In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.
Froehlich, J. M., Seiliez, I., Gabillard, J. C., Biga, P. R. Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages. J. Vis. Exp. (86), e51354, doi:10.3791/51354 (2014).
Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.
Considerable understanding of mammalian myogenesis has been obtained through the recapitulation of this process in both primary mouse (Mus musculus) myoblast cultures and the well-described mouse-derived cell line, C2C125. Beginning in the 1950s6, these cultures have led to much advancement in the understanding of the murinemyogenic program and, by extension, myogenesis in other vertebrates. Additionally, single cell myofiber explant techniques have increased out understanding of interactions between satellite cells and surrounding myofibers7-9.Cell cultures are particularly attractive for investigations of myogenesis due to the short time from precursor to differentiated cell10, relative ease of transfection for RNAi11-14, transgenic15,16 and overexpression studies14,17,18 , in vitro expansion followed by in vivo transplantation18-20, and even comparison of myogenic precursor cells and their regulating agents across taxa21,22. While differences due to the artificial environment of the culture system have been described5,23 , these in vitro systems have proven to be indispensible to our dissection of the intricate program governing the formation of multinucleated, terminally differentiated myofibersfrom mononucleated proliferative progenitor cells known as myosatellite cells (MSCs) among the mammals.
Outside of the class Mammalia, however, the conservation and/or divergence of mechanisms controlling myogenesis are poorly understood, largely due to the difficulty in culturing myogenic precursor cells (MPCs) and myoblasts from various taxa. Indeed, primary myoblast cultures have only been described in three birds24-26, one reptile27, a few amphibians28-30, and some fishes1,3,4,31-33. Continuous myogenic cell lines from vertebrates other than rodents34-36are even more rare, with the only non-mammalian myogenic cell line being derived from Japanese quail (Cortunix japonica), QM737. Despite many attempts at immortalization, a teleost myogenic cell line remains elusive and a protocol for efficient transfection of these cells was only published this year15. Thus, clear and well-optimized protocols for culturing primary MPCs and myoblasts from a variety of vertebrates are very much needed to not only further expand our knowledge of the evolution of the myogenic program, but to employ the power of comparative physiology to make breakthroughs in the treatment of human skeletal muscle diseases and disorders.
While the literature contains many reports of MPC/myoblast isolation38-49, it is common for authors to describe the protocols for such isolations in brief, often incomplete, formats. Further, the most instructive protocols reported have been developed for mice50-53, and some of these rely on antibody selection54,55 or fluorescence transgenes56,57, making these protocols unusable or impractical inmost non-rodent species utilized by muscle biologists. With little known about piscine, amphibian, and reptile myogenesis, a detailed and thorough protocol, described with audiovisual guidance and with demonstrated efficiency in distantly related species, would be most helpful to the field.
First described by Powell and colleagues in 198958, the following protocol was initially developed to isolate MPCs and myoblasts from salmonid fishes (namely, rainbow trout, Oncorhynchus mykiss, and Atlantic salmon, Salmo salar) and some larger cyprinids (i.e. goldfish, Carassiusauratusauratus). In 2000, Fauconneau and Paboeuf optimized a primary myoblast culture for rainbow trout59, and minoroptimizations made that protocol utilizable in several smaller minnows of the Danioninae clade (zebrafish, Danio rerio, and giant danio, Devario aequipinnatus)32 due to the many genetic tools available for zebrafish work and thus its close relatives. Teleost fish are attractive organisms for study due to their divergent growth strategy (at least in most species). Large salmonids, like most fishes, grow indeterminately, with growth potential unfettered by an asymptote at maturity, even in old age60-62. Unlike zebrafish, large danionins such as the giant danio63 and moustached danio display growth potentials typical of teleost fish, making their direct juxtaposition an ideal platform for understanding whether MPC cell fate choice plays a role in skeletal muscle hyperplasia versus hypertrophy.
Likewise, we have demonstrated that this protocol can be used with mice and axolotls, with relatively high cell yield and viability indices. Urodele salamanders, such as the Mexican axolotl (Ambystomamexicanum),possess the remarkable ability to regenerate tissues, including entire limbs and tails64-66. This characteristic makes these amphibians interesting models of skeletal muscle wasting and aging. Using the protocol described below, a similar approach can be undertaken as has been done in many fish species, providing an even wider comparative context for such studies. As many truly comparative biologists appreciate, the most meaningful advances in basic biology and translational biomedicine can be made when data are analyzed within the widest spectrum (here, the entire vertebrate lineage).
Ethics Statement: All experimentation involving vertebrate animals described herein was approved in advance by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham and is consistent with guidelines established by the Office of Laboratory Animal Welfare, National Institutes of Health of the U.S. Department of Health and Human Services.
Prepare the base medium as follows: 9 mM NaHCO3 (1.51 g per 2 L), 20 mM HEPES (9.53 g per 2 L) in DMEM (13.48 g/L; 26.96 g per 2 L).
Determine pH and adjust to 7.40 with HCl or NaOH.
Determine osmolality and adjust to ~ 300 mOsm with NaCl (if needed).
Filter sterilize using (2) 1 L vacuum sterilization systems and store at 4 °C protected from light for up to 2-3 months.
NOTE: See Tables 1 and 2 for complete reagent, tools, consumables, and equipment information.
Prepare poly-L-lysine-treated plates by dissolving 5 mg of γ-irradiated poly-L-lysine (> 300,000 mw) in 50 ml of ultrapure sterile water. Mix gently by inversion for 10 min at room temperature, ensuring full dissolution of lysine polymers.
Pipette the necessary amount of poly-L-lysine solution into each well, according to the plate type (Table 3). Allow plates to stand in laminar flow hood for 5 minutes.
Next, remove poly-L-lysine solution and wash twice with sterile water. Allow plates to air dry in laminar flow hood for 20 minutes.
Gently swirl the solution to ensure full well coverage. Seal plates with laboratory tape and place in incubator for 24 hours. The incubator should be set to appropriate temperature for species being used (see Table 7), and no additional gases need to be added to incubator.
NOTE: Laminin must be allowed to adhere to plates for 24 hours before seeding cells. Failure to do so will result in poor to no cell adherence.
Prepare media as described in Table 4. Complete media is best prepared the day of use.
In preparation for tissue isolation, assemble the following items and autoclave: 300-500 ml beaker(s); glass Petri dishes; scalpel handles; forceps (fine and coarse); dissection scissors; and several sheets of water-repellant autoclave paper.
NOTE: For each person assisting with the dissection process, (2) scalpel handles, (1) forceps (type depending on personal preference), (1) dissection scissors, and (5-10) sheets of water-repellant autoclave paper are sufficient.
