HIV-1 Nef protein and the Nef-associating kinase PAK2 in cell signaling

HIV- eli immuunikatovirustartuntaa kantaa maailmassa arviolta yli 30 miljoonaa ihmistä. Suomessa ilmoituksia HIV-positiivisista henkilöistä oli vuoden 2010 alkuun mennessä kirjattu runsas kolme tuhatta, ja määrä on viime vuosina lisääntynyt noin kahdellasadalla HIV-potilaalla vuosittain. Vaikka HIV:n lääkehoito on viime vuosina kehittynyt merkittävästi ja sairauden etenemistä pystytään tehokkaasti hidastamaan, ei virusta kuitenkaan pystytä nykyisillä lääkkeillä häätämään elimistöstä kokonaan. HIV infektoi pääasiassa ihmisen immuunijärjestelmän kannalta keskeisiä CD4(+) T-soluja sekä makrofageja. Lisääntyessään virus pystyy myös sekä suoraan että epäsuorasti tuhoamaan CD4(+) T-soluja eli auttajalymfosyyttejä. Tämän seurauksena on potilaan immuunipuolustuksen vähittäinen heikkeneminen ja HIV-infektion kehittyminen varsinaiseen AIDS-vaiheeseen, sekä useat siihen liittyvät sairaudet kuten kasvaimet ja muut tulehdukset. Nef on yksi HI-viruksen tuottamista virusproteiineista, ja keskeinen tekijä HIV-infektion kehittymiselle. Useat Nef-proteiinin toiminnot edistävät viruksen selviytymistä ja lisääntymistä potilaan elimistössä, ja infektion etenemisen onkin havaittu hidastuvan viallisen tai puuttuvan Nef-proteiinin seurauksena sekä luonnollisen HIV-infektion yhteydessä ihmisellä että kokeellisen SIV-infektion yhteydessä rhesusapinoilla. Nef ei itsessään ole entsymaattisesti aktiivinen proteiini, mutta sen kyky sitoutua solun omiin proteiineihin ja vaikuttaa niiden toimintaan selittää Nef:n vaikutusten laajan kirjon. Yksi tunnetuista Nef:iin sitoutuvista soluproteiineista on p21-aktivoituva kinaasi 2 (PAK2). Tämän työn tarkoituksena on ollut selvittää Nef/PAK2-kompleksin muodostumiseen vaikuttavia tekijöitä molekyylitasolla, sekä tämän kompleksin toimintaa solunsisäisessä viestinnässä. Tutkimuksessa kuvattiin uusi signalointimekanismi, joka mahdollistaa PAK2:n voimakkaan aktivoitumisen yhdistämällä kaksi viestintäreittiä. Molekyylibiologisia menetelmiä käyttäen tutkittiin myös PAK2:n aktivaatioon liittyvien proteiinin rakenteellisten muutosten, pienten GTPaasi-proteiinien sekä PAK2 kinaasin oman entsymaattisen aktiivisuuden merkitystä Nef/PAK2-kompleksin muodostumiselle. Proteiinikompleksin solunsisäistä sijaintia tutkittaessa sen havaittiin lokalisoituvan solussa tietyille, soluviestinnän kannalta keskeisille solukalvon ns. lipidilautta-alueille. GTPaaseja aktivoivan Vav1-proteiinin havaittiin osallistuvan Nef/PAK2-kompleksin muodostumiseen sekä kompleksin kulkeutumiseen solun kalvorakenteisiin. Lisäksi työssä tutkittiin Nef-molekyylin oletettua dimerisaatiopintaa, jonka vastakkaisesti varautuneilla aminohapoilla havaittiin olevan oleellinen merkitys Nef-Nef dimeerin muodostumiselle, jonka puolestaan todettiin olevan edellytys Nef/PAK2-kompleksin muodostumiselle sekä Nef:n välittämän HIV-partikkeleiden infektiivisyyden lisääntymiselle. Tulokset antavat uutta tietoa siitä, kuinka HIV käyttää solun omia signalointireittejä hyväkseen. HIV-1 Nef-proteiinin toiminnan kannalta tärkeiden vuorovaikutusten ymmärtäminen tarjoaa uusia hyödyntämismahdollisuuksia kohdennetulle lääkekehitykselle.Nef is an accessory protein encoded by the human and simian immunodeficiency viruses (HIV/SIV) and is a major factor in promoting viral pathogenesis. Several cellular functions of Nef, including downregulation of the surface molecules CD4 and MHC I, increase in viral infectivity, and Nef-mediated effects on intracellular signaling favor survival and propagation of the virus. Nef does not possess any intrinsic enzymatic activity; instead, it functions by interacting with a number of endogenous proteins within the host cell. One of the well-characterized Nef-associating protein partners and a central molecule in this study is p21-associating kinase 2 (PAK2). Previously, association of the HIV-1 Nef with PAK2 has been proposed to play a role in T cell activation, viral replication, apoptosis and progression of HIV infection to AIDS. Despite intense research, there is no consensus on the relevance of Nef-PAK2 association and its function in virus pathogenesis. Furthermore, selective association of Nef with a minute but highly active subpopulation of total cellular PAK2 has raised questions on the molecular requirements of Nef-PAK2 association. This work elucidated the interplay of Nef and PAK2 in cellular signaling by studying the mechanism of PAK2 activation and its role in Nef association. To clarify the role of additional proteins during Nef-PAK2 complex formation, the involvement of p21-GTPases and guanine nucleotide exchange factor (GEF) proteins was studied. Finally, the functional role of charge distribution within the putative Nef interaction surface was characterized. Here, we describe a new signaling pathway that potentiates PAK2 activation by converging signal transduction via Src tyrosine kinases with p21-GTPase-induced PAK activation. The p21-GTPase-induced conformational opening of PAK2 was associated with the ability of PAK2 to interact with Nef, and their association was stabilized by reduced kinase activity. The Nef-PAK2 (NAK) complex was specifically localized to detergent-resistant membranes (DRM), and Vav1 was identified as a GEF component of Nef-PAK2 complex, recruited to the DRMs by Nef. The reciprocal charges present at the putative dimer interface (PDI) of Nef were indispensable for Nef-dependent increase in viral infectivity. In addition, several other Nef activities, including NAK association, membrane localization, and activation of Hck were coordinated by charged residues at the Nef PDI. In conclusion, our results provide new insight on the cellular signaling upstream of Nef-PAK2 association, as well as on the molecular domains and binding partners that are important for the formation of the Nef-PAK2 complex. In addition, our data elucidate how Nef functions are related to charge distribution at the PDI surface. Detailed knowledge on the molecular mechanisms involved in Nef-PAK2-mediated signaling may provide clues for potential target sites that can be used to develop HIV inhibitory drugs

