Proteomics | SDS PAGE | Applications of Proteomics | Biology Bug

What is Proteomics?

Proteomics is the branch of molecular biology which is the large scale study of proteomes. But what is proteome? A proteome is set of proteins produced in an organism, a system or biological context. Proteome can be studied in specific organism (human, rat) or can be studied in specific organ (liver, pancreas). The proteome is not constant because it differs from cell to cell and changes over a period of time.

Importance of proteome analysis

Proteins are not only the important macromolecules in our body but also the most common effectors of disease pathogenesis and determinants of treatment response. But analysis of proteins is technically challenging and hence we face troubles to develop effective therapeutics. However analysis of DNA and RNA have some role in the prediction of protein function but these results do not always correlate with protein functions. Proteomics help us to overcome these challenges and also in the prediction of protein functions at different levels like post translational modification, domains, motif etc. Despite the inherent limitations of proteomic methodologies, as many as 115 protein based assays have been approved for use by regulatory agencies and commercialize with the great success.

SDS-PAGE or Gel elctrophoresis

Gel based proteomic is the most popular and versatile method of global protein separation and quantification. This is mature approach to screen the protein expression at the large scale. Based on two biochemical characteristics of proteins, two dimensional electrophoresis combines isoelectric focusing which separates proteins according to their isoelectric point and SDS PAGE which separates them further according to their molecular mass. The next typical steps of flow of gel based proteomics are spots visualization and evaluation, expression analysis and finally the protein identification by mass spectrometry. For the study of differentially expressed proteins, two dimensional electrophoresis allows simultaneously to detect, quantify and compare up to thousand protein spots isoform including post translational modifications in the same gel and in a wide range of biological systems.

First dimension

Proteins are amphoteric molecules i.e. they carry both positive and negative charge; hence the net charge on proteins is zero depending upon their amino acid composition. The isoelectric point of protein is the specific pH at which the net charge of protein is zero. Proteins are positively charged at pH values below their isoelectric point and are negatively charged at pH values above their isoelectric point. IEF is an electrophoretic separation based on this specific characteristics of proteins.

Basically the first dimension of two dimensional electrophoresis is achieved with a strip. It is a dry gel that is formed by the polymerization of acrylamide monomers, linked by bis-acrylamide with molecules of covalently linked immobilin, immobilins are chemical components that are derived from  acrylamide and have additional ionizable non amphoteric functions. Immobilins of various pKa can create am immobilized pH gradient inside the gel.

The strip acrylamide gels are dried and cast on a plastic backing. They are rehydrated in a solution containing a pI- corresponding cocktail of carrier ampholytes and with the correct amount of the proteins in the solubilization buffer. The carrier ampholytes are amphoteric molecules with high buffering capacity near their pI.

When an electric field is applied, the negatively charged molecules (proteins and ampholytes) move towards the anode (red electrode) and the positively charged molecules move towards cathode (black electrode). When the proteins are aligned according to their isoelectric point, the global net charge is zero and the protein is unable to move and is then focused. Focusing is achieved with a dedicated apparatus that is able to deliver up to 8000-10000 V, but with a limitation in current intensity to reduce heat.

The equilibration step is critical for 2DE. In this step the strips are with sodium dodecyl sulfate (SDS), an anionic detergent that can denature proteins and form a negatively charged protein complex. The amount of SDS bound to a protein is directly proportional to the mass of protein. Thus, protein that are completely covered by negative charges are separated on the basis of molecular mass.

Second dimension

The SDS denatured and reduced proteins are separated according to an apparent molecular weight, in comparison with a molecular weight maker. Equilibrated strips are embedded with 1% low melting point agarose in TRIS/Glycine/ SDS running buffer and 0.01% bromophenol blue on the top of second dimension gel. When the bromophenol blue migration front reaches the bottom of the gel, the second dimension is finished and the acrylamide gel can be removed from the glass plates.

