A Bioinformatics Approach to Clinical Sequencing (Lecture)

Dr. Rhodes has a 12-year track record in cancer genomics and bioinformatics, with a significant focus on building systems to enable cutting-edge cancer research. He founded a successful cancer genomics and informatics company, Compendia Bioscience, recently acquired by Life Technologies. To build Compendia, he recruited and trained a talented team of 30 clinical scientists, bioinformaticists, cancer biologists, data curators and software engineers. As a graduate student, he led the development of Oncomine (Rhodes et al, PNAS, 2004; Rhodes et al, Nature Genetics, 2005), a cancer microarray database and data-mining application. Through the use of Oncomine, he contributed to several important discoveries, including the discovery of gene fusions in prostate cancer (Tomlins, Rhodes, et al., Science, 2005), SPINK1 in prostate cancer (Tomlins, Rhodes et al, Cancer Cell, 2008) and AGTR1 in breast cancer (Rhodes et al, PNAS, 2009). Compendia has since extensively expanded the Oncomine platform, and Oncomine is now widely recognized in academia and pharma as the leading cancer genomics data mining resource, with several hundred citations, and 15,000+ users. As Head of Medical Science Informatics at Life Technologies, he has turned his focus to next-generation sequencing data analysis, and leads several collaborations with pharmaceutical companies aimed at discovering novel oncogenic mutations and gene fusions from thousands of exomes and transcriptomes from The Cancer Genome Atlas. In his role as an Adjunct Assistant Professor at the U. of Michigan, he contributes to cancer genome analyses of prostate cancers (Grasso et al, Nature, 2012) and participates in and advises the clinical sequencing initiative MI-ONCOSEQ. (Ref:Bioconference)


DNA Fingerprinting Animation

The chemical structure of everyone's DNA is the same. The only difference between people (or any animal) is the order of the base pairs. There are so many millions of base pairs in each person's DNA that every person has a different sequence.
Using these sequences, every person could be identified solely by the sequence of their base pairs. However, because there are so many millions of base pairs, the task would be very time-consuming. Instead, scientists are able to use a shorter method, because of repeating patterns in DNA.

These patterns do not, however, give an individual "fingerprint," but they are able to determine whether two DNA samples are from the same person, related people, or non-related people. Scientists use a small number of sequences of DNA that are known to vary among individuals a great deal, and analyze those to get a certain probability of a match.

The Southern Blot is one way to analyze the genetic patterns which appear in a person's DNA. Performing a Southern Blot involves:

1. Isolating the DNA in question from the rest of the cellular material in the nucleus. This can be done either chemically, by using a detergent to wash the extra material from the DNA,or mechanically, by applying a large amount of pressure in order to "squeeze out" the DNA.

2. Cutting the DNA into several pieces of different sizes. This is done using one or more restriction enzymes.

3. Sorting the DNA pieces by size. The process by which the size separation, "size fractionation," is done is called gel electrophoresis. The DNA is poured into a gel, such as agarose, and an electrical charge is applied to the gel, with the positive charge at the bottom and the negative charge at the top. Because DNA has a slightly negative charge, the pieces of DNA will be attracted towards the bottom of the gel; the smaller pieces, however, will be able to move more quickly and thus further towards the bottom than the larger pieces. The different-sized pieces of DNA will therefore be separated by size, with the smaller pieces towards the bottom and the larger pieces towards the top.

4. Denaturing the DNA, so that all of the DNA is rendered single-stranded. This can be done either by heating or chemically treating the DNA in the gel.

5. Blotting the DNA. The gel with the size-fractionated DNA is applied to a sheet of nitrocellulose paper, and then baked to permanently attach the DNA to the sheet. The Southern Blot is now ready to be analyzed.

In order to analyze a Southern Blot, a radioactive genetic probe is used in a hybridization reaction with the DNA in question. If an X-ray is taken of the Southern Blot after a radioactive probe has been allowed to bond with the denatured DNA on the paper, only the areas where the radioactive probe binds will show up on the film. This allows researchers to identify, in a particular person's DNA, the occurrence and frequency of the particular genetic pattern contained in the probe.

variable number tandem repeat

A variable number tandem repeats (VNTR) is a short nucleotide sequence ranging from 14 to 100 nucleotides long that is organized into clusters of tandem repeats, usually repeated in the range of between 4 and 40 times per occurrence. Clusters of such repeats are scattered on many chromosomes. Each variant is an allele and they are inherited codominantly.

Coupled with Polymerase chain reactions, VNTRs have been very effective in forensic crime investigations. When VNTRs are cut out, on either side of the sequence, by restriction enzymes and the results are visualized with a gel electrophoresis, a pattern of bands unique to each individual is produced. The number of times that a sequence is repeated varies between different individuals and between maternal and paternal loci of an individual. The likelihood of two individuals having the same band pattern is extremely improbable. Southern blotting is also used to visualize the repeat numbers on the chromosomes. Once the tandem repeat has been found, identification of possible restriction sites on either side of the repeats are carried out. Using restriction enzymes will break the DNA into the repeat sequences plus a little on each end. The number of repeats will determine the length of the fragment of DNA. The repeat sequence itself can be used as a probe, or if the repeat is not long enough, a sequence from the upstream or downstream side can be used. The probe can either be radioactive or have a biotinylated linker for a fluorescent molecule.

