The authors thank the Department of Ornithology and Mammalogy, California Academy of Sciences, San Francisco, the Museum of Vertebrate Zoology, University of California, Berkeley, and the Museum of the North, University of Alaska, Fairbanks, for making their collections available for this research.

The present study was conducted using specimens from the Department of Ornithology and Mammalogy, California Academy of Sciences, San Francisco, the Museum of Vertebrate Zoology, University of California, Berkeley, and the Museum of the North, University of Alaska, Fairbanks. Permission to examine skull specimens and publish works from the data was obtained from the museums that own and manage each collection.
1. Specimen selection and documentation
<ol>
	<li>Document specimen information, including identification numbers, species, sex, and location of origin. 
	NOTE: The number of specimens held within a certain collection, as well as the specimen details, may be available through the Arctos Collaborative Collection Management Solution (see Table of Materials). For the present study, skulls from the Northern elephant seal (Mirounga angustirostris), California bobcat (Lynx rufus californicus), grey fox (Urocyon cinereoargenteus), Northern fur seal (Callorhinus ursinus), Southern sea otter (Enhydra lutris nereis), California mountain lion (Puma conolor cougar), and kit fox (Vulpes macrotis) are considered.</li>
	<li>Examine the skull for completeness of the anatomical structures. Do not include severely fragmented skulls such that normal anatomical structures are unrecognizable without extensive reconstruction.</li>
	<li>If possible, estimate the specimen&#39;s age at the time of death based on the closure of cranial sutures. Consult pertinent literature for the time of cranial suture closure as this varies for each target species.</li>
	<li>Replace loose teeth with their corresponding alveoli. Use published anatomical descriptions of study species to help recognize the tooth type of each loose tooth25,26,27,28,29,30.</li>
</ol>
2. Anatomical and developmental findings
<ol>
	<li>Successionally inspect each dental quadrant and record the presence or absence of teeth.</li>
	<li>Classify the loss of each tooth as congenitally absent versus acquired tooth loss versus artifactually absent. Examine the area of the missing tooth for a smooth margin of bone (congenital), an empty alveolus with remodeling alveolar bone (acquired), or an empty but sharply outlined alveolus (artifactual).</li>
	<li>Document any persistent deciduous teeth or supernumerary teeth (Figure 1).</li>
	<li>Examine each tooth crown&#39;s shape and any visible root structure. Document the number of roots by examining the loose teeth out of their alveoli. 
	NOTE: For teeth that cannot be removed from their alveolus, supernumerary roots can be identified by an additional protuberance, usually on the palatal or lingual aspect of the tooth, or via dental radiographs (Figure 2).</li>
	<li>Document the presence of enamel hypoplasia, characterized by thinning or absence of the tooth&#39;s white, reflective enamel layer, revealing the rougher, tan-yellow dentine surface.</li>
</ol>
3. Periodontal status
<ol>
	<li>Use a metal or plastic periodontal probe and explorer (see Table of Materials) to ascertain the texture of the alveolar bone for evidence of periodontitis31. 
	NOTE: There are no soft tissues, so gingivitis cannot be diagnosed, but increased vascularization, as evidenced by a larger number of vascular foramina, indicates early periodontitis (Figure 3).</li>
	<li>Test for the presence of furcation involvement or exposure by attempting to insert the periodontal probe between the roots of each multirooted tooth. Depending on the number of roots, multiple areas may be needed to test for furcation.</li>
	<li>Use a standardized protocol to identify the stages of progressively worsening periodontitis (Table 1)32.</li>
</ol>
4. Fractured teeth and periapical lesions
<ol>
	<li>Examine each tooth for fracture, indicated by the loss of tooth substance with sharp edges (Figure 4).</li>
	<li>Determine if each fracture is complicated or uncomplicated by attempting to insert the explorer into the pulp chamber from the fractured site. Complicated fractures are indicated by the explorer tip falling into the pulp chamber.</li>
	<li>Record each fracture type based on a standardized classification system (Table 2)33.</li>
