Cloud-based simulation studies in R - A tutorial on using doRedis with Amazon spot fleets.

Simulation studies are helpful in testing novel statistical methods. From a computational perspective, they constitute embarrassingly parallel tasks. We describe parallelization techniques in the programming language R that can be used on Amazon's cloud-based infrastructure. After a short conceptual overview of the parallelization techniques in R, we provide a hands-on tutorial on how the doRedis package in conjunction with the Redis server can be used on Amazon Web Services, specifically running spot fleets. The tutorial proceeds in seven steps, ie, (1) starting up an EC2 instance, (2) installing a Redis server, (3) using doRedis with a local worker, (4) using doRedis with a remote worker, (5) setting up instances that automatically fetch tasks from a specific master, (6) using spot-fleets, and (7) shutting down the instances. As a basic example, we show how these techniques can be used to assess the effects of heteroscedasticity on the equal-variance t-test. Furthermore, we address several advanced issues, such as multiple conditions, cost-management, and chunking.

[Testing the comparability of free-field and HDA200 headphone measurements for the Freiburg speech test]. / Überprüfung der Vergleichbarkeit von Freifeld- und HDA200-Kopfhörermessungen für den Freiburger Sprachtest.

BACKGROUND: In clinical practice, the standardized Freiburg speech test is presented via headphones as well as via loudspeakers. To achieve comparable results with both presentation modes, the headphone measurements are equated to the free-field situation. The free-field correction is determined by subjective loudness balance measurements and realized by a free-field equalizer network and a broadband frequency correction value during headphone calibration. Using the Freiburg speech test, this study tested the comparability of free-field and HDA200 headphone measurements. PATIENTS AND METHODS: In 15 normal hearing subjects, the Freiburg speech intelligibility test was performed using both loudspeakers and HDA200 headphones. RESULTS: The 50% speech reception threshold measured using HDA200 headphones was on average 5.1 dB lower than for loudspeakers and lay below the standardized reference values. CONCLUSION: The desired comparability to free-field measurements could not be shown for the HDA200 headphones. The improved intelligibility via HDA200 is probably caused by the fact that the calibration correction factor of 4 dB is too high. This should be checked by the PTB.

Repression of the gene encoding the TGF-beta type II receptor is a major target of the EWS-FLI1 oncoprotein.

Chromosomal translocations resulting in the expression of chimaeric transcription factors are frequently observed in tumour cells, and have been suggested to be a common mechanism in human carcinogenesis. Ewing sarcoma and related peripheral primitive neuroectodermal tumours share recurrent translocations that fuse the gene EWSR1 (formerly EWS) from 22q-12 to FLI1 and genes encoding other ETS transcription factors (which bind DNA through the conserved ETS domain). It has been shown that transduction of the gene EWSR1-FLI1 (encoding EWS-FLI1 protein) can transform NIH3T3 cells, and that mutants containing a deletion in either the EWS domain or the DNA-binding domain in FLI1 lose this ability. This indicates that the EWS-FLI1 fusion protein may act as an aberrant transcription factor, but the exact mechanism of oncogenesis remains unknown. Because ETS transcription factors regulate expression of TGFBR2 (encoding the TGF-beta type II receptor, TGF-beta RII; Refs 9,14), a putative tumour suppressor gene, we hypothesized that TGFBR2 may be a target of the EWS-FLI1 fusion protein. We show here that Ewing sarcoma [corrected] (ES) cell lines with the EWSR1-FLI1 fusion have reduced TGF-beta sensitivity, and that fusion-positive ES cells and primary tumours both express low or undetectable levels of TGFBR2 mRNA and protein product. Co-transfection of FLI1 and the TGFBR2 promoter induces promoter activity, whereas EWSR1-FLI1 leads to suppression of TGFBR2 promoter activity and FLI1-induced promoter activity. Introduction of EWSR1-FLI1 into cells lacking the EWSR1-FLI1 fusion suppresses TGF-beta RII expression, whereas antisense to EWSR1-FLI1 in ES cell lines positive for this gene fusion restores TGF-beta RII expression. Furthermore, introduction of normal TGF-beta RII into ES cell lines restores TGF-beta sensitivity and blocks tumorigenicity. Our results implicate TGF-beta RII as a direct target of EWS-FLI1.

[Application of new speech intelligibility tests in silence for the assessment of acquired hearing losses]. / Der Einsatz neuer Testverfahren zur Erfassung des Sprachverstehens in Ruhe bei der Begutachtung erworbener Schwerhörigkeiten.