In looking at the VNTR data, two basic principles can be relied on:

Tissue Matching
- both VNTR bands must match. If the two samples are from the same individual, he must have exactly the same binding pattern.

Inheritance Matching- the matching bands must follow the rules of inheritance. In matching an individual with his parents, a person must have one band that matches from each parent. If the relationship is further, such as a grandparent, then the matches must be consistent with the relatedness.

VNTR evidence is considered to be exclusionary, which means that a mismatch (or no match at all) sample can be excluded from the genetic relationship of the original sample trying to be matched.

There are two principal families of VNTR: minisatellites and microsatelites. The former are sequences of 11-16 bp repeated 1000 times. They are important because they are highly repetitive and dispersed into the genome. In humans, they are present in 60 autosomic loci and can be examined by digesting the DNA and hybriding with a monolocus probe or with another probe derived from a sequence that is common to each locus. The other members of the VNTR family are the microsatellites or STR (short tandem repeats).They are represented by short sequences of 100-200 bp given by the repetition of 1-6 bp sequences. They cannot be digested, so they are amplified by a multiplex PCR. Parental investigation with these kind of markers are non suitable between consanguineous, because electrophoresis profiles will be very similar. So it is possible to examine only one locus. In this way the system is perfect: one allele derives from the mother and the other one from the father. Microsatellites have many uses: they can be used in forensics, genetic variability and parentage studies.

G-Protein Signal-coupled Intracellular signal Transduction

The cAMP signal transduction contains 5 main characters: stimulative hormone receptor (Rs) or inhibitory hormone receptor (Ri); stimulative regulative G-protein (Gs) or inhibitory regulative G-protein (Gi); Adenylyl cyclase; Protein Kinase A (PKA); and cAMP phosphodiesterase.

Stimulative hormone receptor (Rs) is a receptor that can bind with stimulative signal molecules, while inhibitory hormone (Ri) is a receptor that can bind with inhibitory signal molecules.

Stimulative regulative G-protein is a G protein-linked to stimulative hormone receptor (Rs) and its α subunit upon activation could stimulate the activity of an enzyme or other intracellular metabolism. On the contrary, inhibitory regulative G-protein is linked to an inhibitory hormone receptor and its α subunit upon activation could inhibit the activity of an enzyme or other intracellular metabolism.

The Adenylyl cyclase is a 12-transmembrane glycoprotein that catalyzes ATP to form cAMP with the help of cofactor Mg2+ or Mn2+. The cAMP produced is a second messenger in cellular metabolism and is an allosteric activator to Protein kinase A.

Protein kinase A is an important enzyme in cell metabolism due to its ability to regulate cell metabolism by phosphorylating specific committed enzymes in the metabolic pathway. It can also regulate specific gene expression, cellular secretion, and membrane permeability. The protein enzyme contains two catalytic subunits and two regulatory subunits. When there is no cAMP,the complex is inactive. When cAMP binds to the regulatory subunits, their conformation is altered, causing the dissociation of the regulatory subunits, which activates protein kinase A and allows further biological effects.

cAMP phosphodiesterase is an enzyme that can degrade cAMP to 5'-AMP, which terminates the signal.

Oncogenic Activation Receptor Tyrosine Kinases

Oncogenic activation receptor tyrosine kinases

Mitochondrial DNA

Mitochondrial DNA (mtDNA) is the DNA located in organelles called mitochondria. Most other DNA present in eukaryotic organisms is found in the cell nucleus. Nuclear and mitochondrial DNA are thought to be of separate evolutionary origin, with the mtDNA being derived from the circular genomes of the bacteria that were engulfed by the early ancestors of today's eukaryotic cells. Each mitochondrion is estimated to contain 2-10 mtDNA copies.[1] In the cells of extant organisms, the vast majority of the proteins present in the mitochondria (numbering approximately 1500 different types in mammals) are coded for by nuclear DNA, but the genes for some of them, if not most, are thought to have originally been of bacterial origin, having since been transferred to the eukaryotic nucleus during evolution. In most multicellular organisms, mtDNA is inherited from the mother (maternally inherited).

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Mechanisms for this include simple dilution (an egg contains 100,000 to 1,000,000 mtDNA molecules, whereas a sperm contains only 100 to 1000), degradation of sperm mtDNA in the fertilized egg, and, at least in a few organisms, failure of sperm mtDNA to enter the egg. Whatever the mechanism, this single parent (uniparental) pattern of mtDNA inheritance is found in most animals, most plants and in fungi as well. mtDNA is particularly susceptible to reactive oxygen species generated by the respiratory chain due to its close proximity. Though mtDNA is packaged by proteins and harbors significant DNA repair capacity, these protective functions are less robust than those operating on nuclear DNA and therefore thought to contribute to enhanced susceptibility of mtDNA to oxidative damage. Mutations in mtDNA cause maternally inherited diseases and are thought to be a major contributor to aging and age-associated pathology.
In humans (and probably in metazoans in general), 100-10,000 separate copies of mtDNA are usually present per cell (egg and sperm cells are exceptions). In mammals, each circular mtDNA molecule consists of 15,000-17,000 base pairs, which encode the same 37 genes: 13 for proteins (polypeptides), 22 for transfer RNA (tRNA) and one each for the small and large subunits of ribosomal RNA (rRNA). This pattern is also seen among most metazoans, although in some cases one or more of the 37 genes is absent and the mtDNA size range is greater. Even greater variation in mtDNA gene content and size exists among fungi and plants, although there appears to be a core subset of genes that are present in all eukaryotes (except for the few that have no mitochondria at all). Some plant species have enormous mtDNAs (as many as 2,500,000 base pairs per mtDNA molecule) but, surprisingly, even those huge mtDNAs contain the same number and kinds of genes as related plants with much smaller mtDNAs.

