JoVE Neuroscience is a multidisciplinary section devoted to investigations of the structure, function, physiology, and pathophysiology of the brain and nervous system. Included methodologies range from molecular and cellular level studies to full central and peripheral neural systems. Potential treatment platforms and surgical techniques for neurological diseases and disorders are also presented in this section.

Neuroscience

JoVE Medicine serves as a connection between basic research and clinical applications. Articles may present medical procedures, case studies or clinical trial methodologies. Included studies are often modeled towards predictive, preventative, personalized, and practical approaches to treatment of disease. The goal of research methodologies in this section is to improve overall patient care and prognosis.

Medicine

JoVE Immunology and Infection includes content from a variety of fields working toward a shared initiative of improving global health. Techniques evaluating the biological response to pathogens from the molecular to organismal level are presented as well as therapeutic agents and their efficacy in treating disease.

Immunology and Infection

JoVE Genetics publishes video articles demonstrating experimental approaches to investigate gene function and regulation. This includes methods to study gene expression, mutagenesis, epigenetics, genetic determinants of development, genetic disorders, population genetics and evolution.

Genetics

JoVE Environment is dedicated to research methodologies that address environmental concerns and seek to better understand Earth's ecosystem.  Special consideration is given to experimental methodologies that assess society’s impact on the environment, suggest solutions for protecting Earth’s resources, and develop sustainable fuel sources. Moving towards an environmentally conscious planet is a collaborative effort with contributions from both life and physical scientists.

Environment

JoVE Engineering encompasses a broad range of experimental and instrumental techniques utilized in physics research. Investigations in this area strive to address and answer a broad range of scientific questions, such as device mechanisms and efficiencies, using physical tools. This approach often requires a combination of specialties, and research in this area tends to be interdisciplinary with contributions from mechanical, electrical, and chemical engineers.

Engineering

JoVE Developmental Biology publishes research methodologies in the field of developmental biology. This includes methods that are used to study biological development in vitro and in vivo at the molecular, cellular, tissue, organ and whole organism levels.

Developmental Biology

JoVE Chemistry surveys a broad scope of chemical disciplines. This collection includes fundamental and applied research that investigates molecular interactions, properties, and structure spanning in the following core divisions: analytical, organic, organometallic, inorganic, bioinorganic, and biochemistry. This section also includes the design and preparation of materials and small molecules for advanced or unique applications.

Chemistry

JoVE Cancer Research illustrates experimental approaches in biomedical research and clinical practice aimed at understanding, detecting, treating and preventing cancer. The video articles in this section demonstrate methods to study the cellular and molecular mechanisms of cancer, animal models of cancer research, development of new therapies and diagnostics, tumor imaging, chemical and radiation treatments, surgical procedures and improvements in therapy. These video articles offer researchers and clinical practitioners an efficient way to learn the technologies and experimental methods in cancer research.

Cancer Research

JoVE Biology welcomes all general biology research methodologies. Content in this section canvases all fields of cell, molecular, and organismal biology, ranging from new applications of standard techniques to novel approaches aimed at understanding the functions of life and living organisms. This diverse section includes, but is not limited to, techniques in physical biology, cellular biochemistry, genetics, physiology, systems biology and a combination of eukaryotic and prokaryotic model systems.

Biology

JoVE Bioengineering merges both physical and life sciences to understand and predict biological processes. Applying physical science tools to life science questions allow for the discovery of better technologies to measure, diagnose, and clinically treat disease.

Bioengineering

JoVE Biochemistry publishes experiments that investigate the structure, function, and interactions of biological molecules. The video articles in this section demonstrate methods to study nucleic acids, proteins, carbohydrates and lipids as well as enzyme function, protein folding, molecular interactions and biochemical reactions.

Biochemistry

JoVE Behavior documents observational and experimental techniques that seek to understand human and animal behavior. Work in this field includes analysis of genetic causes and physiological responses to internal or external stimuli as well as the implicated brain regions.

