Transcriptome studies are revealing the complex gene expression basis of limb regeneration in the primary salamander model - Ambystoma mexicanum (axolotl). To better understand this complexity, there is need to extend analyses to additional salamander species. Using microarray and RNA-Seq, we performed a comparative transcriptomic study using A. mexicanum and two other ambystomatid salamanders: A. andersoni, and A. maculatum. Salamanders were administered forelimb amputations and RNA was isolated and analyzed to identify 405 non-redundant genes that were commonly, differentially expressed 24 h post amputation. Many of the upregulated genes are predicted to function in wound healing and developmental processes, while many of the downregulated genes are typically expressed in muscle. The conserved transcriptional changes identified in this study provide a high-confidence dataset for identifying factors that simultaneous orchestrate wound healing and regeneration processes in response to injury, and more generally for identifying genes that are essential for salamander limb regeneration.

Tissue regeneration is associated with complex changes in gene expression and post-translational modifications of proteins, including transcription factors and histones that comprise chromatin. We tested 172 compounds designed to target epigenetic mechanisms in an axolotl ( Ambystoma mexicanum ) embryo tail regeneration assay. A relatively large number of compounds (N = 55) inhibited tail regeneration, including 18 histone deacetylase inhibitors (HDACi). In particular, romidepsin, an FDA-approved anticancer drug, potently inhibited tail regeneration when embryos were treated continuously for 7 days. Additional experiments revealed that romidepsin acted within a very narrow, post-injury window. Romidepsin treatment for only 1-minute post amputation inhibited regeneration through the first 7 days, however after this time, regeneration commenced with variable outgrowth of tailfin tissue and abnormal patterning. Microarray analysis showed that romidepsin altered early, transcriptional responses at 3 and 6-hour post-amputation, especially targeting genes that are implicated in tumor cell death, as well as genes that function in the regulation of transcription, cell differentiation, cell proliferation, pattern specification, and tissue morphogenesis. Our results show that HDAC activity is required at the time of tail amputation to regulate the initial transcriptional response to injury and regeneration.

Among tetrapods, only salamanders can regenerate their limbs and tails throughout life. This amazing regenerative ability has attracted the attention of scientists for hundreds of years. Now that large, salamander genomes are beginning to be sequenced for the first time, omics tools and approaches can be used to integrate new perspectives into the study of tissue regeneration. Here we argue the need to move beyond the primary salamander models to investigate regeneration in other species. Salamanders at first glance come across as a phylogenetically conservative group that has not diverged greatly from their ancestors. While salamanders do present ancestral characteristics of basal tetrapods, including the ability to regenerate limbs, data from fossils and data from studies that have tested for species differences suggest there may be considerable variation in how salamanders develop and regenerate their limbs. We review the case for expanded studies of salamander tissue regeneration and identify questions and approaches that are most likely to reveal commonalities and differences in regeneration among species. We also address challenges that confront such an initiative, some of which are regulatory and not scientific. The time is right to gain evolutionary perspective about mechanisms of tissue regeneration from comparative studies of salamander species.

We sought to determine the skeletal muscle genome-wide DNA methylation and mRNA responses to one bout of lower load (LL) versus higher load (HL) resistance exercise. Trained college-aged males (n = 11, 23 ± 4 years old, 4 ± 3 years self-reported training) performed LL or HL bouts to failure separated by one week. The HL bout (i.e., 80 Fail) consisted of four sets of back squats and four sets of leg extensions to failure using 80% of participants estimated one-repetition maximum (i.e., est. 1-RM). The LL bout (i.e., 30 Fail) implemented the same paradigm with 30% of est. 1-RM. Vastus lateralis muscle biopsies were collected before, 3 h, and 6 h after each bout. Muscle DNA and RNA were batch-isolated and analyzed using the 850k Illumina MethylationEPIC array and Clariom S mRNA microarray, respectively. Performed repetitions were significantly greater during the 30 Fail versus 80 Fail (p < 0.001), although total training volume (sets x reps x load) was not significantly different between bouts (p = 0.571). Regardless of bout, more CpG site methylation changes were observed at 3 h versus 6 h post exercise (239,951 versus 12,419, respectively; p < 0.01), and nuclear global ten-eleven translocation (TET) activity, but not global DNA methyltransferase activity, increased 3 h and 6 h following exercise regardless of bout. The percentage of genes significantly altered at the mRNA level that demonstrated opposite DNA methylation patterns was greater 3 h versus 6 h following exercise (~75% versus ~15%, respectively). Moreover, high percentages of genes that were up- or downregulated 6 h following exercise also demonstrated significantly inversed DNA methylation patterns across one or more CpG sites 3 h following exercise (65% and 82%, respectively). While 30 Fail decreased DNA methylation across various promoter regions versus 80 Fail, transcriptome-wide mRNA and bioinformatics indicated that gene expression signatures were largely similar between bouts. Bioinformatics overlay of DNA methylation and mRNA expression data indicated that genes related to “Focal adhesion,” “MAPK signaling,” and “PI3K-Akt signaling” were significantly affected at the 3 h and 6 h time points, and again this was regardless of bout. In conclusion, extensive molecular profiling suggests that post-exercise alterations in the skeletal muscle DNA methylome and mRNA transcriptome elicited by LL and HL training bouts to failure are largely similar, and this could be related to equal volumes performed between bouts.

The host epigenetic landscape rapidly changes during SARS-CoV-2 infection, and evidence suggest that severe COVID-19 is associated with durable scars to the epigenome. Specifically, aberrant DNA methylation changes in immune cells and alterations to epigenetic clocks in blood relate to severe COVID-19. However, a longitudinal assessment of DNA methylation states and epigenetic clocks in blood from healthy individuals prior to and following test-confirmed non-hospitalized COVID-19 has not been performed. Moreover, the impact of mRNA COVID-19 vaccines upon the host epigenome remains understudied. Here, we first examined DNA methylation states in the blood of 21 participants prior to and following test-confirmed COVID-19 diagnosis at a median time frame of 8.35 weeks; 756 CpGs were identified as differentially methylated following COVID-19 diagnosis in blood at an FDR adjusted p-value &lt; 0.05. These CpGs were enriched in the gene body, and the northern and southern shelf regions of genes involved in metabolic pathways. Integrative analysis revealed overlap among genes identified in transcriptional SARS-CoV-2 infection datasets. Principal component-based epigenetic clock estimates of PhenoAge and GrimAge significantly increased in people over 50 following infection by an average of 2.1 and 0.84 years. In contrast, PCPhenoAge significantly decreased in people fewer than 50 following infection by an average of 2.06 years. This observed divergence in epigenetic clocks following COVID-19 was related to age and immune cell-type compositional changes in CD4+ T cells, B cells, granulocytes, plasmablasts, exhausted T cells, and naïve T cells. Complementary longitudinal epigenetic clock analyses of 36 participants prior to and following Pfizer and Moderna mRNA-based COVID-19 vaccination revealed that vaccination significantly reduced principal component-based Horvath epigenetic clock estimates in people over 50 by an average of 3.91 years for those who received Moderna. This reduction in epigenetic clock estimates was significantly related to chronological age and immune cell-type compositional changes in B cells and plasmablasts pre- and post-vaccination. These findings suggest the potential utility of epigenetic clocks as a biomarker of COVID-19 vaccine responses. Future research will need to unravel the significance and durability of short-term changes in epigenetic age related to COVID-19 exposure and mRNA vaccination.