Application of Proteomics

Mining

Expression profiling

Network mapping

Protein modification

Mining

It is a process of analysing and identifying all the protein samples. Mining is one of the ultimate excercise in proteomics where one simply resolves proteins to the greatest extent possible. It uses MS with associated database and software tools to identify what exactly is found. The several approaches of mining offer the ability to confirm

Expression profiling

Protein Expression profiling is identification of proteins at different level of stages of organsims or cells eg. Development or disease state. Also to analyze the expression due to some genetic, chemical or physical stimulus eg drug. Expression profiling is actually a specialzed form of mining. It is used especially in the cases where two different stages are compared to see which proteins are expressing differently. This technique is used to detect the potential targets for drug therapy and disease.

Protein network mapping

It is an approach to determine how exactly proteins react with each other in a living system. Protein carry out their function in close association with other proteins. They involve in signal transduction, complex biosynthetic and degradation pathways. Most of the protein protein interaction has been studied in vitro. Then why network mapping is important? Proteomic approaches offer the opportunity to characterize more complex network through creative pairing of affinity capture techniques with analytical proteomics methods. They used to identify the components of multiprotein complexes. Multiple complexes are involved in point to point signal transduction pathways in cells. This technique helps to understand all the components in single pathway.

Protein modifications

Another application of proteomics analysis is to identify how and where the proteins are modified. There are many common post translational modifications which can govern the structure, function and turnover of protein. Also many chemicals, environmental factors or drugs can influence the modification of protein. These modified proteins can be detected with antibiodies but the precise sequence sites of specific modification are not known. Proteomics approaches offer the best  means of establishing both the nature and the sequence of posttranslational modifications. These approaches will provide of chemical modifications in domain.

External links

  • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2873371/
  • https://academic.oup.com/hmg/article/25/R2/R182/2198199
  • https://www.ebi.ac.uk/training/online/course/proteomics-introduction-ebi-resources/what-proteomics

Types and causes of moles- Biology Bug

What are moles?

Moles are the growth on the skin which are brown, black or red colored lesions which can appear alone or exist in groups. They can appear anywhere on your body including the eyes, nose, hands, legs, chest, back or even the genitals. The medical term for the moles is melanocytic nevus (visible chronic lesions on skin). Some people describe them as imperfection on the body while most of the beauty experts consider them as beauty mark. Most of the times moles appear in early childhood and during first 25 years. But have you ever thought what the factors which cause mole are? Let’s discuss some of them.

What causes mole?

  • Exposure to sun
  • Genetic factors
  • Hormonal imbalance

Exposure to sun

Every individual gets a distinct skin color because of different melanin levels. Melanin is a pigment which is produced by specialized skin cells called as melanocytes. These melanocytes produces more melanin when exposed to sun in order to protect the skin from harmful radiation. This is one of the reason why we get tan when exposed to the sun. Moles are concentrated clumps of melanocytes, which is why they look darker. A normal individual can have 15-30 moles. However the people with more number of moles have higher risk of melanoma. Melanoma is a type of cancer that develops from melanocytes. But most of the moles are non-carcinogenic in nature.

Role of genetics

The role of genetics is not well understood for mole development. However the study for melanoma has been widely published and hence we can relate it to development of benign moles. DNA present in the skin cells contains several genes. There can be mutation, hereditary factors or exposure to harmful radiation can result into variation of certain genes which includes FGFR3, PIK3CA, HRAS, and BRAF. Variation in these genes can lead to benign moles. For example, mutation in BRAF gene leads to production of an altered protein that causes melanocytes to aggregate into moles. 

Hormonal imbalance

Hormones can be imbalanced because of several reasons including pregnancy, menopause, puberty or medications. During pregnancy the level of progesterone and estrogen vary each month. Also there is small variation in amount of MSH i.e. melanocyte stimulating hormone which is produced by pituitary gland. MSH hormones stimulate the melanin production in melanocytes. Hence you can see dark or tan patches on the skin in case of pregnant ladies. It frequently occurs during the menopause since the hormonal level is fluctuating continuously and results into increase in either the size of mole or number of moles.

Types of moles

There are basically three types of moles that are symmetrical, irregular and regular. But in medicine it is divided into several types according to their location, shape and time of appearance.

Common moles

It is about 5mm large with distincy edges and dome like surface with brown pigmentation. They are found on the skin when exposed to the sun.

Atypical

This type of mole shows irregular symptoms. They have blurry borders and can vary in color. They are comparatively larger than the common moles. Also they can be flat or raised lesions. If a person is having too many atypical moles then the risk of skin cannot be neglected. The person should go for regular examination.

