Human gut homeostasis and regeneration: the role of the gut microbiota and its metabolites.

The healthy human gut is a balanced ecosystem where host cells and representatives of the gut microbiota interact and communicate in a bidirectional manner at the gut epithelium. As a result of these interactions, many local and systemic processes necessary for host functionality, and ultimately health, take place. Impairment of the integrity of the gut epithelium diminishes its ability to act as an effective gut barrier, can contribute to conditions associated to inflammation processes and can have other negative consequences. Pathogens and pathobionts have been linked with damage of the integrity of the gut epithelium, but other components of the gut microbiota and some of their metabolites can contribute to its repair and regeneration. Here, we review what is known about the effect of bacterial metabolites on the gut epithelium and, more specifically, on the regulation of repair by intestinal stem cells and the regulation of the immune system in the gut. Additionally, we explore the potential therapeutic use of targeted modulation of the gut microbiota to maintain and improve gut homeostasis as a mean to improve health outcomes.

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.

Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach.

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga. We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A2, kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species.

Salamanders: The molecular basis of tissue regeneration and its relevance to human disease.

Salamanders are recognized for their ability to regenerate a broad range of tissues. They have also have been used for hundreds of years for classical developmental biology studies because of their large accessible embryos. The range of tissues these animals can regenerate is fascinating, from full limbs to parts of the brain or heart, a potential that is missing in humans. Many promising research efforts are working to decipher the molecular blueprints shared across the organisms that naturally have the capacity to regenerate different tissues and organs. Salamanders are an excellent example of a vertebrate that can functionally regenerate a wide range of tissue types. In this review, we outline some of the significant insights that have been made that are aiding in understanding the cellular and molecular mechanisms of tissue regeneration in salamanders and discuss why salamanders are a worthy model in which to study regenerative biology and how this may benefit research fields like regenerative medicine to develop therapies for humans in the future.

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.

Polyplexes System to Enhance the LL-37 Antimicrobial Peptide Expression in Human Skin Cells.

Inefficient autologous tissue recovery in diverse skin injuries increases the susceptibility of patients to infections caused by multiresistant microorganisms, resulting in a high mortality rate. Nonviral transfection is an attractive alternative for these patients, where genetically modified cells incorporated into skin substitutes could release additional antimicrobial agents into the native skin. In this work, we have modulated the conditions of using a nonviral system for transfection of primary human keratinocytes and fibroblasts, consisting of a polymer/plasmid DNA (pDNA) complex called polyplex and its effects on the expression of LL-37 antimicrobial peptide. Linear and branched polyethylenimine (PEI) polymers in different weight concentrations were varied for evaluating the formation and colloidal characteristics of the polyplexes. The PEI/pDNA polyplexes with 19 nitrogen/phosphate ratio are nanometric particles (400 and 250 nm with linear and branched PEI, respectively) exhibiting positive surface (+30 ± 2 mV). Both kinds of polyplexes allowed the expression of a reporter gene and increased the human cathelicidin antimicrobial peptide gene expression in transfected keratinocytes and fibroblasts; however, greater cytotoxicity was observed when polyplexes formed with branched PEI were used. Moreover, cell culture supernatants from transfected cells with linear PEI/pDNA polyplexes showed enhanced antimicrobial activity (decrease of bacterial growth in 95.8%) against a Staphylococcus aureus strain in vitro. The study of the PEI/pDNA polyplexes formation allowed us to develop an improved transfection strategy of skin cells, promoting the production of LL-37 antimicrobial peptide. In the future, this strategy could be used for the construction of skin substitutes to prevent, reduce, or eliminate bacterial infections. Impact statement The results of this study contribute to the understanding of the polyplexes system in the genetic modification of skin cells and its effects on the expression of the LL-37 antimicrobial peptide. In the future, three-dimensional skin substitutes built with these cells could be an efficient way to decrease bacterial growth and prevent the infections in skin wounds.

Venomic, Transcriptomic, and Bioactivity Analyses of Pamphobeteus verdolaga Venom Reveal Complex Disulfide-Rich Peptides That Modulate Calcium Channels.

Pamphobeteus verdolaga is a recently described Theraphosidae spider from the Andean region of Colombia. Previous reports partially characterized its venom profile. In this study, we conducted a detailed analysis that includes reversed-phase high-performance liquid chromatography (rp-HPLC), calcium influx assays, tandem mass spectrometry analysis (tMS/MS), and venom-gland transcriptome. rp-HPLC fractions of P. verdolaga venom showed activity on CaV2.2, CaV3.2, and NaV1.7 ion channels. Active fractions contained several peptides with molecular masses ranging from 3399.4 to 3839.6 Da. The tMS/MS analysis of active fraction displaying the strongest activity to inhibit calcium channels showed sequence fragments similar to one of the translated transcripts detected in the venom-gland transcriptome. The putative peptide of this translated transcript corresponded to a toxin, here named ω-theraphositoxin-Pv3a, a potential ion channel modulator toxin that is, in addition, very similar to other theraphositoxins affecting calcium channels (i.e., ω-theraphotoxin-Asp1a). Additionally, using this holistic approach, we found that P. verdolaga venom is an important source of disulfide-rich proteins expressing at least eight superfamilies.

Polyplex System Versus Nucleofection for Human Skin Cell Transfection and Effect of Internal Ribosome Entry Site Sequence.

Nonviral transfection has important implications on gene therapy because of its safety. In particular, polyfection and nucleofection are two widely used systems for nonviral gene delivery. Their potential depends on the transfection efficiency achieved, which is influenced in turn by the type of cells transfected and by the plasmid that carries the gene of interest. The efficiency of transfection by polyfection or nucleofection in human fibroblasts and keratinocytes was evaluated in this study. Transfections were performed with plasmids containing a gene of interest (human cathelicidin antimicrobial peptide) and two reporter genes (red or green fluorescent protein) that included or not an internal ribosome entry site (IRES). The efficiency was measured by flow cytometry in terms of percentage of cells expressing the reporter gene; viability of transfected cells was also evaluated. It was found that nucleofection was more efficient than polyplexes for transfecting fibroblasts, while no significant differences were found between both systems of transfection when applied to keratinocytes. Regarding the viability of fibroblasts after transfection, values were high in both systems. In contrast, keratinocytes were more sensitive to nucleofection. It was also noted that both types of cells decreased reporter gene expression when IRES sequence was located upstream of the reporter gene, suggesting a negative effect on the expression of this gene. These results confirm that the transfection efficiency depends on the type of cells and the system used.

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.

Sustitutos cutáneos desarrollados por ingeniería de tejidos / Skin substitutes developed by tissue engineering

La piel es un tejido complejo vulnerable a procesos que alteran su estructura, integridad y funcionalidad como, por ejemplo, quemaduras, heridas crónicas y diversas enfermedades congénitas. Los avances tecnológicos en la fabricación de biomateriales y en el cultivo de células han permitido la producción de sustitutos cutáneos que han sido una alternativa terapéutica para algunas de estas complicaciones. Esta revisión pretende actualizar los aspectos generales, composición, perspectivas futuras y de aplicación de los principales sustitutos cutáneos que se ofrecen actualmente en el mercado internacional. Igualmente, presentará algunas experiencias del Grupo de Ingeniería de Tejidos y Terapias Celulares (GITTC) de la Facultad de Medicina de la Universidad de Antioquia.

The skin is a complex tissue vulnerable to different processes that may alter its structure, integrity and functionality, among them: burns, chronic wounds and various congenital diseases. Technological advances in biomaterials manufacture and cell culture have allowed the production of skin substitutes thus providing an alternative therapy for some of these complications. This review aims to update the general aspects, composition, future prospects and implementation of the most common skin substitutes currently available in the international market. Some experiences of the Tissue Engineering and Cellular Therapy Group (GITTC), at the University of Antioquia, Faculty of Medicine, in Medellín, Colombia, are also presented.