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Lipids are required as integral building blocks of cells to support cellular structures and functions. The intricate mechanisms underpinning lipid homeostasis are essential for the health and maintenance of the central nervous system. Here we summarize the recent advances in dissecting the effect of lipid metabolism on cognitive function and its age-associated decline by reviewing relevant studies ranging from invertebrate model organisms to mammals including human.

Experimental models play a pivotal role in biomedical research, facilitating the understanding of disease mechanisms and the development of novel therapeutics. This is particularly true for neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and motor neuron disease, which present complex challenges for research and therapy development. In this work, we review the recent literature about experimental models and motor neuron disease. We identified three main categories of models that are highly studied by scientists. In fact, experimental models for investigating these diseases encompass a variety of approaches, including modeling the patient’s cell culture, patient-derived induced pluripotent stem cells, and organoids. Each model offers unique advantages and limitations, providing researchers with a range of tools to address complex biological questions. Here, we discuss the characteristics, applications, and recent advancements in terms of each model system, highlighting their contributions to advancing biomedical knowledge and translational research.

Neural activity is heterogeneous across different cortical areas and can change during learning. Here, we describe a protocol for longitudinal in vivo two-photon calcium imaging with an ultra-large cranial window that exposes most of the dorsal cortex in head-fixed mice. The large cranial window allows optical access to any dorsal cortical areas in individual mice. This protocol enables longitudinal tracking of neural activity from various cortical areas at cellular resolution to understand the cortical computations during behavioral tasks. For complete details on the use and execution of this protocol, please refer to Hattori et al. (2019), and Hattori and Komiyama, 2022a.

Myelin provides physical, neurotrophic, and metabolic support for axonal integrity. The thickness of CNS (central nervous system) myelin sheath is usually

Mechanistic toxicology seeks to identify the molecular and cellular mechanisms by which toxicants exert their deleterious effects. One powerful approach is to generate mutations in genes that respond to a particular toxicant, and then test how such mutations change the effects of the toxicant. CRISPR is a rapid and versatile approach to generate mutations in cultured cells and in animal models. Many studies use CRISPR to generate short insertions or deletions in a target gene and then assume that the resulting mutation, such as a premature termination codon, causes a loss of functional protein. However, recent studies demonstrate that this assumption is flawed. Cells can compensate for short insertion and deletion mutations, leading toxicologists to draw erroneous conclusions from mutant studies. In this review, we will discuss mechanisms by which a mutation in one gene may be rescued by compensatory activity. We will discuss how CRISPR insertion and deletion mutations are susceptible to compensation by transcriptional adaptation, alternative splicing, and rescue by maternally derived gene products. We will review evidence that measuring levels of messenger RNA transcribed from a mutated gene is an unreliable indicator of the severity of the mutation. Finally, we provide guidelines for using CRISPR to generate mutations that avoid compensation. [ABSTRACT FROM AUTHOR]

Philosophers examining mechanistic explanations in biology have identified heuristic strategies scientists use in discovering mechanisms. This paper examines the heuristic strategy of investigating phylogenetically distant model organisms, using research on sleep in fruit flies as an example. At the time sleep was discovered in flies in 2000 next to nothing was known about mechanisms regulating sleep in flies and what they could reveal about those in us. One relatively straightforward line of research focused on homologous genes in flies and humans, using those in flies to understand what roles their homologs played in controlling sleep in us. But other research focused on a higher level of organization—the neural networks involved in homeostatic and circadian control of sleep. This raises a puzzle—given that fly and vertebrate brains are organized very differently, how could sleep regulation in flies serve as an informative model of vertebrate sleep? I argue that the basic design of mechanisms such as those regulating sleep can be conserved even as the composition of the mechanism changes and that researchers can hope to use the designs deciphered in flies as heuristic models for understanding sleep in humans. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton plays a critical role in cell architecture and the control of fundamental processes including cell division, migration and survival. The dynamics and organisation of F-actin have been widely studied in a breadth of cell types on classical two-dimensional (2D) surfaces. Recent advances in optical microscopy have enabled interrogation of these cytoskeletal networks in cells within threedimensional (3D) scaffolds, tissues and in vivo. Emerging studies indicate that the dimensionality experienced by cells has a profound impact on the structure and function of the cytoskeleton, with cells in 3D environments exhibiting cytoskeletal arrangements that differ to cells in 2D environments. However, the addition of a third (and fourth, with time) dimension leads to challenges in sample preparation, imaging and analysis, necessitating additional considerations to achieve the required signal-to-noise ratio and spatial and temporal resolution. Here, we summarise the current tools for imaging actin in a 3D context and highlight examples of the importance of this in understanding cytoskeletal biology and the challenges and opportunities in this domain. [ABSTRACT FROM AUTHOR]

