Cartography of rhodopsin-like G protein-coupled receptors across vertebrate genomes

We conduct a cartography of rhodopsin-like non-olfactory G protein-coupled receptors in the Ensembl database. The most recent genomic data (releases 90-92, 90 vertebrate genomes) are analyzed through the online interface and receptors mapped on phylogenetic guide trees that were constructed based on a set of similar to 14.000 amino acid sequences. This snapshot of genomic data suggest vertebrate genomes to harbour 142 clades of GPCRs without human orthologues. Among those, 69 have not to our knowledge been mentioned or studied previously in the literature, of which 28 are distant from existing receptors and likely new orphans. These newly identified receptors are candidates for more focused evolutionary studies such as chromosomal mapping as well for in-depth pharmacological characterization. Interestingly, we also show that 37 of the 72 human orphan (or recently deorphanized) receptors included in this study cluster into nineteen closely related groups, which implies that there are less ligands to be identified than previously anticipated. Altogether, this work has significant implications when discussing nomenclature issues for GPCRs.Peer reviewe

Orexin receptor agonist Yan 7874 is a weak agonist of orexin/hypocretin receptors and shows orexin receptor-independent cytotoxicity

Two promising lead structures of small molecular orexin receptor agonist have been reported, but without detailed analyses of the pharmacological properties. One of them, 1(3,4-dichloropheny1)-242-imino-3-(4-methylbenzy1)-2,3-dihydro-1H-benzo[climidazol-1-yl] ethan-1-ol (Yan 7874), is commercially available, and we set out to analyze its properties. As test system we utilized human OX1 and OX2 orexin receptor -expressing Chinese hamster ovary (CHO) K1 cells as well as control CHO-K1 and neuro-2a neuroblastoma cells. Gq-coupling was assessed by measurement of intracellular Ca2+ and phospholipase C activity, and the coupling to G(i) and G(s) by adenylyl cyclase inhibition and stimulation, respectively. At concentrations above 1 pM, strong Ca' and low phospholipase C responses to Yan 7874 were observed in both OX1- and OX2-expressing cells. However, a major fraction of the response was not mediated by orexin receptors, as determined utilizing the nonselective orexin receptor antagonist N-biphenyl-2-y1-1-{[(1-methyl-1H-benzimidazol-2-y1) sulfanyl]acetyl}-L-prolinamide (TCS 1102) as well as control CHO-K1 cells. Yan 7874 did not produce any specific adenylyl cyclase response. Some experiments suggested an effect on cell viability by Yan 7874, and we thus analyzed this. Within a few hours of exposure, Yan 7874 markedly changed cell morphology (shrunken, rich in vacuoles), reduced growth, promoted cell detachment, and induced necrotic cell death. The effect was equal in cells expressing orexin receptors or not. Thus, Yan 7874 is a weak partial agonist of orexin receptors. It also displays strong off -target effects in the same concentration range, culminating in necrotic cell demise. This makes Yan 7874 unsuitable as orexin receptor agonist.Peer reviewe

Synthesis of 7β-hydroxy-8-ketone opioid derivatives with antagonist activity at mu- and delta-opioid receptors

Despite extensive years of research, the direct oxidation of the 7,8-double bond of opioids has so far received little attention and knowledge about the effects of this modification on activity at the different opioid receptors is scarce. We herein report that potassium permanganate supported on iron(II) sulfate heptahydrate can be used as a convenient oxidant in the one-step, heterogeneous conversion of Delta(7.8)-opioids to the corresponding 7 beta-hydroxy-8-ketones. Details of the reaction mechanism are given and the effects of the substituent at position 6 of several opioids on the reaction outcome is discussed. The opioid hydroxy ketones prepared are antagonists at the mu- and delta-opioid receptors. Docking simulations and detailed structure-activity analysis revealed that the presence of the 7 beta-hydroxy-8-ketone functionality in the prepared compounds can be used to gain activity towards the delta opioid receptor. The 7 beta-hydroxy-8-ketones prepared with this method can also be regarded as versatile intermediates for the synthesis of other opioids of interest. (C) 2018 Elsevier Masson SAS. All rights reserved.Peer reviewe

Prolyl oligopeptidase inhibition by KYP-2407 increases alpha-synuclein fibril degradation in neuron-like cells

