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Florida State University

13 ARTICLES PUBLISHED IN JoVE

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Biology

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography
Stephanie A. Zlatic 1, Pearl V. Ryder 1, Gloria Salazar 2, Victor Faundez 1
1Department of Cell Biology, Emory University, 2Department of Medicine, Division of Cardiology, Emory University

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

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Neuroscience

The FlyBar: Administering Alcohol to Flies
Kim van der Linde 1, Emiliano Fumagalli 2, Gregg Roman 2, Lisa C. Lyons 1
1Department of Biological Science, Florida State University, 2Department of Biology and Biochemistry, Biology of Behavior Institute, University of Houston

Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

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Environment

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti
Kathryn M. Jones 1, Hajeewaka C. Mendis *1, Clothilde Queiroux *1
1Department of Biological Science, Florida State University

Growth of Medicago truncatula plants in symbiosis with the nitrogen-fixing bacteria Sinorhizobium meliloti in individual, sterile microcosms made from standard laboratory plates permits frequent examination of root systems and nodules without compromising sterility. Plants can be maintained in these growth chambers for up to 9 weeks.

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Chemistry

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro
Rafaela G. Feresin 1, Shirin Pourafshar 2,3, Jingwen Huang 2, Yitong Zhao 2, Bahram H. Arjmandi 2,3, Gloria Salazar 2,3
1Department of Dietetics and Nutrition, University of Arkansas for Medical Sciences, 2Department of Nutrition, Food and Exercise Sciences, Florida State University, 3Center for Advancing Exercise and Nutrition Research on Aging (CAENRA), Florida State University

This work details a step-by-step method to prepare polyphenol-rich extracts from freeze-dried berry powder. In addition, it provides a thorough description of how to use these polyphenol-rich extracts in cell culture in the presence of the peptide hormone angiotensin II (Ang II) using Vascular Smooth Muscle Cells (VSMCs).

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Bioengineering

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition
Jeremy J. Flint 1,2, Kannan Menon 2,3, Brian Hansen 4, John Forder 2,3,5, Stephen J. Blackband 1,2,6
1Department of Neuroscience, University of Florida, 2McKnight Brain Institute, University of Florida, 3Department of Biomedical Engineering, University of Florida, 4Center for Functionally Integrative Neuroscience, Aarhus University, 5Department of Radiology, University of Florida, 6National High Magnetic Field Laboratory, Florida State University

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

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Neuroscience

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages
Alyssa J Rolfe 1, Dale B Bosco 1, Erynn N Broussard 1, Yi Ren 1
1Department of Biomedical Sciences, College of Medicine, Florida State University

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

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Genetics

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions
Stefan Schoenfelder *1, Biola-Maria Javierre *1,2, Mayra Furlan-Magaril 1,3, Steven W. Wingett 1,4, Peter Fraser 1,5
1Nuclear Dynamics Programme, The Babraham Institute, Babraham Research Campus, 2IJC Building, Campus ICO-Germans Trias i Pujol, Josep Carreras Leukemia Research Institute, 3Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 4Bioinformatics Group, The Babraham Institute, Babraham Research Campus, 5Department of Biological Science, Florida State University

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

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Chemistry

Heterogeneous Removal of Water-Soluble Ruthenium Olefin Metathesis Catalyst from Aqueous Media Via Host-Guest Interaction
Cheoljae Kim 1, Hoyong Chung 1
1Department of Chemical and Biomedical Engineering, Florida State University

A removable water-soluble N-heterocyclic carbene (NHC) ligand in aqueous media via host-guest interaction has been developed. We demonstrated representative olefin metathesis reactions in water as well as in dichloromethane. Via either host-guest interaction or extraction, the residual ruthenium (Ru) catalyst was as low as 0.14 ppm after the reaction.

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JoVE Journal

Decomposing the Variance in Reading Comprehension to Reveal the Unique and Common Effects of Language and Decoding
Barbara R. Foorman 1, Yaacov Petscher 1
1Florida Center for Reading Research, Florida State University

Here we present a protocol for decomposing the variance in reading comprehension into the unique and common effects of language and decoding.

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Environment

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures
Malak M. Tfaily *1,2, Rachel M. Wilson *3, Heather M. Brewer 1, Rosalie K. Chu 1, Heino M. Heyman 4, David W. Hoyt 1, Jennifer E. Kyle 5, Samuel O. Purvine 1
1Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 2Department of Soil, Water and Environmental Science, University of Arizona, 3Department of Earth Ocean and Atmospheric Sciences, Florida State University, 4Bruker Daltonics Inc., 5Biological Sciences Division, Pacific Northwest National Laboratory

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

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Behavior

Using Cholesky Decomposition to Explore Individual Differences in Longitudinal Relations between Reading Skills
Florina Erbeli 1, Aaron R. Campbell 1, Sara A. Hart 2
1Department of Educational Psychology, Texas A&M University, 2Department of Psychology and Florida Center for Reading Research, Florida State University

This paper demonstrates use of the gold standard method in behavioral genetics, the Cholesky decomposition method, to estimate unique, overlapping genetic and environmental influences on different variables to answer longitudinally motivated research questions.

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Medicine

A Preclinical Model of Exertional Heat Stroke in Mice
Michelle A. King 1, Jamal M. Alzahrani 1, Thomas L. Clanton 1, Orlando Laitano 1,2
1Department of Applied Physiology and Kinesiology, College of Health and Human Performance, University of Florida, 2Department of Nutrition and Integrative Physiology, College of Health and Human Sciences, Florida State University

The protocol describes the development of a standardized, repeatable, preclinical model of exertional heat stroke (EHS) in mice free from adverse external stimuli such as electric shock. The model provides a platform for mechanistic, preventative, and therapeutic studies.

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Neuroscience

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation
Qiwei Fan *1, Xiaotan Zhang *2, Yuan Wang 3, Xiaoyu Wang 1
1Division of Histology & Embryology, Key Laboratory for Regenerative Medicine of the Ministry of Education, College of Medicine, Jinan University, 2Department of Clinical Pathology, First Affiliated Hospital of Jinan University, 3Program in Neuroscience, Department of Biomedical Sciences, College of Medicine, Florida State University

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

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