miR-15a-5p and miR-21-5p contribute to chemoresistance in cytogenetically normal acute myeloid leukaemia by targeting PDCD4, ARL2 and BTG2

Cytarabine and daunorubicin are old drugs commonly used in the treatment of acute myeloid leukaemia (AML). Refractory or relapsed disease because of chemotherapy resistance is a major issue. microRNAs (miRNAs) were incriminated in resistance. This study aimed to identify miRNAs involved in chemoresistance in AML patients and to define their target genes. We focused on cytogenetically normal AML patients with wild-type NPM1 without FLT3-ITD as the treatment of this subset of patients with intermediate-risk cytogenetics is not well established. We analysed baseline AML samples by small RNA sequencing and compared the profile of chemoresistant to chemosensitive AML patients. Among the miRNAs significantly overexpressed in chemoresistant patients, we revealed miR-15a-5p and miR-21-5p as miRNAs with a major role in chemoresistance in AML. We showed that miR-15a-5p and miR-21-5p overexpression decreased apoptosis induced by cytarabine and/or daunorubicin. PDCD4, ARL2 and BTG2 genes were found to be targeted by miR-15a-5p, as well as PDCD4 and BTG2 by miR-21-5p. Inhibition experiments of the three target genes reproduced the functional effect of both miRNAs on chemosensitivity. Our study demonstrates that miR-15a-5p and miR-21-5p are overexpressed in a subgroup of chemoresistant AML patients. Both miRNAs induce chemoresistance by targeting three pro-apoptotic genes PDCD4, ARL2 and BTG2.     

Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize

Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.     

Evidence for the dimerization of human regulator of G-protein signalling 5 (RGS5).

RGS5 is a R4 type RGS that regulates GPCR signalling. Using western blot, we detected RGS5 as a specific 23 kDa protein in cells overexpressing RGS5. A 42 kDa band representing a possible RGS5 dimer was also detected. Given that GPCRs and their associated proteins form complexes involving multiple protein-protein interactions, we investigated the possibility that the 42 kDa band represents an RGS5-RGS5 dimer. RGS5 dimerization was confirmed by the analysis of a GFP tagged RGS5 fusion in yeast and with two-hybrid assays. Analysis of RGS5 in HEK293A cells suggests that the dimer may serve a regulatory function since it is longer lived than the monomer.     

The amino-terminal extensions of the longer Sendai virus C proteins modulate pY701-Stat1 and bulk Stat1 levels independently of interferon signaling

The Sendai virus (SeV) C proteins are known to interact with Stat1 to prevent interferon (IFN)-induced pY701-Stat1 formation and IFN signaling. Nevertheless, pY701-Stat1 levels paradoxically increase during SeV infection. The C proteins also induce bulk Stat1 instability in some cells, similar to rubulavirus V proteins. We have found that SeV infection increases pY701-Stat1 levels even in cells in which bulk Stat1 levels strongly decrease. Remarkably, both the decrease in bulk Stat1 levels and the increase in pY701-Stat1 levels were found to be independent of the IFN signaling system, i.e., these events occur in mutant cells in which various components of the IFN signaling system have been disabled. Consistent with this, the C-induced decrease in Stat1 levels does not require Y701 of Stat1. We present evidence that C interacts with Stat1 in two different ways, one that prevents IFN-induced pY701-Stat1 formation and IFN signaling that has already been documented, and another that induces pY701-Stat1 formation (while decreasing bulk Stat1 levels) in a manner that does not require IFN signaling. These two types of Stat1 interaction are also distinguishable by C gene mutations. In particular, the IFN signaling-independent Stat1 interactions specifically require the amino-terminal extensions of the longer C proteins. The actions of the SeV C proteins in counteracting the cellular antiviral response are clearly more extensive than previously appreciated.     

Co-culture of human fibroblasts,smooth muscle and endothelial cells promotes osteopontin induction in hypoxia

Arteriovenous fistulas (AVFs) are the preferred vascular access for haemodialysis of patients suffering from end-stage renal disease, a worldwide public health problem. However, they are prone to a high rate of failure due to neointimal hyperplasia and stenosis. This study aimed to determine if osteopontin (OPN) was induced in hypoxia and if OPN could be responsible for driving AVF failure. Identification of new factors that participate in remodelling of AVFs is a challenge. Three cell lines representing the cells of the three layers of the walls of arteries and veins, fibroblasts, smooth muscle cells and endothelial cells, were tested in mono- and co-culture in vitro for OPN expression and secretion in normoxia compared to hypoxia after silencing the hypoxia-inducible factors (HIF-1α, HIF-2α and HIF-1/2α) with siRNA or after treatment with an inhibitor of NF-kB. None of the cells in mono-culture showed OPN induction in hypoxia, whereas cells in co-culture secreted OPN in hypoxia. The changes in oxygenation that occur during AVF maturation up-regulate secretion of OPN through cell-cell interactions between the different cell layers that form AVF, and in turn, these promote endothelial cell proliferation and could participate in neointimal hyperplasia.     

Structural and thermodynamic bases for the design of pure prolactin receptor antagonists: X-ray structure of Del1-9-G129R-hPRL

Competitive antagonists of the human prolactin (hPRL) receptor are a novel class of molecules of potential therapeutic interest in the context of cancer. We recently developed the pure antagonist Del1-9-G129R-hPRL by deleting the nine N-terminal residues of G129R-hPRL, a first generation partial antagonist. We determined the crystallographic structure of Del1-9-G129R-hPRL, which revealed no major change compared with wild type hPRL, indicating that its pure antagonistic properties are intrinsically due to the mutations. To decipher the molecular bases of pure antagonism, we compared the biological, physicochemical, and structural properties of numerous hPRL variants harboring N-terminal or Gly(129) mutations, alone or combined. The pure versus partial antagonistic properties of the multiple hPRL variants could not be correlated to differences in their affinities toward the hPRL receptor, especially at site 2 as determined by surface plasmon resonance. On the contrary, residual agonism of the hPRL variants was found to be inversely correlated to their thermodynamic stability, which was altered by all the Gly(129) mutations but not by those involving the N terminus. We therefore propose that residual agonism can be abolished either by further disrupting hormone site 2-receptor contacts by N-terminal deletion, as in Del1-9-G129R-hPRL, or by stabilizing hPRL and constraining its intrinsic flexibility, as in G129V-hPRL.     

Influence of 6 or 8-substitution on the antiviral activity of 3-phenethylthiomethylimidazo[1,2-a]pyridine against human cytomegalovirus (HCMV) and varicella-zoster virus (VZV)

The synthesis of original imidazo[1,2-a]pyridines bearing a phenethylthiomethyl side chain at the 3 position and a (hetero)aryl substituent on the 6 or 8 position, and their antiviral activities are reported. From the synthesized compounds, the 6-halogeno and 6-phenylimidazo[1,2-a]pyridine derivatives 4c-d and 5b were the most potent against human cytomegalovirus (CMV) and/or varicella-zoster virus (VZV), whereas several other congeners (i.e., 5e, 5g, 5i, 5l, 5n, 5p, 5q, and 5t), while less potent, were equally or more selective in their inhibitory activity against both VZV and CMV. These compounds showed similar activity against thymidine kinase competent (TK(+)) and deficient (TK(-)) VZV strains, demonstrating a mechanism of action independent of the viral thymidine kinase.     

Identification and SAR of a new series of thieno[3,2-d]pyrimidines as Tpl2 kinase inhibitors

We report here the synthesis and SAR of a new series of thieno[3,2-d]pyrimidines as potent Tpl2 kinase inhibitors. The proposed binding mode suggests the potential flipped binding mode depending on the substitution. Biacore studies show evidence of binding of these molecules to the protein kinase. The kinome inhibition profile of these molecules suggests good selectivity.     

LINGO1 variants in the French-Canadian population

Essential tremor (ET) is a complex genetic disorder for which no causative gene has been found. Recently, a genome-wide association study reported that two variants in the LINGO1 locus were associated to this disease. The aim of the present study was to test if this specific association could be replicated using a French-Canadian cohort of 259 ET patients and 479 ethnically matched controls. Our genotyping results lead us to conclude that no association exists between the key variant rs9652490 and ET (P_corr = 1.00).