In addition to the autoclaved items above, assemble 70% ethanol, a weight balance, sterile 50 ml conical tubes (number depends on size of culture), and ice.
Before beginning, ensure that a sufficient stock of fish is available.
NOTE: The age of the fish to be used is of critical importance. While fish of two different species may be of similar weights, a younger fish of one species will possess more MPCs than an older fish of another species. As a general rule, younger fish, especially when working with salmonids, are best. For danionins, fish as old as one year can be utilized optimally, while salmonids of age 4-6 months or younger (up to 15 g) are optimal.
For each 5 g of tissue to be dissected, aliquot 25 ml of isolation medium into a sterile 50 ml conical tube.
Weigh, record mass, and number the tube(s). Place tubes on ice.
Euthanize 2-3 fish at a time by immersion in > 300 mg/L sodium bicarbonate-buffered tricaine methanesulfonate. Allow opercular movement to cease for > 5 minutes and then submerse fish in 70% ethanol (contained in the sterile beakers) for 30 seconds.
Remove fish from 70% ethanol and place on water-repellant autoclave paper. Directly behind the opercula, use a scalpel to make a superficial, shallow incision. Remove the scales and skin by grasping the skin at the aforementioned incision and pull toward tail of fish.
Following skin removal, excise the epaxial, fast-glycolytic 'white' muscle of the fish from both sides (avoiding the slow-oxidative 'red' muscle located near the lateral line) and place in isolation medium. Discard the remainder of the fish as required by institution.
NOTE: While it is entirely possible to culture MPCs originating from red muscle, nearly every publication using teleost MPCs has utilized cells isolated from the epaxial myotome.
Repeat steps 2.4-2.6 until a sufficient quantity of muscle is obtained. During dissection, ensure that pooled muscle tissue remains on ice to maintain cell viability.
Pour one tube of 25 ml/5 g of muscle tissue into a glass Petri dish. Using two scalpel handles with attached #10 scalpel blades, mince tissue to a slurry or purée consistency. A "back-and-forth" motion of pulling the scalpel blades past each other works best.
NOTE: While tissue homogenizers may seem appropriate, the number of viable cells will be dramatically reduced, if not abolished.
Using a 25 ml serological pipette and serological pipettor, remove the slurried tissue and replace in the original conical tube.
Repeat steps 3.1-3.2 until all tissue in all tubes has been mechanically dissociated.
Centrifuge tissue for 5 minutes at 300 x g and 10 °C.
Following centrifugation, discard the 25 ml of media supernatant with a 25 ml serological pipette, vacuum aspirator, or by careful decanting.
Add 25 ml of wash medium to each tube, resuspend tissue, and recentrifuge as above.
Wash twice with wash medium, removing the supernatant each time.
During step 3.4, prepare collagenase solution by combining 0.22 g of Type IV collagenase and 44 ml of base medium (or sufficient quantity at 0.11 g/0.22 ml).
Stir in a beaker for 10-15 minutes at 4 °C. Filter sterilize with a 50 ml syringe and a 0.45 μm syringe filter.
NOTE: More than one syringe filter may be needed, depending on the collagenase preparation. Collagenase obtained from vendors other than Worthington Biochemical poses more difficulty during filtration. Collagenase is used to dissociate peptide bonds in collagen that constitute the endomysium. This digestion will remove that protective barrier of the myofibers.
For each g of muscle tissue, resuspend tissue in a 0.2% collagenase solution. For 5 g of muscle tissue, add 10 ml of collagenase solution and 15 ml of base medium.
Add 10 μl of PSF per ml of collagenase solution/base medium (e.g. 250 μl to 25 ml). Ensure that tissue is well suspended, as this will affect the efficiency of the enzymatic digestion.
Incubate muscle tissue in collagenase for 60 minutes at 18 °C with gentle rocking. Depending on the degree of mechanical dissociation and species, collagenase digestion may be extended to 90 minutes, although this may affect cell viability.
Following collagenase digestion, centrifuge conical tubes at 300 x g for 5 minutes at 12 °C. Discard the supernatant and resuspend tissue in 25 ml of wash medium.
Recentrifuge at 300 x g for 5 minutes at 12 °C. Repeat with washing medium a second time.
After washing tissue twice, resuspend muscle tissue in 25 ml of wash medium and triturate with a 10 ml serological pipette until the tissue/medium homogenate passes in and out of the pipette with relative ease. 5-10 triturations is usually sufficient.
Repeat with a 5 ml serological pipette and then a 16 gauge metal cannula attached to a syringe.
Centrifuge conical tubes at 300 x g for 5 minutes at 12 °C. Decant supernatant.
During the above 5 minute centrifugation, prepare the trypsin digestion solution by combining 0.25 g of trypsin with 5 ml of base medium.
Stir at 4 °C for 10-15 minutes and filter sterilize with a 20 ml syringe and 0.45 μm syringe filter.
Retrieve conical tubes and add 100 μl of trypsin solution and 4.9 ml of dissociation medium for every 1 g of muscle tissue to every tube. For example, add 500 μl of trypsin solution and 24.5 ml of dissociation medium to 5 g of muscle tissue.
NOTE: Trypsin is a serine protease that will separate myogenic precursor cells from the basal lamina.
Resuspend tissue in the trypsin/dissociation medium well. Incubate for 20 minutes at 18 °C.
Following the first trypsin incubation, centrifuge conical tubes at 300 x g for 1 min at 12 °C.
Neutralize trypsin by combining the supernatants with isolation medium at a concentration of 1 volume of supernatant to 4 volumes of isolation medium. Store at 4 °C during the second trypsin digestion.
To the remaining pellet of tissue, repeat steps 4.12-4.15, combining the supernatant following step 4.15 with the supernatant/isolation medium from the first trypsin digestion.
ALTERNATIVELY: The collagenase and trypsin digestions may be combined to maximize cell yields.
To do this, triturate the collagenase digest using a metal cannula (6 in length and 16 gauge works best) attached to a 20 ml syringe until the homogenate easily passes through the cannula.
To the homogenate, add 500 μl of trypsin solution (prepared in step 4.10) and incubate at 18 °C for 20 minutes.
Following the incubation, centrifuge conical tubes at 300 x g for 1 minute at 12 °C.
Neutralize trypsin by combining the supernatants with isolation medium at a concentration of 1 volume of supernatant to 4 volumes of isolation medium.
Store at 4 °C during the second trypsin digestion.
Proceed with step 4.18 as with the standard protocol.
Dispense the final supernatant/isolation medium mixture into 50 ml conical tubes and centrifuge at 300 x g for 10 minutes at 12 °C.
Remove the supernatants taking care not to disturb the cell pellets. Pipette 2 ml of complete medium into each tube, dissolving each cell pellet.