Growing evidence emphasizes insufficient clearance of pathological alpha-synuclein (alpha SYN) aggregates in the progression of Parkinson's disease (PD). Consequently, cellular degradation pathways represent a potential therapeutic target. Prolyl oligopeptidase (PREP) is highly expressed in the brain and has been suggested to increase alpha SYN aggregation and negatively regulate the autophagy pathway. Inhibition of PREP with a small molecule inhibitor, KYP-2407, stimulates autophagy and reduces the oligomeric species of alpha SYN aggregates in PD mouse models. However, whether PREP inhibition has any effects on intracellular alpha SYN fibrils has not been studied before. In this study, the effect of KYP2407 on alpha SYN preformed fibrils (PFFs) was tested in SH-SY5Y cells and human astmcytes. Immunostaining analysis revealed that both cell types accumulated alpha SYN PFFs intracellularly but KYP-2047 decreased intracellular alpha SYN deposits only in SH-SY5Y cells, as astrocytes did not show any PREP activity. Western blot analysis confirmed the reduction of high molecular weight alpha SYN species in SH-SY5Y cell lysates, and secretion of aSYN from SH-SY5Y cells also decreased in the presence of KYP-2407. Accumulation of alpha SYN inside the SH-SY5Y cells resulted in an increase of the auto-lysosomal proteins p62 and LC3BII, as well as calpain 1 and 2, which have been shown to be associated with PD pathology. Notably, treatment with KYP-2407 significantly reduced p62 and LC3BII levels, indicating an increased autophagic flux, and calpain 1 and 2 levels returned to normal in the presence of KYP-2407. Our findings indicate that PREP inhibition can potentially be used as therapy to reduce the insoluble intracellular alpha SYN aggregates

Säilörehun säilöntäopas

Tämä opas on luotu A-Tuottajat Oy:n, Osuuskunta Pohjanmaan Lihan ja Luonnonvarakeskuksen toteuttaman Tuottava nautatilan nurmi -kehityshankkeen piirissä toimivien tahojen ja yhteistyökumppaneiden kanssa. Tämä hanke saa rahoitusta Manner-Suomen maaseudun kehittämisohjelmasta. Oppaan tavoitteena on tuoda kootusti esille ajantasainen tarkastelu säilörehuntuotannon laatutekijöistä ja säilöntäprosesseista ja miten näitä tekijöitä voi kehittää sekä mitkä asiat vaikuttavat saatuun lopputulokseen.202

Differences in brain changes between adults with childhood-onset epilepsy and controls: A prospective population-based study

PurposeTo determine the impact of childhood-onset uncomplicated epilepsy (COE) on brain aging over 50-year prospective follow-up.MethodsA population-based cohort of 41 aging subjects with COE and their 46 matched controls participated in a detailed in-person prospective assessment in 2012 and 2017 to characterize ongoing changes in the aging brain.ResultsThe mean age of the COE participants was 63.2 years (SD 4.14, median 63.2, range 55.8–70.6) and 63.0 years (mean, SD 4.13, median 63.3, range 56.0–69.9) years for controls. Neurologic signs were significantly more common in COE participants not in remission (p = .015), and the most frequent abnormalities were cerebellar signs (p p = .008) and cerebellar signs in particular (p = .018) were significantly more common in focal than in generalized epilepsies. MRI white matter abnormalities were significantly associated with absence of vocational education (p = .011), and MRI hippocampal atrophy in COE subjects was associated with arterial hypertension versus normal blood pressure (p = .017). In the combined study cohort of COE subjects and controls, presenting neurologic signs increased both in the subjects and in the controls from the 2012 to 2017 study.ConclusionsAt ultra-long-term follow-up, clinical and neuroimaging findings show tendencies to brain aging that is more accelerated in COE participants with active adult childhood-onset epilepsy, and particularly in focal epilepsy.</div

Mitoxantrone, pixantrone, and mitoxantrone (2-hydroxyethyl)piperazine are toll-like receptor 4 antagonists, inhibit NF-κB activation, and decrease TNF-alpha secretion in primary microglia

Toll-like receptor 4 (TLR4) recognizes various endogenous and microbial ligands and is an essential part in the innate immune system. TLR4 signaling initiates transcription factor NF-κB and production of proinflammatory cytokines. TLR4 contributes to the development or progression of various diseases including stroke, neuropathic pain, multiple sclerosis, rheumatoid arthritis and cancer, and better therapeutics are currently sought for these conditions. In this study, a library of 140 000 compounds was virtually screened and a resulting hit-list of 1000 compounds was tested using a cellular reporter system. The topoisomerase II inhibitor mitoxantrone and its analogues pixantrone and mitoxantrone (2-hydroxyethyl)piperazine were identified as inhibitors of TLR4 and NF-κB activation. Mitoxantrone was shown to bind directly to the TLR4, and pixantrone and mitoxantrone (2- hydroxyethyl)piperazine were shown to inhibit the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNFα) in primary microglia. The inhibitory effect on NF-κB activation or on TNFα pro-duction was not mediated through cytotoxity at ≤ 1 μM concentration for pixantrone and mitoxantrone (2- hydroxyethyl)piperazine treated cells, as assessed by ATP counts. This study thus identifies a new mechanism of action for mitoxantrone, pixantrone, and mitoxantrone (2-hydroxyethyl)piperazine through the TLR4.Peer reviewe

