Mechanism of action
In pharmacology, the term mechanism of action refers to the specific biochemical interaction through which a drug substance produces its pharmacological effect. A mechanism of action includes mention of the specific molecular targets to which the drug binds, such as an enzyme or receptor. Receptor sites have specific affinities for drugs based on the chemical structure of the drug, as well as the specific action that occurs there. Drugs that do not bind to receptors produce their corresponding therapeutic effect by interacting with chemical or physical properties in the body. Common examples of drugs that work in this way are laxatives. In comparison, a mode of action describes functional or anatomical changes, at the cellular level, resulting from the exposure of a living organism to a substance. Elucidating the mechanism of action of novel drugs and medications is important for several reasons: In the case of anti-infective drug development, the information permits anticipation of problems relating to clinical safety.
Drugs disrupting the cytoplasmic membrane or electron transport chain, for example, are more to cause toxicity problems than those targeting components of the cell wall or 70S ribosome, structures which are absent in human cells. By knowing the interaction between a certain site of a drug and a receptor, other drugs can be formulated in a way that replicates this interaction, thus producing the same therapeutic effects. Indeed, this method is used to create new drugs, it can help identify which patients are most to respond to treatment. Because the breast cancer medication trastuzumab is known to target protein HER2, for example, tumors can be screened for the presence of this molecule to determine whether or not the patient will benefit from trastuzumab therapy, it can enable better dosing because the drug's effects on the target pathway can be monitored in the patient. Statin dosage, for example, is determined by measuring the patient’s blood cholesterol levels, it allows drugs to be combined in such a way that the likelihood of drug resistance emerging is reduced.
By knowing what cellular structure an anti-infective or anticancer drug acts upon, it is possible to administer a cocktail that inhibits multiple targets thereby reducing the risk that a single mutation in microbial or tumor DNA will lead to drug resistance and treatment failure. It may allow other indications for the drug to be identified. Discovery that sildenafil inhibits phosphodiesterase-5 proteins, for example, enabled this drug to be repurposed for pulmonary arterial hypertension treatment, since PDE-5 is expressed in pulmonary hypertensive lungs. Bioactive compounds induce phenotypic changes in target cells, changes that are observable by microscopy, which can give insight into the mechanism of action of the compound. With antibacterial agents, for example, the conversion of target cells to spheroplasts can be an indication that peptidoglycan synthesis is being targeted, filamentation of target cells can be an indication that FtsZ or DNA is being targeted. In the case of anticancer agents, bleb formation can be an indication that the compound is disrupting the plasma membrane.
A current limitation of this approach is the time required to manually generate and interpret data, but advances in automated microscopy and image analysis software may help resolve this. Direct biochemical methods include methods in which a protein or a small molecule, such as a drug candidate, is labeled and is traced throughout the body; this proves to be the most direct approach to find target protein that will bind to small targets of interest, such as a basic representation of a drug outline, in order to identify the pharmacophore of the drug. Due to the physical interactions between the labeled molecule and a protein, biochemical methods can be used to determine the toxicity and the mechanism of action of the drug. Computation inference methods are used to predict protein targets for small molecule drugs based on computer based pattern recognition. However, this method could be used for finding new targets for existing or newly developed drugs. By identifying the pharmacophore of the drug molecule, the profiling method of pattern recognition can be carried out where a new target is identified.
This provides an insight at a possible mechanism of action, as it is known what certain functional components of the drug are responsible for interacting with a certain area on a protein, leading to a therapeutic effect. Omics based methods use omics technologies, such as reverse genetics and genomics and proteomics, to identify the potential targets of the compound of interest. Reverse genetics and genomics approaches, for instance, uses genetic perturbation in combination with the compound to identify genes whose knockdown or knockout abolishes the pharmacological effect of the compound. On the other hand and proteomics profiles of the compound can be used to compare with profiles of compounds with known targets. Thanks to computation inference, it is possible to make hypothesis of the mechanism of action of the compound, which can be subsequently tested. There are many drugs. One example is aspirin; the mechanism of action of aspirin involves irreversible inhibition of the enzyme cyclooxygenase.