Background Consistent with postmortem findings in patients, most animal models for schizophrenia (SCZ) present abnormal levels of parvalbumin (PV), a marker of fast-spiking GABAergic interneurons, in the prefrontal cortex (PFC) and hippocampus (HIP). However, there are discrepancies in the literature. PV reductions lead to a functional loss of PV interneurons, which is proposed to underly SCZ symptoms. Given its complex etiology, different categories of animal models have been developed to study SCZ, which may distinctly impact PV levels in rodent brain areas. Study Design We performed a quantitative meta-analysis on PV-positive cell number/density and expression levels in the PFC and HIP of animal models for SCZ based on pharmacological, neurodevelopmental, and genetic manipulations. Results Our results confirmed that PV levels are significantly reduced in the PFC and HIP regardless of the animal model. By categorizing into subgroups, we found that all pharmacological models based on NMDA receptor antagonism decreased PV-positive cell number/density or PV expression levels in both brain areas examined. In neurodevelopmental models, abnormal PV levels were confirmed in both brain areas in maternal immune activation models and HIP of the methylazoxymethanol acetate model. In genetic models, negative effects were found in neuregulin 1 and ERBB4 mutant mice in both brain regions and the PFC of dysbindin mutant mice. Regarding sex differences, male rodents exhibited PV reductions in both brain regions only in pharmacological models, while few studies have been conducted in females. Conclusion Overall, our findings support deficits in prefrontal and hippocampal PV interneurons in animal models for SCZ. [ABSTRACT FROM AUTHOR]

This research paper focused on the monitoring of marine sites using mussels, which are highly valuable organisms in assessing environmental health. However, a significant challenge arises when determining the appropriate size of mussels for monitoring purposes. The objective of this study was to examine the levels of Cd, Pb, As, and Co in three different size classes of two mussel species, Mytilus galloprovincialis and Perna perna, collected from three sites along the Algerian coast, each exhibiting varying degrees of pollution. At each of the study sites, a total of thirty individuals from small, medium, and large size classes of mussels were collected during four different time periods. The mussels were then dissected, and the concentrations of Cd, Pb, As, and Co were measured in the entire flesh of the mussels using ICP-MS. Across the various study sites, the concentrations of cadmium, lead, arsenic, and cobalt ranged from 0.06 to 1.32 mg/kg, 0.09 to 12.56 mg/kg, 4.23 to 18.31 mg/kg, and 0.11 to 1.85 mg/kg, respectively. Interestingly, the distribution of these metals in the three different size classes of mussels followed a consistent pattern at all the study sites. Large mussels exhibited higher concentrations, while small and medium-sized mussels displayed lower levels. These findings highlight substantial spatial and temporal variations in metal concentrations within the studied sites. [ABSTRACT FROM AUTHOR]

The demand for respiratory disease and dynamic breathing studies has continuously driven researchers to update the pulmonary bronchial tree’s morphology model. This study aims to construct a bronchial tree morphology model efficiently and effectively with practical algorithms. We built a performance index system using failure branch rate, volume ratio, and coefficient of variation of terminal volumes to evaluate the model performance. We optimized the parameter settings and found the best options to build the morphology model, and we constructed a 14th-generation bronchial tree model with a decent performance index. The dimensions of our model closely matched published data from anatomic in vitro measurements. The proposed model is adjustable and computable and will be used in future dynamic breathing simulations and respiratory disease studies. [ABSTRACT FROM AUTHOR]

Simple Summary: The study of the innate immune system's (IIS) properties is one of the key tasks of modern immunology, since it could help enhance the quality of therapy and drug discovery. This system remains understudied for nonmammalians, and teleost fish are promising model organisms for fundamental studies of IIS organization. The recent single-cell transcriptomics (scRNAseq) experiments carried out on zebrafish and other teleosts brought fundamental knowledge about their innate immunity organization. The aim of this review is to summarize information about the available IIS-related scRNAseq experiments for teleosts and outline further perspectives for studies in this field. We found 89 scRNAseq datasets for zebrafish in different stages and organs suitable for the further meta-analysis of IIS properties and six experiments for other teleosts. Therefore, the obtained data for zebrafish could be further generalized and compared with that of mammals, while for other teleosts, we still have a large gap in knowledge. We believe that in the coming years, new scRNAseq data obtained for nonmodel teleosts will be of particular value and interest in the context of animal immunology. The innate immune system is the first line of defense in multicellular organisms. Danio rerio is widely considered a promising model for IIS-related research, with the most amount of scRNAseq data available among Teleostei. We summarized the scRNAseq and spatial transcriptomics experiments related to the IIS for zebrafish and other Teleostei from the GEO NCBI and the Single-Cell Expression Atlas. We found a considerable number of scRNAseq experiments at different stages of zebrafish development in organs such as the kidney, liver, stomach, heart, and brain. These datasets could be further used to conduct large-scale meta-analyses and to compare the IIS of zebrafish with the mammalian one. However, only a small number of scRNAseq datasets are available for other fish (turbot, salmon, cavefish, and dark sleeper). Since fish biology is very diverse, it would be a major mistake to use zebrafish alone in fish immunology studies. In particular, there is a special need for new scRNAseq experiments involving nonmodel Teleostei, e.g., long-lived species, cancer-resistant fish, and various fish ecotypes. [ABSTRACT FROM AUTHOR]

Anxiety disorders are common and can be debilitating, with effective treatments remaining hampered by an incomplete understanding of the underlying genetic etiology. Improvements have been made in understanding the genetic influences on mouse behavioral models of anxiety, yet it is unclear the extent to which genes identified in these experimental systems contribute to genetic variation in human anxiety phenotypes. Leveraging new and existing large‐scale human genome‐wide association studies, we tested whether sets of genes previously identified in mouse anxiety‐like behavior studies contribute to a range of human anxiety disorders. When tested as individual genes, 13 mouse‐identified genes were associated with human anxiety phenotypes, suggesting an overlap of individual genes contributing to both mouse models of anxiety‐like behaviors and human anxiety traits. When genes were tested as sets, we did identify 14 significant associations between mouse gene sets and human anxiety, but the majority of gene sets showed no significant association with human anxiety phenotypes. These few significant associations indicate a need to identify and develop more translatable mouse models by identifying sets of genes that "match" between model systems and specific human phenotypes of interest. We suggest that continuing to develop improved behavioral paradigms and finer‐scale experimental data, for instance from individual neuronal subtypes or cell‐type‐specific expression data, is likely to improve our understanding of the genetic etiology and underlying functional changes in anxiety disorders. [ABSTRACT FROM AUTHOR]

Emphatic claims of a "microbiome revolution" aside, the study of the gut microbiota and its role in organismal development and evolution is a central feature of so-called postgenomics; namely, a conceptual and/or practical turn in contemporary life sciences, which departs from genetic determinism and reductionism to explore holism, emergentism and complexity in biological knowledge-production. This paper analyses the making of postgenomic knowledge about developmental symbiosis in Drosophila melanogaster by a specific group of microbiome scientists. Drawing from both practical philosophy of science and Science and Technology Studies, the paper documents epistemological questions of artefactuality and representativeness of model organisms as they emerge in the day-to-day labour producing and being produced by the "microbiome revolution." Specifically, the paper builds on all the written and editorial exchanges involved in the troubled publication of a research paper studying the symbiotic role of the microbiota in the flies' development. These written materials permit us to delimit the network of justifications, evidence, standards of knowledge-production, trust in the tools and research designs that make up the conditions of possibility of a postgenomic fact. More than reframing the organism as a radically novel multiplicity of reactive genomes, we conclude, doing postgenomic research on the microbiota and symbiosis means producing a story that deviates from the scripts embedded into the sociotechnical experimental systems of post-Human Genome Project life sciences. [ABSTRACT FROM AUTHOR]

Invasive fungal diseases have profound effects upon human health and are on increase globally. The World Health Organization (WHO) in 2022 published the fungal priority list calling for improved public health interventions and advance research. Drosophila melanogaster presents an excellent model system to dissect host-pathogen interactions and has been proved valuable to study immunopathogenesis of fungal diseases. In this review we highlight the recent advances in fungal-Drosophila interplay with an emphasis on the recently published WHO’s fungal priority list and we focus on available tools and technologies.