Behavior

Biomimetic lipid membranes in the form of giant unilamellar vesicles (GUVs) are commonly used to mimic cellular membrane behavior because of the ease of protein reconstitution inside GUVs, visualization, as well as understanding cellular membrane-protein dynamics. However, cell membranes comprise lipid rafts (or domains) arising from the presence of different lipids in the cellular membrane. Such increased complexity in model systems can be incorporated to result into phase separated GUVs, where lipid composition can be finely tuned. While encapsulation methods for the generation of homogeneous GUVs are widely known, methods to encapsulate proteins within phase separated GUVs are limited. Here, this protocol presents a simplified one-pot production of phase separated GUVs, comprised of liquid-disordered (Ld) and liquid ordered (Lo) domains, efficiently encapsulating different cytoskeletal proteins, i.e., FtsZ and actin, making the method a versatile tool for minimal cell production. Specifically, this approach uses an emulsion transfer protocol to produce GUVs with a high encapsulation efficiency. In this method, a lipid-monolayer is first generated by emulsifying a protein solution in a lipid/oil mixture, where lipids of varying phase transition temperatures are chosen to yield phase separation in the resultant GUVs. This emulsion is transferred gently on top of a lipid-in-oil solution in another tube, resulting in the formation of a water-oil interface. The solution is then centrifuged at elevated temperatures (ideally at 37 &#176;C to retain protein activity), after which GUVs are collected for imaging. This method simplifies the in vitro reconstitution of cytoskeletal proteins within phase separated GUVs without using a cumbersome laboratory setup, and thus serves as a convenient method for studying the mechanics of cytoskeletal-membrane interactions in confinement.

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In vitro Reconstitution of Cytoskeletal Networks inside Phase Separated Giant Unilamellar Vesicles (GUVs)

Biomimetic lipid membranes in the form of giant unilamellar vesicles (GUVs) are commonly used to mimic cellular membrane behavior because of the ease of ...

The Type VI Secretion System (T6SS) is a crucial mechanism mediating intercellular interactions in Gram-negative bacteria, particularly in pathogenic species such as Acinetobacter baumannii. Previous studies have shown that the large plasmid pAB3 in the A. baumannii ATCC 17978 strain encodes a TetR-like protein that inhibits the expression of core T6SS genes. In contrast, the WTR- strain, which lacks pAB3, can stably express and secrete the T6SS effector protein Hcp and exhibits the ability to kill E. coli. The tssM gene, one of the core genes of T6SS, is essential for its activity; its deletion directly leads to the inactivation of T6SS. However, traditional T6SS activity detection methods, such as killing assays, suffer from low throughput and insufficient sensitivity. To address these limitations, we have developed quantitative detection methods based on fluorescent labeling.
To improve T6SS activity detection, we developed three fluorescent labeling methods: (1) A quantitative detection method based on Luciferase labeling, which is characterized by high specificity, sensitivity, and reproducibility, making it suitable for high-throughput analysis; (2) A detection method based on green fluorescent protein (GFP) labeling, which, despite being susceptible to environmental interference, offers the advantage of high throughput; (3) Flow cytometry detection, which can quantitatively assess bacterial viability but is operationally complex and costly. After a comprehensive comparison, the Luciferase-based labeling method proved to be the most accurate, sensitive, and user-friendly. When applied to 20 clinical isolates of A. baumannii, this method was confirmed to rapidly and accurately evaluate T6SS activity.

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Establishment and Comparison of Fluorescence-Based T6SS Activity Detection Methods in Acinetobacter baumannii

The Type VI Secretion System (T6SS) is a crucial mechanism mediating intercellular interactions in Gram-negative bacteria, particularly in pathogenic ...

This protocol introduces a unique methodology for investigating the stability of emulsions and the dynamic disintegration of emulsion droplets during enzymatic digestion using dual-trap optical tweezers. The process involves preparing the emulsion, introducing the enzyme, and configuring a force measurement system to monitor inter-droplet forces in real time. By employing dual-trap optical tweezers, the method enables precise manipulation and observation of emulsion droplets, facilitating the measurement of forces between droplets during enzymatic disintegration. This experimental technique offers significant insights into the interactions and stability of emulsions during digestion, with particular relevance to research in food science, pharmaceuticals, and other industries involving organic emulsions. The method is adaptable and applicable to various optical tweezer setups and different types of emulsions or enzymes, serving as a versatile tool for exploring mechanical properties and disintegration processes across a range of fields. Furthermore, this visual approach provides the advantage of real-time monitoring of force dynamics, opening new avenues for studying emulsion behavior in diverse applications.

real-time-force-measurement-between-emulsion-droplets-during

Real-Time Force Measurement Between Emulsion Droplets During Enzymatic Breakdown

This protocol introduces a unique methodology for investigating the stability of emulsions and the dynamic disintegration of emulsion droplets during ...

