Changes in the expression level of the genes involved in the innate and adaptive immunity of divers

771-778From time immemorial, humans had engaged in breath-hold diving. Developing the scuba (self-contained underwater breathing apparatus) in the last century has made humans increase the capabilities and efficiency of diving. Shallow diving is usually without side effects, but there may be a series of side effects called Decompression Sickness (DCS), which can even lead to severe neurological damages and death in deep and long dives. Scuba diving and its complications alter the pattern of many genes expression involved in innate and adaptive immunity. Researchers have reported various types of these changes in both the genomes of healthy and sick divers. This study surveyed the ten gene expression levels imported into immune responses like apoptosis and inflammation by real-time PCR in Iranian professional fit divers in steady-state. These genes were: Interleukins (IL-6, IL-8, IL-10), Tumor Necrosis Factor (TNFα), complement C3 (C3α), Tumor Necrosis Factor Receptor Type 1 - Associated Death Domain (TRADD), bradykinin receptor B2 (BDKRB2), rennin (REN), arachidonate 5-lipoxygenase (ALOX5), and prostaglandin-endoperoxide synthase 2 (PTGS2). The results showed that the expression levels of TNFα, ALOX5, TRADD, and interleukin genes increased, but PTGS2, REN, and C3α genes' expression levels did not change much. BDKRB2 gene expression level also decreased

Comparative and temporal transcriptome analysis of peste des petits ruminants virus infected goat peripheral blood mononuclear cells

Peste des petits ruminanats virus (PPRV), a morbillivirus causes an acute, highly contagious disease – peste des petits ruminants (PPR), affecting goats and sheep. Sungri/96 vaccine strain is widely used for mass vaccination programs in India against PPR and is considered the most potent vaccine providing long-term immunity. However, occurrence of outbreaks due to emerging PPR viruses may be a challenge. In this study, the temporal dynamics of immune response in goat peripheral blood mononuclear cells (PBMCs) infected with Sungri/96 vaccine virus was investigated by transcriptome analysis. Infected goat PBMCs at 48 h and 120 h post infection revealed 2540 and 2000 differentially expressed genes (DEGs), respectively, on comparison with respective controls. Comparison of the infected samples revealed 1416 DEGs to be altered across time points. Functional analysis of DEGs reflected enrichment of TLR signaling pathways, innate immune response, inflammatory response, positive regulation of signal transduction and cytokine production. The upregulation of innate immune genes during early phase (between 2-5 days) viz. interferon regulatory factors (IRFs), tripartite motifs (TRIM) and several interferon stimulated genes (ISGs) in infected PBMCs and interactome analysis indicated induction of broad-spectrum anti-viral state. Several Transcription factors – IRF3, FOXO3 and SP1 that govern immune regulatory pathways were identified to co-regulate the DEGs. The results from this study, highlighted the involvement of both innate and adaptive immune systems with the enrichment of complement cascade observed at 120 h p.i., suggestive of a link between innate and adaptive immune response. Based on the transcriptome analysis and qRT-PCR validation, an in vitro mechanism for the induction of ISGs by IRFs in an interferon independent manner to trigger a robust immune response was predicted in PPRV infection

Evaluation of mRNA markers for estimating blood deposition time : towards alibi testing from human forensic stains with rhythmic biomarkers

