<p>Reliable methods to analyze food for the presence of almond are important – not only for those allergic to almond, but also for monitoring the compliance with labelling regulations (EG directive 2003/89). Until now the Swedish National Food Administration has used methods like rocket immunoelectrophoresis and real-time PCR to detect almond in food. These methods are, however, not sensitive enough for protecting the most sensitive individuals. Therefore, the performance of a commercial ELISA kit was tested with regard to specificity/cross reactivity and limit of detection for almond both in solution and in different matrixes.</p><p>The limit of quantitation was at least 3,1 ppm (mg/kg) in solution and similar concentrations were measured in bisquits and chocolate. The ELISA method was about 100-fold more sensitive than rocket immunoelectrophoresis and PCR.</p><p>The specificity of the test kit was evaluated against a number of different nuts and seeds. No important cross reactivity was found. The antibodies against almond used in the kit can not differentiate between almond and apricot kernel. For such purposes the PCR method could be used.</p>

<p>Invasive Candida infection is an increasing cause of morbidity and mortality in the immunocompromised patient. Molecular diagnosis based on genomic amplification methods, such as real time PCR, has been reported as an alternative to conventional culture for early detection of invasive candidiasis. The template DNA extraction step has been the major limitation in most reported nucleic acid based assays, due to problems in breaking fungal cell walls and incomplete purification in PCR inhibitor substances.</p><p>The aim of this study was to compare enzymatic cell wall disruption using recombinant lyticase with mechanical disruption using glass beads. The QIAamp tissue kit was compared with two automated DNA extraction robots, the BioRobot M48 and NucliSens easyMAG, to determine their sensitivity, reliability and duration for DNA release of C. albicans. Mechanical cell wall disruption shortened and facilitated the extraction procedure, but the quantity of released DNA was significantly lower than when enzymatic cell wall disruption was used. Use of robots did not significantly shorten the DNA extraction time, compared with manual DNA extraction. However the NucliSens easyMAG resulted in a higher yield of target DNA compared to the BioRobot M48 and the manual QIAamp tissue kit.</p> / <p>Invasiva svampinfektioner är ett stort problem hos patienter med dåligt immunförsvar. Förekomst av invasiva svampinfektioner har ökat under senare år och medför hög dödlighet. En svampinfektion som inte snabbt diagnostiseras och behandlas kan bli livshotande om patientens kondition är dålig. Candida albicans är den vanligaste orsaken till invasiva svampinfektioner. Med traditionell svampidentifiering kan det ta dagar till veckor att isolera och artbestämma svampen. En snabbare metod att detektera Candida är att använda sig av molekylärbiologiska metoder som påvisar svampens arvsmassa, DNA. Svampar har en cellvägg som är svår att bryta ner och därför är DNA extraktionssteget ett av de mest rapporterade problemen vid DNA svampdiagnostik.</p><p>Syftet med denna studie var att jämföra enzymatisk och mekanisk cellväggsnedbrytning av C. albicans med hjälp av enzymet lyticase respektive glaskulor. Vi jämförde också en manuell metod med två automatiska robotar för att bestämma deras känslighet, tillförlitlighet och tidsåtgång för DNA-extraktion från C. albicans. De slutsatser som nåtts är att den enzymatiska cellväggsnedbrytningen var känsligare men betydligt mer tidskrävande än den mekaniska cellväggsnedbrytningen. Denna studie visade även att en av de automatiska systemen extraherade signifikant mer DNA än den manuella metoden.</p>

Bovine Spongiform Encephalopathy (BSE) belongs to a group of neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) which affect many species. From 1986 more than 184,000 cattle in the UK have been confirmed to be infected with this disease, and in Canada total losses to the economy reached $6 billion. This study examines the gene expression in three major innate immunity components: complement system, toll-like receptors, interleukins, and selected proteins of their signaling pathways. Quantitative real time polymerase chain reaction analyses were performed on caudal medulla samples to identify differentially expressed genes between non-exposed and orally challenged animals. In general, immune genes were down-regulated in comparison to non-challenged animals during first 12 months of disease with a tendency to be up-regulated at terminal stage of BSE. The results from this study provide a basis for further research on the mechanisms modifying immune responses and altering progression of the disease. / Animal Science

Genotyping can be used to link genetic variation among individuals to certain diseases or conditions. Some known disorders and states that are dependent on single nucleotide polymorphism (SNPs) are lactose intolerance, venous thrombosis, hereditary hemochromatosis and the difference in sensibility among people to metabolise drugs. In this project a new kit, TaqManÒ Sample-to-SNP KitÔ for extraction of DNA and preparation of the extract for genotyping with real-time PCR and allelic discrimination, was evaluated. QIAamp® DNA Blood Biorobot® MDx Kit was used as the reference method. The purpose of the comparison was to find a method that makes DNA extraction from blood samples cheaper and faster, but with the same reliability as the reference procedure. The results of the evaluation showed a complete agreement of the genotype results between the methods tested, which means that the new method was as reliable as the reference method. The costs of reagents and material would be reduced with 52% if the new method is adopted, that alone would result in a cost reduction of 144 000SEK a year with a sample volume of 650 samples/month. The time for DNA extraction would also be reduced with the new procedure.

Aromatase is the enzyme that catalysis the conversion of androgens into estrogens. It´s a member of P450 cytochrome family and is encoded by the CYP19-gene. The enzyme aromatase has an important role in regulating physiological and behavioral sexual mechanisms. This includes for instance activation, motivation and maintenance of the reproductive behaviors. The sexual behavior is affected by a complex series of events that requires the connection of endogenous hormonal and neurochemical changes with social interactions, especially between the opposite sexes. The aim of the present study was to examine how social interactions effect the aromatase expression and activity in the guppy brain. Guppy males were introduced into four different social conditions: Isolated, all male conditions, heterospecific (with zebrafish females) and conspecific female guppies. The focal males were kept under these conditions for two respectively four days. The sexual behavior, of each of the focal males was recorded daily during 10 minutes. The males with the guppy females showed, in contrast to the males in the other groups, a high frequency of reproductive behaviors. The brains of the focal males were collected and the brain aromatase activity was measured using tritiated water assay. I have also tried to analyze the gene-expression of aromatase with RT-PCR. However I was unable to analyze the results with the RT-PCR, because of possible primer-dimerization. Due to the limited time schedule, we were not able to solve the problem. ANOVA performed on the aromatase activity, revealed no significant difference between the different treatment groups. The variance was highest in the zebrafish category and lowest in the isolated males. There was no significant correlation between the mean number of reproductive behaviors and the aromatase activity in males that were together with guppy females. The results do not support the hypothesis that social interactions can affect the brain aromatase activity in guppy males.

PCR is a rapid, reproducible method for nucleic acid detection. However, this technology displays significant deficiencies when applied in clinical microbiology. This work’s aim was to improve current diagnostics and provide sensitive and quantitative real-time PCRs. Paper I describes the development of a sensitive and specific quantitative real-time PCR for the detection of Streptococcus pneumoniae, based on the Spn9802 DNA fragment. Applied to nasopharyngeal aspirates from 166 pneumonia patients, Spn9802 PCR had a sensitivity of 94% and a specificity of 98%. In Paper II the performance of a ply gene PCR for identification of pneumococcal lower respiratory tract infection (LRTI) was evaluated on bronchoalveloar lavage fluids. At the detection limit 103 genome copies/mL, 89% sensitivity but only 43% specificity was achieved. Paper III shows that S. pneumoniae DNA is detectable in plasma from acutely febrile patients. Sensitivities were low (26-42%) for detection of pneumococcal pneumonia, for bacteraemic pneumococcal pneumonia they were 60-70%. Paper IV describes evaluation of four PCR targets for Haemophilus influenzae detection. A real-time PCR based on the P6 gene was developed and applied to 166 CAP patients, using cut-off of 104 genome copies/mL the assay had a sensitivity of 97% and a specificity of 96%. In paper V, the two real-time PCRs presented in papers I and IV were combined with a PCR for detection of Neisseriae meningitidis. The analytical sensitivity of this multiplex real-time PCR was not affected by using a mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae) in single tubes. Applied to 156 LRTI patients, this PCR had sensitivities over 90% for S. pneumoniae and H. influenzae, and specificities of 89% and 96%, respectively. In conclusion, real-time PCR assays are useful for the diagnosis of S. pneumoniae and H. influenzae. They enable detection after antibiotic installation, and quantification increases the etiological specificity of pneumonia.

Reliable methods to analyze food for the presence of almond are important – not only for those allergic to almond, but also for monitoring the compliance with labelling regulations (EG directive 2003/89). Until now the Swedish National Food Administration has used methods like rocket immunoelectrophoresis and real-time PCR to detect almond in food. These methods are, however, not sensitive enough for protecting the most sensitive individuals. Therefore, the performance of a commercial ELISA kit was tested with regard to specificity/cross reactivity and limit of detection for almond both in solution and in different matrixes. The limit of quantitation was at least 3,1 ppm (mg/kg) in solution and similar concentrations were measured in bisquits and chocolate. The ELISA method was about 100-fold more sensitive than rocket immunoelectrophoresis and PCR. The specificity of the test kit was evaluated against a number of different nuts and seeds. No important cross reactivity was found. The antibodies against almond used in the kit can not differentiate between almond and apricot kernel. For such purposes the PCR method could be used.

Picropodophyllin (PPP) is an analogue of the anti tumour lignan podophyllotoxin with the unique ability to selectively inhibit the receptor of Insulin like growth factor 1(IGF-1). IGF-1 is believed to play an important part in development of the endometrium facing implantation. With PPP treated mice, studies can be made to measure gene expression from tissue of both treated and untreated mice to compare the role of IGF-1 regarding ovulation, implantation and receptivity. The aim of this study was to analyze gene expression of some steroid hormone receptors and cytokines in ovaries from mice treated with PPP. In this study, seven mice were treated with PPP at different times and tissue was collected. PCR-primers for cDNA sequences of estrogene receptor α, estrogene receptor β, progesterone receptor A, progesterone receptor B, growth hormone receptor, interleukin 1 α, interleukin 1 β, tumour necrosis factor α and androgen receptor were used. Real Time PCR was run with the samples and gene expression was measured. The results of this study showed that the inhibition of IGF-1 receptor interacted with IGF-1 which lead to altered levels of estrogene receptor alpha, progesterone receptor, growth hormone receptor and androgen receptor that can decrease ovulation. The results also showed the differences in gene products between treated and untreated samples, suggesting that IGF-1 plays an important role regarding ovulation. / Studier med hjälp av den selektiva insulinlika tillväxtfaktor 1 receptorn (IGF-1R) antagonisten; picropodof?phyllin (PPP), hur samspelet mellan livmoderslemhinnan och implantationsprocessen, samt hur ovulationen påverkas av insulinlika tillväxtfaktorn 1 (IGF-1) kan nu utföras. IGF-1 tros ha en viktig roll för den reproduktiva processen, där den påverkar ovulation, implantation och embryoutveckling. IGF-familjen består av tre ligander; insulin, IGF-1 och IGF-2. IGF transporteras bundet till bindarprotein (IGFBP). Medlemmarna i IGF receptorfamiljen kan binda IGF-1, IGF-2 och insulin fast med olika affinitet. PPP som är en cykloligan, är en analog från podofyllotoxin och fungerar som en syntetisk IGF-1 receptorantagonist, som selektivt inhiberar receptorns aktivitet. PPP tros även kunna nedreglera genexpression av receptorn. Tre tidigare projektarbeten har utförts på vävnader från möss injicerade med PPP. Tyngdpunkterna i dessa arbeten har legat på immunhistokemiska studier av IGF-1 i reproduktionsorgan från möss, uttryck av IGF-1, dess receptor och bindarprotein 1 i ovarier och uterus efter behandling med PPP. I denna studie användes vävnad samt cDNA från sju möss behandlade med PPP, i olika stadier av reproduktionen samt även icke behandlade möss. Studiens syfte var att med sanntids-PCR jämföra genuttryck från östrogenreceptor α och β, progesteronreceptor A och B, tillväxthormonreceptor, Interleukin 1 α och β, ’tumor necrosis’ faktor α samt androgenreceptor i vävnad från PPP-behandlade och obehandlade möss och genom de erhållna resultaten från ovarievävnaden utläsa effekten på ovulationen och från uterusvävnaden effekten på implantation och receptivitet. Studieresultaten visade att IGF-1s frånvaro gav förändrade nivåer av genprodukter, som medförde minskad ovulationen. Studien visade att IGF-1s roll vid ovulationen var väsentlig.

Transporters dynamically expressed at the mammary gland transport critical nutrients into the breast milk of nursing mothers to meet the nutritional demands of the suckling infant. However, xenobiotics may interact with these transporters to potentially alter the nutrient composition of milk and compromise neonatal nutrition. The aim of the present study was to quantitatively evaluate the constitutive expression of various nutrient transporters in whole mammary gland tissue and mammary epithelial organoids (MEO) isolated from female Sprague-Dawley rats at various stages of pregnancy, lactation, and involution. Furthermore, the studys aim was to determine if appropriately cultured mammary epithelial organoids (MEO) maintain in vivo transporter expression to lay down critical groundwork for the development of an in vitro screening tool assessing xenobiotic-nutrient transporter interactions. The following transporters were evaluated using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR): multidrug resistance protein (Mdr) 1a, 1b; multidrug resistance-like protein (Mrp) 1; organic cation transporter (Oct) 1; organic cation/carnitine transporter (Octn) 1, 2, and 3; concentrative nucleoside transporter (Cnt) 1, 2, and 3; equilibrative nucleoside transporter (Ent) 1, 2, and 3; nucleobase transporter (Ncbt) 1 and 2; oligopeptide transporter (Pept) 1 and 2; methotrexate carrier (Mtx) 1; divalent metal transporter (Dmt) 1; and the milk protein ?-casein. Transporter expression patterns in MEO differed from whole tissue for ?-actin, Mdr1a, Mdr1b, Oct1, Octn3, Ent3, Cnt1, Cnt3, Ncbt1, Pept2, Mtx1, and ?-casein. This brings into question whether whole mammary gland tissue is truly appropriate for an understanding of transporter expression in the mammary epithelium. Nevertheless, four general transporter expression patterns emerged in isolated MEO: decline throughout lactation (Mdr1a, Mdr1b, Mrp1 & Dmt1), increase throughout lactation (Cnt1 & Octn3), increase in early lactation (Oct1, Octn2, Ent1, Cnt2, Cnt3, Pept2 & Mtx1) and constant expression throughout lactation (Octn1, Ent2, Ent3, Ncbt1, Ncbt2 & Pept1). These expression patterns will provide insight into the critical windows of nutrient delivery to the breast milk to provide adequate nutritional stimuli to the suckling infant. Furthermore, MEO cultured in an extracellular matrix-rich environment maintained transporter expression at the mRNA level, which underscores the potential of the primary MEO in vitro model system as a screening tool for xenobiotic-transporter interactions at the mammary gland. Transporter expression patterns in MEO were unique for each transporter evaluated. This information accompanied by an in vitro screening tool may allow for predictions of xenobiotic interference with breast milk composition to help safeguard infant health.

Winter wheat (<i>Triticum aestivum</i> L.), seeded in the fall, cold acclimates when exposed to low fall temperatures. Growth resumes in spring, culminating in early summer harvest. Winter wheat yield is generally 20-25% higher than spring wheat. However, winter damage/kill can reduce its yield. A better understanding of the cold acclimation/tolerance process could help in the development of improved breeding strategies for winter wheat hardiness. Transcriptional activators and specific cold regulated (COR) genes are induced as a result of exposure to low temperatures. Thus, the objective of this study was to determine the quantitative expression of three COR genes (Wcs120, Wcor410 and Wcor14b) and one transcriptional activator (WCBF1) in field-grown wheat using real-time PCR and to establish any association with LT50 (temperature at which 50% of plants are killed). Winter Norstar (vrn-A1/vrn-A1), spring Manitou (Vrn-A1/Vrn-A1) and two near-isogenic lines (Spring Norstar (Vrn-A1/vrn-A1) and Winter Manitou (vrn-A1/vrn-A1), respectively) were used in these studies. Plants were sampled on three dates (Sept. 29, Oct. 12 and Oct. 26) in the fall of 2004. Accumulation of WCBF1 transcripts was highest in Norstar, but in all four genotypes there was an increase in transcripts by the second sampling date, followed by a decline on the third sampling date. Wcs120 transcripts increased from the first to the third sampling date in Norstar, Spring Norstar and Winter Manitou, but increased to the second sampling date and decreased by the third in Manitou. For Wcor14b, generally there was an increase to the second sampling date, followed by a decrease or steady levels on the third. Wcor410 showed a similar pattern, except for Spring Norstar wherein transcript levels increased by the third sampling date. With the exception of Wcor410 in Manitou, the Vrn-A1 locus affected gene expression in all genotypes. However, only Wcs120 expression followed the low-temperature tolerance pattern in these genotypes.