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Rotating Wall Vessel Bioreactor

Abstract
Recently there are significant amount of research work undergoing about tissue engineering and bioreactor designing. Therefore, there are so may research paper published around the world. It may use embryonic stem cell, mesenchymal stem cell, tissue graft or other animal spaces tissues or cell for development of human and animal medicine treatment. In that case there should be some ethics and laws to control the usage of the tissue or cell in the medical treatment. Some government organizations and private sector by independently or by joining do some research work about the tissue engineering and bioreactor designing. The cardiovascular system is the major disease problem in the human and animal medicine treatment. In recent decade there are cell and tissue engineering and the bioreactor designing involving treating the cardiovascular disease condition. Researchers may try to develop heart valve, wall and blood vessel etc. Hole in the heart is complex congenital heart diseases, in new born babies and leading causes of mortality. The treatment of this kind of the cardiovascular disease only performed surgery correction, the very painful after the surgery at tolerate by baby. When correcting the hole, that must have closed properly otherwise it lead to another problem to the young one, but measurement of the diameter of the hole is very difficult and correction also very difficult. In the recent decade there is stem cell therapy and the tissue engineering has rapidly developed. By using stem cell and tissue culture there are so many researches and development of the treatment about cardiovascular system. Myocardial tissue engineering developed the heart tissue by using the stem cells in three-dimensional matrices of biodegradable polymers scaffold is the innovation of the myocardial constructs and cardiovascular treatment.
Introduction The heart is the most important organs in the human body. It transports blood to the organs, tissues, and cells of the body. Blood delivers oxygen and nutrients to every cell and removes the carbon dioxide and waste products excreted by those cells. A “Holes in the Heart” is an opening in the septum between atria or ventricles of heart, this is congenital condition. 8-10/1000 live born babies has congenital defects in the heart. This condition occurred during the baby’s heart does not develop inside the womb; no specific cause for this condition, but some increase risk of being born. If mother had German measles or toxoplasmosis during pregnancy, or if she has diabetes, or if someone else in her family was born with a heart complaint. A hole in the heart may be noticed in the first few months of life – or even before the baby is born, sometimes a hole is not found until a person is much older. This often happens when the hole is between the upper chambers of heart. It may notice person are feeling a bit short of breath and don’t know why. But sometimes there are no complaints at all. Because of the hole, the flow of blood through the heart is abnormal. This makes noises in the heart, so a doctor can find the hole by listening to the heart with a stethoscope. If the doctor hears a murmur, this tells the doctor there could be a hole. If the doctor thinks there is a hole, person will have an echocardiogram ultrasound test of heart. Sometimes the hole isn’t found until a person is much older – when they notice they are feeling tired and breathless and can’t find a reason for it. Some holes are so small that they cause no problem and are left alone. Some holes in small babies may close by themselves: if the cardiologist thinks this is likely, he will not close it immediately, but wait for some time to see if it has closed by itself, by repeating an echo. Other holes must be closed, either because they are already a problem, or because they will cause a problem in the future.
There are three different types:
Atrial Septal Defect (ASD): this is a hole in the wall between the atria (interatrial septum). This causes more blood to flow to the lungs and may not have any symptoms; the excess flow can damage the lungs. If the hole is small, and doesn’t affect the function of heart, there’s no need to fix it.
Ventricular Septal Defect (VSD): this is a hole in the interventricular septum or wall between two lower chambers (RV and LV). If it’s large, can change the mechanics in heart. This makes the heart work harder than it should and can enlarge it. If the hole is small, and doesn’t affect the function of heart, there’s no need to fix it.
Atrioventricular Septal Defect: this is a large hole in the middle of heart between the atria and ventricles. Some people with this condition only have one valve between the atria and ventricles – instead of two. This defect can also damage the lungs by allowing too much blood to flow to the lungs. Although this condition is uncommon, t can be found in babies born with Down’s syndrome.
VSDs are the commonest lesion about 25-30% of all congenital heart defects whereas ASD are about 5-8% of them. Another point to remember is that all of us are born with small ASD. However, VSD is never found in normal heart. The only treatment available was surgical closure. Though the ultimate outcome was good, these children had to inevitably suffer the pain, scar and long hospital stay. There are two ways to do this. The first way is via an operation called catheterisation. This is when a cardiologist puts a tube into leg that goes up towards heart. Then put a device through that tube so that it fits into the hole. When it’s in the right place, the device opens like a little umbrella, and blocks the hole. The device stays inside forever. This is not possible, because of the size, shape or position of the hole. In these more complicated situations, a surgeon will perform an operation where he puts a patch over the hole directly. If holes have between the two pump chambers of the heart that stay open, will need antibiotic treatment at certain times. This might be before having other operations or serious treatment at the dentist. Most patients who have ASD/VSD corrections go on to lead perfectly normal lives. Person will be followed-up for a short period, but if everything’s OK after a year, won’t need to worry about it ever again. It also doesn’t increase the chances of having any other heart-related issues in the future but should take regular exercise and aim for a healthy diet. After correction of the hole in the heart there are low risk for structural degeneration, thrombo-embolism and endocarditis and growth potential for paediatric patient. From 1970s onwards, a group of cardiologists started thinking differently. They experimented on animals by creating holes in their hearts and then tried closing them without surgery. Gradually they replicated the whole procedure on humans. For the last twenty years, nonsurgical closure or device closure has been the normal.