Congenital moles

They are also known as congenital nevi. They appear at the time of birth or within first year of baby’s life. These moles can be caused by melanocyte cells in middle layer of skin, outer layer of skin or both. They can be referred as birthmarks.

Acquired moles

Acquired moles are moles that appear during childhood and adulthood. Most of these moles are benign and pose no risk, although sometimes they can turn into cancerous moles with age. This type of mole is the most common, and repeated sun exposure usually causes it. These moles appear during early childhood or sometimes in adulthood. This type of mole generally do not pose any risk in future. They are one of the commonly seen moles.

Junctional melanocytic moles

They occur due to accumulation of melanocytes where the dermis and epidermis meet. They are slightly raised with regular borders and dark pigmentation (range from tan to dark brown). As the age increases these moles can migrate down to deeper layers of skin.

Intradermal moles

They are skin colored moles and their pigmentation is not as dark as junctional ones because they are located in the middle layer of your skin. They usually develop during late childhood or throughout the adulthood.

 Now remembering all the types of moles with their unique qualities can be little difficult. Hence doctors have introduced ABCDE method.

A: Asymmetrical

B: Irregular border

C: Multiple color

D: Bigger diameter

E:  Evolving, new or changing

External Links:

  • https://www.webmd.com/skin-problems-and-treatments/guide/moles-freckles-skin-tags
  • https://www.healthline.com/health/new-mole#types
  • https://www.medicinenet.com/moles/article.htm#what_are_moles
  • https://ghr.nlm.nih.gov/primer/traits/moles
  • https://www.skinvision.com/articles/types-of-skin-moles-and-how-to-know-if-they-re-safe

Thanks for reading. Tell me which type of mole you’ve seen? Comment down below.

Nail growth |process|why nails keep growing – Biology Bug

Do you ever realize that your nails never stop growing. But how do they grow even after cutting? Let me tell you that they are growing before you were born. Nails are hardened structure made of dead cells. They grow by the process of keratinization. Keratin is a protein which is responsible for our hair and nail growth. The article discuss about why there is continuous growth of nails and what are the factors associated with it.

Key points: nail growth, structure of nail, process of growth, role of keratin

Keratin is a protein made by our skin cells. As the cells push upwards to the skin they die and becomes harden. This process is known as keratinization. However there are several other factors which contribute to the growth of nails. Amino acids are required to synthesize any protein in your body. Similarly the amino acids which are required for synthesizing keratin are dependent on amount of Sulfur present in your body. Vitamin A is essential for absorbing this protein. Vitamin C works with zinc to create a protein called collagen. This protein develop healthy connective tissue in your finger and rest of the body. Collagen is also known to be skin tightening and glowing agent. Before we discuss the process in details, let us understand some of facts regarding the nail growth.

1) Growth of the nails depends upon the season. They grow faster in summer than any other season. 2) Fingernails grow faster than toenails. 3) Men show higher rate of growth than women.

Note: Technically nails aren’t growing tissue, hence they do not grow. They are results of those cells which stopped growing due to their genetic mechanism.

Structure of Nail:

The structure includes these important parts,

  1. Matrix
  2. Cuticle
  3. Lunula
  4. Nail bed
  5. Nail plate
  6. Hyponichium
  7. Nail grooves

Matrix: This is the root of the nail. Its function is to make new cells. These new cells pushed up by the old one through the skin. This part contains lots of nerves and blood vessels which are essential for cell reproduction. Technically it is the only living part of the nails at initial stage but ultimately results into dead cells which forms nail.

Cuticle: This is a small sliver of skin where the nail grows out of the finger. It basically protects the matrix from any germs or infections

Lunula: it is a small whitish curved like structure that sometime you see at the bottom of the nails. It can be said as a part of the matrix. Most of the time it is under the skin. Therefore not every individual can see the lunula.

Nail bed: This area lies under nail plate. Nail plate is the visible part of nails which is the result of keratinization.

Hyponichium: this is the line where nail plate separates from nail bed.

Nail grooves: These are the grooves at the side of your free edge which gives direction for nail growth.