Summary: Septic cardiomyopathy is associated with high mortality in septic patients, characterized by reversible systolic and diastolic dysfunction. It is essential to monitor cardiac function and hemodynamic changes in septic animals. Here, we present a protocol to monitor cardiac function and hemodynamics in septic rodents. We describe steps for performing cecal ligation and puncture on rodents to induce sepsis, acquiring two-dimensional echocardiographic and M-mode ultrasonic images, and assessing mean arterial pressure in septic animals.For complete details on the use and execution of this protocol, please refer to Zhang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: A subpopulation of pancreatic beta cells becomes senescent during type 1 diabetes (T1D) progression, and removal of these populations protects against T1D in mice. Here, we present a protocol to measure senescence in murine pancreatic islet cells through analysis of senescence-associated β-galactosidase activity. We describe steps for staining with the fluorogenic substrate C12FDG and analysis by flow cytometry. Increased cell size is another marker of senescence and can also be concurrently measured in the same experiment.For complete details on the use and execution of this protocol, please refer to Lee et al.1 and Helman et al.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Multiple patch-clamp recordings and morphological reconstruction are powerful approaches for neuronal microcircuitry dissection and cell type classification but are challenging due to the sophisticated expertise needed. Here, we present a protocol for applying these techniques to neurons in the medial entorhinal cortex (MEC) of mice. We detail steps to prepare brain slices containing MEC and perform simultaneous multiple whole-cell recordings, followed by procedures of histological staining and neuronal reconstruction. We then describe how we analyze morphological and electrophysiological features.For complete details on the use and execution of this protocol, please refer to Shi et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Despite optimal multimodal treatment including surgical resection, 50%–80% of high-grade soft tissue sarcoma (STS) patients metastasize. Here, we present a protocol for the generation and use of post-surgical minimal residual disease models to investigate metastatic relapse in STS patient-derived xenografts. We describe steps for orthotopic engraftment of high-grade STS patient-derived tumor tissue. We then detail procedures for primary tumor resection with broad, negative resection margins and follow-up until metastases using MRI.For complete details on the use and execution of this protocol, please refer to Fischer et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Ovariectomy, involving the surgical removal of ovaries, and estradiol replacement facilitate the understanding of sexual dimorphism-related physiological changes, encompassing reproductive biology, metabolism, and hormone-related diseases. In this study, we present a protocol for conducting ovariectomy and estradiol replacement in mice. We describe steps for performing sham and ovariectomy operations, outline preoperative preparations, and provide details on postoperative care, including analgesia administration and the removal of surgical clips. Additionally, we elaborate on the procedures for performing vehicle and estradiol injections.For complete details on the use and execution of this protocol, please refer to Luengo-Mateos et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The efficacy of chimeric antigen receptor (CAR) T cell immunotherapy is limited by insufficient infiltration and activation of T cells due to the immunosuppressive tumor microenvironment. Preclinical studies with optimized mouse CAR T cells in immunocompetent mouse cancer models will help define the mechanisms underlying immunotherapy resistance. Here, we present a protocol for preparing mouse T cells and generating CAR T cells. We then detail procedures for testing their therapeutic efficacy and tracking them in a syngeneic mouse glioma model.For complete details on the use and execution of this protocol, please refer to Zhang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Postnatal development of the uterus involves the specification of undifferentiated epithelium into uterine-type epithelium. That specification is regulated by stromal-epithelial interactions as well as intrinsic cell-specific transcription factors and gene regulatory networks. Here, we present a co-culture system to study the effects of stromal-derived factors on epithelial cell growth and differentiation into organoids. First, we describe epithelial cell isolation and organoid growth characterization. Second, we detail a co-culture system that allows the study of stromal-derived paracrine factors on epithelial development.For complete details on the use and execution of this protocol, please refer to Rizo et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Functional studies in post-embryonic Xenopus tadpoles are challenging because embryonic perturbations often lead to developmental consequences, such as lethality. Here, we describe a high-throughput protocol for tail vein injection to introduce fluorescent tracers into tadpoles, which we have previously used to effectively