	<li>Examine the teeth for evidence of periapical lesions, characterized by expansion of the alveolar bone in the region of the apex of the tooth root with evidence of increased vascularization (Figure 5). Fenestration over the expansion may or may not be present.</li>
</ol>
5. Attrition/abrasion
<ol>
	<li>Examine each tooth for attrition/abrasion, indicated by the loss of tooth substance with a smooth, glassy appearance and rounded edges (Figure 6).</li>
	<li>Use the explorer to determine pulp chamber exposure from the abraded region by attempting to insert the explorer tip into the pulp chamber.</li>
	<li>Classify the degree of attrition/abrasion on each tooth using a standardized classification system (Table 3)11. 
	&#8203;NOTE: Some species may be more susceptible to attrition/abrasion than others due to their natural behavior. As such, staging the severity of attrition/abrasion may be indicated, or simply recording the presence or absence of attrition/abrasion may suffice.</li>
</ol>
6. Temporomandibular joint pathology
<ol>
	<li>Inspect the bone components of the TMJ, including the head of the mandible of the condylar process, and the mandibular fossa of the squamous part of the temporal bone, for evidence of temporomandibular joint pathology, ruling out artifacts such as post-mortem trauma (e.g., &#34;drawer damage&#34; or preparation artifacts)31 (Figure 6).</li>
	<li>Independently inspect the mandibular head and fossa on both sides and use a semiquantitative scoring system for osteoarthritis (OA) to classify the lesions associated with each bone (Table 4)34.</li>
</ol>
7. Trauma
<ol>
	<li>Examine the skull for any evidence of traumatic injury. 
	&#8203;NOTE: Chronic traumatic injury may be differentiated from acute traumatic injury based upon the sharpness of the fracture edges and any evidence of osseous remodeling. Also, note that some wild species are subject to gunshot injuries, and entry/exit wounds can be recognized, as well as remnants of projectiles.</li>
</ol>
8. Checking of other parameters
<ol>
	<li>Depending on the species, examine the skull for additional abnormalities.</li>
	<li>Check for tooth resorption or dental hard tissue loss due to idiopathic odontoclastic destruction, which is most commonly seen in felids and can be diagnosed radiographically by a loss of radiodensity with or without a loss of periodontal ligaments space19,21,35. 
	NOTE: Tooth resorptive lesions can also be felt with a dental explorer as a roughness at the cervical region of the tooth, but radiographic confirmation is necessary for diagnosis.</li>
	<li>Check for echinochromasia, dark purple staining of hard tissues of the skull, since the consumption of certain species of pigmented echinoderms can be seen in otters11.</li>
	<li>Check for dental caries, which are decay of the tooth surface linked to the metabolism of dietary carbohydrates from acidogenic bacteria36. 
	NOTE: Carious lesions can be discovered by probing the occlusal surface of the teeth with a dental probe to reveal any pitting or irregularities17,18.</li>
	<li>Document evidence of osteomyelitis or neoplasia, characterized by irregular productive/destructive bone lesions37. Record these data descriptively and include objective measurements of any lesions.</li>
</ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diets+high+in+fruits+and+low+in+gum+exudates+promote+the+occurrence+and+development+of+dental+disease+in+pygmy+slow+loris+(Nycticebus+pygmaeus).">Diets high in fruits and low in gum exudates promote the occurrence and development of dental disease in pygmy slow loris (Nycticebus pygmaeus).</a> Zoo Biology. 34 (6), 547-553 (2015).</li><li>Kahle, P., Ludolphy, C., Kierdorf, H., Kierdorf, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+anomalies+and+lesions+in+Eastern+Atlantic+harbor+seals,+Phoca+vitulina+vitulina+(Carnivora,+Phocidae),+from+the+German+North+Sea.">Dental anomalies and lesions in Eastern Atlantic harbor seals, Phoca vitulina vitulina (Carnivora, Phocidae), from the German North Sea.</a> PLoS One. 13 (10), 1-25 (2018).</li><li>Ludolphy, C., Kahle, P., Kierdorf, H., Kierdorf, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis+of+the+temporomandibular+joint+in+the+Eastern+Atlantic+harbour+seal+(Phoca+vitulina+vitulina)+from+the+German+North+Sea:+A+study+of+the+lesions+seen+in+dry+bone.">Osteoarthritis of the temporomandibular joint in the Eastern Atlantic harbour seal (Phoca vitulina vitulina) from the German North Sea: A study of the lesions seen in dry bone.</a> BMC Veterinary Research. 14 (1), 1-14 (2018).</li></ol>

The authors have no conflicts of interest to disclose.

The anatomy of the teeth and jaws is a quintessential example of divergent evolution and is a true reflection of a species&#39; natural history, behavior, and health status. An individual&#39;s oral health may play directly into their survival and fitness. The current study outlines a systematic, reproducible, and detailed manner of assessing the dental health and TMJ abnormalities of museum specimens that may reflect pathology in live populations.
Despite dental diseases universally affecting...

The development of jaws and teeth marks a critical time point in the evolution and development of vertebrates. While jaws initially developed as part of a mechanism of respiration in aquatic and marine species, teeth offered a new manner of apprehending and processing prey items1,2. Since the development of jaws and teeth, organisms have evolved innumerable variations in anatomy that correspond to their function and reflect the ecologic role to which they belong. Due to their mineralized nature, teeth and skulls represent a bounty of information that persists in the environment and fossil record and can offer myriad insights into the ecology, health status, and behavior of individuals and, by extension, species.
The acquisition of information pertaining to the teeth and jaws of animals and characterizing form and pathology has many benefits. Recognizing common disease processes can improve conservation efforts of wild species and optimize the care of captive animals3,4,5. For example, information gleaned from museum skull specimens has been used to make inferences on the exposure of the Baltic grey seal (Halichoerus grypus) and harbor seals (Phoca vitulina) to environmental pollutants such as organochlorines over time6,7, although a causative relationship between orofacial lesions and pollutants has not been confirmed. Furthermore, diseases of the oral cavity are some of the most prevalent diseases in domestic species, and understanding the oral health status of wild species may advance the clinical medicine and management of domestic species8,9.
As animals have developed such variation in normal craniofacial shape and dentition, it can be challenging to characterize and compare these aspects between species. Understanding the ecology and natural behavior of an organism, as well as its typical environment, is imperative before attempting to examine its skull. Doing so will drive the formation of questions and hypotheses about a particular species&#39; dentition and inevitably enrich the conclusions from data analysis. For example, recognizing that the typical diet of the Southern sea otter (Enhydra lutris nereis) includes hard-shelled mollusks, crustaceans, and echinoderms is essential to contextualize the degree and effect of attrition and/or abrasion of the teeth10,11. Although one can assume the likelihood that an individual of a species will develop certain dental diseases, it is critical to have a systematic, precise, and reproducible protocol for evaluating dental pathology. This should include an appraisal of occlusion, anatomical and developmental findings, periodontal disease, endodontal findings, and temporomandibular joint (TMJ)&#160;pathology. Developing such a protocol with similar statistical analysis will allow for a detailed comparison of dental and TMJ disease from species to species. A systematic method has been utilized to characterize dental and temporomandibular joint pathology in many mammalian species and has proven to be translatable to organisms with diverse forms11,12,13,14,15,16,17,18,19,20,21,22,23,24.
To compare future data on additional species, it is important to have an accepted method for assessing diseases of the teeth and jaws that can be applied to a variety of species. This article aims to detail a standardized and organized approach for assessing the dental and TMJ&#160;pathology of skull specimens.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Arctos Collaborative Collection Management Solution</td>
			<td>https://arctosdb.org</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposible Nitrile Gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double-Ended Dental Explorer/Probe, #2 Handle</td>
			<td>Hu Friedy</td>
			<td>541-5860</td>
			<td></td>
		</tr>
		<tr>
			<td>High resolution digital camera</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light source</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnifying glass (Optional)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Magnification Loupes (Optional)</td>
			<td>Surgitel</td>
			<td>EVC00TTL</td>
			<td></td>
		</tr>
	</tbody>
</table>

systematic assessment of mammalian skull specimens for dental and temporomandibular joint pathology

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental pathology, and anatomic variations in many mammalian species. Studying the teeth and jaws of an array of species can present a challenge requiring attention to detail and understanding of a species&#39; normal anatomy. In the present article, a systematic and precise protocol for examining skull specimens is discussed that has been applied to a variety of mammals to define characteristic diseases in the oromaxillofacial region. The procedure outlined is simultaneously precise, repeatable, and adaptable to the highly differing skull and tooth shapes and anatomy across species. Specifically, specimens are examined for missing teeth, periodontal disease, endodontal disease, TMJ pathology, and anatomical variations. Results gleaned from research on museum specimens may reflect the natural history, health, and disease status of individuals and species. Furthermore, these data can inform ecological and conservation research efforts, as well as the care of captive individuals.

The current protocol results in a combination of objective and semi-subjective data, and the positive outcome depends on the accurate and repeatable assessment of specimens. Multiple observers with knowledge of the normal anatomy of the target species and an understanding of general dental and maxillofacial pathology ideally must be present to assess each specimen to minimize bias systematically. The assessment of each specimen must be discussed, and a consensus needs to be obtained. No threshold for the number of specim...

Watch this Scientific Journal Video about Systematic Assessment of Mammalian Skull Specimens for Dental and Temporomandibular Joint Pathology at JoVE.com

Systematic Assessment of Mammalian Skull Specimens for Dental and Temporomandibular Joint Pathology

The present protocol describes the techniques for the systematic assessment of skull specimens to characterize anatomical and developmental variations and abnormalities of the teeth, periodontal disease, endodontal disease, and temporomandibular joint pathology.

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental ...

systematic-assessment-mammalian-skull-specimens-for-dental

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1.3K Views.  University of California-Davis. The present protocol describes the techniques for the systematic assessment of skull specimens to characterize anatomical and developmental variations and abnormalities of the teeth, periodontal disease, endodontal disease, and temporomandibular joint pathology.

Video: Systematic Assessment of Mammalian Skull Specimens for Dental and Temporomandibular Joint Pathology

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...