This mechanism of action is specific to aspirin, is not constant for all nonsteroidal anti-inflammatory drugs. Rather, aspirin is the only NSAID that irreversibly inhibits COX-1; some drug mechanisms of action are still unknown. However
Non-homologous end joining
Non-homologous end joining is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair, which requires a homologous sequence to guide repair; the term "non-homologous end joining" was coined in 1996 by Haber. NHEJ utilizes short homologous DNA sequences called microhomologies to guide repair; these microhomologies are present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are compatible, NHEJ repairs the break accurately. Imprecise repair leading to loss of nucleotides can occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to hallmarks of tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. In budding yeast, homologous recombination dominates when the organism is grown under common laboratory conditions.
When the NHEJ pathway is inactivated, double-strand breaks can be repaired by a more error-prone pathway called microhomology-mediated end joining. In this pathway, end resection reveals short microhomologies on either side of the break, which are aligned to guide repair; this contrasts with classical NHEJ, which uses microhomologies exposed in single-stranded overhangs on the DSB ends. Repair by MMEJ therefore leads to deletion of the DNA sequence between the microhomologies. Many species of bacteria, including Escherichia coli, lack an end joining pathway and thus rely on homologous recombination to repair double-strand breaks. NHEJ proteins have been identified in a number of bacteria, including Bacillus subtilis, Mycobacterium tuberculosis, Mycobacterium smegmatis. Bacteria utilize a remarkably compact version of NHEJ in which all of the required activities are contained in only two proteins: a Ku homodimer and the multifunctional ligase/polymerase/nuclease LigD. In mycobacteria, NHEJ is much more error prone than in yeast, with bases added to and deleted from the ends of double-strand breaks during repair.
Many of the bacteria that possess NHEJ proteins spend a significant portion of their life cycle in a stationary haploid phase, in which a template for recombination is not available. NHEJ may have evolved to help these organisms survive DSBs induced during desiccation. Corndog and Omega, two related mycobacteriophages of Mycobacterium smegmatis encode Ku homologs and exploit the NHEJ pathway to recircularize their genomes during infection. Unlike homologous recombination, studied extensively in bacteria, NHEJ was discovered in eukaryotes and was only identified in prokaryotes in the past decade. In contrast to bacteria, NHEJ in eukaryotes utilizes a number of proteins, which participate in the following steps: In yeast, the Mre11-Rad50-Xrs2 complex is recruited to DSBs early and is thought to promote bridging of the DNA ends; the corresponding mammalian complex of Mre11-Rad50-Nbs1 is involved in NHEJ, but it may function at multiple steps in the pathway beyond holding the ends in proximity.
DNA-PKcs is thought to participate in end bridging during mammalian NHEJ. Eukaryotic Ku is a heterodimer consisting of Ku70 and Ku80, forms a complex with DNA-PKcs, present in mammals but absent in yeast. Ku is a basket-shaped molecule. Ku may function as a docking site for other NHEJ proteins, is known to interact with the DNA ligase IV complex and XLF. End processing involves removal of damaged or mismatched nucleotides by nucleases and resynthesis by DNA polymerases; this step is not necessary if the ends are compatible and have 3' hydroxyl and 5' phosphate termini. Little is known about the function of nucleases in NHEJ. Artemis is required for opening the hairpins that are formed on DNA ends during VJ recombination, a specific type of NHEJ, may participate in end trimming during general NHEJ. Mre11 has nuclease activity, but it seems to be involved in homologous recombination, not NHEJ; the X family DNA polymerases Pol λ and Pol μ fill gaps during NHEJ. Yeast lacking Pol4 are unable to join 3' overhangs that require gap filling, but remain proficient for gap filling at 5' overhangs.
This is because the primer terminus used to initiate DNA synthesis is less stable at 3' overhangs, necessitating a specialized NHEJ polymerase. The DNA ligase IV complex, consisting of the catalytic subunit DNA ligase IV and its cofactor XRCC4, performs the ligation step of repair. XLF known as Cernunnos, is homologous to yeast Nej1 and is required for NHEJ. While the precise role of XLF is unknown, it interacts with the XRCC4/DNA ligase IV complex and participates in the ligation step. Recent evidence suggests that XLF promotes re-adenylation of DNA ligase IV after ligation, recharging the ligase and allowing it to catalyze a second ligation. In yeast, Sir2 was identified as an NHEJ protein, but is now known to be required for NHEJ only because it is required for the transcription of Nej1; the choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails.
