Fibrosis-related transcriptome unveils a distinctive remodeling matrix pattern in penetrating ileal Crohn's disease.

BACKGROUND AND AIMS: Stricturing (B2) and penetrating (B3) ileal Crohn's disease have been reported to present similar levels of histopathological transmural fibrosis. This study aimed to compare the fibrosis-related transcriptomic profiles of penetrating and stricturing ileal Crohn's disease. METHODS: Using Nanostring technology and comparative bioinformatics, we analyzed the expression of 787 fibrosis-related genes in 36 ileal surgical specimens, 12 B2 and 24 B3, the latter including 12 cases with associated stricture(s) (B3s) and 12 without (B3o). Quality control of extracted RNA was performed according to Nanostring parameters and principal component analysis for the distribution analysis. For the selection of the differentially expressed genes a p-adjusted <0.05 and Fold Change ≤-1.5 or ≥ 1.5 was adopted. qPCR and immunohistochemistry analyses were used to validate selected differentially expressed genes. RESULTS: We included 34 patients with B2 and B3 phenotypes, balanced for age at diagnosis, age at surgery, gender, Crohn's disease localization, perianal disease and therapy. Inflammation and fibrosis histopathological scoring were similar in all cases. B2 and B3 groups showed a very good clustering regarding 30 significantly differentially expressed genes, all being remarkably upregulated in B3. More than half of these genes were involved in Crohn's disease fibrogenesis, while eight differentially expressed genes were so in other organs. The most significantly active biologic processes and pathways in penetrating disease were response to TGFßand matrix organization and degradation, as validated by immunohistochemistry. CONCLUSIONS: Despite the histopathological similarities in fibrosis between stricturing and penetrating ileal Crohn's disease, their fibrosis-related transcriptomic profiles are distinct. Penetrating disease exhibits a distinctive transcriptomic landscape related to enhanced matrix remodeling.

Adapter dimer contamination in sRNA-sequencing datasets predicts sequencing failure and batch effects and hampers extracellular vesicle-sRNA analysis.

Small RNA (sRNA) profiling of Extracellular Vesicles (EVs) by Next-Generation Sequencing (NGS) often delivers poor outcomes, independently of reagents, platforms or pipelines used, which contributes to poor reproducibility of studies. Here we analysed pre/post-sequencing quality controls (QC) to predict issues potentially biasing biological sRNA-sequencing results from purified human milk EVs, human and mouse EV-enriched plasma and human paraffin-embedded tissues. Although different RNA isolation protocols and NGS platforms were used in these experiments, all datasets had samples characterized by a marked removal of reads after pre-processing. The extent of read loss between individual samples within a dataset did not correlate with isolated RNA quantity or sequenced base quality. Rather, cDNA electropherograms revealed the presence of a constant peak whose intensity correlated with the degree of read loss and, remarkably, with the percentage of adapter dimers, which were found to be overrepresented sequences in high read-loss samples. The analysis through a QC pipeline, which allowed us to monitor quality parameters in a step-by-step manner, provided compelling evidence that adapter dimer contamination was the main factor causing batch effects. We concluded this study by summarising peer-reviewed published workflows that perform consistently well in avoiding adapter dimer contamination towards a greater likelihood of sequencing success.

Malaria: looking for selection signatures in the human PKLR gene region.

The genetic component of susceptibility to malaria is both complex and multigenic and the better-known protective polymorphisms are those involving erythrocyte-specific structural proteins and enzymes. In vivo and in vitro data have suggested that pyruvate kinase deficiency, which causes a nonspherocytic haemolytic anaemia, could be protective against malaria severity in humans, but this hypothesis remains to be tested. In the present study, we conducted a combined analysis of Short Tandem Repeats (STRs) and Single Nucleotide Polymorphisms (SNPs) in the pyruvate kinase-encoding gene (PKLR) and adjacent regions (chromosome 1q21) to look for malaria selective signatures in two sub-Saharan African populations from Angola and Mozambique, in several groups with different malaria infection outcome. A European population from Portugal, including a control and a pyruvate kinase-deficient group, was used for comparison. Data from STR and SNP loci spread along the PKLR gene region showed a considerably higher differentiation between African and Portuguese populations than that usually found for neutral markers. In addition, a wider region showing strong linkage disequilibrium was found in an uncomplicated malaria group, and a haplotype was found to be associated with this clinical group. Altogether, this data suggests that malaria selective pressure is acting in this genomic region.

Identification of mouse inbred strains through mitochondrial DNA single-nucleotide extension.

Inbred mouse strains are used as model organisms for biomedical research in laboratories throughout the world. The most widely used of these strains had their genome sequenced recently, and phylogenetic studies have been performed, namely, based on mitochondrial DNA (mtDNA). This has allowed determining that few polymorphisms distinguish the mtDNAs of the common inbred strains, but a high number of differences are observed among the wild-derived strains. Taking advantage of these observations, we here present a single base extension typing strategy that, with only a pair of multiplex reactions, allows the distinction between common inbred and wild-derived mice strains, and provides the identification of ten different common inbred and six wild-derived mice mtDNA haplotypes. Given that all the animals inside a strain present the same mtDNA, this strategy allows a rapid identification of the strains without the need for probability calculations. We further test this approach in an island population of wild mice, which provides both an indication on its applicability in wild mice, and a comparison of evolutionary processes on inbred and wild mice that are restricted to a limited space. Rapid genotyping methods that allow the distinction of the different strains are important for both the distinction of materials such as tissue and cell collections and to identify the origin of new strains. Moreover, it may also prove valuable in forensic identification of materials collected in laboratory accidents, as well as in cases of scientific fraud.