G protein receptor

G protein-coupled receptors (GPCRs), also known as seven transmembrane domain receptors, 7TM receptors, heptahelical receptors, and G protein-linked receptors (GPLR), comprise a large protein family of transmembrane receptors that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. G protein-coupled receptors are found only in eukaryotes, including yeast, plants, choanoflagellates, and animals. The ligands that bind and activate these receptors include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, and vary in size from small molecules to peptides to large proteins. G protein-coupled receptors are involved in many diseases, but are also the target of around half of all modern medicinal drugs
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These proteins receive chemical signals from outside the cell and pass the signal into the cell, so that the cell can respond to the signal. The structures of the endogenous ligands for GPCRs are exceptionally diverse. They include biogenic amines such as norephnephrine and serotonine, peptides, glycoproteins, lipids, nucleotides, ions, and proteases. The sensation of exogenous stimuli, such as light, odors, and taste, is also mediated by this class of receptors. Activation of the receptor causes an effector inside the cell to produce a second signal chemical, which eventually triggers the cell to react to the original external chemical signal.
A ligand, in this case Norepinepherine (NE), binds to the receptor and induces a conformational change. This conformational change activates the a/b complex. The complex is bound to GDP while it is inactive. GTP replaces GDP, thus activating the Alpha subunit. The activated Alpha subunit undergoes a conformational change and activates Adenylate Cyclase. Once the Adenylate Cyclase is activated, it is then able to convert ATP. The products of ATP conversion are c-AMP and two phosphate molecules. c-AMP is a second messenger used in many processes required for cell survival and growth.

What is Endocytosis

Endocytosis is a process whereby cells absorb material (molecules such as proteins) from the outside by engulfing it with their cell membrane. It is used by all cells of the body because most substances important to them are large polar molecules, and thus cannot pass through the hydrophobic plasma membrane. The function of endocytosis is the opposite of exocytosis.
The absorption of material from the outside environment of the cell is commonly divided into two processes: phagocytosis and pinocytosis.

Phagocytosis (literally, cell-eating) is the process by which cells ingest large objects, such as cells which have undergone apoptosis, bacteria, or viruses. The membrane folds around the object, and the object is sealed off into a large vacuole known as a phagosome.

Pinocytosis (literally, cell-drinking) is a synonym for endocytosis. This process is concerned with the uptake of solutes and single molecules such as proteins.

Receptor-mediated endocytosis is a more specific active event where the cytoplasm membrane folds inward to form coated pits. These inward budding vesicles bud to form cytoplasmic vesicles.

Endocytosis pathways

There are three types of endocytosis: namely, macropinocytosis, clathrin-mediated endocytosis, and caveolar endocytosis.

Macropinocytosis is the invagination of the cell membrane to form a pocket which then pinches off into the cell to form a vesicle filled with extracellular fluid (and molecules within it). The filling of the pocket occurs in a non-specific manner. The vesicle then travels into the cytosol and fuses with other vesicles such as endosomes and lysosomes.

Clathrin-mediated endocytosis is the specific uptake of large extracellular molecules such as proteins, membrane localized receptors and ion-channels. These receptors are associated with the cytosolic protein clathrin which initiates the formation of a vesicle by forming a crystalline coat on the inner surface of the cell's membrane.

Caveolae consist of the protein caveolin-1 with a bilayer enriched in cholesterol and glycolipids. Caveolae are flask shaped pits in the membrane that resemble the shape of a cave (hence the name caveolae). Uptake of extracellular molecules are also believed to be specifically mediated via receptors in caveolae.

Clathrin-mediated endocytosis

The major route for endocytosis in most cells, and the best understood, is that mediated by the molecule clathrin. This large protein assists in the formation of a coated pit on the inner surface of the plasma membrane of the cell. This pit then buds into the cell to form a coated vesicle in the cytoplasm of the cell. In so doing, it brings into the cell not only a small area of the surface of the cell but also a small volume of fluid from outside the cell.

Vesicles selectively concentrate and exclude certain proteins during formation and are not representative of the membrane as a whole. AP2 adaptors are multisubunit complexes that performs this function at the plasma membrane. The best understood receptors which are found concentrated in coated vesicles of mammalian cells are the LDL receptor (which removes LDL from the blood circulation), the transferrin receptor (which brings ferric ions bound by transferrin into the cell) and certain hormone receptors (such as that for EGF).

At any one moment, about 2% of the plasma membrane of a fibroblast is made up of coated pits. As a coated pit has a life of about a minute before it buds into the cell, a fibroblast takes up its surface by this route about once every 50 minutes. Coated vesicles formed from the plasma membrane have a diameter of about 100nm and a life time measured in a few seconds. Once the coat has been shed, the remaining vesicle fuses with endosomes and proceeds down the endocytic pathway. The actual budding-in process, whereby a pit is converted to a vesicle, is carried out by clathrin assisted by a set of cytoplasmic proteins which includes dynamin and adaptors such as adaptin.