Adult life heart muscle cells do not proliferate, if there is damage or injuries happened to the heart, functional tissue try to form the non-functional scar tissue. In 1996, 98 Klug et al. Suggest that development of human Embryonic stem cell derived cardiomyocytes help for therapy of cardiac disease.
There are some experiments done by using stem cells. Stem cells are the cell ability for self-renewal and the potential for differentiating into mature cell types. The embryonic stem cells can give rise to almost every mature cell type, while adult stem cells are classified as restricted to differentiation into only few types of mature cells. The mesenchymal stem cell can only differentiate to one specific mature cell type, are referred to as precursor cells. First clinical applications of stem cells for cardiac regeneration comprised cell transplantation trials. These trials were less successful than promising preclinical studies; these efforts initiated intense research activities providing new insight into the mechanisms of tissue growth and differentiation. Cardiac tissue engineering is focused on three different organ subunits: the myocardium, valves, and vessels. These three compounds of the heart can already be replaced by artificial or biological transplant constructs with their respective limitations, like assist devices, commercial heart valves, autologous coronary bypasses, etc. When developing the heart tissue must consider produced cardiomyocytes, vascular endothelial cell and the smooth muscles cell. Engineering these tissues must compete with the durability, efficiency and safety of existing substitutes and be affordable at the same time. Tissue engineering is the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ, includes an in vitro.
During designing of bioreactor, physiochemical environment maintain is very impotent, that will help to kept high quality of the stem cells and high degree of reproducibility of the cells. But must make sure cell culture has developed under sterile environment and sufficient nutrition and waste product exchange throughout the medium and clean and maintain the medium. After that, design some mechanical and hydrodynamic force to compression or expansion of the developing tissue, like shear stress to the tissue. Then maintain steady flow of media in pulsatile manner and reduce the excessive turbulence in the fluid flow rate. Other than that, must provide the low volume capacity for effective use for growth factors and medium, also select the fabricate material compatible with the heart tissue or stem cell. The bioreactor designing for heart tissue development must determine some specific design and functional requirement. Both biomechanical and biochemical factors affect the growth of the cell therefore essential to create some control mechanism by stimulate the physiological environment for heart cell growth, like pulsatile forces, pressure, flow rate, compression, expansion, shear stress, frequency, stroke rate and stroke volume. Other than that, when creating heart tissue must consider cardiac flow rate and pressure. When consider the design the bioreactor, there are nutrition, oxygen, carbon dioxide, waste product, pH, temperature and humidity are main important biochemical controls affect the growth of the cell. There than the flow rate, volume, shear stress, pressure, resistance and compliance like biomechanical controls also involved in the cell growth. Therefore, specific bioreactors are need for the growth of the stem cell. Because inside the body, cells are always stimulated by mechanical, electrical and chemical signals, these influencing their behaviour. In fact, biological tissues adapt their structure and composition to surrounding specific and functional demands. By putting cells alone or only in contact with materials in culture medium is not enough to obtain a functional tissue. In vivo, the heart valves are subject to a unique combination of mechanical stimuli, including flexure, shear stress, and tension (Vesely and Boughner 1989). In growth of the embryonic stem cells require temperature, partial pressure of oxygen, partial pressure of carbon dioxide, pH, and shear force and biochemical conditions of their micro- and macro-environment. Then try to find homogeneous and constant conditions for micro- and macro-environment for the entire cells population. By uneven cell distribution, lack of nutrition and oxygen and insufficient extracellular matrix production cause some limitation to bioreactor scaffold in stem cell culture. Therefore, get rid of those must make to stem cell onto polymers, which will increase the mechanical strength of the heart tissue construction and develop subsequent tissue formation.
