Biotech World

Thursday, January 12, 2017

Genetics of sickle cell

Sickle cell disease (SCD) is an autosomal (could affect both male and female) recessive inherited disorder that is caused by genetic mutation in the β hemoglobin (as hemoglobin has 2 subunits: 2 α chains and 2 β chains) gene found on chromosome 11. Hemoglobin transports oxygen from lungs to other parts of the body such as liver, muscle etc. So mutation in hemoglobin (HbB) gene leads to the formation of sickle shaped abnormal hemoglobin S (HbS) or sickle hemoglobin that results in sickle cell disease or sickle cell anemia. Red blood cells with normal hemoglobin (HbA) can smoothly move through the blood vessels but HbS containing blood cells cannot move normally and become stiff, harder, less flexible and pile up and block the flow of blood through vessels. This blockage results in damaging of vital tissue and organs (vasoocclusion) such as lungs, spleen, kidney and liver and causes related pain. Also the sickle or crescent shaped red blood cells polymerize at reduced oxygen tension and die prematurely that results in hemolytic anemia or sickle cell anemia [1].

SCD is a monogenic disorder that results from the missense mutation that substitutes thymine for adenine in the 6th codon of the β chain gene (GAG to GTG) that causes coding of glutamic acid by valine at 6th amino acid position (Glu6val) of the β chain of hemoglobin [2]. Other types of SCD such as sickle hemoglobin C disease (HbSC) and sickle β thalassemia (HbS β) result from coinheritance of HbS with other abnormal β hemoglobin chain variants. SCD is recessive genetic disease i.e. 2 genes for the HbS must be inherited from the parents in order to get the disease. If the person has just one copy of the mutated gene and one normal gene then they are sickle cell trait (who are mostly normal but usually carrier of the SCD) but if the individual has 2 copies of mutated gene (Hb S) that results in SCD. When both parents are sickle cell trait then their child has 25% chance to have two defective genes and suffer from SCD, 50% chance to have one defective gene and develop sickle cell trait and 25 % chance to inherit two normal genes and being unaffected by the gene mutation and diseases [3].

Due to its shape and increased stickiness sickle cells/sickle RBCs adhere to endothelium and express a bunch of adhesion molecules such as CD36, CD18, ICAM 4 (intercellular cell adhesion molecule), P selectin etc. which causes increased microvascular transit times and vaso occlusion. Sickle RBC survives for only 10-20 days (where normal RBCs survives for 90-120 days) and then hemolysis occurs intravascularly that results in releasing of plasma free hemoglobin (PFH) and arginase to plasma [4]. These cause endothelial injury including proinflammatory stress, scavenging NO (nitric oxide) and degradation of arginine (substrate for NO synthesis) which results in low level of NO production and development of pulmonary artery hypertension and severe acute chest syndrome (which is the major cause of mortality for SCD patients). As vaso occlusion damages the spleen of people with SCD they have very low level of immunity (because of low level of serum IgM molecule) and they have increased risk of certain types of bacterial infection such as Mycoplasma pneumoniae, E. coli, Staphylococcus aureus etc. Children with SCD have painful spleen enlargement due to large number of sickle cells which is known as ‘splenic sequestration’ and they also have dactylitis (pain and swelling in hand and feet) [5].

Hemoglobin electrophoresis/cellulose acetate electrophoresis, isoelectric focusing (higher resolution), HPLC (High performance liquid chromatography) are the procedures for diagnosis of SCD. Treatment of SCD includes regular blood transfusion, antibiotics, gene therapy/gene editing, bone marrow transplantation from healthy genetically compatible sibling donor, and hematopoietic stem cell transplantation in severe cases. A new drug called Hydroxycarbamide/hydroxyurea (brand name Droxia which is FDA approved) which is basically an antitumor drug is currently used for SCD [6]. This drug stimulates fetal hemoglobin (that is found only in newborns) production which helps to prevent the sickling of red blood cells and cause improved red cell survival and reduction of white blood cell, reticulocyte and platelet counts. But this drug has significant toxicity including myelosuppression so patients treated with this drug should be monitored closely with routine CBCs and reticulocyte count. Recently Global Blood Therapeutics from South San Francisco developed a pill named gbt440 which can prevent the sickling of red blood cells [7].

