Biotech World

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:

Saturday, January 30, 2016

CRISPR and genome editing

CRISPRs or Clustered Regularly Interspaced Short Palindromic Repeats are short prokaryotic DNA sequences (~ 20 nt) that are part of bacterial adaptive immune system. Recent research in genomics suggests that these CRISPR could change/edit the DNA sequence at exact location on a chromosome of almost any type of living being, including humans, very fast. CRISPR gene editing technology is much easier, cheaper and faster than other gene editing methods including Zn fingers and TALENs (Transcription Activator Like Effector Nucleases). There are 3 types of CRISPR mechanisms among which type II is the most studied one. The key components of CRISPR gene editing are Cas9 protein / Csn1 endonuclease along with the guide RNA (crRNA and trRNA) [1]. In CRISPR pathway first the foreign DNA is integrated into the CRISPR locus and the loci are then transcribed to produce crRNA. These crRNA then guide the Cas9 / RNAse III family endonuclease to edit the target genome or to destroy the invading DNA in sequence specific way [2].

This CRISPER or Cas9 based genetic technology has various applications in biotechnology and medical research for gene knock out, gene repression/activation, genetic screening, genomic loci imaging and purification [3], epigenetic modifications, transcriptional regulation, etc. This powerful gene editing method has huge potential to treat genetic disorders and cancer, to produce genetically modified crops, to engineer the ecosystems through gene drive, to produce transgenic animal model for biomedical research, in drug development and most recently (in April 2015) to edit human embryonic stem cells (though it triggered ethical debate). In 2014 researchers from MIT first used CRISPER editing in mice to treat a metabolic disease tyrosinaemia. In January 2016, scientists from Duke University successfully used CRISPER genetic technology to treat DMD (Duchenne Muscular Dystrophy, a genetic disorder that causes muscle breakdown due to mutation in dystrophin gene) in mouse model [4]. As Cas9 could correct the causative mutation this CRISPER/Cas9 genome editing technology may direct an exciting future in therapeutics to treat monogenic recessive disorders/genetic diseases [5]. Examples of such disorders include sickle cell anaemia, cystic fibrosis, DMD, retinitis etc. Though scientists need to overcome many hurdles this Cas9 based genome editing technology has powerful future solution for effective genome modification that lead to novel inventions in biomedical and genomic research.

References:

1. https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

2. http://www.nature.com/nature/journal/v482/n7385/fig_tab/nature10886_F1.html

3.CRISPR/Cas9 Guide: https://www.addgene.org/CRISPR/guide/

3.http://www.sciencealert.com/crispr-gene-editing-tool-used-to-treat-genetic-disease-in-an-animal-for-the-first-time

5. Patrick D. Hsu, Eric S. Lander, Feng Zhang, Development and Applications of CRISPR-Cas9 for Genome Engineering, DOI: http://dx.doi.org/10.1016/j.cell.2014.05.010

Friday, January 15, 2016

RNA silencing in plant biotechnology

RNA silencing or post transcriptional gene silencing (PTGS) plays an important biological role in plants which includes the following:

defense against viruses
epigenetic control of chromatin modifications
control of the expression of developmentally regulated genes
regulation of biotic & abiotic stress, and
defense against nonviral pathogens and insects.

RNA silencing pathways or RNA interference (RNAi) are mediated by small homologous RNA molecules that are 21 to 24 nt long. These RNA molecules are processed by dicers or dicer-like endonucleases (DCL) and RNA dependent RNA polymerases (RDRs). They bind with AGO (Argonaute) proteins to form RISC (RNA induced silencing complex) and repress/modify the gene expression at transcriptional, post transcriptional and translational level. RNA silencing pathways involve various types of small RNAs which includes

micro RNAs (mi RNAs) that have important biological role in plants including auxin regulator and accumulator of transcription factors that involves in plant development [1]
endogenous trans acting si RNAs/TAS (ta-siRNAs)
natural cis antisense transcripts associated si RNAs (nat si RNAs), and
heterochromatic si RNAs (hc si RNA).