To develop a bioreactor to provide cyclic flexural stimulation, to demonstrate the operation of the bioreactor and sterility maintenance and to evaluate the effects of unidirectional flexure on the effective stiffness of bioresorbable polymeric scaffolds which have been used extensively in the tissue engineering of the heart tissue. Therefore, must design the devices for closed controlled environment in which biological and/or biochemical processes are developed maintained pH, temperature, pressure, nutrient supply and waste removal, with high degree of reproducibility of the heart valve. Therefore, bioreactors are particularly crucial for the regeneration of complex 3D tissues. The bioreactor was designed using 3D software. The structural element of the device was machined from polysulfone; chosen for its excellent thermal and chemical stability; and abrasion-resistant acrylic; which provides good optical transparency. Culture medium was Dulbecco’s Modified Eagle’s Medium with 4.5 g/L glucose and L-glutamine supplemented with 10% fetal bovine serum. Antibiotics were excluded to assess the intrinsic ability of the bioreactor to maintain sterility. When developing scaffold use the degradable material and permanent materials as in artificial implants and in use of cells. Then preparing scaffold must test in vitro and in vivo how they hypotheses of scaffold and cell interaction, scaffold effect on tissue growth and 3D environment effect on stem cell differentiation. Scaffold materials consisted of a non-woven mesh of polyglycolic acid(PGA) fibers dip-coated with poly-4-hydroxybutyrate (P4HB), and a non- woven, 50:50 blend, mesh of PGA and poly-L-lactic acid (PLLA) fibers dip-coated with P4HB. The PGA and PGA/PLLa scaffold had an approximate fiber diameter of 0.012-0.015 mm and density of 69mg/ml. Rectangular scaffold sample were cut to size (approximately 25×7.5x2mm) and dipped briefly into a solution of P4HB in tetrahydrofuran (1% wt/vol), resulting in a P4HB coating following solvent evaporation. P4HB is a bioresorbable thermoplastic that allows for scaffold to be moulded into any shape. Scaffold were cold gas sterilized with ethylene oxide prior to use.
The use of ‘bioreactors’, chambers which provide the flow of nutrient media for the development and culture of heart valves construct, to provide an environment which as closely as possible mimics the natural in vivo conditions. These bioreactors have been designed for pulsatile flow, driven by a pulsatile pump, which leads to the exertion of only a positive pressure. This is not the case in vivo, as during the cardiac cycle the positive pressure exerted by fluid force is slightly counterbalanced by a little vacuum. Stem cells grow in vitro under bioreactor conditions must provide the nutrient and they produced the nitrogen contain waste product, but they sensitive to the nitrogenated waste product. This will be varying with the tissue and that will change the shear stresses effecting on the tissue. The oxygen pressure is maintained at set constant value with calculated volume of solution added every time to the medium. Other way round maintained the carbon dioxide pressure at set constant value with calculated volume of waste product removed from the medium. Oxygen is most important nutrients for cells in all aerobic metabolic cycles. It is the limiting nutrient in successful tissue growth in vitro, sufficient amounts of oxygen to the surface of the cells mainly because of the poor solubility of oxygen in culture media. In that case hypo-oxygen or hyper-oxygen stresses will be concern the stem cell culture causes of programmed cell death or apoptosis. Therefore, adjust the stem cell for the anaerobic cell metabolism with low oxygen tension (40 mmHg) and low pH or for aerobic cell metabolism with higher oxygen tension (80mmHg) and high pH. Then living tissue is sensitive to pH changes in the medium, during maintain of the oxygen level must maintain the pH also. Other than that glucose and lactate are providing to cell metabolic process. Therefore, they act as the indicator for cells activity.
In the bioreactor environment stem cell proliferates and increased the mass that leads the limitation of the final size of the tissue grow. Other than that, there are spaces to pass oxygen and nutrient throughout the scaffold otherwise this also leads to limitation of the tissue growth. Therefore, bioreactor must design to proper diffusion of oxygen and the nutrient and mass proliferation; cell will survive and proliferate within 150-200µm distance.
Shear stress will affect the tissue culture growth, most of the stem cell responds to it. They are proliferating according the orientation of the flow direction. In that case stem cell can aggregates by using higher shear stress that can be used for tissue function and viability. If design the rotating bioreactor that can decrease the shear stress and avoids the contact between the cells and the wall of the bioreactor, chamber must permanently rotate with one direction and control to forming uniformed growth of the tissue. But if design the non-rotating bioreactor then must create the specific mechanical stress applied on the cell culture, by perfusion solution can passed through the cell tissues by flow through the culture chamber. Some experiments were demonstrated that the shears stress 0.1 dyn/cm2 was ideal for stem cell to growth. If that exceeds the shears stress 1 dyn/cm2 were damaged the cells and the shears 0.01 dyn/cm2 were insufficient to promote the growth.
Bioreactors have developed functional heart tissue in vitro environment over specific biochemical and physical signals known to regulate cell differentiation, by improving the formation of the heart tissue by proving uniformed mixing pattern, transported the nutrient to enhance the cell growth and hydrodynamic or mechanical stimulation for stem cell to develop. Simple static flasks or a magnetically stirred flask is not suitable environment for 3-dimensional heart tissue scaffolds to develop. To develop the lowest possible homogeneous cell number for heart tissue, must grow the cell with uniform and efficient of porous scaffolds. When compare the cells seeding into mixed petri dishes yield with the static loading of the cell into the scaffolds has thicker constructs and more spatially uniformed distribution of cells. By seeding in rotating vessels or mixed flask must maintain a uniformed suspension of isolated cells and provide a relative velocity between cells and the scaffold during seeding. Dynamic seeding using mixed flasks will show to achieve seeding efficiencies approaching 100% but led to cell densities higher at the scaffold periphery. Therefore, when design bioreactor; must provide the scaffold perfusion with a cell suspension in alternate directions, which lead to the more homogenous seeding on a variety of scaffold with potential yield. Once the cells are associated with the scaffold, cell-polymer constructs can be cultured in bioreactors applying specific regimes of fluid flow.
