scholarly journals Effect of inulin on the human gut microbiota: stimulation ofBifidobacterium adolescentisandFaecalibacterium prausnitzii

2008 ◽  
Vol 101 (4) ◽  
pp. 541-550 ◽  
Author(s):  
Carlett Ramirez-Farias ◽  
Kathleen Slezak ◽  
Zoë Fuller ◽  
Alan Duncan ◽  
Grietje Holtrop ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bifidobacterium Longum ◽  
Control Period ◽  
Bifidobacterium Bifidum ◽  
Bifidobacterium Adolescentis ◽  
Food Ingredients ◽  
Bifidobacterium Animalis ◽  
Bacterial Groups

Prebiotics are food ingredients that improve health by modulating the colonic microbiota. The bifidogenic effect of the prebiotic inulin is well established; however, it remains unclear which species ofBifidobacteriumare stimulatedin vivoand whether bacterial groups other than lactic acid bacteria are affected by inulin consumption. Changes in the faecal microbiota composition were examined by real-time PCR in twelve human volunteers after ingestion of inulin (10 g/d) for a 16-d period in comparison with a control period without any supplement intake. The prevalence of most bacterial groups examined did not change after inulin intake, although the low G+C % Gram-positive speciesFaecalibacterium prausnitziiexhibited a significant increase (10·3 % for control periodv.14·5 % during inulin intake,P = 0·019). The composition of the genusBifidobacteriumwas studied in four of the volunteers by clone library analysis. Between three and fiveBifidobacteriumspp. were found in each volunteer.Bifidobacterium adolescentisandBifidobacterium longumwere present in all volunteers, andBifidobacterium pseudocatenulatum,Bifidobacterium animalis,Bifidobacterium bifidumandBifidobacterium dentiumwere also detected. Real-time PCR was employed to quantify the four most prevalentBifidobacteriumspp.,B. adolescentis,B. longum,B. pseudocatenulatumandB. bifidum, in ten volunteers carrying detectable levels of bifidobacteria.B. adolescentisshowed the strongest response to inulin consumption, increasing from 0·89 to 3·9 % of the total microbiota (P = 0·001).B. bifidumwas increased from 0·22 to 0·63 % (P < 0·001) for the five volunteers for whom this species was present.

scholarly journals Detection of Bifidobacterium animalis subsp. lactis (Bb12) in the Intestine after Feeding of Sows and Their Piglets

2008 ◽  
Vol 74 (20) ◽  
pp. 6338-6347 ◽  
Author(s):  
Gloria Solano-Aguilar ◽  
Harry Dawson ◽  
Marta Restrepo ◽  
Kate Andrews ◽  
Bryan Vinyard ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Protein Biosynthesis ◽  
Bifidobacterium Longum ◽  
Elongation Factor ◽  
Single Copy ◽  
Bifidobacterium Animalis ◽  
Gene Copies ◽  
Intestinal Contents ◽  
High Level

ABSTRACT A real-time PCR method has been developed to distinguish Bifidobacterium animalis subspecies in the gastrointestinal tracts of pigs. Identification of a highly conserved single-copy tuf gene encoding the elongation factor Tu involved in bacterial protein biosynthesis was used as a marker to differentiate homologous Bifidobacterium animalis subsp. lactis (strain Bb12) from Bifidobacterium animalis subsp. animalis, as well as Bifidobacterium suis, Bifidobacterium breve, Bifidobacterium longum, several species of Lactobacillus, and Enterococcus faecium. Real-time PCR detection of serially diluted DNA extracted from a pure culture of Bb12 was linear for bacterial numbers ranging from 10 to 10,000 tuf gene copies per PCR (r 2 = 0.99). Relative differences in Bb12 bacterial numbers in pigs fed daily with Bb12 were determined after detection of Bb12 tuf gene copies in DNA extracted from the intestinal contents. Piglets treated with Bb12 immediately after birth maintained a high level of Bb12 in their large intestines with continuous daily administration of Bb12. Piglets born to Bb12-treated sows during the last third of their gestation and also treated with Bb12 at birth (T/T group) had a higher number of Bb12 organisms per gram of intestinal contents compared to placebo-treated piglets born to placebo-treated sows (C/C group), Bb12-treated sows (T/C group), or piglets born to placebo sows but treated with Bb12 immediately after birth (C/T group). In addition, there was a significant increase in gene expression for Toll-like receptor 9 (TLR9) in piglets from the T/T group, with no change in TLR2 and TLR4. These findings suggest that the tuf gene represents a specific and functional marker for detecting Bifidobacterium animalis subsp. lactis strain Bb12 within the microbiota of the intestine.

scholarly journals Exploring the Diversity of the Bifidobacterial Population in the Human Intestinal Tract

2009 ◽  
Vol 75 (6) ◽  
pp. 1534-1545 ◽  
Author(s):  
Francesca Turroni ◽  
Elena Foroni ◽  
Paola Pizzetti ◽  
Vanessa Giubellini ◽  
Angela Ribbera ◽  
...  
Keyword(s):  
Bifidobacterium Longum ◽  
Bifidobacterium Bifidum ◽  
Selective Medium ◽  
Rrna Gene ◽  
Bifidobacterium Adolescentis ◽  
Fecal Samples ◽  
Bifidobacterium Animalis ◽  
Human Intestinal Microbiota ◽  
Health Promoting ◽  
Internally Transcribed Spacer

ABSTRACT Although the health-promoting roles of bifidobacteria are widely accepted, the diversity of bifidobacteria among the human intestinal microbiota is still poorly understood. We performed a census of bifidobacterial populations from human intestinal mucosal and fecal samples by plating them on selective medium, coupled with molecular analysis of selected rRNA gene sequences (16S rRNA gene and internally transcribed spacer [ITS] 16S-23S spacer sequences) of isolated colonies. A total of 900 isolates were collected, of which 704 were shown to belong to bifidobacteria. Analyses showed that the culturable bifidobacterial population from intestinal and fecal samples include six main phylogenetic taxa, i.e., Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis, Bifidobacterium pseudolongum, Bifidobacterium breve, and Bifidobacterium bifidum, and two species mostly detected in fecal samples, i.e., Bifidobacterium dentium and Bifidobacterium animalis subp. lactis. Analysis of bifidobacterial distribution based on age of the subject revealed that certain identified bifidobacterial species were exclusively present in the adult human gut microbiota whereas others were found to be widely distributed. We encountered significant intersubject variability and composition differences between fecal and mucosa-adherent bifidobacterial communities. In contrast, a modest diversification of bifidobacterial populations was noticed between different intestinal regions within the same individual (intrasubject variability). Notably, a small number of bifidobacterial isolates were shown to display a wide ecological distribution, thus suggesting that they possess a broad colonization capacity.

scholarly journals Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

2002 ◽  
Vol 68 (5) ◽  
pp. 2420-2427 ◽  
Author(s):  
Teresa Requena ◽  
Jeremy Burton ◽  
Takahiro Matsuki ◽  
Karen Munro ◽  
Mary Alice Simon ◽  
...  
Keyword(s):  
Real Time ◽  
16S Rdna ◽  
Quantitative Pcr ◽  
Gene Sequence ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bifidobacterium Longum ◽  
Bifidobacterium Adolescentis ◽  
Fecal Samples ◽  
Real Time Quantitative Pcr ◽  
Pcr Targeting

ABSTRACT Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.

scholarly journals The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Dong Chen ◽  
Yaqin Wang ◽  
Feiya Yang ◽  
Adili Keranmu ◽  
Qingxin Zhao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Bioinformatic Analysis ◽  
Biological Functions ◽  
Quantitative Real Time Pcr ◽  
Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

scholarly journals Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

2020 ◽  
pp. 15-34
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Tissue Regeneration ◽  
Real Time Pcr ◽  
Molecular Level ◽  
Northern Blotting ◽  
Tissue Constructs ◽  
Low Sensitivity

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

(-)-Epigallocatechin Gallate Promotes MicroRNA 145 Expression against Myocardial Hypoxic Injury through Dab2/Wnt3a/β-catenin

2020 ◽  
Vol 48 (02) ◽  
pp. 341-356
Author(s):  
Chiu-Mei Lin ◽  
Wei-Jen Fang ◽  
Bao-Wei Wang ◽  
Chun-Ming Pan ◽  
Su-Kiat Chua ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Real Time ◽  
Real Time Pcr ◽  
Myocardial Infarct ◽  
Epigallocatechin Gallate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rat Cardiomyocytes

MicroRNA 145 (miR-145) is a critical modulator of cardiovascular diseases. The downregulation of myocardial miR-145 is followed by an increase in disabled-2 (Dab2) expression in cardiomyocytes. (-)-epigallocatechin gallate (EGCG) is a flavonoid that has been evaluated extensively due to its diverse pharmacological properties including anti-inflammatory effects. The aim of this study was to investigate the cardioprotective effects of EGCG under hypoxia-induced stress in vitro and in vivo. The hypoxic insult led to the suppression of miR-145 expression in cultured rat cardiomyocytes in a concentration-dependent manner. Western blotting and real-time PCR were performed. In rat myocardial infarction study, in situ hybridization, and immunofluorescent analyses were adopted. The western blot and real-time PCR data revealed that hypoxic stress with 2.5% O2 suppressed the expression of miR-145 and Wnt3a/[Formula: see text]-catenin in cultured rat cardiomyocytes but augmented Dab2. Treatment with EGCG attenuated Dab2 expression, but increased Wnt3a and [Formula: see text]-catenin in hypoxic cultured cardiomyocytes. Following in vivo myocardial infarction (MI) study, the data revealed the myocardial infarct area reduced by 48.5%, 44.6%, and 48.5% in EGCG (50[Formula: see text]mg/kg) or miR-145 dominant or Dab2 siRNA groups after myocardial infarction for 28 days, respectively. This study demonstrated that EGCG increased miR-145, Wnt3a, and [Formula: see text]-catenin expression but attenuated Dab2 expression. Moreover, EGCG ameliorated myocardial ischemia in vivo. The novel suppressive effect was mediated through the miR-145 and Dab2/Wnt3a/[Formula: see text]-catenin pathways.

195 ALTERED mRNA TRANSCRIPT EXPRESSION OF IN VITRO- VERSUS IN VIVO-MATURED PORCINE OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 210
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
K. M. Whitworth ◽  
W. G. Spollen ◽  
S. M. Blake ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Real Time ◽  
Real Time Pcr ◽  
Genetic Background ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Illumina Genome Analyzer ◽  
Similar Genetic Background

In contrast to oocytes matured in vitro, porcine embryos that result from in vivo maturation and fertilization have a high developmental competence and readily make the transition from oocyte to blastocyst. This observation led us to investigate the transcript profile differences between in vivo- and in vitro-matured porcine oocytes. For the in vivo-matured group, oviducts of 3 gilts of similar genetic background were flushed 2 days after detection of standing oestrus. MII oocytes were collected in pools of 10 and snap frozen in liquid nitrogen for RNA isolation. The in vitro-matured oocytes were obtained by euthanizing 3 gilts, again with a similar genetic background and recovering the ovaries. Follicles (2 to 8 mm in size) were aspirated and oocytes with multiple layers of cumulus cells and uniform cytoplasm were placed in M-199 supplemented with LH, FSH and epidermal growth factor for 42 h. Upon maturation, cumulus cells were stripped and the healthy MII oocytes were collected in pools of 10 and snap frozen. Total RNA was extracted from 3 pools of 10 oocytes for both treatments using an All prep DNA/RNA micro isolation kit (Qiagen, Valencia, CA, USA). Complementary DNA was synthesized using oligo (dT′) primed reverse transcriptase with superscript III (Invitrogen, Carlsbad, CA, USA). Second-strand cDNA was synthesized using DNA polymerase I and sequenced using Illumina Genome Analyzer II. All reads were aligned to a custom-built porcine transcriptome. There were over 18 million reads in the 2 maturation groups that tiled to the 34 433-member transcriptome: 1317 transcripts were detected with a P ≤ 0.1 (Students t-test), a minimum of 7 reads in at least 1 of the treatments and ≥2-fold difference. Real-time PCR was used on selected transcripts. Comparative CT Method was used on an IQ real-time PCR system with the Bio–Rad SYBR green mix. Statistical differences were determined using the Proc general linear model procedure of SAS (SAS Institute Inc., Cary, NC) and means separated with a l.s.d. (P ≤ 0.05). The misrepresented transcripts from the sequencing data were also characterized using the functional annotation tool DAVID. Twelve pathways were overrepresented in the in vitro-matured oocytes (the top 4 are pathways to cancer, spliceosome, cell cycle and ubiquitin-mediated proteolysis). Eight pathways were underrepresented in the in vitro-matured oocytes (the top 4 are cytoskeleton regulation, T-cell receptor signaling pathway, ubiquitin-mediated proteolysis and cell cycle). Eight transcripts were selected for real-time PCR. ZP2 was higher in the in vitro-matured oocytes as determined by both sequencing and real time. ATG4, HSP90, UBAP2 and SOX4 were not different, regardless of assay. SLC7A3, MRPS36 and PDHX2 were not different based on sequencing, but based on real-time MRPS36 and PDHX2, were higher in the in vivo group and SLC7A3 was higher in the in vitro group. In conclusion, there is an abundance of misregulated transcripts and altered pathways in in vitro-matured oocytes. This dataset is a tool that may provide clues to improve the in vitro maturation process so that in vitro-matured oocytes will be more like their in vivo-matured counterparts, thus improving developmental competence. Funded by Food for the 21st Century.

scholarly journals A SYBR green I real-time polymerase chain reaction (PCR) assay for detection and quantification of Trichomonas gallinae

2020 ◽  
Vol 119 (11) ◽  
pp. 3909-3913
Author(s):  
Zaida Rentería-Solís ◽  
Tran Nguyen-Ho-Bao ◽  
Shahinaz Taha ◽  
Arwid Daugschies
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Small Subunit ◽  
Sybr Green ◽  
Pcr Assay ◽  
Standard Curve ◽  
Trichomonas Gallinae ◽  
Host Interaction

Abstract Trichomonas gallinae are parasitic flagellates of importance in wild and domestic birds. The parasite is worldwide distributed, and Columbine birds are its main host. Current research focuses mostly on epidemiological and phylogenetic studies. However, there is still a lack of knowledge regarding parasite-host interaction or therapy development. Real-time PCR is a useful tool for diagnostic and quantification of gene copies in a determined sample. By amplification of a 113-bp region of the 18S small subunit ribosomal RNA gene, a SYBR green-based real-time PCR assay was developed. A standard curve was prepared for quantification analysis. Assay efficiency, linearity, and dissociation analysis were successfully performed. Specificity, sensibility, and reproducibility analysis were tested. This assay could be a useful tool not only for diagnostic purposes but also for future in vivo and in vitro T. gallinae studies.

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