Kannustimet tuottavat tulosta : Sylvia-hankekokonaisuuden vaikuttavuuden arviointi

Tässä arviointiraportissa tarkastellaan työ- ja elinkeinoministeriön AMIF-rahoitteisen Sylvia-hankekokonaisuuden (2015–2018) vaikuttavuutta. Hankkeiden tavoitteena on ollut lisätä kuntapaikkojen saatavuutta kansainvälistä suojelua saaneille ja tehostaa pakolaisten kuntiin sijoittamista myöntämällä kunnille taloudellisia lisätukia sekä edistää pakolaistyötä tekevien osaamista ja verkostoitumista. Arvioinnin tulosten mukaan lisätuilla on tuettu pakolaisten vastaanoton käynnistämistä, jatkuvuutta ja kehittämistä kunnissa. Lisätukien avulla on muodostettu hätätapauksia vastaanottavien kuntien verkosto. Erityisesti hankkeen tuet on koettu tärkeiksi pienissä kunnissa. Kyselyyn vastanneista kunnista 74 % koki lisätuet tarpeellisiksi myös jatkossa. Suurin osa kunnista oli käyttänyt lisätukia henkilöstökuluihin ja koulutukseen sekä muihin kotouttamista tukeviin toimiin. Lisätuet vahvistivat joissain tapauksissa yhteistyötä kolmannen sektorin kanssa. Valtaosa arviointiin osallistuneista koki, että kuntien tulisi myös jatkossa voida päättää lisätukien kohdentamisesta. Arvioinnin perusteella suositellaan pidempää hankekautta, lisää joustavuutta vakioyksikkökustannuksiin ja tuen käytön kriteereihin sekä mahdollisimman proaktiivista ja selkeää tiedotusta kunnille. Arvioinnin mukaan hankekokonaisuus on onnistunut tavoitteessaan lisätä kuntapaikkojen saatavuutta ja tehostaa pakolaisten kuntiin sijoittamista. Tulosten perusteella lisätukirahoitus ja osaamisen tukeminen kunnissa on tarpeen jatkossakin. TEM:n yhteyshenkilö: Alue- ja kaupunkikehitys, kotouttaminen / Hanna-Maria Hyttinen, puh. 02950 4720

In situ production of vitamin B12 and dextran in soya flour and rice bran: A tool to improve flavour and texture of B12-fortified bread

