Analyse von zwei strukturell ähnlichen aber funktionell unterschiedlichen viralen K+-Kanälen: Strukturelle Ursache der inhärenten Einwärtsgleichrichtung

Viren besitzen kodierende Sequenzen für Kaliumkanäle (K+-Kanäle), welche die gleichen strukturellen und funktionellen Merkmale wie komplexere K+-Kanäle aus Pro- und Eukaryonten aufweisen. So wie Letztere verfügen sie ebenfalls über Transmembran-Helices (TMs), eine kanonische Porenregion (Poren-Loop), welche zwei TMs miteinander verbindet und die typische Signatursequenz beinhaltet. In einigen Fällen sind auch sehr kurze zytoplasmatische C- und N-Termini vorhanden. Die bisher bekannten viralen K+-Kanäle sind alle aus zwei TMs, welche über die Porenregion verbunden sind, aufgebaut. Anders als die baugleichen Kir-Kanäle zeichnen sich die viralen Kanäle jedoch über eine sehr geringe Größe aus. In Abwesenheit von großen zytosolischen Domänen sind sie in der Lage mit weniger als 100 Aminosäuren funktionelle Kanäle zu bilden. Wegen der Kombination aus robuster Kanalfunktion und geringer Größe eignen sie sich hervorragend als Modellsystem, um grundlegende Struktur- und Funktionsanalysen durchzuführen, welche auf komplexere K+-Kanäle übertragen werden können. In dieser Arbeit wurden die Kmpv-Kanäle (K+-Kanal Micromonas pusilla virus), welche zu der Familie der Phycodnaviren gehören, untersucht. KmpvSP1, welcher aus dem Micromonas pusilla virus SP1 isoliert wurde, zeigt nach Expression in HEK293-Zellen sowohl in der whole-cell Konfiguration, als auch nach Rekonstitution in einen planaren Lipid-Bilayer eine Einwärtsgleichrichtung. Diese Gleichrichtung ist nicht, wie bei Kir-Kanälen, auf einen intrazellulären Block zurückzuführen. KmpvSP1 leitet auch in einer reinen K+-Lösung fast ausschließlich Einwärtsstrom, woraus zu schließen ist, dass die Gleichrichtung eine intrinsische Eigenschaft des Kanalproteins ist. Obwohl die molekularen Mechanismen der Gleichrichtung unterschiedlich sind, zeigt KmpvSP1 dennoch einige charakteristische Funktionseigenschaften von Kir-Kanälen. Wie bei den letztgenannten Kanälen verschiebt sich nach einer Veränderung der externen K+-Konzentration die Gleichrichtung mit dem Umkehrpotential. Auch im Falle des viralen Kanals hängt dabei die Leitfähigkeit von der Quadratwurzel der externen K+-Konzentration ab. Ein zweiter viraler Kanal aus dieser Familie, Kmpv1 (K+-Kanal Micromonas pusilla virus 1), welcher eine sehr ähnliche Aminosäure-Sequenz zu KmpvSP1 aufweist, zeigt ein völlig anderes elektrophysiologisches Verhalten. Dieser Kanal hat keine Gleichrichtereigenschaften, sondern zeigt eine ohmsche K+-Leitfähigkeit. Kmpv1 weist ferner eine niedrigere Sensitivität für die typischen K+-Kanalblockern Ba2+ und Cs+ auf und besitzt eine deutlich höhere Leitfähigkeit für Rb+ als K+. Im Vergleich dazu ist der Einwärtsgleichrichter KmpvSP1 sehr sensitiv für Ba2+ und Cs+ und leitet deutlich weniger Rb+ als K+. Aufgrund der elektrophysiologischen Unterschiede wurden die beiden viralen K+-Kanäle als Werkzeug benutzt, um die Struktur-Funktionsbeziehung, die für die unterschiedlichen Funktionseigenschaften verantwortlich sind, zu analysieren. Durch kombinierte Mutations- und Funktionsanalysen kann die unterschiedliche K+- und Rb+-Leitfähigkeit und die unterschiedliche Sensitivität für Cs+ auf die TMs zurückgeführt werden. Diese geht auf den Befund zurück, dass eine Chimäre, welche die TMs aus KmpvSP1 und die Pore aus Kmpv1 vereint, eine niedrige Rb+-Leitfähigkeit, eine hohe Sensitivität für Cs+ und eine deutliche Einwärtsgleichrichtung für K+ zeigt. Das entspricht den funktionellen Eigenschaften von KmpvSP1, also dem Teil des Kanals, der die TMs zu der Chimäre beisteuert. Für die hohe Ba2+-Sensitivität in KmpvSP1 kann hingegen die Struktur der Pore und zwar die Aminosäure Serin (53) hinter dem GYG-Motiv verantwortlich gemacht werden. Durch die Substitution dieser einen Aminosäure durch Phenylalanin, welche Kmpv1 an vergleichbarer Stelle trägt, kann die Sensitivität der Mutante auf das Niveau der Sensitivität des Wildtyp-Kanals Kmpv1 gesenkt werden. Der Mechanismus der Einwärtsgleichrichtung von KmpvSP1 kann in dieser Arbeit nicht vollständig geklärt werden. Die Experimente schließen jedoch die Pore als primäre Struktur für die Gleichrichtung aus. Ein Austausch der Pore in KmpvSP1, durch die entsprechende Domäne des Kanals mit ohmscher Leitfähigkeit, führt nicht zum Verlust der Gleichrichtung. In weiteren Untersuchungen wurden diejenigen Aminosäuren, in denen sich der KmpvSP1 Kanal von Kmpv1 unterscheidet, so mutiert, dass sie dem Kmpv1 gleichen. Aus der Analyse von 22 funktionellen Punktmutanten ergab sich nur eine Mutante, KmpvSP1 F72I, bei der die makroskopischen Ströme zwar sehr klein, aber nicht mehr einwärtsgleichrichtend waren. Nach einer funktionellen Rekonstitution der Mutante in planaren Lipid-Bilayern zeigte der Kanal, anders als der Wildtyp-Kanal, Kanalfluktuationen im positiven Spannungsbereich. Auch wenn diese Mutation das Schaltverhalten des Kanals verändert, kann die Aminosäure F72 nicht als einzige Ursache für die Gleichrichtung interpretiert werden. Auch die F72I Mutante zeigt immer noch eine Einwärtsgleichrichtung mit einer höheren Offenwahrscheinlichkeit bei negativen im Vergleich zu positiven Spannungen. Zudem hat die gleiche Mutation in einem nahe verwandten K+-Kanal von KmpvSP1 dem KmpvPL1, keinerlei Auswirkung auf die Gleichrichtereigenschaften

