Abstract Zebrafish can regenerate the heart by proliferation of cardiomyocytes. While the innate immune response and wound re-vascularization are pre-requisites for cardiomyocyte regeneration, little is known about signals linking early injury responses with the initiations of regenerative programs in cardiomyocytes. Here we show that mTOR (mechanistic target of rapamycin) signaling is rapidly activated in response to heart injury in many cell types of the heart including endothelial cells, cardiomyocytes and macrophages, but surprisingly not in neutrophils. We find that mTORC2 regulates macrophage recruitment to the wound, while mTORC1 is required for wound debris clearance by macrophages. In addition, mTOR signaling is required for wound re-vascularization. Interestingly, it also appears to directly regulate cardiomyocyte dedifferentiation and proliferation, making mTOR signaling a central hub for regenerative responses. Anabolic mTOR signaling acts as potent inhibitor of catabolic autophagy in many systems. Yet, we observed upregulation of autophagy within border zone cardiomyocytes where mTOR signaling is active. We show that mTOR signaling limits, but does not block autophagy, and that autophagic flux is regulated by both inhibitory mTOR signaling and stimulatory JNK and MEK pathways. Our results indicate that a fine-balanced anabolic and catabolic injury response is essential for zebrafish heart regeneration. Furthermore, they reveal interesting differences in the regulation of mTOR signaling and autophagy between the regenerative zebrafish heart and non-regenerative mammalian hearts.

Abstract Zebrafish bone regeneration is a highly efficient process, enabling the complete restoration of an amputated fin within few weeks. The hallmark of this epimorphic regeneration is the formation of a blastema atop of a bony fin ray. Osteoblasts near the injury site dedifferentiate and migrate off the bone to contribute to the developing blastema. We show that an injury or a blastema alone is not sufficient to trigger off-bone migration of osteoblasts. Surprisingly, we found that blastema cells themselves possess intrinsic migratory properties. Moreover, when multiple injury sites are present, a preferential distal migration could be observed. We conclude that multiple injuries are hierarchical organized, and that injuries with the highest regenerative potential take priority.

BACKGROUND:Cell migration is a fundamental biological process essential for embryonic development and hematopoiesis. In a shRNA screen, we identified the TRAM-LAG1-CLN8 domain-containing transmembrane protein TMEM56 as a previously uncharacterized regulator of stromal cell-derived factor 1 (SDF-1)-mediated cell migration. This study investigates the molecular mechanisms underlying TMEM56 function. RESULTS:TMEM56 is expressed in both murine embryonic and adult tissues, with enrichment in hematopoietic stem and erythroid progenitor cells. Lipidomic analysis reveals that TMEM56 modulates ceramide metabolism, particularly affecting levels of hexosylated ceramides. Co-immunoprecipitation assays indicate that TMEM56 physically interacts with ceramide synthase 2 (CerS2), suggesting a role in lipid signaling pathways. CONCLUSION:Our findings identify TMEM56 as a key regulator of cell migration, linking lipid metabolism with hematopoietic and developmental processes. These results provide novel insights into the molecular mechanisms governing migration.

