Neutralizing Aptamers from Whole-Cell SELEX Inhibit the RET Receptor Tyrosine Kinase

Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance

Blocking α2δ-1 Subunit Reduces Bladder Hypersensitivity and Inflammation in a Cystitis Mouse Model by Decreasing NF-kB Pathway Activation

Bladder pain is frequently associated with bladder inflammation, as in conditions like interstitial cystitis (IC), for which current analgesic therapies have limited efficacy. The antinociceptive effect of alpha-2-delta (α2δ) ligands on inflammation-associated visceral pain like that experienced in cystitis has been poorly investigated. To investigate the effect of pregabalin (PGB), an α2δ ligand, we evaluated its impact on mechanical hyperalgesia in a mouse model of cystitis induced by cyclophosphamide (CYP). We further studied its effect on inflammation and NF-kB pathway activation. Acute cystitis was induced by intraperitoneal injection of 150 mg kg-1 of CYP in C57Bl/6J male mice. PGB was subcutaneously injected (30 mg kg-1) 3 h after CYP injection. The effect of PGB on CYP-induced mechanical referred hyperalgesia (abdominal Von Frey test), inflammation (organ weight, cytokine production, α2δ subunit level, NF-kB pathway activation) were assessed 1 h after its injection. In parallel, its effect on cytokine production, α2δ subunit level and NF-kB pathway activation was assessed in vitro on peritoneal exudate cells (PECs) stimulated with LPS. PGB treatment decreased mechanical referred hyperalgesia. Interestingly, it had an anti-inflammatory effect in the cystitis model by reducing pro-inflammatory cytokine production. PGB also inhibited NF-kB pathway activation in the cystitis model and in macrophages stimulated with LPS, in which it blocked the increase in intracellular calcium. This study shows the efficacy of PGB in hypersensitivity and inflammation associated with cystitis. It is therefore of great interest in assessing the benefit of α2δ ligands in patients suffering from cystitis

The Cleavage/Polyadenylation Activity Triggered by a U-rich Motif Sequence Is Differently Required Depending on the Poly(A) Site Location at Either the First or Last 3′-Terminal Exon of the 2′-5′ Oligo(A) Synthetase Gene

Abstract Background Among the different mechanisms involved in irritable bowel syndrome ( IBS ) physiopathology, visceral hypersensitivity seems to play a key role. It involves sensitization of the colonic primary afferent fibers, especially through an overexpression of ion channels. The aims of this translational study were to investigate the colonic expression of Ca v 3.2 calcium channels and their involvement in an animal model of colonic hypersensitivity, and to assess their expression in the colonic mucosa of symptomatic IBS patients. Methods This bench‐to‐bed study combined a preclinical experimental study on mice and a case–control clinical study. Preclinical studies were performed on wild‐type and Ca v 3.2‐ KO mice. Colonic sensitivity and Ca v 3.2 expression were studied after a low‐dose treatment of dextran sodium sulfate ( DSS 0.5%). Regarding the clinical study, colonic biopsies were performed in 14 IBS patients and 16 controls during a colonoscopy to analyze the mucosal Ca v 3.2 expression. Key results Wild‐type, but not Ca v 3.2‐ KO , mice developed visceral hypersensitivity without colonic inflammation, after 0.5% DSS treatment. A significant increase of Ca v 3.2 mRNA ( p = 0.04) was found in the colon of low‐dose DSS ‐treated wild‐type ( WT ) mice compared to their controls. In human colonic biopsies, the Ca v 3.2 mRNA level was significantly higher in the IBS group compared to the control group ( p = 0.01). The immunofluorescence staining revealed their protein expression in colonic mucosa, particularly in nerve fibers. Conclusions & inferences This translational study supports the involvement of the calcium channels Ca v 3.2 in abdominal pain, as observed in IBS patients. It opens new therapeutic perspectives based on molecules specifically blocking these channels

Iodide and T4 kinetics in plasma, thyroid gland and skin of 10-day-old rats: effects of iodine deficiency

The effects of iodine deficiency on the peripheral metabolism of thyroid hormone in immature rat were evaluated by measuring the kinetics of iodide and thyroxine (T 4 ) in control and iodine-deficient 10-day-old rats. Iodine-deficient pups were obtained by giving the mother drinking water containing perchlorate; this anion is not transferred but prevents iodine transfer in the mother's milk. Labelled iodocompounds were measured in plasma, thyroid and skin for 48 h following intravenous injection of Na 131 I plus [ 125 I]T 4 . Data were interpreted by compartmental analysis. The iodide plasma clearance rate, plasma equivalent distribution volume, plasma concentration, production and iodine thyroid content of iodine-deficient rats were significantly lower (−29%, −31%, −84%, −89% and −87% respectively) than in control 10-day-old rats. The iodide thyroid uptake was reduced (−47%) but remained higher than the release of iodine as T 4 . Cutaneous iodine was lost much more quickly by iodine-deficient pups than by control pups, explaining the decreased iodide distribution volume. The parameters of T 4 metabolism were not changed by iodine deficiency, except for a slight but significant reduction of the thyroxinemia and T 4 pools (−13%). Thence, T 4 production was not significantly changed (about 8 pmol/h in control and iodine-deficient rats). The labelled T 4 curves in iodine-deficient and control skin were superimposed and the patterns of labelled T 3 derived from [ 125 I]T 4 were identical. Thus, the skin of immature rats converts T 4 to T 3 ; this process was not disturbed by iodine deficiency. The thyroid function of immature rats is particularly resistant to iodine deficiency, but the mechanisms remain unknown

Behavioral, Cellular and Molecular Responses to Cold and Mechanical Stimuli in Rats with Bilateral Dopamine Depletion in the Mesencephalic Dopaminergic Neurons

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Assessment of citalopram and escitalopram on neuroblastoma cell lines. Cell toxicity and gene modulation

International audienceSelective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of-107 (p<2.26 10-7),-24.1 (p<5.6 10-9) and-17.7 (p<1.2 10-7). CCNE1, AURKA, IGF2, MYCN and ERBB2 were more moderately down-regulated by both molecules. Glioma markers E2F1, DAPK1 and CCND1 were down-regulated. Citalopram displayed more powerful action with broader and distinct spectrum of action than escitalopram