This study was supported by grant 27.011.001 by the Netherlands Organization for Scientific Research (NWO) Forensic Science Program, Erasmus MC University Medical Center Rotterdam, by the EU 6th Framework project EUCLOCK (018741), UK Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/I019405/1, and by a previous grant from the Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO) within the framework of the Forensic Genomics Consortium Netherlands (FGCN). D.J.S. is a Royal Society Wolfson Research Merit Award holder.Determining the time a biological trace was left at a scene of crime reflects a crucial aspect of forensic investigations as - if possible - it would permit testing the sample donor's alibi directly from the trace evidence, helping to link (or not) the DNA-identified sample donor with the crime event. However, reliable and robust methodology is lacking thus far. In this study, we assessed the suitability of mRNA for the purpose of estimating blood deposition time, and its added value relative to melatonin and cortisol, two circadian hormones we previously introduced for this purpose. By analysing 21 candidate mRNA markers in blood samples from 12 individuals collected around the clock at 2 h intervals for 36 h under real-life, controlled conditions, we identified 11 mRNAs with statistically significant expression rhythms. We then used these 11 significantly rhythmic mRNA markers, with and without melatonin and cortisol also analysed in these samples, to establish statistical models for predicting day/night time categories. We found that although in general mRNA-based estimation of time categories was less accurate than hormone-based estimation, the use of three mRNA markers HSPA1B, MKNK2 and PER3 together with melatonin and cortisol generally enhanced the time prediction accuracy relative to the use of the two hormones alone. Our data best support a model that by using these five molecular biomarkers estimates three time categories, i.e., night/early morning, morning/noon, and afternoon/evening with prediction accuracies expressed as AUC values of 0.88, 0.88, and 0.95, respectively. For the first time, we demonstrate the value of mRNA for blood deposition timing and introduce a statistical model for estimating day/night time categories based on molecular biomarkers, which shall be further validated with additional samples in the future. Moreover, our work provides new leads for molecular approaches on time of death estimation using the significantly rhythmic mRNA markers established here.PostprintPeer reviewe

PLoS One

IntroductionIrritable Bowel Syndrome (IBS) and Inflammatory Bowel Disease (IBD) can profoundly affect quality of life and are influenced by stress and resiliency. The impact of mind-body interventions (MBIs) on IBS and IBD patients has not previously been examined.MethodsNineteen IBS and 29 IBD patients were enrolled in a 9-week relaxation response based mind-body group intervention (RR-MBI), focusing on elicitation of the RR and cognitive skill building. Symptom questionnaires and inflammatory markers were assessed pre- and post-intervention, and at short-term follow-up. Peripheral blood transcriptome analysis was performed to identify genomic correlates of the RR-MBI.ResultsPain Catastrophizing Scale scores improved significantly post-intervention for IBD and at short-term follow-up for IBS and IBD. Trait Anxiety scores, IBS Quality of Life, IBS Symptom Severity Index, and IBD Questionnaire scores improved significantly post-intervention and at short-term follow-up for IBS and IBD, respectively. RR-MBI altered expression of more genes in IBD (1059 genes) than in IBS (119 genes). In IBD, reduced expression of RR-MBI response genes was most significantly linked to inflammatory response, cell growth, proliferation, and oxidative stress-related pathways. In IBS, cell cycle regulation and DNA damage related gene sets were significantly upregulated after RR-MBI. Interactive network analysis of RR-affected pathways identified TNF, AKT and NF-\uce\ubaB as top focus molecules in IBS, while in IBD kinases (e.g. MAPK, P38 MAPK), inflammation (e.g. VEGF-C, NF-\uce\ubaB) and cell cycle and proliferation (e.g. UBC, APP) related genes emerged as top focus molecules.ConclusionsIn this uncontrolled pilot study, participation in an RR-MBI was associated with improvements in disease-specific measures, trait anxiety, and pain catastrophizing in IBS and IBD patients. Moreover, observed gene expression changes suggest that NF-\uce\ubaB is a target focus molecule in both IBS and IBD\ue2\u20ac\u201dand that its regulation may contribute to counteracting the harmful effects of stress in both diseases. Larger, controlled studies are needed to confirm this preliminary finding.Trial RegistrationClinicalTrials.Gov NCT02136745R01DP000339/DP/NCCDPHP CDC HHS/United States25927528PMC441576

Kuolema kuittaa univelat? : effects of cumulative sleep loss on immune functions and lipid metabolism