Combine cells suspended in complete medium in one 50 ml conical tube.
Rinse each tube with 1 ml of complete medium, combining with the pool of cells resuspended previously.
Triturate with a metal cannula and syringe 5-10 times.
Filter cell suspension through a 100 μm cell strainer, rinsing with complete medium.
Filter the cell suspension through a 40 μm cell strainer.
Add sufficient complete medium to 50 ml and centrifuge at 300 x g for 10 minutes at 15 °C.
Following the final centrifugation in step 4.25, remove supernatant by careful decanting or with a serological pipette. Resuspend the cell pellet in 5 ml of complete medium.
With cells fully resuspended, remove 20 μl of the cell suspension and place in a 1.5 ml microcentrifuge tube.
Add 5 μl of Trypan blue dye and wait 5 minutes. Using a hemocytometer, determine the number of viable cells.
Upon determination, dilute cells to the needed concentration. Teleost MPCs are best seeded at 150,000-200,000 cells per cm2. For a six-well plating strategy, diluting cells to 1.5 x 106 - 2.0 x 106 per ml supports sufficient proliferation and differentiation.
Retrieve poly-L-lysine-treated, laminin-coated plates and seed cells. (See Table 5 for plate-specific dilutions and plating volumes.) NOTE: Table 6 depicts average number of cells isolated per gram of muscle tissue isolated from various teleost species. Rainbow trout (O. mykiss) exhibit fewer MPCs per gram of muscle tissue than do zebrafish (D. rerio) and will thereby require more fish to isolate from for proper seeding.
ALTERNATIVELY: Some laboratories have adopted a protocol that calls for allowing MPCs to adhere to the culture substrata for 40 minutes.
Then rinse plates with washing media to remove any loosely attached and non-adherent cells. Warning: This step is very important to avoid "non-specific" adhesion of other cells (e.g. fibroblasts) to the culture substrata.
Add complete media and place cells in the incubator and care for as detailed below.
Twenty-four hours post-seeding, myogenic precursor cells (MPCs) should be visible attached to the laminin substratum (see Figures 1a and 1d). Following seeding, cells (MPCs) adopt a spindle-like shape, indicative of this cell type (Figure 1) and are MyoD1+ (Figure 2). In Danio species, MPCs appear to be more compact with smaller bipolar processes than do MPCs from Oncorhynchus and Salmo species. However, over four days of culture, MPCs from all piscine species examined adopt similar morphologies and look distinctly like myoblasts (Figures 1b and 1e). Complete media should be removed, and MPCs should be washed twice with wash medium. Myofibroblasts can contaminate myogenic cultures and are readily distinguishable from MPCs/myoblasts by their star-like morphology (versus the spindle shape of the myoblast). Washing of the plates greatly improves the removal of such fibroblasts.
In the species described here (see Table 7), daily media changes produce the best results. MPCs and progeny (myoblasts and nascent myotubes) should be washed once or twice prior to the addition of fresh complete media. Alternatively, media changes can be reduced to every other day after the cells have reached the definitive myoblast stage (approximately day 4 of culture). Myotubes should form within 2-3 days (Figures 1c and 1f) and be myogenin+ (Figure 2), especially when cultured in differentiation medium (see below). While periods of weeks to months have been reported in several mammalian species, piscine MPCs and myoblasts do not appear to tolerate such periods of time. Cells proliferate for the first six days of culture (Figure 3), with subsequent differentiation between days 7 and 9-11 of culture. Afterward, cells senescence or apoptose.
The myogenic nature of MPCs and their progeny isolated using this protocol has been determined3,32,33 based on gene expression. Unlike mammalian culture systems, proliferation of MPCs and myoblast progeny is relatively low during the initial days of culture (when compared to systems utilizing primary murine myoblasts or C2C12), with rapid increases detected as the cells form immature myotubes during days 6-9 of culture in Danio species32. Reports from rainbow trout have utilized a differentiation medium (common in mammalian myoblast cultures, but not required in piscine systems) and indicate that proliferation indices decrease as expected with myotube formation33. In our hands, the resultant myotubes can remain in culture for up to 9-11 days (Danio species) and 11-13 days (Oncorhynchus species) before cellular senescence or apoptosis occurs.
Table 1. Detailed reagent list with preferred manufacturer and catalog number.
Table 2. Consumables, tools, and equipment needed for cell culture.
Table 3. Optimized volumes for coating cell culture plates with poly-L-lysine and laminin.
Table 4. Different media preparations for isolation and culture of myogenic precursor cells (MPCs).
Table 5. Recommended dilutions and plating volumes by cell culture plate well size.
Table 6. Average number of myogenic precursor cells isolated from 1 gram of muscle tissue from various teleost species: Danio rerio (zebrafish), Daniodangila (moustacheddanio), Devario aequipinnatus (giant danio), and Oncorhynchus mykiss (rainbow trout).
* Lower temperatures support lower proliferation rates.
Table 7. Recommended incubation temperatures for piscine and amphibian myogenic precursor cells (MPCs).
Figure 1. Representative bright field images of MPCs (leftmost), myoblasts (middle), and early myotubes (rightmost) from two species, rainbow trout (Oncorhynchus mykiss, top) and zebrafish (Danio rerio, bottom).
Figure 2. Representative immunocytochemical staining of MPCs (a, c) and myotubes (b, d) isolated and cultured from giant danio (Devario aequipinnatus, top) and rainbow trout (Oncorhynchus mykiss, bottom). In culture, myoblasts are MyoD1+ positive (a, c) as visualized using commercially available MyoD1+ antibodies (danio: C-20, Santa Cruz Biotechnology; trout: NB100-80899, Novus Biologicals). Additionally, as myoblasts differentiate, they express myogenin (b, d) as visualized using commercially available myogenin antibodies (danio: M-225; trout: SC-567; both from Santa Cruz Biotechnology).
Figure 3. Representative cell proliferation data using BrdU to measure cell proliferation rates over time in culture. Data obtained from MPCs isolated and cultured from giant danio67.
The myogenic program, in whichever species examined, can be most easily studied through an in vitro system. Indeed, upon isolation, myogenic precursor cells (MPCs) in fish or myosatellite cells (MSCs) in mammals readily enter this highly regulated process involving the proliferation, cell cycle withdrawal, and terminal differentiation of myoblasts and the fusion of those myoblasts into nascent myotubes. The general lack of transgenic gene reporter strains of piscine species (with the possible exception of the zebrafish67 and rainbow trout69) constrainsin vivo work of MPC/MSC activation, proliferation, and differentiation, and thus the in vitro system presented here is an attractive platform for studies in fish species.
