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

Implant-associated infections represent a significant clinical challenge, as they arise when pathogenic microorganisms infiltrate the site of medical implants, such as joint replacements, catheters, or pacemakers. These infections can lead to severe complications, including implant failure, prolonged hospitalization, and the need for additional surgical interventions. To investigate these critical issues, murine models provide an invaluable platform for studying implant-associated infections and evaluating therapeutic strategies against bacterial biofilms, yielding insights that may ultimately improve clinical outcomes for patients afflicted by infected implants. The commonly employed materials in these studies include titanium, polyethylene, and stainless steel, which closely mimic those used in clinical practice. The experimental design generally involves a surgical procedure to implant the device, which is often performed subcutaneously to simulate joint or skin-related infections. Following implantation, a biofilm-forming bacterial strain, such as Staphylococcus aureus or Escherichia coli, is inoculated at the site to initiate infection, thereby establishing a model that mirrors the pathophysiology of human infections. Researchers have rigorously assessed the effectiveness of various antibiotic regimens against established biofilm infections, employing both systemic and localized delivery methods. Investigations have focused on novel antimicrobial agents, biofilm-disrupting compounds, and innovative therapeutic approaches aimed at augmenting the host immune response. Efficacy evaluation encompasses quantifying bacterial loads on the implant and surrounding tissue, alongside assessing reductions in biofilm thickness and density posttreatment. Clinical symptomatology, including changes in swelling and pain, serves as an additional indicator of therapeutic success. Furthermore, overall survival and morbidity rates among murine subjects provide critical insights into the safety and effectiveness of the evaluated treatments. It is imperative that all studies adhere to rigorous ethical guidelines and secure necessary institutional approvals, ensuring that findings can be reliably replicated across different laboratories. This methodological rigor is essential for validating results and advancing our understanding of effective treatments for implant-associated infections.

animal-model-of-implant-associated-infections-in-mice

Animal Model of Implant-Associated Infections in Mice

Implant-associated infections represent a significant clinical challenge, as they arise when pathogenic microorganisms infiltrate the site of medical ...

Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), and Ureaplasma urealyticum (UU) are common pathogens associated with sexually transmitted diseases (STDs). NG is the causative agent of gonorrhea, which can result in various disorders of the genitourinary system, including epididymitis, prostatitis, cervicitis, and infertility. CT is a microorganism responsible for non-gonococcal urethritis and pelvic inflammatory disease. UU is associated with non-gonococcal urethritis, cervicitis, prostatitis, and infertility. Current molecular technologies for the analysis of NG, CT, and UU are often complex and time-consuming. Rapid molecular testing for these pathogens may facilitate the early diagnosis of STDs. The RNA amplification lateral flow assay (RGT) is a method that does not require nucleic acid extraction. In this process, samples are lysed to release nucleic acid fragments, which are then amplified through reverse transcription and transcription. The amplified RNA product is recognized and captured by specific probes, forming an RNA-detection probe-gold probe complex that can be immobilized on a nitrocellulose membrane via lateral flow to produce visible bands. The entire procedure takes less than 1 h. The aim of this study was to evaluate the efficacy of RGT for the rapid detection of NG, CT, and UU. A total of 1,416 samples were included. The consistency of NG, CT, and UU when analyzed by RGT compared to the polymerase chain reaction (PCR) fluorescence probing method was 99.03% (307/310), 99.38% (159/160), and 99.02% (303/306), respectively. The consistency of NG, CT, and UU when analyzed by RGT compared to gene sequencing was 99.17% (238/240), 98.95% (188/190), and 98.30% (173/176), respectively. Compared with the pathogen isolation culture method, the detection rates for NG, CT, and UU assays were 100.00% (9/9, 17/17, 8/8). The high sensitivity and specificity, ease of use, and reduced time requirements make this assay ideal for the rapid and accurate detection of NG, CT, and UU.

rapid-identification-neisseria-gonorrhoeae-chlamydia-trachomatis

Rapid Identification of Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum by RNA Amplification Lateral Flow Assay

Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), and Ureaplasma urealyticum (UU) are common pathogens associated with sexually transmitted diseases ...

