Comparison of potency between histone deacetylase inhibitors trichostatin A and valproic acid on enhancing in vitro development of porcine somatic cell nuclear transfer embryos

Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer (SCNT) embryos. Such modification includes an increase in histone acetylation. Histone deacetylase inhibitors (HDACi), such as trichostatin A (TSA) and valproic acid (VPA), have been known to maintain a high cellular level of histone acetylation. Hence, treatment of nuclear transfer embryos with HDACi may increase the efficiency of cloning. The present study attempted direct comparison of TSA and VPA with regard to the potency of enhancement of in vitro development in porcine SCNT embryos. Reconstructed oocytes using fetal fibroblasts were cultured in PZM-3 containing no HDACi (control), 5 mM VPA, or 50 nM TSA for 24 h, and another 5 d thereafter without HDACi. The frequency of blastocyst formation was significantly higher (P<0.05) in embryos treated with VPA than the frequencies with TSA and without HDACi (125/306, 40.8% vs. 94/313, 30.2% vs. 80/329, 23.4%). In addition, VPA treatment significantly increased (P<0.05) the number of inner cell mass (ICM) cells compared with the control (15.6 ± 1.7 vs. 10.8 ± 2.6), whereas no differences were observed between the TSA treatment and control groups (12.9 ± 3.0 vs. 10.8 ± 2.6). The present study demonstrates that VPA enhances in vitro development of porcine SCNT embryos, particularly by an increase in blastocyst formation and in the number of ICM cells, suggesting that VPA may be more potent than TSA in supporting developmental competence of cloned embryos.     

Identification of a mutation associated with fatal Foal Immunodeficiency Syndrome in the Fell and Dales pony

The Fell and Dales are rare native UK pony breeds at risk due to falling numbers, in-breeding, and inherited disease. Specifically, the lethal Mendelian recessive disease Foal Immunodeficiency Syndrome (FIS), which manifests as B-lymphocyte immunodeficiency and progressive anemia, is a substantial threat. A significant percentage (～10%) of the Fell ponies born each year dies from FIS, compromising the long-term survival of this breed. Moreover, the likely spread of FIS into other breeds is of major concern. Indeed, FIS was identified in the Dales pony, a related breed, during the course of this work. Using a stepwise approach comprising linkage and homozygosity mapping followed by haplotype analysis, we mapped the mutation using 14 FIS-affected, 17 obligate carriers, and 10 adults of unknown carrier status to a ～1 Mb region (29.8 - 30.8 Mb) on chromosome (ECA) 26. A subsequent genome-wide association study identified two SNPs on ECA26 that showed genome-wide significance after Bonferroni correction for multiple testing: BIEC2-692674 at 29.804 Mb and BIEC2-693138 at 32.19 Mb. The associated region spanned 2.6 Mb from ～29.6 Mb to 32.2 Mb on ECA26. Re-sequencing of this region identified a mutation in the sodium/myo-inositol cotransporter gene (SLC5A3); this causes a P446L substitution in the protein. This gene plays a crucial role in the regulatory response to osmotic stress that is essential in many tissues including lymphoid tissues and during early embryonic development. We propose that the amino acid substitution we identify here alters the function of SLC5A3, leading to erythropoiesis failure and compromise of the immune system. FIS is of significant biological interest as it is unique and is caused by a gene not previously associated with a mammalian disease. Having identified the associated gene, we are now able to eradicate FIS from equine populations by informed selective breeding.     

Universality of thermodynamic constants governing biological growth rates

Background

Mathematical models exist that quantify the effect of temperature on poikilotherm growth rate. One family of such models assumes a single rate-limiting ‘master reaction’ using terms describing the temperature-dependent denaturation of the reaction''s enzyme. We consider whether such a model can describe growth in each domain of life.

Methodology/Principal Findings

A new model based on this assumption and using a hierarchical Bayesian approach fits simultaneously 95 data sets for temperature-related growth rates of diverse microorganisms from all three domains of life, Bacteria, Archaea and Eukarya. Remarkably, the model produces credible estimates of fundamental thermodynamic parameters describing protein thermal stability predicted over 20 years ago.

Conclusions/Significance

The analysis lends support to the concept of universal thermodynamic limits to microbial growth rate dictated by protein thermal stability that in turn govern biological rates. This suggests that the thermal stability of proteins is a unifying property in the evolution and adaptation of life on earth. The fundamental nature of this conclusion has importance for many fields of study including microbiology, protein chemistry, thermal biology, and ecological theory including, for example, the influence of the vast microbial biomass and activity in the biosphere that is poorly described in current climate models.     