Successful culture of MPC/MSCs, no matter the species, is highly dependent on a) rigorous attention to sterility; b) thorough mechanical dissociation of skeletal muscle tissue; and c) optimization of enzymatic digestion. During the initial steps of muscle tissue isolation, great care must be taken to ensure that the necessary quantity of tissue is excised without contamination. It is of utmost importance that fish (or any animals being utilized, for that matter) are properly disinfected in 70% ethanol and for sufficient time. In our hands, 30 seconds works best; shorter periods of time can result in contamination and longer, loss of tissue integrity and cell viability. Ethanol can be used as a fixative, so care must be taken not to dehydrate the fish before dissection. Dissection can be done outside of a laminar flow cell culture hood; however, we recommend that this process be done within a hood to minimize any potential bacterial or fungal contamination.
While the mechanical dissociation described above may seem crude and/or tedious at first, it is critical to the isolation procedure. Two large scalpel blades, pulled past each other in a sliding motion (as shown in the video protocol), produces the best results. Once finished, the consistency of the homogenate should be that of a slurry or purée, and easily collected with a wide-bore serological pipette (i.e. 25 ml). Simply, the better the mechanical dissociation, the higher the MPC/MSC yield and the better the resulting culture. Poor dissociation will hinder enzymatic digestion and decrease cell yield. Although it may be tempting to consider, the use of electric tissue homogenizers dramatically lowers cell viability despite its obvious convenience, at least with piscine MPCs.
As with all protocols, optimization of the procedure detailed above is often necessary. This has proven true in our work with several danionin species. Results from our laboratory indicate that smaller danionins (e.g. the zebrafish) yield more MPCs per gram of skeletal muscle than do larger species (e.g. giant or moustached danio). However, this is only realized if a higher concentration of collagenase (0.3%) is used with tissue from smaller species. In our hands, a concentration of 0.2% is appropriate for salmonid species (e.g. rainbow trout; cutthroat trout [Oncorhynchusclarki], Chinook salmon [Oncorhynchustschawytscha]), the larger danionins, axolotl (Ambystomamexicanum) limb and tail, and even mouse skeletal muscle. Likewise, care must be taken to select the appropriate collagenase. In our experience, type IV collagenase preserves cell viability far better than collagenases recommended for fibrous tissues such as bone and muscle (i.e. collagenase type II)70. While digestion may be more complete in a shorter time period, cell membrane receptor integrity is likely compromised by increased tryptic activity in these preparations. With respect to trypsin, our laboratory has always utilized the cruder trypsin preparation purified from porcine pancreas, rather than the 'purer' preparations available. In our culture activities with various species, we have noticed little beneficial effect of varying trypsin concentrations.
Trituration is critical to this protocol. It both dissociates the fibrous matrix of skeletal muscle and increases surface area for tryptic digestion all the while manually disrupting the structural integrity of the myofibers. When performing the triturations, successively using smaller bore pipettes and cannulas is much easier than using the cannula and syringe straightaway. Proceeding directly to triturations with a cannula and syringe can result in plugged cannulas and/or excessive pressure being applied to cells. Applying tryptic digestion without adequate trituration will dramatically reduce cell yields.
Once isolated, the culture process is quite straightforward. Most publications to date have utilized a protocol with one media for both proliferation and differentiation33,71-75, including our own; however, more recent articles written by several French investigators have described a two media protocol, with separate based media and serum content for proliferation and differentiation33. This 'newer' protocol more closely mimics those used in the culture of murine MSCs and myoblastsand appears to enhance proliferation of early stage myoblasts33 and may be more appropriate to control proliferation and/or differentiation of cells. However, without extensive characterization of gene expression, it is difficult to conclude whether such a 'proliferation' media also conserves a more 'myoblast-like' state than does the traditional one media methodology. Previous publications describing gene expression, whether at the transcript or protein level, reported using the traditional one media protocol3. Alternatively, a single media (DMEM described herein) can be supplemented with differing concentrations of fetal bovine serum (FBS): 10% for proliferation and 2% for differentiation.
While investigators employing mammalian cell culture systems may be acclimated to using carbon dioxide atmospheres for the cultivation of these cells, the intrinsic differences between terrestrial and aquatic gas exchange call for a different approach when culturing piscine or water-confined urodele cells, including MPCs. Media detailed here are buffered with a piperazine-derived, zwitterionic organic compound [HEPES: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid] and sodium bicarbonate (NaHCO3). Thus, an incubator with gas injection is not needed or required for culture of these cells. More importantly, such incubators should possess the ability to cool rather than heat, as the temperatures needed to culture piscine and urodele MPCs range between 18-26 °C. Further, salmonid MPCs can be cultured at lower temperatures if needed, further highlighting the need for a cooling incubator. Additionally, we have found (as have other investigators, as communicated previously) that sealing of culture plates is necessary to preserve cellular viability. Simply wrapping the interface between the culture lid and plate with standard laboratory tape is sufficient to realize this goal.
While passaging of both primary MPCs/MSCs/myoblasts is common in mammalian cell culture, it does not appear to be possible with piscine cells, at least before the late Mb stage (around day 6 of culture). Therefore, the appropriate number of cells sufficient to support proliferation and to reach the goals of the experiment must be seeded at the initiation of the culture. Further, if less than expected cell yields are obtained, it is not possible to propagate cells and then replate the progeny later. We have attributed this characteristic to the increased reliance on the extracellular matrix (ECM) of piscine MPC/myoblasts. Alternatively, it is possible that this is an artifact of the laminin substrate and thus further empirical determination of ECM components necessary for piscine MPC/Mb proliferation and differentiation is warranted. We do note, however, that several of our collaborators have successfully removed late-stage myoblasts (~ day 6 of culture) from the culture substratum and replated these cells (J. M. Froehlich and P. R. Biga, personal communication).
Using this protocol or one similar to it, experimentation involving RT-PCR3,71-74,76-78, Western blot32,79-81, immunocytochemistry32,33, proliferation assays32,33,59, gene transfection15, morphometric analysis78, toxicology screening82, and chromatin immunoprecipitation (ChIP; J. M. Froehlich and P. R. Biga, unpublished results) has been done. However, further descriptions of additional in vitro protocols, namely those involving passaging and clonal propagation, are very much needed. Indeed, the Rodgers laboratory15 made significant advancements in the culture of piscine MPCs/myoblasts by optimizing a protocol for inducing transfection of rainbow trout myoblasts. Based on this methodology, further investigations involving RNAi or overexpression of skeletal muscle-specific targets, especially those postulated to be involved with the lifelong growth trajectories of teleost fishes, are not only possible, but may lead to significant advancements in the aquaculture and biomedical fields of science.
