Human enterovirus group B viruses rely on vimentin dynamics for efficient processing of viral non-structural proteins

We report that several viruses from the human enterovirus group B cause massive vimentin rearrangements during lytic infection. Comprehensive studies suggested that viral protein synthesis was triggering the vimentin rearrangements. Blocking the host cell vimentin dynamics with IDPN did not significantly affect the production of progeny viruses and only moderately lowered the synthesis of structural proteins such as VP1. In contrast, the synthesis of the non-structural proteins 2A, 3C, and 3D was drastically lowered. This led to attenuation of the cleavage of the host cell substrates PABP and G3BP1 and reduced caspase activation, thus leading to prolonged cell survival. Furthermore, the localization of the proteins differed in the infected cells. Capsid protein VP1 was found diffusely around the cytoplasm, whereas 2A and 3D followed vimentin distribution. Based on protein blotting, lower amounts of non-structural proteins did not result from proteasomal degradation, but from lower synthesis without intact vimentin cage structure. In contrast, inhibition of Hsp90 chaperone activity, which regulates P1 maturation, lowered the amount of VP1, but had less effect on 2A. The results suggest that, the vimentin dynamics regulate viral non-structural protein synthesis while having no effect on structural protein synthesis or overall infection efficiency. The results presented here shed new light on differential fate of structural and non-structural proteins of enteroviruses, having consequences on host cell survival. Importance A virus needs the host cell in order to replicate and produce new progeny viruses. For this, the virus takes over the host cell and modifies it to become a factory for viral proteins. Irrespective of the specific virus family, these proteins can be divided into structural and non-structural proteins. Structural proteins are the building blocks for the new progeny virions, whereas the non-structural proteins orchestrate the take-over of the host cell and its functions. Here we have shown a mechanism that viruses exploit in order to regulate the host cell. We show that viral protein synthesis induces vimentin cages, which promote production of specific viral proteins that eventually control apoptosis and the host cell death. This study specifies vimentin as the key regulator of these events and indicates that viral proteins have different fates in the cells depending on their association with vimentin cages.peerReviewe

Talin-mediated force transmission and talin rod domain unfolding independently regulate adhesion signaling

Talin protein is one of the key components in integrin-mediated adhesion complexes. Talins transmit mechanical forces between beta-integrin and actin, and regulate adhesion complex composition and signaling through the force-regulated unfolding of talin rod domain. Using modified talin proteins, we demonstrate that these functions contribute to different cellular processes and can be dissected. The transmission of mechanical forces regulates adhesion complex composition and phosphotyrosine signaling even in the absence of the mechanically regulated talin rod subdomains. However, the presence of the rod subdomains and their mechanical activation are required for the reinforcement of the adhesion complex, cell polarization and migration. Talin rod domain unfolding was also found to be essential for the generation of cellular signaling anisotropy, since both insufficient and excess activity of the rod domain severely inhibited cell polarization. Utilizing proteomics tools, we identified adhesome components that are recruited and activated either in a talin rod-dependent manner or independently of the rod subdomains. This study clarifies the division of roles between the force-regulated unfolding of a talin protein (talin 1) and its function as a physical linker between integrins and the cytoskeleton.Peer reviewe

Cancer associated talin point mutations disorganise cell adhesion and migration

Talin-1 is a key component of the multiprotein adhesion complexes which mediate cell migration, adhesion and integrin signalling and has been linked to cancer in several studies. We analysed talin-1 mutations reported in the Catalogue of Somatic Mutations in Cancer database and developed a bioinformatics pipeline to predict the severity of each mutation. These predictions were then assessed using biochemistry and cell biology experiments. With this approach we were able to identify several talin-1 mutations affecting integrin activity, actin recruitment and Deleted in Liver Cancer 1 localization. We explored potential changes in talin-1 signalling responses by assessing impact on migration, invasion and proliferation. Altogether, this study describes a pipeline approach of experiments for crude characterization of talin-1 mutants in order to evaluate their functional effects and potential pathogenicity. Our findings suggest that cancer related point mutations in talin-1 can affect cell behaviour and so may contribute to cancer progression

Surface modification of silicate, borosilicate, and phosphate bioactive glasses to improve/control protein adsorption : PART II

