Proteogenomics for Non-model Ocean-Derived Fungi.

Most biological and biomedical experiments are designed and studied using the most common model organisms (MOs) like humans, mice, Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, worms, fruit flies, zebrafish, and Arabidopsis thaliana. These model organisms have been extensively studied and have a well-established set of genetic, physiological, and other tools available for research. In contrast, non-model organisms (NMOs) are those that are not traditionally used in scientific research and do not have a well-established set of genetic or other biological tools available for their study. The majority of MOs are associated with land habitats but rarely with ocean environments. The ocean forms the largest portion of our planet, yet ocean-derived organisms are the least explored, and these organisms are primarily NMOs. However, these are thrilling living entities, such as ocean-derived fungi (ODF). These ODFs are a diverse group of fungi that live in different ocean sectors, including the ocean, estuaries, and coastal ecosystems. These fungi are found to colonize and adapt to different substrates. They are important decomposers in marine ecosystems, breaking down dead organic matter and recycling nutrients. ODFs have adapted to survive in the unique and challenging conditions of the ocean environment, including high salt concentrations, low nutrient availability, and exposure to waves and currents. ODFs are potent producers of natural compounds with pharmaceutical and industrial applications, such as antibiotics, anticancer agents, antivirals, and enzymes for industrial processes. ODFs are an exciting group of fungi; however, these are the least studied because of the nonavailability of MOs from this group. Hence, there is a massive scope of expanding our current knowledge about ODFs, their genetic traits, potential future drug-producing capabilities, and lifestyle traits.With the advent of next-generation DNA sequencing, there is huge potential for the characterization of the genetic material of ODF as NMOs. Parallel proteomic methods also pose huge potential. A marriage of NGS and proteomic methods generates a new avenue called proteogenomics, which focuses on better annotation of existing genomic data. Both methods are getting cheaper and accessible to the research community for studying the proteogenomics of NMOs. Herein, the proteogenomic protocol development and data analyses are illustrated for the ocean-derived fungus Scopulariopsis brevicaulis.

Protocol to induce cell labeling in vivo and to trace labeled hematopoietic cells using mouse models.

A combination of hematopoietic stem cell (HSC)- and endothelial cell (EC)-lineage-tracing mouse models enables us to determine blood cell origins. We present a protocol to induce cell labeling in vivo and to trace labeled hematopoietic cells to segregate their origins. We describe the steps for harvesting various hematopoietic tissues, antibody staining, and analyzing the Tomato+ percentages within each immune cell population. We also show how to estimate HSC- and EC-derived percentages of the target cell populations. For complete details on the use and execution of this protocol, please refer to Kobayashi et al.1.

Promising tools into oxidative stress: A review of non-rodent model organisms.

Oxidative stress is a crucial concept in redox biology, and significant progress has been made in recent years. Excessive levels of reactive oxygen species (ROS) can lead to oxidative damage, heightening vulnerability to various diseases. By contrast, ROS maintained within a moderate range plays a role in regulating normal physiological metabolism. Choosing suitable animal models in a complex research context is critical for enhancing research efficacy. While rodents are frequently utilized in medical experiments, they pose challenges such as high costs and ethical considerations. Alternatively, non-rodent model organisms like zebrafish, Drosophila, and C. elegans offer promising avenues into oxidative stress research. These organisms boast advantages such as their small size, high reproduction rate, availability for live imaging, and ease of gene manipulation. This review highlights advancements in the detection of oxidative stress using non-rodent models. The oxidative homeostasis regulatory pathway, Kelch-like ECH-associated protein 1-Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2), is systematically reviewed alongside multiple regulation of Nrf2-centered pathways in different organisms. Ultimately, this review conducts a comprehensive comparative analysis of different model organisms and further explores the combination of novel techniques with non-rodents. This review aims to summarize state-of-the-art findings in oxidative stress research using non-rodents and to delineate future directions.

Protocol for investigating light/dark locomotion in larval stage zebrafish using a standardized behavioral assay.

Zebrafish (Danio rerio) larvae offer a unique avenue for high-throughput in vivo investigation. The light/dark locomotor assay is widely used but lacks experimental consistency. Here, we present a protocol for a standardized light/dark assay by describing the steps for plating, acclimatizing larvae, performing the assay, and preparing drug exposure solutions. We also detail procedures for substance exposure and data analysis. For complete details on the use and execution of this protocol, please refer to Hillman et al.1.

Protocol for inducing varying TBI severity in a mouse model using a closed-head, weight-drop, impact-induced acceleration mechanism.