Selecting rotation wall vessels bioreactor The bioreactors are used for proliferation of cells on a small or large scale, to generate 3D tissue constructs, a certain process must occur. That case the cells are proliferated in a bioreactor to provide the quantity of cells needed. The cell loses their specialized characteristics during the process of proliferation is the problem. Therefore, microcarrier culture used for improves cell expansion significantly and that mixed the bioreactor system well. After the cell proliferation they must associate with enhanced heart tissue formation. In above process cells must receive proper nutrition and a stable environment. There for controlled the temperature, optimum pH, sufficient substrate, water, salts, vitamins, and oxygen. The Rotating-wall vessel culture is the best bioreactor for culturing constructs stained intensely, and homogeneously for scaffold for their cross-sectional area. Inside the bioreactor a dynamic flow generated by a rotating fluid environment is an alternative and efficient way to reduce diffusion limitations of nutrients and wastes. The rotation produced the low level of the shear stress to the cells, creating mechanical stimulation. Other than that, there are other mechanical forces that affect the cells during growth, like mechanical compression, hydrodynamic pressure, and fluid flow. They will affect the magnitude, frequency, and duty of the bioreactor cycle.
To control the free-falling state adjusted the rotation speed, it protects the fragile tissue by decreasing the shear stress and avoiding the contact between cells and the walls of the bioreactor. During 1990s NASA scientist did some research about the microgravity involved in to the cell tissue of the mammals. They used the closed tubular cylinder forms the system’s cell culture chamber, which filled with a liquid medium where the cell grows on micron-size beads. The chamber has rotated along the horizontal axis; in that case they allowed the cell to develop in an environment like the free fall of microgravity. They supply oxygen and nutrition through a porous wall in the chamber, as same way they removed the waste product and the carbon dioxide. The rotating wall vessel bioreactor is providing the conditions of weightlessness for microbes by growing them inside of a slowly rotating liquid-filled chamber. The process of the rotation liquid has counteracted with slow sedimentation of the cell by creating a constant free fall of the cells through the culture medium. While rotation cell gets a slight sheer stress from liquid, lead to avoid the flattened on the bottom of the container. The scientist used the clear shell for allowed to check growth and cylindrical filter holds on the centre for supply the oxygen and nutrition and removed the carbon dioxide and waste products. And, they insure the fluid rotation without shear stress would leads to destroy the cells. They noticed rotation vessels did not cancelled the gravity, but that maintain the cells in continual free fall environment inside the shell.
“Bioreactors for the application of physical forces to engineered cartilage tissues. In the rotating wall vessel system (A), the rotational speed is adjusted so that the drag force of the medium (Fd) is balanced by the centrifugal (Fc) and gravitational (Fg) forces. The constructs are thus maintained in a tumble-slide regime and the resulting dynamic laminar flow enhances the production and accumulation of cartilaginous extracellular matrix. Specific culture chambers (B) have been developed for the application of direct deformation to engineered constructs. Chambers include wells to allocate tissue constructs (I), a magnetic bar for medium stirring (II), an inlet/outlet port for medium change (III), a cover lid to maintain sterility (IV), and micrometer screws to accurately establish the contact position between the plungers and each specimen (V).”
The cell seeding is effects of shaking speed and initial cell concentration in suspension on cell culture medium, therefore cell seeding must do in efficiency. In that case initial seeding density and cell distribution within the scaffolds must understand. Initially cell concentration is low, in that time seeding efficiency and initial density will decreased with increasing shaking speed. But high initial cell concentration that will reverse the result. All the different cell concentration uniformity of the cell distribution decreased with increased shaking speed. But under the same shaking intensity were observed with on significant differences in uniformity between cells with different initial concentration. In vitro the tissue engineering of heart tissue structures is to develop combined cell seeding and perfusion system. Cell seeding is consisting of whole system, that incorporated into the perfusion system and air-driven respirator pump connected to the bioreactor. Therefore, cell culture medium is closed-loop system that will continuously circulate. Therefore, scientist developed a cell seeding device for static and dynamic seeding of vascular cells onto a polymeric vascular scaffold and a closed-loop perfuse bioreactor for long term vascular conditioning. By using cell seeding chamber can be easily connected to the bioreactor, which have combines continuous pulsatile perfusion and mechanical stimulation to the tissue -engineered conduct. In that scientist adjust the stroke volume, the stroke rate, and inspiration/ expiration time of the ventilator allow various pulstile flows and different levels of pressure.