Sleep is an essential physiological function. It is conserved across the animal kingdom; no animal species studied has been shown not to sleep. The timing of sleep and wakefulness is regulated by two processes. The circadian process works like a clock and accounts for the timing of sleep and arousal, synchronised by the light dark rhythm. The homeostatic process acts like an hourglass balancing the amount of sleep vs wakefulness. In case waking is prolonged, the homeostatic sleep need drives more sleep to take place, i.e. the sleep rebound. The homeostatic aspect of sleep can be studied by restricting sleep in laboratory conditions and assessing the effects on e.g. cognitive functions and physiological processes. Sleep is not merely a function of the brain, occurring in the brain and for the brain. The brain acts in concert with other organs and tissues in physiological and pathophysiological processes. During the recent decades, epidemiological studies have suggested that there is a connection between short or insufficient sleep with higher mortality. Increased risk for cardiovascular diseases, atherosclerosis, type II diabetes, and obesity has been reported in individuals who sleep less than the average. Laboratory studies have partly supported these findings, suggesting a causative role of sleep loss in the development of metabolic diseases, particularly type II diabetes. Experimental sleep restriction has been shown to alter glucose metabolism towards insulin resistance. Studies on the effects of sleep loss on lipid metabolism have been more inconclusive. Sleep is tightly interconnected with the immune system. Experimental sleep restriction increases proinflammatory cytokines, which in turn promote sleep. Atherosclerosis is the pathophysiological process underlying ischaemic heat disease and stroke, the two leading causes of death worldwide. The immune system plays a major role in the development of this metabolic disease characterised by plaque-formation in the arterial walls. Altered cholesterol transport by low density and high density lipoproteins (LDL and HDL) triggers an immune response involving macrophages and other white blood cells. Chronic low-grade inflammation has been shown to in turn predict future cardiovascular diseases. Thus, the development of atherosclerosis is a complex process with both metabolic and immunological components. In the current thesis, I have investigated the effects of sleep loss on gene expression and metabolites in the blood, focusing on changes that may participate in the development of cardiovascular diseases, especially atherosclerosis. Short-term sleep loss was studied in carefully controlled laboratory conditions with an experimental protocol simulating a working week with restricted sleep (4 h sleep/night for 5 nights; N=21). Sleep loss occurring chronically in real-life conditions was assessed in two Finnish epidemiological cohorts. Subjective sleep insufficiency (SSI) was estimated using questionnaire information on sleep, and a prevalence of 16-18% was found in these samples (FINRISK2007/DILGOM; N=518; SSI 16%, and Young Finns Study, YFS; N=2221; SSI 18%). In both the experimental and epidemiological samples, whole-genome expression profiles were assessed with RNA microarrays and serum lipoprotein profiles with NMR metabolomics. Immune response-related gene pathways were enriched among transcripts with higher expression in experimental sleep loss. Pathways involved in reverse cholesterol transport (RCT) were down-regulated in both experimental and epidemiological sleep loss. Concentration of large high density lipoprotein (HDL) particles was lower in subjects with SSI, even though in experimental sleep loss the low density lipoproteins (LDL) decreased. Up-regulation of low-grade inflammation-related pathways, and down-regulation of RCT-related pathways with decreased serum large HDL in chronic sleep loss may participate in the development of cardiovascular diseases, such as atherosclerosis. Experimental and epidemiological sleep studies in human volunteers can complement each other, but still yield information mostly from blood samples. Other methods are needed to elucidate mechanisms involving e.g. the liver. As sleep is a complex phenomenon involving synchronised activity of neuronal networks and integration with other systems and organs, it is not feasible to be studied in vitro, i.e. in cultured cells. Thus, animal models are needed to further study the effects of sleep loss at the level of molecular mechanisms. Zebrafish is a small diurnal vertebrate whose genome has been sequenced. It has a short generation time, readily available genetic tools, and it is well suited for in vivo imaging studies thanks to its transparent larval stage. Sleep or sleep-like states have been reported in this species using behavioural criteria. According to these studies, adult and larval zebrafish exhibit behavioural quiescence periods with circadian timing and increased arousal threshold. However, the homeostatic sleep rebound after prolonged wakefulness has not been unquestionably proven in this species. To confirm sleep homeostasis and validate this model for further studies on the effects of sleep loss, I developed a method for naturally prolonging the waking activity of zebrafish larvae. After 6 hours of this water flow protocol applied during the night, the larvae showed less responses to sensory stimuli than control larvae. Thus, I suggest that zebrafish larvae do have sleep homeostasis and they can be a useful model to study the sleep loss-related mechanisms involved in disease development.Aristoteles aprikoi jo antiikin Kreikassa 300-luvulla eaa., onko uni mielen, ruumiin vai kenties molempien toiminto. Aivoihin keskittyvät teoriat ovat sittemmin olleet unitutkimuksessa pitkälti vallalla. Viime vuosikymmeninä kiinnostus unen ja kehon toimintojen yhteyttä kohtaan on kuitenkin herännyt uudelleen. Immuunijärjestelmän on havaittu olevan tiiviissä vuoropuhelussa unen säätelyn kanssa. Tulehdusta välittävät tekijät (proinflammatoriset sytokiinit) lisääntyvät univajeessa. Toisaalta nämä samat viestimolekyylit myös lisäävät unta, kuten arkielämässä saattaa bakteeri- tai virusinfektioiden yhteydessä havaita. Vaikka univajetilassa ei ole infektiota, se näyttää aiheuttavan elimistössä puolustusreaktion. Alkuperäinen immuunijärjestelmän aktivaation laukaiseva tekijä ei ole kuitenkaan tiedossa. Väestötutkimuksissa on havaittu yhteys lyhyen tai riittämättömän unen ja kohonneeseen kuolleisuuden välillä. Myös sydän- ja verisuonitautiriski on joidenkin tutkimusten mukaan keskimääräistä korkeampi vähän nukkuvilla. Näiden epidemiologisten löydösten selittäjiksi on ehdotettu monia tekijöitä univajeen yhteydessä usein esiintyvistä epäterveellisistä elintavoista fysiologisiin tekijöihin. Kokeelliset tutkimukset ovat osin tukeneet väestötason havaintoja. Univajeen on osoitettu mm. nostavan verenpainetta ja ajavan hiilihydraattiaineenvaihduntaa insuliiniresistenssin suuntaan lisäten kakkostyypin diabeteksen riskiä. Rasva-aineenvaihduntaa on tutkittu vähemmän, ja tulokset ovat olleet ristiriitaisia. Tämän väitöskirjatutkimuksen osatöissä selvitin univajeen aiheuttamia muutoksia ihmisen immuunijärjestelmässä ja aineenvaihdunnassa. Tutkin univajeen vaikutuksia sekä tarkoin kontrolloiduissa kokeellisissa olosuhteissa (laboratoriossa simuloitu vähäuninen työviikko, unta 4 h/yö viiden yön ajan, N=21) että väestötasolla (kansallisen FINRISKI 2007 -terveystutkimuksen osaotos, N=472, sekä Lasten Sepelvaltimotaudin Riskitekijät -aineiston 2007-aikapiste, N=2221). Keskityin erityisesti muutoksiin, jotka saattavat osallistua sydän- ja verisuonitautien kehitykseen. Tulokset osoittivat, että unen rajoittaminen kokeellisesti terveillä koehenkilöillä aktivoi immuunijärjestelmän geenien ilmentymisen tasolla. Kolesterolin