Direct observation of nanocrystal-induced enhancement of tensile ductility in a metallic glass composite

Bulk metallic glasses (BMGs) have attracted wide interest, but their successful application is hindered by their low ductility at room temperature. Therefore, the use of composites of a BMG matrix with crystalline secondary phases has been proposed to overcome this drawback. In the present work we demonstrate the fabrication of a tailored BMG nanocomposite containing a high density of monodisperse nanocrystals with a size of around 20 nm using a combination of mechanical and thermal treatment of Cu36Zr48Al8Ag8 well below the crystallization temperature. Direct observations of the interaction of the nanocrystals with a shear band during in situ deformation in a transmission electron microscope demonstrate that the achieved nanocomposite has the potential to inhibit catastrophic fracture in tension. This demonstrates that a sufficient number of nanoscale structural heterogeneities can be a route towards BMG composites with superior mechanical properties

Associação entre ingesta de leite materno e desenvolvimento de retinopatia da prematuridade

Purpose: To evaluate the possible protective effect of breast milk against retinopathy of prematurity by comparing the amount of breast milk received by patients who developed retinopathy of prematurity and those who did not and to determine both the required minimum amount of breast milk and the time of life during which neonates need to receive breast milk for this effect to be significant. Methods: Cohort study of newborns with a birth weight of <1500 g or gestational age of <32 weeks, or both, born between January 2011 and October 2014 and hospitalized within the first 24 h of life in the Hospital Criança Conceição Neonatal Intensive Care Unit in Porto Alegre, RS, Brazil. Results: The prevalence of retinopathy of prematurity of any degree was 31% (100 of 323 patients) and that of severe retinopathy of prematurity was of 9% (29 of 323 patients). The median amounts of breast milk received daily by patients with and without retinopathy of prematurity were 4.9 mL/kg (interquartile range, 0.3-15.4) and 10.2 mL/kg (1.5-25.5), respectively. The amount of breast milk received in the first 6 weeks of life was inversely associated with the incidence of both retinopathy of prematurity of any degree and severe retinopathy of prematurity in the univariate analyses. However, the statistical significance was maintained only during the sixth week of life in a per-period multivariate analysis controlling for confounding factors. Conclusions: Small amounts of breast milk are inadequate to prevent retinopathy of prematurity in premature newborns at risk for the disease.Objetivos: Avaliar o possível efeito protetor do leite materno contra a retinopatia da prematuridade, através da comparação da quantidade de leite materno recebida entre os pacientes que desenvolveram retinopatia da prematuridade e aqueles livres da doença. Tentar determinar a quantidade mínima necessária e o momento em que o recém-nascido precisa receber o leite materno para que esse efeito seja significativo. Métodos: Estudo de coorte observacional incluindo recém-nascidos com peso de nascimento inferior a 1500 gramas e/ou com idade gestacional inferior a 32 semanas, nascidos no período de janeiro de 2011 a outubro de 2014 e internados nas primeiras 24 horas de vida na UTI Neonatal do Hospital da Criança Conceição em Porto Alegre. Resultados: A prevalência da retinopatia da prematuridade em qualquer grau foi de 31% (100 casos em 323 pacientes) e a de retinopatia da prematuridade grave foi de 9% (29 casos em 323 pacientes). A mediana da quantidade de leite materno recebida pelos pacientes foi de 10,2 mL/kg/dia entre os pacientes sem retinopatia da prematuridade (amplitude interquartil 1,5-25,5) e de 4,9 mL/kg/dia entre os pacientes com retinopatia da prematuridade (0,3-15,4). A quantidade de leite materno recebida nas primeiras seis semanas de vida foi inversamente associada à incidência de retinopatia da prematuridade em qualquer grau e de retinopatia da prematuridade grave nas análises univariadas, mas a significância estatística não se manteve após análise multivariada para controle de fatores confundidores na maioria dos períodos avaliados, exceto na sexta semana de vida. Conclusão: Pequenas quantidades de leite materno não são suficientes para prevenção de retinopatia da prematuridade em recém-nascidos com de risco para a doença

A small viral potassium ion channel with an inherent inward rectification

Some algal viruses have coding sequences for proteins with structural and functional characteristics of pore modules of complex K+ channels. Here we exploit the structural diversity among these channel orthologs to discover new basic principles of structure/function correlates in K+ channels. The analysis of three similar K+ channels with ≤ 86 amino acids (AA) shows that one channel (Kmpv1) generates an ohmic conductance in HEK293 cells while the other two (KmpvSP1, KmpvPL1) exhibit typical features of canonical Kir channels. Like Kir channels, the rectification of the viral channels is a function of the K+ driving force. Reconstitution of KmpvSP1 and KmpvPL1 in planar lipid bilayers showed rapid channel fluctuations only at voltages negative of the K+ reversal voltage. This rectification was maintained in KCl buffer with 1 mM EDTA, which excludes blocking cations as the source of rectification. This means that rectification of the viral channels must be an inherent property of the channel. The structural basis for rectification was investigated by a chimera between rectifying and non-rectifying channels as well as point mutations making the rectifier similar to the ohmic conducting channel. The results of these experiments exclude the pore with pore helix and selectivity filter as playing a role in rectification. The insensitivity of the rectifier to point mutations suggests that tertiary or quaternary structural interactions between the transmembrane domains are responsible for this type of gating

Impact of medical practice guidelines on the assessment of patients with acute coronary syndrome without persistent ST segment elevation

Objective. To assess the impact of introducing clinical practice guidelines on acute coronary syndrome without persistent ST segment elevation (ACS) on patient initial assessment. Design. Prospective before-after evaluation over a 3-month period. Setting. The emergency ward of a tertiary teaching hospital. Patients. All consecutive patients with ACS evaluated in the emergency ward over the two 3-month periods. Intervention. Implementation of the practice guidelines, and the addition of a cardiology consultant to the emergency team. Main outcome measures. Diagnosis, electrocardiogram interpretation, and risk stratification after the initial evaluation. Results. The clinical characteristics of the 328 and 364 patients evaluated in the emergency ward for suspicion of ACS before and after guideline implementation were similar. Significantly more patients were classified as suffering from atypical chest pain (39.6% versus 47.0%; P = 0.006) after guideline implementation. Guidelines availability was associated with significantly more formal diagnoses (79.9% versus 92.9%; P < 0.0001) and risk stratification (53.7% versus 65.4%, P < 0.0001) at the end of initial assessment. Conclusion. Guidelines implementation, along with availability of a cardiology consultant in the emergency room had a positive impact on initial assessment of patients evaluated for suspicion of ACS. It led to increased confidence in diagnosis and stratification by risk, which are the first steps in initiating effective treatment for this common conditio

Hospital waste: impact on Allium cepa cells

The dumping of chemical substances, from urban, industrial and hospital waste, is the main responsible for the high concentrations of toxic substances in the environment. The adverse effects and risks that these substances may cause to the ecosystem have become the target of research in the scientific environment. The chemical substances present in tailings can be metabolized, stored as well as interact directly with the DNA of eukaryotic cells promoting pre-oncogenic events. To assess the impact of so-called emerging contaminants (EDC's) on the genome of eukaryotes, it is possible to use micronucleus (MN) and nuclear abnormalities (NA) tests through onion root (Allium cepa) meristematic cells. The objective of this research was to investigate the cellular, genotoxic and clastogenic alterations in onion root meristematic cells, through exposure in three hospital waste sampling efforts at three different times. The collected samples were analyzed in a gas chromatograph coupled to a mass spectrometer (GC-MS QP 2010) in order to qualify the constituents present in the tailings. By analyzing semi-permanent slides stained by the Shiff method, the nuclear and micronucleus abnormalities were computed in 3000 Allium cepa meristematic cells exposed to hospital waste for 96 hours. The results obtained show that the calculated MI (Mitotic Index) of the cells submitted to the waste were significantly lower when compared to the negative control group. There is also an important correlation between mitotic index and MN appearance. In the GC-MS analysis, several main peaks were observed and different retention times were identified. In treatment 1 T (3.44) and treatment 2, T (3.47), Propoxur was one of the products found that offers clastogenic potential as it is a synthetic carbamate, composed of aromatic ether and acetylcholinesterase inhibitor, widely used as a pesticide. The results show that the waste from the hospital under study has cytotoxic and genotoxic potential

ROS- and Radiation Source-Dependent Modulation of Leukocyte Adhesion to Primary Microvascular Endothelial Cells

Anti-inflammatory effects of low-dose irradiation often follow a non-linear dose–effect relationship. These characteristics were also described for the modulation of leukocyte adhesion to endothelial cells. Previous results further revealed a contribution of reactive oxygen species (ROS) and anti-oxidative factors to a reduced leukocyte adhesion. Here, we evaluated the expression of anti-oxidative enzymes and the transcription factor Nrf2 (Nuclear factor-erythroid-2-related factor 2), intracellular ROS content, and leukocyte adhesion in primary human microvascular endothelial cells (HMVEC) upon low-dose irradiation under physiological laminar shear stress or static conditions after irradiation with X-ray or Carbon (C)-ions (0–2 Gy). Laminar conditions contributed to increased mRNA expression of anti-oxidative factors and reduced ROS in HMVEC following a 0.1 Gy X-ray and 0.5 Gy C-ion exposure, corresponding to reduced leukocyte adhesion and expression of adhesion molecules. By contrast, mRNA expression of anti-oxidative markers and adhesion molecules, ROS, and leukocyte adhesion were not altered by irradiation under static conditions. In conclusion, irradiation of endothelial cells with low doses under physiological laminar conditions modulates the mRNA expression of key factors of the anti-oxidative system, the intracellular ROS contents of which contribute at least in part to leucocyte adhesion, dependent on the radiation source

Modulation of Differentiation and Bone Resorbing Activity of Human (Pre-) Osteoclasts After X-Ray Exposure

Low-dose radiotherapy (LD-RT) is a local treatment option for patients with chronic degenerative and inflammatory diseases, in particular musculoskeletal diseases. Despite reported analgesic and anti-inflammatory effects, cellular and molecular mechanisms related to osteoimmunological effects are still elusive. Here we test the hypothesis that X-irradiation inhibits the differentiation of precursor osteoclasts into mature osteoclasts (mOC) and their bone resorbing activity. Circulating monocytes from healthy donors were isolated and irradiated after attachment with single or fractionated X-ray doses, comparable to an LD-RT treatment scheme. Then monocytes underwent ex vivo differentiation into OC during cultivation up to 21 days, under conditions mimicking the physiological microenvironment of OC on bone. After irradiation, apoptotic frequencies were low, but the total number of OC precursors and mOC decreased up to the end of the cultivation period. On top, we observed an impairment of terminal differentiation, i.e. a smaller fraction of mOC, reduced resorbing activity on bone, and release of collagen fragments. We further analyzed the effect of X-irradiation on multinucleation, resulting from the fusion of precursor OC, which occurs late during OC differentiation. At 21 days after exposure, the observation of smaller cellular areas and a reduced number of nuclei per mOC suggest an impaired fusion of OC precursors to form mOC. Before, at 14 days, the nuclear translocation of Nuclear Factor Of Activated T Cells 1 (NFATc1), a master regulator of osteoclast differentiation and fusion, was decreased. In first results, obtained in the frame of a longitudinal LD-RT study, we previously reported a pain-relieving effect in patients. However, in a subgroup of patients suffering from Calcaneodynia or Achillodynia, we did not observe a consistent decrease of established blood markers for resorption and formation of bone, or modified T cell subtypes involved in regulating these processes. To assess the relevance of changes in bone metabolism for other diseases treated with LD-RT will be subject of further studies. Taken together, we observed that in vitro X-irradiation of monocytes results in an inhibition of the differentiation into bone-resorbing OC and a concomitant reduction of resorbing activity. The detected reduced NFATc1 signaling could be one underlying mechanism