Nettimuskari : Muskarilaulut tutuiksi kotiväelle verkon välityksellä

Opinnäytetyömme pääpainona oli tuottaa Jyväskylän ammattikorkeakoulun Muskaristudiolle verkkotyötila, jota pystytään jatkossa hyödyntämään aikuisen ja lapsen yhteisenä muskari työtilana. Kokeilu suunnattiin Muskaristudion Taikas-Nelosten ryhmälle, nimikkeellä Nettimuskari. Pyrimme selvittämään, voiko lapsen musiikillista kehitystä tukea verkko-opetuksella. Internet on yleistynyt viime vuosina ja pyrimmekin hyödyntämään verkko-opetusta myös meidän alallamme. Nykyään Internetin ja tietotekniikan hallinta on yksi edellytys työnteossa alalla kuin alalla. Olemme opinnäytetyömme aikana tutustuneet Internetin tarjoamiin musiikin verkko-opetusmateriaaleihin lapsille, nuorille ja aikuisille. Työssä pohdimme lapsille suunnatun musiikin verkko-opetuksen hyötyjä ja haittoja muskaristudiolaisten vanhemmille teetettyjen kyselyjen avulla. Lisäksi pohdimme Nettimuskarin tuottamisen eri vaiheita. Opinnäytetyömme tuloksena kyselyjen perusteella oli, että Nettimuskarista oltiin kiinnostuneita. Alussa materiaalia oli vähän, mutta videoista pidettiin ja niitä hyödynnettiin kotona laulaen, leikkien ja kuunnellen. Nettimuskari oli onnistunut kokeilu, ja sen toivottiin olevan käytössä myös tulevaisuudessa.The aim of the thesis was to produce an online workspace for the Muskaristudio of Jamk University of Applied Sciences which can be used as a shared music workspace by adults and children. This experiment was targeted at Muskaristudio’s Magical- Fourth group, called Nettimuskari. The purpose was to examine if it was possible to support a child’s musical growth with online learning material. The Internet has increased its popularity in the last few years, and the aim was to utilize online teaching also in this field. Nowadays skills related to the internet and information technology are a basic qualification in any field of work. During the thesis process the authors familiarized themselves with music online-workspaces for children, youngsters and adults offered by the Internet. Thesis we dealt with the benefits and drawbacks of children’s online music workspaces with the help of surveys directed at the children’s parents. The thesis also described the different phases of producing Nettimuskari. Based on surveys, the results of the thesis were that Nettimuskari was interesting. At the beginning there was marginally material but the videos were liked and used by singing, playing and listening at home. Nettimuskari was successful experiment and it was requested to be able to use in future

Molecular evolution of G protein-coupled receptors : insights into the orexin system

