COMET Assay for Detection of DNA Damage During Axolotl Tail Regeneration.

The purpose of this chapter is to evaluate DNA damage during axolotl tail regeneration using an alkaline comet assay. Our method details the isolation of cells from regenerating and non-regenerating tissues and the isolation of peripheral blood for single-cell gel electrophoresis. Also, we detail each of the steps for the development of the comet assay technique which includes mounting the isolated cells on an agarose matrix, alkaline electrophoresis, and DNA damage detection.

Ramosin: The First Antibacterial Peptide Identified on Bolitoglossa ramosi Colombian Salamander.

The discovery and improvements of antimicrobial peptides (AMPs) have become an alternative to conventional antibiotics. They are usually small and heat-stable peptides, exhibiting inhibitory activity against Gram-negative and Gram-positive bacteria. In this way, studies on broad-spectrum AMPs found in amphibians with the remarkable capability to regenerate a wide array of tissues are of particular interest in the search for new strategies to treat multidrug-resistant bacterial strains. In this work, the use of bioinformatic approaches such as sequence alignment with Fasta36 and prediction of antimicrobial activity allowed the identification of the Ramosin peptide from the de novo assembled transcriptome of the plethodontid salamander Bolitoglossa ramosi obtained from post-amputation of the upper limb tissue, heart, and intestine samples. BLAST analysis revealed that the Ramosin peptide sequence is unique in Bolitoglossa ramosi. The peptide was chemically synthesized, and physicochemical properties were characterized. Furthermore, the in vitro antimicrobial activity against relevant Gram-positive and Gram-negative human pathogenic bacteria was demonstrated. Finally, no effect against eukaryotic cells or human red blood cells was evidenced. This is the first antibacterial peptide identified from a Colombian endemic salamander with interesting antimicrobial properties and no hemolytic activity.

Post-amputation reactive oxygen species production is necessary for axolotls limb regeneration.

Introduction: Reactive oxygen species (ROS) represent molecules of great interest in the field of regenerative biology since several animal models require their production to promote and favor tissue, organ, and appendage regeneration. Recently, it has been shown that the production of ROS such as hydrogen peroxide (H2O2) is required for tail regeneration in Ambystoma mexicanum. However, to date, it is unknown whether ROS production is necessary for limb regeneration in this animal model. Methods: forelimbs of juvenile animals were amputated proximally and the dynamics of ROS production was determined using 2'7- dichlorofluorescein diacetate (DCFDA) during the regeneration process. Inhibition of ROS production was performed using the NADPH oxidase inhibitor apocynin. Subsequently, a rescue assay was performed using exogenous hydrogen peroxide (H2O2). The effect of these treatments on the size and skeletal structures of the regenerated limb was evaluated by staining with alcian blue and alizarin red, as well as the effect on blastema formation, cell proliferation, immune cell recruitment, and expression of genes related to proximal-distal identity. Results: our results show that inhibition of post-amputation limb ROS production in the A. mexicanum salamander model results in the regeneration of a miniature limb with a significant reduction in the size of skeletal elements such as the ulna, radius, and overall autopod. Additionally, other effects such as decrease in the number of carpals, defective joint morphology, and failure of integrity between the regenerated structure and the remaining tissue were identified. In addition, this treatment affected blastema formation and induced a reduction in the levels of cell proliferation in this structure, as well as a reduction in the number of CD45+ and CD11b + immune system cells. On the other hand, blocking ROS production affected the expression of proximo-distal identity genes such as Aldha1a1, Rarß, Prod1, Meis1, Hoxa13, and other genes such as Agr2 and Yap1 in early/mid blastema. Of great interest, the failure in blastema formation, skeletal alterations, as well as the expression of the genes evaluated were rescued by the application of exogenous H2O2, suggesting that ROS/H2O2 production is necessary from the early stages for proper regeneration and patterning of the limb.

Ambystoma mexicanum, A MODEL ORGANISM IN DEVELOPMENTAL BIOLOGY AND REGENERATION: A COLOMBIAN EXPERIENCE / Ambystoma mexicanum, un organismo modelo en biología del desarrollo y regeneración: experiencia colombiana

ABSTRACT Ambystoma mexicanum is a urodele amphibian endemic to Xochimilco Lake in Mexico, it belongs to the salamander family Ambystomatidae. This species has frequently been used as model organism in developmental biology and regeneration laboratories around the world due to its broad regenerative capacities and adaptability to laboratory conditions. In this review we describe the establishment of the first colony of axolotls in Colombia to study tissue regeneration and our perspectives on the use A. mexicanum as a model organism in Colombia are discussed emphasizing its possible uses in regeneration and developmental biology.

RESUMEN Ambystoma mexicanum es un anfibio urodelo endémico del lago Xochimilco en México, perteneciente a la familia de salamandras Ambystomatidae. Esta especie se ha empleado frecuentemente como organismo modelo en laboratorios de biología del desarrollo y regeneración alrededor del mundo, dadas sus amplias capacidades regenerativas y adaptabilidad en condiciones de laboratorio. En esta revisión, se describe el establecimiento de la primera colonia de ajolotes en Colombia, para adelantar estudios de regeneración de tejidos, y se discuten las perspectivas de A. mexicanum como organismo modelo en el país, enfatizando sus posibles usos en regeneración y biología del desarrollo.

Hydrogen peroxide is necessary during tail regeneration in juvenile axolotl.

BACKGROUND: Hydrogen peroxide (H2 O2 ) is a key reactive oxygen species (ROS) generated during appendage regeneration among vertebrates. However, its role during tail regeneration in axolotl as redox signaling molecule is unclear. RESULTS: Treatment with exogenous H2 O2 rescues inhibitory effects of apocynin-induced growth suppression in tail blastema cells leading to cell proliferation. H2 O2 also promotes recruitment of immune cells, regulate the activation of AKT kinase and Agr2 expression during blastema formation. Additionally, ROS/H2 O2 regulates the expression and transcriptional activity of Yap1 and its target genes Ctgf and Areg. CONCLUSIONS: These results show that H2 O2 is necessary and sufficient to promote tail regeneration in axolotls. Additionally, Akt signaling and Agr2 were identified as ROS targets, suggesting that ROS/H2 O2 is likely to regulate epimorphic regeneration through these signaling pathways. In addition, ROS/H2 O2 -dependent-Yap1 activity is required during tail regeneration.

In Silico Selection and Evaluation of Pugnins with Antibacterial and Anticancer Activity Using Skin Transcriptome of Treefrog (Boana pugnax).

In order to combat bacterial and cancer resistance, we identified peptides (pugnins) with dual antibacterial l-anticancer activity from the Boana pugnax (B. pugnax) skin transcriptome through in silico analysis. Pugnins A and B were selected owing to their high similarity to the DS4.3 peptide, which served as a template for their alignment to the B. pugnax transcriptome, as well as their function as part of a voltage-dependent potassium channel protein. The secondary peptide structure stability in aqueous medium was evaluated as well, and after interaction with the Escherichia coli (E. coli) membrane model using molecular dynamics. These pugnins were synthesized via solid-phase synthesis strategy and verified by Reverse phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry. Subsequently, their alpha-helix structure was determined by circular dichroism, after which antibacterial tests were then performed to evaluate their antimicrobial activity. Cytotoxicity tests against cancer cells also showed selectivity of pugnin A toward breast cancer (MFC7) cells, and pugnin B toward prostate cancer (PC3) cells. Alternatively, flow cytometry revealed necrotic cell damage with a major cytotoxic effect on human keratinocytes (HaCaT) control cells. Therefore, the pugnins found in the transcriptome of B. pugnax present dual antibacterial-anticancer activity with reduced selectivity to normal eukaryotic cells.

Mitochondrial transplant to replenish damaged mitochondria: A novel therapeutic strategy for neurodegenerative diseases?

Neurodegenerative diseases are currently some of the most debilitating and incurable illness, including highly prevalent disorders, such as Alzheimer's and Parkinson's disease. Despite impressive advances in understanding the molecular basis of neurodegenerative diseases, several clinical trials have failed in identifying drugs that successfully delay or stop disease progression. New targets are likely necessary to successfully combat these devastating diseases. In this chapter, we review the evidence indicating that impairment in the cellular energy machinery in the form of mitochondrial damage and dysfunction may be at the root of neurodegeneration. We also propose that transplant of functional isolated mitochondria may overcome the energetic damage and delay the progression of neurodegenerative diseases.

Limb regeneration in salamanders: the plethodontid tale.

Salamanders are the only vertebrates that can regenerate limbs as adults. This makes them ideal models to investigate the cellular and molecular mechanisms of tissue regeneration. Ambystoma mexicanum and Nothopthalmus viridescens have long served as primary salamander models of limb regeneration, and the recent sequencing of the axolotl genome now provides a blueprint to mine regeneration insights from other salamander species. In particular, there is a need to study South American plethodontid salamanders that present different patterns of limb development and regeneration. A broader sampling of species using next-generation sequencing approaches is needed to reveal shared and unique mechanisms of regeneration, and more generally, the evolutionary history of salamander limb regeneration.

Peptides with Dual Antimicrobial-Anticancer Activity: Strategies to Overcome Peptide Limitations and Rational Design of Anticancer Peptides.

Peptides are naturally produced by all organisms and exhibit a wide range of physiological, immunomodulatory, and wound healing functions. Furthermore, they can provide with protection against microorganisms and tumor cells. Their multifaceted performance, high selectivity, and reduced toxicity have positioned them as effective therapeutic agents, representing a positive economic impact for pharmaceutical companies. Currently, efforts have been made to invest in the development of new peptides with antimicrobial and anticancer properties, but the poor stability of these molecules in physiological environments has triggered a bottleneck. Therefore, some tools, such as nanotechnology and in silico approaches can be applied as alternatives to try to overcome these obstacles. In silico studies provide a priori knowledge that can lead to the development of new anticancer peptides with enhanced biological activity and improved stability. This review focuses on the current status of research in peptides with dual antimicrobial-anticancer activity, including advances in computational biology using in silico analyses as a powerful tool for the study and rational design of these types of peptides.

A tree frog (Boana pugnax) dataset of skin transcriptome for the identification of biomolecules with potential antimicrobial activities.

Increases in the prevalence of multiply resistant microbes have necessitated the search for new molecules with antimicrobial properties. One noteworthy avenue in this search is inspired by the presence of native antimicrobial peptides in the skin of amphibians. Having the second highest diversity of frogs worldwide, Colombian anurans represent an extensive natural reservoir that could be tapped in this search. Among this diversity, species such as Boana pugnax (the Chirique-Flusse Treefrog) are particularly notable, in that they thrive in a diversity of marginal habitats, utilize both aquatic and arboreal habitats, and are members of one of few genera that are known to mount a robust immunological response against the fungus Batrachochytrium dendrobatidis, which has decimated the population of frogs worldwide. To search for molecules with potential antimicrobial activity, we have assembled and annotated a reference transcriptome from the skin of four wild captured B. pugnax from Antioquia, Colombia. Analysis of potential antimicrobial and immunological components was performed using ontology analyses, we identified several antimicrobial chemokines with particularly strong potential for exhibiting broadscale antimicrobial activities, as well as several genes related to rapid alteration of transcriptional (KRAB zinc finger protein) and phosphorylation (MAPK) responses to exogenous stressors. We also found eight families of transmembrane transport proteins, including sodium, potassium and voltage-dependent calcium channels, which will be invaluable in future studies aimed at more precisely defining the diversity and function of cationic antimicrobial peptides with alpha-helical structures. These data highlight the utility of frogs such as Boana pugnax in the search of new antimicrobial molecules. Moreover, the molecular datasets presented here allow us to expand our knowledge of this species and illustrate the importance of preserving the vast potential of Colombian biodiversity for the identification of useful biomolecules.

A de novo reference transcriptome for Bolitoglossa vallecula, an Andean mountain salamander in Colombia.

The amphibian order Caudata, contains several important model species for biological research. However, there is need to generate transcriptome data from representative species of the primary salamander families. Here we describe a de novo reference transcriptome for a terrestrial salamander, Bolitoglossa vallecula (Caudata: Plethodontidae). We employed paired-end (PE) illumina RNA sequencing to assemble a de novo reference transcriptome for B. vallecula. Assembled transcripts were compared against sequences from other vertebrate taxa to identify orthologous genes, and compared to the transcriptome of a close plethodontid relative (Bolitoglossa ramosi) to identify commonly expressed genes in the skin. This dataset should be useful to future comparative studies aimed at understanding important biological process, such as immunity, wound healing, and the production of antimicrobial compounds.

Using transcriptomics to enable a plethodontid salamander (Bolitoglossa ramosi) for limb regeneration research.

BACKGROUND: Tissue regeneration is widely distributed across the tree of life. Among vertebrates, salamanders possess an exceptional ability to regenerate amputated limbs and other complex structures. Thus far, molecular insights about limb regeneration have come from a relatively limited number of species from two closely related salamander families. To gain a broader perspective on the molecular basis of limb regeneration and enhance the molecular toolkit of an emerging plethodontid salamander (Bolitoglossa ramosi), we used RNA-Seq to generate a de novo reference transcriptome and identify differentially expressed genes during limb regeneration. RESULTS: Using paired-end Illumina sequencing technology and Trinity assembly, a total of 433,809 transcripts were recovered and we obtained functional annotation for 142,926 non-redundant transcripts of the B. ramosi de novo reference transcriptome. Among the annotated transcripts, 602 genes were identified as differentially expressed during limb regeneration. This list was further processed to identify a core set of genes that exhibit conserved expression changes between B. ramosi and the Mexican axolotl (Ambystoma mexicanum), and presumably their common ancestor from approximately 180 million years ago. CONCLUSIONS: We identified genes from B. ramosi that are differentially expressed during limb regeneration, including multiple conserved protein-coding genes and possible putative species-specific genes. Comparative analyses reveal a subset of genes that show similar patterns of expression with ambystomatid species, which highlights the importance of developing comparative gene expression data for studies of limb regeneration among salamanders.

Allosteric mechanism of action of the therapeutic anti-IgE antibody omalizumab.

Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE.

Seletalisib: Characterization of a Novel, Potent, and Selective Inhibitor of PI3Kδ.

Phosphoinositide 3-kinases (PI3K) are key signaling enzymes regulating cellular survival, development, and function. Expression of the PI3Kδ isoform is largely restricted to leukocytes and it plays a key role in immune cell development and function. Seletalisib is a novel small-molecule inhibitor of PI3Kδ that was evaluated in biochemical assays, cellular assays of adaptive and innate immunity, and an in vivo rat model of inflammation. Our findings show that seletalisib is a potent, ATP-competitive, and selective PI3Kδ inhibitor able to block protein kinase B (AKT) phosphorylation following activation of the B-cell receptor in a B-cell line. Moreover, seletalisib inhibited N-formyl peptide-stimulated but not phorbol myristate acetate-stimulated superoxide release from human neutrophils, consistent with a PI3Kδ-specific activity. No indications of cytotoxicity were observed in peripheral blood mononuclear cells (PBMCs) or other cell types treated with seletalisib. Findings from cellular assays of adaptive immunity demonstrated that seletalisib blocks human T-cell production of several cytokines from activated T-cells. Additionally, seletalisib inhibited B-cell proliferation and cytokine release. In human whole blood assays, seletalisib inhibited CD69 expression upon B-cell activation and anti-IgE-mediated basophil degranulation. Seletalisib showed dose-dependent inhibition in an in vivo rat model of anti-CD3-antibody-induced interleukin 2 release. Collectively, these data characterize seletalisib as a selective PI3Kδ inhibitor and potential therapeutic candidate for the treatment of B-cell malignancies and autoimmune diseases driven by dysregulated proinflammatory cytokine secretion.

Limb regeneration in a direct-developing terrestrial salamander, Bolitoglossa ramosi (Caudata: Plethodontidae): Limb regeneration in plethodontid salamanders.

Appendage regeneration is one of the most compelling phenomena in regenerative biology and is extensively studied in axolotls and newts. However, the regenerative capacity in other families of salamanders remains poorly described. Here we characterize the limb regeneration process in Bolitoglossa ramosi, a direct-developing terrestrial salamander of the plethodontid family. We (1) describe the major morphological features at different stages of limb regeneration, (2) show that appendage regeneration in a terrestrial salamander varies from other amphibians and (3) show that limb regeneration in this species is considerably slower than in axolotls and newts (95 days post-amputation for complete regeneration) despite having a significantly smaller genome size than axolotls or newts.

Mechanism of Action of Secreted Newt Anterior Gradient Protein.

Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.

Maintaining plethodontid salamanders in the laboratory for regeneration studies.

Limb regeneration studies have been extensively carried out in species of Ambystomatidae and Salamandridae families. So far limited research has been conducted in species belonging to the Plethodontidae family, where some of the species differs from other salamander families due to their direct development, thus absence of a larval life. Here, we describe a protocol to maintain the plethodontid salamanders of genus Bolitoglossa species under laboratory conditions to perform regeneration studies.

Generation of aneurogenic larvae by parabiosis of salamander embryos.

Limb regeneration of salamanders is nerve dependent, and the removal of the nerves in early stages of limb regeneration severely curtails the proliferation of the blastemal cells and growth of the regenerate. The removal of the neural tube from a developing salamander embryo results in an aneurogenic larva and the aneurogenic limb (ANL) develops independently without innervation. Paradoxically, the limb in an ANL is capable of regeneration in a nerve-independent manner. Here, we describe a detailed method for the generation of ANL in the spotted salamander, Ambystoma maculatum, for regeneration studies.

Human immunoglobulin E flexes between acutely bent and extended conformations.

Crystallographic and solution studies have shown that IgE molecules are acutely bent in their Fc region. Crystal structures reveal the Cɛ2 domain pair folded back onto the Cɛ3-Cɛ4 domains, but is the molecule exclusively bent or can the Cɛ2 domains adopt extended conformations and even 'flip' from one side of the molecule to the other? We report the crystal structure of IgE-Fc captured in a fully extended, symmetrical conformation and show by molecular dynamics, calorimetry, stopped-flow kinetic, surface plasmon resonance (SPR) and Förster resonance energy transfer (FRET) analyses that the antibody can indeed adopt such extended conformations in solution. This diversity of conformational states available to IgE-Fc offers a new perspective on IgE function in allergen recognition, as part of the B-cell receptor and as a therapeutic target in allergic disease.

The aneurogenic limb identifies developmental cell interactions underlying vertebrate limb regeneration.

The removal of the neural tube in salamander embryos allows the development of nerve-free aneurogenic limbs. Limb regeneration is normally nerve-dependent, but the aneurogenic limb regenerates without nerves and becomes nerve-dependent after innervation. The molecular basis for these tissue interactions is unclear. Anterior Gradient (AG) protein, previously shown to rescue regeneration of denervated limbs and to act as a growth factor for cultured limb blastemal cells, is expressed throughout the larval limb epidermis and is down-regulated by innervation. In an aneurogenic limb, the level of AG protein remains high in the epidermis throughout development and regeneration, but decreases after innervation following transplantation to a normal host. Aneurogenic epidermis also shows a fivefold difference in secretory gland cells, which express AG protein. The persistently high expression of AG in the epithelial cells of an aneurogenic limb ensures that regeneration is independent of the nerve. These findings provide an explanation for this classical problem, and identify regulation of the epidermal niche by innervation as a distinctive developmental mechanism that initiates the nerve dependence of limb regeneration. The absence of this regulation during anuran limb development might suggest that it evolved in relation to limb regeneration.