ABSTRACT Simian immunodeficiency viruses (SIVs) have crossed from apes to humans at least four times, but only one event gave rise to the AIDS pandemic. The host barriers that pandemic HIV-1 group M (major) strains overcame to spread efficiently in humans remain poorly understood. To identify such barriers, we performed CRISPR-Cas9 screens driven by the replication efficiency of SIVcpz, the chimpanzee precursor of HIV-1. Guide RNA libraries targeting more than 500 human genes encoding potential antiviral factors were inserted into the replication-competent SIVcpz MB897 molecular clone, which is phylogenetically closely related to HIV-1 group M strains. Propagation in Cas9-expressing human SupT1 T cells significantly enriched for sgRNAs targeting AXIN1, CEACAM3, CD72, EHMT2, GRN, HMOX1, HMGA1, ICAM2, IFITM2, MEFV, PCED1B, SGOL2, SMARCA4, SUMO1, and TMEM173. These hits only partially overlapped with those identified in analogous HIV-1-based screens, indicating virus-specific restriction profiles. Functional analyses confirmed that IFITM2 (interferon-induced transmembrane protein 2), PCED1B (PC-esterase domain-containing protein 1B), MEFV (Mediterranean fever protein, pyrin/TRIM20), and AXIN1 (Axis inhibition protein 1) restrict replication of the analyzed SIVcpz strains but not HIV-1 group M strains in primary human CD4+ T cells. These findings reveal previously unrecognized host factors that limit SIVcpz replication in human cells and highlight barriers that at least some HIV-1 group M strains overcame during adaptation for pandemic spread.IMPORTANCEFour independent transmission events of simian immunodeficiency viruses from chimpanzees and gorillas to humans gave rise to human immunodeficiency virus type 1, but only one led to the global AIDS pandemic. Understanding which adaptations allowed the pandemic HIV-1 M strains to spread efficiently in humans remains a key question in virus evolution and public health. In this study, we engineered replication-competent SIVcpz constructs carrying more than 1,500 single-guide RNAs to identify antiviral genes in Cas9-expressing cells. This approach revealed several cellular factors that restrict SIVcpz but not the pandemic HIV-1 M strains analyzed in primary human T cells. These findings provide new insights into antiviral defense mechanisms and the adaptations that most likely contributed to the efficient spread of HIV-1.

Abstract Angicin is a class IId bacteriocin produced by Streptococcus anginosus with activity against Gram-positive pathogens, including Listeria monocytogenes and vancomycin-resistant Enterococcus faecium . While the mannose phosphotransferase system (Man-PTS) has been identified as a receptor in L. monocytogenes , its role in streptococci and the structural determinants of Angicin activity remain unclear. Here, we demonstrate that the Man-PTS is required for Angicin susceptibility in Streptococcus constellatus . A transposon mutant ( manM ::ISS1) showed complete resistance to Angicin and impaired mannose utilization. Structure–activity relationship analysis of truncated and modified peptides localized antimicrobial activity to the C-terminal region, although none of the variants matched the activity of the full-length peptide. Angicin induced membrane depolarization and pore formation in target bacteria. Residual activity in Man-PTS-impaired L. monocytogenes suggests an additional receptor-independent effect at higher concentrations. In vivo toxicity analysis using zebrafish embryos showed low toxicity at active concentrations. These findings identify the Man-PTS as a receptor for Angicin in streptococci and define structural features associated with its antimicrobial activity.

ABSTRACT Late endosome-dependent viruses, including filo- and arenaviruses, rely on host endolysosomal trafficking for productive infection. Here, we used a dual-colour Vesicular stomatitis virus (VSV) based pseudoparticle screen of CytoSorb-derived fractions to identify inhibitors of the Zaire Ebolavirus glycoprotein (GP)-mediated entry. Iterative chromatographic purification and mass spectrometry identified Laudanosine, a degradation product of the clinically used neuromuscular blocker Atracurium, as the antiviral compound. Laudanosine specifically inhibited entry mediated by Ebola, Marburg, Lymphocytic choriomeningitis and Lassa virus glycoproteins without affecting VSV-G-dependent entry. Importantly, Laudanosine inhibited authentic Ebola virus infection without detectable cytotoxicity in cell culture and embryonic zebrafish. Molecular dynamics simulations suggest stable association of Laudanosine with the allosteric inhibitory pocket of the lysosomal two-pore channel (TPC2). Consistently, Laudanosine impairs autophagic flux and disrupts endolysosomal trafficking. Together, our findings identify Laudanosine as a previously unrecognised inhibitor of TPC2-dependent entry of highly lethal viral pathogens.