kuljetukseen osallistuvat geenit olivat vähemmän aktiivisia univajeisilla sekä kokeellisessa univajeessa että väestöaineistoissa. Veren lipoproteiinitasoissa kokeellinen univaje laski LDL-partikkeleiden määrää, kun taas väestötasolla suuria HDL-partikkeleita oli vähemmän riittämättömästi nukkuvilla. Ehdotan, että immuunijärjestelmän aktivoituminen univajeessa muuttaa aineenvaihdunnan säätelyä. Tämä näkyy lyhyellä aikavälillä LDL:ien vähentymisenä, mikä perinteisesti tulkitaan sydän- ja verisuonitautiriskiä vähentäväksi. Univajeen ja tulehdustilan kroonituessa kolesteroliaineenvaihdunta saattaa kuitenkin kääntyä epäedulliseen suuntaan ja altistaa sydän- ja verisuonitautien kehittymiselle yhdessä muiden riskitekijöiden kanssa. Kokeelliset ja väestötason tutkimukset täydentävät toisiaan tarjoten tietoa sekä valvotuista laboratorio-olosuhteista että tosielämän pitkäaikaisvaikutuksista. Ihmisillä tehtävissä tutkimuksissa saadaan yleensä kuitenkin tietoa lähinnä verinäytteistä. Vaikka veressä kiertävät valkosolut ovatkin monien sairauksien keskiössä, myös muut elimet, kuten maksa, ovat olennaisia näiden monimutkaisten prosessien säätelyssä. Koska uni liittyy hermoverkkojen yhteistoimintaan ja laajemmin aivojen ja muiden elimien yhteistoimintaan ei sen tutkiminen in vitro eli soluviljelmissä petrimaljoilla ole mielekästä. Täten eläinmallit ovat tarpeen, kun selvitetään univajeen ja tautiriskin yhteyden taustalla vaikuttavia molekyylitason mekanismeja. Aristoteles pohti, nukkuvatko kaikki eläimet ja onko eri lajien nukkuminen samankaltaista ja lähtöisin samasta tarpeesta. Hän esitti kalojen käyttäytymisen havainnointiin perustuen, että kalojen voidaan todeta nukkuvan. Havainnot perustuivat kalojen ajoittaiseen paikallaanoloon, johon liittyi myös aistien osittainen sulkeminen. Tähän yhdistyi myös lajityypillisiä nukkumisasentoja ja -paikkoja. 1980-luvulla esitetyt käyttäytymiskriteerit unen määrittämiseen ilman aivosähkökäyrää (EEG) perustuvat samantyyppiseen havainnointiin. Näillä kriteereillä on raportoitu nukkumista tai nukkumisenkaltaisia tiloja monenlaisilta eläinlajeilta, mukaan lukien kaloilta, banaanikärpäsiltä ja sukkulamadoilta. Seeprakala on biolääketieteellisessä tutkimuksessa verrattain paljon käytetty mallieläin, jonka genomi on sekvensoitu. Lajin etuina tutkimuksessa ovat mm. lyhyt sukupolvien väli, poikasvaiheen läpinäkyvyys sekä geneettinen muokattavuus. Tämän päiväaktiivisen selkärankaisen on myös käyttäytymiskriteerien perusteella ehdotettu nukkuvan. Seeprakalalla on valveen ja unen ajoituksesta vastaava sirkadiaaninen järjestelmä eli vuorokausirytmi. Unen säätelyn toinen kulmakivi, homeostaattinen unipaine, vastaa unen ja valveen määrän tasapainosta. Valveen pitkittyessä unipaine kasvaa, minkä tuloksena voidaan havaita enemmän ja/tai syvempää unta. Tätä kutsutaan korvausuneksi, ja se voidaan havaita esimerkiksi kohonneena kynnyksenä reagoida aistiärsykkeisiin. Tätä ns. homeostaattista sleep reboundia ei ollut kiistattomasti todistettu seeprakalalta. Tässä työssä kehitin seeprakalan poikasille luonnollisen menetelmän valveen pitkittämiseen ja reaktioiden mittaamiseen. Menetelmän avulla sain osoitettua, että kalanpoikaset, joiden unta oli rajoitettu yön aikana, reagoivat vähemmän kuin verrokit. Tämän tulkitsen merkiksi homeostaattisesta sleep reboundista. Seeprakalan poikaset soveltuvat tämän jälkeen ihmisainaistoissa saamieni tulosten tarkempiin mekanismitason tutkimuksiin. Mm. kolesterolin kulkeutumista voi seurata in vivo eli elävässä eläimessä ja saada tarkempaa tietoa univajeen aiheuttamista kolesteroliaineenvaihdunnan muutoksista. Kroonistuessaan univaje saattaa ylläpitää elimistössä matala-asteista tulehdustilaa ja muuttaa kolesteroliaineenvaihdunnan säätelyä, ja siten osallistua sydän- ja verisuonitautien kehittymiseen. Seeprakalan poikaset voivat tarjota mahdollisuuksia taustalla vaikuttavien molekyylitason mekanismien tarkempiin jatkotutkimuksiin