Coated pits and vesicles were first seen in thin sections of tissue in the electron microscope by Thomas Roth and Keith Porter in 1964. The importance of them for the clearance of LDL from blood was discovered by R. G Anderson, Michael S. Brown and Joseph L. Goldstein in 1976. Coated vesicles were first purified by Barbara Pearse, who discovered the clathrin coat molecule, also in 1976.

Understanding Bipolar Disorder

Bipolar Disorder encompasses a wide spectrum of symptoms and is classified according to the types of mood episodes exhibited, including: manic, hypomanic, major depressive and mixed episodes.

Bipolar I disorder involves a manic or mixed episode in contrast to Bipolar II disorder, which involves at least one major depressive episode and at least one hypomanic episode, but no full manic or mixed episodes.

Bipolar Disorder should be differentiated from Major Depressive Disorder (MDD), which is diagnosed when a patient experiences one or more major depressive episodes without any lifetime episodes of hypomania or mania.

Depicted here is a life chart (or mood chart), which follows the patient's lifetime history of mood episodes. This permits the identification of mood episodes that are the most prevalent and important to prevent.

In this patient, as with many patients with bipolar disorder, depressive episodes become the more prominent aspect of the illness as the person ages.
Several morphometric differences have been observed in the brains of Bipolar Disorder patients relative to healthy subjects.

White matter hyperintensities and reduction in grey matter volume, identified with MRI, have been described in patients with Bipolar Disorder.
Increased ventricular size and decreased frontal cortical area volumes may also be observed in Bipolar Disorder patients.

The pathophysiology of Bipolar Disorder encompasses environmental, behavioural, neuronal, cellular, and molecular levels.
At the molecular level, aberrant signaling cascades alter synaptic plasticity. Strong evidence supporting the importance of second messenger signaling has come from studying the targets of mood stabilizing drugs such as lithium.
GSK-3 and IP3 signaling cascades are known to mediate axonogenesis, synaptogenesis, neuronal growth and cone spreading. Other downstream effects may also be involved.
The heritability of bipolar disorder is around 80%. Monozygotic twins are reported to have a higher incidence of developing Bipolar Disorder, approximately 40%, whereas the incidence is only 10% in dizygotic twins.
Although the process of developing bipolar disorder likely arises from complex interactions between genes and environmental factors, the specific genes that contribute to this risk are not known with certainty. Variations of several genes have been identified as potential contributors to the pathophysiology of bipolar disorder.
Among the identified genes are those associated with serotonin signaling (SLC6A4, TPH2), dopamine signaling (SLC6A3, DRD4), glutamate transmission (DAOA, DTNBP1), and cell maintenance and growth (NRG1, BDNF, DISC1).
The most significant environmental triggers of mood episodes among patients with bipolar disorder include use of drugs with mood-altering properties, changes in circadian rhythm, and life stressors.
Successful management of bipolar disorder requires particular attention to minimizing the effects of these influences.

Immunotherapy: Boosting the immune system to fight cancer

The concept of 'teaching' the immune system to recognize and destroy cancer cells is over a century old, but the development of immunotherapeutic strategies for cancer was slow for many decades. However, much has been learned about the immune system in the meantime, and with the recent approval of two new immunotherapeutic anticancer drugs and several drugs in late-stage development, a new era in anticancer immunotherapy is beginning.

The video takes an audio-visual journey through the different approaches that are being investigated to harness the immune system to treat cancer.

Nature video on how brain sees

At the micro-scale the brain is a mess; a thick tangle of nerve cells connected at synapses. Mapping just a tiny portion of this mess, a few hundred cells, is a huge challenge. You have to wonder if it's worth the effort. But seeing exactly how brain cells are wired together is giving us new insights into brain function. The researchers who made the 3D maps in this video discovered a new type of cell and worked out how insects see movement. If you've ever tried to swat a fly you'll know how good they are at sensing motion!

Immunology in the Gut Mucosa

The gut microbiota has an essential role in stimulating the development and maintainance of the intestinal immune system – in fact, without the gut microbiome the human body would be unable to protect its health from being affected by all environmental pathogens that enter the body, e.g. through our daily food. The cellular and molecular mechanisms by which this is achieved are nicely illustrated in a video animation that Nature Immunology has released with the expertise of world-reknown immunologist Tom MacDonald from London:

Fluoroquinolones: Mechanisms of Action and Resistance

In this animation, we demonstrate the biology of DNA replication leading to bacterial cell division in a gram positive bacterium, such as S. pneumoniae. The DNA is shown as a circular double strand within the bacterial cell. Like the DNA of all living organisms, it contains the unique genetic code for all of the proteins required for bacterial survival. Bacteria replicate by a process known as binary fission whereby one bacterium separates into 2 new daughter cells. However, before this can occur, the bacterium must make an identical copy of its complete circular DNA.
 In this animation, we demonstrate the biology of DNA replication leading to bacterial cell division in a gram positive bacterium, such as S. pneumoniae. The DNA is shown as a circular double strand within the bacterial cell. Like the DNA of all living organisms, it contains the unique genetic code for all of the proteins required for bacterial survival. Bacteria replicate by a process known as binary fission whereby one bacterium separates into 2 new daughter cells. However, before this can occur, the bacterium must make an identical copy of its complete circular DNA. DNA replication requires that the two strands of DNA separate so that the genetic code of the bacterium can be read and a new complimentary strand can be created for each of the original strands. To accomplish this, various enzymes known as helicases break the hydrogen bonds between the bases in the two DNA strands, unwind the strands from each other, and stabilize the exposed single strands, preventing them from joining back together. The points at which the two strands of DNA separate to allow replication of DNA are known as replication forks. The enzymes DNA polymerase then move along each strand of DNA, behind each replication fork synthesizing new DNA strands (in red) complementary to the original ones. As the replication forks move forward, positive superhelical twists in the DNA begin to accumulate ahead of them. In order for DNA replication to continue, these superhelical twists must be removed. The bacterial enzyme, DNA gyrase, which is also known as topoisomerase II, is responsible for removing the positive superhelical twists so that DNA replications can procede. DNA gyrase is an essential bacterial enzyme composed of two A and two B subunits which are products of the gyrA and gyrB genes. This enzyme has other important functions which affect the initiation of DNA replication and transcription of many genes. With the combined involvement of these enzymes, an entire duplicate copy of the bacterial genome is produced as the 2 replication forks move in opposite directions around the circular DNA genome. Eventually, as the 2 replication forks meet, 2 new complete chromosomes have been made, each consisting of 1 old and 1 new strand of DNA. This is referred to as semi-conservative replication. In order to allow the 2 new interlinked chromosomes to come apart, another bacterial enzyme is needed which is known as topoisomerase IV. This enzyme is structurally related to DNA gyrase and is coded for by the parC and parE genes. Topoisomerase IV allows for the 2 new inter-linked chromosomes to separate so that they can be segregated into 2 new daughter bacterial cells. Complete separation of bacterial cells Fluoroquinolones. First mechanism of action -- inhibition of DNA gyrase. Fluoroquinolones act by inhibiting the activity of both the DNA gyrase and the topoisomerase IV enzymes. For most gram negative bacteria, DNA gyrase is the primary fluoroquinolone target. Fluoroquinolones have been shown to bind specifically to the complex of DNA gyrase and DNA rather than to DNA gyrase alone. As a result of this binding, quinolones appear to stabilize the enzyme-DNA complexes which in turn results in breaks in the DNA that are fatal to the bacterium. A second mechanism of fluoroquinolone action is shown here. With some exceptions, topoisomerase IV is the primary target of fluoroquinolone action in most gram positive bacteria such as Staphylococci and Streptococci, with DNA gyrase being a secondary target. The separation of 2 new interlinked daughter strands of circular DNA is disrupted. The final result on the bacteria, however, is the same. Bacterial replication is disrupted and the bacterium breaks apart. The relative potency of different fluoroquinolone antibiotics (and thus their spectrum of activity) is dependent in part on their affinity for either DNA gyrase or topoisomerase IV or both. One of the most common mechanisms by which bacteria acquire resistance to fluoroquinolones is by spontaneously occurring mutations in chromosomal genes that alter the target enzymes -- DNA gyrase and topoisomerase IV or both. The frequency with which these spontaneous mutations occurs may be in the range of 10-6. The effect of mutations on the activity of an individual fluoroquinolone will vary depending on the number of mutations, the location of the mutations and which target enzyme is affected. If a mutation occurs (either in the gyrA or gyrB gene) that alters DNA gyrase and results in a reduced affinity of the fluoroquinolone antibiotic for this enzyme, the organism will become resistant.

Macrolides: Mechanisms of Action and Resistance

The macrolides are a group of drugs (typically antibiotics) whose activity stems from the presence of a macrolide ring, a large macrocyclic lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. The lactone rings are usually 14-, 15-, or 16-membered. Macrolides belong to the polyketide class of natural products.
Macrolides are protein synthesis inhibitors. The mechanism of action of macrolides is inhibition of bacterial protein biosynthesis, and they are thought to do this by preventing peptidyltransferase from adding the peptidyl attached to tRNA to the next amino acid (similarly to chloramphenicol) as well as inhibiting ribosomal translocation. Another potential mechanism is premature dissociation of the peptidyl-tRNA from the ribosome.
Macrolide antibiotics do so by binding reversibly to the P site on the subunit 50S of the bacterial ribosome. This action is considered to be bacteriostatic. Macrolides tend to accumulate within leukocytes, and are, therefore, transported into the site of infection.

Diffuse panbronchiolitis
The macrolide antibiotics erythromycin, clarithromycin, and roxithromycin have proven to be an effective long-term treatment for the idiopathic, Asian-prevalent lung disease diffuse panbronchiolitis (DPB). The successful results of macrolides in DPB stems from controlling symptoms through immunomodulation (adjusting the immune response), with the added benefit of low-dose requirements.
With macrolide therapy in DPB, great reduction in bronchiolar inflammation and damage is achieved through suppression of not only neutrophil granulocyte proliferation but also lymphocyte activity and obstructive secretions in airways.[6] The antimicrobial and antibiotic effects of macrolides, however, are not believed to be involved in their beneficial effects toward treating DPB. This is evident, as the treatment dosage is much too low to fight infection, and in DPB cases with the occurrence of the macrolide-resistant bacterium Pseudomonas aeruginosa, macrolide therapy still produces substantial anti-inflammatory results


The primary means of bacterial resistance to macrolides occurs by post-transcriptional methylation of the 23S bacterial ribosomal RNA. This acquired resistance can be either plasmid-mediated or chromosomal, i.e., through mutation, and results in cross-resistance to macrolides, lincosamides, and streptogramins (an MLS-resistant phenotype).
Two other types of acquired resistance rarely seen include the production of drug-inactivating enzymes (esterases or kinases), as well as the production of active ATP-dependent efflux proteins that transport the drug outside of the cell.
Azithromycin has been used to treat strep throat (Group A streptococcal (GAS) infection caused by Streptococcus pyogenes) in penicillin-sensitive patients, however macrolide-resistant strains of GAS are not uncommon. Cephalosporin is another option for these patients.

