Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression

<p>Abstract</p> <p>Background</p> <p>NOD2 is an innate immune receptor for the bacterial cell wall component muramyl-dipeptide. Mutations in the leucine-rich repeat region of NOD2, which lead to an impaired recognition of muramyl-dipeptide, have been associated with Crohn disease, a human chronic inflammatory bowel disease. Tissue specific constitutive and inducible expression patterns of NOD2 have been described that result from complex regulatory events for which the molecular mechanisms are not yet fully understood.</p> <p>Results</p> <p>We have identified two novel exons of the <it>NOD2 </it>gene (designated exon 1a and 1b), which are spliced to the canonical exon 2 and constitute the 5' untranslated region of two alternative transcript isoforms (i.e. exon 1a/1b/2 and exon 1a/2). The two novel transcripts are abundantly expressed and seem to comprise the majority of NOD2 transcripts under physiological conditions. We confirm the expression of the previously known canonical first exon (designated exon 1c) of the gene in unstimulated mononuclear cells. The inclusion of the second alternative exon 1b, which harbours three short upstream open reading frames (uORFs), is downregulated upon stimulation with TNF-α or under pro-inflammatory conditions in the inflamed intestinal mucosa <it>in vivo</it>. Using the different 5' UTR splice forms fused to a firefly luciferase (LUC) reporter we demonstrate a rapamycin-sensitive inhibitory effect of the uORFs on translation efficacy.</p> <p>Conclusion</p> <p>The differential usage of two alternative promoters in the <it>NOD2 </it>gene leads to tissue-specific and context-dependent <it>NOD2 </it>transcript isoform patterns. We demonstrate for the first time that context-dependent alternative splicing is linked to uORF-mediated translational repression. The results suggest complex parallel control mechanisms that independently regulate NOD2 expression in the context of inflammatory signaling.</p

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Radiological Outcomes of Femoral Head Resection in Patients with Cerebral Palsy: A Retrospective Comparative Study of Two Surgical Procedures

Background: We conducted this study to compare postoperative radiological outcomes of two surgical procedures (femoral head resection (FHR) and femoral head cap plastic surgery (FCP)) in patients with CP and hip dislocation. Methods: CP patients with Gross Motor Function Classification Score (GMFCS) IV or V, who underwent either FHR or FCP between 2007 and 2018 at Heidelberg University Hospital in Germany, were included. Most participants underwent postoperative traction in an attempt to prevent telescoping. Besides the above-mentioned objectives, we examined the association between telescoping and spasmolytic use, traction weight, and traction duration. Results: Thirty-eight CP patients were included, of whom 15 (25 hips) underwent FHR and 23 (30 hips) underwent FCP. Heterotopic ossification (grades I, II, and III) occurred in 80% and 83.3% of patients in the FHR and FCP groups, respectively. Telescoping occurred in 18.68 and 31.99% of patients in the FHR and FCP groups, respectively (p = 0.999). Other complications were similar between both groups. Conclusions: The postoperative outcomes of FHR and FCP are similar in terms of telescoping, heterotopic ossification, and complications. Although telescoping was encountered more in the FCP group, no significant difference from the FHR group was found. We noted that the weight of traction could reduce the development of telescoping

Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression-0

<p><b>Copyright information:</b></p><p>Taken from "Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression"</p><p>http://www.biomedcentral.com/1471-2164/8/472</p><p>BMC Genomics 2007;8():472-472.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2228316.</p><p></p>in bold print. The productive ATG used as an alternative translation start in exon 2 is marked by an asterisk. (B) Graphical representation of the alternatively spliced isoforms from the two alternate promoters. STP, stop codon of the uORFs. Below is a representation of the alternatively translated protein isoforms depicted by single letter amino acid code. (1) denotes the original start in the canonical exon 1, (2) denotes the alternative start in exon 2 used by the two novel transcript isoforms. (C) Primary monocytes were treated with TNF-α (10 ng/ml) for 12 h. NOD2 was amplified as outlined and analyzed on agarose gels (D) Amplification of NOD2 in a human multiple tissue panel. Note the preponderance of the short isoform exon1a/2 in the leukocytes. (E) Densitometric analysis of RT-PCR experiments from colonic biopsies of healthy controls and inflamed tissue from Crohn disease patients. Depicted is the ratio between the short (exon1a/2) and the long (exon 1a/1b/2) isoform of NOD2 (**p < 0.002; *p < 0.05, Student's T-test, colonic samples from = 8 healthy controls and = 11 inflamed Crohn disease patients;= 5 ileal samples from healthy controls and = 5 inflamed ileal samples from Crohn disease patients)

Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression-2

<p><b>Copyright information:</b></p><p>Taken from "Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression"</p><p>http://www.biomedcentral.com/1471-2164/8/472</p><p>BMC Genomics 2007;8():472-472.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2228316.</p><p></p> of the uAUGs (ΔATG1.2.3 and wildtype). Note the significant reduction of the inhibitory effect of the uORFs after rapamycin treatment (1 μM). Values represent mean ± SD calculated from three independent experiments. RLU, relative luciferase units, determined by dual luciferase assay *p < 0.05, Student's T-Test)

Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression-1

<p><b>Copyright information:</b></p><p>Taken from "Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression"</p><p>http://www.biomedcentral.com/1471-2164/8/472</p><p>BMC Genomics 2007;8():472-472.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2228316.</p><p></p>ining only exon 1a and alt 5' UTR NOD2 b containing exon 1a and 1b) cloned in front of a luciferase reporter gene (pGL Basic) ATG1-3 denote the uAUGs and STP the respective stop codons. (B) Constitutive luciferase activities of the two constructs in transfected HEK293 cells. (C) Sequential deletion of the uAUGs (e.g. ΔATG 1 denotes deletion of the first uAUG) abolishes the inhibitory activity of the long alternative 5'UTR (alt 5' UTR NOD2 b). All values correspond to mean ± SD calculated from at least three independent experiments. RLU, relative luciferase units, determined by dual luciferase assay *p < 0.05, **p < 0.01, Student's T-Test)

IgE/FcεRI-Mediated Antigen Cross-Presentation by Dendritic Cells Enhances Anti-Tumor Immune Responses

Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated allergies and cancer, implying tumor-protective properties of IgE. However, the underlying immunologic mechanisms remain poorly understood. Antigen cross-presentation by dendritic cells (DCs) is of key importance for anti-tumor immunity because it induces the generation of cytotoxic CD8+ T lymphocytes (CTLs) with specificity for tumor antigens. We demonstrate that DCs use IgE and FcεRI, the high-affinity IgE receptor, for cross-presentation and priming of CTLs in response to free soluble antigen at low doses. Importantly, IgE/FcεRI-mediated cross-presentation is a distinct receptor-mediated pathway because it does not require MyD88 signals or IL-12 induction in DCs. Using passive immunization with tumor antigen-specific IgE and DC-based vaccination experiments, we demonstrate that IgE-mediated cross-presentation significantly improves anti-tumor immunity and induces memory responses in vivo. Our findings suggest a cellular mechanism for the tumor-protective features of IgE and expand the known physiological functions of this immunoglobulin