Discussion When selecting of the scaffold consider the biocompatibility, reproducibility, biodegradability, ability to be processed to complex shapes, ability to support cell growth and proliferation and mechanical properties of materials. Other than that, scaffolds must have similar electrical and functional activity with create systolic force. The limited availability of the incubator space; the place where the multiple bioreactors place, in this space multiple bioreactors must be places. Development of the stem cell is temperature depended process, any cells grow at body temperature in optimal level therefore temperature must maintain in that level as possible. The bioreactor design must set the temperature parameter to monitor the temperature. If inside temperature changes by increased or decreased then that must alarm on, then it can adjust manually.
Sterility is very important throughout the development of the heart tissue. We used flask and glass vessels with threaded fitting, which is cheap and proved to maintain perfect sterility. To reduce the risk of contamination, make sure all connections before sterilisation and sterilize bottles with correction solutions connected to the vessel, by using either alcohol or stem. The tubing can be placed into the pump head easily after the sterilization. Because contamination of the medium lead to the growth of the heart tissue. Therefore, bioreactor must develop as a semi-closed system.
Maintain the small cell culture medium all the time, easy replace the balance amount of the cell culture medium for requirement created by cell seeded as soon as possible. If require in addition to that easy seeding of the additional cells. Maintain the oxygen level in the medium is very essential; therefore, reassure the amount of the oxygen in the medium is enough for the development of the stem cells. When we maintain the pH level in the medium that passively adjust oxygen level in the medium, by enrich the medium with CO2 level up to 5%. The biocompatible substances must use when the designing process of the bioreactor, those substance will not kill the stem cell during the tissue growth. There are many analytic parameters, those must monitor regularly with some sensory methods to alarm if there are any changes occur in the media and correct it manually. Any design bioreactor can have ability to experiment several times with longer period. If there are any alternations, like change the cell culture medium with ingredients needed or changer scaffold materials change those and can perform the process easily. By using roller pump can sucks the cell culture medium from the bioreactor, which leads to stress of the scaffold. This help to stem cell growth towards the heart tissue.
This bioreactor must use inside the hospital, for treat each of the hole in the heart patient therefore this must produce low cost heart tissue for the patient. Other than that, there should be very low laboratory involvement and convince for patient and the surgeon. When using this kind of tissue engineering think some social highlight that affect the both quality and quantity of the life. Some religious background this technology is some bad for the life, ethical concern there are some extent to do those kinds of experiment. But medical point of view this is the good solution for treatment of the patient without suffering. In that case be careful of handling with stem cell and other, that will lead to caused critical threat to handler.
Conclusions The developed bioreactor has set sterility at least week, with working tool for conducting experiments regarding heart tissue growth. The growth of the heart tissue helps to develop entire heart, which can helpful to many heart diseases. Nutrition concentration must keep in mind when performing the bioreactor process. When the time of the replacing the medium nutrition concentration must maintain, also try to minimize the number of time replacement the medium.
Acknowledgment
I would like to thank Professor Alicia El Haj, Dr. Nicholas R. Forsyth and Dr. Ying Yang for their support and guidance in completing this study.
I would like to special thank Dr. Sun Tao for his support and guidance in completing this study.

Analysis of Proteins in Fish Muscle Tissue

Introduction In vertebrates, the muscular system is an anatomical organ system controlled through the nervous system. Derived from the mesodermal layer of embryonic germ cells, these contractile tissues-of skeletal, smooth, or cardiac origin-are responsible for blood circulation, internal organ function, heat production, and organ protection.[1] With the skeletal system integrated, voluntary and reflexive movement, as well as posture and body position, become possible. Surrounded by an epimysium, skeletal muscles are composed of many long muscle fibers lined with endomysium, which are bound together by perimysium into bundles called fascicles.[2] Within these myocytes, there are smaller strands of myofibrils that contain myofilaments (or sarcomeres) – the basic unit of a striated muscle tissue. These repeating sarcomeres contract in response to nerve signals by means of sliding filaments: actin and myosin. The thin filaments consist of two chains of spherical actin proteins twisted in a helical conformation and troponin as a contraction regulator.[2] Each actin molecule has a myosin-binding site that is covered by tropomyosin during muscle relaxation. Having a head and tail region, myosin II proteins generally form the thick filaments with its six polypeptide chains and can cross bridge with actin filaments due to their elasticity and contractibility properties. Specifically, the motor domain of its two heavy chains adopt an α-helical coiled coil configuration and couple ATP hydrolysis with its motion while its two light chains-which wrap around the neck region of each heavy chain at the IQ sequence motif-have regulatory roles[1]. Although this major multi-subunit protein has remained greatly stabile across the animal kingdom over time, myosin light chains have undergone evolutionary divergences for different species; however, the essential structure and functions have remained highly conserved.[3] Caused by genetic mutations, only favorable variations are passed through – this process allows for specialization, speciation, and evolution that eventually increases survival ability: DNA (genes) ® RNA® Protein ® Trait ® Evolution. Protein gel electrophoresis and western blotting can be used to compare myosin light chains of different species by identifying any commonalities or alterations in specific subunits. Since proteins reflect changes in the gene pool, the phenotype and function as well as form of an organism can be identified, allowing for the study of their physiological adaptations to the environment. Through comparative proteomics-defined as the analysis of differentially expressed proteins with comparison between at least two protein profiles-changes in the proteome that have been caused by development, diseases, and the environment can be identified – allowing for assessment of biological variability and dataset comparability.[4]
The objective of this lab was to extract proteins from unknown samples of fish muscle tissue and then qualitatively analyze this protein mixture by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) twice. The protein bands of the first gel-representing the total amount of proteins found in the tissue homogenate-were stained and visualized at 595nm with the Bio-Safe Coomassie Blue G-250 dye at 595nm while the fractionated proteins of the second gel were electroblotted onto a nitrocellulose membrane via Western blotting – where the specific protein of interest was selectively immuno-detected by chemiluminescence with a horseshoe radish peroxidase-linked secondary antibody. [3,4] Accordingly, the goal of this report is to identify the different types of proteins found in fish muscle-specifically of shark, tilapia, skitter, and salmon-required for muscle contraction and movement and to establish whether they are highly conserved or variable across all animal species. Consequently, information about the environment, niche, or physiological stresses faced by the organism can be elucidated as specific protein modifications that alter muscle function and performance work to increase their fitness and adaptiveness.[2] Differences in proteins may reveal information about the evolutionary relationships among various organisms and by understanding this diversity in the natural world, many biological problems can be solved to improve the quality of human life.