References:

Friday, December 16, 2016

Vitamin D and longevity

The main function of vitamin D is to maintain the calcium and phosphorus levels in blood. However recent research found that it also has a huge impact on longevity. New studies found that vitamin D enhances lifespan and promotes protein homeostasis via stress response and cellular detoxification genes skn-1, ire-1 and xbp-1. The circulatory form of vitamin D is 25 hydroxyvitamin D [25(OH)D3]/calcidiol which utilizes longevity genes to increase lifespan and blocks the accumulation of toxic insoluble proteins (e.g. human β amyloids) that are associated with various age-related diseases such as Parkinson's, Alzheimer's, cognitive impairment, diabetes, heart disease, autoimmune disorders, and even cancer. Recent research (on C. elegans) from Buck institute of biological research discovered that vitamin D could increase lifespan by 33% and slow down the aging related protein misfolding because human age-related diseases are very often connected with vitamin D deficiency [1].

Vitamin D (Vitamin D3 or cholecalciferol and Vitamin D2 or ergocalciferol) is a fat soluble hormone that is produced naturally in the body from 7-dehydro-cholesterol by either ultraviolet light of sun (at the wavelength of 290 to 315 nm) or dietary intake [2]. Then it is transported to the liver/hepatocytes (where it is stored) by vitamin D binding protein (DBP) and hydroxylized by the enzyme 25 hydroxylase to form the 25 (OH) D3 (the serum vitamin D). The active form of vitamin D is calcitriol which acts as a hormone that is controlled by parathyroid hormone (PTH). This calcitriol mediates its biological function by binding with vitamin D receptor (VDR) of white blood cells or T cells or B cells. But the most important function of calcitriol is controlling phosphorus magnesium and calcium homeostasis with the help of PTH to maintain normal levels of calcium, phosphorus and magnesium level in the blood. PTH stimulates calcitriol to increase the calcium absorption by the intestine and reabsorption by the kidneys and also stimulate the calcium release from the bone. Without calcitriol calcium absorption would be only 15% and phosphorus would be 60% but calcitriol increases calcium absorption by 30- 40% and phosphorus level by 80% and then deposits these mineral crystals onto the collagen fibres of the osteoid protein matrix. Vitamin D regulates synthesis of calbindine, a cell membrane protein that binds with calcium and help to opens the Ca 2+ channels and by this mechanism ⅓ of daily calcium is absorbed by body daily. Also calcitriol stimulates normal bone growth, bone remodelling, bone metabolism and mineralization process [3].

Epidemiological studies showed that increased level of vitamin D intake either by sunlight or from supplements could reduce all causes of mortality in general population. After the age of 62-65 years there is a decline of intestinal calcium absorption rate due to decreased production of gastric acid and lower level of vitamin D/reduced numbers of intestinal VDRs that results in a lower amount of intra luminal ionized calcium, progressive loss of bone mass (that results in osteoporosis), changes of duodenal mucosa, increased level of PTH (secondary hyperparathyroidism), progressive decline of renal functions (due to reduced efficiency of renal 1 α hydroxylase enzyme). Children, elders (because of reduction of 7-dehydro-cholesterol only 25% of vitamin D3 is synthesized in a 70 years old) and people with darker skin (because most of the UVB rays are absorbed by melanin) are at high risk of vitamin D deficiency. According to NIH 15 mins of direct exposure to sunlight twice a week without sunscreen (because it reduces vitamin D synthesis) is sufficient to maintain optimum serum vitamin D level [4]. Several studies found that daily intake of 600-1000 IU vitamin D can protect skeleton, improve muscle functions, prevent rickets and other vitamin D related disorders in children and increase longevity. Recent studies on human LTL (leukocyte telomere length) found that people with high blood calcidiol level had low C reactive protein and longer LTL that results in 5 years of aging difference which means vitamin D could prolong lifespan by 5 years.

References:

Wednesday, November 30, 2016

Piezo - the mechanosensor

Piezo proteins (piezo1 and piezo2) are evolutionarily conserved transmembrane proteins (24-40 domains) which are involved in mechanotransduction in mammalian cells and function as mechanosensor. Piezo proteins are 2500-2800 amino acids long and are identified as mechanically activated ion channels(MA) / mechanosensitive channels that are encoded by FAM 38 genes. Piezo proteins mediate mechanosensory transduction i.e. conversion of mechanical forces into biological signals which is a very important physiologic process for all types of mammalian cells. This mechanotransduction regulates vital processes in mammals including embryonic development, blood pressure regulation, various sensations such as touch, hearing, pain, proprioception, urine flow regulation, cell migration, proliferation and elongation, bladder distension, vascular tone regulation, sensation of shear stress etc. All organisms have mechanosensitive channels which are directly gated by forces to convert mechanical stimuli into electrical signals in mechanosensory transduction. The 3 important mechanical sensory modalities are touch, hearing and proprioception that are mediated by mechanosensory channels. These channels open very rapidly with short latency (<5 milliseconds) and directly gated by forces [1].

Piezo1 is expressed with high levels in erythrocytes, endothelial cells and periodontal ligament cells in skin, lung, bladder, and kidney. Piezo2 is mostly expressed in sensory trigeminal ganglia (TG) and dorsal root ganglia (DRG), Merkel cells (epidermal mechanoreceptor involved in touch), lung and bladder. Piezo 1 acts as an endothelial sensor of blood flow, promotes endothelial cell organization, regulates erythrocyte volume, maintains structural integrity of red cells, detects urothelial extension during bladder distension, regulates stretch activated calcium pathway and also acts as an osmoreceptor in erythrocytes [2]. Piezo2 is involved in mechanosensation (such as light, touch and noxious stimuli) and somatosensation through cutaneous mechanoreceptor.

Mutation in human piezo protein results in various disorders including hereditary xerocytosis (a dominant disorder of erythrocyte dehydration results from missense mutation in piezo1) and different types of neuromuscular disorders such as distal arthrogryposis type 5 (another dominant disorder characterized by skeletal muscle contractures and restrictive lung disease), Gordon syndrome (piezo 2 mutation) etc. A new study from Scripps research institute suggests that Piezo1 could help to design better pain medication and future therapies for blood disorders and hypertension because piezo proteins can control the sensation of touch / sense force by detecting tension in the cell membrane [3].

References:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018681/

http://www.jbc.org/content/early/2014/10/10/jbc.R114.612697.full.pdf

http://www.sci-news.com/biology/piezo-protein-senses-touch-04364.html

Sunday, September 18, 2016

Protein engineering for Disease Control

Protein engineering is the method of production and design of new protein from natural protein/amino acid sequence using recombinant DNA technology. Nowadays protein engineering has huge impact on biotechnology and biopharmaceuticals as it has a potential to control and treat chronic diseases as protein is the workhorse of human body. Protein engineering includes various methods such as site directed mutagenesis, x-ray crystallography, DNA shuffling, random mutagenesis, homology modeling, cell surface and phage display technology, flow cytometry, molecular dynamics, computational protein design etc. Using these methods protein engineering alters protein structure to achieve functional changes such as decreased product inhibition, better substrate sensitivity, higher catalytic rates, desired cofactor use and reduced substrate competition [1]. The proteins engineered by this method can be used as therapeutic proteins to treat and control diseases which makes these very important in pharmaceutical and biotech industry.

Cancer treatment: Conventional protein engineering along with recombinant DNA technology offers intriguing possibilities for development of multifunctional and smart drug vehicles at nanoscale for the treatment of cancer and other genetic diseases. Currently cancer research involves development of specific agents for targeted delivery of imaging probes and drugs to different tumor sites. Phage display is a powerful protein engineering technology that involves selection and cloning of peptides that are displayed on the surface of bacteriophage [2]. This phage display technology isolates tumor homing peptides by in vivo phage display library screening against tumor vasculature that have huge potential as targeting probes for tumor molecular imaging and drug delivery. Protein engineering involves production of recombinant immunotoxins by fusion of variable regions of “cancer specific antibodies” with the truncated bacterial or plant toxins [3]. These immunotoxins have been shown to cause the regression of human tumor xenografts grown in mouse model. Protein engineering also utilizes complement invasion to increase complement fixing or to enhance complement dependent cell cytotoxicity (CDC) or to reverse complement resistance for cancer therapy, because cancer cells show overexpression of complement inhibitory proteins such as CD46, CD55, and CD59 in breast, lungs and other types of cancer [4].

Cardiovascular Therapeutics: Protein engineering also has a huge impact on cardiovascular therapeutics/cardiac regeneration and disease treatment advancement to specifically enhance the efficacy of molecules for cardiac repair. Recently a number of engineered proteins have been used to treat cardiovascular diseases in clinical trials such as tumor necrosis factor antagonists etanercept (Enbrel), Atrial natriuretic peptide and B-type natriuretic peptide (BNP), Insulin-like growth factor-1 (IGF-1), stromal cell–derived factor-1α (SDF-1), Granulocyte colony–stimulating factor (G-CSF), IL receptor antagonists (tocilizumab), erythropoietin (EPO), Neuregulin (NRG) etc [6]. Sarcomeric protein is the functional unit for myocyte contraction and the cardiomyopathies are caused by mutation in sarcomeric genes. Recent research suggests that stoichiometric replacement of sarcomeric proteins is a potential gene therapy approach to replace mutant proteins, alter sarcomeric responses, or neutralize altered sarcomeric function in cardiac disease.

Alzheimer and other amyloid disease advancement: Protein engineering involves Alzheimer’s research - the most common dementia in older people that start with memory loss and caused by nerve damage in brain. All amyloid diseases such as Parkinson’s, Alzheimer’s have a unique abnormally folded peptide structure/amyloid protein (also called fibrils) [5]. Recently University of Washington’s bioengineers developed a synthetic protein called alpha sheet/affibody protein that complements the toxic structure of amyloid proteins and blocks/neutralizes these proteins to prevent the amyloid fibrils from forming. This approach would be very helpful for diagnosis and specific therapies for Alzheimer’s and other amyloid diseases.

References:

Wednesday, August 24, 2016

Side effects of tea

Though tea intake has several positive health effects as I wrote in my last post, it has some negative effects too.

Consumption of high concentrations of tea polyphenols results in adverse effects such as nausea, stomach ache, heartburn, muscle pain, dizziness etc. Moderate amounts of caffeine consumption from tea (more than 300 mg to 400 mg) may have adverse effects on health. Black and green tea may also inhibit iron bioavailability/iron absorption - so have negative effects on anemic patients. Some tea contains higher amounts of caffeine such as black tea, oolong tea which should be taken under certain limit. Also some types of black, green, oolong, white, pu-erh and matcha tea, that grow in China, may contain heavy metals such as lead (Pb), aluminium (Al), arsenic (As), cadmium (Cd), mercury (Hg) and fluoride (F-) that have negative impact on health such as birth defects, osteomalacia and neurodegenerative disorders as all heavy metals are neurotoxic material. China grown tea have alarming level of heavy metals due to use of coal fired power plant (which provides about 70% of China’s energy) and the pollutants derived from these coal plants contaminate the tea plants [1]. A recent joint research study by University of Alberta and Lulea University of Technology in Sweden found that tea contains toxic elements that might be risky for pregnant and nursing women [2]. Heavy metals and other toxic components from tea can result in various types of birth defects, dampen brain development and cause chromosomal anomalies in the unborn child of pregnant women. A recent Canadian study found that Chinese oolong tea had highest level of arsenic, lead and cadmium than other types of regular or organic green tea, white tea and black tea. Tea can be contaminated with heavy metals even if it is USDA organic because organic tea may not be coated with pesticides but they could be tainted by the heavy metals from the water and soil. Also the high fluoride concentration of tea can result in dental and skeletal fluorosis [3] because mature tea leaves are fluoride accumulator.