The micro RNAs are produced by DCL1 and binds with AGO1 to cleave target mRNA and repress the translation. The ta-si RNA is produced by DCL4 and RDR6 to cleave TAS gene and control the gene expression in plants [2]. The hc si RNAs are produced by plant specific transcript POL4, RDR2 and DCL3 and then binds with AGO4, AGO6 and AGO9 to direct DNA methylation. Through these RNA silencing pathways plants are defended against invaded viral nucleic acid/transgenes. During this defense mechanism viral RNA is diced by DCL4&DCL2 to produce the si RNAs to degrade the viral RNA [3].

RNAi is now widely used in industrial plant biotechnology for production of disease and pathogen resistant plants, for generation of male sterility to produce hybrid seeds, for crop production with improved nutritional contents and in various other aspects. Industrially RNAi has also been used to modify the metabolic pathways of plants to enhance nutrient accumulation and decrease toxin generation. For example, lyc gene and DET1 gene in tomato are engineered by RNAi to increase lycopene (antioxidant), flavonoid and beta carotene concentration. FAD2 and SAD1 gene in canola, cotton, and peanut plant is manipulated by RNAi to increase the oleic acid and stearic acid concentration [4]. In 2015 a group of scientists at Australia's Commonwealth Scientific and Industrial Research Organization (CSIRO) explained the use of RNAi to increase the healthier monounsaturated fatty acid (MUFA) level in flaxseeds [5]. CSIRO used RNAi to reduce the function of 3 genes in cottonseed that usually convert oleic acid to bad fatty acids. This resulted in high oleic acid containing cottonseed oil that is free from cholesterol raising trans fatty acids [6].

Other than plants RNAi technology is also applied for genome analysis and drug target validation for therapeutic development and hence it is termed as "breakthrough in biotech industry".

References:

Friday, December 25, 2015

Astrocyte research may lead to development of novel and improved treatments of autism/neurodevelopmental disorders

Astrocytes are star shaped glial cells in CNS (central nervous system) of mammalian brain which are derived from neural stem cells during early embryogenesis and express the marker Glial Fibrillary Acidic Protein GFAP. These astrocytes play an important role in pathogenesis of autism and other neurodevelopmental disorders such as Rett syndrome and FXS (Fragile X Syndrome) that result from synaptic defects. Astrocytes can modulate synaptogenesis (by releasing molecular signals such as thrombospondin/TSP that specifically increases number of excitatory synapses) during their developmental maturation in CNS and play an active role in synaptic physiology in the human brain [1]. In adult CNS astrocytes serve a number of regional functions such as axon guidance, control blood brain barrier and blood flow, synaptic support (regulation of synapse function and synaptic remodeling), maintaining brain homeostasis, regulating neuronal signaling, neuronal migration, protecting neurons from oxidative damage and determining the fate of endogenous neural precursors. Astrocytes also remove excess glutamate from extracellular space and supply glutamine to maintain glutamatergic neurotransmission. Glutamate transporter dysregulation results in pathogenesis of FXS and other neurodevelopmental disorders. [2]

As astrocytes play essential roles in synaptic mechanisms astrocyte dysfunction contributes to behavioral disorders. Recent research found microglial activation, high level of GFAP (around 3 folds than normal) and neuroinflammation in individuals with autism spectrum disorders (ASD) which results in gliosis, reactive injury and disturbed neuronal migration during early gestation [3].

Another neurodevelopmental disorder Rett syndrome is characterized by mutation in MECP2 (methyl CpG binding protein 2), or loss of MECP2 gene which plays an important role (can modulate the expression of thousands of genes) in brain cells and astrocytes to provide physical and functional support for neurons [4]. Rett and ASD have some similar symptoms including learning and memory problem, repetitive behavior and lack of social interaction though Rett affects mainly girls and ADS affects boys.

Fragile X syndrome, the most commonly inherited form of mental impairment is caused by glial cell dysfunction and transcriptional silencing of FMRP (Fragile X mentally retarded protein) gene by hypermethylation and CGG nucleotide repetition in FMR1 gene that turns off the gene.

