Distorted copper homeostasis with decreased sensitivity to cisplatin upon chaperone Atox1 deletion in Drosophila

Copper is an integral part of a number of proteins and thus an essential trace metal. However, free copper ions can be highly toxic and every organism has to carefully control its bioavailability. Eukaryotes contain three copper chaperones; Atx1p/Atox1 which delivers copper to ATP7 transporters located in the trans-Golgi network, Cox17 which provides copper to the mitochondrial cytochrome c oxidase, and CCS which is a copper chaperone for superoxide dismutase 1. Here we describe the knockout phenotype of the Drosophila homolog of mammalian Atox1 (ATX1 in yeast). Atox1−/− flies develop normally, though at reduced numbers, and the eclosing flies are fertile. However, the mutants are unable to develop on low-copper food. Furthermore, the intestinal copper importer Ctr1B, which is regulated by copper demand, fails to be induced upon copper starvation in Atox1−/− larvae. At the same time, intestinal metallothionein is upregulated. This phenotype, which resembles the one of the ATP7 mutant, is best explained by intestinal copper accumulation, combined with insufficient delivery to the rest of the body. In addition, compared to controls, Drosophila Atox1 mutants are relatively insensitive to the anticancer drug cisplatin, a compound which is also imported via Ctr1 copper transporters and was recently found to bind mammalian Atox

Characterization of MtnE, the fifth metallothionein member in Drosophila

Metallothioneins (MTs) constitute a family of cysteine-rich, low molecular weight metal-binding proteins which occur in almost all forms of life. They bind physiological metals, such as zinc and copper, as well as nonessential, toxic heavy metals, such as cadmium, mercury, and silver. MT expression is regulated at the transcriptional level by metal-regulatory transcription factor1 (MTF-1), which binds to the metal-response elements (MREs) in the enhancer/promoter regions of MT genes. Drosophila was thought to have four MT genes, namely, MtnA, MtnB, MtnC, and MtnD. Here we characterize a new fifth member of Drosophila MT gene family, coding for metallothionein E (MtnE). The MtnE transcription unit is located head-to-head with the one of MtnD. The intervening sequence contains four MREs which bind, with different affinities, to MTF-1. Both of the divergently transcribed MT genes are completely dependent on MTF-1, whereby MtnE is consistently more strongly transcribed. MtnE expression is induced in response to heavy metals, notably copper, mercury, and silver, and is upregulated in a genetic background where the other four MTs are missin

Polyglutamine tracts as modulators of transcriptional activation from yeast to mammals

Microsatellite repeats are genetically unstable and subject to expansion and shrinkage. A subset of them, triplet repeats, can occur within the coding region and specify homomeric tracts of amino acids. Polyglutamine (polyQ) tracts are enriched in eukaryotic regulatory proteins, notably transcription factors, and we had shown before that they can contribute to transcriptional activation in mammalian cells. Here we generalize this finding by also including evolutionarily divergent organisms, namely, Drosophila and baker's yeast. In all three systems, Gal4-based model transcription factors were more active if they harbored a polyQ tract, and the activity depended on the length of the tract. By contrast, a polyserine tract was inactive. PolyQs acted from either an internal or a C-terminal position, thus ruling out a merely structural ‘linker' effect. Finally, a two-hybrid assay in mammalian cells showed that polyQ tracts can interact with each other, supporting the concept that a polyQ-containing transcription factor can recruit other factors with polyQ tracts or glutamine-rich activation domains. The widespread occurrence of polyQ repeats in regu­latory proteins suggests a beneficial role; in addition to the contribution to transcriptional activity, their genetic instability might help a species to adapt to changing environmental conditions in a potentially reversible manne

Using Business Intelligence Tools to Support Medical Validation of Laboratory Tests

Modern clinical laboratories have to confirm that the procedures used for specific tests are reliable and valid. There are several sources of errors and interferences that can invalidate the results. Medical validation refers to the plausibility check of the test results. Implausible results indicate that something might went wrong with a sample retrieved from the patient, e.g., the blood sample got contaminated with another fluid, which requires re-examination. Here, we describe how an integrated R-based business intelligence (BI) tool can be developed that increase the efficiency of the medical validation at the Institute of Clinical Chemistry (ICC) of the University Hospital Zurich. A BI software environment allowed us to digitalize steps in the validation process that were manually done in Excel worksheets, e.g., importing the data, calculating percentiles, and producing graphical outputs

Head to head evaluation of the analytical performance of two commercial methotrexate immunoassays and comparison with liquid chromatography-mass spectrometry and the former fluorescence polarization immunoassay

AbstractBackground: Monitoring of plasma drug levels is mandatory in patients receiving high-dose methotrexate. This study evaluated the analytical performance of the novel Architect and the established ARK™ methotrexate immunoassay (running on the Roche Cobas© c502 analyzer) in comparison with liquid chromatography-mass spectrometry (LC-MS) and the TDx/TDxFLx Methotrexate II assay. Methods: Imprecision and linearity were verified for the Architect and ARK assay according to CLSI EP15-A3 and EP6-A guidelines, respectively. The reported limit of quantitation (0.04 μmol/L) was tested for both assays according to the CLSI EP17-A2 guideline. Correlation and agreement between the different assays were evaluated using residual plasma samples (n=153). Results: Total imprecision was <6.3% and <9.5% for the Architect and ARK assay, respectively. The claimed linearity and limit of quantitation were confirmed for the Architect assay. For the ARK assay, imprecision at the limit of quantitation was <18% with a positive bias resulting in a high total error up to 58%, and hence the linearity could not be confirmed. Both assays showed strong correlations with the TDX assay and LC-MS but a positive bias of 12.2% and 20.5% in comparison to LC-MS for the Architect and ARK assay, respectively. For the ARK assay this bias increased dramatically for samples with concentrations towards the limit of quantitation. Conclusions: The Architect assay is suitable for monitoring plasma methotrexate, but the ARK assay showed unsatisfactory performance in the analysis of low concentrated samples. Unlike the TDX assay, both assays require manual dilution of samples at higher concentrations, which delays sample processing in clinical routine

Navigiert oder konventionell in der Acetabulumchirurgie

Hintergrund Behandlungsprinzip der gelenkerhaltenden Therapie von Acetabulumfrakturen ist die anatomische Reposition der gelenktragenden Elemente und die interne Osteosynthese. Um den vorderen und den hinteren Pfeiler gegeneinander zu stabilisieren, wird die infraazetabuläre Schraube (IAS) im klinischen Alltag regelhaft eingesetzt. Ziel Ziel der vorliegenden Studie ist es, die Lage der IAS im infraazetabulären Korridor nach navigierter Platzierung mit der nach Freihandplatzierung zu vergleichen. Material und Methode Die Lage der Schraube wurde bei 42 Patienten mithilfe multiplanarer Rekonstruktionen evaluiert. Bei 30 Patienten wurde diese freihandplatziert, bei 12 Patienten mittels bildgestützter Navigation. Neben der Vermessung der Schraubenlage wurden demografische Daten, Operationszeit, Strahlenbelastung sowie Blutverlust erhoben. Ergebnisse Der überwiegende Teil der Patienten war männlich (86 %), das mediane Alter lag bei 67 Jahren und der mediane BMI bei 25 kg/m2. Die mediane Operationszeit betrug 166 min, und die mediane Blutverlustmenge lag bei 900 ml. Die adjustierten Werte in der gesamten Stichprobe bezüglich der Position der Schrauben lagen bei: Abstand Schraube zum Knorpel Mittelwert (MW) = 3,8 mm, Abstand Schraube zum Korridorzentrum MW = 3,5 mm, Winkel Schraube zum Korridor MW = 1,4°. Die zwei Gruppen unterschieden sich nicht in den demografischen Parametern sowie in der Genauigkeit der Positionierung der Schrauben (p-Werte > 0,05). In der navigierten Gruppe zeigten sich eine längere Strahlungszeit und höhere Strahlendosis im Vergleich zur Gruppe ohne Navigation (p-Werte < 0,001). Schlussfolgerung Die beiden Verfahren sind bei entsprechender Erfahrung hinsichtlich der Genauigkeit vergleichbar. Hinsichtlich weiterer perioperativer Parameter wie Strahlenbelastung und geplanter Operationsdauer sollten auch patientenbezogene Faktoren berücksichtigt werden

Implementation of targeted next-generation sequencing for the diagnosis of drug-resistant tuberculosis in low-resource settings: a programmatic model, challenges, and initial outcomes

Targeted next-generation sequencing (tNGS) from clinical specimens has the potential to become a comprehensive tool for routine drug-resistance (DR) prediction of Mycobacterium tuberculosis complex strains (MTBC), the causative agent of tuberculosis (TB). However, TB mainly affects low- and middle-income countries, in which the implementation of new technologies have specific needs and challenges. We propose a model for programmatic implementation of tNGS in settings with no or low previous sequencing capacity/experience. We highlight the major challenges and considerations for a successful implementation. This model has been applied to build NGS capacity in Namibia, an upper middle-income country located in Southern Africa and suffering from a high-burden of TB and TB-HIV, and we describe herein the outcomes of this process

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio