Role of Intestinal Microbiota in Ulcerative Colitis

More documents

Recommendations

Info

in 1 x TAE at 60 °C for 16 h at 36 V, 28 mA, stained with ethidium bromide for 15 min, distained for 20 min, and viewed by UV‐B trans illumination at 302 nm (Bio‐Rad). Analysis of DGGE patterns The obtained DGGE patterns were analyzed using Bionumerics software version 5.0 (Applied Maths, Sint‐Martens‐Latem, Belgium). The triplicates taken from each fecal sample were analyzed to assess similarity of the three extractions. DGGE gels were normalized by an assigned marker (developed in our laboratory). A cluster analysis was performed based on Dice coefficient of similarity (weighted) with the unweighted pair group method with arithmetic averages clustering algorithm. Excision, cloning and sequencing of selected bands from DGGE gels Bands of specific interest from the human fecal microbiota profiles were excised from DGGE gels with a sterile razor, placed in 40 µL sterile water and incubated at 4 °C overnight for diffusion of DNA into the water. For sequencing of bands retrieved from the gels, the eluted DNA was used in a PCR with HDA1/2 primers without GC‐clamp (4 min at 94°C, 20 cycles consisting of 30 s at 94°C, 30 s at 56°C, and 1 min at 68°C, and finally 7 min at 68°C). Subsequently, the PCR products were directly cloned into pCR® 4‐TOPO (Invitrogen, Taastrup, Denmark) according to the manufacturer's instructions, and electroporated into electrocompetent E. coli TOP10 cells (Invitrogen) with a single pulse (2500 V, 400 Ω, 25 µF) by use of a Gene Pulser apparatus (Bio‐Rad). Plasmid DNA was isolated from the cells using the Qiagen Mini Spin Prep kit, and subjected to PCR (HDA1/2‐GC) as earlier described. The PCR product was run on a DGGE gel to check the purity and confirm the melting behaviour of the excised band. The inserts were sequenced by GATC (Konstanz, Germany) using primers T3 and T7. The obtained sequences were compared to known sequences in the Ribosomal Database (RDP, Michigan State University, Release 10), and aligned using BLAST (blastn) and the GenBank database. Quantitative PCR assay conditions Quantitative PCR was performed on an ABI Prism 7900 HT from Applied Biosystems. The amplification reactions were carried out in a total volume of 20 μL containing; 10 μL (EXPRESS SYBR ® GreenER TM qPCR SuperMix, ROX, Invitrogen), primers (each at 200 nM concentration) 6
(Eurofins MWG Synthesis GmbH), 2 μL template DNA, and Nuclease‐free water (Qiagen) purified for PCR. The amplification program consisted of one cycle at 50°C for 2 min; one cycle at 95°C for 10 min; 40 cycles at 95°C for 15 sec and 60°C for 1 min; and finally one cycle of melting curve analysis for amplicon specificity at 95°C for 15 sec, 60°C for 20 sec and increasing ramp rate by 2% until 95° for 15 sec. Quantitative PCR primers The primers specific to regions of the 16S rRNA genes of selected bacterial phyla‐ and groups, are listed in Table 1, while the species specific primers targeting either the 16S rRNA genes or the 16S‐ 23S rRNA intergenic spacer region are listed in Table 2. Genus‐ and species specific primers for amplification of Alistipes spp. and Bacteroides uniformis were designed using 16S rRNA gene sequences from the EMBL‐EBI database (http://www.ebi.ac.uk/ebisearch). Sequences were aligned by the BioEdit software (version 7.0.5.3; Ibis Biosciences, Carlsbad, CA) using ClusterW multiple alignment. The target‐specific sites were assessed by FastPCR Software (version 6.1.9; Primer Digital Ltd., Helsinki, Finland). Specificity of the primers was evaluated in silico using the nucleotide BLAST, blastn algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The two primer sets were tested to confirm amplification of the 16S rRNA genes from the genomic DNA of Bac. uniformis (DSM 6597) and Alistipes spp., respectively. Additionally, the primer sets were tested to confirm that they did not amplify the 16S rRNA genes from species belonging to other phyla. Quantitative PCR data handling The relative quantities of gene targets encoding 16S rRNA gene sequences of the bacterial taxa were calculated using the 2 delta‐delta Ct method according to Pfaffl [38], assuming primer efficiency at 1.0. The calculated results were analyzed as ratio of species specific 16S rRNA gene density relative to total bacterial 16S rRNA gene density in order to correct data for difference in total DNA concentration between individual samples. Standard curves were created using serial 10‐fold dilutions of bacterial DNA extracted from one of the feces samples for all primer sets. Analysis of the standard curves allowed verification of PCR efficiency (limit 1.0±10%) for the chosen PCR conditions. All results were calculated as means of duplicate determinations, equal values required. 7