Process of Growth

  • The process starts with the DNA and the gene which codes for keratin. Especially in the case of nails there is one gene called as CRATIN which is actively present in our nail cells.
  • This gene will code for the protein i.e. keratin which will be more produced near the nail area.
  • Now the matrix is continuously producing cells because it is rich in blood vessels. The keratin is present in every cell until the cell undergoes a death cycle.
  • As more new cells are produced, the older ones get pushed up by the new cells. This is a sensitive phase where a mineral balance (zinc, iron, cobalt) and hormonal balance is required (minerelocorticoids, thyroxine etc.)
  • The keratin protein in the cells grows harder and beyond a certain point there are no longer living cells with nuclei.
  • The growing hard structure continues to from a nail plate. Most of the growth takes place underside the nail plate and that area is called as germinal matrix. The outer layer of germinal matrix is a half-moon shaped structure that is lunula.
  • The nail plate is loosely attached to the germinal matrix. It also held by some skin at the bottom and at its sides. As it grows the nail will acquire a concave shape.

 Why do we have nails?

The nails are flattened version of claws. It is one of the important features of primates. If you observe the arboreal animals like apes, monkeys, lemurs etc. possess the same feature. Humans are primates and nails are our distinguished character. They are nothing but dead cells and hence not really useful as claws.

Do nails grow after death?

No. it is clearly a myth. Fingernails might appear long after death. It is not because they are still growing but the skin around that area is retracted. If you have carefully observed that the skin and other soft tissues starts shrinking after death. It is an optical illusion which makes the nails look bigger than the ordinary size.

Can they stop growing in certain cases?

This depends on various factors. One of the common reason which we usually see around us is the habit of biting nails. This in certain cases can damage the nail bed or matrix or lunula. If any sensitive area of the nail is damaged because of biting, injury, accident or by using sharpened objects then there is a possibility that the nail might stop growing.

External links:

  1. https://www.livestrong.com/article/181963-what-makes-your-hair-nails-grow/
  2. https://www.aad.org/public/kids/nails/how-nails-grow
  3. https://www.nytimes.com/2015/08/25/science/the-inexorable-forces-of-nail-growth.html
  4. https://classroom.synonym.com/the-process-of-nail-growth-12079176.html
  5. https://nailbees.com/nail-structure

Thank you for reading. Did you find this article helpful? Mention in the comments.

How does heat produce in body?-The Biology Bug

This article gives an idea about what exactly heat in your body is? How does it produced and regenerate? What is thermal regulation?

Key points: Endotherms, heat generation in body, organs involved in production of heat, excess production, cooling agents

When you get pimples or boils on your face, sometimes rashes on your body, headache, hair fall  or fatigue, then I am sure you all must have heard by someone in the house saying that there is too much heat in your body. This is absolutely correct because all you’re experiencing are the symptoms of increased heat in your body. But have you ever thought what this heat is? Where does it come from? How is it generated or produced in someone’s body? Further article explains the process of heat generation by various parts in human body.

Let us first understand there are two categories of animals when it comes to thermoregulation. Endotherms which are warm blooded animals and ectotherms that are cold blooded animals. Humans are endotherms which means they have to adjust the body temperature according to the environment unlike the ectotherms which includes fishes, amphibians, reptiles etc. Human body tries to maintain that with the help of several metabolic processes. Heat is one of the byproduct of the metabolic processes which occur simultaneously.

The body finds its own way to release the heat outside and controls thermoregulation. It finds the ways to release excess of heat by skin, acid production or high rate of burning calories. Human body has several sources for production of heat. One of the major sources is liver but it also includes kidneys, muscles and brain. Highest metabolic activity can be seen in the liver. Liver has more than fifty functions to do at regular intervals. The heat generated in the liver is basically due to cellular respiration. Since liver is a large organ, there are many cells that contribute to the heat. Also sometimes when old blood cells are split, they release the bonding energy which again leads to heat production. It is observed that blood leaving from the liver and brain is comparatively warmer than the normal blood.

Now when we talk about various organs in the body which are responsible for the process, it is important to note that there is one organelle which is present in almost every organ of the body which is mitochondrion. This is called as powerhouse of the cell and basic function of this organelle is to produce ATP. Therefore during the production of ATP i.e. energy, some amount of heat is released.     