The diversity of G protein-coupled receptors is a fundamental element in this thesis. GPCRs are the largest family of membrane proteins, with about 800 in humans. While the human GPCR repertoire is well described, in other species, especially in non-mammals, GPCRs are not tracked to the individual subtype level in large-scale genomic studies. The diversity of GPCRs in non-human vertebrates was studied in the first publication. The study classified 142 rhodopsin-like non-olfactory GPCRs without human orthologue, 69 of which were reported for the first time. The study also points out inconsistencies in the GPCR nomenclature system and reveals a pool of yet-to-be studied receptors. Understanding the repertoire of GPCRs in non-human species might also be useful for many areas of science, such as pharmacology, ecotoxicology and evolutionary biology. For more insight into the orexin system, two small-molecule orexin receptor agonists were pharmacologically characterised in the second and third publications. Nag26 was confirmed to be a potent and almost full agonist of orexin receptors, but the selectivity for orexin receptor type-2 was not as strong as reported (20-fold against 70-fold). Yan7874 was also confirmed to be an orexin receptor agonist, but only partial and weak with high off-target activity. Neither of these compounds is likely to be suitable for further drug development as such. These studies also display the challenge in the development of small-molecule agonists for peptide-bound GPCRs. Finally, Ciona intestinalis putative orexin receptor was studied, and its functionality was verified in recombinant cells. Homology models of C. intestinalis orexin receptor revealed a highly similar binding cavity as in the human OX2 orexin receptor. Thus, the functionality of the receptor was studied in Ca2+ elevation assay, and the receptor was verified to be functional and binding to human orexin peptides. Further database mining resulted in the identification of putative C. intestinalis prepro-orexin and orexin peptide that was shown to bind to a plasma membrane of C. intestinalis orexin receptor-expressing cells. Solving the function of the orexin system in distinct species such as C. intestinalis might be interesting from an evolutionary point of view but also essential in extending the knowledge of the orexin system in humans. This thesis was conducted collaboratively with the Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy and Department of Veterinary Biosciences, Faculty of Veterinary Medicine, at the University of Helsinki.G-proteiinikytkentäisten reseptorien moninaisuus on keskiössä tässä väitöskirjatyössä. G-proteiinikytkentäiset reseptorit ovat suurin yhtenäinen kalvoreseptorien ryhmä, ja ihmisellä on noin 800 G-proteiinikytkentäistä reseptoria. Toisin kuin ihmisen reseptorien kohdalla, muiden lajien edustajien (varsinkin nisäkkäiden ulkopuolelta) reseptorijoukkoa on harvoin jäljitetty alatyyppitasolle asti laajoissa genomitutkimuksissa. Tämän väitöskirjatyön ensimmäinen julkaisu perehtyy G-proteiinikytkentäisten reseptorien moninaisuuteen selkärankaisilla lajeilla. Tutkimus luokittelee 142 rodopsiini-reseptorien ryhmään kuuluvaa G-proteiinikytkentäistä reseptoria ilman humaania ortologia. Näistä reseptoreista 69 esitetään tässä työssä ensimmäistä kertaa. Lisäksi tutkimus osoittaa reseptorien epäjohdonmukaisuuksia nimeämissysteemissä, sekä esittelee vielä tutkimattomia reseptoreja. Laajempi ymmärrys eri lajien reseptorirepertuaarista voi olla hyödyllinen monilla tieteenaloilla, kuten farmakologiassa, ekotoksikologiassa ja evoluutiobiologissa. Tässä väitöskirjatyössä tutkittiin myös ihmisen G-proteiinikytkentäisiä reseptoreja. Kahden pienmolekyylioreksiinireseptoriagonistin ominaisuudet luokiteltiin farmakologisesti tämän väitöskirjan toisessa ja kolmannessa julkaisussa. Tutkimus vahvisti pienmolekyyli Nag26:n olevan potentti, lähes täysagonisti ja selektiivinen tyypin-2 oreksiinireseptorille, joskaan ei yhtä voimakkaasti selektiivinen kuin aikaisemmin on raportoitu. Pienmolekyyli Yan7874:n vahvistettiin olevan myös oreksiinireseptoriagonisti, mutta vain osittainen ja heikko. Lisäksi, Yan7874 indusoi soluissa epäspesifisiä vaikutuksia. On siis epätodennäköistä, että kumpikaan näistä yhdisteistä tarjoaisi sellaisenaan potentiaalia pidemmälle lääkekehitykselle. Nämä tutkimukset osoittavat osaltaan pienmolekyyliagonistien kehityksen haasteita peptideihin sitoutuviin G-proteiinikytkentäistiin reseptoreihin. Lopuksi, tässä työssä tutkittiin selkärangattoman Ciona intestinalis –lajin putatiivista oreksiinireseptoria, ja sen toiminnallisuus vahvistettiin ilmentämällä sitä rekombinanteissa soluissa. Homologiamallien perusteella reseptorin sitoutumistaskun osoitettiin muistuttavan ihmisen tyyppi-2 oreksiinireseptorin sitoutumistaskua. Oreksiinireseptorin toimivuus vahvistettiin reseptori-riippuvaisella solunsisäisen kalsiumin mobilisaatiolla, ja sitovan humaaneja oreksiinipeptidejä. Lisäksi tietokantalouhinta johti putatiivisen C. intestinalis oreksiini-prekursorin ja oreksiinipeptidin tunnistamiseen. Tässä väitöskirjatyössä tunnistettiin ensimmäistä kertaa C. intestinalis –lajin oreksiini-prekursori ja osoitettiin, että putatiivinen oreksiinipeptidi sitoutuu C. intestinalis oreksiinireseptoria expressoivien solujen solukalvolle. Oreksiinisysteemin toiminnallisuus evolutiivisesti etäisillä lajeilla, kuten C. intestinalis –lajilla, on mielenkiintoinen tutkimuskohde evoluutiobiologian kannalta. Lisäksi se voi tarjota tietoa kokonaisvaltaiseen oreksiinisysteemin ymmärtämiseen myös ihmisillä. Tämä väitöskirja on toteutettu yhteistyössä Helsingin Yliopiston Farmaseuttisen Kemian ja Teknologian osaston (Farmasian Tiedekunta) ja Eläinlääketieteellisten Biotieteiden osaston (Eläinlääketieteellinen Tiedekunta) kanssa