Glioma Cell Death

Glioma is a type of cancer that starts in the brain or spine. It is called a glioma because it arises from glial cells. The most common site of gliomas is the brain.

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Symptoms of gliomas depend on which part of the central nervous system is affected. A brain glioma can cause headaches, nausea and vomiting, seizures, and cranial nerve disorders as a result of increased intracranial pressure. A glioma of the optic nerve can cause visual loss. Spinal cord gliomas can cause pain, weakness, or numbness in the extremities. Gliomas do not metastasize by the bloodstream, but they can spread via the cerebrospinal fluid and cause "drop metastases" to the spinal cord.

In this video Non-adherent cell treated with metal colloid based agent to induce apoptosis.

Muscle Architecture

Muscle architecture is the physical arrangement of muscle fibers at the macroscopic level that determines a muscle’s mechanical function. There are several different muscle architecture types including: parallel, pinnate and hydrostats. Force production and gearing vary depending on the different geometries of the muscle. Some parameters used in architectural analysis are muscle length (Lm), fiber length (Lf), pennation angle (θ), and physiological cross-sectional area (PCSA)

Architecture types

Parallel and pennate (also known as pinnate) are two main types of muscle architecture. A third subcategory, muscular hydrostats, can also be considered. Architecture type is determined by the direction in which the muscle fibers are oriented relative to the force-generating axis. The force produced by a given muscle is proportional to the cross-sectional area, or the number of parallel sarcomeres present.
The parallel muscle architecture is found in muscles where the fibers are parallel to the force-generating axis.These muscles are often used for fast or extensive movements and can be measured by the anatomical cross-sectional area (CSA). Parallel muscles can be further defined into three main categories: strap, fusiform, or fan-shaped.
Strap muscles are shaped like a strap or belt and have fibers that run longitudinally to the contraction direction.[4] These muscles have broad attachments compared to other muscle types and can shorten to about 40%-60% of their resting length. Strap muscles, such as the laryngeal muscles, have been thought to control the fundamental frequency used in speech production, as well as singing. Another example of this muscle is the longest muscle in the human body, the sartorius.
Fusiform muscles are wider and cylindrically shaped in the center and taper off at the ends. This overall shape of fusiform muscles is often referred to as a spindle. The line of action in this muscle type runs in a straight line between the attachment points which are often tendons. Due to the shape, the force produced by fusiform muscles is concentrated into a small area. An example of this architecture type is the biceps brachii in humans.
The fibers in fan-shaped muscles converge at one end (typically at a tendon) and spread over a broad area at the other end. Because of this, some consider muscles with this relative shape to be in a separate architecture type known as convergent muscle. Fan-shaped muscles, such as the pectoralis major in humans, have a weaker pull on the attachment site compared to other parallel fibers due to their broad nature. These muscles are considered versatile because of their ability to change the direction of pull depending on how the fibers are contracting.
Typically, fan-shaped muscles experience varying degrees of fiber strain. This is largely due to the different lengths and varying insertion points of the muscle fibers. Studies on ratfish have looked at the strain on fan-shaped muscles that have a twisted tendon. It has been found that strain becomes uniform over the face of a fan-shaped muscle with the presence of a twisted tendon.
Unlike in parallel muscles, pennate fibers are at an angle to the force-generating axis (pennation angle) and usually insert into a central tendon.Because of this structure, fewer sarcomeres can be found in series, resulting in a shorter fiber length. This further allows for more fibers to be present in a given muscle; however, a trade-off exists between the number of fibers present and force transmission. The force produced by pennate muscles is greater than the force produced by parallel muscles.Since pennate fibers insert at an angle, the anatomical cross-sectional area cannot be used as in parallel fibered muscles. Instead, the physiological cross-sectional area (PCSA) must be used for pennate muscles. Pennate muscles can be further divided into uni-, bi- or multipennate.

Transcriptional regulation

Regulation of transcription controls when transcription occurs and how much RNA is created. Transcription of a gene by RNA polymerase can be regulated by at least five mechanisms:
Specificity factors alter the specificity of RNA polymerase for a given promoter or set of promoters, making it more or less likely to bind to them (i.e. sigma factors used in prokaryotic transcription).
Repressors bind to non-coding sequences on the DNA strand that are close to or overlapping the promoter region, impeding RNA polymerase's progress along the strand, thus impeding the expression of the gene.
General transcription factors These transcription factors position RNA polymerase at the start of a protein-coding sequence and then release the polymerase to transcribe the mRNA.
Activators enhance the interaction between RNA polymerase and a particular promoter, encouraging the expression of the gene. Activators do this by increasing the attraction of RNA polymerase for the promoter, through interactions with subunits of the RNA polymerase or indirectly by changing the structure of the DNA.
Enhancers are sites on the DNA helix that are bound to by activators in order to loop the DNA bringing a specific promoter to the initiation complex.
Silencers are regions of DNA that are bound by transcription factors in order to silence gene expression. The mechanism is very similar to that of enhancers.