Bioactive glasses (BGs) are characterized by high biocompatibility and bioactivity and are particularly promising for bone tissue regeneration. Once implanted, the BGs interact with the environment and adsorb chemical moieties and biomolecules. Proteins in body fluids are critical for the success of implants, because the adsorption of specific proteins can either promote or inhibit the adhesion of surrounding tissue or other factors such as bacteria. Controlling protein adsorption by tailoring the surface properties of implanted biomaterials is fundamental. This can determine the fate of the implant. In the current study, four BG compositions (two silicates, one borosilicate, and one phosphate glass) and three model proteins (fibronectin, chimeric avidin, and streptavidin) were considered. Each BG was surface pretreated, and the adsorption of fluorescently labeled fibronectin, chimeric avidin, or streptavidin was monitored. Untreated surfaces were used as controls. The amount and spatial distribution of each protein were estimated by confocal microscopy in fluorescence modality, followed by protein clustering analysis. Although streptavidin was not adsorbed efficiently on any of the considered substrates, BGs were successfully coated with fibronectin and chimeric avidin. Both proteins showed different affinities and surface distributions as functions of the implemented pretreatment on each substrate.publishedVersionPeer reviewe

Clathrin-independent entry of baculovirus triggers uptake of E. coli in non-phagocytic human cells

Peer reviewe

Suoramyynti liiketoimintamahdollisuutena

Tämän opinnäytetyön tarkoituksena oli tutkia suoramyyntiä liiketoimintamahdollisuutena. Suoramyynnillä tarkoitetaan tuotteiden ja palvelujen henkilökohtaista myyntiä suoraan kuluttajalle yleensä kodeissa tai työpaikoilla. Suoramyynti on perinteinen ja hyväksi koettu kauppatapa. Opinnäytetyössä selvitettiin suoramyynnin aloittamista harkitsevalle henkilölle asioita, joita hänen tulee ottaa huomioon ennen toimintansa aloittamista. Työssä käytettiin esimerkkeinä Suomessa suoramyyntikonseptilla toimivia yrityksiä. Opinnäytetyön aihe valikoitui tekijän omasta kiinnostuksesta aiheeseen. Aihe on myös ajankohtainen, sillä suoramyyntiala on iso ja kasvava sektori Euroopassa. Yli kuusi miljoonaa ihmistä työskentelee suoramyynnin parissa EU:ssa. Perustiedot esimerkkiyrityksistä saatiin niiden verkkosivuilta ja lisätietoa hankittiin yritysten myyntiedustajilta. Näin saatiin tietoa myyntiedustajien näkökulmista suoramyynnistä ja esimerkkiyrityksistä. Työssä käytettiin laadullista tutkimusmenetelmää. Tutkimus toteutettiin yksilöhaastatteluina. Haastattelumenetelmänä käytettiin virtuaalista haastattelua sähköpostitse. Haastateltavat tavoitettiin sosiaalisen median kautta sekä Hämeenlinnassa 13. – 14.8.2016 järjestetyiltä Elomessuilta. Haastattelut toteutettiin syksyn 2016 aikana. Työn lähdemateriaalina käytettiin aiheeseen liittyvää kirjallisuutta ja verkkosivuja sekä lainsäädäntöä. Työssä selvitetään aluksi suoramyyntiin liittyviä käsitteitä ja kotikutsumyyntikonseptia. Omaa liiketoimintaa aloitettaessa on huomioitava eri asioita, kuten alkuinvestoinnit, toiminimen perustaminen, verotus ja kirjanpitovelvollisuus. Näitä asioita työssä on esitelty yleisellä tasolla. Suoramyyntiin liittyviä sääntöjä tuodaan esiin, sillä jokaisen suoramyyjän tulee olla niistä tietoinen ja toimia niiden mukaisesti. Suoramyyntiyrityksestä ja sen tuotteista ei synny helposti julkista brändiä, sillä markkinointi on ulkoistettu itsenäisille suoramyyjille perinteisten markkinointikanavien sijaan. Teorian sekä haastatteluiden avulla työssä kartoitettiin, mitä yrityksen brändi ja brändäys merkitsee suoramyyjän työssä. Haastatelluilta selvitettiin lisäksi, mitä hyviä ja huonoja puolia suoramyyntityössä on. Haastateltavat kokevat saavansa suoramyynnistä erilaisia hyötyjä. Myyntipalkkioiden lisäksi he saavat jakaa hyväksi kokemaansa tuotetta, saavat omaa aikaa ja luovat uusia ihmissuhteita. Opinnäytetyön tutkimusosiossa esitellään 20 esimerkkiyritystä. Työssä selvitettiin, kuinka niissä voi aloittaa myyntiedustajana. Jokaisen yritysesittelyn kohdalla selvitettiin myyjän työn osalta mahdolliset aloituskustannukset, tuleeko pitää omaa varastoa, pitääkö olla y-tunnus ja kuinka palkkio työstä muodostuu.The purpose of this thesis was to examine direct selling as a business opportunity. Direct selling is personal sales of products and services directly to consumers, usually in their homes or workplaces. Direct selling is a traditional and time-tested way of commerce. The thesis explains to the person considering to start direct selling what things he will need to take into account before starting a business. This thesis introduces companies which function in Finland and use direct sales concept. The subject of this thesis was chosen by my own interest in the topic. Also I find the topic to be current, because direct selling industry is a large and growing sector in Europe. More than six million people are working in direct selling industry in the EU. The general information of example companies was obtained on their websites and additional information was obtained from companies sales representatives. This gave information of direct selling and of the companies from the sales representatives’ points of view. Qualitative research method was used in this thesis. The study was conducted with individual interviews. The method of these interviews was virtual interview via e-mail. The interviewees were reached through social media and from Elomessut, which were held in Hämeenlinna 13. - 14. August 2016. The interviews were conducted during the autumn of 2016. The used source material are topic -related literature, web sites and legislation. At the beginning of this thesis is declared definitions of direct selling and home sales concept. When starting your own business you have to take into account various things, such as the starting investments, the establishment of the company, taxation and accounting obligation. These things are presented on a general level. The rules related to direct selling are adduced, as each direct seller should be aware of and act upon them. It is hard to arise a public brand for direct sales company and its products, because marketing is outsourced to independent direct sellers instead of traditional marketing channels. This thesis explores what the company brand and branding means for sales representatives in their work. Interviewees tell about the pros and cons of working among direct selling. The interviewees feel that they gain a variety of benefits for direct selling. In addition to sales commissions they get to share their good experiences of the product, will receive their own time and create new relationships. The research section of the thesis presents 20 example companies. It is described how you can start as a sales representative in them. In each company presentation it is described are there any start-up costs, whether you need to keep your own stock, whether you need to have a business ID and how the reward of the work is formed

