Distinct features of the regenerating heart uncovered through comparative single-cell profiling.

Adult humans respond to heart injury by forming a permanent scar, yet other vertebrates are capable of robust and complete cardiac regeneration. Despite progress towards characterizing the mechanisms of cardiac regeneration in fish and amphibians, the large evolutionary gulf between mammals and regenerating vertebrates complicates deciphering which cellular and molecular features truly enable regeneration. To better define these features, we compared cardiac injury responses in zebrafish and medaka, two fish species that share similar heart anatomy and common teleost ancestry but differ in regenerative capability. We used single-cell transcriptional profiling to create a time-resolved comparative cell atlas of injury responses in all major cardiac cell types across both species. With this approach, we identified several key features that distinguish cardiac injury response in the non-regenerating medaka heart. By comparing immune responses to injury, we found altered cell recruitment and a distinct pro-inflammatory gene program in medaka leukocytes, and an absence of the injury-induced interferon response seen in zebrafish. In addition, we found a lack of pro-regenerative signals, including nrg1 and retinoic acid, from medaka endothelial and epicardial cells. Finally, we identified alterations in the myocardial structure in medaka, where they lack primordial layer cardiomyocytes and fail to employ a cardioprotective gene program shared by regenerating vertebrates. Our findings reveal notable variation in injury response across nearly all major cardiac cell types in zebrafish and medaka, demonstrating how evolutionary divergence influences the hidden cellular features underpinning regenerative potential in these seemingly similar vertebrates.

Distinct features of the regenerating heart uncovered through comparative single-cell profiling.

Adult humans respond to heart injury by forming a permanent scar, yet other vertebrates are capable of robust and complete cardiac regeneration. Despite progress towards characterizing the mechanisms of cardiac regeneration in fish and amphibians, the large evolutionary gulf between mammals and regenerating vertebrates complicates deciphering which cellular and molecular features truly enable regeneration. To better define these features, we compared cardiac injury responses in zebrafish and medaka, two fish species that share similar heart anatomy and common teleost ancestry but differ in regenerative capability. We used single-cell transcriptional profiling to create a time-resolved comparative cell atlas of injury responses in all major cardiac cell types across both species. With this approach, we identified several key features that distinguish cardiac injury response in the non-regenerating medaka heart. By comparing immune responses to injury, we found altered cell recruitment and a distinct pro-inflammatory gene program in medaka leukocytes, and an absence of the injury-induced interferon response seen in zebrafish. In addition, we found a lack of pro-regenerative signals, including nrg1 and retinoic acid, from medaka endothelial and epicardial cells. Finally, we identified alterations in the myocardial structure in medaka, where they lack embryonic-like primordial layer cardiomyocytes, and fail to employ a cardioprotective gene program shared by regenerating vertebrates. Our findings reveal notable variation in injury response across nearly all major cardiac cell types in zebrafish and medaka, demonstrating how evolutionary divergence influences the hidden cellular features underpinning regenerative potential in these seemingly similar vertebrates.

Adaptive duplication and genetic diversification of protein kinase R contribute to the specificity of bat-virus interactions.

Several bat species act as asymptomatic reservoirs for many viruses that are highly pathogenic in other mammals. Here, we have characterized the functional diversification of the protein kinase R (PKR), a major antiviral innate defense system. Our data indicate that PKR has evolved under positive selection and has undergone repeated genomic duplications in bats in contrast to all studied mammals that have a single copy of the gene. Functional testing of the relationship between PKR and poxvirus antagonists revealed how an evolutionary conflict with ancient pathogenic poxviruses has shaped a specific bat host-virus interface. We determined that duplicated PKRs of the Myotis species have undergone genetic diversification, allowing them to collectively escape from and enhance the control of DNA and RNA viruses. These findings suggest that viral-driven adaptations in PKR contribute to modern virus-bat interactions and may account for bat-specific immunity.

Diarrheal pathogens trigger rapid evolution of the guanylate cyclase-C signaling axis in bats.

The pathogenesis of infectious diarrheal diseases is largely attributed to enterotoxins that cause dehydration by disrupting intestinal water absorption. We investigated patterns of genetic variation in mammalian guanylate cyclase-C (GC-C), an intestinal receptor targeted by bacterially encoded heat-stable enterotoxins (STa), to determine how host species adapt in response to diarrheal infections. Our phylogenetic and functional analysis of GC-C supports long-standing evolutionary conflict with diarrheal bacteria in primates and bats, with highly variable susceptibility to STa across species. In bats, we further show that GC-C diversification has sparked compensatory mutations in the endogenous uroguanylin ligand, suggesting an unusual scenario of pathogen-driven evolution of an entire signaling axis. Together, these findings suggest that conflicts with diarrheal pathogens have had far-reaching impacts on the evolution of mammalian gut physiology.

Recurrent Loss-of-Function Mutations Reveal Costs to OAS1 Antiviral Activity in Primates.

Immune responses counteract infections but also cause collateral damage to hosts. Oligoadenylate synthetase 1 (OAS1) binds double-stranded RNA from invading viruses and produces 2'-5' linked oligoadenylate (2-5A) to activate ribonuclease L (RNase L), which cleaves RNA to inhibit virus replication. OAS1 can also undergo autoactivation by host RNAs, a potential trade-off to antiviral activity. We investigated functional variation in primate OAS1 as a model for how immune pathways evolve to mitigate costs and observed a surprising frequency of loss-of-function variation. In gorillas, we identified a polymorphism that severely decreases catalytic function, mirroring a common variant in humans that impairs 2-5A synthesis through alternative splicing. OAS1 loss-of-function variation is also common in monkeys, including complete loss of 2-5A synthesis in tamarins. The frequency of loss-of-function alleles suggests that costs associated with OAS1 activation can be so detrimental to host fitness that pathogen-protective effects are repeatedly forfeited.

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome.

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena's germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.

Recombinant rubistatin (r-Rub), an MVD disintegrin, inhibits cell migration and proliferation, and is a strong apoptotic inducer of the human melanoma cell line SK-Mel-28.

Disintegrins are low molecular weight peptides isolated from viper venom. These peptides bind to integrin receptors using a conserved binding motif sequence containing an RGD or similar motif. As a consequence, disintegrins can inhibit platelet aggregation and inhibit cell migration, proliferation, and initiate apoptosis in cancer cell lines. Rubistatin is a MVD disintegrin cloned from a Crotalus ruber ruber venom gland. The biological activity of MVD disintegrins is poorly understood. Recombinant rubistatin (r-Rub) was cloned into a pET32b plasmid and expressed in reductase-deficient Escherichia coli. Expression was induced with IPTG and the resulting fusion peptide was affinity purified, followed by thrombin cleavage, and removal of vector coded sequences. r-Rub peptide inhibited ADP-induced platelet aggregation by 54% ± 6.38 in whole blood. We assessed the ability of r-Rub to initiate apoptosis in three human cancer cell lines. Cultures of SK-Mel-28, HeLA, and T24 cells were grown for 24 h with 2.5 µM r-Rub followed by Hoechst staining. Chromatin fragmentation was observed in treated SK-Mel-28, but not in T24 or HeLA cells. A TUNEL assay revealed that 51.55% ± 5.28 of SK-Mel-28 cells were apoptotic after 18 h of treatment with 3.5 µM of r-Rub. Cell migration and proliferation assays were performed in order to further characterize the biological effects of r-Rub on SK-Mel-28 cells. At 3 µM, r-Rub inhibited cell migration by 44.4% ± 0.5, while at 3.5 µM it was able to inhibit cell proliferation by 83% ± 6.0.