The widespread accessibility of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology has made gene targeting in primary cells a routine method for evaluating gene function in T cells. Given the cost and limited availability of knockout (KO) mouse strains, testing preliminary hypotheses involving gene function in T cells can be prohibitive using gene-targeted animal models. However, using commercially available resources, including predesigned guide RNAs (gRNAs), researchers can conveniently generate gene-targeted na&#239;ve T cells that can be used for T cell activation and differentiation studies. 
Here we outline a protocol for using nucleofection-delivered CRISPR-Cas9 ribonucleoprotein complexes (RNPs) to efficiently generate gene KO murine na&#239;ve CD4 T cells that can be used to evaluate gene function in CD4 T cell differentiation, in vitro. Isolation of na&#239;ve CD4 T cells from mouse secondary lymphoid organs, followed by nucleofection with Cas9-gRNA complexes ensures gene KO is initiated before downstream T cell activation, offering a strategic advantage over retroviral-mediated gRNA delivery, which typically requires preactivation of T cells, preventing the evaluation of effects in na&#239;ve T cells. Furthermore, this nucleofection-based method bypasses potential developmental issues associated with gene KO animals. 
Following Cas9-gRNA delivery, we describe protocols for studying CD4 T cell differentiation into Th1, Th2, Th17, and Treg lineages using in vitro polarization. In addition, this protocol is adaptable to using gene-targeted CD4 or CD8 T cells for numerous downstream applications, including other T cell activation studies in vitro and adoptive transfer studies in vivo. The use of CRISPR-Cas9 methods has streamlined our ability to evaluate gene function in T cells and allows for the routine KO of many genes of interest, freeing researchers from limitations associated with studying gene KO animals.

investigating-murine-cd4-t-cell-differentiation-using-crispr-cas9

Investigating Murine CD4 T Cell Differentiation Using CRISPR-Cas9 Ribonucleoprotein Complex-mediated Gene Ablation

The widespread accessibility of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology has made gene targeting in primary cells ...

Psoriasis is a common, chronic, relapsing, and inflammatory skin condition characterized by thickened skin infiltration, scales, and erythema. The pathogenesis of the disease is highly complex, involving the interplay of various factors such as genetics, immunity, and environmental factors. To investigate the mechanisms underlying psoriasis, researchers have developed multiple animal models of the disease. However, the variability in methodologies has presented challenges for constructing these models consistently. In this study, we established and comprehensively evaluated a highly efficient and stable psoriasis mouse model using imiquimod (IMQ). 8-week-old SPF-grade female C57BL/6J mice were used to establish the psoriasis mouse model. After one week of acclimatization, mice with irritated or damaged skin in the depilated areas were excluded. Starting on day two, 5% IMQ cream (62.5 mg/d) was applied daily to the depilated neck and back regions for seven consecutive days. During the experiment, daily measurements of body weight, skin photography, skin scoring, and behavioral observations were conducted. Post-experiment, spleen index determination, skin histological analysis, serum cytokine detection, and skin mRNA level detection were performed. The results showed that mice in the IMQ group developed typical psoriasis-like symptoms, including erythema, scales, and skin thickening, accompanied by increased scratching behavior, weight loss, elevated spleen index, and increased skin thickness. And significant changes in the levels of TNF-&#945;, IL-23, and IL-17A in serum, as well as the mRNA levels of IL-23, IL-17A, IL-17F, and IFN-&#947; in the skin. Serum cytokine levels (TNF-&#945;, IL-23, IL-17A) and skin mRNA expression of pro-inflammatory markers (IL-23, IL-17A, IL-17F, IFN-&#947;) were notably upregulated. In contrast, the control group exhibited no such alterations. By optimizing the IMQ induction duration and standardizing the administration protocol, while dynamically monitoring skin changes, this study aims to provide a standardized framework for researchers to construct reliable psoriasis models.

establishment-of-psoriasis-mouse-model-by-imiquimod

Establishment of Psoriasis Mouse Model by Imiquimod

Psoriasis is a common, chronic, relapsing, and inflammatory skin condition characterized by thickened skin infiltration, scales, and erythema. The ...

Ocular mucous membrane pemphigoid (MMP) presents as chronic cicatrizing conjunctivitis that, if left untreated, can cause blindness. Timely diagnosis and immunosuppressive therapy prevent disease progression. Direct immunofluorescence staining (DIF) of the conjunctival biopsy for immune complex deposition in the basement membrane is diagnostic of MMP. The biopsy positivity rate depends on biopsy characteristics, laterality, old age, and disease activity. The current methods paper describes the technique of harvesting the conjunctival biopsy and using optimal cutting temperature medium-filled cartridges for sample transfer and block preparation. A biopsy is performed from a non-scarred, uninvolved, or relatively less-involved region of the conjunctiva in both eyes. The usual biopsy size is 4-5 mm long, 2-3 mm wide, and should have tiny underlying subconjunctival tissue so that the basement membrane is not missed. We recommend performing periodic acid Schiff staining to confirm the presence of the basement membrane. Using a specified protocol, the cryosections from biopsies are stained with fluorescent-conjugated antibodies IgG, IgM, IgA, and complement factors C3c and C4c. A positive biopsy shows the green positive fluorescence in the basement membrane, and even if one of the antibodies is positive, it is MMP positive. A negative biopsy does not rule out pemphigoid; however, a positive one confirms the MMP and differentiates it from drug-induced cicatrizing conjunctivitis or pseudopemphigoid.

technique-conjunctival-biopsy-direct-immunofluorescence-for

Technique of Conjunctival Biopsy and Direct Immunofluorescence for Diagnosing Mucous Membrane Pemphigoid

Ocular mucous membrane pemphigoid (MMP) presents as chronic cicatrizing conjunctivitis that, if left untreated, can cause blindness. Timely diagnosis and ...

Tuberculosis is a deadly disease, and the emergence of antibiotic drug resistance in the causative agent bacterium, Mycobacterium tuberculosis, worsens treatment outcomes. Precise and rapid drug resistance identification through sequencing technologies is needed to improve tuberculosis patient outcomes through tailored therapeutic regimens. The DNA extraction method is critical for downstream molecular assays and is complicated by the tough cell wall of Mycobacterium, the low bacillary load of many clinical samples, and the complexity of the sputum matrix. There are numerous M. tuberculosis DNA extraction methods reported, but there is currently no gold standard. Furthermore, few of these methods are shown to work consistently, and many are not suitable for low-resource and high-burden tuberculosis settings. Consequently, laboratories frequently introduce their own procedure modifications, resulting in significant method variability. Here, we present a cost-effective, rapid, and standardized protocol for Mycobacterial DNA extraction from both clinical sputum and culture that produces DNA suitable for qPCR, and which should be considered for use in clinical diagnostics laboratories.

mycobacterial-dna-extraction-using-bead-beating-custom-buffer

Mycobacterial DNA Extraction using Bead Beating in Custom Buffer Followed by NGS Workflow

Tuberculosis is a deadly disease, and the emergence of antibiotic drug resistance in the causative agent bacterium, Mycobacterium tuberculosis, worsens ...

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has significantly impacted global public health and the economy. This study aimed to investigate the seroprevalence of IgG antibodies against SARS-CoV-2 among healthy blood donors in Jeddah, Saudi Arabia, to better understand the dynamics of COVID-19 transmission during three distinct periods: pre-lockdown, during-lockdown, and post-lockdown. The seroprevalence was assessed concerning demographic factors such as gender, age, blood group, and nationality. A cross-sectional study was conducted from December 2019 to December 2020. A total of 3,825 blood samples from healthy donors were screened using an in-house enzyme-linked immunosorbent assay (ELISA) for anti-SARS-CoV-2 IgG antibodies detection. Positive samples were further confirmed using a micro-neutralization (MN) assay. Of 3,825 serum samples, 173 (4.5%) were tested positive by ELISA, and 147 samples were confirmed positive by MN. All samples collected before the lockdown (December 2019 to February 2020) were negative for IgG antibodies. During the lockdown period (March 2020 to June 2020), 44 out of 1,482 samples (2.97%) were tested positive. After the lockdown was eased (July 2020 to December 2020), the positivity rate increased to 12.15% (103 out of 848 samples). The overall seroprevalence rate among healthy blood donors was 3.84%, with significantly higher rates among non-Saudi and male donors (p = 0.00). The findings suggest that COVID-19 restrictions implemented by the Saudi authorities were effective in reducing the initial spread of the virus. However, the marked increase in seroprevalence over time suggests that asymptomatic or mildly symptomatic individuals may pose a significant risk for continued transmission. Continuous monitoring of seroprevalence is essential to control the spread of COVID-19.

covid-19-seroprevalence-test-for-igg-antibody-levels-among-healthy

COVID-19 Seroprevalence Test for IgG Antibody Levels Among Healthy Donors Across Different Pandemic Phases in Jeddah

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has significantly impacted global public ...