inject morpholinos and molecular antagonists. We describe steps for safely positioning tadpoles onto agarose double-coated plates, draining media, injecting into the ventral tail vein, rehydrating plates, and sorting tadpoles by fluorescence with minimal injury for high-throughput experiments.For complete details on the use and execution of this protocol, please refer to Kakebeen et al.,1 Patel et al.,2 and Patel et al.,3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Monosodium urate (MSU) crystal deposition in articular joints and bursal tissue causes acute joint inflammation, which is a hallmark of gout. Here, we describe the steps necessary to create a subcutaneous air pouch on the back of mice that resembles this bursa-like space with a synovial lining-like membrane. We then detail the injection of MSU crystals into this pouch, which induces a localized inflammatory response reminiscent of gout and approaches to quantify the inflammatory response.For complete details on the use and execution of this protocol, please refer to Devi et al. (2023),1 de Almeida et al. (2022),2 and Ratsimandresy et al. (2017).3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a protocol for estimating nuclear transport parameters in single cells. We describe steps for performing four consecutive fluorescence recovery after photobleaching experiments, fitting the obtained data to an ordinary differential equations model, and statistical analysis of the fittings using a specialized R package. This protocol permits the estimation of import and export rates, nuclear or cytosolic fixed fractions, and total number of molecules.For complete details on the use and execution of this protocol, please refer to Durrieu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Precise, on-target CRISPR-Cas9 genome editing has been shown in Schistosoma mansoni, involving both non-homology end joining and homology-directed repair pathways. Here, we present a multiplexed CRISPR-Cas9 protocol for large transgene integration into the S. mansoni genome. We describe steps for deploying multiplexed ribonucleoprotein complexes (RNPs) and donor DNA preparation. We then detail procedures for introducing RNPs into schistosome eggs by square-wave electroporation in the presence of a 5′ phosphorothioate-modified double-stranded donor transgene.For complete details on the use and execution of this protocol, please refer to Ittiprasert et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: While brain regions function in coordination to mediate diverse behaviors, techniques allowing simultaneous monitoring of many deep brain regions remain limited. Here, we present a multi-fiber recording protocol that enables simultaneous recording of fluorescence signals from multiple brain regions in freely behaving mice. We describe steps for assembling a multi-fiber array and patch cord, implantation, and recording. We then detail procedures for data extraction and visualization. This protocol enables a comprehensive view of the neural activity at the network level.For complete details on the use and execution of this protocol, please refer to Guo et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Atomic force microscope (AFM) is a powerful and versatile tool to determine the physical properties of cells. The force-distance curves obtained from AFM experiments can be used to determine the stiffness and viscoelastic properties of cells. Here, we present a protocol for the determination of viscoelasticity from live cells such as Drosophila hemocytes or mouse embryonic stem cells using AFM. This protocol has potential application in determining the physical properties of cells in healthy and diseased conditions.For complete details on the use and execution of this protocol, please refer to Mote et al. (2020),1 and Singh et al. (2023).2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Studying fetal hematopoiesis is challenging as hematopoiesis transitions from the liver to bone marrow. Obtaining human samples is not possible, and small animal models may not provide sufficient biological material. Here, we present a protocol for isolating hematopoietic cells from the nonhuman primate fetal liver and bone. We describe steps for using cells from the same fetus for fluorescence lifetime imaging microscopy to measure metabolism, assessing cellular function, and flow cytometry for immunophenotyping at the single-cell level.For complete details on the use and execution of this protocol, please refer to Nash et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Neoblasts are the only cells capable of proliferation in planarians. The traditional flow cytometry protocol using Hoechst inhibits the cell cycle. Here, we present a protocol for culturing and functionally manipulating planarian neoblasts using SiR-DNA-based flow cytometry. We describe steps for cell dissociation and staining, flow cytometry, and cell collection and culture. We then detail procedures for Nanoluciferase mRNA transfection. This protocol facilitates further investigations into the pluripotency and regeneration mechanisms within neoblasts.For complete details on the use and execution of this protocol, please refer to Lei et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Cholecystokinin (CCK) is the most abundant neuropeptide that broadly regulates the physiological status of animals. Here, we present a two-color laser theta burst stimulation (L-TBS) protocol for simultaneous activation of Schaffer collateral and perforant pathway in the hippocampus of CCK Cre mice. We describe steps for heterosynaptic long-term potentiation induction by L-TBS. This technique allows for the examination of the neurotransmitter roles in synaptic modulation and facilitates the exploration of pathological mechanisms in genetic models of brain disorders in mice.For complete details on the use and execution of this protocol, please refer to Su et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The isolated perfused kidney is a classic ex vivo preparation for studying renal physiology in general and vascular function. Here, we present a protocol for assessing myogenic tone in isolated mouse kidneys as well as vasodilatory and vasoconstrictive responses, expressed as perfusion pressure. We describe steps for pre-operative preparation, kidney and renal artery isolation, and connection of renal artery with glass cannula. We then detail how to measure pressure changes in perfused kidneys and the myogenic tone.For complete details on the use and execution of this protocol, please refer to Cui et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a protocol for marking engram cells to efficiently measure reactivation levels and their projection pathways. We describe steps for genetic manipulation utilizing transgenic mice and viral infections, labeling engram cells, and a modified version of CLARITY, a tissue-clearing technique. This protocol can be adapted to various research inquiries that involve assessing the overlap of cell populations and uncovering novel long-range connectivity pathways.For complete details on the use and execution of this protocol, please refer to Refaeli et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The whole kidney marrow (WKM) is the site for hematopoiesis in the adult zebrafish. Here, we present a protocol for analyzing hematopoietic lineages in the WKM of adult zebrafish. We describe steps for the isolation of hematopoietic cells from the WKM, the downstream analysis of total marrow cellularity, and analysis of cell populations by flow cytometry. We then detail procedures for May-Grünwald-Giemsa staining for analysis of cellular morphology and phenotyping.For complete details on the use and execution of this protocol, please refer to Mahony et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Forced metastasis models, those in which the step of intravasation is bypassed, can be used to investigate the mechanisms underlying metastasis and evaluate potential therapeutic targets. Here, we present a protocol for using three forced models of lung and liver metastasis to generate metastasis within 3–4 weeks in approximately 99% of injected mice. We describe steps for cancer cell preparation, mouse analgesia and anesthesia; injecting through intrasplenic, intraportal, and intravenous techniques; and daily evaluation of metastasis.For complete details on the use and execution of this protocol, please refer to Giannou et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The isolation of sufficient amounts of intact nuclei is essential to obtain high-resolution maps of chromatin accessibility via assay for transposase-accessible chromatin using sequencing (ATAC-seq). Here, we present a protocol for tag-free isolation of nuclei from both cell walled and cell wall-deficient strains of the green model alga Chlamydomonas reinhardtii at a suitable quality for ATAC-seq. We describe steps for nuclei isolation, quantification, and downstream ATAC-seq. This protocol is optimized to shorten the time of isolation and quantification of nuclei. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: To investigate underlying mechanisms for cancer metastasis and promising therapies in animal models, spontaneous metastasis models can be used to recreate metastasis development. Here, we present three mouse models of spontaneous lung and/or liver metastasis induction. We describe steps for cancer cell preparation, mouse analgesia, and three injection techniques (subcutaneous, intracecal, and intramucosal). We then detail procedures for evaluating metastasis. Most of these models generate metastasis in a time span of 4 weeks in the majority of injected mice.For complete details on the use and execution of this protocol, please refer to Giannou et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Whole-organism-based screen holds promise for discovering biologically active compounds. However, high-content imaging is challenging due to the difficulty of positioning live animals and individual variability of neuron counts. Here, we present a protocol to identify neuroprotective compounds for dopaminergic neurons in zebrafish using an image-based small-molecule screen. We describe steps for raising larvae, agarose embedding, and treatment to induce neurodegeneration. We then detail procedures for live confocal imaging, image processing, and data analysis.For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Ribosome profiling is a sequencing technique that provides a global picture of translation across a genome. Here, we present iRibo, a software program for integrating any number of ribosome profiling samples to obtain sensitive inference