DSBs that have not been resected can be rejoined by NHEJ, but resection of a few nucleotides inhibits NHEJ and commits the break to repair by recombination. NHEJ is act
Physcomitrella patens, the spreading earthmoss, is a moss used as a model organism for studies on plant evolution and physiology. Physcomitrella patens is an early colonist of exposed mud and earth around the edges of pools of water. P. patens has a disjunct distribution in temperate parts of the world, with the exception of South America. The standard laboratory strain is the "Gransden" isolate, collected by H. Whitehouse from Gransden Wood, in Cambridgeshire in 1962. Mosses share fundamental genetic and physiological processes with vascular plants, although the two lineages diverged early in land-plant evolution. A comparative study between modern representatives of the two lines may give insight into the evolution of mechanisms that contribute to the complexity of modern plants. In this context, P. patens is used as a model organism. P. patens is one of a few known multicellular organisms with efficient homologous recombination. Meaning that an exogenous DNA sequence can be targeted to a specific genomic position to create knockout mosses.
This approach is called reverse genetics and it is a powerful and sensitive tool to study the function of genes and, when combined with studies in higher plants such as Arabidopsis thaliana, can be used to study molecular plant evolution. The targeted deletion or alteration of moss genes relies on the integration of a short DNA strand at a defined position in the genome of the host cell. Both ends of this DNA strand are engineered to be identical to this specific gene locus; the DNA construct is incubated with moss protoplasts in the presence of polyethylene glycol. As mosses are haploid organisms, the regenerating moss filaments can be directly assayed for gene targeting within 6 weeks using PCR methods; the first study using knockout moss appeared in 1998 and functionally identified ftsZ as a pivotal gene for the division of an organelle in a eukaryote. In addition, P. patens is used in biotechnology. Examples are the identification of moss genes with implications for crop improvement or human health and the safe production of complex biopharmaceuticals in moss bioreactors.
By multiple gene knockout Physcomitrella plants were engineered that lack plant-specific post-translational protein glycosylation. These knockout mosses are used to produce complex biopharmaceuticals in a process called molecular farming; the genome of P. patens, with about 500 megabase pairs organized into 27 chromosomes, was sequenced in 2008. Physcomitrella ecotypes and transgenics are stored and made available to the scientific community by the International Moss Stock Center; the accession numbers given by the IMSC can be used for publications to ensure safe deposit of newly described moss materials. Like all mosses, the lifecycle of P. patens is characterized by an alternation of two generations: a haploid gametophyte that produces gametes and a diploid sporophyte where haploid spores are produced. A spore develops into a filamentous structure called protonema, composed of two types of cells – chloronema with large and numerous chloroplasts and caulonema with fast growth. Protonema filaments grow by tip growth of their apical cells and can originate side branches from subapical cells.
Some side-branch initial cells can differentiate into buds rather than side branches. These buds give rise to gametophores, more complex structures bearing leaf-like structures and the sexual organs: female archegonia and male antheridia. P. patens is monoicous, meaning that male and female organs are produced in the same plant. If water is available, flagellate sperm cells can swim from the antheridia to an archegonium and fertilize the egg within; the resulting diploid zygote originates a sporophyte composed of a foot and capsule, where thousands of haploid spores are produced by meiosis. P. patens is an excellent model in which to analyze repair of DNA damages in plants by the homologous recombination pathway. Failure to repair double-strand breaks and other DNA damages in somatic cells by homologous recombination can lead to cell dysfunction or death, when failure occurs during meiosis, it can cause loss of gametes; the genome sequence of P. patens has revealed the presence of numerous genes that encode proteins necessary for repair of DNA damages by homologous recombination and by other pathways.
PpRAD51, a protein at the core of the homologous recombination repair reaction, is required to preserve genome integrity in P. patens. Loss of PpRAD51 causes marked hypersensitivity to the double-strand break-inducing agent bleomycin, indicating that homologous recombination is used for repair of somatic cell DNA damages. PpRAD51 is essential for resistance to ionizing radiation; the DNA mismatch repair protein PpMSH2 is a central component of the P. patens mismatch repair pathway that targets base pair mismatches arising during homologous recombination. The PpMsh2 gene is necessary in P. patens to preserve genome integrity. Genes Ppmre11 and Pprad50 of P. patens encode components of the MRN complex, the principal sensor of DNA double-strand breaks. These genes are necessary for accurate homologous recombinational repair of DNA damages in P. patens. Mutant plants defective in either Ppmre11 or Pprad50 exhibit restricted growth and development, enhanced sensitivity to UV-B and bleomycin-induced DNA damage compared to wild-type plants.
P. patens was first described by Johann Hedwig in his 1801 work Species Muscorum Frondosorum, under the name Phascum patens. Physcomitrella is sometimes treated as a synonym of the genus Aphanorrhegma, in which case P. patens is known as Aphanorrhegma patens. The generic name Physcomitrella implies a resemblance to
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology, used to determine the order of the four bases: adenine, guanine and thymine; the advent of rapid DNA sequencing methods has accelerated biological and medical research and discovery. Knowledge of DNA sequences has become indispensable for basic biological research, in numerous applied fields such as medical diagnosis, forensic biology and biological systematics; the rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of complete DNA sequences, or genomes, of numerous types and species of life, including the human genome and other complete DNA sequences of many animal and microbial species. The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of fluorescence-based sequencing methods with a DNA sequencer, DNA sequencing has become easier and orders of magnitude faster.
DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions, full chromosomes, or entire genomes of any organism. DNA sequencing is the most efficient way to indirectly sequence RNA or proteins. In fact, DNA sequencing has become a key technology in many areas of biology and other sciences such as medicine and anthropology. Sequencing is used in molecular biology to study genomes and the proteins they encode. Information obtained using sequencing allows researchers to identify changes in genes, associations with diseases and phenotypes, identify potential drug targets. Since DNA is an informative macromolecule in terms of transmission from one generation to another, DNA sequencing is used in evolutionary biology to study how different organisms are related and how they evolved; the field of metagenomics involves identification of organisms present in a body of water, dirt, debris filtered from the air, or swab samples from organisms. Knowing which organisms are present in a particular environment is critical to research in ecology, epidemiology and other fields.
Sequencing enables researchers to determine which types of microbes may be present in a microbiome, for example. Medical technicians may sequence genes from patients to determine if there is risk of genetic diseases; this is a form of genetic testing. DNA sequencing may be used along with DNA profiling methods for forensic identification and paternity testing. DNA testing has evolved tremendously in the last few decades to link a DNA print to what is under investigation; the DNA patterns in fingerprint, hair follicles, etc. uniquely separate each living organism from another. Testing DNA is a technique which can detect specific genomes in a DNA strand to produce a unique and individualized pattern; every living organism created has a one of a kind DNA pattern, which can be determined through DNA testing. It is rare that two people have the same DNA pattern, therefore DNA testing is successful; the canonical structure of DNA has four bases: thymine, adenine and guanine. DNA sequencing is the determination of the physical order of these bases in a molecule of DNA.
However, there are many other bases. In some viruses, cytosine may be replaced by hydroxy methyl glucose cytosine. In mammalian DNA, variant bases with methyl groups or phosphosulfate may be found. Depending on the sequencing technique, a particular modification, e.g. the 5mC common in humans, may or may not be detected. Deoxyribonucleic acid was first discovered and isolated by Friedrich Miescher in 1869, but it remained understudied for many decades because proteins, rather than DNA, were thought to hold the genetic blueprint to life; this situation changed after 1944 as a result of some experiments by Oswald Avery, Colin MacLeod, Maclyn McCarty demonstrating that purified DNA could change one strain of bacteria into another. This was the first time. In 1953, James Watson and Francis Crick put forward their double-helix model of DNA, based on crystallized X-ray structures being studied by Rosalind Franklin – and without crediting her. According to the model, DNA is composed of two strands of nucleotides coiled around each other, linked together by hydrogen bonds and running in opposite directions.