The authors would like to extend many thanks to Drs. Josep Planas and Juan Castillo for their professional expertise in the development and application of this culture protocol to small fishes and amphibians. Thanks are also due to the countless individuals who have tirelessly assisted with the dissection and dissociation of muscle tissue from many fish (both in species and number), including Matthew Charging, Delci Christensen, Zachary Fowler, Brooke Franzen, Nathan Froehlich, Kira Marshall, Ben Meyer, Ethan Remily, and Sinibaldo Romero. This work was supported by University of Alabama at Birmingham Department of Biology start-up funds, Center for Protease Research NIH Grant # 2P20 RR015566, NIH NIAMS Grant # R03AR055350, and NDSU Advance FORWARD NSF Grant #HRD-0811239 to PRB. Support was also provided by the UAB Nutrition Obesity Research Center award # P30DK056336, NIH NIDDK. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
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Scott, I. C., Tomlinson, W., Walding, A., Isherwood, B., Dougall, I. G. Large-scale isolation of human skeletal muscle satellite cells from post-mortem tissue and development of quantitative assays to evaluate modulators of myogenesis. Journal of Cachexia, Sarcopenia and Muscle. 10-1007 (2013).
Baquero-Perez, B., Kuchipudi, S. V., Nelli, R. K., Chang, K. C. A simplified but robust method for the isolation of avian and mammalian muscle satellite cells. BMC Cell Biology. 13, (2012).
Lu, A., et al. Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics. Gene Therapy. 15, (15), 1116-1125 (2008).
Rouger, K., et al. Progenitor cell isolation from muscle-derived cells based on adhesion properties. The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 55, (6), 607-618 (2007).
Michal, J., et al. Isolation and characterization of canine satellite cells. In vitro cellular & Developmental Biology Animal. 38, 467-480 (2002).
McFarland, D. C., et al. Isolation and characterization of myogenic satellite cells from the muscular dystrophic hamster. Tissue & Cell. 32, (3), 257-265 (2000).
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Doumit, M. E., Merkel, R. A. Conditions for isolation and culture of porcine myogenic satellite cells. Tissue & Cell. 24, (2), 253-262 (1992).
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Pasut, A., Oleynik, P., Rudnicki, M. A. Isolation of muscle stem cells by fluorescence activated cell sorting cytometry. Methods in Molecular Biology. 798, 53-64 (2012).
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Musaro, A., Barberi, L. Isolation and culture of mouse satellite cells. Methods in Molecular Biology. 633, 101-111 (2010).
Sherwood, R. I., et al. Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle. Cell. 119, (4), 543-554 (2004).
Yi, L., Rossi, F. Purification of progenitors from skeletal muscle. J. Vis. Exp. (49), (2011).
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Bosnakovski, D., et al. Prospective isolation of skeletal muscle stem cells with a Pax7 reporter. Stem Cells. 26, (12), 3194-3204 (2008).
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Powell, R. L., Dodson, M. V., Cloud, J. G. Cultivation and differentiation of satellite cells from skeletal muscle of the rainbow trout Salmo gairdneri. Journal of Experimental Zoology. 250, (3), 333-338 (1989).
Fauconneau, B., Paboeuf, G. Effect of fasting and refeeding on in vitro muscle cell proliferation in rainbow trout (Oncorhynchus mykiss). Cell and Tissue Research. 301, (3), 459-463 (2000).
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Johnston, I. A., Bower, N. I., Macqueen, D. J. Growth and the regulation of myotomal muscle mass in teleost fish. The Journal of Experimental Biology. 214, 1617-1628 (2011).
Mommsen, T. P. Paradigms of growth in fish. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology. 129, (2-3), 207-219 (2001).
Biga, P. R., Goetz, F. W. Zebrafish and giant danio as models for muscle growth: determinate vs. indeterminate growth as determined by morphometric analysis. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 291, (5), 1327-1337 (2006).
Roy, S., Gatien, S. Regeneration in axolotls: a model to aim for! Experimental Gerontology. 43, (11), 968-973 (2008).
Echeverri, K., Tanaka, E. M. Ectoderm to mesoderm lineage switching during axolotl tail regeneration. Science. 298, (5600), 1993-1996 (2002).
Tanaka, E. M., Reddien, P. W. The cellular basis for animal regeneration. Developmental Cell. 21, (1), 172-185 (2011).
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Seger, C., et al. Analysis of Pax7 expressing myogenic cells in zebrafish muscle development, injury, and models of disease. Developmental Dynamics: an Official Publication of the American Association of Anatomists. 240, (11), 2440-2451 (2011).
Gabillard, J. C., Ralliere, C., Sabin, N., Rescan, P. Y. The production of fluorescent transgenic trout to study in vitro myogenic cell differentiation. BMC Biotechnology. 10, (2010).
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Cross and Cutlass: The Word of God!
"Afternoon, MaryLu." Oh, and hi to -- Laura and Debbie.
Wonderful Scripture verse .... and a great photo !!!!!!! The details and adventure in the picture .... beautiful !
Magnificent picture, MaryLu, and that verse certainly makes clear the importance of the Word!! Thank you!!
Hi ladies!! Thanks for your comments!! Merry Christmas!


text:
International Dragon Association
American Dragon Association
Canadian Dragon Association
Dragons in North America
John Foster Dulles Cup Toronto 1960s
The first Canadian Dragon was launched on English Bay in Vancouver in 1951. The fleet in Canada was given a boost by H.R.H. Prince Philip who presented the CYA with the Duke of Edinburgh Trophy, first won in 1953 by Spence Hanna of the RCYC in Toronto. In 1954 H.R.H. Prince Philip brought his Dragon, Bluebottle (a wedding present from the Island Sailing Club of Cowes) to Canada where he raced it in Montreal, Hamilton and Toronto against local Dragon sailors from Canada and the US.
In the US the first fleets were started in the early 50s in the North East, the South, the Pacific North West and California. Many were Norwegian or UK built. In 1950 Roland yacht yard of Norway shipped a Dragon to a trade fair in Houston. After the event it was taken to the Southern Yacht Club in New Orleans and it inspired a couple of local sailors to travel to the UK to buy two more Dragons in 1954. In 1951 Lars Lyngnes imported 10 Dragons from Norway to Seattle to start a fleet there. They started with sail number 10, assuming there were about 10 on the east coast.
Olympic Years
Williwaw 68 Olympics winner
The Dragon class has long been one of the premier one design racing yachts in the world. The selection of the Dragon as an Olympic class for the 1948 Games ensured that the design would spread worldwide.