Teas that grow in Japan such as organic green tea or matcha green tea have a risk of contamination with radioactive elements due to nuclear disaster after the 2011 tsunami [6]. Surprisingly organic green tea/organic matcha tea have higher level (30 %) of toxic components or heavy metal concentrations than the regular one. ConsumerLab.com study estimates that a cup of matcha green tea contain 30 times more lead than a cup of regular green tea [4]. As per research Camelia sinensis (tea tree) is a “hyperaccumulator” type tree that has a built in molecular mechanism to extract metals from the soil and accumulates them in its leaves [5]. Older leaves have highest metal concentration (20 times higher) than the younger leaves. Also higher steeping time (more than 4 minutes) leads to increased level of lead and aluminium (potential neurotoxin linked to Alzheimers) concentration.

Due to the contamination of tea with toxic material/heavy metal it is recommended that children and pregnant women should be very careful about the type of the tea they are drinking. The African red rooibos tea is generally safe for pregnant women and they are full of antioxidants. Though tea consumption is a healthy alternative of water it should be taken under certain limit as it has some negative impact on health that you cannot ignore.

References:

Sunday, July 31, 2016

Tea : The ancient beverage

Tea is the most consumed beverage in the world that has several health effects from bone health to weight loss. According to American Journal of Clinical Nutrition (AJCN) the antioxidants from the tea have helpful benefits in human health such as prevention of chronic illness, treatment of mood disorder, weight loss etc. Following are the health effects of tea:

Improved bone health: Recent research has found a positive connection between drinking green tea and osteoporosis which is a major health problem nowadays for elderly people especially women (because of their low estrogen level after menopause). Green tea and its bioactive compounds polyphenols/catechins (collectively known as green tea polyphenols or GTP), such as EGCG (Epigallocatechin gallate), ECG(Epicatechin gallate), EC (Epicatechin) and EGC (Epigallocatechin), may decrease the risk of bone fracture by improving the bone mineral density (BMD) and support osteoblastic (osteoblasts are bone forming cells) activities. Green tea has more bone health benefits/osteoprotective effects than other teas such as black tea or oolong tea due to decreased oxidative stress, high concentration of antioxidant enzymes and decreased expression of proinflammatory mediators [1]. Other than catechins tea also has flavonoids (such as phytoestrogen, isoflavone and lignans), caffeine and dietary fluoride that also can increase osteoporotic progression and improve BMD.

Weight loss: Due to its high concentration of caffeine and polyphenols and antioxidants tea consumption is an effective way for weight loss. The most effective weight loss tea includes oolong tea, white tea, mint tea, red rooibos tea and matcha green tea which helps in weight reduction by increasing fat metabolism and by boosting lipolysis (fat breakdown) and blocking adipogenesis (fat cell formation). The African red rooibos tea contains an unique and powerful flavonoid aspalathin that can reduce the stress hormones which trigger hunger and fat storage [2]. The catechin contents (EGCG) of Japanese green matcha tea is 30 times higher than regular green tea which makes it a very effective weight loss tea. Though its caffeine concentration is also way high, so it may not be applicable to everyone.

