Die Rolle von IQGAP3 in der Zellzyklusprogression

The cell cycle is a critical process for organism development and tissue homeostasis. In addition, it is required for organ regeneration and its mis-regulation is known to lead to cancer. Thus, tight regulation of the cell cycle is of fundamental importance. In the heart, cardiomyocytes proliferate during early mammalian development while they reach a post-mitotic state after birth. This state can be reversed in postnatal day 3 cardiomyocytes by treating them with fibroblast growth factor 1 (FGF1) and an inhibitor of p38 mitogen-activated protein kinase (p38i). In addition tumors derived from cardiomyocytes are extremely rare in adults. These characteristics make cardiomyocytes a good model to identify novel cell cycle regulators. Therefore, the aim of this PhD thesis was to identify a new cell cycle regulator, by comparing gene expression data from heart development and cardiomyocyte cell cycle reentry. Candidate genes from the heart development database were chosen from a cluster of similar temporal expression profiles enriched in mitotic genes. Subsequently, it was assessed which of the candidate genes is reexpressed upon FGF1/p38i stimulation. A literature-based analysis identified Iqgap3 as a possible candidate gene. Immunofluorescence analyses showed that IQGAP3 is mainly expressed in mitosis. It concentrates at the cleavage furrow, at the stem body of the midbody and later at the midbody remnant due to its N-terminal part. Gene silencing experiments have demonstrated that IQGAP3 is required for cell cycle progression because its depletion results in a decreased proliferation rate and a delay in S and G2/M phase progression. Time-lapse movies indicated that IQGAP3 depletion caused a mitotic delay from pro-metaphase to abscission. However, IQGAP3-depleted HeLa cells did not exhibit any alterations in the localization of proteins known to be important for cytokinesis suggesting that IQGAP3 is not required for cytokinesis, also because no binucleation was observed. Closer analyses of time-lapse movies have highlighted the presence of mitotic multipoles in IQGAP3-depleted HeLa cells. In addition, FISH analysis has demonstrated that IQGAP3-depleted cell cultures accumulate over time aneuploid cells. These data demonstrate that IQGAP3 is a novel cell cycle factor playing a possible role in chromosome segregation preventing aneuploidy.Der Zellzyklus ist ein kritischer Prozeß für die Entwicklung eines Organismus und die Gewebshomöostase. Zudem ist er für die Organregeneration erforderlich und seine Fehlregulation führt bekannter Weise zu Krebs. Daher ist die enge Regulierung des Zellzyklus von grundlegender Bedeutung. Im Herzen proliferieren Herzmuskelzellen während der frühen Säugetierentwicklung während sie nach der Geburt einen post-mitotischen Zustand einnehmen. Dieser Zustand kann in postnatalen Tag 3 Herzmuskelzellen durch Behandlung mit Fibroblasten-Wachstumsfaktor 1 (FGF1) und einem Inhibitor der p38 Mitogen-aktivierten Proteinkinase (p38i) umgekehrt werden. Ferner sind Tumore die von Herzmuskelzellen abgeleitet sind bei Erwachsenen extrem selten. Diese Eigenschaften machen Herzmuskelzellen zu einem guten Modell, um neue Zellzyklusregulatoren zu identifizieren. Daher war das Ziel dieser Doktorarbeit einen neuen Zellzyklus-Regulator zu identifizieren durch den Vergleich von Genexpressionsdaten aus der Herzentwicklung und Herzmuskelzellzyklus-Wiedereintritt. Kandidatengene aus der Herzentwicklungsdatenbank wurden aus einem Cluster zeitlich ähnlicher Expressionsprofile ausgewählt, das in mitotischen Genen angereichert war. Anschließend wurde untersucht, welche der Kandidatengene nach FGF1/p38i Stimulation reexprimiert werden. Eine Literatur-basierte Analyse identifizierte Iqgap3 als mögliches Kandidatengen. Immunfluoreszenz-Analysen zeigten, daß IQGAP3 hauptsächlich in der Mitose exprimiert wird. Es konzentriert sich aufgrund seines N-terminalen Teils an der Teilungsfurche, am Flemming-Körper und später am Rest des Flemming-Körpers. Gen-Silencing-Experimente zeigten, dass IQGAP3 für die Zellzyklusprogression benötigt wird, da seine Depletierung in einer verringerten Proliferationsrate und einer Verzögerung der S und G2/M-Progression resultierte. Lebendzellbeobachtungen deuteten darauf hin, dass IQGAP3 Depletierung eine mitotische Verzögerung von Prometaphase bis zur Abschnürung verursacht. Allerdings wiesen IQGAP3-depletierte HeLa-Zellen keine Veränderungen in der Lokalisierung von für die Zellteilung wichtigen Proteinen auf. Dies deutete darauf hin, daß IQGAP3 nicht für die Zytokinese erforderlich ist, da auch keine Binukleation beobachtet wurde. Eine nähere Analyse von Lebendzellbeobachtungen hat das Vorhandensein von mitotischen Multipolen in IQGAP3-depletierten HeLa-Zellen hervorgehoben. Darüber hinaus hat eine FISH Analyse gezeigt, daß IQGAP3-depletierte Zellkulturen über die Zeit aneuploide Zellen akkumulieren. Diese Daten zeigen, daß IQGAP3 ein neuer Zellzyklus-Faktor ist, der möglicherweise eine Rolle in der Chromosomensegregation spielt um Aneuploidie zu verhindern

Mediterranean jellyfish as novel food: effects of thermal processing on antioxidant, phenolic, and protein contents

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Concepts Are More than Percepts: The Case of Action Verbs

Several regions of the posterior-lateral-temporal cortex (PLTC) are reliably recruited when participants read or listen to action verbs, relative to other word and nonword types. This PLTC activation is generally interpreted as reflecting the retrieval of visual-motion features of actions. This interpretation supports the broader theory, that concepts are comprised of sensory–motor features. We investigated an alternative interpretation of the same activations: PLTC activity for action verbs reflects the retrieval of modality-independent representations of event concepts, or the grammatical types associated with them, i.e., verbs. During a functional magnetic resonance imaging scan, participants made semantic-relatedness judgments on word pairs varying in amount of visual-motion information. Replicating previous results, several PLTC regions showed higher responses to words that describe actions versus objects. However, we found that these PLTC regions did not overlap with visual-motion regions. Moreover, their response was higher for verbs than nouns, regardless of visual-motion features. For example, the response of the PLTC is equally high to action verbs (e.g., to run) and mental verbs (e.g., to think), and equally low to animal nouns (e.g., the cat) and inanimate natural kind nouns (e.g., the rock). Thus, PLTC activity for action verbs might reflect the retrieval of event concepts, or the grammatical information associated with verbs. We conclude that concepts are abstracted away from sensory–motor experience and organized according to conceptual properties.Psycholog

A new type of Na+-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif

Abstract: The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen. Author Summary: Essential cellular processes such as biosynthesis, transport, and motility are sustained by the energy released in the hydrolysis of ATP, the universal energy carrier in living cells. Most ATP in the cell is produced by a membrane-bound enzyme, the ATP synthase, through a rotary mechanism that is coupled to the translocation of ions across the membrane. The majority of ATP synthases are energized by transmembrane electrochemical gradients of protons (proton-motive force), but a number of organisms, including some important human pathogens, use gradients of sodium ions instead (sodium-motive force). The ion specificity of ATP synthases is determined by a membrane-embedded sub-complex, the c-ring, which is the smallest known biological rotor. The functional mechanism of the rotor ring and its variations among different organisms are of wide interest, because of this enzyme's impact on metabolism and disease, and because of its potential for nanotechnology applications. Here, we characterize a previously unrecognized type of Na+-driven ATP synthase from the opportunistic human pathogen Fusobacterium nucleatum, which is implicated in periodontal diseases. We analyzed this ATP synthase and its rotor ring through a multi-disciplinary approach, combining cell-growth and biochemical assays, X-ray crystallography and computer-simulation methods. Two crystal structures of the membrane rotor were solved, at low and high pH, revealing an atypical ion-recognition motif mediated by two carboxylate side-chains. This motif is shared by other human pathogens, such as Mycobacterium tuberculosis or Streptococcus pneumonia, whose ATP synthases are targets of novel antibiotic drugs. The implications of this ion-recognition mode on the mechanism of the ATP synthase and the cellular bioenergetics of F. nucleatum were thus examined. Our results provide the basis for future pharmacological efforts against this important pathogen

Wildfires Impact Assessment on PM Levels Using Generalized Additive Mixed Models

Abstract: Wildfires are relevant sources of PM emissions and can have an important impact on air pollution and human health. In this study, we examine the impact of wildfire PM emissions on the Piemonte (Italy) air quality regional monitoring network using a Generalized Additive Mixed Model. The model is implemented with daily PM10 and PM2.5 concentrations sampled for 8 consecutive years at each monitoring site as the response variable. Meteorological data retrieved from the ERA5 dataset and the observed burned area data stored in the Carabinieri Forest Service national database are used in the model as explanatory variables. Spline functions for predictive variables and smooths for multiple meteorological variables’ interactions improved the model performance and reduced uncertainty levels. The model estimates are in good agreement with the observed PM data: adjusted R2 range was 0.63–0.80. GAMMs showed rather satisfactory results in order to capture the wildfires contribution: some severe PM pollution episodes in the study area due to wildfire air emissions caused peak daily levels up to 87.3 µg/m3 at the Vercelli PM10 site (IT1533A) and up to 67.7 µg/m3 at the Settimo Torinese PM2.5 site (IT1130A)

The sense of sounds: brain responses to phonotactic frequency, phonological grammar and lexical meaning

