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12 ARTICLES PUBLISHED IN JoVE

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Biology

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis
Mark F Brady 1, Jorge Coronel 2, Robert H Gilman 3, David AJ Moore 4
1The Warren Alpert Medical School of Brown University, 2Laboratorio de Investigacion de Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, 3Department of International Health, Johns Hopkins Bloomberg School of Public Health, 4Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College London

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

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JoVE Journal

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.
Mulenga Musapa 1, Taida Kumwenda 1, Mtawa Mkulama 1, Sandra Chishimba 1, Douglas E. Norris 2, Philip E. Thuma 1, Sungano Mharakurwa 1
1Malaria Institute at Macha, 2Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

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Medicine

A Simple Method of Mouse Lung Intubation
Sandhya Das 1, Kelvin MacDonald 2, Herng-Yu Sucie Chang 1, Wayne Mitzner 1
1Department of Environmental Health Sciences, Program in Respiratory Biology and Lung Disease, Johns Hopkins Bloomberg School of Public Health, 2Department of Pediatrics, Oregon Health Sciences University

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

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Immunology and Infection

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy
Sabriya Stukes 1, Arturo Casadevall 1
1Department of Microbiology and Immunology, Albert Einstein College of Medicine

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

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Biology

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity
Nathachit Limjunyawong 1, Jonathan Fallica 1, Amritha Ramakrishnan 2, Kausik Datta 3, Matthew Gabrielson 1, Maureen Horton 1, Wayne Mitzner 1
1Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, 2Department of International Health, Johns Hopkins University Bloomberg School of Public Health, 3Department of Medicine, Johns Hopkins University School of Medicine

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

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JoVE Core

Measurement of the Pressure-volume Curve in Mouse Lungs
Nathachit Limjunyawong 1, Jonathan Fallica 1, Maureen R. Horton 1, Wayne Mitzner 1
1Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung’s structural changes with developing pathology.

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Medicine

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research
Nathachit Limjunyawong 1, Jason Mock 2, Wayne Mitzner 1
1Bloomberg School of Public Health, Environmental Health Sciences, Johns Hopkins University, 2Department of Medicine, Pulmonary Diseases and Critical Care Medicine, University of North Carolina School of Medicine

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

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Biology

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy
Shuo Li *1,2, Sumana Raychaudhuri *1, Shigeki Watanabe 1,3
1Department of Cell Biology, Johns Hopkins School of Medicine, 2Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, 3Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine

We developed a novel technique in electron microscopy, "flash-and-freeze," that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

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Developmental Biology

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model
Stephen R Wellard *1, Jessica Hopkins *1, Philip W. Jordan 1
1Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

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Education

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II
Grace H. Hwang *1, Jessica L. Hopkins *1, Philip W. Jordan 1
1Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

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Immunology and Infection

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body
Quigly Dragotakes 1, Arturo Casadevall 1
1Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

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Biology

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes
Mabel Taracena 1,2, Catherine Hunt 1, Pamela Pennington 3, Deborah Andrew 4,5, Marcelo Jacobs-Lorena 5,6, Ellen Dotson 1, Michael Wells 5,7,8
1Division of Parasitic Diseases and Malaria, Entomology Branch, Centers for Disease Control and Prevention, 2Department of Entomology, Cornell University, 3Centro de Estudios en Biotecnologia, Universidad del Valle de Guatemala, 4Department of Cell Biology, Johns Hopkins School of Medicine, 5Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, 6Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health and Malaria Research Institute, 7Department of Cell Biology, Johns Hopkins School of Medicine, 8Biomedical Sciences Department, Idaho College of Osteopathic Medicine

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

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