Each strand is composed of four complementary nucleotides – adenine, cytosine and thymine – with an A on one strand always paired with T on the other, C always paired with G. They proposed such a structure allowed each strand to be used to reconstruct the other, an idea central to the passing on of hereditary information between generations; the foundation for sequencing proteins was first laid by the work of Frederick Sanger who by 1955 had completed the sequence of all the amino acids in insulin, a small protein secreted by the pancreas. This provided the first conclusive evidence that proteins were chemical entities with a specific molecular pattern rather than a random mixture of material suspended in fluid. Sanger's success in sequencing insulin electrified x-ray crystallographers, including Watson and Crick who by now were trying to understand how DNA directed the formation of proteins within a cell. Soon after attending a series of lectures given by Frederick Sanger in October 1954, Crick began to develo
Essential genes are those genes of an organism that are thought to be critical for its survival. However, being essential is dependent on the circumstances in which an organism lives. For instance, a gene required to digest starch is only essential if starch is the only source of energy. Systematic attempts have been made to identify those genes that are required to maintain life, provided that all nutrients are available; such experiments have led to the conclusion that the required number of genes for bacteria is on the order of about 250–300. These essential genes encode proteins to maintain a central metabolism, replicate DNA, translate genes into proteins, maintain a basic cellular structure, mediate transport processes into and out of the cell. Most genes convey selective advantages and increased fitness. Two main strategies have been employed to identify essential genes on a genome-wide basis: directed deletion of genes and random mutagenesis using transposons. In the first case, individual genes are deleted from the genome in a systematic way.
In transposon-mediated mutagenesis transposons are randomly inserted in as many positions in a genome as possible, aiming to inactivate the targeted genes. Insertion mutants that are still able to survive or grow are not in essential genes. A summary of such screens is shown in the table. Table 1. Essential genes in bacteria. Mutagenesis: targeted mutants are gene deletions. Methods: Clones indicate single gene deletions, population indicates whole population mutagenesis, e.g. using transposons. Essential genes from population screens include genes essential for fitness. ORFs: number of all open reading frames in that genome. Notes: mutant collection available. Only partial dataset available. Includes predicted gene essentiality and data compilation from published single-gene essentiality studies. Project in progress. Deduced by comparison of the two gene essentiality datasets obtained independently in the P. aeruginosa strains PA14 and PAO1. The original result of 271 essential genes has been corrected to 261, with 31 genes that were thought to be essential being in fact non-essential whereas 20 novel essential genes have been described since then.
Counting genes with essential domains and those that lead to growth-defects when disrupted as essential, those who lead to growth-advantage when disrupted as non-essential. Involved a saturated mutant library of 14 replicates, with 84.3% of possible insertion sites with at least one transposon insertion. In Saccharomyces cerevisiae 15-20% of all genes are essential. In Schizosaccharomyces pombe 4,836 heterozygous deletions covering 98.4% of the 4,914 protein coding open reading frames have been constructed. 1,260 of these deletions turned out to be essential. Similar screens are more difficult to carry out in other multicellular organisms, including mammals, due to technical reasons, their results are less clear. However, various methods have been developed for the nematode worm C. elegans, the fruit fly, zebrafish. A recent study of 900 mouse genes concluded that 42% of them were essential although the selected genes were not representative. Gene knockout experiments are not possible or at least not ethical in humans.
However, natural mutations have led to the identification of mutations that lead to early embryonic or death. Note that many genes in humans are not essential for survival but can cause severe disease when mutated; such mutations are catalogued in the Online Mendelian Inheritance in Man database. In a computational analysis of genetic variation and mutations in 2,472 human orthologs of known essential genes in the mouse, Georgi et al. found strong, purifying selection and comparatively reduced levels of sequence variation, indicating that these human genes are essential too. While it may be difficult to prove that a gene is essential in humans, it can be demonstrated that a gene is not essential or not causing disease. For instance, sequencing the genomes of 2,636 Icelandic citizens and the genotyping of 101,584 additional subjects found 8,041 individuals who had 1 gene knocked out. Of the 8,041 individuals with complete knock-outs, 6,885 were estimated to be homozygotes, 1,249 were estimated to be compound heterozygotes.