Another way in which the body produces heat is by hyperthyroidism. Thyroid gland is an endocrine gland which produces certain hormones like T3 and T4. When there is excess production of these hormones, the body starts getting warmer. Thyroid hormonal imbalance can lead to excess heat production. In response to external environment hypothalamus sends a signal to thyroid gland which increases metabolic activity and makes more energy.

What causes excess of heat?

Let us understand one thing that body continuously produces heat to maintain the body temperature and to respond the external environment. But in few cases when the production is not controlled and body can not regular thermal conditions.

Micro-organism

There can be multiple reasons for that which include bacteria or any micro-organism which elicits the immune response. In order to kill that organism body increases the temperature. Therefore fever is one of the immune response to react against foreign substance. The only concern in this case is when body goes on increasing the temperature and starts damaging the other cells. Therefore we need to visit the doctor. Similarly we can give an example of mosquito bite. In response to that bite, body produces excess heat in that specific area and it becomes reddish in color.

Medicines

As we have already discussed that liver is one of the major source for producing heat. It is because all the food i.e. complex substances that we eat goes to the liver, breaks down to the simple substances and then distributed in body. Our liver acts as a checkpoint for all the substances that we eat or drink. Since liver has never encountered medicines at regular basis, it needs to cross check them. It is called as a first pass effect when some amount of medicine is lost due to the liver action. During this effect a lot of metabolic processes occur which contribute to the heat production. Liver is already producing heat via several metabolic processes. In addition to that the medicine causes more heat and the amount of heat depends on the number of medicines. More medicines causes higher metabolic rate and produces lots of heat in the body.    

Food

Generally the food which takes more time to digest or has high amounts of carbohydrates and proteins tend to produce more heat in the body. The body has to spend lot of energy for breaking down the high molecular weight substances. As a results lots of metabolic activity takes place and lead to excess heat production. It also applies to the oily or especially saturated fatty substances. This type of food has more amount of fats but generally body has a tendency to store the fats in adipose tissues and can be useful in starving conditions. But at the same time some of the fats are broken down and involved in metabolic pathways. Hence they also contribute to the heat.

How to reduce heat naturally?

  • According to Ayurveda one should drink plenty amount of water. As we all know that water is a universal solvent and can dissolve almost anything. It also act as a cooling agent.
  • Yogurt and buttermilk are the best option to reduce heat in your body. These substances are slightly acidic in nature and contribute to neutralize the effect of heat.
  • The best way to reduce heat is to consume sweeteners rather than using sugars. Therefore consumption of honey can be benefecial to reduce heat.

Thanks for reading! Did you find this article helpful? Comment down below!

Structure of nephron and Formation of Urine|Process of excretion

Key points: kidneys, nephron, structure of nephron, process of urine formation.

Kidneys play an essential role in excretion. They are bean shaped, reddish brown organs which are covered by renal capsule. There is a con caved side of a kidney that has a depression where a renal artery enters and a renal vein exits towards uterus. The kidneys are located at the rear wall of abdominal cavity.

Internal structure of kidney

Kidney has an outer dark cortical region, an inner medullary region and a funnel shaped renal pelvis region.

  • Renal cortex: This region contains glomeruli of nephrons. It appears to be dark and dotted due to the presence of malpighian bodies.
  • Renal medulla: It is a region just inner to renal cortex. It contains renal tubules, collecting ducts and blood vessels present in the form of pyramids. The conical shape of pyramid projects into pelvis.
  • Renal pelvis: It is large funnel shaped space behind the medulla region. Urine is collected in renal pelvis and passed down to uterus.
Internal structure of kidney

NOTE: Each kidney consists of large number of filtering units called as ‘nephrons’. It has approximately 1.3 million nephrons.

Each nephron originates in the cortex region and extends into medulla region.

