Endogenous gpdh-1 transcriptional reporters as new tools for the study of the osmotic stress response.

In vivo monitoring of gpdh-1 gene expression using standard transcriptional reporters is a powerful and commonly used tool for genetic dissection of the osmotic stress response in C. elegans . Like all transgene reporters, these gpdh-1 reporters have important limitations that restrict their utility. To overcome these limitations, we created three different gpdh-1 reporters using CRISPR/Cas9 methods to insert several variants of GFP into the endogenous gpdh-1 locus. These new strains provide a more powerful and accurate tool for the analysis of gpdh-1 regulatory pathways.

Protein Kinase D2 Modulates Cell Cycle By Stabilizing Aurora A Kinase at Centrosomes.

Aurora A kinase (AURKA) is a master cell-cycle regulator that is often dysregulated in human cancers. Its overexpression has been associated with genome instability and oncogenic transformation. The protein kinase D (PKD) family is an emerging therapeutic target of cancer. Aberrant PKD activation has been implicated in tumor growth and survival, yet the underlying mechanisms remain to be elucidated. This study identified, for the first time, a functional crosstalk between PKD2 and Aurora A kinase in cancer cells. The data demonstrate that PKD2 is catalytically active during the G2-M phases of the cell cycle, and inactivation or depletion of PKD2 causes delay in mitotic entry due to downregulation of Aurora A, an effect that can be rescued by overexpression of Aurora A. Moreover, PKD2 localizes in the centrosome with Aurora A by binding to γ-tubulin. Knockdown of PKD2 caused defects in centrosome separation, elongated G2 phase, mitotic catastrophe, and eventually cell death via apoptosis. Mechanistically, PKD2 interferes with Fbxw7 function to protect Aurora A from ubiquitin- and proteasome-dependent degradation. Taken together, these results identify PKD as a cell-cycle checkpoint kinase that positively modulates G2-M transition through Aurora A kinase in mammalian cells.Implications: PKD2 is a novel cell-cycle regulator that promotes G2-M transition by modulating Aurora A kinase stability in cancer cells and suggests the PKD2/Aurora A kinase regulatory axis as new therapeutic targets for cancer treatment. Mol Cancer Res; 16(11); 1785-97. ©2018 AACR.

Molecular analysis of benzimidazole-resistance associated SNPs in Haemonchus contortus populations of Uruguay.

Haemonchus contortus is one of the most important parasite nematodes of small ruminants around the world and causes great economic losses in livestock production. Control of gastrointestinal nematode infections, like haemonchosis, relies mainly on anthelmintic drugs, but its excessive and inappropriate use has caused serious drug resistance issues in many countries, including Uruguay, where sheep production occupies an important place in the country's economy. Benzimidazole (BZ) anthelmintics have been used for decades to treat sheep against H. contortus infection and resistance to this anthelmintic group has been widely described. Molecularly, BZ resistance in H. contortus has been correlated with single nucleotide polymorphisms (SNPs) of the ß-tubulin isotype 1 gene at codon 200 and 167 (both TTC to TAC, F167Y and F200Y) and at codon 198 (GAA to GCA, E198A).The aim of this work was to explore the presence of these tubulin SNPs in H. contortus adult worms recovered from sheep abomasa from a slaughterhouse in Uruguay. The mean resistant allelic frequencies at positions F167Y and F200Y were 20.25 and 47.45%, respectively, for worms recovered from naturally infected sheep slaughtered in 2013, while those that were slaughtered in 2014 presented only F200Y SNP with a frequency of 86.89%. Also H. contortus Kirby adult worms (anthelmintic- susceptible McMaster isolate), recovered from artificially infected sheep, were analyzed as reference for comparative purposes This analysis showed susceptible genotype at 167 and 198 position, and a low level of the resistance allele at the 200 position (3.66%). This is the first study for the presence of SNPs in the isotype-1 ß-tubulin gene of H. contortus populations in Uruguay, which is consistent with the previous epidemiological studies carried out through the method of fecal egg count reduction test (FECRT), thus confirming the serious resistance levels to BZ anthelmintics also in this country.