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Background/Objectives : Tuberculosis (TB) is one of the leading infectious causes of death worldwide, highlighting the importance of identifying new anti-TB agents. In previous research, our team identified antimycobacterial activity in Kielmeyera membranacea leaf extract; therefore, this study aims to conduct further exploration of its potential. Methods : Classical chromatography was applied for fractionation and spectrometric techniques were utilized for chemical characterization. For in vitro tests, samples were assessed against Mycobacterium tuberculosis and Mycobacterium marinum . The toxicity and efficacy of active samples were evaluated in vivo using different zebrafish models. Chemogenomics studies were applied to predict the isolated active compound's potential mode of action. Results : We performed fractionation of K. membranacea ethanolic extract (EE) and then its dichloromethane fraction (DCM), and the biflavonoid podocarpusflavone A (PCFA) was isolated and identified as a promising active compound. The EE and PCFA were found to be non-toxic to zebrafish larvae and were able to inhibit M. tuberculosis growth extracellularly. Additionally, PCFA demonstrated antimycobacterial activity within infected macrophages, especially when combined with isoniazid. In addition, the EE, DCM, and PCFA have shown the ability to inhibit M. marinum's growth during in vivo zebrafish larvae yolk infection. Notably, PCFA also effectively countered systemic infection established through the caudal vein, showing a similar inhibitory activity profile to rifampicin, both at 32 µM. A reduction in the transcriptional levels of pro-inflammatory cytokines confirmed the infection resolution. The protein tyrosine phosphatase B (PtpB) of M . tuberculosis , which inhibits the macrophage immune response, was predicted as a theoretical target of PCFA. This finding is in agreement with the higher activity observed for PCFA intracellularly and in vivo on zebrafish, compared with the direct action in M. tuberculosis . Conclusions : Here, we describe the discovery of PCFA as an intracellular inhibitor of M. tuberculosis and provide evidence of its in vivo efficacy and safety, encouraging its further development as a combination drug in novel therapeutic regimens for TB.

Dendritic hydrogels based on carbosilane crosslinkers are promising drug delivery systems, as their amphiphilic nature improves the compatibility with poorly water-soluble drugs. In this work, we explored the impact of the complementary polymer on the amphiphilic properties of the dendritic network. Different polymers were selected as precursors, from the highly lipophilic propylene glycol (PPG) to the hydrophilic polyethylene glycol (PEG), including amphiphilic Pluronics L31, L35 and L61. The dithiol polymers reacted with carbosilane crosslinkers through UV-initiated thiol-ene coupling (TEC), and the resultant materials were classified as non-swelling networks (for PPG, PLU L31 and PLU L61 ) and high-swelling hydrogels (for PEG and PLU L35 ). The hydrogels exhibited thermo-responsive properties, shrinking at higher temperatures, and exhibited an intriguing drug release pattern due to internal nanostructuring. Furthermore, we fine-tuned the dendritic crosslinker, including hydroxyl and azide pendant groups in the focal point, generating functional networks that can be modified through degradable (ester) and non-degradable (triazol) bonds. Overall, this work highlighted the crucial role of the amphiphilic balance in the design of dendritic hydrogels with thermo-responsive behavior and confirmed their potential as functional networks for biomedical applications.

Ancient bony fishes had heterocercal tails, like modern sharks and sturgeons, with asymmetric caudal fins and a vertebral column extending into an elongated upper lobe. Teleost fishes, in contrast, developed a homocercal tail characterized by two separate equal-sized fin lobes and the body axis not extending into the caudal fin. A similar heterocercal-to-homocercal transition occurs during teleost ontogeny, although the underlying genetic and developmental mechanisms for either transition remain unresolved. Here, we investigated the role of hox13 genes in caudal fin formation as these genes control posterior identity in animals. Analysis of expression profiles of zebrafish hox13 paralogs and phenotypes of CRISPR/Cas9-induced mutants showed that double hoxb13a and hoxc13a mutants fail to form a caudal fin. Furthermore, single mutants display heterocercal-like morphologies not seen since Mesozoic fossil teleosteomorphs. Relaxation of functional constraints after the teleost genome duplication may have allowed hox13 duplicates to neo- or subfunctionalize, ultimately contributing to the evolution of a homocercal tail in teleost fishes.

The emergence of antibiotic resistance is a serious global health problem. There is an incessant demand for new antimicrobial drugs and materials that can address this global issue from different angles. Dendritic hydrogels have appeared as a promising strategy. A family of bifunctional amphiphilic carbosilane dendrimers was designed and employed as nanosized cross-linking points for the synthesis of high-swelling hydrogels using the highly efficient Thiol-Ene click reaction for their preparation. Both stoichiometric and off-stoichiometric conditions were studied, generating hydrogels with pendant hydroxyl or alkene moieties. These hydrogels were found to be tunable antibacterial materials. They can easily be postmodified with relevant antibiotic moieties through covalent attachment on the hydroxyl or alkene pendant groups, generating ammonium-decorated networks with temperature and pH-responsive properties. Additionally, they can efficiently encapsulate drugs with poor solubility in water, like ciprofloxacin, and perform a sustained release over time, as demonstrated in preliminary assays against Staphylococcus aureus ., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Existing drug treatment against tuberculosis is no match against the increasing number of multi-drug resistant strains of its causative agent, Mycobacterium tuberculosis ( Mtb ). A better understanding of how mycobacteria subvert the host immune defenses is crucial for developing novel therapeutic strategies. A potential approach is enhancing the activity of the autophagy machinery, which can direct bacteria to autophagolysosomal degradation. However, the interplay specifics between mycobacteria and the autophagy machinery must be better understood. Here, we analyzed live imaging data from the zebrafish model of tuberculosis to characterize mycobacteria-autophagy interactions during the early stages of infection in vivo. For high-resolution imaging, we microinjected fluorescent Mycobacterium marinum ( Mm ) into the tail fin tissue of zebrafish larvae carrying the GFP-LC3 autophagy reporter. We detected phagocytosed Mm clusters and LC3-positive Mm -containing vesicles within the first hour of infection. LC3 associations with these vesicles were transient and heterogeneous, ranging from simple vesicles to complex compound structures, dynamically changing shape by fusions between Mm -containing and empty vesicles. LC3- Mm -vesicles could adopt elongated shapes during cell migration or alternate between spacious and compact morphologies. LC3- Mm -vesicles were also observed in cells reverse migrating from the infection site, indicating that the autophagy machinery fails to control infection before tissue dissemination.

Modeling human infectious diseases using the early life stages of zebrafish provides unprecedented opportunities for visualizing and studying the interaction between pathogens and phagocytic cells of the innate immune system. Intracellular pathogens use phagocytes or other host cells, like gut epithelial cells, as a replication niche. The intracellular growth of these pathogens can be counteracted by host defense mechanisms that rely on the autophagy machinery. In recent years, zebrafish embryo infection models have provided in vivo evidence for the significance of the autophagic defenses and these models are now being used to explore autophagy as a therapeutic target. In line with studies in mammalian models, research in zebrafish has shown that selective autophagy mediated by ubiquitin receptors, such as p62, is important for host resistance against several bacterial pathogens, including Shigella flexneri , Mycobacterium marinum , and Staphylococcus aureus . Furthermore, an autophagy related process, Lc3-associated phagocytosis (LAP), proved host beneficial in the case of Salmonella Typhimurium infection but host detrimental in the case of S. aureus infection, where LAP delivers the pathogen to a replication niche. These studies provide valuable information for developing novel therapeutic strategies aimed at directing the autophagy machinery towards bacterial degradation.

Neutrophils are a major component of the innate immune response and the most abundant circulating cell type in humans and zebrafish. The CXCL12/CXCR4 ligand receptor pair plays a key role in neutrophil homeostasis, controlling definitive hematopoiesis and neutrophil release into circulation. Neutrophils overexpressing CXCR4 respond by migrating towards sources of CXCL12, which is abundant in hematopoietic tissues. However, the physiological role of CXCL12/CXCR4 signaling during inflammatory responses remains unknown. Here, we show that zebrafish mutants lacking functional CXCL12a or CXCR4b show disrupted granulopoiesis in the kidney and increased number of circulating neutrophils. Additionally, CXCL12a and CXCR4b mutants display exacerbated recruitment of neutrophils to wounds and not to infections, and migrating neutrophils to wounds show increased directionality. Our results show that CXCL12a/CXCR4b signaling antagonizes wound-induced inflammatory signals by retaining neutrophils in hematopoietic tissues as a part of a balance between both inflammatory and anti-inflammatory cues, whose dynamic levels control neutrophils complex migratory behavior.

In vertebrates, damage to mechanosensory hair cells elicits an inflammatory response, including rapid recruitment of macrophages and neutrophils. While hair cells in amniotes usually become permanently lost, they readily regenerate in lower vertebrates such as fish. Damage to hair cells of the fish lateral line is followed by inflammation and rapid regeneration; however the role of immune cells in this process remains unknown. Here, we show that recruited macrophages are required for normal regeneration of lateral line hair cells after copper damage. We found that genetic ablation or local ablation using clodronate liposomes of macrophages recruited to the site of injury, significantly delays hair cell regeneration. Neutrophils, on the other hand, are not needed for this process. We anticipate our results to be a starting point for a more detailed description of extrinsic signals important for regeneration of mechanosensory cells in vertebrates. J. Cell. Biochem. 117: 1880-1889, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)