Dedifferentiation of mature cells is an essential mechanism of source cell formation for regeneration of many systems, including the zebrafish fin. Here, we use bulk and single cell RNASeq to show that osteoblast injury responses involve rapid and extensive transcriptional reprogramming, yielding a cell state that shares characteristics with embryonic osteoblasts, but also expresses many regeneration-specific genes. One gene characterizing this state is the canonical Wnt ligand wnt10a. Using genetic and transgenic perturbations, we demonstrate that wnt10a-dependent Wnt/β-catenin signaling cell-autonomously induces osteoblast dedifferentiation. Loss of wnt10a or of Wnt/β-catenin activity blocks the dedifferentiation program, whereas wnt10a overexpression enhances dedifferentiation and is sufficient to induce it even without injury. Wnt/β-catenin signaling promotes dedifferentiation by suppressing NF-κB activity, placing it upstream of known cues whose loss causes dedifferentiation. Notably, wnt10a overexpression also stimulates cardiomyocyte dedifferentiation during zebrafish heart regeneration, revealing a conserved role in activating source-cell formation during regeneration. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, 257897648, 68236468, 450627322, 251293561, 316249678, 51420450

Metastatic breast carcinoma (BC) cells are prone to spreading in the bone microenvironment, leading to a vicious cycle between local osteoclast-mediated osteolysis and tumor progression. Therefore, the targeted pharmacological down-modulation of BC cell proliferation as well as osteoclast differentiation and hyperactivity might represent a promising treatment option. We developed a multifunctional peptide nanocarrier combining bioactive EPI-X4 peptides and the Rho-inhibiting C3bot enzyme from Clostridium botulinum. C3bot is preferentially internalized into the cytosol of monocytic cells, including osteoclasts, where it inhibits Rho-mediated signal transduction. However, Rho-mediated cellular processes like migration and cell division can also be inhibited in non-monocytic cells if C3bot is delivered into their cytosol by a nanocarrier. To accomplish this, we designed a supramolecular transporter where one molecule of biotinylated C3bot and three biotinylated entities of the human EPI-X4 peptide-derived CXCR4 antagonist JM173 are assembled on avidin as a central platform. This modular transport system (JM173)3-Avi-C3 down-modulated osteoclast formation and hyperactivity and delivered the therapeutic cargo C3bot successfully into the cytosol of breast cancer cells, where it inhibited Rho.

Pertussis, also known as whooping cough, is a highly infectious respiratory disease caused by the bacterium Bordetella pertussis. The bacterial virulence factor, pertussis toxin (PT), is associated with the manifestation of the characteristic symptoms of pertussis and the severe form of this disease. Increasing case numbers and the lack of treatment options highlight the need to develop novel pharmacological strategies, e.g., the generation of specific PT inhibitors. Recently, we identified the endogenous human protein α1-antitrypsin (α1AT) as an inhibitor of PT from a screening of a human hemofiltrate protein/peptide library. In the present work, we tested an in-house α1AT peptide bank to identify an α1AT region with anti-PT activity. Then, we compared the sequences of the positive hits from the peptide bank with all known α1AT fragments in the hemofiltrate samples to find new active peptides. In total, 36 peptides were tested for their PT inhibition, leading to the identification of an endogenous α1AT fragment, α1AT HF, derived from hemofiltrate with anti-PT activity. This peptide had no toxic effects on HeLa cells and in vivo on zebrafish embryos, rendering it an attractive lead compound for further evaluation to treat pertussis in the future.

The ability of heart tissue to repair itself after injury has fascinated scientists for decades[1][1],[2][2]. Researchers have long studied the internal body clock, or circadian rhythm, for its role in coordinating daily cycles of metabolism and cell activity[3][3],[4][4], but its relevance to heart repair has remained unknown. This study explores, for the first time, whether natural daily rhythms influence heart regeneration—a process driven by cardiomyocyte proliferation. We discovered that DNA replication, mitosis, oxidative phosphorylation, and glycolysis follow a precise daily order in regenerating zebrafish hearts. Disrupting core clock gene expression abolishes the rhythms of glycolysis and mitosis, preventing cardiomyocyte cell cycle progression and regeneration. Insulin-resistant Astyanax mexicanus cavefish, which have adapted to dark caves, similarly show a loss of mitosis rhythm and cardiomyocyte cell cycle progression, which we find is caused by reduced glycolysis. Despite this reduction, glycolysis rhythm displays a larger amplitude in cavefish—a pattern recapitulated in insulin-resistant zebrafish. Insulin resistance resets metabolic rhythms to the morning, which is equally detrimental to regeneration. Here, we show that successful cardiac regeneration depends on synchronised clock and glucose rhythms, which together orchestrate the cell cycle events essential for cardiomyocyte proliferation and tissue repair. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4

In contrast to mammals, adult zebrafish achieve complete heart regeneration via proliferation of cardiomyocytes. Surprisingly, we found that regenerating cardiomyocytes experience DNA replication stress, which represents one reason for declining tissue regeneration during aging in mammals. Pharmacological inhibition of ATM and ATR kinases revealed that DNA damage response signaling is essential for zebrafish heart regeneration. Manipulation of Bone Morphogenetic Protein (BMP)-Smad signaling using transgenics and mutants showed that BMP signaling alleviates cardiomyocyte replication stress. BMP signaling also rescues neonatal mouse cardiomyocytes, human fibroblasts and human hematopoietic stem and progenitor cells (HSPCs) from replication stress. DNA fiber spreading assays indicate that BMP signaling facilitates re-start of replication forks after replication stress-induced stalling. Our results identify the ability to overcome replication stress as key factor for the elevated zebrafish heart regeneration capacity and reveal a conserved role for BMP signaling in promotion of stress-free DNA replication.

Peptides hold great promise for safe and effective treatment of viral infections. However, their use is often constrained by limited efficacy and high production costs, especially for long or complex peptide chains. Here, we used ReaxFF molecular dynamics (MD) simulations to optimize the size and activity of VIRIP (Virus Inhibitory Peptide), a naturally occurring 20-residue fragment of α1-antitrypsin that binds the HIV-1 GP41 fusion peptide (FP), thereby blocking viral fusion and entry into host cells. Specifically, we used the NMR structure of the complex between an optimized VIRIP derivative (VIR-165) and the HIV-1 gp41 FP for ReaxFF-guided in silico analysis, evaluating the contribution of each amino acid in the interaction of the inhibitor with its viral target. This approach allowed us to reduce the size of the HIV-1 FP inhibitor from 20 to 10 amino acids (2.28-1.11 kDa). HIV-1 infection assays showed that the size-optimized VIRIP derivative (soVIRIP) retains its broad-spectrum anti-HIV-1 capability and is nontoxic in the vertebrate zebrafish model. Compared to the original VIRIP, soVIRIP displayed more than 100-fold higher antiviral activity (IC50 of ∼120 nM). Thus, it is more potent than a dimeric 20-residue VIRIP derivative (VIR-576) that was proven safe and effective in a phase I/II clinical trial. Our results show that ReaxFF-based MD simulations represent a suitable approach for the optimization of therapeutic peptides.

Zebrafish have a lifelong cardiac regenerative ability after damage, whereas mammals lose this capacity during early postnatal development. This study investigated whether the declining expression of growth factors during postnatal mammalian development contributes to the decrease of cardiomyocyte regenerative potential. Besides confirming the proliferative ability of neuregulin 1 (NRG1), interleukin (IL)1b, receptor activator of nuclear factor kappa-Β ligand (RANKL), insulin growth factor (IGF)2, and IL6, we identified other potential pro-regenerative factors, with BMP7 exhibiting the most pronounced efficacy. Bmp7 knockdown in neonatal mouse cardiomyocytes and loss-of-function in adult zebrafish during cardiac regeneration reduced cardiomyocyte proliferation, indicating that Bmp7 is crucial in the regenerative stages of mouse and zebrafish hearts. Conversely, bmp7 overexpression in regenerating zebrafish or administration at post-mitotic juvenile and adult mouse stages, in vitro and in vivo following myocardial infarction, enhanced cardiomyocyte cycling. Mechanistically, BMP7 stimulated proliferation through BMPR1A/ACVR1 and ACVR2A/BMPR2 receptors and downstream SMAD5, ERK, and AKT signaling. Overall, BMP7 administration is a promising strategy for heart regeneration.

The resurfacing of cutaneous wounds in mammals takes up to several weeks, but in zebrafish complete coverage is achieved within hours. New work uncovers that the rapid wound healing on zebrafish body surfaces involves the mobilization of fin-resident epithelial cells.

One hallmark of aging is a decline in tissue regeneration, which can be caused by DNA replication stress. Whether highly regenerative species like zebrafish are immune from such hindrances to replication is unknown. In contrast to most mammals, adult zebrafish achieve complete heart regeneration via cell cycle entry and proliferation of mature cardiomyocytes. We found that cycling cardiomyocytes experience replication stress, which is induced by the demands of regeneration, but does not occur during physiological heart growth. Since zebrafish cardiomyocyte regeneration is remarkably efficient, heart regeneration appears to depend on elevated capabilities to overcome replication stress. Indeed, pharmacological inhibition of ATM and ATR kinases revealed that DNA damage response signaling is essential for heart regeneration. Using inducible overexpression of ligands and inhibitors of the Bone Morphogenetic Protein (BMP)-Smad pathway, combined with analysis of genetic mutants, we found that BMP signaling alleviates cardiomyocyte replication stress. In the absence of BMP signaling, cardiomyocytes become arrested in the S-phase of the cell cycle, which prevents progression to mitosis and results in heart regeneration failure. Interestingly, BMP signaling can also rescue neonatal mouse cardiomyocytes and human fibroblasts from hydroxyurea-induced replication stress. DNA fiber spreading assays in human cancer cells and human hematopoietic stem and progenitor cells (HSPCs) indicate that BMP signaling acts directly on replication dynamics by accelerating DNA replication fork progression and by facilitating their re-start after replication stress-induced stalling. Our results identify the ability to overcome replication stress as key factor for the elevated heart regeneration capacity in zebrafish. Notably, the conserved capability of BMP signaling to promote stress-free DNA replication might unlock new avenues towards anti-aging and pro-regenerative applications in humans.### Competing Interest StatementThe authors have declared no competing interest.

Zebrafish has a remarkable and lifelong ability for cardiac regeneration after severe damage, whereas mammals lose their innate capacity for heart regeneration during early postnatal development. This study aimed to investigate whether the decreased production of growth factors during postnatal mammalian development contributes to the exit of cardiomyocytes from the cell cycle and the reduction in cardiac regenerative ability. We identified growth factors with declining expression levels during early postnatal life in the mouse model and assessed the pro-proliferative ability of these factors on neonatal murine primary cardiomyocytes in vitro . Our findings confirmed the previously reported pro-proliferative effects of NRG1, IL1b, RANKL, IGF2 and IL6, while also identifying novel potential pro-regenerative growth factors. Among them, BMP7 exhibited the most pronounced efficacy. Bmp7 knockdown interfered with the proliferation of neonatal mouse cardiomyocytes in culture and adult bmp7 mutant zebrafish displayed reduced cardiomyocyte proliferation during heart regeneration, indicating that Bmp7 is crucial for cardiomyocyte proliferation in the regenerative stages of mouse and zebrafish hearts. Conversely, bmp7 overexpression was sufficient to boost cardiomyocyte cycling in regenerating zebrafish hearts, while BMP7 administration stimulated mouse cardiomyocyte cycling at postnatal-day-7, when cardiomyocytes ceased to proliferate, and enhanced cardiomyocyte regeneration in vivo in adult mice following myocardial infarction. Mechanistically, BMP7-induced proliferation was mediated by type I BMP receptors BMPR1A and ACVR1, and type II receptors ACVR2A and BMPR2. Downstream signalling involved SMAD5, ERK and AKT. In conclusion, the administration of BMP7 holds promise as a strategy to stimulate heart regeneration following cardiac injury.

EPI-X4, a natural peptide CXCR4 antagonist, shows potential for treating inflammation and cancer, but its short plasma stability limits its clinical application. We aimed to improve the plasma stability of EPI-X4 analogues without compromising CXCR4 antagonism. Our findings revealed that only the peptide N-terminus is prone to degradation. Consequently, incorporating d-amino acids or acetyl groups in this region enhanced peptide stability in plasma. Notably, EPI-X4 leads 5, 27, and 28 not only retained their CXCR4 binding and antagonism but also remained stable in plasma for over 8 h. Molecular dynamic simulations showed that these modified analogues bind similarly to CXCR4 as the original peptide. To further increase their systemic half-lives, we conjugated these stabilized analogues with large polymers and albumin binders. These advances highlight the potential of the optimized EPI-X4 analogues as promising CXCR4-targeted therapeutics and set the stage for more detailed preclinical assessments.