This study aimed to develop a fermentation process that allows the concomitant production of dextran (a texture-enhancing agent) and vitamin B12 (in situ fortification) for bread applications. Mixed fermentation of soya flour or rice bran using Propionibacterium freudenreichii DSM 20271 and Weissella confusa A16 with added sucrose resulted in substantial quantities of dextran (5.6-5.8% dry matter) and B12 (7.9-8.9 mu g/100 g fresh weight), together with antifungal metabolites (e.g. acetic and propionic acids). In addition to an extended mould-free shelf life, the bread containing 50% (dough weight) fermented soya flour or rice bran not only contained adequate levels of B12 but also exhibited improved texture and sensory quality compared to the control, such as a higher loaf volume, a softer crumb, and a more cohesive and moister mouthfeel. The mixed fermentation reduced the beany or cooked-rice flavour but increased sour and cheesy flavours and aftertaste. Dextran pro-duced during the fermentation process exhibited a masking effect on the beany and sour notes and aftertaste, consistently with reduced levels of green or grassy volatiles (e.g. hexanal, heptanal, (E,E)-2,4-decadienal, and 2-pentylfuran). Overall, the mixed fermentation method using P. freudenreichii DSM 20271 and W. confusa A16 showed potential for B12 fortification of bread products with high sensory quality.Peer reviewe

Maternal Hypertensive Pregnancy Disorders and Mental and Behavioral Disorders in the Offspring : a Review

Purpose of Review We review here recent original research and meta-analytic evidence on the associations of maternal hypertensive pregnancy disorders and mental and behavioral disorders in the offspring. Recent Findings Seven meta-analyses and 11 of 16 original research studies published since 2015 showed significant associations between maternal hypertensive pregnancy disorders and offspring mental and behavioral disorders. Evidence was most consistent in meta-analyses and high-quality cohort studies. The associations, independent of familial confounding, were observed on different mental and behavioral disorders in childhood and schizophrenia in adulthood. Preterm birth and small-for-gestational age birth emerged as possible moderators and mediators of the associations. Cross-sectional and case-control studies yielded inconsistent findings, but had lower methodological quality. Summary Accumulating evidence from methodologically sound studies shows that maternal hypertensive pregnancy disorders are associated with an increased risk of mental and behavioral disorders in the offspring in childhood. More studies on adult mental disorders are needed.Peer reviewe

The Impact of Early Life Stress on Anxiety Symptoms in Late Adulthood

Early life stress (ELS) may increase the risk of anxiety throughout the life course. Whether this effect extends to late adulthood is poorly known. In our study comprising 1872 participants from the Helsinki Birth Cohort Study born in 1934-1944, we investigated the association of various forms of ELS and their accumulation with self-reported anxiety symptoms at the age of 65-77 years. Data on childhood socioeconomic status and separation from parents were based on national registers for all participants. Information on self-reported emotional and physical trauma, parental divorce, and death of a family member in childhood was obtained from 1277 participants. We found that experiencing emotional trauma, physical trauma, and low socioeconomic status in childhood were associated with increased anxiety symptoms in late adulthood [B = 0.44 (95% CI = 0.31-0.58); B = 0.33 (95%CI = 0.20-0.46); B = 0.10 (95%CI = 0.01-0.19), respectively]. These associations remained significant even after controlling for other forms of ELS. Accumulation of early life stress also increased the levels of late-adulthood anxiety symptoms and the risk of anxiety regarded as clinically significant. Screening for potentially stressful childhood experiences in elderly populations may help identifying individuals with increased anxiety symptoms and planning preventive and therapeutic interventions for those exposed to ELS.Peer reviewe

The Fate of Bacteriophages in Recirculating Aquaculture Systems (RAS)—Towards Developing Phage Therapy for RAS