Infectious Entry Pathway of Enterovirus B Species

Enterovirus B species (EV-B) are responsible for a vast number of mild and serious acute infections. They are also suspected of remaining in the body, where they cause persistent infections contributing to chronic diseases such as type I diabetes. Recent studies of the infectious entry pathway of these viruses revealed remarkable similarities, including non-clathrin entry of large endosomes originating from the plasma membrane invaginations. Many cellular factors regulating the efficient entry have recently been associated with macropinocytic uptake, such as Rac1, serine/threonine p21-activated kinase (Pak1), actin, Na/H exchanger, phospholipace C (PLC) and protein kinase Cα (PKCα). Another characteristic feature is the entry of these viruses to neutral endosomes, independence of endosomal acidification and low association with acidic lysosomes. The biogenesis of neutral multivesicular bodies is crucial for their infection, at least for echovirus 1 (E1) and coxsackievirus A9 (CVA9). These pathways are triggered by the virus binding to their receptors on the plasma membrane, and they are not efficiently recycled like other cellular pathways used by circulating receptors. Therefore, the best “markers” of these pathways may be the viruses and often their receptors. A deeper understanding of this pathway and associated endosomes is crucial in elucidating the mechanisms of enterovirus uncoating and genome release from the endosomes to start efficient replication.peerReviewe

Surface Modification of Bioactive Glass Promotes Cell Attachment and Spreading

Phosphate glasses have several advantages over traditional silicate-based bioglasses but are inferior in the crucial step of cell attachment to their surface. Here, as a proof of concept, we analyze fibroblast attachment to the phosphate glass surface subjected to basic treatment and silanization. Silicate (S53P4)- and phosphate (Sr50)-based bioactive glasses were either untreated or surface-treated with basic buffer and functionalized with silane. The surface-treated samples were studied as such and after fibronectin was adsorbed on to their surface. With both glass types, surface treatment enhanced fibroblast adhesion and spreading in comparison to the untreated glass. The surface-treated Sr50 glass allowed for cell adhesion, proliferation, and spreading to a similar extent as seen with S53P4 and borosilicate control glasses. Here, we show that surface treatment of bioactive glass can be used to attract cell adhesion factors found in the serum and promote cell–material adhesion, both important for efficient tissue integration.publishedVersionPeer reviewe