BACKGROUND: Previous studies comparing the Freiburg speech tests (FST) currently used in Germany to assess the severity of hearing loss with two modern speech intelligibility tests [the Göttingen sentence test (GöSa) and the monosyllabic rhyme test devised by von Wallenberg and Kollmeier (WaKo)] have indicated that a replacement of the old procedure would be possible. The current study investigates the consequences of the modern test procedures for the estimation of reduction in earning capacity resulting from hearing loss, and considers the optimal presentation levels for the WaKo test. METHODS: The FST, GöSa, and WaKo speech intelligibility tests were performed on 29 volunteers with a hearing impairment. All tests were conducted in silence. The presentation levels for the Freiburg monosyllabic test were 60, 80, and for some participants also 100 dB SPL. The WaKo test was carried out once at 45, 65, and 85 dB SPL (a reduction of 15 dB relative to the FST) and again at 40, 60 and 80 dB SPL (reduction of 20 dB relative to the FST). RESULTS: A consideration across all presentation levels revealed that the best correlation match between the two monosyllabic tests was achieved at a 20-dB reduction in presentation level for the WaKo test relative to the FST. On average, the application of modern procedures and the different options for level reduction had only a minor effect on the quantitative assessment of reduction in earning capacity.

Construction of an active humidity regulation setup for NMR/MRI-Observation and simulation of the controlled evaporation of sessile water droplets.

Controlling and improving processes like for example the production of organic semiconductors via printing depends on understanding the interplay of wetting and evaporation of complex fluids. Therefore, examination of the time dependent composition of complex fluid droplets during wetting or evaporation is of interest. The evaporation rate of sessile droplets containing largely water depends on the vapor pressures of the individual components and on the humidity (or partial pressure) of the surrounding gas phase. Hence, for a complete picture of an evaporation process and the comparability of the results of different measurements, it is essential to measure and control the humidity and temperature in the measurement compartment. Accordingly, climate chambers are available in different scales to fit a variety of techniques like contact angle goniometry to obtain results in a controlled atmosphere. We recently reported the application of MRI (Magnetic Resonance Imaging) and spatially resolved NMR (Nuclear Magnetic Resonance) spectroscopy for the examination of the evaporation of sessile droplets on surfaces in 10 mm NMR tubes. These are considered to be closed compartments. Here, we present an apparatus to a) measure and b) control the relative humidity within the sample compartment of the NMR setup by introducing preconditioned gas into the NMR tube. We monitored the evaporation of water droplets using RARE images and compared the volume decay with a) a simple diffusive evaporation model and b) with detailed FEM (finite element numerical model) simulations using COMSOL for validation. We find three evaporation regimes depending on the flow rate as well as on the distance of the gas outlet and the evaporating droplet. In one of the sample configurations tested the evaporation takes place in such a way that it can be described with the help of the simple diffusive model without convection. Thus, the presented approach opens comparative measurements with other methods as well as the observation of droplet evaporation in very dry or very humid environments with and without the influence of convection. Finally, using PRESS spectra, it is shown that the evaporation rate of water from a water/DMSO droplet can be controlled. This shows how the setup presented here can be used to study the evaporation of droplets of more complex mixtures.

[Göttingen sentence in noise for different audiogram classes]. / Göttinger Satztest im Störgeräusch für verschiedene Gruppen von Schwerhörigkeit.

BACKGROUND: In addition to the standardized Freiburg sentence test in silence validated speech tests in noise are available. They are mainly used to test the benefit of hearing systems in everyday-life situations. In diagnostics tests in noise can also give additional information of the patients' hearing impairment. The determined speech reception threshold (SRT) depends on the degree of hearing loss and the used speech and noise test material. Aim of this study was to analyze the results of the Göttingen sentence test in noise in relation to different audiogram classes. MATERIAL AND METHODS: The Göttingen sentence test in stationary noise (65 dB) was performed for 135 patients with different degrees of hearing losses. Based on the air-conducted threshold all ears were categorized to an audiogram class by an automated procedure. RESULTS: For the mild hearing losses the results of the Göttingen sentence test increased with increasing pure-tone-average (PTA) and the values are smaller than 0 dB SNR with a small standard deviation (± 1-2 dB SNR). For the moderate hearing losses values of 5 dB SNR with a standard deviation of 6 dB SNR were achieved on average. Patients with a high hearing loss achieved values higher than 20 dB SNR. CONCLUSION: For the mild hearing losses the results were located in a small range. However, they can give important information about the patient's hearing impairment mainly appears in a noisy environment. With increasing hearing loss also the reduced hearing in silence affects the results achieved with the Göttingen sentence test in noise. Therefore the hearing in silence has to be taken into account at the interpretation of the results.

Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles.

Here, to study lipid-protein interactions that contribute to the biogenesis of regulated secretory vesicles, we have developed new approaches by which to label proteins in vivo, using photoactivatable cholesterol and glycerophospholipids. We identify synaptophysin as a major specifically cholesterol-binding protein in PC12 cells and brain synaptic vesicles. Limited cholesterol depletion, which has little effect on total endocytic activity, blocks the biogenesis of synaptic-like microvesicles (SLMVs) from the plasma membrane. We propose that specific interactions between cholesterol and SLMV membrane proteins, such as synaptophysin, contribute to both the segregation of SLMV membrane constituents from plasma-membrane constituents, and the induction of synaptic-vesicle curvature.

[Hearing loss determination and assessment of the reduction in earning capacity taking speech discrimination tests in noise into account]. / Hörverlustbestimmung und MdE-Abschätzung unter Einbezug von Sprachverständlichkeitsmessungen im Störgeräusch.

BACKGROUND: In Germany the guideline for the hearing loss quantification and the resulting assessment of the reduction in earning capacity is the Königsteiner Merkblatt (KM). The quantification depends on the results of the tone and speech audiogram in silence. However, speech discrimination tests in noise may provide additional information about the impairment of the ENT patients. Especially, the impairment of persons with a slight hearing loss, characterized by high-frequency hearing loss, in noisy environment can not be indicated by the measurement in silence, which is proposed in the KM. METHODS UND PATIENTS: The Göttingen sentence test in noise was applied as a supplement to the routine ENT procedures of the medical estimate in 135 test persons. Based on these measurement results a table for the percentage hearing loss in noise is defined. Furthermore, an integration of the hearing loss in noise in the assessment of the reduction in earning capacity is proposed in addition to the hearing loss in silence. RESULTS: Using the newly introduced hearing loss for speech in noise, a suitable assessment for persons with a slight hearing loss is achieved. By integrating it into the assessment procedure additionally to the hearing loss in silence, the hearing impairment of all patients can be rated depending on their speech test results in silence and in noise. In comparison to the results obtained by the KM, the new procedure does not lead to higher values for the proposed reduction in earning capacity in general, but instead seems to be equally suitable for all groups of patients.

Assembly of myelin by association of proteolipid protein with cholesterol- and galactosylceramide-rich membrane domains.

Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid-protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a low-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids. PLP associated with the CIMF after leaving the endoplasmic reticulum but before exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP with myelin lipids in CIMF was supported by the finding that it was efficiently cross-linked to photoactivable cholesterol, but not to phosphatidylcholine, which is underrepresented in both myelin and CIMF. Furthermore, depletion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocytes abolished the association of PLP with CIMF. Thus, PLP may be recruited to myelin rafts, represented by CIMF, via lipid-protein interactions. In contrast to oligodendrocytes, after transfection in BHK cells, PLP is absent from isolated CIMF, suggesting that PLP requires specific lipids for raft association. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycosphingolipids, a large fraction of PLP no longer associates with rafts. Formation of a cholesterol- and galactosylceramide-rich membrane domain (myelin rafts) may be critical for the sorting of PLP and assembly of myelin in oligodendrocytes.

MRI and localised NMR spectroscopy of sessile droplets on hydrophilic, hydrophobic and superhydrophobic surfaces - Examination of the chemical composition during evaporation.

Evaporation of droplets is a process important in many different areas of science, technology and also everyday life. The understanding of droplet evaporation of homogeneous and heterogeneous substance mixtures is important, for example, to explain the formation of coffee stains or to optimize the results in offset printing. For a detailed understanding of the evaporation of complex mixtures from structured surfaces, such as inks used in offset printing, a time-resolved analysis of the droplet composition is essential. Measurement of (local) concentrations may deepen the understanding of wetting phenomena and their connection with transport phenomena. Therefore, we demonstrate in this paper that magnetic resonance methods can be used to (a) image sessile droplets on structured surfaces and (b) investigate their composition in a time-resolved manner. First it is shown that water droplets on superhydrophobic, hydrophobic and hydrophilic surfaces, despite the large liquid/gas interface, can be imaged well and without interfering artefacts using RARE. Further, the signals are examined in localised PRESS NMR spectra with respect to line shape and quantifiability. Finally, it is demonstrated that non-localised NMR spectra can be used to track the droplet composition during evaporation.

Induction of TrkB by retinoic acid mediates biologic responsiveness to BDNF and differentiation of human neuroblastoma cells. Eukaryotic Signal Transduction Group.

