphological and biochemical signs of apoptosis after 4 h, which were further increased after 72 h. VEGF significantly attenuated apoptosis as demonstrated by DAPI staining, TUNEL flow cytometry and electron microscopy. Caspase-3, Bax, Fas, and 85-kDa apoptosis-related cleavage fragments were clearly reduced by VEGF. After 72 h, most surviving endothelial cells had assembled to three-dimensional tubular structures. Simulated weightlessness induced apoptosis and increased the amount of ECMP. VEGF develops a cell-protective influence on endothelial cells exposed to simulated microgravity.

Mechanisms of apoptosis after ischemia and reperfusion: role of the renin-angiotensin system.

BACKGROUND: Apoptosis plays a key role in the pathogenesis of cardiac diseases. We examined the influence of the renin-angiotensin system (RAS) on different regulators of apoptosis using an isolated hemoperfused working porcine heart model of acute ischemia (2 h), followed by reperfusion (4 h). METHODS AND RESULTS: 23 porcine hearts were randomized to 5 groups: hemoperfused non-infarcted hearts (C), infarcted hearts (MI: R. circumflexus), infarcted hearts treated with quinaprilat (Q), infarcted hearts treated with angiotensin-I (Ang I), and infarcted hearts treated with angiotensin-I and quinaprilat (QA). Fas, Bax, bcl-2 and p53 proteins were increased in MI hearts and further elevated by Ang I. Quinaprilat reduced Bax and p53. Bcl-2 was elevated in Q and reduced in QA. An early upregulation of caspase-3 gene and protein expression was detected in MI and Ang I hearts compared to C. Q reduced caspase-3 gene expression, but had no effect on caspase-3 and Fas protein. CONCLUSIONS: These data suggest that the RAS plays a pivotal role in cardiac apoptosis which is the early and predominant form of death in myocardial infarction. Ischemia/reperfusion induces programmed cell death via extrinsic and intrinsic pathways. Early treatment with quinaprilat attenuated cardiomyocyte apoptosis.

Therapeutic short hairpin RNA expression in the liver: viral targets and vectors.

Over 500 million people worldwide are infected with one or more different and unrelated types of human hepatitis virus. Such individuals are at a high risk of developing acute or chronic hepatic disease, and ultimately dying from sequelae. Although a vaccine is available for hepatitis A and B virus, treatment options for chronically infected patients are limited, and particularly ineffective in case of hepatitis C virus (HCV) infection. A promising new avenue currently being explored is to harness the power of RNA interference for development of an antiviral therapy. The timing to pursue this particular approach is excellent, with the first in vivo animal models for HCV infection becoming available, and the technology for liver-specific expression of short hairpin RNAs advancing at a rapid pace. Here, we critically review these important current developments, and discuss the next steps to bring this novel approach into the clinics.

From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy.

Gene transfer vectors based on the human adeno-associated virus serotype 2 (AAV-2) have been developed and tested in pre-clinical studies for almost 20 years, and are currently being evaluated in clinical trials. So far, all these studies have provided evidence that AAV-2 vectors possess many properties making them very attractive for therapeutic gene delivery to humans, such as a lack of pathogenicity or toxicity, and the ability to confer long-term gene expression. However, there is concern that two restrictions of AAV-2 vectors might limit their clinical use in humans. First, these vectors are rather inefficient at transducing some cells of therapeutic interest, such as liver and muscle cells. Second, gene transfer might be hampered by neutralizing anti-AAV-2 antibodies, which are highly prevalent in the human population. In efforts to overcome both limitations, an increasing number of researchers are now focusing on the seven other naturally occurring serotypes of AAV (AAV-1 and AAV-3 to -8), which are structurally and functionally different from AAV-2. To this end, several strategies have been devised to cross-package an AAV-2 vector genome into the capsids of the other AAV serotypes, resulting in a new generation of "pseudotyped" AAV vectors. In vitro and in vivo, these novel vectors were shown to have a host range different from AAV-2, and to escape the anti-AAV-2 immune response, thus underscoring the great potential of this approach. Here the biology of the eight AAV serotypes is summarized, existing technology for pseudotyped AAV vector production is described, initial results from pre-clinical evaluation of the vectors are reviewed, and finally, the prospects of these promising novel tools for human gene therapy are discussed.

Characteristics of sensitometric curves of radiographic films.

A new type of radiographic film, EDR (extended dose range) film, has been recently become available for film dosimetry. It is particularly attractive for composite isodose verification of intensity modulated radiation therapy because of its low sensitivity relative to the more common Kodak XV film. For XV film, the relationship between optical density and dose, commonly known as the sensitometric curve, depends linearly on the dose at low densities. Unlike XV film, the sensitometric curve of EDR film irradiated by megavoltage x rays is not linearly dependent on the dose at low densities. In this work, to understand the mechanisms governing the shape of the sensitometric curves, EDR film was studied with kilovoltage x rays, 60Co gamma rays, megavoltage x rays, and electron beams. As a comparison, XV film was also studied with the same beams mentioned above. The model originally developed by Silberstein [J. Opt. Soc. Am. 35, 93-107, 1945)] is used to fit experimental data. It is found that the single hit model can be used to predict the sensitometric curve for XV films irradiated by all beams used in this work and for EDR films exposed to kilovoltage x rays. For EDR film irradiated by 60Co gamma rays, megavoltage x rays, and electron beams, the double hit model is used to fit the sensitometric curves. For doses less than 100 cGy, a systematic difference between measured densities and that predicted by the double hit model is observed. Possible causes of the observed differences are discussed. The results of this work provide a theoretical explanation of the sensitometric behavior of EDR film.