Structure of Nephron

A nephron is also called as uriniferous tubule which is a functional unit of kidney. Each nephron filters a part of blood and produces a small amount of urine. It consists of two parts,

  1. Malpighian body
  2. Long coiled tubule

Malpighian body

Malpighian body is also divided into two main parts:  a) Glomerulus b) Arterioles   c) Bowman’s capsule

a) Glomerulus

It is like a capillary network that receives its blood from afferent arteriole from renal circulation. The glomerular blood pressure provides the driving force for fluid and solutes to be filtered out and into the space made by Bowman’s capsule. The remainder of the blood which is not filtered

into glomerulus passes into the narrower efferent arteriole. It then moves to vasa recta which are collecting capillaries.  

b) Afferent and efferent arteriole

Afferent arteriole supplies blood to the glomerulus. A group of specialized cells known as juxtaglomerular cells are located around the afferent arteriole, where it enters the renal corpuscle. The efferent arteriole drains the glomerulus. Specialized cells lies between two arterioles and those are called macula densa. The juxtaglomerular cells and macula densa together forms a juxtaglomerular apparatus.

Bowman’s capsule

Bowman’s capsule surrounds the glomerulus and is composed of visceral and parietal layers. The visceral layer lies beneath the thickened glomerular basement membrane and it is made of podocytes. The parietal layer is lined by single layer of squamous epithelium. Between the visceral and parietal layer is Bowman’s space, into which the filtrate enters after passing through podocytes. Any small molecules such as water, glucose, salts and urea pass freely into Bowman’s space but cells, platelets and large proteins cannot pass through it. As a result the filtrate leaving the Bowman’s capsule is very similar to blood plasma in composition.

c) Long coiled tubule

It consists of three important parts as follows;

  • Proximal convoluted tubule
  • Loop of Henle
  • Distal convoluted tubule

Its various parts are modified for absorption of salts and water from the blood. It opens into collecting duct. The capillaries that take blood out from glomerulus, form a network all over the long tubule and finally unite to form a renal vein.

Formation of Urine

Urine formation in nephron involves three basic steps

Ultrafiltration

Selective reabsorption

Tubular secretion

Single nephron: mechanism of urine formation

Ultrafiltration

Blood enters the afferent arteriole and flows into glomerulus. Blood in the glomerulus has both filterable and non-filterable blood components. The blood flows under pressure in the renal artery. Also in the glomerulus the efferent arteriole leaving the glomerulus is narrower than afferent arterioles. Therefore this pressure helps the fluid to filter out through thin capillary walls of glomerulus. The filterable components include water and dissolved molecules while non filterable components are blood cells and proteins. The glomerular filtrate is not the same consistency as urine. Ultrafiltration is purely a physical process and energy for filtration is derived from the hydrostatic pressure of blood.

Reabsorption

Sodium chloride reabsorbed into the system increases the osmolarity of blood in comparison to the glomerular filtrate. This reabsorption process allows water to pass from the glomerular filtrate back into the circulatory system. Glucose and amino acids are reabsorbed into circulatory system. If too much glucose appears in the glomerular filtrate it increases the osmolarity of filtrate, causing water to be released into the urine rather than reabsorbed by the circulatory system. Frequent urination and unexplained thirst are warning signs of diabetes, due to water not being reabsorbed. Glomerular filtrate has now been separated into two forms reabsorbed filtrate and non-reabsorbed filtrate. Non reabsorbed filtrate is now known as tubular fluid as it passes through the collecting duct to be processed into urine. Simultaneously any excretory wastes that were not filtered out at the glomerulus are put back into the tubule by the capillaries.

Secretion

Finally at the end of the tubule, as a result of all the mechanisms that are ultrafiltration, selective reabsorption, tubular secretion the filtrate contains urea, other wastes, excess salts and small amount of water. This fluid is now called as urine. It is drained into the collecting duct. A collecting duct collects urine from several nephrons. Finally all collecting ducts drain urine into a space called renal pelvis in the kidney, from where it passes out into the ureter, and then to urinary bladder where it is stored till excreted out of the body.

Micturition

Micturition is the process by which the urinary bladder empties when it becomes filled. This process involves two main steps: First, the bladder fills progressively until the tension on its wall rises above a threshold level. This elicits the second step, which is a nervous reflex called the micturition reflex that empties the bladder or if this fails, at least causes a conscious desire to urinate.

Conclusion

In ultra filtration process blood pressure forces the fluid and small molecules like glucose, salts and amino acids are filtered. Selective re-absorption there is active re-absorption of useful molecules such as water, glucose and salts. In case of tubular secretion the filtrate contains all the excretory waste and small amount of water.

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