Retinoic acid (RA) induces the neuronal differentiation of many human neuroblastoma cell lines. In this study, we show that RA treatment of neuroblastoma cells induces the expression of TrkB, the receptor for the neurotrophins BDNF, NT-3, and NT-4/5. BDNF addition to RA-treated SH-SY5Y neuroblastoma cells stimulated the tyrosine phosphorylation of TrkB and neuronal differentiation. RA treatment of KCNR neuroblastoma cells, which constitutively express BDNF mRNA, resulted in the expression of TrkB and differentiation in the absence of added BDNF. Finally, in 15N neuroblastoma cells, which express BDNF mRNA but do not differentiate in response to RA, RA induced only a truncated form of TrkB. 15N cells transfected with full-length TrkB differentiated in the absence of RA. These results indicate that RA induces the neuronal differentiation of neuroblastoma cells by modulating the expression of neurotrophin receptors.

Downregulation of Bim by brain-derived neurotrophic factor activation of TrkB protects neuroblastoma cells from paclitaxel but not etoposide or cisplatin-induced cell death.

Chemoresistance and increased expression of TrkB and brain-derived neurotrophic factor (BDNF) are biomarkers of poor prognosis in tumors from patients with neuroblastoma (NB). Previously, we found BDNF activation of TrkB through PI3K/Akt protects NB from etoposide/cisplatin-induced cell death. In this study, the role of Bim, a proapoptotic protein, was investigated. Bim was involved in paclitaxel but not etoposide or cisplatin-induced cell death in NB cells. Pharmacological and genetic studies showed that BDNF-induced decreases in Bim were regulated by MAPK and not PI3K/Akt pathway. Both MAPK and PI3K pathways were involved in BDNF protection of NB cells from paclitaxel-induced cell death, while PI3K predominantly mediated BDNF protection of NB cells from etoposide or cisplatin-induced cell death. These data indicate that different chemotherapeutic drugs induce distinct death pathways and growth factors utilize different signal transduction pathways to modulate the effects of chemotherapy on cells.

Protein secretion: puzzling receptors.

All known sorting receptors for soluble cargo in the secretory pathway are transmembrane proteins. For sorting to the regulated pathway, however, a subpopulation of secretory proteins, associated with the membrane but not membrane-spanning, appears to link cargo and membrane in storage granule biogenesis.

Differential protooncogene expression characterizes histopathologically indistinguishable tumors of the peripheral nervous system.

We have found highly predictable patterns of protooncogene expression in cell lines and tumor tissue of neuroblastoma (NB), a tumor of the peripheral nervous system (PNS). These patterns make it possible to recognize two different genetically definable subgroups among histopathologically indistinguishable tumors. Additionally, we have identified a difference in neurotransmitter biosynthetic enzyme activity in these two subgroups of NB. The patterns of protooncogene expression and neurotransmitter biosynthetic enzymes suggests that these tumors arise in different cells of the PNS.

Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation.

We detected expression of the c-myb proto-oncogene, which was initially thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage, in human tissues of neuronal origin. Since the level of c-myb expression declined during fetal development, we studied the regulation of its expression in human neuroblastoma cell lines induced to differentiate by retinoic acid. The expression of c-myb declined during the maturation of neuroblastoma cells, and this change was mediated by a decrease in c-myb transcription.

Distribution and transport of cholesterol in Caenorhabditis elegans.

Cholesterol transport is an essential process in all multicellular organisms. In this study we applied two recently developed approaches to investigate the distribution and molecular mechanisms of cholesterol transport in Caenorhabditis elegans. The distribution of cholesterol in living worms was studied by imaging its fluorescent analog, dehydroergosterol, which we applied to the animals by feeding. Dehydroergosterol accumulates primarily in the pharynx, nerve ring, excretory gland cell, and gut of L1-L3 larvae. Later, the bulk of dehydroergosterol accumulates in oocytes and spermatozoa. Males display exceptionally strong labeling of spermatids, which suggests a possible role for cholesterol in sperm development. In a complementary approach, we used a photoactivatable cholesterol analog to identify cholesterol-binding proteins in C. elegans. Three major and several minor proteins were found specifically cross-linked to photocholesterol after UV irradiation. The major proteins were identified as vitellogenins. rme-2 mutants, which lack the vitellogenin receptor, fail to accumulate dehydroergosterol in oocytes and embryos and instead accumulate dehydroergosterol in the body cavity along with vitellogenin. Thus, uptake of cholesterol by C. elegans oocytes occurs via an endocytotic pathway involving yolk proteins. The pathway is a likely evolutionary ancestor of mammalian cholesterol transport.

