Snezana Kojic

Adult zebrafish efficiently regenerate skeletal muscle following different types of injury; however, the molecular programs involved in repair after extensive cryoinjury remain to be comprehensively characterized. Here, we explored the transcriptomic response of adult zebrafish skeletal muscle at 7 days post cryoinjury (dpci), a stage marked by ongoing tissue clearance, progenitor expansion, myogenic differentiation, and nascent myofiber formation, and compared it with phase-matched stab wound injury. Cryoinjury induced a broad transcriptional response, with 5,330 differentially expressed genes. Integrated enrichment and protein-protein interaction analyses revealed that, at 7 dpci, zebrafish skeletal muscle functions as an integrated regenerative system in which immune remodeling, progenitor expansion, myogenic differentiation, extracellular matrix reconstruction, mechanotransduction, biosynthetic adaptation, proteostasis, and intracellular trafficking operate simultaneously. In parallel, mature sarcomeric and oxidative metabolic programs were suppressed, consistent with ongoing tissue reconstruction and structural immaturity. Comparison with stab-wounded skeletal muscle revealed substantial transcriptional conservation, as 612 of 717 stab-wound-responsive genes (85%) were also differentially expressed after cryoinjury. Shared upregulated genes formed coherent functional modules related to proliferation, extracellular matrix organization and signaling, immune regulation, muscle differentiation, and protein processing. Thus, distinct injury modalities converge on a common regenerative program, while cryoinjury elicits a quantitatively broader transcriptional response. These findings support a conserved regenerative architecture of adult zebrafish skeletal muscle repair, in which interconnected biological modules act coordinately, with the breadth of transcriptional engagement reflecting regenerative demand.

This work builds upon our previous “repopulation upon shattering” (RUS) studies in the yeast-like microbe Ustilago maydis, focusing mainly on a deeper understanding of the suicidal type of death caused by self-generated hypoxia (SGH). The key findings are: (1) the massive loss of viability induced by SGH occurs via programmed cell death (PCD), (2) the PCD-released materials are more beneficial for the surviving cells than the nutrients liberated from cells that die through necrotic processes, (3) products of PCD can stimulate growth even in the presence of growth-suppressive necromass, (4) the beneficial effects of substances released through PCD extend to two other species within the family Ustilaginaceae, and (5) U. maydis exhibited a remarkable ability to withstand the negative effects of non-PCD necromass. The third and fifth points are of special note. The third indicates that PCD-derived materials are essential for critical initial regrowth in post-stress environments where there is a high presence of non-PCD necromass. As the revitalized population strengthens, it would correspondingly enhance the communal ability to exploit more challenging nutrients. The robust resilience of U. maydis survivors against non-PCD necromass could represent an important fitness determinant, mediating competitive success in mixed microbial communities where non-PCD necromass imposes selective pressure. Thus, while providing valuable insights, the study also underscores the need for further research, especially in understanding the molecular underpinnings of the RUS phenomenon.

This work builds upon our previous “repopulation upon shattering” (RUS) studies in the yeast-like microbe Ustilago maydis, focusing mainly on a deeper understanding of the suicidal type of death caused by self-generated hypoxia (SGH). The key findings are: 1) the massive loss of viability induced by SGH occurs via programmed cell death (PCD), 2) the PCD-released materials are more beneficial for the surviving cells than the nutrients liberated from cells that die through necrotic processes, 3) products of PCD can stimulate growth even in the presence of growth-suppressive necromass, and 4) the beneficial effects of substances released through PCD extend to two other species within the family Ustilaginaceae. Additionally, U. maydis exhibited a remarkable ability to withstand the negative effects of non-PCD necromass. These results suggest PCD-released materials are essential for critical initial regrowth in post-stress environments where there is a high presence of non-PCD necromass. As the revitalized population strengthens, it would correspondingly enhance the communal ability to exploit more challenging nutrients. Alongside with the robust resilience of U. maydis survivors against non-PCD necromass, the PCD mode of dying may improve the fitness of this microorganism in harsh and fluctuating environments, warranting further investigation.

This study identifies ankrd1a as a novel modulator of skeletal muscle repair in adult zebrafish. Revealing its dual role in newly forming and intact myofibers near injury, the work highlights ankrd1a’s function in fine-tuning myogenic differentiation during repair. We position ankrd1a as both a marker of myofibers that repopulate wounded tissue and a player in coordination of adaptive responses essential for effective muscle tissue recovery.

In contrast to humans, zebrafish have a remarkable ability to regenerate injured heart through a highly orchestrated process involving all cardiac structures. To replace the lost myocardium, resident cardiomyocytes (CMs) dedifferentiate and proliferate, invading the injured area. The response of the myocardium is preceded by the activation of the epicardium and endocardium, which form active scaffolds to provide mechanical and paracrine support to guide regeneration. New CMs use protrusions to migrate and invade fibrotic injured tissue, to replace it with functional myocardium. Here, we investigated the expression profile of the stress-responsive ankrd1a gene in different cardiac structures, at key time points during regeneration, aiming to gain insight into its precise roles during zebrafish heart regeneration. In the TgBAC(ankrd1a:EGFP) reporter line, transgene upregulation was restricted to the myocardium, initiated as early as 15 hours post-cryoinjury, and consistently marked CMs bordering the injury or scar area during regeneration. Transcriptome profiling and immunostaining revealed a potential role of ankrd1a in regulating CMs’ dedifferentiation, as well as changes in expression of genes associated with antigen presentation and extracellular matrix composition in the ankrd1a mutant. Our results indicate that the ankrd1a is dispensable for ventricle regeneration after cryoinjury and may be considered as a marker and fine-tuner in the healing process of injured cardiac muscle.

Ankrd2, a mechanoresponsive protein primarily studied in muscle physiology, is emerging as a player in cancer progression. This study investigates the functional role of Ankrd2 in osteosarcoma cells, revealing its critical involvement in cell proliferation and response to chemotherapeutic drugs. We showed that Ankrd2 knockdown impairs the activation of PI3K/Akt and ERK1/2 pathways, reduces levels of cell cycle regulators including cyclin D1 and cyclin B, and counteracts the expression of nuclear lamin A and lamin B, disrupting nuclear morphology and DNA integrity. Strikingly, the loss of Ankrd2 enhances the sensitivity of osteosarcoma cells to doxorubicin and cisplatin, highlighting Ankrd2 potential as a therapeutic target to improve chemotherapeutic efficacy. Defining a novel mechanistic role for Ankrd2 in promoting tumor progression, we propose that Ankrd2 reduction could be exploited as an adjuvant strategy to enhance the efficacy of chemotherapy, offering new therapeutic opportunities for OS treatment.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Respecting the age of the patients and the tumor aggressiveness, investigation of the molecular mechanisms of RMS tumorigenesis is directed toward the identification of novel therapeutic targets. To contribute to a better understanding of the molecular pathology of RMS, we investigated ankyrin repeat domain 1 (ANKRD1), designated as a potential marker for differential diagnostics. In this study, we used three RMS cell lines (SJRH30, RD, and HS-729) to assess its expression profile, intracellular localization, and turnover. They express wild-type ANKRD1, as judged by the sequencing of the open reading frame. Each cell line expressed a different amount of ANKRD1 protein, although the transcript level was similar. According to western blot analysis, ANKRD1 protein was expressed at detectable levels in the SJRH30 and RD cells (SJRH30 > RD), but not in the HS-729, even after immunoprecipitation. Immunocytochemistry revealed nuclear and cytoplasmic localization of ANKRD1 in all examined cell lines. Moreover, the punctate pattern of ANKRD1 staining in the nuclei of RD and HS-729 cells overlapped with coilin, indicating its association with Cajal bodies. We have shown that RMS cells are not able to overexpress ANKRD1 protein, which can be attributed to its proteasomal degradation. The unsuccessful attempt to overexpress ANKRD1 in RMS cells indicates the possibility that its overexpression may have detrimental effects for RMS cells and opens a window for further research into its role in RMS pathogenesis and for potential therapeutic targeting.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Respecting the age of the patients and the tumor aggressiveness, investigation of the molecular mechanisms of RMS tumorigenesis is directed toward the identification of novel therapeutic targets. To contribute to a better understanding of the molecular pathology of RMS, we investigated ankyrin repeat domain 1 (ANKRD1), designated as a potential marker for differential diagnostics. In this study, we used three RMS cell lines (SJRH30, RD, and HS-729) to assess its expression profile, intracellular localization, and turnover. They express wild-type ANKRD1, as judged by the sequencing of the open reading frame. Each cell line expressed a different amount of ANKRD1 protein, although the transcript level was similar. According to Western blot analysis, ANKRD1 protein was expressed at detectable levels in the SJRH30 and RD cells (SJRH30>RD), but not in the HS-729, even after immunoprecipitation. Immunocytochemistry revealed nuclear and cytoplasmic localization of ANKRD1 in all examined cell lines. Moreover, the punctate pattern of ANKRD1 staining in the nuclei of RD and HS-729 cells overlapped with coilin, indicating its association with Cajal bodies. We have shown that RMS cells are not able to overexpress ANKRD1 protein, which can be attributed to its proteasomal degradation. The unsuccessful attempt to overexpress ANKRD1 in RMS cells indicates the possibility that its overexpression may be lethal for RMS cells and opens a window for therapeutic targeting.

Bronchial epithelial cells are exposed to environmental influences, microbiota, and pathogens and also serve as a powerful effector that initiate and propagate inflammation by the release of pro-inflammatory mediators. Recent studies suggested that lung microbiota differ between inflammatory lung diseases and healthy lungs implicating their contribution in the modulation of lung immunity. Lactic acid bacteria (LAB) are natural inhabitants of healthy human lungs and also possess immunomodulatory effects, but so far, there are no studies investigating their anti-inflammatory potential in respiratory cells. In this study, we investigated immunomodulatory features of 21 natural LAB strains in lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Our results show that several LAB strains reduced the expression of pro-inflammatory cytokine and chemokine genes. We also demonstrated that two LAB strains, Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22, effectively attenuated LPS-induced nuclear factor-κB (NF-κB) nuclear translocation. Moreover, BGZLS10-17 and BGPKM22 reduced the activation of p38, extracellular signal-related kinase (ERK), and c-Jun amino-terminal kinase (JNK) signaling cascade resulting in a reduction of pro-inflammatory mediator expressions in BEAS-2B cells. Collectively, the LAB strains BGZLS10-17 and BGPKM22 exhibited anti-inflammatory effects in BEAS-2B cells and could be employed to balance immune response in lungs and replenish diminished lung microbiota in chronic lung diseases.