Pseudomonas aeruginosa is a major human pathogen, particularly in chronic wound infections and chronic pulmonary infections (especially in patients with cystic fibrosis (CF)). Chronic bacterial infections are refractory to antibiotic treatments and there is an urgent need for in vivo chronic infection models amenable to drug screening to develop efficient therapies. Here, we describe a protocol of infection by a P. aeruginosa clinical isolate from a CF patient, expressing constitutively the Green Fluorescent Protein (GFP), for generating a persistent wound infection in zebrafish larvae. Tail fin-injured embryos are immersed in a bacterial suspension for 1.5 h, washed, and monitored for bacterial load for 3 days. The bacterial burden was quantified daily by counting fluorescent colony-forming units (CFU) from lysed infected larvae, which allowed&#160;a persistent infection.&#160;Moreover, persistent P. aeruginosa bacteria were refractory to antibiotic treatment. This novel in vivo model of persistent P. aeruginosa infection offers opportunities to evaluate the efficacy of innovative treatments against chronic infections.

modeling-persistent-pseudomonas-aeruginosa-infection-wounded

Modeling Persistent Pseudomonas aeruginosa Infection in Wounded Zebrafish Larvae

Pseudomonas aeruginosa is a major human pathogen, particularly in chronic wound infections and chronic pulmonary infections (especially in patients with ...

The chicken embryo has emerged as a popular in vivo model with increasing application in biomedical research due to its simplicity, affordability, and adaptability in the study of various biological phenomena. This model has been used to investigate microbial pathogenicity and is becoming a useful tool to study bacterial dormancy. The viable but non-culturable (VBNC) state is a dormant state in which bacteria become metabolically quiescent and resistant to cultivation to preserve their viability in harsh environments. Under favorable conditions, VBNC bacteria can wake up back into a metabolically active and culturable state. Bacterial pathogens that switch to a VBNC state, such as the foodborne listeriosis-causing Listeria monocytogenes, are a public health concern, as they elude detection by conventional growth-dependent methods and can recover their virulence upon revival. This urges a better understanding of the conditions and mechanisms driving the revival of VBNC pathogens. The method presented here showcases the chicken embryo as an efficient in vivo model to revive VBNC L. monocytogenes back into a culturable status. Where in vitro revival attempts, largely based on nutritional replenishing, were unproductive, this protocol succeeds in promoting the reactivation of cell wall-deficient VBNC forms of L. monocytogenes generated by starvation in mineral water. Importantly, the results obtained underline the requirement of the embryo for the revival of VBNC L. monocytogenes, indicating an important role of embryo-associated factors in this process. Other potential uses for this method include the screening and identification of bacterial factors implicated in the mechanisms of VBNC state revival. This model can thus provide insight into the molecular workings of bacterial dormancy, whose knowledge is critical to reduce&#160;the public health risks entailed by undetectable pathogens.

chicken-embryo-as-an-vivo-model-to-revive-viable-but-non-culturable

Chicken Embryo as an In Vivo Model to Revive Viable but Non-Culturable Pathogens

The chicken embryo has emerged as a popular in vivo model with increasing application in biomedical research due to its simplicity, affordability, and ...

The in vitro culture of T cells is a critical method for studying immune responses, viral infections, and potential therapeutic strategies. However, no established protocols for culturing avian T cells in vitro have been reported to date. In this study, we present a protocol for the first time, utilizing the highly pathogenic avian influenza (HPAIV) H5N1 virus as a model. Here, 4-week-old ducks were infected with the virus, and memory T lymphocytes were isolated 28 days post-infection to culture H5N1-specific duck T lymphocytes, enabling the investigation of the specific immune response to the H5N1 virus. Key steps in the protocol include isolating memory peripheral blood mononuclear cells (PBMCs) from infected ducks, activating antigen-presenting cells (APCs) with an optimal multiplicity of infection (MOI = 5) for 6 h, and preparing T cell culture media supplemented with recombinant IL-2 and other specific additives. T-cell proliferation, cytokine secretion, and cytotoxic activity were closely monitored throughout the process. Additionally, we established an intracellular cytokine staining protocol to quantify IFN-&#947; secretion in duck T cells. This involved generating a hybridoma cell line expressing IgG3&#954; antibodies specific to duck IFN-&#947;, followed by successful antibody purification. The purified antibody was diluted 1:10 and applied in flow cytometry to precisely measure IFN-&#947; secretion. This method offers a reliable tool for evaluating duck T cell responses and lays the foundation for future investigations into avian viral immunology.

in-vitro-culture-for-h5n1-specific-duck-t-cells-detection-immune

In Vitro Culture for H5N1-Specific Duck T Cells and Detection of Immune Responses Using Intracellular Cytokine Staining Method

The in vitro culture of T cells is a critical method for studying immune responses, viral infections, and potential therapeutic strategies. However, no ...

Journal - immunology and infection