Aquaculture production has increased tremendously during the last decades, and new techniques have been developed, e.g., recirculating aquaculture systems (RAS). In RAS, the majority of water volume is circulated via mechanical and biological filters and reused in the tanks. However, the prevention and treatment of diseases in these systems are challenging, as the pathogens spread throughout the system, and the addition of chemicals and antibiotics disrupts the microbiome of the biofilters. The increasing antibiotic resistance has made phage therapy a relevant alternative for antibiotics in food production. Indeed, as host-specific and self-replicating agent they might be optimal for targeted pathogen eradication in RAS. We tested the survival and spread of Flavobacterium columnare -infecting phage FCL-2 in recirculating aquaculture fish farm with rainbow trout (Oncorhynchus mykiss) in a fully controlled study. After a single addition, phage persisted in water samples collected from tank, fixed bed, moving bed, and aeration unit up to 14 days, and in the water of rearing tanks, rainbow trout mucus, and bioreactor carrier media from the fixed and moving bed biofilters for 21 days. Furthermore, phage adsorbed preferentially to moving bed carrier media, which contained biofilm attached and from which higher phage numbers were recovered. This study shows phages as a potent strategy for maintaining biosecurity in RAS systems.Aquaculture production has increased tremendously during the last decades, and new techniques have been developed, e.g., recirculating aquaculture systems (RAS). In RAS, the majority of water volume is circulated via mechanical and biological filters and reused in the tanks. However, the prevention and treatment of diseases in these systems are challenging, as the pathogens spread throughout the system, and the addition of chemicals and antibiotics disrupts the microbiome of the biofilters. The increasing antibiotic resistance has made phage therapy a relevant alternative for antibiotics in food production. Indeed, as host-specific and self-replicating agent they might be optimal for targeted pathogen eradication in RAS. We tested the survival and spread of Flavobacterium columnare -infecting phage FCL-2 in recirculating aquaculture fish farm with rainbow trout (Oncorhynchus mykiss) in a fully controlled study. After a single addition, phage persisted in water samples collected from tank, fixed bed, moving bed, and aeration unit up to 14 days, and in the water of rearing tanks, rainbow trout mucus, and bioreactor carrier media from the fixed and moving bed biofilters for 21 days. Furthermore, phage adsorbed preferentially to moving bed carrier media, which contained biofilm attached and from which higher phage numbers were recovered. This study shows phages as a potent strategy for maintaining biosecurity in RAS systems.Peer reviewe

Maternal early-pregnancy body mass index-associated metabolomic component and mental and behavioral disorders in children

Maternal pre-pregnancy obesity and/or higher body mass index (BMI) have been associated with neurodevelopmental and mental health adversities in children. While maternal metabolomic perturbations during pregnancy may underpin these associations, the existing evidence is limited to studying individual metabolites, not capturing metabolic variation specific to maternal BMI, and not accounting for the correlated nature of the metabolomic measures. By using multivariate supervised analytical methods, we first identified maternal early-pregnancy BMI-associated metabolomic component during pregnancy. We then examined whether this component was associated with mental and behavioral disorders in children, improved the prediction of the child outcomes over maternal BMI, and what proportion of the effect of maternal BMI on the child outcomes this component mediated. Early-pregnancy BMI of 425 mothers participating in the PREDO study was extracted from the national Medical Birth Register. During pregnancy, mothers donated up to three blood samples, from which a targeted panel of 68 metabolites were measured. Mental and behavioral disorders in children followed-up from birth until 8.4-12.8 years came from the Care Register for Health Care. Of the 68 metabolites averaged across the three sampling points, 43 associated significantly with maternal early-pregnancy BMI yielding a maternal early-pregnancy BMI-associated metabolomic component (total variance explained, 55.4%; predictive ability, 52.0%). This metabolomic component was significantly associated with higher hazard of any mental and behavioral disorder [HR 1.45, 95%CI(1.15, 1.84)] and relative risk of having a higher number of co-morbid disorders [RR 1.43, 95%CI(1.12, 1.69)] in children. It improved the goodness-of-model-fit over maternal BMI by 37.7-65.6%, and hence the predictive significance of the model, and mediated 60.8-75.8% of the effect of maternal BMI on the child outcomes. Maternal BMI-related metabolomic perturbations during pregnancy are associated with a higher risk of mental and behavioral disorders in children. These findings may allow identifying metabolomic targets for personalized interventions.Peer reviewe

Flavor challenges in extruded plant-based meat alternatives: A review

Demand for plant-based meat alternatives has increased in recent years due to concerns about health, ethics, the environment, and animal welfare. Nevertheless, the market share of plant-based meat alternatives must increase significantly if they are to support sustainable food production and consumption. Flavor is an important limiting factor of the acceptability and marketability of plant-based meat alternatives. Undesirable chemosensory perceptions, such as a beany flavor, bitter taste, and astringency, are often associated with plant proteins and products that use them. This study reviewed 276 articles to answer the following five research questions: (1) What are the volatile and nonvolatile compounds responsible for off-flavors? (2) What are the mechanisms by which these flavor compounds are generated? (3) What is the influence of thermal extrusion cooking (the primary structuring technique to transform plant proteins into fibrous products that resemble meat in texture) on the flavor characteristics of plant proteins? (4) What techniques are used in measuring the flavor properties of plant-based proteins and products? (5) What strategies can be used to reduce off-flavors and improve the sensory appeal of plant-based meat alternatives? This article comprehensively discusses, for the first time, the flavor issues of plant-based meat alternatives and the technologies available to improve flavor and, ultimately, acceptability.Peer reviewe

Maternal depressive symptoms during and after pregnancy are associated with poorer sleep quantity and quality and sleep disorders in 3.5-year-old offspring

Objective: Maternal depressive symptoms during pregnancy have been associated with poor offspring sleep. Yet, it remains unknown whether depressive symptoms throughout pregnancy are more harmful to the child than depressive symptoms only during certain time periods in pregnancy, whether associations are specific to pregnancy stage, whether maternal symptomatology after pregnancy mediates or adds to the prenatal effects, and whether any effects are specific to some child sleep characteristics. Methods: A total of 2321 mothers from the Prediction and Prevention of Pre-eclampsia and Intrauterine Growth Restriction (PREDO) study completed the Center for Epidemiological Studies Depression Scale biweekly between gestational weeks thorn days 12 + 0/13 + 6 and 38 + 0/39 + 6. At child's mean age of 3.5 (standard deviation = 0.7) years, mothers completed the Beck Depression Inventory-II and answered questions on child sleep quantity and quality using the Brief Infant Sleep Questionnaire (BISQ) and sleep disorders using the Sleep Disturbance Scale for Children. Results: Maternal depressive symptoms showed high stability throughout pregnancy. Children of mothers with clinically significant symptomatology throughout pregnancy had shorter mother-rated sleep duration, longer sleep latency, higher odds for waking up two or more times during the night and for total and several specific sleep disorders. These associations were robust to covariates. However, maternal depressive symptoms at the child follow-up fully mediated the associations with sleep duration and awakenings, partially mediated those with sleep latency and disorders, and added to the effects on sleep disorders. Conclusion: Maternal depressive symptoms throughout pregnancy are associated with mother-rated child sleep quantity, quality, and disorders. Maternal depressive symptoms at child follow-up mediate and add to the prenatal adverse effects on child sleep characteristics. (C) 2018 Elsevier B.V. All rights reserved.Peer reviewe

Identification of proprotein convertase substrates using genome-wide expression correlation analysis

Identification of proprotein convertase substrates using genome-wide expression correlation analysis Turpeinen, Hannu Kukkurainen, Sampo Pulkkinen, Kati Kauppila, Timo Ojala, Kalle Hytonen, Vesa P Pesu, Marko England BMC genomics BMC Genomics. 2011 Dec 20;12:618. engABSTRACT: BACKGROUND: Subtilisin/kexin-like proprotein convertase (PCSK) enzymes have important regulatory function in a wide variety of biological processes. PCSKs proteolytically process at a target sequence that contains basic amino acids arginine and lysine, which results in functional maturation of the target protein. In vitro assays have showed significant biochemical redundancy between the seven family members, but the phenotypes of PCSK deficient mice and patients carrying an inactive PCSK allele argue for a specific biological function. Modeling the structures of individual PCSK enzymes has offered little insights into the specificity determinants. However, previous studies have shown that there can be a coordinated expression between a PCSK and its target molecule. Here, we have surveyed the putative PCSK target proteins using genome-wide expression correlation analysis and cleavage site prediction algorithms. RESULTS: We first performed a gene expression correlation analysis over the whole genome for all PCSK enzymes. PCSKs were found to cluster differently based on the strength of correlations. The screen for putative PCSK target proteins showed a significant enrichment (p-values from 1.2e-4 to <1.0e-10) of putative targets among the most positively correlating genes for most PCSKs. Interestingly, there was no enrichment in putative targets among the genes that correlated positively with the biologically redundant PCSK7, whereas PCSK5 showed an inverse correlation. PCSKs also showed a highly variable degree of shared target genes that were identified by expression correlation and cleavage site prediction. Multiple alignments were used to evaluate the putative targets to pinpoint the important residues for the substrate recognition. Finally, we validated our approach and identified biochemically PAPPA1 and ADAMTS6 as novel targets for FURIN proteolytic activity. CONCLUSIONS: Most PCSK enzymes display strong positive expression correlation with predicted target proteins in our genome-wide analysis. We also show that expression correlation screen combined with a cleavage site-prediction analysis can be used to identify novel bona fide target molecules for PCSKs. Exploring the positively correlating genes can thus offer additional insights into the biology of proprotein convertases.Peer reviewe