The class grew quickly in North America, with Dragon fleets spread all across the country in every one of the major sailing centers. There were active fleets in the Pacific Northwest, southern and northern California, Long Island Sound, the northern Gulf Coast, and on the Great Lakes. Both US and Canadian sailors also traveled and raced internationally, a tradition that continues to this day. At the height of it's popularity as a racing class in the United States, American sailors were among the best in the world, with Lowell North winning a Bronze at the Tokyo Olympics in 1964, and New Orleans native G.S. (Buddy) Friedrichs II winning Gold at the Olympics in Acapulco, Mexico. In the final Olympic Competition for the Dragon class, in Kiel, Germany in 1972, Don Cohan of Philadelphia took home the Olympic Bronze medal.
2008 Interport Royal Vancouver YC
In 1973 Borge Børresen got approval from the class to begin building in fibreglass (metal spars were introduced in 1970). During the 70s and early 80s fibreglass Dragons were build by Marty Godsil in the Pacific Northwest and O.E. Schuppe in Ontario, Canada.
The class has active racing fleets in the Great Lakes and the Pacific Northwest. The Vancouver fleet has twelve Dragons at the Royal Vancouver Yacht Club and half a dozen Dragons in the area. The Great Lakes region has Dragon Fleets in Toronto and Cleveland. And there are small clusters of Dragons in California, the Northeast, and along the Gulf Coast.
For 60 years Dragons have been sailing our shores, and the same qualities of design and performance that attracted people to her then continue to do so now. The same beautiful lines drawn by Johan Anker in 1928 that grab your eye and hold it still appeal to our love of beauty, and her performance on the water.
Read more history about North American Dragon Trophies.
2018 IDA Magazine
ADA & CDA online AGM
Minutes of 2018 IDA AGM
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redpajama_set_name: RedPajamaCommonCrawl


text:
World, Americas
Protests take place as anti-immigrant crackdown slated to begin in USA
Published : Jul 14, 2019, 9:23 am IST
Updated : Jul 14, 2019, 9:23 am IST
Trump renewed his demands for the crackdown this week, confirming that the raids would begin from Sunday.
The exercise will include 'collateral' deportations, which means that undocumented individuals who are not the targets of ICE officials will also face arrests if found at the site of a raid. (Photo: ANI)
Washington: Protests are being held across major cities in the United States against President Donald Trump's policies, especially after the leader announced the start of a crackdown on undocumented immigrants by the Immigration and Customs Enforcement (ICE) from Sunday.
Trump first suggested mass deportations last month, which were cancelled after he talked with House Speaker Nancy Pelosi, according to Al Jazeera.
He, again, renewed his demands for the crackdown this week, confirming that the raids would begin from Sunday, around two days ago.
The crackdown will target around 2,000 undocumented people who have already received orders across 10 US cities, including New York and Chicago, as per The New York Times.
The exercise will include "collateral" deportations, which means that undocumented individuals who are not the targets of ICE officials will also face arrests if found at the site of a raid.
"We are focused on criminals as much as we can before we do anything else," Trump had said on Friday while announcing the raids.
Even though the US President has said that the raids will mainly target criminals, justice groups expect the crackdown to include recently arrived undocumented people who escaped poverty and violence back home.
Many have pointed out that Trump's announcement has spread panic amongst migrant communities, who are bracing to expect the worst.
Lakshmi Sridaran, the co-executive of a group named South Asian Americans Leading Together (SAALT) pointed out that ICE has already been carrying out such raids at a smaller scale, which go undetected.
She cited the example of an Indian restaurant in Washington which was targeted by ICE last week, resulting in the detainment of several employees.
"Something like this might not have even made the news had it not been for a community member letting us know...It could've just very easily slipped under the radar. To me, what's even more frightening and insidious is that this is actually happening like this on a more low-key level all the time throughout the year...these larger scale raids are just an escalation of something that's already happening on a daily basis," Al Jazeera quoted Sridaran as saying.
Tags: anti immigrant, ice
Location: United States, Washington
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While it is probably better to run a PSGI based server, than a CGI-based server it can be also very useful to learn how to write CGI scripts. Especially if you need to maintain one.
This article will help you set up an Apache web server to run CGI scripts.
We will configure a Digital Ocean droplet, using Ubuntu 13.10 64bit.
After creating the Droplet, ssh to the server and update the installed packaged to the latest and then reboot the machine.
Once the machine restarted ssh to it again and and install the MPM preforking version of Apache2.
Now you can browse to the web-site by pointing your browser to the IP address of the machine. In my case it was http://107.170.93.222/ Please note, some browsers will not work properly if you don't put the http:// in front of the IP address.
This is the content of the /var/www/index.html file on the server.
You can edit that file, and reload the page in the browser.
This is your first CGI-script in Perl.
Now we need to configure the Apache web server to server it properly.
This will tell Apache to read the configuration files again.
Then we can brows to http://107.170.93.222/cgi-bin/echo.pl (after replacing the IP address by yours) and you will see it shows the word "hi".
Congratulations, your first CGI script is running.
This simple CGI script will show exactly the same content no matter who access it and when. Let's make a little change to the script so that we can see it is really generated dynamically.
After changing the script if you access the page now it will display the current time on the server. If you reload the page, it will show the new current time.
If when you access the http://107.170.93.222/cgi-bin/echo.pl URL you see the content of the script instead of the word "hi" then you probably put the cgi-bin directory inside the /var/www and/or probably forgot to create the symbolic links for the cgid.* files. Move the cgi-bin directory outside the /var/www, update the configuration files, set up the symbolic links, and reload the server.
The above lines in the error.log, probably indicated that the script does not have the Unix executable bit.
This probably means that the script died before it managed to print the Content-type. The error.log, probably contains the exception that was not caught just before the above line.
You can also turn of the buffering of STDOUT by setting $| to a true value.
I am not sure, but I think Premature end of script headers is the same as End of script output before headers.
In most of the situations a reload should be enough, but maybe not when a module is enabled/disabled.
in the error log, then maybe the ScriptAlias line is missing, or not pointing to the proper directory.
If you get a 403 Forbidden error, the probably the Directory directive was not correctly configure or does not point to the correct path.
That's it. Hopefully, you have your first CGI script in Perl running.
Of course using PSGI is much more modern and much more flexible than CGI. For that you might want to check out the Perl Dancer, or the Mojolicious frameworks. They provide a better experience.


text:
SXSW Online Unveils First Wave Of Programming Including Willie Nelson, Samantha Bee, Barry Jenkins, Cynthia Erivo And More
Like many festivals, South by Southwest (SXSW) is going online this year, which is a seamless transition considering the Austin-based festival has always included a large digital component. SXSW Online, which takes place March 16-20, has unveiled its initial wave of Featured Speakers and over 230 conference sessions and this includes music legend Willie Nelson as Keynote.
