Biotech World: 2016

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.

References:

Friday, May 27, 2016

Understanding the zika virus

Zika virus disease is a mosquito borne disease that has currently become epidemic in the world. The zika virus (ZIKV) is a mosquito borne pathogen that is transferred to human sexually or through the bite of infected Aedes mosquitoes. Pregnant women and women who want to conceive are at higher risk to be infected by zika because the transplacental transmission of zika virus leads to serious brain damage, microcephaly (abnormally small head) and other birth defects to the fetus. Zika virus can also cause a rare autoimmune neurological disorder Guillain-Barre syndrome that affects the chest muscle cells in adult men and results in paralysis.

Zika virus is an enveloped (by lipids and E glycoproteins) single stranded RNA virus that belongs to flavivirus genus (the same genus for West Nile virus and dengue virus). The virus attaches to the host cell receptors by the E glycoprotein and then through endocytosis the viral RNA is released into the host cytoplasm [1]. Then the ZIKV targets the neural stem cells called cortical neural precursors which are responsible for the formation of cerebral cortex (responsible for brain functions) of the brain. These neural stem cells become the factories for the zika viral replication. ZIKV also interferes with gangliosides that are crucial for brain development, neurogenesis, synaptogenesis, synaptic transmission and cell proliferation [2]. Recent research in UCSD using a 3D stem cell based first trimester human brain model revealed that zika virus may cause microcephaly by hijacking human immune system [3]. Zika virus infection in pregnant women stops the neonatal brain development and results in birth of babies with abnormally small head known as microcephaly. UCSD scientists discovered that zika virus activates a cell bound protein TLR3 which is responsible for turning off the genes that are required to develop various human brain parts from the neural stem cells. Binding of TLR3 with the viral RNA results in organoid shrinkage/brain damage (microcephaly) and apoptosis in developing fetuses.

Zika viral infection symptoms include fever and rash conjunctivitis, muscle and joint pain that lasts up to 7 days [4]. Currently there is no permanent treatment for zika virus infection/microcephaly in pregnant women. Recent research by the virologists from KU Leuven in Belgium suggests that an experimental antiviral drug against hepatitis C virus can slow down the zika infection in mice [5]. Also the TLR3 inhibitor is a big future in zika therapeutics.

Brazil became the epicenter of zika outbreak where 5600 cases of confirmed or suspected cases of microcephaly is reported by World Health Organization and the virus is spreading to Latin America and Caribbean.

References:

1. http://emedicine.medscape.com/article/2500035-overview

2. http://www.ncbi.nlm.nih.gov/pubmed/27001187

3. https://www.sciencedaily.com/releases/2016/05/160506132202.htm

4. http://www.who.int/features/qa/zika/en/

5. http://www.sciencenewsline.com/summary/2016051714440016.html

Saturday, April 30, 2016

Listeriosis: an important public health concern in the United States

Listeriosis is a fatal foodborne opportunistic bacterial infection caused by a genus of gram positive bacteria called Listeria. Listeria is found in soil, water and in some animals including cattle and poultry. Individuals with weakened immunity system especially T cell mediated immunity such as pregnant women and older people are at highest risk of listeriosis. These bacteria have 6 species among which Listeria monocytogenes is the disease causing one in humans. Listeria bacteria do not form spores, they don't have capsules, and they are motile at 10 to 25 degrees Centigrade. Listeriosis occurs by ingestion of contaminated raw and industrially processed food that contains L. monocytogenes pathogen.These bacteria can survive the food processing technology, can tolerate high salt concentration and low pH and are able to multiply in refrigeration temperature which makes this pathogen a serious threat to food industry.

As listeriosis is a foodborne infection the primary site of entry of the bacteria is the gastrointestinal tract of the host and then the liver and the CNS. L. monocytogenes is intracellular parasite that can survive in macrophages to invade non phagocytic cells such as epithelial cells, hepatocytes and endothelial cells where they undergo intracellular proliferation [1]. The hemolysin/hly gene is the virulence factor that leads the Listeria bacteria for intracellular survival. Other virulence factors are also involved in listeria pathogenesis such as cell to cell adhesion protein internlanin (In1A & In1B) for epithelial cell invasion and ActA for cell to cell spread. Internlanin binds with the host cell membrane receptors E cadherin (transmembrane glycoprotein), C1q complement fraction receptor, ECM (Heparin Sulfate Proteoglycan/HSPG) and MET receptor for hepatocyte growth factor (HGF) [2]. After phagocytosis the bacterial phagolysosome listeriolysin O (cholesterol dependent pore forming toxins/CDTX) enter the host cytoplasm and multiply there. In the cytoplasm bacterial surface protein Act A initiate actin formation through which listeria spread cell to cell which results in infection. Studies suggest that iron metal plays an important role in regulation of virulence gene expression in L. monocytogenes [3]. Recent research also identifies an auxiliary protein secretion system (SecA2) that secretes autolytic enzymes that promotes pathogenesis in L. monocytogenes [4].

Consumption of raw fruit, vegetables and meat, unpasteurized milk and milk products, smoked seafood, processed ready to eat food may result in listeriosis. CDC estimates that approximately 1600 illnesses and 260 deaths due to listeriosis occur annually in the United States. In 2011 147 illnesses, 33 deaths and one miscarriage occurred due to consumption of contaminated cantaloupe from a single farm [5]. Symptoms of listeriosis include fever, chill, severe body and headaches, diarrhea, vomiting and influenza type symptoms, which can be treated by antibiotics in most of the cases but may be life-threatening in some cases [6].

