Transkriptiotekijöiden GATA4 ja NKX2-5 yhteisvaikutukseen kohdennettujen isoksatsoli-amidi yhdisteiden suunnittelu ja kehitys

Acute myocardial infarction is a life-threatening condition that occurs as a result of reduced blood flow in the cardiac muscle, eventually leading to tissue damage. In infarcted areas, cardiomyocytes have insufficient ability to proliferate and replace the injured cells, which is associated with a deficient pumping capacity. A strictly regulated combinatorial interplay of transcription factors, e.g., GATA4, NKX2-5, TBX5, and MEF2C, orchestrates cardiac type gene expression during the cardiomyocyte differentiation and maturation processes. The aim of the present study was to (i) characterize the protein-protein interaction of the cardiac transcription factors GATA4-NKX2-5, (ii) evaluate the chemical agents that modify the synergy of GATA4-NKX2-5 in vitro, (iii) examine the capacity of the lead compound to promote myocardial repair in vivo after myocardial infarction and other cardiac injuries and (iv) study the structural features of the compound important for metabolism and cytotoxicity. Integration of the experimental mutagenic data with computational modeling suggests that the structural architecture of the GATA4-NKX2-5 interaction resembles the protein structure of the conserved DNA binding domain of nuclear receptors. Fragment-based screening, reporter gene-based optimization and pharmacophore searching were utilized to identify the most potent lead compound targeting the GATA4-NKX2-5 interaction: N-[4- (diethylamino)phenyl]-5-methyl-3-phenylisoxazole-4-carboxamide. This compound presented anti-hypertrophic effects in vitro and cardioprotective effects in vivo. In addition, structural analysis of the lead compound revealed the signature molecular features for metabolism and cytotoxicity. Current drug treatments are able to delay, but not prevent the progress of the heart failure; therefore, modulators of protein-protein interactions of key transcription factors may represent a novel class of pharmaceuticals for cardiac remodeling and repair.Geenien ilmentymisen säätelijöihin kohdennettujen yhdisteiden suunnittelu ja kehitys Sydäninfarkti on henkeä uhkaava verenkierron häiriö, joka syntyy veren virtauksen äkillisen vähentymisen seurauksena sydänlihaksessa aiheuttaen kudosvaurion. Vaurioituneen sydänlihaskudoksen kyky uusiutua tai korvata kuolleet sydänlihassolut uusilla on puutteellinen, ja tämän seurauksena sydämen pumppauskyky heikkenee. Transkriptiotekijöiden GATA4, NKX2-5, TBX5 ja MEF2C muodostamat ja koordinoimat proteiinikompleksit säätelevät sydänsolujen geenien ilmenemistä solujen elinkaaren aikana. Väitöskirjatyön tavoitteena oli (i) karakterisoida geeninsäätelytekijöiden GATA4-NKX2-5 molekyylirakenteet ja niiden keskinäinen vuorovaikutus, (ii) seuloa kemiallisia yhdisteitä, jotka muokkaavat GATA4-NKX2-5 proteiinikompleksin aikaansaamaa geeniaktivaatiota, (iii) tutkia johtoyhdisteen vaikutuksia in vivo sydäninfarktia ja painekuormitusta kuvaavissa eläinmalleissa, ja (iv) tutkia johtoyhdisteen molekyylirakenteen yhteyttä yhdisteen aineenvaihduntaan ja soluja tuhoavaan vaikutukseen. Väitöskirjatyö osoittaa molekyylimallinnuksen ja kokeellisen työn perusteella, että geeninsäätelytekijöiden GATA4 ja NKX2-5 muodostama proteiinikompleksi jäljittelee tumareseptoriperheeseen kuuluvien proteiinien laskostumista. Laskennallisten- ja kokeellisten mallien avulla seulottiin ja optimoitiin sitoutumisvoimakkuudeltaan lupaavin GATA4-NKX2-5 proteiinikompleksin toimintaan vaikuttava johtoyhdiste: N-[4-(dietyyliamino)fenyyli]-5-metyyli-3-fenyyli-isoksatsoli-4-karboksamidi. Johtoyhdisteellä havaittiin solu- ja eläinmalleissa solujen kasvua estäviä vaikutuksia in vitro ja sydäntä suojaavia vaikutuksia in vivo. Väitöskirjatyö osoitti lisäksi aktiivisten molekyylien rakenneominaisuuksia, jotka keskeisesti vaikuttavat yhdisteiden aineenvaihduntaan ja soluja tuhoavaan vaikutukseen. Nykyinen lääkehoito hidastaa, mutta ei pysäytä sydänlihasvaurioon liittyvän kroonisen sydämen vajaatoiminnan etenemistä. Lääkevaikutuksen kohdentaminen sydämen keskeisten transkriptiotekijöiden yhteisvaikutukseen avaa uuden mahdollisen tutkimuslinjan sydänlihasvaurion estossa ja korjauksessa

Data synchronization in web-based liquid software solutions

When a computer application is designed with a liquid software approach it means that the application can be used on multiple devices and on multiple environments. In addition the user can move from one device to another and the state of the application seamlessly transfers when switching between devices. It also means that the persistent data used by the program is synchronized between the devices. Using the program on multiple devices can either be sequential or simultaneous and there can be more than one user using the same application. In this thesis the liquid software approach was tested in a web application to see what kind of challenges the browser environment would have. The focus of the application was on synchronizing persistent data between devices. The web application approach was chosen because it can be run on all platforms that have support for a modern browser. In order to test it a proof of concept application was made. The requirements for the application were that it would not require third party add-ons and that the core design would not be tied to any third party system. Different technologies were tested and according to the tests the three most important technologies were Push API, service workers and IndexedDB. They all are new web technologies and their standardization is not yet finished. The standards are being developed by W3C, WHATWG and IETF. Persistent data synchronization that satisfies the requirements of a liquid software is possible to develop for the browser environment. Web browsers still face challenges such as that the browser does not have access to low level services of the underlying operating system which for some applications would be mandatory. Also if the liquid software approach is to be combined with an Internet of things application then the web-runtime might not be possible to be used with all of the devices

Work Stress and Satisfaction with Leadership Among Nurses Encountering Patient Aggression in Psychiatric Care : A Cross-Sectional Survey Study

We examined the associations between work stress (job strain, effort-reward imbalance), perceived job insecurity, workplace social capital, satisfaction with leadership and working hours in relation to the likelihood of encountering patient aggression (overall exposure, assaults on ward property, mental abuse, physical assaults). We conducted a cross-sectional survey for nurses (N = 923) in 84 psychiatric units. Both work stress indicators were associated higher odds for different types of patient aggression. Poorer satisfaction with leadership was associated with higher odds for overall exposure to patient aggression. These findings were robust to adjustment for several nurse and work characteristics, and unit size.Peer reviewe

Domain-Independent Inhibition of CBP/p300 Attenuates alpha-Synuclein Aggregation

Neurodegenerative diseases are associated with failed proteostasis and accumulation of insoluble protein aggregates that compromise neuronal function and survival. In Parkinson's disease, a major pathological finding is Lewy bodies and neurites that are mainly composed of phosphorylated and aggregated alpha-synuclein and fragments of organelle membranes. Here, we analyzed a series of selective inhibitors acting on multidomain proteins CBP and p300 that contain both lysine acetyltransferase and bromodomains and are responsible for the recognition and enzymatic modification of lysine residues. By using high-affinity inhibitors, A-485, GNE-049, and SGC-CBP30, we explored the role of two closely related proteins, CBP and p300, as promising targets for selective attenuation of alpha-synuclein aggregation. Our data show that selective CBP/p300 inhibitors may alter the course of pathological alpha-synuclein accumulation in primary mouse embryonic dopaminergic neurons. Hence, drug-like CBP/p300 inhibitors provide an effective approach for the development of high-affinity drug candidates preventing alpha-synuclein aggregation via systemic administration.Peer reviewe

Work Stress and Satisfaction with Leadership Among Nurses Encountering Patient Aggression in Psychiatric Care: A Cross-Sectional Survey Study

We examined the associations between work stress (job strain, effort-reward imbalance), perceived job insecurity, workplace social capital, satisfaction with leadership and working hours in relation to the likelihood of encountering patient aggression (overall exposure, assaults on ward property, mental abuse, physical assaults). We conducted a cross-sectional survey for nurses (N = 923) in 84 psychiatric units. Both work stress indicators were associated higher odds for different types of patient aggression. Poorer satisfaction with leadership was associated with higher odds for overall exposure to patient aggression. These findings were robust to adjustment for several nurse and work characteristics, and unit size.</p

GATA4-targeted compound exhibits cardioprotective actions against doxorubicin-induced toxicity in vitro and in vivo : establishment of a chronic cardiotoxicity model using human iPSC-derived cardiomyocytes

Doxorubicin is a widely used anticancer drug that causes dose-related cardiotoxicity. The exact mechanisms of doxorubicin toxicity are still unclear, partly because most in vitro studies have evaluated the effects of short-term high-dose doxorubicin treatments. Here, we developed an in vitro model of long-term low-dose administration of doxorubicin utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Moreover, given that current strategies for prevention and management of doxorubicin-induced cardiotoxicity fail to prevent cancer patients developing heart failure, we also investigated whether the GATA4-targeted compound 3i-1000 has cardioprotective potential against doxorubicin toxicity both in vitro and in vivo. The final doxorubicin concentration used in the chronic toxicity model in vitro was chosen based on cell viability data evaluation. Exposure to doxorubicin at the concentrations of 1-3 mu M markedly reduced (60%) hiPSC-CM viability already within 48 h, while a 14-day treatment with 100 nM doxorubicin concentration induced only a modest 26% reduction in hiPCS-CM viability. Doxorubicin treatment also decreased DNA content in hiPSC-CMs. Interestingly, the compound 3i-1000 attenuated doxorubicin-induced increase in pro-B-type natriuretic peptide (proBNP) expression and caspase-3/7 activation in hiPSC-CMs. Moreover, treatment with 3i-1000 for 2 weeks (30 mg/kg/day, i.p.) inhibited doxorubicin cardiotoxicity by restoring left ventricular ejection fraction and fractional shortening in chronic in vivo rat model. In conclusion, the results demonstrate that long-term exposure of hiPSC-CMs can be utilized as an in vitro model of delayed doxorubicin-induced toxicity and provide in vitro and in vivo evidence that targeting GATA4 may be an effective strategy to counteract doxorubicin-induced cardiotoxicity.Peer reviewe

Conformal Titanium Nitride in a Porous Silicon Matrix: a Nanomaterial for In-Chip Supercapacitors

Today's supercapacitor energy storages are typically discrete devices aimed for printed boards and power applications. The development of autonomous sensor networks and wearable electronics and the miniaturisation of mobile devices would benefit substantially from solutions in which the energy storage is integrated with the active device. Nanostructures based on porous silicon (PS) provide a route towards integration due to the very high inherent surface area to volume ratio and compatibility with microelectronics fabrication processes. Unfortunately, pristine PS has limited wettability and poor chemical stability in electrolytes and the high resistance of the PS matrix severely limits the power efficiency. In this work, we demonstrate that excellent wettability and electro-chemical properties in aqueous and organic electrolytes can be obtained by coating the PS matrix with an ultra-thin layer of titanium nitride by atomic layer deposition. Our approach leads to very high specific capacitance (15 F/cm$^3$), energy density (1.3 mWh/cm$^3$), power density (up to 214 W/cm$^3$) and excellent stability (more than 13,000 cycles). Furthermore, we show that the PS-TiN nanomaterial can be integrated inside a silicon chip monolithically by combining MEMS and nanofabrication techniques. This leads to realisation of in-chip supercapacitor, i.e., it opens a new way to exploit the otherwise inactive volume of a silicon chip to store energy

Nuclear Receptor-Like Structure and Interaction of Congenital Heart Disease-Associated Factors GATA4 and NKX2-5

Aims Transcription factor GATA4 is a dosage sensitive regulator of heart development and alterations in its level or activity lead to congenital heart disease (CHD). GATA4 has also been implicated in cardiac regeneration and repair. GATA4 action involves combinatorial interaction with other cofactors such as NKX2-5, another critical cardiac regulator whose mutations also cause CHD. Despite its critical importance to the heart and its evolutionary conservation across species, the structural basis of the GATA4-NKX2-5 interaction remains incompletely understood. Methods and Results A homology model was constructed and used to identify surface amino acids important for the interaction of GATA4 and NKX2-5. These residues were subjected to site-directed mutagenesis, and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5. The studies identify 5 highly conserved amino acids in the second zinc finger (N272, R283, Q274, K299) and its C-terminal extension (R319) that are critical for physical and functional interaction with the third alpha helix of NKX2-5 homeodomain. Integration of the experimental data with computational modeling suggests that the structural arrangement of the zinc finger-homeodomain resembles the architecture of the conserved DNA binding domain of nuclear receptors. Conclusions The results provide novel insight into the structural basis for protein-protein interactions between two important classes of transcription factors. The model proposed will help to elucidate the molecular basis for disease causing mutations in GATA4 and NKX2-5 and may be relevant to other members of the GATA and NK classes of transcription factors.Peer reviewe

GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line

BackgroundPharmacological modulation of cell fate decisions and developmental gene regulatory networks holds promise for the treatment of heart failure. Compounds that target tissue-specific transcription factors could overcome non-specific effects of small molecules and lead to the regeneration of heart muscle following myocardial infarction. Due to cellular heterogeneity in the heart, the activation of gene programs representing specific atrial and ventricular cardiomyocyte subtypes would be highly desirable. Chemical compounds that modulate atrial and ventricular cell fate could be used to improve subtype-specific differentiation of endogenous or exogenously delivered progenitor cells in order to promote cardiac regeneration.MethodsTranscription factor GATA4-targeted compounds that have previously shown in vivo efficacy in cardiac injury models were tested for stage-specific activation of atrial and ventricular reporter genes in differentiating pluripotent stem cells using a dual reporter assay. Chemically induced gene expression changes were characterized by qRT-PCR, global run-on sequencing (GRO-seq) and immunoblotting, and the network of cooperative proteins of GATA4 and NKX2-5 were further explored by the examination of the GATA4 and NKX2-5 interactome by BioID. Reporter gene assays were conducted to examine combinatorial effects of GATA-targeted compounds and bromodomain and extraterminal domain (BET) inhibition on chamber-specific gene expression.ResultsGATA4-targeted compounds 3i-1000 and 3i-1103 were identified as differential modulators of atrial and ventricular gene expression. More detailed structure-function analysis revealed a distinct subclass of GATA4/NKX2-5 inhibitory compounds with an acetyl lysine-like domain that contributed to ventricular cells (%Myl2-eGFP+). Additionally, BioID analysis indicated broad interaction between GATA4 and BET family of proteins, such as BRD4. This indicated the involvement of epigenetic modulators in the regulation of GATA-dependent transcription. In this line, reporter gene assays with combinatorial treatment of 3i-1000 and the BET bromodomain inhibitor (+)-JQ1 demonstrated the cooperative role of GATA4 and BRD4 in the modulation of chamber-specific cardiac gene expression.ConclusionsCollectively, these results indicate the potential for therapeutic alteration of cell fate decisions and pathological gene regulatory networks by GATA4-targeted compounds modulating chamber-specific transcriptional programs in multipotent cardiac progenitor cells and cardiomyocytes. The compound scaffolds described within this study could be used to develop regenerative strategies for myocardial regeneration.Peer reviewe

99mTechnetium Sestamibi-123Iodine Scintigraphy Is More Accurate Than 99mTechnetium Sestamibi Alone before Surgery for Primary Hyperparathyroidism

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