Fluorescent quantitative PCR of Mycobacterium tuberculosis for differentiating intestinal tuberculosis from Crohn's disease

Intestinal tuberculosis (ITB) and Crohn's disease (CD) are granulomatous disorders with similar clinical manifestations and pathological features that are often difficult to differentiate. This study evaluated the value of fluorescent quantitative polymerase chain reaction (FQ-PCR) for Mycobacterium tuberculosis (MTB) in fecal samples and biopsy specimens to differentiate ITB from CD. From June 2010 to March 2013, 86 consecutive patients (38 females and 48 males, median age 31.3 years) with provisional diagnoses of ITB and CD were recruited for the study. The patients' clinical, endoscopic, and histological features were monitored until the final definite diagnoses were made. DNA was extracted from 250 mg fecal samples and biopsy tissues from each patient. The extracted DNA was amplified using FQ-PCR for the specific MTB sequence. A total of 29 ITB cases and 36 CD cases were included in the analysis. Perianal disease and longitudinal ulcers were significantly more common in the CD patients (P<0.05), whereas night sweats, ascites, and circumferential ulcers were significantly more common in the ITB patients (P<0.05). Fecal FQ-PCR for MTB was positive in 24 (82.8%) ITB patients and 3 (8.3%) CD patients. Tissue PCR was positive for MTB in 16 (55.2%) ITB patients and 2 (5.6%) CD patients. Compared with tissue FQ-PCR, fecal FQ-PCR was more sensitive (X2=5.16, P=0.02). We conclude that FQ-PCR for MTB on fecal and tissue samples is a valuable assay for differentiating ITB from CD, and fecal FQ-PCR has greater sensitivity for ITB than tissue FQ-PCR.

The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P<0.0001). Moreover,FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs=0.317, P=0.006), hemoglobin levels (rs=0.210, P=0.049), and percentage of peripheral blood blasts (rs=0.256, P=0.027), but inversely correlated with hemoglobin levels in the control group (rs=–0.391, P<0.0001). AML patients with high CD34+ expression showed significantly higherFAMLF-CS expression than those with low CD34+ expression (P=0.041). Our results showed thatFAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

Study of bacteria Gluconobacter sp.: isolation, purification, phenotypic and molecular identification

The importance of the study of acetic bacteria, on species of the Gluconobacter genus is based on its industrial application, as these possess the capacity of bioconversion of sorbitol to sorbose, enabling the process of vitamin C production. The study involved samples collected in industries of soft drinks, flowers, fruits and honey, followed by purification, phenotypic identification, molecular identification with the use of primer defined from Nucleotide Sequence Database consultation. Strains preserved were identified as members of the Acetobacteraceae family, Gluconobacter genus. 110 strains had been isolated of substrate: Pyrostegia venusta (ker-gawler), honey, Vitis vinifera (grape), Pyrus communis (pear), Malus sp. (apple) and in two samples of soft drinks. Of this total 57 strains had been recovered in manitol medium (manitol, yeast extract, peptone), 12 in YMG medium (glucose, manitol, yeast extract, ethanol, acetic acid), 41 in enrichment medium (De Ley and Swings) and later in the GYC medium (glucose, yeast extract and calcium carbonate). 68 strains were identified as Gram negative bacilli rods. Of these, 31 were characterized biochemically as belonging to the Acetobacteriaceae family as they were catalase positive, oxidase negative and producers of acid from glucose. The characterization of these strains was complemented with the biochemistry tests: gelatin liquefaction, nitrate reduction, indole and H2S production, oxidation of ethanol to acetic acid and molecular tests for genus identification. Only eight strains were characterized as pertaining to the Gluconobacter genus. The strains are maintained in collection cultures at the Microbiology Laboratory of the Biology Department at the São Paulo State University (UNESP) in Assis, stored in malt extract at -196 ºC.

Detection of enterotoxins produced by B. cereus through PCR analysis of ground and roasted coffee samples in Rio de Janeiro, Brazil

Coffee is one of the most appreciated drinks in the world. Coffee ground is obtained from the fruit of a small plant that belongs to the genus Coffea. Coffea arabica and Coffea canephora robusta are the two most commercially important species. They are more commonly known as arabica and robusta, respectively. Two-thirds of Coffea arabica plants are grown in South and Central America, and Eastern Africa - the place of origin for this coffee species. Contamination by microorganisms has been a major matter affecting coffee quality in Brazil, mainly due to the harvesting method adopted. Brazilian harvests are based on fruits collected from the ground mixed with those that fall on collection cloths. As the Bacillus cereus bacterium frequently uses the soil as its environmental reservoir, it is easily capable of becoming a contaminant. This study aimed to evaluate the contamination and potential of B. cereus enterotoxin genes encoding the HBL and NHE complexes, which were observed in strains of ground and roasted coffee samples sold in Rio de Janeiro. The PCR (Polymerase Chain Reaction) results revealed high potential of enterotoxin production in the samples. The method described by Speck (1984) was used for the isolation of contaminants. The investigation of the potential production of enterotoxins through isolates of the microorganism was performed using the B. cereus enterotoxin Reverse Passive Latex Agglutination test-kit (BCET-RPLA, Oxoid), according to the manufacturer's instructions. The potential of enterotoxin production was investigated using polymerase chain reaction (PCR) methods for hblA, hblD and hblC genes (encoding hemolysin HBL) and for nheA, nheB and nheC genes (encoding non-hemolytic enterotoxin - NHE). Of all the 17 strains, 100% were positive for at least 1 enterotoxin gene; 52.9% (9/17) were positive for the 3 genes encoding the HBL complex; 35.3% (6/17) were positive for the three NHE encoding genes; and 29.4% (5/17) were positive for all enterotoxic genes.

Standardization of a multiplex PCR for the identification of coagulase-positive Staphylococcus

The enterotoxigenic species Staphylococcus aureus, S. hyicus and S. intermedius show very similar characteristics, making their identification through conventional microbiological methods difficult. This study aimed at the development of a Multiplex PCR (mPCR) for the identification of S. aureus, S. intermedius and S. hyicus using the nuc gene as the target sequence. The results obtained suggest that the set of primers used was specific for the three species of Staphylococcus evaluate with a detection limit of 10² CFU.mL-1.

Use of real-time PCR to evaluate two DNA extraction methods from food

The DNA extraction is a critical step in Genetically Modified Organisms analysis based on real-time PCR. In this study, the CTAB and DNeasy methods provided good quality and quantity of DNA from the texturized soy protein, infant formula, and soy milk samples. Concerning the Certified Reference Material consisting of 5% Roundup Ready® soybean, neither method yielded DNA of good quality. However, the dilution test applied in the CTAB extracts showed no interference of inhibitory substances. The PCR efficiencies of lectin target amplification were not statistically different, and the coefficients of correlation (R²) demonstrated high degree of correlation between the copy numbers and the threshold cycle (Ct) values. ANOVA showed suitable adjustment of the regression and absence of significant linear deviations. The efficiencies of the p35S amplification were not statistically different, and all R² values using DNeasy extracts were above 0.98 with no significant linear deviations. Two out of three R² values using CTAB extracts were lower than 0.98, corresponding to lower degree of correlation, and the lack-of-fit test showed significant linear deviation in one run. The comparative analysis of the Ct values for the p35S and lectin targets demonstrated no statistical significant differences between the analytical curves of each target.

Extraction of DNA from honey and its amplification by PCR for botanical identification

The physiochemical and biological properties of honey are directly associated to its floral origin. Some current commonly used methods for identification of botanical origin of honey involve palynological analysis, chromatographic methods, or direct observation of the bee behavior. However, these methods can be less sensitive and time consuming. DNA-based methods have become popular due to their simplicity, quickness, and reliability. The main objective of this research is to introduce a protocol for the extraction of DNA from honey and demonstrate that the molecular analysis of the extracted DNA can be used for its botanical identification. The original CTAB-based protocol for the extraction of DNA from plants was modified and used in the DNA extraction from honey. DNA extraction was carried out from different honey samples with similar results in each replication. The extracted DNA was amplified by PCR using plant specific primers, confirming that the DNA extracted using the modified protocol is of plant origin and has good quality for analysis of PCR products and that it can be used for botanical identification of honey.

Sequence-characterized amplified region (SCAR) technique used for variety discrimination in vinca (Catharanthus roseus (L.) G.Don)

Sequence-Characterized Amplified Region (SCAR) appears as a useful technique for genetic purity testing and variety discrimination, applicable to species in which some other techniques have failed. In particular, this technique is very attractive with species in which RAPD results were not consistent. The RAPD polymorphic bands were cloned, sequenced and from the sequence information, primers pairs for normal PCR were developed. Since the probability of obtaining successful SCAR primers from RAPD polymorphic bands was about 50%, a larger number of RAPD polymorphic bands are needed to develop sufficient SCAR primers for varietal discrimination in vinca. In addition, the efficiency of the SCAR technique is strongly affected by the quality of DNA extracted from seeds. The SCAR banding patterns obtained from vinca seed were consistent and repeatable making the results reliable for genetic purity testing and variety discrimination. The SCAR technique is simple, fast, relatively inexpensive and allows the use of DNA extracted from dry seeds, which is very important in a seed-quality evaluating program

Aikaerotteisesti fluoresenssia mittaavan reaaliaikaisen PCR-laitteen kehitys

Kvantitatiivinen reaaliaikainen polymeraasiketjureaktio (engl. polymerase chain reaction, PCR) on osoittautunut käyttäjäystävällisimmäksi menetelmäksi nukleiinihapposekvenssien kvantitoimisessa. Tätä menetelmää voidaan herkistää pienempien DNA-pitoisuuksien havaitsemiseen käyttämällä hyväksi aikaerotteista fluorometriaa (engl. time-resolved fluorometry, TRF) ja luminoivia lantanidileimoja, joiden fluoresenssin pitkän eliniän ansiosta emission mittaus voidaan suorittaa vasta hetki virittävän valopulssin jälkeen, jolloin lyhytikäinen taustasäteily ehtii sammua. Tuloksena saadaan korkea signaali-taustasuhde. Tämän diplomityön tarkoituksena oli rakentaa TRF:än pystyvä reaaliaikainen PCR-laite, sillä tällaista laitetta ei ole markkinoilla tarjolla. Laite rakennettiin kehittämällä lämpökierrätin ja yhdistämällä se valmiiseen TRF:än kykenevään mittapäähän. Mittapään ja lämpökierrättimen hallitsemiseksi kehitettiin myös tietokoneohjelma. Valon tuottamiseksi ja mittaamiseksi haluttiin käyttää edullisia komponentteja, joten työssä käytettiin valmiin mittapään optiikkaa, jossa viritys tapahtuu hohtodiodilla (engl. light-emitting diode, LED) ja lantanidileiman emission mittaus fotodiodilla (engl. photodiode, PD) tai valomonistinputkella (engl. photomultiplier tube, PMT). Myös mittapään suorituskykyä tutkittiin. Työtä varten kehitettiin lämpökierrätin, joka koostui Peltier-elementillä lämmitettävästä PCR-putkitelineestä ja lämpökannesta. Mittalaitteen suorituskyvyn tutkimiseen käytettiin kelaattikomplementaatioon perustuvaa PCR-tuotteen havaitsemismenetelmää. Kelaattikomplementaatio perustuu kahteen erilliseen oligonukleotidimolekyyliin, joista toiseen on sidottu lantanidi-ioni ja toiseen valoa absorboiva ligandirakenne, jotka yhdessä muodostavat fluoresoivan kokonaisuuden. Kehitetyn lämpökierrättimen todettiin olevan tarpeeksi tarkka sekä tehokas ja sen lämmitys- ja jäähdytysnopeuden maksimeiksi saatiin 2,6 °C/sekunti. Detektorina käytetyn PD:n ei todettu olevan tarpeeksi herkkä emission havainnoimiseksi ja se korvattiin laitteessa PMT:llä. Käytetyllä PCR-määrityksellä kynnyssykleiksi (engl. threshold cycle, Ct) sekä kehitetylle että referenssilaitteelle saatiin 28,4 käyttämällä samaa 100 000 kopion DNA:n aloitusmäärää. Työssä osoitettiin, että on mahdollista kehittää edullisia komponentteja käyttävä, TRF:än pystyvä, reaaliaikainen PCR-laite, joka kykenee vastaavaan Ct-arvoon kuin vertailulaite. PD:n herkkyys ei kuitenkaan riittänyt. Tulokset olivat lupaavia, sillä LED- ja PD-teknologiat kehittyvät ja markkinoille on tullut myös muita komponentteja, joiden avulla on tulevaisuudessa mahdollista kehittää vielä herkempi laite.

Probing MST1 Kinase Sequence and Structure for Rational Drug Discovery (Targeting diabetes by blocking protein-protein interactions)

Apoptotic beta cell death is an underlying cause majorly for type I and to a lesser extent for type II diabetes. Recently, MST1 kinase was identified as a key apoptotic agent in diabetic condition. In this study, I have examined MST1 and closely related kinases namely, MST2, MST3 and MST4, aiming to tackle diabetes by exploring ways to selectively block MST1 kinase activity. The first investigation was directed towards evaluating possibilities of selectively blocking the ATP binding site of MST1 kinase that is essential for the activity of the enzymes. Structure and sequence analyses of this site however revealed a near absolute conservation between the MSTs and very few changes with other kinases. The observed residue variations also displayed similar physicochemical properties making it hard for selective inhibition of the enzyme. Second, possibilities for allosteric inhibition of the enzyme were evaluated. Analysis of the recognized allosteric site also posed the same problem as the MSTs shared almost all of the same residues. The third analysis was made on the SARAH domain, which is required for the dimerization and activation of MST1 and MST2 kinases. MST3 and MST4 lack this domain, hence selectivity against these two kinases can be achieved. Other proteins with SARAH domains such as the RASSF proteins were also examined. Their interaction with the MST1 SARAH domain were evaluated to mimic their binding pattern and design a peptide inhibitor that interferes with MST1 SARAH dimerization. In molecular simulations the RASSF5 SARAH domain was shown to strongly interact with the MST1 SARAH domain and possibly preventing MST1 SARAH dimerization. Based on this, the peptidic inhibitor was suggested to be based on the sequence of RASSF5 SARAH domain. Since the MST2 kinase also interacts with RASSF5 SARAH domain, absolute selectivity might not be achieved.

Functional genomics of O-glucosyltransferases from Concord grape (Vitis labrusca)

Grape (Vitis spp.) is a culturally and economically important crop plant that has been cultivated for thousands of years, primarily for the production of wine. Grape berries accumulate a myriad of phenylpropanoid secondary metabolites, many of which are glucosylated in plantae More than 90 O-glucosyltransferases have been cloned and biochemically characterized from plants, only two of which have been isolated from Vitis spp. The world-wide economic importance of grapes as a crop plant, the human health benefits associated with increased consumption of grape-derived metabolites, the biological relevance of glucosylation, and the lack of information about Vitis glucosyltransferases has inspired the identification, cloning and biochemical characterization of five novel "family 1" O-glucosyltransferases from Concord grape (Vitis labrusca cv. Concord). Protein purification and associated protein sequencIng led to the molecular cloning of UDP-glucose: resveratrollhydroxycinnamic acid O-glucosyltransferase (VLRSGT) from Vitis labrusca berry mesocarp tissue. In addition to being the first glucosyltransferase which accepts trans-resveratrol as a substrate to be characterized in vitro, the recombinant VLRSGT preferentially produces the glucose esters of hydroxycinnamic acids at pH 6.0, and the glucosides of trans-resveratrol and flavonols at 'pH 9.0; the first demonstration of pH-dependent bifunctional glucosylation for this class of enzymes. Gene expression and metabolite profiling support a role for this enzyme in the bifuncitonal glucosylation ofstilbenes and hydroxycinnamic acids in plantae A homology-based approach to cloning was used to identify three enzymes from the Vitis vinifera TIGR grape gene index which had high levels of protein sequence iii identity to previously characterized UDP-glucose: anthocyanin 5-0-glucosyltransferases. Molecular cloning and biochemical characterization demonstrated that these enzymes (rVLOGTl, rVLOGT2, rVLOGT3) glucosylate the 7-0-position of flavonols and the xenobiotic 2,4,5-trichlorophenol (TCP), but not anthocyanins. Variable gene expression throughout grape berry development and enzyme assays with native grape berry protein are consistent with a role for these enzymes in the glucosylation of flavonols; while the broad substrate specificity, the ability of these enzymes to glucosylate TCP and expression of these genes in tissues which are subject to pathogen attack (berry, flower, bud) is consistent with a role for these genes in the plant defense response. Additionally, the Vitis labrusca UDP-glucose: flavonoid 3-0-glucosyltransferase (VL3GT) was identified, cloned and characterized. VL3GT has 96 % protein sequence identity to the previously characterized Vitis vinifera flavonoid 3-0-glucosyltransferase (VV3GT); and glucosylates the 3-0-position of anthocyanidins and flavonols in vitro. Despite high levels of protein sequence identity, VL3GT has distinct biochemical characteristics (as compared to VV3GT), including a preference for B-ring methylated flavonoids and the inability to use UDP-galactose as a donor substrate. RT-PCR analysis of VL3GT gene expression and enzyme assays with native grape protein is consistent with an in planta role for this enzyme in the glucosylation of anthocyanidins,but not flavonols. These studies reveal the power of combining several biochemistry- and molecular biology-based tools to identify, clone, biochemically characterize and elucidate the in planta function of several biologically relevant O-glucosyltransferases from Vitis spp.

Evolutionary synthesis of stochastic gene network models using feature-based search spaces

A feature-based fitness function is applied in a genetic programming system to synthesize stochastic gene regulatory network models whose behaviour is defined by a time course of protein expression levels. Typically, when targeting time series data, the fitness function is based on a sum-of-errors involving the values of the fluctuating signal. While this approach is successful in many instances, its performance can deteriorate in the presence of noise. This thesis explores a fitness measure determined from a set of statistical features characterizing the time series' sequence of values, rather than the actual values themselves. Through a series of experiments involving symbolic regression with added noise and gene regulatory network models based on the stochastic 'if-calculus, it is shown to successfully target oscillating and non-oscillating signals. This practical and versatile fitness function offers an alternate approach, worthy of consideration for use in algorithms that evaluate noisy or stochastic behaviour.

Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.

Innate immunity genes as determinants of resistance/susceptibility to human disease : studies in leukemia patients

La leucémie lymphoblastique aiguë des cellules Pré-B (B-ALL) reste le type de cancer le plus souvent diagnostiqué chez les enfants. Des études ont montré que des déterminants génétiques jouent un rôle important dans la susceptibilité/résistance au développement de ce cancer. À cet égard, les gènes Killer-cell Immunoglobulin-like Receptor (KIR) sont d'une importance particulière. Ces gènes sont fortement polymorphiques et codent pour des récepteurs qui contrôlent l’activité fonctionnelle des cellules Natural Killer (NK). Notre hypothèse est que les gènes activateurs des KIR s’associent avec la résistance innée pour développer la B-ALL. Afin d'évaluer cette hypothèse, nous avons entrepris une étude de cas-contrôles chez des enfants canadiens-français dans laquelle nous avons utilisé l'ADN génomique de 100 patients atteints de B-ALL ainsi que l’ADN de 245 individus sains. La présence ou l'absence de chaque gène KIR a été détectée par PCR en utilisant des amorces de séquences spécifiques. Nous avons trouvé que la présence des gènes KIR activateurs est significativement diminuée chez les enfants leucémiques par rapport aux témoins. En outre, le nombre de ces gènes a aussi montré une association significative linéaire avec la résistance au développement d’une B-ALL. Cela suggère des effets additifs de ces gènes permettant de conférer une protection contre ce cancer. Ces résultats pourraient être utiles afin de déceler de façon précoce les enfants ayant un risque de développer cette leucémie. Enfin, des stratégies thérapeutiques basées sur les récepteurs KIR pourraient être envisagées et s'avérer utiles concernant le traitement de ce cancer chez les enfants.

Study of cox1 trans-splicing in Diplonema papillatum mitochondria

Diplonema papillatum est un organisme unicellulaire qui vit dans l’océan. Son génome mitochondrial possède une caractéristique spéciale: tous les gènes sont brisés en de multiples fragments qui s’appellent modules. Chaque module est codé par un chromosome différent. L’expression d’un gène exige des épissages-en-trans qui assemblent un ARN messager complet à partir de tous les modules du gène. Nous avons précédemment montré que le gène cox1 est encodé dans neuf modules avec six Us non encodés entre le module 4 et le module 5 de l’ARN messager mature [1]. Nous n’avons identifié aucune séquence consensus connue de site d’épissage près des modules. Nous spéculons qu’un ARN guide (gRNA) a dirigé l’épissage-en-trans du gène cox1 par un mécanisme qui est semblable à l’édition d’ARN par l’insertion/la suppression des Us chez les kinétoplastides, le groupe sœur des diplonémides. Nous avons trouvé que les six Us sont ajoutés au bout 3’ de l’ARN d’une façon semblable à ceux ajoutés par le TUTase lors de l’édition de l’insertion des Us chez les kinétoplastides. Nous avons construit des profils de gRNA de l’épissage-en-trans avec les expressions régulières basé sur notre connaissance des gRNAs dans l’édition d’ARN chez les kinétoplastides. Selon la complémentarité partielle entre le gRNA et les deux modules adjacents, nous avons généré des amorces pour RT-PCR visant à détecter des séquences qui sont assorties à un des profils de gRNA. Une expérience pilote in vitro n’a pas permis de reconstituer l’épissage-en-trans des modules 3, 4, et 5, suggérant que nous devons améliorer nos techniques.