Springer protocols feed by Molecular Medicine 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Detection and Differentiation Between Peroxynitrite and Hydroperoxides Using Mitochondria-Targeted Arylboronic Acid
The development of boronic probes enabled reliable detection and quantitative analysis of hydrogen peroxide and peroxynitrite. The major product, in which boronate moiety of the probe is replaced by the hydroxyl group, is however common for both oxidants. Here, we describe how ortho-isomer of mitochondria-targeted phenylboronic acid can be used to detect and differentiate peroxynitrite-dependent and peroxynitrite-independent probe oxidation. This method highlights the detection and quantification of both the major, phenolic product and the minor, peroxynitrite-specific nitrated product of probe oxidation. (Source: Springer...
Source: Springer protocols feed by Molecular Medicine - January 1, 2015 Category: Molecular Biology Source Type: news
Live-Cell Assessment of Mitochondrial Reactive Oxygen Species Using Dihydroethidine
Reactive oxygen species (ROS) play an important role in both physiology and pathology. Mitochondria are an important source of the primary ROS superoxide. However, accurate detection of mitochondrial superoxide especially in living cells remains a difficult task. Here, we describe a method and the pitfalls to detect superoxide in both mitochondria and the entire cell using dihydroethidium (HEt) and live-cell microscopy. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - January 1, 2015 Category: Molecular Biology Source Type: news
Simultaneous Quantification of Mitochondrial ATP and ROS Production
Several methods are available to measure ATP production by isolated mitochondria or permeabilized cells but with a number of limitations, depending upon the particular assay employed. These limitations may include poor sensitivity or specificity, complexity of the method, poor throughput, changes in mitochondrial inner membrane potential as ATP is consumed, and/or inability to simultaneously assess other mitochondrial functional parameters. Here we describe a novel nuclear magnetic resonance (NMR)-based assay that can be carried out with high efficiency in a manner that alleviates the above problems. (Source: Springer prot...
Source: Springer protocols feed by Molecular Medicine - January 1, 2015 Category: Molecular Biology Source Type: news
mTRIP: An Imaging Tool to Investigate Mitochondrial DNA Dynamics in Physiology and Disease at the Single-Cell Resolution
Mitochondrial physiology and metabolism are closely linked to replication and transcription of the genome of the organelle, the mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. Here, we describe mTRIP (mitochondrial transcription and replication imaging protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures engaged in mtDNA initiation of replication and global mitochondrial RNA (mtRNA) content at the single-cell level in human cells. In addition, mTRIP can be cou...
Source: Springer protocols feed by Molecular Medicine - January 1, 2015 Category: Molecular Biology Source Type: news
Accurate Measurement of Circulating Mitochondrial DNA Content from Human Blood Samples Using Real-Time Quantitative PCR
We describe a protocol to accurately measure the amount of human mitochondrial DNA (MtDNA) in peripheral blood samples which can be modified to quantify MtDNA from other body fluids, human cells, and tissues. This protocol is based on the use of real-time quantitative PCR (qPCR) to quantify the amount of MtDNA relative to nuclear DNA (designated the Mt/N ratio). In the last decade, there have been increasing numbers of studies describing altered MtDNA or Mt/N in circulation in common nongenetic diseases where mitochondrial dysfunction may play a role (for review see Malik and Czajka, Mitochondrion 13:481–492, 2013). ...
Source: Springer protocols feed by Molecular Medicine - January 1, 2015 Category: Molecular Biology Source Type: news
Mitochondria-Targeted RNA Import
The import of a modest number of nucleus-encoded RNAs into mitochondria has been reported in species ranging from yeast to human. With the advent of high-throughput RNA sequencing, additional nucleus-encoded mitochondrial RNAs are being identified. Confirming the mitochondrial localization of candidate RNAs of interest (e.g., small noncoding RNAs, miRNAs, tRNAs, and possibly lncRNAs and viral RNAs) and understanding their function within the mitochondrion is assisted by in vitro and in vivo import assay systems. Here we describe these two systems for studying mitochondrial RNA import, processing, and functions. (Source: Sp...
Source: Springer protocols feed by Molecular Medicine - January 1, 2015 Category: Molecular Biology Source Type: news
Analysis of Mitochondrial DNA and RNA Integrity by a Real-Time qPCR-Based Method
This chapter describes the use of real-time qPCR to analyze the integrity of mitochondrial nucleic acids quantitatively. The method has low material requirement, is low cost, and can detect modifications with high resolution. The method is specifically designed for mitochondrial RNA and DNA, but can be easily transferred to other high-copy number cases. This procedure describes analyses of brain nucleic acids, but other tissues or cells can be analyzed similarly. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - January 1, 2015 Category: Molecular Biology Source Type: news
Surgical Procedures and Experimental Outcomes of Closed Fractures in Rodent Models
The closed fracture rat model, first described by Bonnarens and Einhorn, has been widely implemented in recent years to characterize various fracture phenotypes and evaluate treatment modalities. Slight modifications in the fixation depth, to reduce surgical error associated with movement/dislocation of the k-wire fixation, were previously described. Here, we describe this method which involves the creation of a medial parapatellar incision, dislocation of the patella, boring an 18 gauge hole through the center of the femur, delivery of an adjunct (if applicable), fixation of the k-wire in the greater trochanter of the fem...
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Induction of Fully Stabilized Cortical Bone Defects to Study Intramembranous Bone Regeneration
Bone is a regenerative tissue with an innate ability to self-remodel in response to environmental stimuli and the need to repair damage. Rodent models of fracture healing, and in particular genetic mouse models, can be used to study the contributions of specific molecular switches to skeletal repair, as well as to recreate and exacerbate biological development and repair mechanisms in postnatal skeletons. Here, we describe methodology for producing fully stabilized, single-cortex defects in mouse femurs to study mechanisms of intramembranous bone regeneration. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Assessment of Knee Joint Pain in Experimental Rodent Models of Osteoarthritis
Pain assessment in animal models of osteoarthritis is integral to interpretation of a model’s utility in representing the clinical condition, and enabling accurate translational medicine. Here we describe two methods for behavioral pain assessments available for use in animal models of experimental osteoarthritic pain: Von Frey filaments and spontaneous activity monitoring. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Mouse Models of Osteoarthritis: Surgical Model of Posttraumatic Osteoarthritis Induced by Destabilization of the Medial Meniscus
The surgical model of destabilization of the medial meniscus (DMM) has become a gold standard for studying the onset and progression of posttraumatic osteoarthritis (OA). The DMM model mimics clinical meniscal injury, a known predisposing factor for the development of human OA, and permits the study of structural and biological changes over the course of the disease. In addition, when applied to genetically modified or engineered mouse models, this surgical procedure permits dissection of the relative contribution of a given gene to OA initiation and/or progression. This chapter describes the requirements for the surgical ...
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
In Vivo Axial Loading of the Mouse Tibia
Noninvasive methods to apply controlled, cyclic loads to the living skeleton are used as anabolic procedures to stimulate new bone formation in adults and enhance bone mass accrual in growing animals. These methods are also invaluable for understanding bone signaling pathways. Our focus here is on a particular loading model: in vivo axial compression of the mouse tibia. An advantage of loading the tibia is that changes are present in both the cancellous envelope of the proximal tibia and the cortical bone of the tibial diaphysis. To load the tibia of the mouse axially in vivo, a cyclic compressive load is applied up to fiv...
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
EPIC-μCT Imaging of Articular Cartilage
Characterization of articular cartilage morphology and composition using microcomputed tomography (microCT) techniques requires the use of contrast agents to enhance X-ray attenuation of the tissue. This chapter describes the use of an anionic iodinated contrast agent at equilibrium with articular cartilage. In this technique, negatively charged contrast agent molecules distribute themselves inversely with respect to the negatively charged proteoglycans (PGs) within the cartilage tissue (Palmer et al. Proc Natl Acad Sci U S A 103:19255–19260, 2006). This relationship allows for assessment of cartilage degradation, as...
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Four-Point Bending Protocols to Study the Effects of Dynamic Strain in Osteoblastic Cells In Vitro
Strain engendered within bone tissue by mechanical loading of the skeleton is a major influence on the processes of bone modeling and remodeling and so a critical determinant of bone mass and architecture. The cells best placed to respond to strain in bone tissue are the resident osteocytes and osteoblasts. To address the mechanisms of strain-related responses in osteoblast-like cells, our group uses both in vivo and in vitro approaches, including a system of four-point bending of the substrate on which cells are cultured. A range of cell lines can be studied using this system but we routinely compare their responses to th...
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Immunohistochemistry of Skeletal Tissues
Immunohistochemistry (IHC) is the process of identifying proteins in tissue sections by incubating the sample with antibodies specific to the protein of interest, and then visualizing the bound antibody using a chromogen. Unlike in situ hybridization, which identifies gene transcripts in cells, IHC identifies the products themselves and provides information about their localization within cells (nuclear, cytoplasmic, or membrane) or extracellular matrix. This can be particularly important in the context of bone and cartilage because they contain many cell types as well as matrix components, each with distinct protein expre...
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