Identification of CASZ1 NES reveals potential mechanisms for loss of CASZ1 tumor suppressor activity in neuroblastoma.

As a transcription factor, localization to the nucleus and the recruitment of cofactors to regulate gene transcription is essential. Nuclear localization and nucleosome remodeling and histone deacetylase (NuRD) complex binding are required for the zinc-finger transcription factor CASZ1 to function as a neuroblastoma (NB) tumor suppressor. However, the critical amino acids (AAs) that are required for CASZ1 interaction with NuRD complex and the regulation of CASZ1 subcellular localization have not been characterized. Through alanine scanning, immunofluorescence cell staining and co-immunoprecipitation, we define a critical region at the CASZ1 N terminus (AAs 23-40) that mediates the CASZ1b nuclear localization and NuRD interaction. Furthermore, we identified a nuclear export signal (NES) at the N terminus (AAs 176-192) that contributes to CASZ1 nuclear-cytoplasmic shuttling in a chromosomal maintenance 1-dependent manner. An analysis of CASZ1 protein expression in a primary NB tissue microarray shows that high nuclear CASZ1 staining is detected in tumor samples from NB patients with good prognosis. In contrast, cytoplasmic-restricted CASZ1 staining or low nuclear CASZ1 staining is found in tumor samples from patients with poor prognosis. These findings provide insight into mechanisms by which CASZ1 regulates transcription, and suggests that regulation of CASZ1 subcellular localization may impact its function in normal development and pathologic conditions such as NB tumorigenesis.

A novel liquid crystalline system for partial alignment of polar organic molecules.

A new system for partial alignment of polar organic molecules to measure residual dipolar couplings in NMR consists of a 1:1 or a 2:1 mixture of water and DMSO including 3-13% n-alkylpentaethylene glycol as the surfactant. Temperature and concentration dependence of the alignment system are investigated and, as examples, the 13C,1H residual dipolar couplings for the amino acid methionine 1 and for an alpha-methylene-gamma-butyrolactone 2 have been obtained and are compared with those obtained from the alignment media consisting of n-alkylpentaethylene glycol, n-alkyl alcohol and water.

In vitro activation of distinct molecular and cellular phenotypes after induction of differentiation in a human neuroblastoma cell line.

In this report we provide evidence for the activation of distinct differentiation pathways during treatment of the neuroblastoma cell line SMS-KCNR with 1 mM dibutyryl cyclic AMP (dbcAMP) and/or 5 microM retinoic acid (RA). Our results show that the adrenal gland specific gene pG2 is induced only during dbcAMP treatment, while RA induces a neuronal phenotype and expression of all neural related genes while decreasing the expression of many chromaffin related genes. Furthermore dbcAMP does not affect the DNA content distribution of SMS-KCNR [G1 = 61.8 +/- 4.1% (SD); S = 20.3 +/- 6.3%; G2-M = 18 +/- 5.4%] despite morphological and molecular signs of cellular differentiation. Conversely, RA arrests cell growth causing a decrease in cells in the growth fraction (S + G2 + M = 15.6 +/- 6.1%) and an increase in cells in G1 (G1 = 84.3 +/- 5%). Using cyclic AMP and RA in combination, we found that RA inhibited expression of adrenal gland specific gene pG2 and induced a neuronal phenotype. Since dbcAMP does not cause a significant G1 block in SMS-KCNR cells we propose that this agent may be able to induce SMS-KCNR only to an intermediate stage of chromaffin differentiation in which cells retain their proliferative potential.

Novel mutations in the p16/CDKN2A binding region of the cyclin-dependent kinase-4 gene.

Mutations in genes that lie in the retinoblastoma pathway have been implicated in the pathogenesis of many tumor types. Two critical components that determine progression from G1 to S include p16/CDKN2A and CDK4. Alterations in p16/CDKN2A have been well documented in multiple cancers, including melanoma. However, changes in CDK4 are apparently more rare. Only two alterations, both at codon 24, have been identified in CDK4: an activating arginine-to-cysteine transition and a germ-line arginine-to-histidine substitution in one French kindred. In a survey of 20 neuroblastomas, 17 uncultured metastatic melanomas, 33 uncultured primary uveal melanomas, 8 colon cancer cell lines, and 20 primary colon cancer samples, we found no evidence of mutations in exon 2 of CDK4. From our cell lines derived from metastatic melanomas, we detected two alterations in the functionally critical exon 2 of CDK4: a lysine-to-glutamine transition at codon 22 and the arginine-to-histidine mutation at codon 24. These findings document several novel changes in the p16-binding region of CDK4.