Page 1:
Role of Intestinal Microbiota in Ul
Page 4 and 5:
Role of Intestinal Microbiota in Ul
Page 6 and 7:
Preface Preface This thesis present
Page 8 and 9:
Summary Summary The microbiota of t
Page 10 and 11:
Dansk sammendrag Dansk sammendrag M
Page 12 and 13:
Introduction and objectives Introdu
Page 14 and 15:
List of Manuscripts Not included in
Page 16 and 17: List of contents List of Centents P
Page 18 and 19: List of Centents Methodology append
Page 21 and 22: 1. The intestinal environment Theor
Page 23 and 24: Theoretical part 5 1. The intestina
Page 25 and 26: 2. The colonic environment Theoreti
Page 27 and 28: Theoretical part 9 2. The colonic e
Page 29 and 30: Table 1: The presence of glycoside
Page 31 and 32: Theoretical part Figure 3: The colo
Page 33 and 34: 3. Inflammatory Bowel disease Theor
Page 35 and 36: Theoretical part 17 3. Inflammatory
Page 37 and 38: Theoretical part 19 4. Modulation o
Page 43 and 44: Table 4: Clinical trials on the pre
Page 45 and 46: Theoretical part 5. Production of p
Page 51: Methodology part
Page 54 and 55: Methodology part 6. Methodology, co
Page 60 and 61: Introduction Methodology part 42 Pa
Page 62 and 63: Abstract Background Detailed knowle
Page 64 and 65: depending the level of disease acti
Page 68 and 69: Statistics PCA were generated by SA
Page 70 and 71: The PCA of the Gram‐positive bact
Page 72 and 73: layer of UC patients and found that
Page 74 and 75: Acknowledgements The authors thank
Page 76 and 77: Table 2 ‐ 16S rRNA gene and 16S
Page 78 and 79: 1. Firmicutes phylum 2. Bacteroidet
Page 80 and 81: Supplementary Figure S1. Dice clust
Page 82 and 83: Reference List 1. Ahmed S, Macfarla
Page 84 and 85: 32. Matsuki T, Watanabe K, Fujimoto
Page 87 and 88: Methodology part Paper 2 Fecal lact
Page 89 and 90: Fecal lactobacilli and bifidobacter
Page 91 and 92: Introduction The mucus layer lining
Page 93 and 94: efore enrolment and there was no si
Page 95 and 96: (Bio‐Rad Labs, Hercules, Californ
Page 97 and 98: Microbial community analysis using
Page 99 and 100: difference from the luminal microbi
Page 101 and 102: that C. coccoides group and C. lept
Page 103 and 104: Table 1 ‐ 16S rRNA gene of phylum
Page 105 and 106: Table 2 ‐ Preference of bacterial
Page 107 and 108: Figure 1. A) Schematic overview of
Page 109 and 110: A. B. Figure 3. Principal component
Page 111 and 112: 15. Fooks LJ, Gibson GR. (2002) In
Page 113 and 114: 47. Ouwehand AC, Suomalainen T, Tol
Page 115 and 116: Methodology part Paper 3 Paper 3 In
Page 117 and 118:
APPLIED AND ENVIRONMENTAL MICROBIOL
Page 119 and 120:
8338 VIGSNÆS ET AL. APPL. ENVIRON.
Page 121 and 122:
Page 123 and 124:
Page 125:
Page 128 and 129:
Methodology part Introduction The a
Page 130 and 131:
Journal of Agricultural and Food Ch
Page 132 and 133:
Page 134 and 135:
Page 136 and 137:
Page 139 and 140:
Methodology part Paper 5 Paper 5 Ma
Page 141 and 142:
Appl Microbiol Biotechnol (2011) 90
Page 143 and 144:
Page 145 and 146:
Page 147 and 148:
Page 149 and 150:
Page 151 and 152:
Page 153 and 154:
Methodology part Paper 6 Tailored e
Page 155 and 156:
Process Biochemistry 46 (2011) 1039
Page 157 and 158:
Table 1 List of enzymes. Enzyme Sou
Page 159 and 160:
J. Holck et al. / Process Biochemis
Page 161 and 162:
Intensity 50 0 C1 175.05 J. Holck e
Page 163 and 164:
J. Holck et al. / Process Biochemis
Page 165:
[29] Bauer S, Vasu P, Persson S, Mo
Page 168 and 169:
Methodology part 150
Page 170 and 171:
Methodology part 152 Appendix 1 hor
Page 172 and 173:
Appendix 3 Methodology part Appendi
Page 174 and 175:
Methodology part 156 Appendix 3
Page 177 and 178:
7. Discussion and perspectives Disc
Page 179 and 180:
Discussion and conclusion 161 7. Di
Page 181 and 182:
Page 183 and 184:
Page 185 and 186:
References References Abreu,M.T., V
Page 187 and 188:
References Derrien,M., Vaughan,E.E.
Page 189 and 190:
References Haarman,M. and Knol,J. (
Page 191 and 192:
References Lantz,P.G., Matsson,M.,
Page 193 and 194:
References Matsuki,T., Watanabe,K.,
Page 195 and 196:
References Pullan,R.D., Thomas,G.A.
Page 197 and 198:
References Tannock,G.W. (2010). Ana
Page 200:
National Food Institute Technical U
show all

Role of Intestinal Microbiota in Ulcerative Colitis

Create successful ePaper yourself

Delete template?

Save as template?