Platelets, or thrombocytes, are small anucleated blood cells that play a crucial role in hemostasis and thrombosis. Defects in platelet functions can cause bleeding or thrombotic events in patients with cardiovascular diseases. Therefore, it is important to characterize platelets phenotypically to be able to assign platelet subpopulations to platelet function. Stimulation of human platelets with platelet agonists and activators induces morphological and physiological changes in platelets, accompanied by changes in the surface receptor population. This leads to functionally diverse platelet subpopulations. Classically defined platelet subpopulations are resting, aggregatory, procoagulant, and apoptotic platelets. To characterize the effect of agonists on platelet subtypes, we established an assay using multi-color flow cytometry consisting of 10 different antibodies and dyes (anti-CD62P, anti-CD63, anti-CD61, anti-CD41, anti-CD42b, anti-Integrin&#945;IIb&#946;3 (clone: PAC-1), anti-CXCR4, anti-ACKR3, Annexin V, Zombie NIR). Isolated human platelets were incubated with platelet agonists and stained with specific fluorophore-conjugated antibodies and dyes. Afterwards, they were measured by flow cytometry. This allows us to define agonist-specific subtypes within the classic four subpopulations. In conclusion, the combination of activation markers (anti-CD62P, anti-CD63, anti-Integrin&#945;IIb&#946;3 (clone: PAC-1)), inflammatory markers (anti-CXCR4, anti-ACKR3) and apoptotic markers (Annexin V, Zombie NIR) that compose the 10-color flow cytometry panel described in this manuscript opens up the possibility to define further platelet subtypes that could be linked to specific platelet function. This method can be applied in basic research on platelet function and physiology, as well as in defining new platelet subtypes in disease models and patient studies.

analyzing-platelet-subpopulations-by-multi-color-flow-cytometry

Analyzing Platelet Subpopulations by Multi-color Flow Cytometry

Platelets, or thrombocytes, are small anucleated blood cells that play a crucial role in hemostasis and thrombosis. Defects in platelet functions can ...

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) virus-like particle (SC2-VLP) method offers a powerful and accessible tool for studying the SARS-CoV-2 life cycle without the need for biosafety level 3 (BSL-3) laboratories. This system effectively mimics critical stages of the viral life cycle, including assembly, genome packaging, and egress, using a luciferase reporter fused to the T20 signal for sensitive and precise detection of viral particle production. SC2-VLPs are generated by co-expressing SARS-CoV-2 structural proteins, including membrane (M), nucleocapsid (N), envelop (E), and spike (S), along with the RNA packaging signal in HEK-293T cells. Unlike traditional virus-like particle systems, the SC2-VLP method ensures accurate quantification and greater fidelity to the natural viral life cycle. Furthermore, compared to lentiviral pseudotyping methods, which are limited to studying viral entry through the incorporation of S protein into HIV-based lentiviral particles, the SC2-VLP system provides a more comprehensive platform for exploring multiple stages of SARS-CoV-2 biology. While this method bypasses the risks of handling live virus and expands accessibility. The SC2-VLP method represents a significant advancement in antiviral research and the development of therapeutic strategies against SARS-CoV-2.

production-sars-cov-2-virus-like-particle-system-to-investigate-viral

Production of a SARS-CoV-2 Virus-Like-Particle System to Investigate Viral Life Cycles In Vitro

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) virus-like particle (SC2-VLP) method offers a powerful and accessible tool for studying ...

High salt intake is a major risk factor for hypertension, and its underlying mechanism may be closely linked to extracellular vesicles (EVs) secreted by gut microbiota. These EVs, produced by gut microbiota, carry various bioactive components that may play a crucial role in the development of hypertension induced by a high-salt diet (HSD). To investigate this mechanism, we developed an efficient extraction method based on density gradient centrifugation to isolate EVs from the gut microbiota of salt-sensitive rats fed an HSD. Through particle size analysis, transmission electron microscopy (TEM), and lipopolysaccharide (LPS) detection, we identified the gradient distribution of gut microbiota EVs and achieved precise extraction. Furthermore, 16S rRNA gene sequencing was employed to analyze the origin and compositional differences of EVs between the normal and HSD groups, revealing the impact of high salt intake on the genetic characteristics of gut microbiota EVs. This study provides valuable tools and scientific insights into the gut microbiota mechanisms underlying salt-induced hypertension and offers new perspectives for the prevention and treatment of related diseases.

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Separation and Differential Characterization of Gut Microbial Extracellular Vesicles in Salt-Sensitive Rats under High-Salt Diet Conditions

High salt intake is a major risk factor for hypertension, and its underlying mechanism may be closely linked to extracellular vesicles (EVs) secreted by ...