of annotated or unannotated translated open reading frames. We describe the process of using iRibo to generate a species’ translatome from a set of ribosome profiling samples using S. cerevisiae as an example.For complete details on the use and execution of this protocol, please refer to Wacholder et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: GCaMP8f is a sensitive genetically encoded Ca2+ indicator that enables imaging of neuronal activity. Here, we present a protocol to perform Ca2+ imaging of the Drosophila neuromuscular junction using GCaMP8f targeted to pre- or postsynaptic compartments. We describe ratiometric Ca2+ imaging using GCaMP8f fused to mScarlet and synaptotagmin that reveals Ca2+ dynamics at presynaptic terminals. We then detail “quantal” imaging of miniature transmission events using GCaMP8f targeted to postsynaptic compartments by fusion to a PDZ-binding motif.For complete details on the use and execution of this protocol, please refer to Li et al.,1 Han et al.,2 Perry et al.,3 and Han et al.4 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Although the male epididymal fat pad is an effective site for islet transplantation, females lack this tissue. Here, we present a protocol to assess the parametrial fat pad (PFP) adjacent to the uterine horn in females as an alternative site for islet transplantation. We describe steps for islet isolation from the pancreas, counting, transplantation into PFP, and monitoring for engraftment. Transplantation into PFP is minimally invasive, time efficient, and supports long-term engraftment of syngeneic islets and rejection of allogeneic islets.For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a protocol for modulating the function of the Cth2 mRNA-binding protein (RBP) in Saccharomyces cerevisiae. We describe steps to amplify and integrate mutations in Cth2 that affect its stability and function. Next, we detail the functional assay to verify the activity of the wild-type and mutant versions of Cth2 in yeast cells. This protocol can be adopted to modify the function of other RBPs with their respective functional mutations.For complete details on the use and execution of this protocol, please refer to Patnaik et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: C. elegans is a bacteria-eating soil-dwelling nematode. Typical cultivation of laboratory-reared populations occurs on bacteria-covered solid media, where they move along with sinusoidal undulations. Nematodes decelerate when they encounter food. Dopaminergic and serotonergic neurotransmission regulate this behavior. Here, we describe the procedure for determining food-dependent locomotion rate of fed and fasting nematodes. We detail steps for assay plate preparation, C. elegans synchronization, and assessment of locomotion. The behaviors we describe provide information regarding the animal’s physiological neuronal and muscular function.For complete details on the use and execution of this protocol, please refer to Petratou et al. (2023)1 and Sawin et al. (2000).2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Hunting in larval zebrafish begins with eye convergence and orienting turns, proceeds to approach swims, and ends with the strike, where larvae consume the prey. Here, we describe a protocol to present UV stimuli to zebrafish, which greatly increases the occurrence of hunting initiation and strikes. We also describe how we record and analyze strike behavior in head-fixed larvae. Our goals are to increase the robustness of prey capture and to allow other labs to implement the strike behavioral assay.For complete details on the use and execution of this protocol, please refer to Khan et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Alpha-synuclein (α-Syn) positron emission tomography (PET) imaging is a valuable approach for diagnosing and monitoring synucleinopathies-related diseases, such as Parkinson disease. Here, we present a protocol for screening potential α-Syn PET tracers using in vitro and ex vivo approaches. We describe steps for employing recombinant pre-formed fibrils and conducting screening procedures on neuronal models, mouse models, and patients’ brain tissue sections to assess the specificity and selectivity of the candidate compounds.For complete details on the use and execution of this protocol, please refer to Xiang et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by ExoELISA and western blot analysis. This allows for EVP cross-validation across different platforms.For complete details on the use and execution of this protocol, please refer to Hoshino et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Quiescent skeletal muscle stem cells (MuSCs) are morphologically and functionally heterogeneous and exhibit different lengths of cellular extensions, which we call protrusions. Here, we present a protocol for ex vivo two-photon imaging of MuSCs in their native environment. We describe steps for muscle dissection, fixation, embedding, imaging, and analysis of datasets. This protocol allows the examination of MuSC morphology and protrusions at the single-cell level as well as stem cell numbers.For complete details on the use and execution of this protocol, please refer to Ma et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The fluorescent non-canonical amino acid tagging (FUNCAT) technique has been