Boosting immunity: Several studies show that tea can positively influence the immune cells, so can boost immunity. Tea EGCG can modulate the production of cytokines (such as IL1, IL6, IL12 and TNF1- alpha) produced by the immune cells such as T cells, B cells, dendritic cells and antigen presenting cells which play key roles in immunity against microbial pathogens and tumors. See my related post for more background on immunity.

For heart attack, stroke and cancer prevention: Tea drinking (even 2 cups/day) supports healthy blood pressure and arterial function and can help to reduce stroke, heart attack, some types of cancer and other cardiovascular disease. The green tea polyphenols and the theaflavin and thearubigins from black tea have antioxidant/free radicals extraction activity that can protect cells from DNA damage caused by reactive oxygen/free radicals. Research shows that green tea catechins can activate detoxification enzymes glutathione S transferase and quinone reductase that may help to protect against tumor development [3].

Digestion and sleeping aid: Tea has a positive impact on digestion (such as green tea, mint tea, chai tea, herbal tea and ginger tea) and sleeping (such as chamomile tea and lavender tea).

Though tea is a healthy alternative of caffeine and calorie containing beverages some tea constituents may have negative impact on health and not advisable for everybody. I will write about this in my next post.

References:

1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2754215/

2. http://www.eatthis.com/5-best-teas-weight-loss

3. http://www.cancer.gov/about-cancer/causes-prevention/risk/diet/tea-fact-sheet

Thursday, June 30, 2016

Vitamin A deficiency

Vitamin A is a collection of fat soluble retinoids (retinal, retinol and retinyl esters) that are involved in immunity, cell growth & differentiation and communication, vision and reproduction. Human takes 2 types of vitamin A, which are preformed vitamin A and provitamin A / carotenoids (alpha & beta carotene and beta cryptoxanthin) that are metabolized intracellularly into active vitamin A [1]. 50-80% vitamin A are cellular RBP (retinol binding protein) / tranthyretin bound in liver and the rest is deposited into kidneys, lungs and adipose tissue as retinyl palmitate [2].

Vitamin A deficiency (VAD) / hypovitaminosis is very common in Africa and South Asia and according to WHO around 2500000 to 5000000 children become blind every year due to VAD among which half of them die within a year after blindness. Pregnant women are also at higher risk for vitamin A deficiency during third trimester. Patients with cystic fibrosis, cancer, pancreatic insufficiency, inflammatory bowel disorder (IBD) are also at increased risk for VAD. The serum retinol concentration is affected by several factors such as infection, RBP synthesis in liver and zinc & iron levels. While zinc deficiency affects retinol transport from liver, iron deficiency affects vitamin A metabolism.

Vitamin A has huge impact on vision so VAD results in various types of vision problem including xeropthalmia, night blindness, corneal inflammation, keratomalacia, corneal xerosis, bitot’s spots (abnormal squamous cell proliferation and keratinization of conjunctiva that results in irregular foamy patches in the white of the eye) etc. Xeropthalmia is characterized by abnormal dryness of cornea and conjunctiva that leads to cessation of lachrymal fluid / tears that results in thick, dehydrated and wrinkled conjunctiva. Keratomalacia is characterized by xerosis and dryness with ulceration and perforation in the cornea that results in eye loss. As retinol plays an important role in rhodopsin (which is a photoreceptor pigment essential to the retinol receptors that are responsible for night vision) formation, VAD also results in night blindness [3].

Recently researchers from Weill Cornell medical college found that VAD may lead to beta cell loss in pancreas that results in reduced insulin production and increased blood glucose levels which are main symptoms of type 2 diabetes [4].

Chicken, egg, whole milk, carrot, orange color fruits, sweet potatoes, spinach, kale and green vegetables are rich in vitamin A and daily 5 servings of these vitamin A rich food should be taken to avoid or reduce VAD. In high risk population mainly in children from 6 months to 5 years, vitamin A supplementation is recommended to reduce morbidity, mortality and blindness.

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