Animal models of traumatic brain injury (TBI) are critical for understanding its complex neuropathology. Here, we present a protocol to induce varying TBI severities in mice using a closed-head, weight-drop model that includes an impact-induced acceleration mechanism. We describe steps for habituation with neurological severity score (NSS) equipment, assessing NSS baseline, performing anesthesia and TBI, assessing NSS post-injury, and analyzing data. This protocol requires no prior surgical intervention and is adaptable for rat studies. For complete details on the use and execution of this protocol, please refer to PhD Dissertation of Javier Allende Labastida1 and Tang et al.2.

Mushroom against Cancer: Aqueous Extract of Fomitopsis betulina in Fight against Tumors.

Background/Objectives: This study investigated the anticancer potential of an aqueous extract of the fungus Fomitopsis betulina. Methods: The study assessed the effect of the extract on nine cancer cell lines, including melanoma (LM-MEL-75), lung cancer (A549), and colorectal cancer (HT29, LoVo), and four normal cell lines. The cytotoxicity of the extract was evaluated using MTT, sulforhodamine-B (SRB), and clonogenic viability assays. Additionally, the study examined the effect of the extract on plant model organisms, garden cress (Lepidium sativum) and common onion (Allium cepa), to further investigate its biological activity. Results: The assays demonstrated selective cytotoxicity of the extract toward cancer cells, while sparing normal cells. The extract induced significant cytotoxic effects at lower concentrations in lung cancer, melanoma, and colon cancer cells, showing promise as a potential anticancer agent. The results also revealed that the extract inhibited seed germination and root growth, suggesting its potential to disrupt cell cycles and induce apoptosis. Conclusions: This study highlights the therapeutic potential of F. betulina and highlights the need for further research to identify the active ingredients and mechanisms underlying its anticancer effects.

Understanding developmental system drift.

Developmental system drift (DSD) occurs when the genetic basis for homologous traits diverges over time despite conservation of the phenotype. In this Review, we examine the key ideas, evidence and open problems arising from studies of DSD. Recent work suggests that DSD may be pervasive, having been detected across a range of different organisms and developmental processes. Although developmental research remains heavily reliant on model organisms, extrapolation of findings to non-model organisms can be error-prone if the lineages have undergone DSD. We suggest how existing data and modelling approaches may be used to detect DSD and estimate its frequency. More direct study of DSD, we propose, can inform null hypotheses for how much genetic divergence to expect on the basis of phylogenetic distance, while also contributing to principles of gene regulatory evolution.

Protocol for monitoring clonal hematopoiesis in transgenic mouse models using multispectral imaging.

Clonal hematopoiesis involves the clonal expansion of hematopoietic cells, potentially progressing into hematological malignancies. Here, we present a protocol for the development and characterization of two mouse models designed to simulate clonal hematopoiesis and acute myeloid leukemia. We describe steps for model generation, monitoring clonal expansion, harvesting, fixation, and staining of bone marrow and spleen tissues. We specifically focus on the visualization and analysis of p53 mutant clonal expansions, providing a comprehensive protocol for studying these phenomena in mouse models. For complete details on the use and execution of this protocol, please refer to Pourebrahim et al.1.

Protocol for synchronized wireless fiber photometry and video recordings in rodents during behavior.

Fiber photometry technique allows investigation of in vivo neural activity during behavior allowing understanding of brain-behavior relationship. Here, we provide a protocol for synchronized wireless fiber photometry and video recordings in rodents during behavior. We explain the detailed steps for stereotaxic virus injection, optic fiber cannula implantation, setup for synchronized fiber photometry and behavioral recording, and analysis of photometry data. These protocol steps can be adapted for various animal models, photometry, and behavioral recording systems. For complete details on the use and execution of this protocol, please refer to Tamboli et al.1 and Amalyan et al.2.

Protocol for detecting genomic insulators in Drosophila using insulator-seq, a massively parallel reporter assay.

Genomic insulators are DNA elements that prevent transcriptional activation of a promoter by an enhancer when interposed. We present a protocol for insulator-seq that enables high-throughput screening of genomic insulators using a plasmid-based massively parallel reporter assay in Drosophila cultured cells. We describe steps for insulator reporter plasmid library generation, transient transfection into cultured cells, and sequencing library preparation and provide a pipeline for data analysis. For complete details on the use and execution of this protocol, please refer to Tonelli et al.1.

Enzymatic tools for mitochondrial genome manipulation.

Mutations in mitochondrial DNA (mtDNA) can manifest phenotypically as a wide range of neuromuscular and neurodegenerative pathologies that are currently only managed symptomatically without addressing the root cause. A promising approach is the development of molecular tools aimed at mtDNA cutting or editing. Unlike nuclear DNA, a cell can have hundreds or even thousands of mitochondrial genomes, and mutations can be present either in all of them or only in a subset. Consequently, the developed tools are aimed at reducing the number of copies of mutant mtDNA or editing mutant nucleotides. Despite some progress in the field of mitochondrial genome editing in human cells, working with model animals is still limited due to the complexity of their creation. Furthermore, not all existing editing systems can be easily adapted to function within mitochondria. In this review, we evaluate the mtDNA editing tools available today, with a particular focus on specific mtDNA mutations linked to hereditary mitochondrial diseases, aiming to provide an in-depth understanding of both the opportunities and hurdles to the development of mitochondrial genome editing technologies.

Comparative Analysis of Canine and Human HtrA2 to Delineate Its Role in Apoptosis and Cancer.

Therapeutically, targeting the pro- and anti-apoptotic proteins has been one of the major approaches behind devising strategies to combat associated diseases. Human high-temperature requirement serine protease A2 (hHtrA2), which induces apoptosis through both caspase-dependent and independent pathways is implicated in several diseases including cancer, ischemic heart diseases, and neurodegeneration, thus making it a promising target molecule. In the recent past, the canine model has gained prominence in the understanding of human pathophysiology that was otherwise limited to the rodent system.  Moreover, canine models in cancer research provide an opportunity to study spontaneous tumors as their size, lifespan, and environmental exposure are significantly closer to that of humans compared to laboratory rodents. Therefore, using HtrA2 as a model protein, comparative analysis has been done to revisit the hypothesis that canines might be excellent models for cancer research. We have performed evolutionary phylogenetic analyses that confirm a close relationship between canine and human HtrA2s. Molecular modeling demonstrates structural similarities including orientation of the catalytic triad residues, followed by in silico docking and molecular dynamics simulation studies that identify the potential interacting partners for canine HtrA2 (cHtrA2). In vitro biophysical and protease studies depict similarities in interaction with their respective substrates as well as transient transfection of cHtrA2 in mammalian cell culture shows induction of apoptosis. This work, therefore, promises to open a new avenue in cancer research through the study of spontaneous cancer model systems in canines.

Mitochondrial Dysfunctions: Genetic and Cellular Implications Revealed by Various Model Organisms.

Mitochondria play a crucial role in maintaining the energy status and redox homeostasis of eukaryotic cells. They are responsible for the metabolic efficiency of cells, providing both ATP and intermediate metabolic products. They also regulate cell survival and death under stress conditions by controlling the cell response or activating the apoptosis process. This functional diversity of mitochondria indicates their great importance for cellular metabolism. Hence, dysfunctions of these structures are increasingly recognized as an element of the etiology of many human diseases and, therefore, an extremely promising therapeutic target. Mitochondrial dysfunctions can be caused by mutations in both nuclear and mitochondrial DNA, as well as by stress factors or replication errors. Progress in knowledge about the biology of mitochondria, as well as the consequences for the efficiency of the entire organism resulting from the dysfunction of these structures, is achieved through the use of model organisms. They are an invaluable tool for analyzing complex cellular processes, leading to a better understanding of diseases caused by mitochondrial dysfunction. In this work, we review the most commonly used model organisms, discussing both their advantages and limitations in modeling fundamental mitochondrial processes or mitochondrial diseases.

Protocol for a non-surgical model of perineural invasion for assessing neural drivers of cancer aggressiveness in mice.

Perineural invasion (PNI) is a significant risk factor for cancer recurrence and metastasis; however, its mechanisms relating to cancer aggressiveness remain poorly understood. Here, we present a protocol for a non-surgical model of PNI in mice using a neurotropic melanoma cell line that migrates from the skin to the sciatic nerve. We describe the steps for cell culture and injection, tumor burden measurements, mouse euthanasia, and tissue dissection. We then detail procedures for sample cross-section and confocal imaging.

Protocol for the acquisition and maturation of oligodendrocytes from neonatal rodent brains.

The generation of an oligodendrocyte primary culture model encompassing the diverse stages of the lineage is essential for the in vitro research of oligodendrocyte physiology and pathophysiology. Here, we provide a protocol for generating oligodendrocytes from the neonatal rodent brain. We describe steps for isolating oligodendrocyte progenitor cells (OPCs) through differential centrifugation, their subsequent expansion, passaging, and differentiation. For complete details on the use and execution of this protocol, please refer to Kim et al.1.

Identification of non-model mammal species using the MinION DNA sequencer from Oxford Nanopore.

Background: The Neotropics harbors the largest species richness of the planet; however, even in well-studied groups, there are potentially hundreds of species that lack a formal description, and likewise, many already described taxa are difficult to identify using morphology. Specifically in small mammals, complex morphological diagnoses have been facilitated by the use of molecular data, particularly from mitochondrial sequences, to obtain accurate species identifications. Obtaining mitochondrial markers implies the use of PCR and specific primers, which are largely absent for non-model organisms. Oxford Nanopore Technologies (ONT) is a new alternative for sequencing the entire mitochondrial genome without the need for specific primers. Only a limited number of studies have employed exclusively ONT long-reads to assemble mitochondrial genomes, and few studies have yet evaluated the usefulness of such reads in multiple non-model organisms. Methods: We implemented fieldwork to collect small mammals, including rodents, bats, and marsupials, in five localities in the northern extreme of the Cordillera Central of Colombia. DNA samples were sequenced using the MinION device and Flongle flow cells. Shotgun-sequenced data was used to reconstruct the mitochondrial genome of all the samples. In parallel, using a customized computational pipeline, species-level identifications were obtained based on sequencing raw reads (Whole Genome Sequencing). ONT-based identifications were corroborated using traditional morphological characters and phylogenetic analyses. Results: A total of 24 individuals from 18 species were collected, morphologically identified, and deposited in the biological collection of Universidad EAFIT. Our different computational pipelines were able to reconstruct mitochondrial genomes from exclusively ONT reads. We obtained three new mitochondrial genomes and eight new molecular mitochondrial sequences for six species. Our species identification pipeline was able to obtain accurate species identifications for up to 75% of the individuals in as little as 5 s. Finally, our phylogenetic analyses corroborated the identifications from our automated species identification pipeline and revealed important contributions to the knowledge of the diversity of Neotropical small mammals. Discussion: This study was able to evaluate different pipelines to reconstruct mitochondrial genomes from non-model organisms, using exclusively ONT reads, benchmarking these protocols on a multi-species dataset. The proposed methodology can be applied by non-expert taxonomists and has the potential to be implemented in real-time, without the need to euthanize the organisms and under field conditions. Therefore, it stands as a relevant tool to help increase the available data for non-model organisms, and the rate at which researchers can characterize life specially in highly biodiverse places as the Neotropics.

Biotic interactions, evolutionary forces and the pan-plant specialized metabolism.

Plant specialized metabolism has a complex evolutionary history. Some aspects are conserved across the green lineage, but many metabolites are unique to certain lineages. The network of specialized metabolism continuously diversified, simplified or reshaped during the evolution of streptophytes. Many routes of pan-plant specialized metabolism are involved in plant defence. Biotic interactions are recalled as major drivers of lineage-specific metabolomic diversification. However, the consequences of this diversity of specialized metabolism in the context of plant terrestrialization and land plant diversification into the major lineages of bryophytes, lycophytes, ferns, gymnosperms and angiosperms remain only little explored. Overall, this hampers conclusions on the evolutionary scenarios that shaped specialized metabolism. Recent efforts have brought forth new streptophyte model systems, an increase in genetically accessible species from distinct major plant lineages, and new functional data from a diversity of land plants on specialized metabolic pathways. In this review, we will integrate the recent data on the evolution of the plant immune system with the molecular data of specialized metabolism and its recognition. Based on this we will provide a contextual framework of the pan-plant specialized metabolism, the evolutionary aspects that shape it and the impact on adaptation to the terrestrial environment.This article is part of the theme issue 'The evolution of plant metabolism'.

Protocol to quantify the activation dynamics of tumor-associated T cells in mice by functional intravital microscopy.

Tumor-associated T cells orchestrate cancer rejection after checkpoint blockade immunotherapy. T cell function depends on dynamic antigen recognition through the T cell receptor (TCR) resulting in T cell activation. Here, we present an approach to quantify the dynamics and magnitude of tumor-associated T cell activation at multiple time points in living mice using the genetically encoded calcium reporter Salsa6f and functional intravital microscopy (F-IVM). Our protocol allows researchers to measure the activation dynamics of various immune cells in vivo. For complete details on the use and execution of this protocol, please refer to Geels et al.1.

Protocol for the neonatal intracerebroventricular delivery of adeno-associated viral vectors for brain restoration of MECP2 for Rett syndrome.

Here, we present a protocol for neonatal intracerebroventricular (ICV) delivery of adeno-associated viral vectors (AAVs), achieving gene therapy for a Rett syndrome mouse model. We describe steps for preparing mouse lines, replacing foster mothers, sex typing, and genotyping. We then detail procedures for ICV delivery and validation through immunofluorescent and immunoblot techniques. This protocol is also applicable to preclinical gene therapy research that targets the neonatal mouse brain for other neurodevelopmental disorders. For complete details on the use and execution of this protocol, please refer to Yang et al.1.