National single-step genomic method that integrates multi-national genomic information.

The aim of this paper was to develop a national single-step genomic BLUP that integrates multi-national genomic estimated breeding values (EBV) and associated reliabilities without double counting dependent data contributions from the different evaluations. Simultaneous use of all data, including phenotypes, pedigree, and genotypes, is a condition to obtain unbiased EBV. However, this condition is not always fully met, mainly due to unavailability of foreign raw data for imported animals. In dairy cattle genetic evaluations, this issue is traditionally tackled through the multiple across-country evaluation (MACE) of sires, performed by Interbull Centre (Uppsala, Sweden). Multiple across-country evaluation regresses all the available national information onto a joint pedigree to obtain country-specific rankings of all sires without sharing the raw data. In the context of genomic selection, the issue is handled by exchanging sire genotypes and by using MACE information (i.e., MACE EBV and reliabilities), as a valuable source of "phenotypic" data. Although all the available data are considered, these "multi-national" genomic evaluations use multi-step methods assuming independence of various sources of information, which is not met in all situations. We developed a method that handles this by single-step genomic evaluation that jointly (1) uses national phenotypic, genomic, and pedigree data; (2) uses multi-national genomic information; and (3) avoids double counting dependent data contributions from an animal's own records and relatives' records. The method was demonstrated by integrating multi-national genomic EBV and reliabilities of Brown Swiss sires, included in the InterGenomics consortium at Interbull Centre, into the national evaluation in Slovenia. The results showed that the method could (1) increase reliability of a national (genomic) evaluation; (2) provide consistent ranking of all animals: bulls, cows, and young animals; and (3) increase the size of a genomic training population. These features provide more efficient and transparent selection throughout a breeding program.

Molecular identification of the rumen flukes Paramphistomum leydeni and Paramphistomum cervi in a concurrent infection of the red deer Cervus elaphus.

Paramphistomosis, caused by paramphistomid flukes, is a gastrointestinal parasitic disease of domestic and wild ruminants. Originally thought to be limited to the tropics and subtropics, the disease has recently been reported in temperate regions. Here we describe the concurrent infection of a red deer doe (Cervus elaphus) with Paramphistomum leydeni and Paramphistomum cervi. This is the first report of P. leydeni in Croatia. Flukes were identified on the basis of morphological keys (tegumental papillae) and sequencing of the internal transcribed spacer region 2 in ribosomal DNA. Our results confirm that the absence of tegumental papillae allows P. cervi to be differentiated morphologically from other paramphistomid species in Europe based on incident light stereomicroscopy. Nevertheless the limitations of morphological identification and taxonomic issues suggest that previous findings on paramphistomid infection should be interpreted carefully. The possible worldwide distribution of these pathogens means that paramphistomosis may be more common and its economic impact greater than previously thought.

PP2A B55α inhibits epithelial-mesenchymal transition via regulation of Slug expression in non-small cell lung cancer.

PP2A B55α, encoded by PPP2R2A, acts as a regulatory subunit of the serine/threonine phosphatase PP2A. Despite a frequent loss of heterozygosity of PPP2R2A in cases of non-small cell lung cancer (NSCLC), research on PP2A B55α's functions remains limited and controversial. To investigate the biological roles of PP2A B55α, we conducted bulk RNA-sequencing to assess the impact of PPP2R2A knockdown using two shRNAs in a NSCLC cell line. Gene set enrichment analysis (GSEA) of the RNA-sequencing data revealed significant enrichment of the epithelial-mesenchymal transition (EMT) pathway, with SNAI2 (the gene encoding Slug) emerging as one of the top candidates. Our findings demonstrate that PP2A B55α suppresses EMT, as PPP2R2A deficiency through knockdown or homozygous or hemizygous depletion promotes EMT and metastatic behavior in NSCLC cells, as evidenced by changes in EMT biomarkers, invasion and migration abilities, as well as metastasis in a tail vein assay. Mechanistically, PP2A B55α inhibits EMT by downregulating SNAI2 expression via the GSK3ß-ß-catenin pathway. Importantly, PPP2R2A deficiency also slows cell proliferation by disrupting DNA replication, particularly in PPP2R2A-/- cells. Furthermore, PPP2R2A deficiency, especially PPP2R2A-/- cells, leads to an increase in the cancer stem cell population, which correlates with enhanced resistance to chemotherapy. Overall, the decrease in PP2A B55α levels due to hemizygous/homozygous depletion heightens EMT and the metastatic or stemness/drug resistance potential of NSCLC cells despite their proliferation disadvantage. Our study highlights the significance of PP2A B55α in EMT and metastasis and suggests that targeting EMT/stemness could be a potential therapeutic strategy for treating PPP2R2A-deficient NSCLC.