In these individuals, a total of 1,171 of the 19,135 human genes were knocked out. It was concluded that these 1,171 genes are non-essential in humans — at least no associated diseases were reported; the exome sequences of 3222 British Pakistani-heritage adults with high parental relatedness revealed 1111 rare-variant homozygous genotypes with predicted loss of gene function in 781 genes. This study found an average of 140 predicted LOF genotypes, including 16 rare heterozygotes, 0.34 rare homozygotes, 83.2 common heterozygotes and 40.6 common homozygotes. Nearly all rare homozygous LOF genotypes were found within autozygous segments. Though most of these individuals had no obvious health issue arising from their defective genes, it is possible that minor health issues may be found upon more detailed examination. A summary of essentiality screens is shown in the table below (mostly based on the Database of Essential Genes. Screens for essential genes have been carr
Stem cells are cells that can differentiate into other types of cells, can divide in self-renewal to produce more of the same type of stem cells. In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the inner cell mass of blastocysts in early embryonic development, adult stem cells, which are found in various tissues of developed mammals. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues. In a developing embryo, stem cells can differentiate into all the specialized cells—ectoderm and mesoderm —but maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues. There are three known accessible sources of autologous adult stem cells in humans: bone marrow, adipose tissue, blood. Stem cells can be taken from umbilical cord blood just after birth. Of all stem cell therapy types, autologous harvesting involves the least risk. By definition, autologous cells are obtained from one's own body, just as one may bank his or her own blood for elective surgical procedures.
Adult stem cells are used in various medical therapies. Stem cells can now be artificially grown and transformed into specialized cell types with characteristics consistent with cells of various tissues such as muscles or nerves. Embryonic cell lines and autologous embryonic stem cells generated through somatic cell nuclear transfer or dedifferentiation have been proposed as promising candidates for future therapies. Research into stem cells grew out of findings by Ernest A. McCulloch and James E. Till at the University of Toronto in the 1960s; the classical definition of a stem cell requires that it possesses two properties: Self-renewal: the ability to go through numerous cycles of cell division while maintaining the undifferentiated state. Potency: the capacity to differentiate into specialized cell types. In the strictest sense, this requires stem cells to be either totipotent or pluripotent—to be able to give rise to any mature cell type, although multipotent or unipotent progenitor cells are sometimes referred to as stem cells.
Apart from this it is said. Two mechanisms ensure that a stem cell population is maintained: 1. Obligatory asymmetric replication: a stem cell divides into one mother cell, identical to the original stem cell, another daughter cell, differentiated; when a stem cell self-renews it does not disrupt the undifferentiated state. This self-renewal demands control of cell cycle as well as upkeep of multipotency or pluripotency, which all depends on the stem cell.2. Stochastic differentiation: when one stem cell develops into two differentiated daughter cells, another stem cell undergoes mitosis and produces two stem cells identical to the original. Potency specifies the differentiation potential of the stem cell. Totipotent stem cells can differentiate into extraembryonic cell types; such cells can construct a viable organism. These cells are produced from the fusion of an sperm cell. Cells produced by the first few divisions of the fertilized egg are totipotent. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells, i.e. cells derived from any of the three germ layers.
Multipotent stem cells can differentiate into a number of cell types, but only those of a related family of cells. Oligopotent stem cells can differentiate into only a few cell types, such as lymphoid or myeloid stem cells. Unipotent cells can produce only one cell type, their own, but have the property of self-renewal, which distinguishes them from non-stem cells. In practice, stem cells are identified by. For example, the defining test for bone marrow or hematopoietic stem cells is the ability to transplant the cells and save an individual without HSCs; this demonstrates. It should be possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into another individual without HSCs, demonstrating that the stem cell was able to self-renew. Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, in which single cells are assessed for their ability to differentiate and self-renew. Stem cells can be isolated by their possession of a distinctive set of cell surface markers.
However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells shall behave in a similar manner in vivo. There is considerable debate as to whether some proposed adult cell populations are stem cells. Embryonic stem cells are the cells of the inner cell mass of a blastocyst, formed prior to implantation in the uterus. In human embryonic development the blastocyst stage is reached 4–5 days after fertilization, at which time it consists of 50–150 cells. ESCs are pluripotent and give rise during development to all derivatives of the three germ layers: ectoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type, they do not contribute to the placenta. During embryonic development the cells of the inner cell mass continuously divide and become more specialized. For example, a portion of the ectoderm in the dorsal part of the embryo specializes as'neurectoderm', which
Cas9 is an RNA-guided DNA endonuclease enzyme associated with the CRISPR adaptive immunity system in Streptococcus pyogenes, among other bacteria. S. pyogenes utilizes Cas9 to memorize and interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking for sites complementary to the 20 basepair spacer region of the guide RNA. If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In this sense, the CRISPR-Cas9 mechanism has a number of parallels with the RNA interference mechanism in eukaryotes. Apart from its original function in bacterial immunity, the Cas9 protein has been utilized as a genome engineering tool to induce site-directed double strand breaks in DNA; these breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination in many laboratory model organisms. Alongside zinc finger nucleases and TALEN proteins, Cas9 is becoming a prominent tool in the field of genome editing.
Cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the guide RNA. Because the target specificity of Cas9 stems from the guide RNA:DNA complementarity and not modifications to the protein itself, engineering Cas9 to target new DNA is straightforward. Versions of Cas9 that bind but do not cleave cognate DNA can be used to localize transcriptional activator or repressors to specific DNA sequences in order to control transcriptional activation and repression. While native Cas9 requires a guide RNA composed of two disparate RNAs that associate to make the guide – the CRISPR RNA, the trans-activating RNA. Cas9 targeting has been simplified through the engineering of a chimeric single guide RNA. Scientists have suggested that Cas9-based gene drives may be capable of editing the genomes of entire populations of organisms. In 2015, Cas9 was used to modify the genome of human embryos for the first time. To survive in a variety of challenging, inhospitable habitats that are filled with bacteriophages and archea have evolved methods to evade and fend off predatory viruses.
This includes the CRISPR system of adaptive immunity. In practice, CRISPR acts as a self-programmable restriction enzyme. CRISPR loci are composed of short, palindromic repeats that occur at regular intervals composed of alternate CRISPR repeats and variable CRISPR spacers between 24-48 nucleotides long; these CRISPR loci are accompanied by adjacent CRISPR-associated genes. In 2005, it was discovered by three separate groups that the spacer regions were homologous to foreign DNA elements, including plasmids and viruses; these reports provided the first biological evidence that CRISPRs might function as an immune system. Cas9 has been used as a genome-editing tool. Cas9 has been used in recent developments in preventing viruses from manipulating hosts’ DNA. Since the CRISPR-Cas9 was developed from bacterial genome systems, it can be used to target the genetic material in viruses; the use of the enzyme Cas9 can be a solution to many viral infections. Cas9 possesses the ability to target specific viruses by the targeting of specific strands of the viral genetic information.
More the Cas9 enzyme targets certain sections of the viral genome that prevents the virus from carrying out its normal function. Cas9 has been used to disrupt the detrimental strand of DNA and RNA that cause diseases and mutated strands of DNA. Cas9 has showed promise in disrupting the effects of HIV-1. Cas9 has been shown to suppress the expression of the long terminal repeats in HIV-1; when introduced into the HIV-1 genome Cas9 has shown the ability to mutate strands of HIV-1. Cas9 has been used in the treatment of hepatitis b through targeting of the ends of certain of long terminal repeats in the hepatitis b viral genome. In addition, Cas9 has been used in human trials in the treatment of cystic fibrosis and oncogenic mutations in human pluripotent stem cells. Cas9 has used to repair the mutations causing cataracts in mice. CRISPR-Cas systems are divided into three major types and twelve subtypes, which are based on their genetic content and structural differences. However, the core defining features of all CRISPR-Cas systems are the cas genes and their proteins: cas1 and cas2 are universal across types and subtypes, while cas3, cas9, cas10 are signature genes for type I, type II, type III, respectively.
Adaptation involves recognition and integration of spacers between two adjacent repeats in the CRISPR locus. The "Protospacer" refers to the sequence on the viral genome. A short stretch of conserved nucleotides exists proximal to the protospacer, called the protospacer adjacent motif; the PAM is a recognition motif, used to acquire the DNA fragment. In type II, Cas9 recognizes the PAM during adaptation in order to ensure the acquisition of functional spacers. CRISPR expression includes the transcription of a primary transcript called a CRISPR RNA, transcribed from the CRISPR locus by RNA polymerase. Specific endoribonucleases cleave the pre-crRNAs into small CRISPR RNAs. Interference involves the crRNAs within a multi-protein complex called CASCADE, which can recognize and base-pair with regions of inserting complementary foreign DNA; the crRNA-foreign nucleic acid complex is cleaved, however if there are mismatches between the spacer and the target DNA, or if there are mutations in the PAM cleavage will not be initiated.