Transcriptional and translational dynamics of the human heart

Die Genexpression wurde bisher hauptsächlich auf Transkriptions- und Proteinebene untersucht, wobei der Einfluss der Translation, die die Proteinhäufigkeit direkt beeinflusst, weitgehend außer Acht gelassen wurde. Um diese Rolle besser zu verstehen, habe ich Ribosomen-Profiling-Daten (Ribo-seq) verwendet, um die Translationsregulation zu untersuchen und neue Translationsvorgänge in 65 linksventrikulären Proben von DCM-Patienten im Endstadium und 15 Nicht-DCM-Kontrollen zu identifizieren. Dieser Datensatz half dabei, die Transkriptions- und Translationsregulation zwischen erkrankten und nicht betroffenen menschlichen Herzen zu sezieren und enthüllte Gene und Prozesse, die rein unter Translationskontrolle stehen. Darüber hinaus habe ich neue kardiale Proteine vorhergesagt, die von langen nicht-kodierenden RNAs (lncRNAs) und zirkulären RNAs (circRNAs) translatiert werden. Computergestützte Analysen dieser evolutionär jungen Proteine legten eine Beteiligung an verschiedenen molekularen Prozessen nahe, mit einer besonderen Anreicherung für den mitochondrialen Energiestoffwechsel. Schließlich identifizierte ich RNA-bindende Proteine (RBPs), deren Expression die Menge der Ziel-mRNA oder die Frequenz der Translationseffizienz (TE) beeinflusst. Für eine Untergruppe von 21 RBPs habe ich die Regulation auf beiden quantitativen Merkmalen beobachtet, was zu einer unterschiedlichen mechanistischen Basis der Expressionskontrolle für unabhängige Gensätze führte. Obwohl die genaue Umschaltung der RBP-Funktion wahrscheinlich durch eine Kombination von mehreren Faktoren erreicht wird, haben wir für drei Kandidaten eine starke Abhängigkeit von der Zielgenlänge und der 5'-UTR-Struktur beobachtet. Diese Arbeit präsentiert einen Katalog von neu identifizierten Translationsereignissen und einen quantitativen Ansatz zur Untersuchung der Translationsregulation im gesunden und kranken menschlichen Herzen.Gene expression has primarily been studied on transcriptional and protein levels, largely disregarding the extent of translational regulation that directly influences protein abundance. To elucidate its role, I used ribosome profiling (Ribo-seq) data, obtained through ribosome profiling, to study translational regulation and identify novel translation events in 65 left ventricular samples of end-stage DCM patients and 15 non-DCM controls. This dataset helped dissect transcriptional and translational regulation between diseased and unaffected human hearts, revealing genes and processes purely under translational control. These would have remained undetected by only looking at the transcriptional level. Furthermore, I predicted novel cardiac proteins translated from long non-coding RNAs (lncRNAs) and circRNAs. Computational analysis of these evolutionary young proteins suggested involvement in diverse molecular processes with a particular enrichment for mitochondrial processes. Finally, I identified RNA-binding proteins (RBPs) whose expression influences target mRNA abundance or translational efficiency (TE) rates. For a subset of 21 RBPs, I have observed regulation on both quantitative traits, which resulted in different mechanistic basis expression control for independent sets of genes. Though the precise switch in RBP function is likely achieved by a combination of multiple factors, for three candidates we have observed a strong dependency on target length and 5’ UTR structure. This work presents a catalogue of newly identified translation events and a quantitative approach to study translational regulation in the healthy and failing human heart