Two outstanding questions in spoken-language comprehension concern (1) the interplay of phonological grammar (legal vs. illegal sound sequences), phonotactic frequency (high- vs. low-frequency sound sequences) and lexicality (words vs. other sound sequences) in a meaningful context, and (2) how the properties of phonological sequences determine their inclusion or exclusion from lexical-semantic processing. In the present study, we used a picture-sound priming paradigm to examine the ERP responses of adult listeners to grammatically illegal sound sequences, to grammatically legal sound sequences (pseudowords) with low- vs. high-frequency, and to real words that were either congruent or incongruent to the picture context. Results showed less negative N1-P2 responses for illegal sequences and low-frequency pseudowords (with differences in topography), but not high-frequency ones. Low-frequency pseudowords also showed an increased P3 component. However, just like illegal sequences, neither low- nor high-frequency pseudowords differed from congruent words in the N400. Thus, phonotactic frequency had an impact before, but not during lexical-semantic processing. Our results also suggest that phonological grammar, phonotactic frequency and lexicality may follow each other in this order during word processing

Multiple sclerosis in the Republic of Moldova: a descriptive study of prevalence and evolution of clinical manifestations

Introducere. Scleroza multiplă (SM) este o boală inflamatorie cronică a sistemului nervos central, care poate duce la invaliditate permanentă la adulţii tineri. Investigarea epidemiologiei SM în Republica Moldova prezintă interes, luând în consideraţie şi faptul că evoluţia naturală a bolii încă nu este influenţată de utilizarea medicamentelor modificatoare a bolii. Material şi metode. În studiu au fost incluşi pacienţii diagnosticaţi cu SM conform criteriilor McDonald 2010, formele clinic şi imagistic definite. Sursele epidemiologice au fost colectate din înregistrările medicilor de familie din diferite regiuni ale ţării, ale neurologilor din spitalele raionale, municipale şi orăşeneşti, din arhivele centrelor de diagnostic republicane de imagistică prin rezonanţă magnetică (IRM), din registrele administrative (centralizate). Au fost înregistraţi parametrii demografici ai pacienţilor şi cei clinici ai maladiei. Rezultate. Şapte sute patruzeci şi şapte de pacienţi cu SM locuiau în Republica Moldova la ziua de prevalenţă (31 decembrie 2012). Astfel, prevalenţa estimată a SM în Republica Moldova este de 21,0 la 100.000 de locuitori (95% CI: 14,8–27,1). Din 724 de cazuri de SM, 460 (63,5%) au fost de sex feminin (vârsta medie – 42,1±11,9 ani) şi 264 (36,5%) – de sex masculin (vârsta medie – 40,8±12,8 ani); în cele 23 de cazuri de SM restante, sexul pacienţilor nu a putut fi identificat în documentele medicale originale. Prevalenţa maximă a SM a fost identificată la grupul de vârstă de 40-49 de ani: la femei – 57,0 la 100.000 locuitori, la bărbaţi – 29,0 la 100.000 locuitori. Distribuţia a 721 de cazuri prevalente pe zone administrative în Republica Moldova, a fost următoarea: 32% – Centru, 13,9% – municipiul Chişinău, 33,8% – Nord, 11,1% – Sud, 2,1% – Unităţile teritoriale din stânga Nistrului şi 7,1% – UTA Găgăuzia. Concluzii. Prevalenţa brută estimată a sclerozei multiple în Republica Moldova a fost de 21,0 cazuri la 100.000 de locuitori, care s-a dovedit a fi mai mică decât în ţările din Europa. Cel mai probabil, prevalenţa SM în Republica Moldova este subestimată din cauza ignorării simptomatologiei precoce a maladiei de către pacient, adresării tardive la neurolog sau, deoarece diagnosticul dat nu este stabilit la adresarea pacientului simptomatic.Introduction. Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system, which can lead to standing disability in young adults. Investigation of MS epidemiology in the Republic of Moldova shows interest, taking into account the fact that the natural evolution of the disease is still not affected by use of the disease modifying drugs. Material and methods. The study included patients diagnosed with MS according to the McDonald criteria 2010, clinical and imagistic defined forms. Epidemiological sources were collected from records of family doctors from different regions of the country, neurologists form district, municipal and city hospitals, investigations archives of magnetic resonance imaging (MRI) of republican diagnostic centers, administrative records (centralized). Collection of cases included: questionnaire of patient assessment with inclusion of demographic and clinical data. Results. 747 MS patients were living in the study area, on the prevalence day, 31 December 2012. A crude prevalence was 21.0 per 100.000 inhabitants (95% CI: 14.8–27.1). From 724 prevalent cases, 460 (63.5%) were females with a mean age of 42.1±11.9 years and 264 (36.5%) were males, mean age of 40. 8±12.8 years. The highest estimates were observed in the age group 40-49, for women, 57.0 per 100.000 inhabitants and, for men, 29.0 per 100.000 inhabitants. The distribution of 721 prevalent cases by administrative areas in the Republic of Moldova was the following: Center – 32%, Chisinau – 13.9%, North – 33.8%, South – 11.1%, Transnistria – 2.1% and UTA Gagauzia – 7.1%. Conclusion. The estimated prevalence of multiple sclerosis in the Republic of Moldova was 21.0 per 100.000 inhabitants. This proved to be lower than in European countries. Also the patient’s neglect of the first appeared signs and symptoms lead to a late addressing to the neurologist doctor. On the other hand, when the patient addresses in time to a doctor, the reason why MS is not detected would be underestimating the diagnosis, which in some cases is established with delay and in the other cases it is not set at all