Search

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., (Copyright: © 2023 by the authors.)

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

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 G 2 -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 G 2 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 G 2 -M transition through Aurora A kinase in mammalian cells. Implications: PKD2 is a novel cell-cycle regulator that promotes G 2 -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 ., (©2018 American Association for Cancer Research.)

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 β-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 β-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., (Copyright © 2018 Elsevier B.V. All rights reserved.)