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Guilherme C de Oliveira, Karina S Kanamori, Maria Auxiliadora-Martins, Claudia C S Chini, Eduardo N Chini
CD38 is a multifunctional enzyme involved in calcium signaling and Nicotinamide Adenine Dinucleotide (NAD+ ) metabolism. Through its major activity, the hydrolysis of NAD+ , CD38 helps maintain the appropriate levels of this molecule for all NAD+ -dependent metabolic processes to occur. Due to current advances and studies relating NAD+ decline and the development of multiple age-related conditions and diseases, CD38 gained importance in both basic science and clinical settings. The discovery and development of strategies to modulate its function and, possibly, treat diseases and improve health span put CD38 under the spotlights...
July 20, 2018: Bio-protocol
Michael S Schappe, Bimal N Desai
After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways-one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al...
July 20, 2018: Bio-protocol
Karina S Kanamori, Guilherme C de Oliveira, Maria Auxiliadora-Martins, Renee A Schoon, Joel M Reid, Eduardo N Chini
Current studies on the age-related development of metabolic dysfunction and frailty are each day in more evidence. It is known, as aging progresses, nicotinamide adenine dinucleotide (NAD+ ) levels decrease in an expected physiological process. Recent studies have shown that a reduction in NAD+ is a key factor for the development of age-associated metabolic decline. Increased NAD+ levels in vivo results in activation of pro-longevity and health span-related factors. Also, it improves several physiological and metabolic parameters of aging, including muscle function, exercise capacity, glucose tolerance, and cardiac function in mouse models of natural and accelerated aging...
July 20, 2018: Bio-protocol
Alina C Teuscher, Collin Y Ewald
Green fluorescent protein (GFP) is widely used as a molecular tool to assess protein expression and localization. In C. elegans , the signal from weakly expressed GFP fusion proteins is masked by autofluorescence emitted from the intestinal lysosome-related gut granules. For instance, the GFP fluorescence from SKN-1 transcription factor fused to GFP is barely visible with common GFP (FITC) filter setups. Furthermore, this intestinal autofluorescence increases upon heat stress, oxidative stress (sodium azide), and during aging, thereby masking GFP expression even from proximal tissues...
July 20, 2018: Bio-protocol
Michael M Wördehoff, Wolfgang Hoyer
Studying the aggregation of amyloid proteins like α-synuclein in vitro is a convenient and popular tool to gain kinetic insights into aggregation as well as to study factors ( e.g. , aggregation inhibitors) that influence it. These aggregation assays typically make use of the fluorescence dye Thioflavin T as a sensitive fluorescence reporter of amyloid fibril formation and are conducted in a plate-reader-based format, permitting the simultaneous screening of multiple samples and conditions. However, aggregation assays are generally prone to poor reproducibility due to the stochastic nature of fibril nucleation and the multiplicity of modulating factors...
July 20, 2018: Bio-protocol
Andrew D Doyle
Rat tail collagen solutions have been used as polymerizable in vitro three-dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. These 3D hydrogels are a relatively inexpensive, simple to use model system that can mimic the in vivo physical characteristics of numerous tissues within the body, namely the skin. While confocal imaging techniques such as fluorescence reflection and two-photon microscopy are able to visualize collagen fibrils during 3D imaging without fluorescence, other imaging modalities require direct conjugation of fluorescent dyes to collagen...
July 5, 2018: Bio-protocol
Yolanda Schaerli
High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants. Here, I describe a protocol for high-throughput screening of bacterial ( E. coli ) microcolonies in gel beads. Single cells are encapsulated into monodisperse water-in-oil emulsion droplets produced with a microfluidic device...
July 5, 2018: Bio-protocol
Sabrina Flütsch, Luca Distefano, Diana Santelia
In this protocol, we describe how to quantify starch in guard cells of Arabidopsis thaliana using the fluorophore propidium iodide and confocal laser scanning microscopy. This simple method enables monitoring, with unprecedented resolution, the dynamics of starch in guard cells.
July 5, 2018: Bio-protocol
Jie Zhu, Deepak A Lamba
Retinal degeneration leads to loss of light-sensing photoreceptors eventually resulting in vision impairment and impose a heavy burden on both patients and the society. Currently available treatment options are very limited and mainly palliative. Ever since the discovery of human pluripotent stem cell technologies, cell replacement therapy has become a promising therapeutic strategy for these patients and may help restore visual function. Reproducibly generating enriched retinal cells including retinal progenitors and differentiated retinal neurons such as photoreceptors using human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells in a dish is an essential first step for developing stem cell-based therapies...
June 20, 2018: Bio-protocol
Hannah Levardon, Lael M Yonker, Bryan P Hurley, Hongmei Mou
Airway basal stem cells are the progenitor cells within the airway that exhibit the capacity to self-renew and give rise to multiple types of differentiated airway epithelial cells. This stem cell-derived epithelium displays organized architecture with functional attributes of the airway mucosa. A protocol has been developed to culture and expand human airway basal stem cells while preserving their stem cell properties and capacity for subsequent mucociliary differentiation. This achievement presents a previously unrealized opportunity to maintain a durable supply of progenitor cells derived from healthy donors to differentiate into human primary airway epithelium for cellular and molecular-based studies...
June 5, 2018: Bio-protocol
Sergey Korshunov, James A Imlay
Bacteria release cysteine to moderate the size of their intracellular pools. They can also evolve hydrogen sulfide, either through dissimilatory reduction of oxidized forms of sulfur or through the deliberate or inadvertent degradation of intracellular cysteine. These processes can have important consequences upon microbial communities, because excreted cysteine autoxidizes to generate hydrogen peroxide, and hydrogen sulfide is a potentially toxic species that can block aerobic respiration by inhibiting cytochrome oxidases...
May 20, 2018: Bio-protocol
Weihuan Wang, Gurnoor Majihail, Cui Lui, Lan Zhou
Osteoblasts are bone marrow endosteum-lining niche cells playing important roles in the regulation of hematopoietic stem cells by secreting factors and cell adhesion molecules. Characterization of primary osteoblasts has been achieved through culture of outgrowth of collagenase treated bone. Immunophenotyping and flow-based analysis of long bone osteoblasts offer a simplified and rapid approach to characterize osteoblasts. We describe a modified procedure of isolating mouse bone marrow osteoblastic cells based on cell surface immunophenotyping...
May 20, 2018: Bio-protocol
Mindy Call, Ewa Anna Meyer, Winston W Kao, Friedrich E Kruse, Ursula Schlötzer-Schrehardt
Stem cells are widely used for numerous clinical applications including limbal stem cell deficiency. Stem cell derived from the bulge region of the hair follicle have the ability to differentiate into a variety of cell types including interfollicular epidermis, hair follicle structures, sebaceous glands and corneal epithelial cells when provided the appropriate cues. Hair follicle stem cells are being studied as a valuable source of autologous stem cells to treat disease. The protocol described below details the isolation and expansion of these cells for eventual clinical application...
May 20, 2018: Bio-protocol
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
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
Zheng Chai, R Jude Samulski, Chengwen Li
Neutralizing antibodies (Nabs) are a major challenge in clinical trials of adeno-associated virus (AAV) vector gene therapy, because Nabs are able to inhibit AAV transduction in patients. We have successfully isolated several novel Nab-escaped AAV chimeric capsids in mice by administrating a mixture of AAV shuffled library and patient serum. These AAV chimeric capsid mutants enhanced Nab evasion from patient serum with a high muscle transduction efficacy. In this protocol, we describe the procedures for selection of the Nab-escaped AAV chimeric capsid, including isolation and characterization of Nab-escaping AAV mutants in mice muscle...
May 5, 2018: Bio-protocol
Emilie Viennois, Anika Tahsin, Didier Merlin
We have developed a protocol to purify RNA from DSS (Dextran Sulfate Sodium)-treated mouse tissues. This method, which prevents downstream inhibition of q-RT-PCR observed in DSS-treated tissues, relies on successive precipitations with lithium chloride.
May 5, 2018: Bio-protocol
Todd V Brennan, Liwen Lin, Xiaopei Huang, Yiping Yang
Murine tumor models have been critical to advances in our knowledge of tumor physiology and for the development of effective tumor therapies. Essential to these studies is the ability to both track tumor development and quantify tumor burden in vivo . For this purpose, the introduction of genes that confer tumors with bioluminescent properties has been a critical advance for oncologic studies in rodents. Methods of introducing bioluminescent genes, such as firefly luciferase, by viral transduction has allowed for the production of tumor cell lines that can be followed in vivo longitudinally over long periods of time...
April 20, 2018: Bio-protocol
Sangho Yu, Heike Münzberg
The fundamental of neuroscience is to connect the firing of neurons to physiological and behavioral outcomes. Chemogenetics enables researchers to control the activity of a genetically defined population of neurons in vivo through the expression of designer receptor exclusively activated by designer drug (DREADD) in specific neurons and the administration of its synthetic ligand clozapine N-oxide (CNO) (Sternson and Roth, 2014). Using stimulatory Gq-coupled DREADD (hM3Dq) in mice, we showed that leptin receptor (LepRb)-expressing neurons in the preoptic area (POA) of the hypothalamus are warm-sensitive neurons that mediate warm-responsive metabolic and behavioral adaptations by reducing energy expenditure and food intake (Yu et al...
April 20, 2018: Bio-protocol
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
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