References

Thursday, March 31, 2016

Restriction enzymes - The most useful tool in Molecular biology

The restriction enzymes/endonucleases (REases) were first discovered by Swiss microbiologist Werner Arber (with Stuart Linn) in 1978 (for which they received Nobel prize) during the study of host controlled restriction of bacteriophases (from E. coli strain). Now it has become the most essential tool in genetic engineering & molecular biology that can cleave the sugar phosphate backbone of the DNA at specific nucleotide sequence (called recognition sequence which is about 4-6 nt long). As example, restriction enzyme HaeII from the bacterium Hemophilus aegypticus cuts the DNA only at particular sequence:

5’ GGCC 3’

3’ CCGG 5’

The recognition sequence length dictates the cutting frequency of the enzyme in a random DNA sequence. Such as enzymes with 6 bp long recognition site will cut the DNA in every 46(i.e. 4096) bp. The recognition site of one RE can be similar with another RE and such REs are called isoschizomers such as SacI and SstI [1]. REs are evolved from bacteria and named by the host, for example, EcoR1 are derived from E. coli strain RY13.

Restriction enzymes are mainly of 4 types on the basis of subunit composition, sequence specificity, cleavage position and cofactor requirements [2].Type I restriction enzymes cleave DNA at random sites that are far from its recognition sequence. The most common (93% of REs) commercially available restriction enzyme is Type II restriction enzyme that cleaves DNA in the presence of Mg2+ within its recognition sequence and it does not require ATP hydrolysis such as HindIII [3]. Type II restriction enzymes are of several types, such as Type IIG (that cleaves outside its recognition sequence such as Acul), type IIP (that cleaves symmetric sequences) and type IIS (that cleaves asymmetric sequences such as FolkI). TypeIII restriction enzyme cleaves outside their recognition sequences and require two sequences with opposite orientation within the same DNA molecule. Type IV restriction enzymes cleave only modified/methylated DNA.

Restriction digestion of DNA double helix results in highly reactive sticky ends or cohesive ends that can be bound by DNA ligase. All restriction enzymes are heat sensitive. Their activity is highly affected by enzyme concentration, sequence context, incubation temperature and buffer composition. Deviation of any of these factors results in ineffective restriction digestion at cognate restriction site or cleavage at non-cognate star sites (Star activity) [4]. Some factors can induce star activity in restriction digestion such as high pH, prolonged reaction time, presence of organic solvents (e.g. DMSO) and glycerol concentration of the reaction mix. Recently New England Biolabs developed High Fidelity engineered restriction endonucleases that have reduced star activity and rapid digestion [5].

Applications:

Cloning utilizes restriction enzymes along with DNA ligase for the study and production of recombinant proteins.
DNA mapping or restriction mapping for the detection of single nucleotide polymorphism and mutation to identify genetic disorder loci
Epigenetic modification study
Synthetic biology utilizes restriction enzymes to create novel technologies such as Bio-BrickTM and Golden Gate assembly.
DNA Library construction by SAGE (Serial Analysis of Gene Expression) to identify mutation in cancer research [6]
Gene editing, DNA sequencing, DNA fingerprinting and in vast area of recombinant DNA and biotechnology

References:

Monday, February 29, 2016

Neglected tropical diseases (NTDs)

Neglected tropical diseases are diverse group of tropical diseases that affect more than 1 billion people including 500 million children in 149 tropical and subtropical countries globally. NTDs are endemic and bacterial and parasitic diseases that affect mainly the world’s poorest communities with inadequate sanitation and absence of clean water. The 7 most common NTDs are found in low and middle-income countries of Asia, Africa and Latin America. The examples of some neglected tropical diseases are as follows:

Chagas disease/American Trypanosomiasis: Named after Brazilian doctor Carlos Chagas this disease is caused by the unicellular protozoan parasite Trypanosoma cruzi and mainly found in the rural areas of Latin America. This parasite is transmitted to human blood by triatomine bugs and result in cardiomyopathy and dysrhythmias.

Buruli ulcer: This disease is usually found in the tropical areas of Africa and Australia and caused by the bacteria Myobacteria ulceran which affects the skin and bone. This bacteria produces plasmid-encoded toxin mycolactone that diffuses into subcutaneous fat and causes progressive necrosis [1].

Dengue: The most common tropical and subtropical disease that affects 400 million people every year is caused by the viruses transmitted mainly by Aedes mosquitoes. Dengue virus is a small single-stranded RNA virus that has four distinct serotype which are transmitted to humans through the bite of infected Aedes mosquitoes mainly A. aegypti.

Cysticercosis: A parasitic infection caused by Taenia solium that infects brain muscle and other tissues.

Echinococcosis: A parasitic infection caused by the small tapeworm Echinococcus granulosus (Cystic Echinococcosis) and Echinococcus multilocularis (Alveolar Echinococcosis/AE) that result in infiltrative liver lesions.

Onchocerciasis/River blindness: An infection caused by the worm O. volvulus and transmitted by black fly (Simulium) and mainly found in Africa resulting in skin lesions and blindness. Ivermectin drug treatment is the most effective method of control of this disease.

Currently there are various researches going on to prevent and control NTDs. Metabolomics study identified small molecules biomarkers (SMBs) in acute phase clinical specimens (blood serum) that differentiate dengue disease outcomes [2]. These metabolites (SMBs) also provide the insight of metabolic pathways, pathogenic and immunologic mechanism associated with dengue disease. Medical research on AE suggests that serial endoscopic balloon dilation and stenting combined with benzimidazole treatment can re-establish and maintain biliary duct patency for many years in AE patients [3]. Currently 13 leading pharmaceutical companies, global health organizations, donor and endemic country governments are partnered with World Health Organization to control, eliminate and reduce the global burden of NTDs.

References: