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Bio-protocol

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https://www.readbyqxmd.com/read/29911127/-ex-vivo-follicle-rupture-and-in-situ-zymography-in-drosophila
#1
Elizabeth M Knapp, Lylah D Deady, Jianjun Sun
Ovulation, the process of releasing a mature oocyte from the ovary, is crucial for animal reproduction. In order for the process of ovulation to occur, a follicle must be fully matured and signaled to rupture from the ovary. During follicle rupture in both mammals and Drosophila , somatic follicle cells are enzymatically degraded to allow the oocyte to be liberated from the follicle. Here, we describe a detailed protocol of our newly developed ex vivo follicle rupture assay in Drosophila , which represents a first assay allowing direct quantification of follicles' capacity to respond to ovulation stimuli and rupture...
May 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29888297/-in-vitro-analysis-of-ubiquitin-like-protein-modification-in-archaea
#2
Xian Fu, Zachary Adams, Julie A Maupin-Furlow
The ubiquitin-like (Ubl) protein is widely distributed in Archaea and involved in many cellular pathways. A well-established method to reconstitute archaeal Ubl protein conjugation in vitro is important to better understand the process of archaeal Ubl protein modification. This protocol describes the in vitro reconstitution of Ubl protein modification and following analysis of this modification in Haloferax volcanii , a halophilic archaeon serving as the model organism.
May 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29780855/magnetic-resonance-imaging-and-histopathological-visualization-of-human-dural-lymphatic-vessels
#3
Seung-Kwon Ha, Govind Nair, Martina Absinta, Nicholas J Luciano, Daniel S Reich
In this protocol, we describe a method to visualize and map dural lymphatic vessels in-vivo using magnetic resonance imaging (MRI) and ex-vivo using histopathological techniques. While MRI protocols for routine imaging of meningeal lymphatics include contrast-enhanced T2-FLAIR and T1- weighted black-blood imaging, a more specific 3D mapping of the lymphatic system can be obtained by administering two distinct gadolinium-based MRI contrast agents on different days (gadofosveset and gadobutrol) and subsequently processing images acquired before and after administration of each type of contrast...
April 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29770355/host-regulated-hepatitis-b-virus-capsid-assembly-in-a-mammalian-cell-free-system
#4
Kuancheng Liu, Jianming Hu
The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for developing antiviral therapies against HBV infection. Although several systems have been developed to study HBV capsid assembly, including heterologous overexpression systems like bacteria and insect cells, in vitro assembly using purified protein, and mammalian cell culture systems, the requirement for non-physiological concentrations of HBc and salts and the difficulty in manipulating host regulators of assembly presents major limitations for detailed studies on capsid assembly under physiologically relevant conditions...
April 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29770354/3d-co-culture-system-of-tumor-associated-macrophages-and-ovarian-cancer-cells
#5
Lingli Long, Mingzhu Yin, Wang Min
Ovarian cancer is fairly unique in that ovarian carcinoma cells can detach and spread directly through peritoneal cavity. It has been unclear, however, how detached cancer cells survive in the peritoneum and form spheroid structure. We have recently reported that there is a strong correlation between Tumor-associated macrophages (TAMs)-associated spheroid and clinical pathology of ovarian cancer, and that TAMs promote spheroid formation and tumor growth at early stages of transcoelomic metastasis in orthotopic mouse models...
April 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29770353/quantification-of-bacterial-twitching-motility-in-dense-colonies-using-transmitted-light-microscopy-and-computational-image-analysis
#6
Benjamin Smith, Jianfang Li, Matteo Metruccio, Stephanie Wan, David Evans, Suzanne Fleiszig
A method was developed to allow the quantification and mapping of relative bacterial twitching motility in dense samples, where tracking of individual bacteria was not feasible. In this approach, movies of bacterial films were acquired using differential interference contrast microscopy (DIC), and bacterial motility was then indirectly quantified by the degree to which the bacteria modulated the intensity of light in the field-of-view over time. This allowed the mapping of areas of relatively high and low motility within a single field-of-view, and comparison of the total distribution of motility between samples...
April 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29795778/sacral-spinal-cord-transection-and-isolated-sacral-cord-preparation-to-study-chronic-spinal-cord-injury-in-adult-mice
#7
Carmelo Bellardita, Maite Marcantoni, Peter Löw, Ole Kiehn
Spinal cord injury (SCI) is characterized by multiple sensory/motor impairments that arise from different underlying neural mechanisms. Linking specific sensory/motor impairments to neural mechanism is limited by a lack of direct experimental access to these neural circuits. Here, we describe an experimental model which addresses this shortcoming. We generated a mouse model of chronic spinal cord injury that reliably reproduces spasticity observed after SCI, while at the same time allows study of motor impairments in vivo and in an in vitro preparation of the spinal cord...
April 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29770352/context-driven-salt-seeking-test-rats
#8
Stephen E Chang, Kyle S Smith
Changes in reward seeking behavior often occur through incremental learning based on the difference between what is expected and what actually happens. Behavioral flexibility of this sort requires experience with rewards as better or worse than expected. However, there are some instances in which behavior can change through non-incremental learning, which requires no further experience with an outcome. Such an example of non-incremental learning is the salt appetite phenomenon. In this case, animals such as rats will immediately seek out a highly-concentrated salt solution that was previously undesired when they are put in a novel state of sodium deprivation...
April 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29770351/a-microfluidic-device-for-massively-parallel-whole-lifespan-imaging-of-single-fission-yeast-cells
#9
Stephen K Jones, Eric C Spivey, James R Rybarski, Ilya J Finkelstein
Whole-lifespan single-cell analysis has greatly increased our understanding of fundamental cellular processes such as cellular aging. To observe individual cells across their entire lifespan, all progeny must be removed from the growth medium, typically via manual microdissection. However, manual microdissection is laborious, low-throughput, and incompatible with fluorescence microscopy. Here, we describe assembly and operation of the multiplexed-Fission Yeast Lifespan Microdissector (multFYLM), a high-throughput microfluidic device for rapidly acquiring single-cell whole-lifespan imaging...
April 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29707607/analysis-of-mitochondrial-structure-in-the-body-wall-muscle-of-caenorhabditis-elegans
#10
Shaarika Sarasija, Kenneth R Norman
Mitochondrial function is altered in various pathologies, highlighting the crucial role mitochondria plays in maintaining cellular homeostasis. Mitochondrial structure undergoes constant fission and fusion in response to changing cellular environment. Due to this, analyzing mitochondrial structure could provide insight into the physiological state of the cell. In this protocol, we describe a method to analyze mitochondrial structure in body wall muscles in the nematode Caenorhabditis elegans, using both transgenic and dye-based approaches...
April 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29707606/measurement-of-ros-in-caenorhabditis-elegans-using-a-reduced-form-of-fluorescein
#11
Shaarika Sarasija, Kenneth R Norman
Oxidative stress is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease. Oxidative stress is a result of a disruption of the equilibrium between antioxidants and oxidants, in favor of oxidants. Since mitochondria are major sites of production and reduction of reactive oxygen species (ROS), measurement of ROS levels can help us determine if mitochondrial functional integrity has been compromised. In this protocol, we describe a method to measure the level of ROS in the nematode Caenorhabditis elegans, using chloromethyl-2,7'-dichlorodihydrofluorescein diacetate (CM-H2 DCFDA)...
April 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29770349/single-step-precision-genome-editing-in-yeast-using-crispr-cas9
#12
Azat Akhmetov, Jon M Laurent, Jimmy Gollihar, Elizabeth C Gardner, Riddhiman K Garge, Andrew D Ellington, Aashiq H Kachroo, Edward M Marcotte
Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29770348/quantitative-live-cell-reporter-assay-for-noncanonical-wnt-activity
#13
Edith P Karuna, Michael W Susman, Hsin-Yi Henry Ho
Noncanonical Wnt signaling functions independently of the β-catenin pathway to control diverse developmental processes, and dysfunction of the pathway contributes to a number of human pathological conditions, including birth defects and metastatic cancer. Progress in the field, however, has been hampered by the scarcity of functional assays for measuring noncanonical Wnt signaling activity. We recently described the Wnt5a-Ror-Kif26b (WRK) reporter assay, which directly monitors a post-transcriptional regulatory event in noncanonical Wnt signaling...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29744374/measurement-of-intracellular-ros-in-caenorhabditis-elegans-using-2-7-dichlorodihydrofluorescein-diacetate
#14
Dong Suk Yoon, Myon-Hee Lee, Dong Seok Cha
Reactive oxygen species (ROS) are generated during normal metabolic processes under aerobic conditions. Since ROS production initiates harmful radical chain reactions on cellular macromolecules, including lipid peroxidation, DNA mutation, and protein denaturation, it has been implicated in a wide spectrum of diseases such as cancer, cardiovascular disease, ischemia-reperfusion and aging. Over the past several decades, antioxidants have received explosive attention regarding their protective potential against these deleterious reactions...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29713655/hydrogen-deuterium-exchange-mass-spectrometry-of-oxygen-sensitive-proteins
#15
Luke Berry, Angela Patterson, Natasha Pence, John W Peters, Brian Bothner
The protocol detailed here describes a way to perform hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) on oxygen sensitive proteins. HDX-MS is a powerful tool for studying the protein structure-function relationship. Applying this technique to anaerobic proteins provides insight into the mechanism of proteins that perform oxygen sensitive chemistry. A problem when using HDX-MS to study anaerobic proteins is that there are many parts that require constant movement into and out of an anaerobic chamber...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29682596/spared-nerve-injury-model-of-neuropathic-pain-in-mice
#16
Joseph Cichon, Linlin Sun, Guang Yang
Experimental models of peripheral nerve injury have been developed to study mechanisms of neuropathic pain in living animals. The spared nerve injury (SNI) model in rodents is a partial denervation model, in which the common peroneal and tibial nerves are injured, producing consistent and reproducible tactile hypersensitivity in the skin territory of the spared, intact sural nerve. SNI-operated mice require less force applied to the affected limb to elicit a withdrawal behavior as compared to sham mice. This effect is observed as early as 2 days after surgery and lasts for at least 1 month...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29651453/bacterial-cell-wall-precursor-phosphatase-assays-using-thin-layer-chromatography-tlc-and-high-pressure-liquid-chromatography-hplc
#17
Manuel Pazos, Christian Otten, Waldemar Vollmer
Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by 'attacking' enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius ...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29644261/detection-and-analysis-of-circular-rnas-by-rt-pcr
#18
Amaresh C Panda, Myriam Gorospe
Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29644260/biochemical-analysis-of-dimethyl-suberimidate-crosslinked-yeast-nucleosomes
#19
Yuichi Ichiakwa, Paul D Kaufman
Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the functional consequences of nucleosome symmetry, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, allowing us to compare cells with single or double H3 alterations...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29644259/rna-cap-methyltransferase-activity-assay
#20
Jackson B Trotman, Daniel R Schoenberg
Methyltransferases that methylate the guanine-N7 position of the mRNA 5' cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32 P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging...
March 20, 2018: Bio-protocol
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