Exceptionally long-lived species, including many bats, rarely show overt signs of aging, making it difficult to determine why species differ in lifespan. Here, we use DNA methylation (DNAm) profiles from 712 known-age bats, representing 26 species, to identify epigenetic changes associated with age and longevity. We demonstrate that DNAm accurately predicts chronological age. Across species, longevity is negatively associated with the rate of DNAm change at age-associated sites. Furthermore, analysis of several bat genomes reveals that hypermethylated age- and longevity-associated sites are disproportionately located in promoter regions of key transcription factors (TF) and enriched for histone and chromatin features associated with transcriptional regulation. Predicted TF binding site motifs and enrichment analyses indicate that age-related methylation change is influenced by developmental processes, while longevity-related DNAm change is associated with innate immunity or tumorigenesis genes, suggesting that bat longevity results from augmented immune response and cancer suppression.

Interactions of leptin and leptin receptors play crucial roles during animal development and regulation of appetite and energy balance. In this study we analyzed expression pattern of a zebrafish leptin receptor gene in both developing and adult zebrafish using in situ hybridization and Q-PCR methods. Zebrafish leptin receptor message (lepr) was detected in all embryonic and larval stages examined, and in adult zebrafish. In embryonic zebrafish, lepr was mainly expressed in the notochord. As development proceeded, lepr expression in the notochord decreased, while its expression in several other tissues, including the trunk muscles and gut, became evident. In both larval and adult brains, large lepr expressing cells were detected in similar regions of the hindbrain. In adult zebrafish, lepr expression was also observed in several other brain regions including the hypothalamic lateral tuberal nucleus, the fish homolog of the arcuate nucleus. Q-PCR experiments confirmed lepr expression in the adult fish brain, and also showed lepr expression in several adult tissues including liver, muscle and gonads. Our results showed that lepr expression was both spatially and temporally regulated.

Objectives: Recent studies have shown that tRNA-derived RNA fragments (tRFs) are novel regulators of post-transcriptional gene expression. However, the expression profiles and their role in post-transcriptional gene regulation in chondrocytes is unknown. Here, we determined tRFs expression profile and explored tRF-3003a role in post-transcriptional gene regulation in IL-1b stimulated chondrocytes. Methods: We used qPCR arrays to determine tRNAs and tRFs expression in age-and sex-matched primary human OA chondrocytes and TC28/I2 cells stimulated with IL-1b. Chondrocytes were transfected with tRNA-Cys GCA overexpression plasmid or tRF-3003a mimic and 3 0 UTR luciferase reporter plasmids of mRNAs harboring predicted tRF target "seed sequence". The AGO-RNA-induced silencing complex (AGO-RISC)-dependent repressive activity of tRF-3003a was determined by siRNA-mediated knockdown of AGO2. Results: IL-1b increased the expression levels of specific tRNAs and of tRF-3003a, a type 3 tRF produced by the cleavage of tRNA-Cys GCA . tRF-3003a "seed sequence" was identified in the 3 0 UTR of JAK3 mRNA and tRNA-Cys GCA overexpression or transfection of a tRF-3003a mimic in chondrocytes downregulated JAK3 expression and significantly reduced the activity of the 3 0 UTR reporter. RIP assay showed enrichment of tRF-3003a into AGO2/RISC in IL-1b treated chondrocytes. The suppressive effect of tRF-3003a on JAK3 3 0 UTR reporter was abrogated with siRNA-mediated depletion of AGO2. Conclusions: We demonstrate that under pathological conditions chondrocytes display perturbations in the expression profile of specific tRNAs and tRFs. Furthermore, a specific tRF namely tRF-3003a can posttranscriptionally regulate JAK3 expression via AGO/RISC formation in chondrocytes. Identification of this novel mechanism may be of value in the design of precision therapies for OA.

Leptin is a pleiotropic protein best known for regulation of appetite and fat storage in mammals. While many leptin orthologs have been identified among vertebrates, an authentic leptin in birds has remained elusive and controversial. Here we identify leptin sequence from the Peregrine falcon, Falco peregrinus (pfleptin), and identify sequences from two other birds (mallard and zebra finch), and ‘missing’ vertebrates (elephant shark, alligator, Indian python, Chinese soft-shelled turtle, and coelacanth). The pattern of genes surrounding leptin (snd1, rbm28) is syntenic between the falcon and mammalian genomes. Phylogenetic analysis of all known leptin protein sequences improves our understanding of leptin’s evolution. Structural modeling of leptin orthologs highlights a highly conserved hydrophobic core in the four-helix cytokine packing domain. A docked model of leptin with the leptin receptor for Peregrine falcon reveals several conserved amino acids important for the interaction and possible coevolution of leptin with its receptor. We also show for the first time, an authentic avian leptin sequence that activates the JAK-STAT signaling pathway. These newly identified sequences, structures, and tools for avian leptin and its receptor will allow elucidation of the function of these proteins in feral and domestic birds.

The processes of lipid deposition and utilization, via the gene leptin (Lep), are poorly understood in taxa with varying degrees of adipose storage. This study examines how these systems may have adapted in marine aquatic environments inhabited by cetaceans. Bowhead (Balaena mysticetus) and beluga whales (Delphinapterus leucas) are ideal study animals- they possess large subcutaneous adipose stores (blubber) and undergo bi-annual migrations concurrent with variations in food availability. To answer long-standing questions regarding how (or if) energy and lipid utilization adapted to aquatic stressors, we quantified variations in gene transcripts critical to lipid metabolism related to season, age and blubber depth. We predicted Leptin tertiary structure conservation and assessed inter-specific variations in Lep transcript numbers between bowheads and other mammals. Our study is the first to identify seasonal and age-related variations in Lep and lipolysis in these cetaceans. While Lep transcripts and protein oscillate with season in adult bowheads reminiscent of hibernating mammals, transcript levels reach up to 10-times higher in bowheads than any other mammal. Data from immature bowheads are consistent with the hypothesis that short baleen inhibits efficient feeding. Lipolysis transcripts also indicate young Fall bowheads and those sampled during Spring months limit energy utilization. These novel data from rarely examined species expand existing knowledge and offer unique insight into how the regulation of Lep and lipolysis has adapted to permit seasonal deposition and maintain vital blubber stores.

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Cetacean evolution was shaped by an extraordinary land-to-sea transition in which the ancestors of whales became fully aquatic. As part of this transition, these mammals evolved unusually thick blubber which acts as a metabolic reservoir as well as an insulator and provides buoyancy and streamlining. This study describes blubber stratification and correlates it to seasonal variation, feeding patterns, and ontogeny in an arctic-adapted mysticete, the bowhead whale (Balaena mysticetus). Bowheads are unique among mammals for possessing the largest known blubber stores. We found that adipocyte numbers in bowheads, like other mammals, do not vary with season or feeding pattern but that adipocyte size and structural fiber densities do vary with blubber depth.

The value of molecular databases for unicellular eukaryotic identification and phylogenetic reconstruction is predicated on the availability of sequences and accuracy of taxonomic identifications that accompany those sequences. Biased representation of sequences is due in part to the differing ability to isolate and culture various groups of protists. Techniques that allow for parallel single-cell morphological and molecular identifications have been reported for a few groups of unicellular protists. We have sought to explore how those techniques can be adapted to work across a greater phylogenetic diversity of taxa. Twelve morphologically diverse and abundant members of limnetic microplankton, including ciliates, dinoflagellates, cryptophytes, stramenopiles, and synurophytes, were targeted for analysis. These cells were captured directly from environmental samples, identified, and prepared for sequence analyses using variations of single-cell extraction techniques depending on their size, mobility, and the absence or presence of the cell wall. The application of these techniques yielded a strong congruence between the morphological and molecular identifications of the targeted taxa. Challenges to the single-cell approach in some groups are discussed. The general ability to obtain DNA sequences and morphological descriptions from individual cells should open new avenues to studying either rare or difficult to culture taxa, even directly at the point of collection (e.g. remote locations or shipboard).