zebrafish-larvae-as-model-to-evaluate-potential-radiosensitizers-or

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Institute of Life Sciences

Activation of the Wnt signalling cascade is primarily based on the interplay between Wnt ligands, their receptors and extracellular modulators. One prominent family of extracellular modulators is represented by the SFRP (secreted Frizzled-related protein) family. These proteins have significant similarity to the extracellular domain of Frizzled receptors, suggesting that they bind Wnt ligands and inhibit signalling. The SFRP-type protein Fz4-v1, a splice variant of the Frizzled-4 receptor found in humans and Xenopus, was shown to augment Wnt/β-catenin signalling, and also interacts with those Wnt ligands that act on β-catenin-independent Wnt pathways.

A secreted splice variant of the Xenopus frizzled-4 receptor is a biphasic modulator of Wnt signalling.

The nucleolar protein 4-like (NOL4L) gene is present on chromosome 20 (20q11.21) in humans. Parts of this gene have been shown to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively. The normal function of NOL4L in humans and other organisms is not well understood. The expression patterns and functions of NOL4L homologs during vertebrate development have not been reported. We sought to address these questions by studying the expression pattern of zebrafish nol4l during embryogenesis. Our data show that Znol4l mRNA is expressed in multiple organs in zebrafish embryos. The sites of expression include parts of the brain, spinal cord, pronephros, hematopoietic cells and gut. 

Nucleolar protein 4-like has a complex expression pattern in zebrafish embryos.

The growth and metastasis of tumors is dependent on angiogenesis. C-type lectins are carbohydrate-binding proteins with a diverse range of functions. The C-type lectin family XIV members are transmembrane glycoproteins, and all four members of this family have been reported to regulate angiogenesis, although the detailed mechanism of action has yet to be completely elucidated. They interact with extracellular matrix proteins and mediate cell-cell adhesion by their lectin-like domain. The aim of the present study was to summarize the available information on the function and mechanism of C-type lectin family XIV in angiogenesis and discuss their potential as targets for cancer therapy.

C-type lectin family XIV members and angiogenesis.

DZIP3/hRUL138 is a poorly characterized RNA-binding RING E3-ubiquitin ligase with functions in embryonic development. Here we demonstrate that DZIP3 is a crucial driver of cancer cell growth, migration, and invasion. In mice and zebrafish cancer models, DZIP3 promoted tumor growth and metastasis. In line with these results, DZIP3 was frequently overexpressed in several cancer types. Depletion of DZIP3 from cells resulted in reduced expression of Cyclin D1 and a subsequent G arrest and defect in cell growth. Mechanistically, DZIP3 utilized its two different domains to interact and stabilize Cyclin D1 both at mRNA and protein levels. Using an RNA-binding lysine-rich region, DZIP3 interacted with the AU-rich region in 3' untranslated region of Cyclin D1 mRNA and stabilized it. Using a RING E3-ligase domain, DZIP3 interacted and increased K63-linked ubiquitination of Cyclin D1 protein to stabilize it. Remarkably, DZIP3 interacted with, ubiquitinated, and stabilized Cyclin D1 predominantly in the G phase of the cell cycle, where it is needed for cell-cycle progression. In agreement with this, a strong positive correlation of mRNA expression between DZIP3 and Cyclin D1 in different cancer types was observed. Additionally, DZIP3 regulated several cell cycle proteins by modulating the Cyclin D1-E2F axes. Taken together, this study demonstrates for the first time that DZIP3 uses a unique two-pronged mechanism in its stabilization of Cyclin D1 to drive cell-cycle and cancer progression. SIGNIFICANCE: These findings show that DZIP3 is a novel driver of cell-cycle and cancer progression via its control of Cyclin D1 mRNA and protein stability in a cell-cycle phase-dependent manner. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/2/315/F1.large.jpg.

RNA-Binding RING E3-Ligase DZIP3/hRUL138 Stabilizes Cyclin D1 to Drive Cell-Cycle and Cancer Progression.

Phosphorylation of the central adaptor protein complex, AP-2 is pivotal for clathrin-mediated endocytosis (CME). Here, we uncover the role of an uncharacterized kinase (BMP-2 inducible kinase-BMP2K) in AP-2 phosphorylation. We demonstrate that BMP2K can phosphorylate AP-2 in vitro and in vivo. Functional impairment of BMP2K impedes AP-2 phosphorylation leading to defects in clathrin-coated pit (CCP) morphology and cargo internalization. BMP2K engages AP-2 via its extended C-terminus and this interaction is important for its CCP localization and function. Notably, endogenous BMP2K levels decline upon functional impairment of AP-2 indicating AP-2 dependent BMP2K stabilization in cells. Further, functional inactivation of BMP2K in zebrafish embryos yields gastrulation phenotypes which mirror AP-2 loss-of-function suggesting physiological relevance of BMP2K in vertebrates. Together, our findings propose involvement of a novel kinase in AP-2 phosphorylation and in the operation of CME.

BMP2K phosphorylates AP-2 and regulates clathrin-mediated endocytosis.

The host immune responses to  a frequent cause of nosocomial infections, are not well understood. We have established a bath immersion model of this infection in zebrafish () larvae. Macrophages play a primary role in the host immune response and are involved in clearance of infection in the larvae.  infection results in upregulation of  There is marked inflammation characterized by heightened NF-κB signaling and elevation of several proinflammatory cytokines. There is rapid upregulation of  and  transcripts, whereas an increase in  levels is relatively more delayed. The IL-6 signaling pathway is further amplified by elevation of IL-6 signal transducer () levels, which negatively correlates with miRNA dre-miR-142a-5p. Enhanced IL-6 signaling is protective to the host in this model as inhibition of the signaling pathway resulted in increased mortality upon  infection. Our study describes the host immune responses to  infection, establishes the importance of IL-6 signaling, and identifies a potential role of miR-142-5p- interaction in this infection model.

IL-6 Signaling Protects Zebrafish Larvae during  Infection in a Bath Immersion Model.

Pancreatic cancer (PC) is highly resistant to chemo and radiotherapy. Radiation-induced fibrosis (RIF) is a major cause of clinical concern for various malignancies, including PC. In this study, we aimed to evaluate the radiosensitizing and anti-RIF potential of fluvastatin in PC. Short-term viability and clonogenic survival assays were used to evaluate the radiosensitizing potential of fluvastatin in multiple human and murine PC cell lines. The expression of different proteins was analyzed to understand the mechanisms of fluvastatin-mediated radiosensitization of PC cells and its anti-RIF effects in both mouse and human pancreatic stellate cells (PSCs). Finally, these effects of fluvastatin and/or radiation were assessed in an immune-competent syngeneic murine model of PC. Fluvastatin radiosensitized multiple PC cell lines, as well as radioresistant cell lines in vitro, by inhibiting radiation-induced DNA damage repair response. Nonmalignant cells, such as PSCs and NIH3T3 cells, were less sensitive to fluvastatin-mediated radiosensitization than PC cells. Interestingly, fluvastatin suppressed radiation and/or TGF-β-induced activation of PSCs, as well as the fibrogenic properties of these cells in vitro. Fluvastatin considerably augmented the antitumor effect of external radiation therapy and also suppressed intra-tumor RIF in vivo. These findings suggested that along with radiation, fluvastatin co-treatment may be a potential therapeutic approach against PC.

Fluvastatin sensitizes pancreatic cancer cells toward radiation therapy and suppresses radiation- and/or TGF-β-induced tumor-associated fibrosis.

Correction to: Fluvastatin sensitizes pancreatic cancer cells toward radiation therapy and suppresses radiation- and/or TGF-β-induced tumor-associated fibrosis.

The lateral line system is a mechanosensory organ of fish and amphibians that detects changes in water flow and is formed by the coordinated action of many signalling pathways. These signalling pathways can easily be targeted in zebrafish using pharmacological inhibitors to decipher their role in lateral line system development at cellular and molecular level. We have identified two uncharacterized proteins, whose mRNA are expressed in the lateral line system of zebrafish. One of these proteins, uncharacterized protein LOC564095 precursor, is conserved across vertebrates and its mRNA is expressed in posterior lateral line primordium (pLLP). The other uncharacterized protein, LOC100536887, is present only in the teleost fishes and its mRNA is expressed in neuromasts. We show that inhibition of retinoic acid (RA) signalling reduces the expression of both of these uncharacterized genes. It is reported that inhibition of RA signalling during gastrulation starting at 7 hours post fertilization (hpf) abrogates pLLP formation, and inhibition of RA signalling at 10 hpf delays the initiation of pLLP migration. Here, we show that inhibition of RA signalling before and during segmentation (9-16 hpf) results in delayed initiation and reduced speed of pLLP migration, as well as inhibition of posterior neuromasts formation.

Expression of two uncharacterized protein coding genes in zebrafish lateral line system.

The current global pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), led to the investigation with clinical, biochemical, immunological, and genomic characterization from patients to understand the pathophysiology of viral infection.

Clinical, Virological, Immunological, and Genomic Characterization of Asymptomatic and Symptomatic Cases With SARS-CoV-2 Infection in India.

SARS-CoV2 infection in patients with comorbidities, particularly T2DM, has been a major challenge globally and has been shown to be associated with high morbidity and mortality. Here, we did whole blood immunophenotyping along with plasma cytokine, chemokine, antibody isotyping, and viral load from oropharyngeal swab to understand the immune pathology in the T2DM patients infected with SARS-CoV2.

Article	Year
A secreted splice variant of the Xenopus frizzled-4 receptor is a biphasic modulator of Wnt signalling. Cell communication and signaling : CCS\| PubMed ID: 24252524	2013
Nucleolar protein 4-like has a complex expression pattern in zebrafish embryos. The International journal of developmental biology\| PubMed ID: 26934290	2016
C-type lectin family XIV members and angiogenesis. Oncology letters\| PubMed ID: 31579078	2019
RNA-Binding RING E3-Ligase DZIP3/hRUL138 Stabilizes Cyclin D1 to Drive Cell-Cycle and Cancer Progression. Cancer research\| PubMed ID: 33067265	2021
BMP2K phosphorylates AP-2 and regulates clathrin-mediated endocytosis. Traffic (Copenhagen, Denmark)\| PubMed ID: 34480404	2021
IL-6 Signaling Protects Zebrafish Larvae during Infection in a Bath Immersion Model. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 34544800	2021
Fluvastatin sensitizes pancreatic cancer cells toward radiation therapy and suppresses radiation- and/or TGF-β-induced tumor-associated fibrosis. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 34773069	2022
Correction to: Fluvastatin sensitizes pancreatic cancer cells toward radiation therapy and suppresses radiation- and/or TGF-β-induced tumor-associated fibrosis. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 34853441	2022
Expression of two uncharacterized protein coding genes in zebrafish lateral line system. The International journal of developmental biology\| PubMed ID: 34881803	2021
Clinical, Virological, Immunological, and Genomic Characterization of Asymptomatic and Symptomatic Cases With SARS-CoV-2 Infection in India. Frontiers in cellular and infection microbiology\| PubMed ID: 34993157	2021
Underlying Co-Morbidity Reveals Unique Immune Signatures in Type II Diabetes Patients Infected With SARS-CoV2. Frontiers in immunology\| PubMed ID: 35572555	2022

Rajeeb K. Swain

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