used to visualize newly synthesized proteins in cell lines and tissues. Here, we present a protocol for measuring protein synthesis in specific cell types in the mouse brain using in vivo FUNCAT. We describe steps for metabolically labeling newly synthesized proteins with azidohomoalanine, which introduces an azide group into the polypeptide. We then detail procedures for binding a fluorophore-conjugated alkyne to the azide group to allow its visualization.For complete details on the use and execution of this protocol, please refer to tom Dieck et al. (2012)1 and Hooshmandi et al. (2023).2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a streamlined Agrobacterium-mediated transformation protocol for jute (Corchorus sp.). We describe steps to pierce and vacuum infiltrate imbibed jute seeds with Agrobacterium suspension. We then detail procedures for selecting transformed seeds by using a hygromycin-B-supplemented medium. This approach can achieve transformation efficiencies of 20.44% ± 1.17% and 15.55% ± 0.58% for tossa (C. olitorius) and white (C. capsularis) jute, respectively. Demanding minimal resources and time, this protocol can elevate genetic engineering research in jute fiber crops.For complete details on the use and execution of this protocol, please refer to Majumder et al. (2020).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Background We have been documenting the biological responses to low levels of radiation (natural background) and very low level radiation (below background), and thus these studies are testing mild external stimuli to which we would expect relatively mild biological responses. We recently published a transcriptome software comparison study based on RNA-Seqs from a below background radiation treatment of two model organisms, E. coli and C. elegans (Thawng and Smith, BMC Genomics 23:452, 2022). We reported DNAstar-D (Deseq2 in the DNAstar software pipeline) to be the more conservative, realistic tool for differential gene expression compared to other transcriptome software packages (CLC, Partek and DNAstar-E (using edgeR). Here we report two follow-up studies (one with a new model organism, Aedes aegypti and another software package (Azenta) on transcriptome responses from varying dose rates using three different sources of natural radiation. Results When E. coli was exposed to varying levels of K40, we again found that the DNAstar-D pipeline yielded a more conservative number of DEGs and a lower fold-difference than the CLC pipeline and DNAstar-E run in parallel. After a 30 read minimum cutoff criterion was applied to the data, the number of significant DEGs ranged from 0 to 81 with DNAstar-D, while the number of significant DEGs ranged from 4 to 117 and 14 to 139 using DNAstar-E and the CLC pipelines, respectively. In terms of the extent of expression, the highest foldchange DEG was observed in DNAstar-E with 19.7-fold followed by 12.5-fold in CLC and 4.3-fold in DNAstar-D. In a recently completed study with Ae. Aegypti and using another software package (Azenta), we analyzed the RNA-Seq response to similar sources of low-level radiation and again found the DNAstar-D pipeline to give the more conservative number and fold-expression of DEGs compared to other softwares. The number of significant DEGs ranged 31–221 in Azenta and 31 to 237 in CLC, 19–252 in DNAstar-E and 0–67 in DNAStar-D. The highest fold-change of DEGs were found in CLC (1,350.9-fold), with DNAstar-E (5.9 -fold) and Azenta (5.5-fold) intermediate, and the lowest levels of expression (4-fold) found in DNAstar-D. Conclusions This study once again highlights the importance of choosing appropriate software for transcriptome analysis. Using three different biological models (bacteria, nematode and mosquito) in four different studies testing very low levels of radiation (Van Voorhies et al., Front Public Health 8:581796, 2020; Thawng and Smith, BMC Genomics 23:452, 2022; current study), the CLC software package resulted in what appears to be an exaggerated gene expression response in terms of numbers of DEGs and extent of expression. Setting a 30-read cutoff diminishes this exaggerated response in most of the software tested. We have further affirmed that DNAstar-Deseq2 gives a more conservative transcriptome expression pattern which appears more suitable for studies expecting subtle gene expression patterns.

The neural crest (NC), also known as the “fourth germ layer”, is an embryonic structure with important contributions to multiple tissue and organ systems. Neural crest cells (NCCs) are subjected to epithelial to mesenchymal transition and migrate throughout the embryo until they reach their destinations, where they differentiate into discrete cell types. Specific gene expression enables this precise NCCs delamination and colonization potency in distinct and diverse locations therein. This review aims to summarize the current experimental evidence from multiple species into the NCCs specifier genes that drive this embryo body axes segmentation. Additionally, it attempts to filter further into the genetic background that produces these individual cell subpopulations. Understanding the multifaceted genetic makeup that shapes NC-related embryonic structures will offer valuable insights to researchers studying organogenesis and disease phenotypes arising from dysmorphogenesis.