Materials and Methods
First, unknown tissue samples from two different fish species were prepared for protein extraction: in a 1.5mL microcentrifuge tube, 250μL of Laemmli (1x SDS) sample buffer was added as well as the minced tissue. After gently agitating the contents by flicking the tube, it was left to incubate at room temperature for five minutes. Next, the tube was centrifuged to pellet the tissue; this allowed for transfer of the supernatant buffer to a new 1.5mL screw cap tube, which was then boiled at 95°C for five minutes. Second, SDS PAGE was performed on two separate precast TGX gels (purchased from Bio-Rad) since both Coomassie Blue staining and Western blotting were required. Refer to the BIO314 experiment 7 lab manual for instructions on how the gel apparatus was assembled with the Mini-Protean gels and tetra cell. When this was completed, the loading scheme for Coomassie staining involved pipetting the protein ladder (Biorad cat #161-0375) in lane 1 (at 7 μL/line) and the actin/myosin standards in lane 6 (at 5 μL/line). The rest of the lanes were used to load the samples (at 10μL/line). The same set-up was done for the immunoblotting gel, except only 5μL/line of each boiled sample was loaded. Refer to the BIO314 experiment 7 lab manual for instructions on how these solutions were loaded. After all of the samples have been loaded, the gel box lid was connected to the electrode assembly by matching the red and black leads with their corresponding electrodes. Then, the leads were plugged into the power supply, which was subsequently turned on and set to run at a constant voltage of 200V. This process was terminated at 30 minutes when the loading dye started to exit the gel. Refer to the BIO314 experiment 7 lab manual for instructions on how the gels were removed.
Third, Bio-Safe Coomassie staining was done on the appropriate gel-with samples loaded at 10μL/line-which was peeled from the plate: it was then inserted into a container of deionized water and washed for 5 minutes on a rocking platform. Afterwards, the gel was transferred to another container with Coomassie staining solution – again, this was left on a rocking platform for 15 minutes. Upon completion, the stained gel was put in deionized water (destaining solution) and the lid was capped onto this container, which was placed onto the rocking platform for 15 minutes. Fourth, the immunoblot was prepared and transferred: with blunt-ended tweezers, the PVDF membrane and bottom stack was placed on the cassette base; the membrane was left facing up. Any air bubbles seen were immediately removed with a blot roller. Since one mini gel was employed, the stack was centered in the cassette. Then, the second gel-with samples loaded at 5μL/line-was peeled from the plate (from the SDS-PAGE step) and stacked over-top of the PVDF membrane. Any air bubbles present were subsequently removed using a blot roller. Next, a second wetted top-ion transfer stack was placed above this gel. This assembled sandwich was rolled thoroughly with a blot roller to prevent any air bubbles from being trapped. Finally, the lid was closed and locked onto the cassette and this was set inside the turbo blotter to initiate the transfer. When the electro-transfer process was finished, the blots were dismantled and stored (at -20°C) according to the instructions written in the BIO314 experiment 7 lab manual. After one week, the Western blot-that had been rocked on a platform with block solution A for 1 hour-was placed into 10mL of blocking solution B and 5μL of primary antibody was added on that solution with swirling; this was incubated for 20 minutes. Upon completion, the gel was washed with 15mL of wash buffer (three times, each with 10 minutes of incubation); then 15mL of blocking solution B and 5μL of secondary antibody was added and incubated at 15 minutes. The three wash steps were repeated. With the wash buffer drained, the membrane was put on a plastic paper protector (with the protein side up) and 400μL of substrate (made by mixing reagent A and B in 1:1 ratio, 200μL each) was spread evenly across the middle of the blot. A plastic protector was then added over it and this was imaged with a digital imager for chemiluminescence detection and analyzed using the BioRad ChemiDOC-MP Imaging System for the molecular weight and signal intensity of the protein bands (refer to the instructions posted on blackboard on how this program was operated).
