Upregulated Genes In Sporadic, Idiopathic Pulmonary Arterial Hypertension

BACKGROUND: To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH). METHODS: Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test. RESULTS: We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase. CONCLUSION: Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively

Functional Screening of Metagenome and Genome Libraries for Detection of Novel Flavonoid-Modifying Enzymes

The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This m etagenome e xtract t hin-layer chromatography a nalysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter . GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis . Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside

A Comparative Metagenome Survey of the Fecal Microbiota of a Breast- and a Plant-Fed Asian Elephant Reveals an Unexpectedly High Diversity of Glycoside Hydrolase Family Enzymes

<div><p>A phylogenetic and metagenomic study of elephant feces samples (derived from a three-weeks-old and a six-years-old Asian elephant) was conducted in order to describe the microbiota inhabiting this large land-living animal. The microbial diversity was examined via 16S rRNA gene analysis. We generated more than 44,000 GS-FLX+454 reads for each animal. For the baby elephant, 380 operational taxonomic units (OTUs) were identified at 97% sequence identity level; in the six-years-old animal, close to 3,000 OTUs were identified, suggesting high microbial diversity in the older animal. In both animals most OTUs belonged to Bacteroidetes and Firmicutes. Additionally, for the baby elephant a high number of Proteobacteria was detected. A metagenomic sequencing approach using Illumina technology resulted in the generation of 1.1 Gbp assembled DNA in contigs with a maximum size of 0.6 Mbp. A KEGG pathway analysis suggested high metabolic diversity regarding the use of polymers and aromatic and non-aromatic compounds. In line with the high phylogenetic diversity, a surprising and not previously described biodiversity of glycoside hydrolase (GH) genes was found. Enzymes of 84 GH families were detected. Polysaccharide utilization loci (PULs), which are found in Bacteroidetes, were highly abundant in the dataset; some of these comprised cellulase genes. Furthermore the highest coverage for GH5 and GH9 family enzymes was detected for Bacteroidetes, suggesting that bacteria of this phylum are mainly responsible for the degradation of cellulose in the Asian elephant. Altogether, this study delivers insight into the biomass conversion by one of the largest plant-fed and land-living animals.</p></div

Rarefaction curves calculated for the feces sample of the three-weeks-old and the six-years-old Asian elephant at 3% genetic distance of 16S rRNA genes.

<p>The curve for the six-years-old elephant comprises 8,014 archaeal sequences which were clustered to 54 OTUs. The sequences were denoised employing Acacia. Chimeric sequences were removed using UCHIME in reference mode with the most recent SILVA SSU database as reference dataset (SSURef 115 NR).</p

Overall coverage of selected cellulolytic GH family genes in the feces samples in relation to their phylogenetic affiliation.

<p><b>A</b>) three-weeks-old elephant, <b>B</b>) six-years-old elephant.</p

Relative abundances of different phyla and classes in the two elephant feces samples.

<p><b>A:</b> Relative abundance of phyla in the feces of the three-weeks-old and the six-years-old Asian elephant based on 16S rRNA gene sequences. For the three-weeks-old elephant no Archaea were observed. <b>B:</b> Phylogenetic comparison on class level between both elephants. Heat map colors indicate the abundances of the respective 16S rRNA genes.</p

Phylogenetic analysis of the elephant feces in comparison with other fecal and intestinal metagenome data sets.

<p>Data indicate the phylogenetic relation based on gene similarities in the metagenome sequences. The percent of sequences assigned to each phylum according to IMG/M ER is shown based on the total number of obtained sequences of each data set. Sequence data for the metagenomes were extracted from the IMG/M ER web page of the US Department of Energy Joint Genome Institute and the respective bioprojects (IMG Genome IDs: Six-years-old Elephant (this study): 3300001598; three-weeks-old Elephant (this study): 3300001919; Green Cockroach: 2228664000; Termite: 3300001544; Dog: 2019105001; Reindeer: 2088090000; Neotropical Beetle: 3300000114; Asian Long-Horned Beetle: 2084038013; Bovine Rumen: 2061766007; Northwest Shipworm: 2189573029; Human stool: 7000000038).</p