Molecular characterization of the maize <em>(Zea mays L.) AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA)</em> gene family

The phytohormone auxin is an important molecular component in plant signal transduction. As an endogenous signaling molecule, auxin controls many aspects of plant development. Members of the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) gene family play an important role in auxin signal transduction. The rum1 (rootless with undetectable meristem 1) gene encodes the Aux/IAA protein ZmIAA29, which is to date the only Aux/IAA member with an assigned function in plant development in maize. It controls the initiation of the embryonic seminal roots and post-embryonic lateral roots of the primary root. Based on the function of rum1 in root development a comprehensive characterization of the Aux/IAA gene family was initiated in this study. At the beginning of this study 31 Aux/IAA genes were known. A homology search for novel Aux/IAA sequences in the latest maize genome assembly version identified three unknown Aux/IAA genes (ZmIAA32 - GRMZM2G366373, ZmIAA33 – GRMZM2G359924, ZmIAA34 – GRMZM2G031615). Phylogenetic reconstructions of the 34 Aux/IAA proteins revealed two classes of Aux/IAA proteins that can be distinguished by alterations in their domain III. Moreover, seven pairs of paralogous maize Aux/IAA proteins were discovered via syntenic comparisons. Comprehensive root-type and tissue-specific expression profiling revealed unique expression patterns of the diverse members of the gene family. The Aux/IAA genes displayed their highest expression in crown roots followed by seminal and primary roots. Lateral roots of the primary root displayed the lowest Aux/IAA expression level. Based on the results of the phylogenetic and expression studies five members of the maize Aux/IAA gene family, ZmIAA2, ZmIAA11, ZmIAA15, ZmIAA20 and ZmIAA33, were functionally characterized. Alternative protein variants were generated via the introduction of specific point mutations in the degron sequence by substituting the first proline by serine or the second proline by leucine. In general, Aux/IAA proteins are short-lived and localized in the nucleus. The five Aux/IAA protein half-life times ranged between ~11 min (ZmIAA2) to ~120 min (ZmIAA15) while the mutated forms were more stable. Subcellular localization studies revealed that ZmIAA2, ZmIAA11 and ZmIAA15 and their mutated forms were exclusively localized in the nucleus, whereas ZmIAA20 and ZmIAA33 were detected in nucleus and cytoplasm. Furthermore, all five maize Aux/IAA proteins were acting as active repressors. In addition, interaction of RUM1 with all five Aux/IAA proteins was detected, but only ZmIAA15 and ZmIAA33 interacted with the RUM1 paralog RUL1. In summary, the analyzed Aux/IAA genes displayed root-, tissue- and development specific expression patterns. Furthermore, the selected Aux/IAA proteins revealed different half-life times together with various activity of their repressor function. All five Aux/IAA proteins were localized in the nucleus; ZmIAA20 and ZmIAA33 were additionally expressed in the cytoplasm. Finally, specific protein interactions were identified between the selected Aux/IAA proteins with RUM1 and RUL1.Dem Phytohormon Auxin ist ein wichtige Komponente der pflanzlichen Signaltransduktion. Als endogener Signalstoff steuert Auxin vielfältige Aspekte der pflanzlichen Entwicklung. Mitglieder der AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) Genfamilie spielen eine zentrale Rolle in der Auxin-Signaltransduktion. In Mais kodiert das Gen rum1 (rootless with undetectable meristem 1) das Aux/IAA Protein ZmIAA29, dem bislang als einzigem Aux/IAA Protein eine Rolle in der Entwicklung zugeschrieben werden konnte. Es kontrolliert die Initiation der embryonalen Seminalwurzeln und der postembryonalen Lateralwurzeln der Primärwurzel. Ausgehend von diesem Befund wurde in dieser Arbeit die Aux/IAA Genfamilie von Mais genauer charakterisiert. Zu Beginn dieser Arbeit waren 31 Aux/IAA Gene im Maisgenom beschrieben. Eine Homologiesuche in der aktuellsten Annotation des Maisgenoms führte zur Identifizierung von drei weiteren Aux/IAA Genen (ZmIAA32 – GRMZM2G366373, ZmIAA33 – GRMZM2G359924 und ZmIAA34 – GRMZM2G031615). Phylogenetische Analysen aller 34 Aux/IAA Proteine ergaben eine strukturelle Aufspaltung des Stammbaums in zwei Klassen aufgrund von Unterschieden der Aminosäuresequenz in der konservierten Domäne III. Insgesamt konnten durch Syntänievergleiche sieben paraloge Aux/IAA Paare bestimmt werden. Die Aux/IAA Gene zeigten unterschiedliche Expressionsmuster in verschiedenen Wurzeltypen. Im Durchschnitt war das Expressionsniveau der Aux/IAA Gene in den Kronwurzeln am höchsten, gefolgt von den Seminal- und Primärwurzeln. Die geringste Expression wurde in den Lateralwurzeln der Primärwurzel gemessen. Basierend auf den Ergebnissen der phylogenetischen Untersuchungen und der Expressionsanalysen wurden die Aux/IAA Gene ZmIAA2, ZmIAA11, ZmIAA15, ZmIAA20 und ZmIAA33 für die weitere funktionelle Charakterisierung ausgewählt. Alternative Varianten dieser Proteine wurden durch zielgerichtete Punktmutationen erzeugt, die dazu führten, dass in den Degron-Sequenzen der fünf Aux/IAA Proteine das primäre Prolin durch Serin und das sekundäre Prolin durch Leuzin ersetzt wurden. Aux/IAA Gene kodieren für kurzlebige Proteine, welche im Kern lokalisiert sind. Die gemessenen Halbwertszeiten der untersuchten wildtypischen Aux/IAA Proteine betrugen zwischen ~11 Minuten (ZmIAA2) und ~120 Minuten (ZmIAA15). Die Punktmutationen führten zu einer deutlichen Stabilisierung der Aux/IAA Proteine im Vergleich zu den Wildtypvarianten. Die Wildtyp- sowie die mutierten Proteine von ZmIAA2, ZmIAA11 und ZmIAA15 wurden ausschließlich im Kern nachgewiesen. Dagegen waren die Proteine ZmIAA20 und ZmIAA33 in Kern und Zytoplasma lokalisiert. Außerdem konnte gezeigt werden, dass alle Aux/IAA Kandidatengene als aktive Repressoren fungieren. Weiterhin konnte eine Wechselwirkung der fünf untersuchten Aux/IAA Proteine mit RUM1 gezeigt werden, jedoch interagierten nur ZmIAA15 und ZmIAA33 mit dessen Paralog RUL1. Zusammenfassend konnte nachgewiesen werden, dass die untersuchten Aux/IAA Gene wurzel-, gewebe- und entwicklungsspezifische Expressionsmuster aufwiesen, darüber hinaus zeigten die von diesen Genen kodierten Proteine unterschiedliche Stabilitäten sowie eine unterschiedlich starke Repressoraktivität. Während alle fünf Aux/IAA Proteine im Kern lokalisiert waren, zeigten ZmIAA20 und ZmIAA33 zusätzlich Expression im Zytoplasma. Schließlich wurden spezifische Protein-Protein-Interaktionen der Aux/IAA Proteine mit RUM1 und RUL1 identifiziert