Results and Discussion
According to the Coomassie-stained gel, the variability in the staining intensity of the protein bands in lanes 2, 3, 4 and 5-for skeletal muscle tissue samples from shark, tilapia, skitter, and salmon-signify the difference in the relative abundance of individual polypeptides in each organism (note that lane 5, band 11 was used as the reference). Influenced by factors such as protein expression and control, these species have generated different quantities of proteins with similar masses in their muscle tissues as they have adapted to specific environmental and biochemical interactions.[5] In figure 1, the potential mass and intensity values of myosin-light chain (MLC) are as follows: shark (15.43kDa at 0.37, 17.65 at 1.71, 20.64 at 1.09, 21.60 at 0.25, 23.05 at 0.69, 23.79 at 0.92, and 25.54 at 1.02); tilapia (15.33kDa at 1.34, 16.42 at 0.75, 19.02 at 0.35, 20.37 at 1.56, 21.47 at 0.34, and 23.79 at 0.36); skitter (15.92kDa at 2.09, 17.99 at 0.94, 20.12 at 0.48, and 23.75 at 0.55) and salmon (16.07kDa at 1.13, 20.12 at 0.31, 21.08 at 0.64, 21.76 at 0.26, and 24.92 at 0.34). Due to selective immunodetection of MLC proteins in Western blotting by a primary antibody, the various protein bands lying in the general MLC range of 15-25kDa in the Coomassie gel can be narrowed to: shark (23.94kDa at 1.33); tilapia (24.47 at 0.70); skitter (24.47 at 0.36); salmon (24.47 at 0.22) and myosin marker (24.47 at 2.40) – all of which resemble the myosin light chain isoform I (>20kDa) as isoforms II (20kDa) and III (15kDa) have lower masses; with a greater variability of myosin, tilapia has an additional band of 20.68kDa at 0.39 that resembles isoform II. [5] The other bands were dismissed as non-specific background interferences (note that lane 4, band 5 was used as the reference for the immunoblot). The high specificity of primary antibodies in probing their target allows for its wide-use in proteomic research as a reliable immunodetection technique; since proteins can indicate evolutionary relatedness or the presence of genetic diseases, their role as biomarkers has allowed for measurements of physiological changes as well as their quantifications.[6] In the appendix, all of the protein bands for the four species have been assigned a protein that corresponds to its molecular weight. From this, it can be denoted that sharks are more closely related to salmons than tilapia and skitters, both of which are tied for second place. However, based on fish phylogeny: sharks and skitters-belonging to the same class called Chondrichthyes-have diverged prior to the class of Actinopterygiis, which include both salmon and tilapia.[7] In terms of classification relative to the “order”, sharks (of Elasmobranchii) have the greatest evolutionary relationship with skitters (of Rajiformes), then salmons (of Salmoniformes), and lastly tilapia (of Perciformes).[7]
As a hexameric ATPase cellular motor protein, myosin is composed of four light chains (MLC)-two non-phosphorylatable essential alkali chains, two phosphorylatable regulatory chains-and two heavy chains (MHC). Specifically, the protein bands of these light chains have a molecular weight as a range from 15 to 25kDa; this diversity in the masses occur largely from alternative RNA splicing mechanisms that generate multiple tissue-/developmental stage-specific isoforms.[7] Although these polymorphic variations do not significantly alter the actin-activated ATPase activity of the myosin-heavy chain, they affect the actin-filament sliding velocities and kinetics-leading to different force-generating abilities.[8] In an evolutionary context, the existence of these hybrid molecules has been adopted by muscles-in response to changing functional demands-to shorten this translocation time in order to increase their overall fitness. Consequently, numerous variants of slow and fast light chains were developed despite the underlying plasticity of striated muscles.[7]
Voluntary muscles are divided into slow twitch and fast twitch muscles. The main difference is that the former “red” muscle contracts for longer periods of time with little force, require an oxygen-rich operating environment, and contain only two distinct light chains while the latter “white” type contracts quickly and powerfully for only short bursts of anaerobic activity as they become exhausted due to lactic acid buildup, have glycogenolytic capacity, and possess three different light chain subunits.[8] Over 90% of swimming muscles from sharks are composed of myotomes that can create massive propulsive forces by contracting their high numbers of white fibres; only a few such as the Great White incorporate bands of red muscle to elevate endurance over strength.[9] Accordingly, this explains why the MLC band on the Western blot has the greatest intensity of 1.33 relative to the other species. Conversely, fish species are generally composed of endothermic red-segmented muscles in their trunk musculature-allowing for their stiff-bodied, slow undulatory swimming motions.[6] Due to their decreased mass of white muscles, MLC bands of tilapia, skitter, and salmon are of lower intensity at 0.70, 0.36, and 0.22 – respectively. Relative to mammals, fish myosins share the same light chain patterns but have higher variability in MLC mass and quantity due to adaptive differences in movement between red and white myofibrils.[6] Since they have larger phylogenetic diversity, there is an enormous range of contraction speeds and swimming styles among homologous muscles.[6] For example, fast twitch muscles of rabbit, sheep, and chicken have three light chain components at 250kDa-whereas only one is found homologous at 180kDa among pike, dogfish, mackerel, angler-fish, and carp.[5] Moreover, their poikilothermic-nature may have contributed to these light chain divergences as they were forced to adjust to fluctuating environment temperatures that required specific muscle responses for survival.[9]
Sources of errors with the techniques employed contributed in hindering the accuracy of the results. First, the amount of protein stained with Coomassie dye varied greatly between the sample replicates since the dye may complex with the anionic detergent in its free cationic form – interfering with protein concentration estimates. Moreover, this dye selectively targets amino acid resides arginine, tryptophan, tyrosine, histidine, and phenylalanine; however, the assay performed responds primarily to arginine residues – eight-times higher than other ones listed above.[2] Second, reproducibility of the sample preparation and protein extraction steps was an issue due to variability among the skills of the student, which may have caused the quantity differences seen among the replicates. For example: if more tissues were added for one specie, the increased concentration of proteins loaded into the lane would be misled for a true difference in expression among or between the species. To overcome these problems: one, an automated protein extraction systems should be employed since its robotic liquid handing technology can control for errors and contaminations – leading to greater reproducibility and accuracy; two, silver staining can be substituted for Coomassie due to its higher sensitivity (0.2ng versus 7ng – respectively); third, adjustable single-/multi-channel Rainin electronic pipettes should be used as its fully automated and repetitive micro-pipetting has superior consistency – allowing for higher throughput work.[4,5,6,9]
Overall, it has been discovered that-irrespective of muscle tissue origin-myosin light chain molecules are heterogeneous in mass and intensity and the existence of phasically active fast muscles versus slow tonic muscles has led to characteristic light chain patterns among different fish species. Based on similarities and divergences in the overall protein content and intensities of the different fish species mentioned above, sharks are deemed to be more closely related to salmons than tilapia and skitters – both of which are tied for second place. However, according to fish phylogeny, sharks and skitters have diverged before salmon and tilapia, leading to an “order” classification of sharks (Chondrichthyes, Elasmobranchii) having the greatest evolutionary relationship with skitters (Chondrichthyes, Rajiformes), then salmons (Actinopterygiis, Salmoniformes), and lastly tilapia (Actinopterygiis, Perciformes). Radical alterations in their muscle proteome may have originated from adaptive responses to environmental stresses-i.e. osmotic, anaerobic, and thermal condition changes- or during symbiosis and development since cells can make different sets of proteins based on its specific spatial-temporal conditions.[5] The inferences made in this lab come with great uncertainty due many accuracy and reproducibility problems. Thus, fluorescence two-dimensional differential gel electrophoresis can be substituted for SDS-PAGE; high-throughput proteomic technologies like micro arrays, mass spectrometry-based methods, protein chips, and reverse-phased protein-microarrays can be used for protein profiling and detection; and hybrid separation-analysis techniques such as reversed-phase chromatography-ESI ionization online analysis systems can be utilized for greater sensitivity, accuracy, and precision – all of which allow an experimenter to draw firmer conclusions.
References
Bandman, E. et al. Developmental Appearance of Myosin Heavy and Light Chain Isoforms in-Vitro and in-Vivo in Chicken Skeletal Muscle. Developmental Biology. 1982, 2, 508-518.
Chatfield, S. Experiment 7: Extraction and Electrophoresis of Proteins: Immunoblot Preparation. BIO 314 Laboratory Manual. 2017.
Chatfield, S. Experiment 8: Development of Immunoblots (Western Blots). BIO 314 Laboratory Manual. 2017.
Focant, B. et al. Subunit Composition of Fish Myofibrils: The Light Chains of Myosin. Journal of Biochemistry. 1976, 110-120.
Lowey, S. et al. Function of Skeletal Muscle Myosin Heavy and Light Chain Isoforms by an in Vitro Motility Assay. The Journal of Biological Chemistry.1993, 268, 20414-20418.
Lowey, S. et al. Light Chains from Fast and Slow Muscle Myosins. Nature. 1971, 81-85.
Syme, D. et al. Red Muscle Function in Stiff-Bodied Swimmers: There and Almost Back Again. Philosophical Transactions of the Royal Society B: Biological Sciences. 2011, 1507-1515.
Tomanek, L. et al. Environmental Proteomics: Changes in the Proteome of Marine Organisms in Response to Environmental Stress, Pollutants, Infection, Symbiosis, and Development. Journal of Animal Science. 2003, 373-390.
Young, R. et al. Structural Analysis of Myosin Genes Using Recombinant DNA Techniques. Journal of Animal Science. 1968, 259-268.

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