The development of genome-wide plasmid libraries using existing genomic repositories serves as a pivotal prerequisite for systematic functional characterization of genes across diverse biological processes. Current high-throughput methodologies for inter-vector DNA fragment transfer, however, necessitate PCR amplification of target sequences prior to cloning, rendering the generation of genome-scale plasmid collections technically demanding and time-intensive. By leveraging a CRISPRshuttle cassette, we developed a new high-throughput cloning method, CRISPR-based shuttle cloning (CRISPRshuttle cloning), which facilitates the transfer of many DNA fragments from donor plasmids sharing identical backbone sequences to a CRISPRshuttle-compatible vector without PCR amplification of the DNA fragments. Here, we present a protocol for CRISPRshuttle. This protocol involves two sequential test tube reactions prior to bacterial transformation. First, target DNA fragments are excised from donor plasmids by Cas9-mediated cleavage of their shared vector backbone sequence. Second, the excised DNA fragments are inserted into linearized CRISPRshuttle-compatible vectors through Gibson assembly. Our results demonstrate that the efficiency of CRISPRshuttle exceeds 94% and that two researchers can generate about 300 plasmids in 7 days using CRISPRshuttle. CRISPRshuttle facilitates efficient, adaptable, and cost-effective DNA fragment transfer between vectors, significantly streamlining genome-wide plasmid library generation.

crispr-based-shuttle-cloning-a-high-throughput-cloning-method

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

The development of genome-wide plasmid libraries using existing genomic repositories serves as a pivotal prerequisite for systematic functional ...

Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. Historically, keratinocyte division behavior was investigated via methods such as clonal analysis, immunostaining, and cell cycle analysis. None of these methods allow for the analysis of keratinocyte behavior at the single-cell level in real time. Over the past decade, groups have utilized live cell imaging to identify keratinocyte stem cells and committed progenitors without the need for labeling. Differences have been identified in each respective group's division behavior, rate of terminal differentiation, and cell cycle duration. Here, a method for keratinocyte live cell imaging with time-lapse photography and its analysis is described. Utilizing unpassaged keratinocytes is recommended for this method to most closely mimic in vivo behavior. Live cell imaging provides a unique ability to study stem cell and committed progenitor behavior at the single cell level and to determine division fates, cell cycle duration, as well as other proliferation metrics.

live-cell-imaging-with-time-lapse-photography-to-study-epidermal

Live Cell Imaging with Time Lapse Photography to Study Epidermal Keratinocyte Proliferation Kinetics

Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. Historically, keratinocyte division behavior was ...

The mitochondrial respiratory chain is crucial for cellular energy metabolism, serving as the core of oxidative phosphorylation. The mitochondrial respiratory chain comprises five enzyme complexes and their interacting supercomplexes. Analysis of the expression and complexes assembly of these proteins is vital to understanding mitochondrial function. This can be studied by combining biochemical and genetic methods in an excellent model organism fission yeast Schizosaccharomyces pombe (S. pombe), which provides a compensatory system to budding yeast for studies of mitochondrial biology. Here, we present a detailed protocol for the isolation of S. pombe mitochondria and analysis of expression levels and complexes assembly of the mitochondrial respiratory proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blue native-PAGE (BN-PAGE). Briefly, mitochondria from the wild-type and gene mutants are purified, and then their complexes are solubilized and subjected to SDS-PAGE/BN-PAGE and immunoblotting. This method enables the characterization of a gene's novel function in the mitochondrial respiratory chain.

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Analysis of the Expression and Complexes Assembly of the Mitochondrial Respiratory Chain Proteins in the Fission Yeast Schizosaccharomyces pombe

The mitochondrial respiratory chain is crucial for cellular energy metabolism, serving as the core of oxidative phosphorylation. The mitochondrial ...

Defining the sequence of events in renal disease is the cornerstone of clinical practice in the nephrologist toolkit. Tissue proteomic analyses are a significant approach to understanding the fundamental physiological and molecular processes of renal pathophysiology. The methods and protocols we present here will allow for the molecular dissection of the kidney in each specific region of interest related to disease sequelae. To determine the effects of disease on specific kidney regions and structures with unique functions, the goals of this protocol are to demonstrate simplified mouse kidney compartmentalization and renal cortical tubule isolation techniques in tandem with streamlined label-free quantitative proteomic workflows. Combining these methods will assist in the identification of perturbed molecular patterns in the whole kidney, medullary compartments, and cortical tubule structures of kidneys, with the ultimate and eventual goal of single-cell proteomics in pathological contexts. Applying these methods in virtually any disease model will be helpful in delineating mechanisms of pathology related to kidney dysfunction.

comparative-proteomic-analysis-whole-kidney-medulla-cortical-tubules

Comparative Proteomic Analysis of Whole Kidney, Medulla, and Cortical Tubules in Diabetic Pathogenesis of Kidney Injury in Mice

Defining the sequence of events in renal disease is the cornerstone of clinical practice in the nephrologist toolkit. Tissue proteomic analyses are a ...

Journal - biochemistry