"No individual has had the cultural impact on, or been more synonymous with the creative vibrancy of Austin than Willie Nelson, and we are incredibly honored to have him as a SXSW Online 2021 Keynote," said Hugh Forrest, Chief Programming Officer. "In addition to dozens of incredible Featured Speakers, today's announcement also includes more than 200 sessions curated from our global community via PanelPicker, and is a key part of the premium content you can expect from SXSW Online 2021."
SXSW has also unveiled its robust roster of featured speakers which includes Samantha Bee, Cynthia Erivo, Taraji P. Henson, Barry Jenkins, Queen Latifah, LL Cool J, Brian Grazer, Matthew McConaughey, among others.
The fest will continue its tradition of exploring what's next across entertainment, culture and technology, presented in a variety of formats. This year the sessions will be organized by themes which includes: A New Urgency, Challenging Tech's Path Forward, Cultural Resilience in the Arts, The Rebirth Of Business, Transforming the Entertainment Landscape, Connection in Disconnection and An Uncharted Future. All are wildly relevant as they specifically focus on this moment in history, provididing a glimpse into what the future could look like.
Read the first wave of programming below.
Musician, author, actor, entrepreneur and activist Willie Nelson. Nelson has earned every conceivable award as a musician and continues to thrive as a relevant and progressive musical and cultural force. In recent years, he has delivered more than a dozen new albums, released a Top 10 New York Times' bestsellers book, again headlined Farm Aid, an event he co-founded in 1985, been honored by the Library of Congress with their Gershwin Prize for Popular Song, received his 5th degree black belt in Gong Kwon Yu Sul, headlined the annual Luck Reunion food and music festival during SXSW, and launched his cannabis companies Willie's Reserve and Willie's Remedy.
Emmy Award-winning host of TBS' Full Frontal with Samantha Bee, Samantha Bee
Founder and Chief Executive Officer of Streamlytics, Angela Benton
Entrepreneur and Virgin Group founder Sir Richard Branson
Latin Grammy-winning? recording artist (singer/songwriter), businesswoman, author and television star Chiquis
Future of Sex founder and podcast host Bryony Cole
Cannabis rights activist Steve DeAngelo
Entrepreneur, author and academic Joost van Dreunen
Co-founder and CEO of Relativity Space Tim Ellis
Emmy-nominated creative director and choreographer Laurieann Gibson
Schwab EVP and Chief Digital Officer Neesha Hathi
Academy Award-nominated and Golden Globe-winning actor, writer and producer Taraji P. Henson in conversation with SELF magazine Editor-in-Chief Carolyn Kylstra
Leadership strategist and author Ann Hiatt
Award-winning design entrepreneur and founder of the creative agency, LAYER, Benjamin Hubert
Award-winning writer and publisher of the Water & Music newsletter Cherie Hu
Author and co-founder and CEO of Affectiva Dr. Rana el Kaliouby
New York Times bestselling author Michael Lewis
Author, entrepreneur and Freeman Chief Design Officer Bruce Mau
Academy Award-winning actor, author, producer, director and philanthropist Matthew McConaughey in conversation with Major League Soccer commissioner Don Garber
Square co-founder Jim McKelvey
International yoga teacher, actress, writer and entrepreneur Adriene Mishler
Mathematician, data scientist and author Cathy O'Neil
Executive Director of Los Angeles Department of Cannabis Regulation Cat Packer
Actor, filmmaker, author, and Olympic athlete Alexi Pappas
Riot Games Chief Diversity Officer Angela Roseboro
Comedian, writer and How to Citizen podcast host Baratunde Thurston
Author, behavioral scientist and modern love expert Logan Ury
Director of NASA's Jet Propulsion Lab (JPL) Dr. Michael Watkins
Quantitative futurist, author and founder and CEO of the Future Today Institute Amy Webb
AI and Great Power Competition: A conversation about the future of Artificial Intelligence and National Security with L Colonel Alexander S. Vindman and Pryon CEO Igor Jablokov.
Aquaculture, Food Systems and Climate Change: A conversation about aquaculture's role in creating sustainable food systems and combating climate change featuring award-winning ocean activist Alexandra Cousteau; four-time James Beard Award-winning TV personality and chef Andrew Zimmern; multi award-winning writer and producer David E. Kelley; and organic seaweed farmer and entrepreneur Sarah Redmond.
Breaking the Sonic Color Line: A discussion about authenticity of voice in media, defeating racial stereotypes in voice acting, the impact of race in audio ads and how the industry can come together and make real change featuring DJ, actress and entrepreneur MC Lyte; Pandora Group Creative Director Roger Sho Gehrmann; and voice-over and television actress Joan Baker
Can VR Create Real Change?: A discussion about creating real change, and direct action through virtual reality with AR/VR producer and director Fifer Garbesi; award-winning immersive artist and professor Gabo Arora; Director of EarthXR Tiffany Kieran; and founder of Emblematic Group Nonny de la Peña.
The Chainsmokers on launching MANTIS VC: Grammy® Award-winning and Billboard Chart topping artist/producer duo, The Chainsmokers, are a dominating musical force with a diverse repertoire of songs that have led them to become one of world's biggest recording artists. Alex Pall and Drew Taggart have expanded The Chainsmokers' empire into film and television, tequila, philanthropy, and most recently their venture capital firm Mantis. Hear their story on how the duo have evolved their music career into so much more.
A Conversation with Icons Queen Latifah and LL COOL J: From the mic to the big screen, award-winning rappers, actors and producers Queen Latifah and LL COOL J have been major forces in the entertainment industry for over three decades. Queen Latifah executive produces and stars as the first female Equalizer, Robyn McCall, in the reimagining of the series Equalizer, and LL COOL J stars as Special Agent Sam Hanna on NCIS: Los Angeles. Join them for a lively, in-depth conversation about their illustrious careers in music, television and movies (in front and behind the cameras), the cultural resonance and timeliness of their series, and much more.
Driving Action & Advocacy Through Online Community: a conversation about online communities, their role in the digital ecosystem, and the value they bring to brands looking to build real connection, advocacy, and drive action, with Reddit COO Jen Wong and media reporter Kerry Flynn
From Moonlight to The Underground Railroad: A Conversation with Barry Jenkins and composer Nicholas Britell: A conversation with Academy Award® winning filmmaker Barry Jenkins and with Academy Award® nominated and Emmy® Award winning composer Nicholas Britell < (Succession), where they will discuss the joy, delicate nuances, challenges and unexpected discoveries from their work together. The pair will talk about their unique creative process in building a singular audiovisual identity with a specific focus on their upcoming Amazon Original limited series, The Underground Railroad, based on Colson Whitehead's Pulitzer Prize winning novel of the same name. Jenkins and Britell first collaborated on Moonlight, which won the Academy Award® for Best Picture. During the making of Moonlight<, the duo formed an inimitable rapport that brought them back together again for If Beale Street Could Talk. The Underground Railroad will stream in more than 240 countries and territories worldwide on Amazon Prime Video in 2021.
Hi, I'm Dave: FXX's DAVE is based on the life of Dave Burd (aka Lil Dicky), and centered on a neurotic man who's convinced himself that he's destined to be one of the best rappers of all time. The critically-acclaimed first season explored ambition, mental illness and masculinity in the world of hip-hop. Join co-creator/executive producer/writer/star Dave Burd (aka Lil Dicky), co-creator/executive producer Jeff Schaffer, executive producer Saladin Patterson and series star GaTa for Dave's first panel at SXSW. Season 1 is available on FX on Hulu; season two will premiere on FXX in 2021.
How GenZ Duets the News on TikTok: Hear about tactics publishers are using to build relationships with young audiences on TikTok, and the content that moves audiences to action with The Washington Post video producer Dave Jorgenson, NowThis politics producer Ian McKenna, and content creator Jackie James.
How to Scale a Mission-Driven Brand: Hear from Supergoop! CEO and Founder Holly Thaggard in conversation with early-stage investor, world-class tennis champion and entrepreneur Maria Sharapova about how to scale a company, innovate a category, and prioritizing doing good.
Inside GENIUS: ARETHA, the Story of the Queen of Soul: A conversation about National Geographic's GENIUS, an Emmy-winning anthology series that dramatizes the fascinating stories of the world's most brilliant innovators, exploring their extraordinary achievements along with their volatile, passionate, and complex personal relationships. Season three will explore Aretha Franklin's musical genius, incomparable career and the immeasurable impact and lasting influence she has had on music and culture around the world. Join executive producer and director Anthony Hemingway; multi award-winning producer and author Brian Grazer, multi award-winning actress and singer Cynthia Erivo; Broadway and television costume designer Jennifer Bryan; and Pulitzer Prize-winning playwright Suzan-Lori Parks to explore the craft and care that goes into honoring her legacy and bringing her story to life.
Leading Safely + Motivating Empathetically: Learn how the hospitality industry have changed their tactics to adapt to the ever-changing health and wellness regulations and lead, motivate and engage their employees, colleagues and communities; featuring Blackberry Farm Vice President of Food & Beverage Andy Chabot; Food & Wine editor-in-chief Hunter Lewis; executive chef and Cúrate Bar de Tapas and La Bodega by Cúrate co-owner Katie Button; and award winning chef and activist Marcus Samuelson.
Making Emotional Connections With Volumetric Video: Hear from three seasoned creatives on the most effective way to make emotional connections through volumetric video with writer, director, and new media artist Illya Szilak; Microsoft's Mixed Reality Capture Studios creative director Jason Waskey; and producer and Atlas V co-founder Antoine Cayrol.
The New Marketplace of Music Royalties: A discussion around maximizing and navigating royalty opportunities for musicians and how the music modernization act will transform the landscape for music royalties with founder and CEO of Sound Royalties Alex Heiche; CMO of The Mechanical Licensing Collective Ellen Truley; entertainment lawyer Robert A. Celetsin; and Grammy award-winning musician and actor Wyclef Jean.
Postcards for Democracy: A Collective Art Demo: Artistic visionaries DEVO cofounder Mark Mothersbaugh and self-proclaimed "musical weirdo" Beatie Wolfe share a love of tangible artforms, in and amongst their futuristic explorations. In light of the threat to our 225 year old postal service, at a time that could jeopardize the democracy of the country, Mothersbaugh and Wolfe joined forces for this collective postcard art demonstration. The aim of this campaign is to encourage as many people as possible to support USPS (if we don't use it we'll lose it!), our right to vote, and democracy as a whole via the power of art.
RIP Live Shows? Concerts in the Time of COVID: A conversation about the ways the live/touring industry are trying to stay afloat, what's working, what isn't, and what still needs to be done to save the music we love, featuring Drift general manager Adam Shore; Panache Booking and Panache Management founder Michelle Cable; and Paradigm Talent Agency Executive, Wilder Records founder and Home School co-founder Tom Windish.
What is Taste?: A discussion about taste, what it is and how science, technology and culture informs it with Cofounder of So Vegan Ben Pook, three-Michelin-starred chef Dominique Crenn, CEO of Motif FoodWorks Jonathan McIntyre, Ph.D, and author and scientist of senses and emotion Rachel Herz, Ph.D.
This article was printed from	https://deadline.com/2021/01/sxsw-online-first-wave-programming-willie-nelson-samantha-bee-barry-jenkins-cynthia-erivo-1234672530/


Childhood or pediatric asthma is one of the most common serious chronic disease in children. Kids suffering from asthma often has difficulty breathing and reduced energy. While astha can't be cured completely, this condition can be controlled with a proper treatment plan.
How can massage help in treating asthma?
Asthma most commonly affects children with a small percentage of adults being affected. Often, parents are concerned about the long-term effects of the drugs that children need to consume to treat their asthmatic conditions. Therefore, these parents are looking for a more natural and holistic way to help their children. Luckily, there's massage for asthma that works just like that..
What makes Traditional Chinese Medicine (TCM) most attractive as a complementary approach for child asthma treatment is that it is customised to the needs of the patient. Their symptoms and presentations are taken into consideration, differentiated between acute and chronic illness. Within each category, the organ or channel disharmony is defined as well. These inputs then guides the practitioner to the appropriate treatment for the patient.
Through gently-placed needles or non-invasive methods like massage, specific acupoints are stimulated which balance the energy, essentially teaching the on body how to heal itself. From a biomedical perspective, stimulation of acupoints helps reduce inflammation, bronchospasm in the case of asthma, and bolster the immune system.
On the other hand, Chinese herbal remedies aid in the reduction of inflammation and bronchospasm too. Apart from that, they also address the root energetic imbalance that is present. So, for pediatric asthma, herbs that tonify the lungs, spleen, and kidney organs would likely be prescribed.
Who should seek for Pediatric TCM for Asthma?
A big number of children who are receiving modern asthma treatments still have poorly controlled asthma. This is why TCM for kids is the best way to control your child's condition. If you are looking for more holistic approach to your child's overall wellness, you may want to consider giving Traditional Chinese Medicine a try.
Where to go for pediatric TCM?
In TCM, healing is based on the restoration of harmony and balance within the body. We are committed in working hand in hand with you in achieving your desired outcome and level of wellness for your child. For pediatric TCM consultation and/or kids massage, contact +65 6779 4266 or enquiries@aegletcm.com to make an appointment.
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