Rising incidences and mortalities have drawn attention to Clostridioides difficile infections (CDIs) in recent years. The main virulence factors of this bacterium are the exotoxins TcdA and TcdB, which glucosylate Rho-GTPases and thereby inhibit Rho/actin-mediated processes in cells. This results in cell rounding, gut barrier disruption and characteristic clinical symptoms. So far, treatment of CDIs is limited and mainly restricted to some antibiotics, often leading to a vicious circle of antibiotic-induced disease recurrence. Here, we demonstrate the protective effect of the human antimicrobial peptide α-defensin-6 against TcdA, TcdB and the combination of both toxins in vitro and in vivo and unravel the underlying molecular mechanism. The defensin prevented toxin-mediated glucosylation of Rho-GTPases in cells and protected human cells, model epithelial barriers as well as zebrafish embryos from toxic effects. In vitro analyses revealed direct binding to TcdB in an SPR approach and the rapid formation of TcdB/α-defensin-6 complexes, as analyzed with fluorescent TcdB by time-lapse microscopy. In conclusion, the results imply that α-defensin-6 rapidly sequesters the toxin into complexes, which prevents its cytotoxic activity. These findings extend the understanding of how human peptides neutralize bacterial protein toxins and might be a starting point for the development of novel therapeutic options against CDIs.

Successful regeneration requires the coordinated execution of multiple cellular responses to injury. In amputated zebrafish fins, mature osteoblasts dedifferentiate, migrate towards the injury, and form proliferative osteogenic blastema cells. We show that osteoblast migration is preceded by cell elongation and alignment along the proximodistal axis, which require actomyosin, but not microtubule (MT) turnover. Surprisingly, osteoblast dedifferentiation and migration can be uncoupled. Using pharmacological and genetic interventions, we found that NF-ĸB and retinoic acid signalling regulate dedifferentiation without affecting migration, while the complement system and actomyosin dynamics affect migration but not dedifferentiation. Furthermore, by removing bone at two locations within a fin ray, we established an injury model containing two injury sites. We found that osteoblasts dedifferentiate at and migrate towards both sites, while accumulation of osteogenic progenitor cells and regenerative bone formation only occur at the distal-facing injury. Together, these data indicate that osteoblast dedifferentiation and migration represent generic injury responses that are differentially regulated and can occur independently of each other and of regenerative growth. We conclude that successful fin bone regeneration appears to involve the coordinated execution of generic and regeneration-specific responses of osteoblasts to injury.

Successful regeneration requires the coordinated execution of multiple cellular responses to injury. In amputated zebrafish fins, mature osteoblasts dedifferentiate, migrate towards the injury and form proliferative osteogenic blastema cells. We show that osteoblast migration is preceded by cell elongation and alignment along the proximodistal axis, which require actomyosin, but not microtubule turnover. Surprisingly, osteoblast dedifferentiation and migration can be uncoupled. Using pharmacological and genetic interventions, we found that NF-κB and retinoic acid signalling regulate dedifferentiation without affecting migration, while the complement system and actomyosin dynamics are required for migration but not dedifferentiation. Furthermore, by removing bone at two locations within a fin ray, we established a trauma model containing two injury sites. We found that osteoblasts dedifferentiate at and migrate towards both sites, while accumulation of osteogenic progenitor cells and regenerative bone formation only occur at the distal-facing injury. Together, these data indicate that osteoblast dedifferentiation and migration represent generic injury responses that are differentially regulated and can occur independently of each other and of regenerative growth. Successful bone regeneration appears to require the coordinated execution of generic and regeneration-specific responses of osteoblast to trauma. ![Figure][1]</img> ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes