Amplifying control RNA for RT-PCR applications by nucleic acid sequence based amplification (NASBA)

Control RNA for RT-PCR applications was amplified by nucleic acid sequence based amplification (NASBA) using the NucliSens (R) Basic Kit. This method was used to construct positive control RNA for enterovirus, insulin, and G-protein RT-PCR, and for interferon-alpha real-time RT-PCR. The primers were designed to amplify identical RNA from RNA templates, which differs from the usual NASBA procedure, where opposite strand RNA is amplified from the target. This "inverse NASBA" method is easy to use and it does not require any expensive special equipment. The amplification reaction is done using a water bath and detection of amplified product by agarose gel electrophoresis. Generated RNA fragments were 195-714 bases long, of positive polarity and the amount of RNA was sufficient for thousands of RT-PCR reactions depending on the sensitivity of the RT-PCR. (c) 2005 Elsevier B.V. All rights reserved

Oura-sormuksen ja Polar-rannekkeen keräämän fyysisen aktiivisuus tiedon vertailu Pohjois-Suomen syntymäkohortti 1986 pilotissa

Tiivistelmä. Työtarkoitus: Tutkimuksen tarkoituksena oli vertailla Oura-sormuksen ja Polar Active-rannekkeen päivittäisen fyysisen aktiivisuuden määrää ja selvittää oliko tutkimushenkilöillä eroja vapaapäivien ja työpäivien aktiivisuudessa. Menetelmät: Tutkimukseen osallistui 30 henkilöä. Yhtäaikaista aktiivisuusdataa saatiin 22 (73 %) henkilöltä ja vapaapäiviä ja työpäiviä pystyttiin vertailemaan 12 (40 %) henkilöltä. Tutkimushenkilöt pitivät molempia aktiivisuusmittareita kahden viikon ajan ja täyttivät tältä ajalta myös päiväkirjaa, johon merkattiin vapaapäivät, työpäivät, ”valot pois”-aika, heräämisaika ja lisätietoihin mm. päiväunet tai mittareiden riisumiset. Tutkimusdataa vertailtiin Microsoft excel-ohjelmalla. Tulokset: Yhtäaikaista dataa saatiin keskimäärin 6,6 päivän ajalta. Mittareiden mittaavat aktiivisuuden korreloivat keskenään, niin aktiivisuustasoittain, kuin kokonaisaktiivisuuden mittauksessa, yksittäisiä suurempia heittoja lukuun ottamatta. Aktiivisuustasojen MET-minuuttien keskimääräiset korrelaatiot olivat tasoittain 1–4: 0,64 (p0,05). Johtopäätökset: Oura-sormuksen ja Polar Active-rannekkeen mittaavat päivittäisen aktiivisuudet korreloivat keskenään, vaikka joitakin eroja yksittäisten mittauspäivien välillä oli. Vapaapäivien ja työpäivien aktiivisuudessa ei ollut merkittävää eroa.Comparison of the physical activity measurements between Oura-ring and Polar-bracelet at the pilot study of the 1986 North Finland birth cohort. Abstract. Objective: The objective of this study was to compare daily physical activity measurements between Oura-ring and Polar-bracelet and figure out if there were differences in physical activity between workday and a rest day. Methods: 30 participants were participated in this study. Simultaneous active data were measured from 22 (73 %) participants and workdays and rest days was compared from 12 (40 %) participants. Participants wore both devices and wrote diaries for two weeks. In diaries participants wrote down workdays, rest days, “lights off”-time, wakeup time and to additional information naps and if the devices were taken off. All the data was compared with Microsoft excel. Results: Average number of days when the data was simultaneous were 6,6. Differences between the devices on the total physical activity and the activity levels were detected, but the average correlation of MET-minutes correlated between Oura and Polar active measurements at the activity levels 1–4 with 0,64 (p0,05). Conclusion: Daily activity measurements between Oura-ring and Polar-bracelet were pretty close each other and only in few individual cases bigger differences were detected. Between workdays and rest days there was not significant differences in physical activity

Molecular analysis of an echovirus 3 strain isolated from an individual concurrently with appearance of islet cell and IA-2 autoantibodies

Growing evidence has implicated members of the genus Enterovirus of the family Picornaviridae in the etiology of some cases of type I diabetes (T1D). To contribute to an understanding of the molecular determinants underlying this association, we determined the complete nucleotide sequence of a strain of echovirus 3 (E3), Human enterovirus B (HEV-B) species, isolated from an individual who soon after virus isolation developed autoantibodies characteristic of T1D. The individual has remained positive for over 6 years for tyrosine phosphatase-related IA-2 protein autoantibodies and islet cell autoantibodies, indicating an ongoing autoimmune process, although he has not yet developed clinical T1D. The sequence obtained adds weight to the observation that recent enterovirus isolates differ significantly from prototype strains and provides further evidence of a role for recombination in enterovirus evolution. In common with most HEV-B species members, the isolate exhibits 2C and VP1 sequences suggested as triggers of autoimmunity through molecular mimicry. However, comparisons with the E3 prototype strain and previously reported diabetogenic and nondiabetogenic HEV-B strains do not reveal clear candidates for sequence features of PicoBank/DM1/E3 that could be associated with autoantibody appearance. This is the first time a virus strain isolated at the time of commencement of beta-cell damage has been analyzed and is an invaluable addition to enterovirus strains isolated previously at the onset of T1D in the search for specific molecular features which could be associated with diabetes induction

PCR inhibition in stool samples in relation to age of infants

Background: PCR is rapidly replacing traditional methods in diagnostic virus laboratories. PCR inhibitors,which are often present in clinical samples, may lead to false negative test results.Objectives: The aim was to study the presence of PCR inhibitors in stool samples collected from 3- to24-month old children.Study design: Total RNA fraction extracted from stool samples was spiked with a standardized amount ofSemliki Forest Virus RNA and amplified using specific PCR primers. The presence of PCR inhibitors wasdetected by a decrease in amplification rate compared to spiked water samples. Inhibition in differentage groups and dietary origin of PCR inhibitors were analyzed by comparing the samples taken duringexclusive and non-exclusive breastfeeding periods. The inactivation of PCR inhibitors was also assessed.Results: Complete inhibition was seen in 12% (13/108) and partial inhibition in 19% (21/108) of the samples.Inhibition was seen in none of the stool samples (0/31) taken from infants younger than 6 monthscompared to 17% of samples (13/77) taken from6 to 24 months old infants (p more common in younger age group. Addition of bovine serum albumin (BSA) into the reaction mixtureseliminated the effect of inhibitors leading to all samples being positive.Conclusions: PCR inhibitors are frequent in stool samples. They may originate from dietary componentsand can lead to false negative PCR results. The addition of BSA to the cDNA and PCR reactions proved tobe an easy and effective method for eliminating the inhibitory effect of these compounds

Analysis of pancreas tissue in a child positive for islet cell antibodies

Conclusions/interpretation These observations suggest that positivity for ICA alone, even when lasting for more than 1 year, is not associated with inflammatory changes in the islets. However, it is most likely that the pancreatic islets were infected by an enterovirus in this child

Recover from a service failure : The differential effects of brand betrayal and brand disappointment on an exclusive brand offering

Brand managers inevitably have to face service failures and respond to them. Undertaking brand recovery is essential as customers might desire to take revenge or spread negative word-of-mouth if they feel betrayed or disappointed by the brand following the service failure. Thus, it is necessary to understand customer responses to brand recovery, which depend on whether they feel betrayed or disappointed (while related, this paper distinguishes these feelings). This research challenges the conventional wisdom by demonstrating that, after presenting customers with an exclusive brand offering during the brand recovery, brand betrayal predicts a positive brand attitude and brand disappointment predicts a negative brand attitude with the service failure. Further, the brand attitude mediates the positive relationship between brand betrayal, positive word-of-mouth, and the likelihood of recommending the brand to others. Thus, the quick recovery that follows an exclusive brand offering positively impacts on the brand relationship among betrayed customers.Peer reviewe

Detection of a low-grade enteroviral infection in the islets of Langerhans of living patients newly diagnosed with type 1 diabetes

Journal ArticleThis is an author-created, uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association (ADA), publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version is available in Diabetes, May 2015, vol. 64, no. 5 pp. 1682-1687 in print and online at http://diabetes.diabetesjournals.org/content/64/5/1682.abstractThe Diabetes Virus Detection study (DiViD) is the first to examine fresh pancreatic tissue at the diagnosis of type 1 diabetes for the presence of viruses. Minimal pancreatic tail resection was performed 3-9 weeks after onset of type 1 diabetes in six adult patients (age 24-35 years). The presence of enteroviral capsid protein 1 (VP1) and the expression of class I HLA were investigated by immunohistochemistry. Enterovirus RNA was analyzed from isolated pancreatic islets and from fresh-frozen whole pancreatic tissue using PCR and sequencing. Nondiabetic organ donors served as controls. VP1 was detected in the islets of all type 1 diabetic patients (two of nine controls). Hyperexpression of class I HLA molecules was found in the islets of all patients (one of nine controls). Enterovirus-specific RNA sequences were detected in four of six patients (zero of six controls). The results were confirmed in various laboratories. Only 1.7% of the islets contained VP1(+) cells, and the amount of enterovirus RNA was low. The results provide evidence for the presence of enterovirus in pancreatic islets of type 1 diabetic patients, which is consistent with the possibility that a low-grade enteroviral infection in the pancreatic islets contributes to disease progression in humans.Academy of FinlandSouth-Eastern Norway Regional HealthAuthorityNovo Nordisk FoundationPEVNET (Persistent Virus Infection in Diabetes Network) Study GroupEuropean Union’s Seventh Framework ProgrammeSwedish Medical Research CouncilDiabetes Wellness FoundationJDR

Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively

Effect of inactivated nature-derived microbial composition on mouse immune system

Introduction: The hygiene hypothesis suggests that decrease in early life infections due to increased societal-level hygiene standards subjects one to allergic and autoimmune diseases. In this report, we have studied the effect of sterilized forest soil and plant-based material on mouse immune system and gut microbiome. Methods: Inbred C57Bl/6 mice maintained in normal sterile environment were subjected to autoclaved forest soil-derived powder in their bedding for 1 h a day for 3 weeks. Immune response was measured by immune cell flow cytometry, serum cytokine enzyme-linked immunoassay (ELISA) and quantitative polymerase chain reaction (qPCR) analysis. Furthermore, the mouse gut microbiome was analyzed by sequencing. Results: When compared to control mice, mice treated with soil-derived powder had decreased level of pro-inflammatory cytokines namely interleukin (IL)-17F and IL-21 in the serum. Furthermore, splenocytes from mice treated with soil-derived powder expressed less IL-1b, IL-5, IL-6, IL-13, and tumor necrosis factor (TNF) upon cell activation. Gut microbiome appeared to be stabilized by the treatment. Conclusions: These results provide insights on the effect of biodiversity on murine immune system in sterile environment. Subjecting mice to soil-based plant and microbe structures appears to elicit immune response that could be beneficial, for example, in type 2 inflammation-related diseases, that is, allergic diseases.Peer reviewe