A species tree for the Australo-Papuan Fairy-wrens and allies (Aves: Maluridae)

We explored the efficacy of species tree methods at the family level in birds, using the Australo-Papuan Fairy-wrens (Passeriformes: Maluridae) as a model system. Fairy-wrens of the genus Malurus are known for high intensities of sexual selection, resulting in some cases in rapid speciation. This history suggests that incomplete lineage sorting (ILS) of neutrally evolving loci could be substantial, a situation that could compromise traditional methods of combining loci in phylogenetic analysis. Using 18 molecular markers (5 anonymous loci, 7 exons, 5 introns, and 1 mitochondrial DNA locus), we show that gene tree monophyly across species could be rejected for 16 of 18 loci, suggesting substantial ILS at the family level in these birds. Using the software Concaterpillar, we also detect three statistically distinct clusters of gene trees among the 18 loci. Despite substantial variation in gene trees, species trees constructed using four different species tree estimation methods (BEST, BUCKy, and STAR) were generally well supported and similar to each other and to the concatenation tree, with a few mild discordances at nodes that could be explained by rapid and recent speciation events. By contrast, minimizing deep coalescences produced a species tree that was topologically more divergent from those of the other methods as measured by multidimensional scaling of trees. Additionally, gene and species trees were topologically more similar in the BEST analysis, presumably because of the species tree prior employed in BEST which appropriately assumes that gene trees are correlated with each other and with the species tree. Among the 18 loci, we also discovered 102 independent indel markers, which also proved phylogenetically informative, primarily among genera, and displayed a ～4-fold bias towards deletions. As suggested in earlier work, the grasswrens (Amytornis) are sister to the rest of the family and the emu-wrens (Stipiturus) are sister to fairy-wrens (Malurus, Clytomyias). Our study shows that ILS is common at the family level in birds yet, despite this, species tree methods converge on broadly similar results for this family.     

Monoclonal TCR-redirected tumor cell killing

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.     

A nuclear import pathway exploited by pathogenic noncoding RNAs

The prevailing view of intracellular RNA trafficking in eukaryotic cells is that RNAs transcribed in the nucleus either stay in the nucleus or cross the nuclear envelope, entering the cytoplasm for function. However, emerging evidence illustrates that numerous functional RNAs move in the reverse direction, from the cytoplasm to the nucleus. The mechanism underlying RNA nuclear import has not been well elucidated. Viroids are single-stranded circular noncoding RNAs that infect plants. Using Nicotiana benthamiana, tomato (Solanum lycopersicum), and nuclear-replicating viroids as a model, we showed that cellular IMPORTIN ALPHA-4 (IMPa-4) is likely involved in viroid RNA nuclear import, empirically supporting the involvement of Importin-based cellular pathway in RNA nuclear import. We also confirmed the involvement of a cellular protein (viroid RNA-binding protein 1 [VIRP1]) that binds both IMPa-4 and viroids. Moreover, a conserved C-loop in nuclear-replicating viroids serves as a key signal for nuclear import. Disrupting C-loop impairs VIRP1 binding, viroid nuclear accumulation, and infectivity. Further, C-loop exists in a subviral satellite noncoding RNA that relies on VIRP1 for nuclear import. These results advance our understanding of subviral RNA infection and the regulation of RNA nuclear import.

RNA C-loop motif is a key signal recognized by the VIRP1 for the nuclear import of pathogenic noncoding RNAs (i.e. nuclear-replicating viroids and possibly a viral satellite RNA), via the IMPORTIN ALPHA-4-based cellular pathway.

IN A NUTSHELL Background: During the course of evolution, eukaryotic cells gained a nuclear envelope to protect their genomes. However, to coordinate diverse biological processes, cellular contents need to communicate between the nucleus and the cytoplasm. The nuclear/cytoplasmic shuttling of proteins has been well studied, but only the nuclear export of RNAs has been analyzed in detail. Nevertheless, increasing evidence has shown that multiple functional RNAs traffic from the cytoplasm to the nucleus, by a yet-to-be-elucidated mechanism. Questions: How can RNA be recognized for nuclear import? Which cellular proteins serve as vehicles for RNA nuclear import? Findings: We used a pathogenic noncoding RNA (potato spindle tuber viroid [PSTVd]) as a model to study RNA nuclear import and found a particular RNA structure (C-loop) that is critical for PSTVd nuclear accumulation. PSTVd C-loop is recognized and bound by the cellular viroid RNA-binding protein 1 (VIRP1). Since nuclear import of proteins often relies on Importin, we performed a screen and identified IMPORTIN ALPHA-4 (IMPa-4) in a complex with PSTVd. Reducing the amount of IMPa-4 in cells inhibited PSTVd nuclear accumulation and infectivity. Interestingly, VIRP1 also relies on IMPa-4 for nuclear accumulation. Therefore, we propose a model that IMPa-4 transports the VIRP1–PSTVd complex into the nucleus. Notably, nearly all nuclear-replicating viroids and a viral satellite RNA contain a C-loop, suggesting that the C-loop is a conserved signal for RNA nuclear import. Next steps: We are interested in identifying the C-loop structure in cellular RNAs. Once we find cellular RNAs with a C-loop, we will test whether those cellular RNAs are transported into the nucleus and explore the biological significance of their nuclear import.