Advances in Transgenic Mouse Models to Study Infections by Human Pathogenic Viruses

Medical research is changing into direction of precision therapy, thus, sophisticated preclinical models are urgently needed. In human pathogenic virus research, the major technical hurdle is not only to translate discoveries from animals to treatments of humans, but also to overcome the problem of interspecies differences with regard to productive infections and comparable disease development. Transgenic mice provide a basis for research of disease pathogenesis after infection with human-specific viruses. Today, humanized mice can be found at the very heart of this forefront of medical research allowing for recapitulation of disease pathogenesis and drug mechanisms in humans. This review discusses progress in the development and use of transgenic mice for the study of virus-induced human diseases towards identification of new drug innovations to treat and control human pathogenic infectious diseases

Measuring and validating the levels of brain-derived neurotrophic factor in human serum

Brain-derived neurotrophic factor (BDNF) secreted by neurons is a significant component of synaptic plasticity. In humans, it is also present in blood platelets where it accumulates following its biosynthesis in megakaryocytes. BDNF levels are thus readily detectable in human serum and it has been abundantly speculated that they may somehow serve as an indicator of brain function. However, there is a great deal of uncertainty with regard to the range of BDNF levels that can be considered normal, how stable these values are over time and even whether BDNF levels can be reliably measured in serum. Using monoclonal antibodies and a sandwich ELISA, this study reports on BDNF levels in the serum of 259 volunteers with a mean value of 32.69 ± 8.33 ng/ml (SD). The mean value for the same cohort after 12 months was not significantly different (N = 226, 32.97 ± 8.36 ng/ml SD, p = 0.19). Power analysis of these values indicates that relatively large cohorts are necessary to identify significant differences, requiring a group size of 60 to detect a 20% change. The levels determined by ELISA could be validated by Western blot analyses using a BDNF monoclonal antibody. While no association was observed with gender, a weak, positive correlation was found with age. The overall conclusions are that BDNF levels can be reliably measured in human serum, that these levels are quite stable over one year, and that comparisons between two populations may only be meaningful if cohorts of sufficient sizes are assembled

Non-communicating syringomyelia: a feature of spinal cord involvement in multiple sclerosis

In patients with multiple sclerosis (MS) non-communicating syringomyelia (NCS) has been described as an incidental finding in case studies and small case series. NCS in MS patients commonly leads to uncertainty particularly as the clinical picture of NCS is variable and surgical therapy may be considered. Up to date little is known about the prevalence and clinical importance of NCS in MS. We report the imaging and clinical characteristics of NCS formations in nine MS patients from a 1 year follow-up study in a representative group of 202 MS (4.5%) patients. Brain and spinal cord MRI was performed as part of a genetic study. NCS did commonly extend the central canal and the cord was slightly distended at the level of the syrinx. The cord and syrinx showed no tendency to change in size or shape over 1 year. Despite thorough search into the clinical history and current clinical status no definite but only minimal indications of symptoms potentially related to the NCS were found. We confirm that NCS may occur in MS patients with spinal cord pathology. It can be a subtle finding without clinical correlates. Syrinx formations are more likely to be a consequence of MS cord pathology than a coincidental findin

Determination of stellar radii from asteroseismic Data

The NASA Kepler mission is designed to find planets through transits. Accurate and precise radii of the detected planets depend on knowing the radius of the host star accurately, which is difficult unless the temperature and luminosity of the star are known precisely. Kepler, however, has an asteroseismology programme that will provide seismic variables that can characterise stellar radii easily, accurately, and extremely precisely. In this paper we describe the Yale-Birmingham (YB) method to determine stellar radii using a combination of seismic and conventional variables, and analyse the effect of these variables on the result. We find that for main-sequence stars, a knowledge of the parallax is not important to get accurate radii using the YB method: we can get results to an accuracy and precision of better than a few percent if we know the effective temperature and the seismic parameters for these stars. Metallicity does not make much difference either. However, good estimates of the effective temperature and metallicity, along with those of the seismic parameters, are essential to determine radii of sub giants properly. On the other hand, for red giants we find that determining radii properly is not possible without a good estimate of the parallax. We find that the so called "surface term" in the seismic data has minimal effect on the inferred radii. Uncertainties in the convective mixing length can matter under some circumstances and can cause a systematic shift in the inferred radii. Blind tests with data simulated to match those expected from the asteroseismic Survey Phase of Kepler show that it will be possible to infer stellar radii successfully using our method.Comment: Submitted to Ap

Association of telomerase activity with radio- and chemosensitivity of neuroblastomas

<p>Abstract</p> <p>Background</p> <p>Telomerase activity compensates shortening of telomeres during cell division and enables cancer cells to escape senescent processes. It is also supposed, that telomerase is associated with radio- and chemoresistance. In the here described study we systematically investigated the influence of telomerase activity (TA) and telomere length on the outcome of radio- and chemotherapy in neuroblastoma.</p> <p>Methods</p> <p>We studied the effects on dominant negative (DN) mutant, wild type (WT) of the telomerase catalytic unit (hTERT) using neuroblastoma cell lines. The cells were irradiated with ⁶⁰Co and treated with doxorubicin, etoposide, cisplatin and ifosfamide, respectively. Viability was determined by MTS/MTT-test and the GI₅₀was calculated. Telomere length was measured by southernblot analysis and TA by Trap-Assay.</p> <p>Results</p> <p>Compared to the hTERT expressing cells the dominant negative cells showed increased radiosensitivity with decreased telomere length. Independent of telomere length, telomerase negative cells are significantly more sensitive to irradiation. The effect of TA knock-down or overexpression on chemosensitivity were dependent on TA, the anticancer drug, and the chemosensitivity of the maternal cell line.</p> <p>Conclusions</p> <p>Our results supported the concept of telomerase inhibition as an antiproliferative treatment approach in neuroblastomas. Telomerase inhibition increases the outcome of radiotherapy while in combination with chemotherapy the outcome depends on drug- and cell line and can be additive/synergistic or antagonistic. High telomerase activity is one distinct cancer stem cell feature and the here described cellular constructs in combination with stem cell markers like CD133, Aldehyddehydrogenase-1 (ALDH-1) or Side population (SP) may help to investigate the impact of telomerase activity on cancer stem cell survival under therapy.</p

Replication-incompetent influenza A viruses armed with IFN-γ effectively mediate immune modulation and tumor destruction in mice harboring lung cancer

Low pathogenic influenza A viruses (IAVs) have shown promising oncolytic potential in lung cancer-bearing mice. However, as replication-competent pathogens, they may cause side effects in immunocompromised cancer patients. To circumvent this problem, we genetically engineered nonreplicating IAVs lacking the hemagglutinin (HA) gene (ΔHA IAVs), but reconstituted the viral envelope with recombinant HA proteins to allow a single infection cycle. To optimize the therapeutic potential and improve immunomodulatory properties, these replication-incompetent IAVs were complemented with a murine interferon-gamma (mIFN-γ) gene. After intratracheal administration to transgenic mice that develop non-small cell lung cancer (NSCLC), the ΔHA IAVs induced potent tumor destruction. However, ΔHA IAVs armed with mIFN-γ exhibited an even stronger and more sustained effect, achieving 85% tumor reduction at day 12 postinfection. In addition, ΔHA-mIFN-γ viruses were proven to be efficient in recruiting and activating natural killer cells and macrophages from the periphery and in inducing cytotoxic T lymphocytes. Most important, both viruses, and particularly IFN-γ-encoding viruses, activated tumor-associated alveolar macrophages toward a proinflammatory M1-like phenotype. Therefore, replication-incompetent ΔHA-mIFN-γ-IAVs are safe and efficient oncolytic viruses that additionally exhibit immune cell activating properties and thus represent a promising innovative therapeutic option in the fight against NSCLC