Quiescence versus apoptosis: Myc abundance determines pathway of exit from the cell cycle.

When exposed to diverse growth conditions in vitro, cells can respond by entering states of proliferation, quiescence, differentiation or apoptosis. While the choices among these states can be influenced by proto-oncogene expression, how these disparate outcomes are achieved remains poorly understood. To address these issues, we have generated rodent fibroblast cell lines that harbor a human c-myc gene under the control of a tetracycline-regulated promoter. When Myc-induced cells are deprived of serum growth factors, they rapidly become apoptotic with the onset of apoptosis preceded by a large, transient increase in cdk2 kinase activity that is associated with the induction of cdc25A phosphatase and the later accumulation of p27Kip1 kinase inhibitor. Surprisingly, serum starvation in the absence of myc overexpression, (which leads to quiescence instead of apoptosis) also causes a marked transient elevation in cdk2 kinase activity, an induction of cdc25A and a delayed increase in p27Kip1. Transient elevations in cdk2 kinase activity and cdc25A abundance are required for cell cycle progression, but it is evident that these changes also precede entry to either apoptosis or quiescence in serum-starved cells. These findings suggest that the pathways to both quiescence and apoptosis share regulatory machinery with cell cycle control mechanisms. In addition, the abundance of Myc protein can be critical in the choices among these cellular states.

Specific isoforms of the resident endoplasmic reticulum protein glucosidase II associate with the CD45 protein-tyrosine phosphatase via a lectin-like interaction.

We have previously demonstrated that CD45 physically associates with the endoplasmic reticulum processing enzyme glucosidase II (GII). GII consists of the catalytic alpha-chain and an associated beta-chain. To gain insight into the basis of the association between CD45 and GII, we examined the biochemical requirements for the interaction. We show that the alpha-subunit is essential for the interaction. Interestingly, only a higher molecular weight form of GIIalpha is capable of associating with CD45 in a competitive situation where multiple GIIalpha isoforms are expressed. Further, transfection studies demonstrate that only isoforms containing the alternatively spliced sequence Box A1 are capable of binding CD45, although all isoforms are catalytically active. The interaction between CD45 and GII is dependent on the active site of GII, is mediated through the carbohydrate on CD45, and can be inhibited with mannose. Taken together, these results suggest that GIIalpha acts as a lectin and binds to CD45 in an exon-dependent manner. This lectin activity of GII may be a novel mechanism for the regulation of CD45 biology and play a role in immune function, possibly by regulating CD45 glycosylation.

Expression of novel ING variants is regulated by thyroid hormone in the Xenopus laevis tadpole.

The candidate tumor suppressor gene, ING1, encodes several protein isoforms as a result of alternative splicing that may possess agonistic and antagonistic roles in the control of cell proliferation and apoptosis. Recently a related gene, ING2, was isolated in human whose expression is increased in adenocarcinomas. Little is known about the cellular function and regulation of these ING family members, but the fact that ING proteins contain a plant homeodomain finger suggests that these proteins may modulate transcription factor-mediated pathways. To elucidate how ING may interact in different tissues to modulate function, we used amphibian metamorphosis as a model system in which a single stimulus, thyroid hormone (TH), initiates tissue-specific proliferation, differentiation, and apoptosis. We have isolated the first Xenopus laevis ING2 and demonstrate that transcript levels increase in response to TH treatment. We provide evidence for the existence of splice variants that are differentially expressed in tissues with different TH-induced fates. Western blots using an antibody directed against the highly conserved C-terminal end of ING proteins reveal a tissue-specific pattern of ING isoform expression in adult Xenopus tissues. Analyses of premetamorphic tadpole tissues show a TH-induced accumulation of ING proteins in tail, whereas the levels in the leg are not affected. This TH-induced accumulation is also observed in serum-free tail organ cultures and is prevented by inhibitors of tail apoptosis. Therefore, this work presents the first link between ING expression and a hormonally regulated nuclear transcription factor-mediated apoptotic response opening the possibility that ING family members may be involved in transducing the signal initiated by TH that determines cell fate.