Recent astrocyte research and development indicates their roles in motor neuron diseases and emphasize the potential of astrocytes/astroglia as therapeutic targets and agents in cell replacement therapy. A provocative study at University of Rochester Medical Center in 2013 found evidence that astrocyte provides restorative benefits of sleep by expending energy to drive water transport channels whose pumping action convects cerebrospinal fluid around neurons. They also found that knocking out the transport channels from astrocytes slows the clearance of neurotoxic molecules such as beta amyloid peptides deposited in Alzheimers disease by 65%. In large brain found in autistic and other psychiatric/neurodevelopmental patients the decreased efficiency of passive diffusion makes astrocyte driven active transport of toxic molecules crucial to survival.

Recently Laurie Doering a professor at Mcmaster University in Canada established a link by co-culturing healthy hippocampal neurons (that are responsible for learning and memory) and astrocytes. According to his research, in co-culture healthy hippocampal neurons exhibited delayed dendritic branching (a process of neural network building) and restricted the development of excitatory synapses. His research (for which he received a passion in science award by New England Biolabs) is actively working to identify new astrocyte based factors for the treatment of neuronal dysfunction using molecular, cellular and behavioral approaches. The interaction between astrocyte and secreted molecules from them and neurons leads to study how astrocyte dependent factors and signaling molecules can modulate the structure and physiology of FXS/ASD neurons [5]. A detailed understanding of how astrocytes regulate neural circuit development and their function in brain may lead to discovery of novel therapeutic treatment of ADS/FXS and other neurodevelopmental disorders.

References:

Wednesday, November 18, 2015

Parkinson’s disease - a common challenge in elderly people

Parkinson's disease is a progressive neurodegenerative disorder named after British doctor James Parkinson where 75% of dopamine producing brain cells in substantia nigra part of brain is damaged and they cannot produce enough dopamine. Dopamine (C8H11NO2) is a neurotransmitter that regulates movement and emotional responses. So, lower dopamine results in the motor symptoms of Parkinson’s disease, such as movement problem, postural instability, tremor, rigidity, and walking problem. Non-motor symptoms include sleep disorder/insomnia, mental/mood disorders, orthostatic hypotension and hallucination/psychosis. Parkinson’s patients also have lewy bodies i.e. abnormally accumulated proteins (mainly alpha synuclein) inside their neuron cells.

Parkinson’s is of 3 types: primary or idiopathic (80%) which has no known cause, secondary which is from exposure to external/environmental toxins such as pesticides, fungicides (maneb), herbicides (paraquat), insecticides (such as permethrin and beta HCH), heavy metals etc., and genetic (15%) which is genetically transferred. The most common genes involved are PARK2 (parkin), LRRK2 (leucine rich repeat kinase 2) and GBA (glucocerebrosidase) [1]. Mutations in any of these genes result in Parkinson’s. Recent research describes neuroepigenetics or epigenetic modifications in brain to explain the unexplained detail of Parkinson’s [2]. Another recent research in University of Copenhagen, Denmark, describes IFNβ gene may play a role in Parkinson’s [3]. A research study from National Center for Biological Sciences, Bangalore, found that Calcium can regulate the Dopamine level in brain cells [4].

Currently there is no permanent cure for Parkinson’s. Medications, exercise/physical therapy and neurosurgery such as deep brain stimulation (when medications can’t control the symptoms) can control the symptoms to some extent. The common medication includes carbidopa-levedopa (Rytary, Sinemet), dopamine agonists (that mimic dopamine effects in brain) such as pramipixole (mirapex) and ropinirole (Requip), MAO-B (monoamine oxidase B) inhibitors (that prevent dopamine breakdown) such as selegiline ( Eldepryl) and rasagiline (Azilect). In 2015 FDA approved carbidopa-levedopa infusion drug Duopa that delivers in gel form directly to small intestine for patients with more advanced stage of Parkinson’s.

References