Eukaryotic translation Animation

Eukaryotic translation is the process by which messenger RNA is translated into proteins in eukaryotes.
Initiation The cap-dependent initiation
Initiation of translation involves the interaction of certain key proteins with a special tag bound to 5'-end of an mRNA molecule. The protein factors bind the small ribosomal subunit (also referred to as the 40S subunit), and these initiation factors hold the mRNA in place. The eukaryotic Initiation Factor 3 (eIF3) is associated with the small ribosomal subunit, and plays a role in keeping the large ribosomal subunit from prematurely binding. eIF3 also interacts with the eIF4F complex which consists of three other initiation factors: eIF4A, eIF4E and eIF4G. eIF4G is a scaffolding protein which directly associates with both eIF3 and the other two components. eIF4E is the cap-binding protein. It is the rate-limiting step of cap-dependent initiation, and is often cleaved from the complex by some viral proteases to limit the cell's ability to translate its own transcripts. This is a method of hijacking the host machinery in favor of the viral (cap-independent) messages. eIF4A is an ATP-dependent RNA helicase, which aids the ribosome in resolving certain secondary structures formed by the mRNA transcript. There is another protein associated with the eIF4F complex called the Poly-A Binding Protein (PABP), which binds the poly-A tail of most eukaryotic mRNA molecules. This protein has been implicated in playing a role in circularization of the mRNA during translation.

This pre-initiation complex (43S subunit, or the 40S and mRNA) accompanied by the protein factors move along the mRNA chain towards its 3'-end, scanning for the 'start' codon (typically AUG) on the mRNA, which indicates where the mRNA will begin coding for the protein. In eukaryotes and archaea, the amino acid encoded by the start codon is methionine. The initiator tRNA charged with Met forms part of the ribosomal complex and thus all proteins start with this amino acid (unless it is cleaved away by a protease in subsequent modifications). The Met-charged initiator tRNA is brought to the P-site of the small ribosomal subunit by eukaryotic Initiation Factor 2 (eIF2). It hydrolyzes GTP, and signals for the dissociation of several factors from the small ribosomal subunit which results in the association of the large subunit (or the 60S subunit). The complete ribosome (80S) then commences translation elongation, during which the sequence between the 'start' and 'stop' codons is translated from mRNA into an amino acid sequence -- thus a protein is synthesized.

The cap-independent initiation

The best studied example of the cap-independent mode of translation initiation in eukaryotes is the Internal Ribosome Entry Site IRES approach. What differentiates cap-independent translation from cap-dependent translation is that cap-independent translation does not require the ribosome to start scanning from the 5' end of the mRNA cap until the start codon. The ribosome can be trafficked to the start site by ITAFs (IRES trans-acting factors) bypassing the need to scan from the 5' end of the untranslated region of the mRNA. This method of translation has been recently discovered, and has found to be important in conditions that require the translation of specific mRNAs, despite cellular stress or the inability to translate most mRNAs. Examples include factors responding to apoptosis, stress-induced responses.

Nanobots replacing neurons

Nanorobotics is the technology of creating machines or robots at or close to the microscopic scale of a nanometres (10-9 metres). More specifically, nanorobotics refers to the still largely hypothetical nanotechnology engineering discipline of designing and building nanorobots. Nanorobots (nanobots, nanoids or nanites) would be typically devices ranging in size from 0.1-10 micrometers and constructed of nanoscale or molecular components. As no artificial non-biological nanorobots have so far been created, they remain a hypothetical concept at this time.

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Another definition sometimes used is a robot which allows precision interactions with nanoscale objects, or can manipulate with nanoscale resolution. Following this definition even a large apparatus such as an atomic force microscope can be considered a nanorobotic instrument when configured to perform nanomanipulation. Also, macroscale robots or microrobots which can move with nanoscale precision can also be considered nanorobots.

Nanomachines are largely in the research-and-development phase, but some primitive molecular machines have been tested. An example is a sensor having a switch approximately 1.5 nanometers across, capable of counting specific molecules in a chemical sample. The first useful applications of nanomachines, if such are ever built, might be in medical technology, where they might be used to identify cancer cells and destroy them. Another potential application is the detection of toxic chemicals, and the measurement of their concentrations, in the environment. Recently, Rice University has demonstrated a single-molecule car which is developed by a chemical process and includes buckyballs for wheels. It is actuated by controlling the environmental temperature and by positioning a scanning tunneling microscope tip. Basic nanomachines are also in use in other areas. Nanotechnology coatings are already being used to make clothing with stain-resistant fibers and are used on swim suits to repel water, reduce friction with the water, and allow swimmers to go faster. Nanotech powders are being used to create high-performance sun-screen lotions and nanoparticles are helping to deliver drugs to targeted tissues in the body.

Beta cells (Insulin Production)

Beta cells (beta-cells, β-cells) are a type of cell in the pancreas in areas called the islets of Langerhans. They make up 65-80% of the cells in the islets.
In mammals, insulin is synthesized in the pancreas within the beta cells (β-cells) of the islets of Langerhans. One to three million islets of Langerhans (pancreatic islets) form the endocrine part of the pancreas, which is primarily an exocrine gland. The endocrine portion only accounts for 2% of the total mass of the pancreas. Within the islets of Langerhans, beta cells constitute 60–80% of all the cells.

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In beta cells, insulin is synthesized from the proinsulin precursor molecule by the action of proteolytic enzymes, known as prohormone convertases (PC1 and PC2), as well as the exoprotease carboxypeptidase E. These modifications of proinsulin remove the center portion of the molecule, or C-peptide, from the C- and N- terminal ends of the proinsulin. The remaining polypeptides (51 amino acids in total), the B- and A- chains, are bound together by disulfide bonds/disulphide bonds. Confusingly, the primary sequence of proinsulin goes in the order "B-C-A", since B and A chains were identified on the basis of mass, and the C peptide was discovered after the others.
Glucose enters the beta cells through the glucose transporter GLUT2
Glucose goes into the glycolysis and the respiratory cycle where multiple high-energy ATP molecules are produced by oxidation Dependent on ATP levels, and hence blood glucose levels, the ATP-controlled potassium channels (K+) close and the cell membrane depolarizes On depolarization, voltage controlled calcium channels (Ca2+) open and calcium flows into the cells An increased calcium level causes activation of phospholipase C, which cleaves the membrane phospholipid phosphatidyl inositol 4,5-bisphosphate into inositol 1,4,5-triphosphate and diacylglycerol. Inositol 1,4,5-triphosphate (IP3) binds to receptor proteins in the membrane of endoplasmic reticulum (ER). This allows the release of Ca2+ from the ER via IP3 gated channels, and further raises the cell concentration of calcium. Significantly increased amounts of calcium in the cells causes release of previously synthesised insulin, which has been stored in secretory vesicles

This is the main mechanism for release of insulin and regulation of insulin synthesis. In addition some insulin synthesis and release takes place generally at food intake, not just glucose or carbohydrate intake, and the beta cells are also somewhat influenced by the autonomic nervous system. The signalling mechanisms controlling this are not fully understood.
Other substances known to stimulate insulin release include amino acids from ingested proteins, acetylcholine, released from vagus nerve endings (parasympathetic nervous system), cholecystokinin[citation needed], released by enteroendocrine cells of intestinal mucosa and glucose-dependent insulinotropic peptide (GIP). Three amino acids (alanine, glycine and arginine) act similarly to glucose by altering the beta cell's membrane potential. Acetylcholine triggers insulin release through phospholipase C, while the last acts through the mechanism of adenylate cyclase.
The sympathetic nervous system (via Alpha2-adrenergic stimulation as demonstrated by the agonists clonidine or methyldopa) inhibit the release of insulin. However, it is worth noting that circulating Epinephrine will activate Beta2-Receptors on the Beta cells in the pancreatic islets to promote insulin release. This is important since muscle cannot benefit from the the raised blood sugar resultant from adrenergic stimulation (increased gluconeogenisis and glycogenolysis from the low blood insulin:glucogon state) unless insulin in present to allow for GLUT-4 translocation in the tissue. So in summary, first through direct innervation, NE inhibits insulin release via alpha2-receptors, then later, circulating Epi from the adrenal medulla will stimulate beta2-receptors thereby promoting insulin release.
When the glucose level comes down to the usual physiologic value, insulin release from the beta cells slows or stops. If blood glucose levels drop lower than this, especially to dangerously low levels, release of hyperglycemic hormones (most prominently glucagon from Islet of Langerhans' alpha cells) forces release of glucose into the blood from cellular stores, primarily liver cell stores of glycogen. By increasing blood glucose, the hyperglycemic hormones correct life-threatening hypoglycemia. Release of insulin is strongly inhibited by the stress hormone norepinephrine (noradrenaline), which leads to increased blood glucose levels during stress.

SDS-Polyacrylamide Gel Electrophoresis

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used in biochemistry, genetics and molecular biology to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight as well as higher order protein folding, posttranslational modifications and other factors).

The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure. This is known as Native PAGE. Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide.
SDS bind in a ratio of approximately 1.4 g SDS per 1.0 g protein (although binding ratios can vary from 1.1-2.2 g SDS/g protein), giving an approximately uniform mass:charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein. A tracking dye may be added to the protein solution to allow the experimenter to track the progress of the protein solution through the gel during the electrophoretic run.
''Polyacrylamide gel (PAG)'' had been known as a potential embedding medium for sectioning tissues as early as 1954. Two independent groups: Davis and Raymond, employed PAG in electrophoresis in 1959. It possesses several electrophoretically desirable features that made it a versatile medium. Polyacrylamide gel separates protein molecules according to both size and charge. It is a synthetic gel, thermo-stable, transparent, strong, relatively chemically inert, can be prepared with a wide range of average pore sizes, can withstand high voltage gradients, feasible to various staining and destaining procedures and can be digested to extract separated fractions or dried for autoradiography and permanent recording. DISC electrophoresis utilizes gels of different pore sizes. The name DISC was derived from the discontinuities in the electrophoretic matrix and coincidentally from the discoid shape of the separated zones of ions (Anbalagan, 1999). There are two layers of gel, namely stacking or spacer gel, and resolving or separating gel.

The denatured proteins are subsequently applied to one end of a layer of polyacrylamide gel submerged in a suitable buffer. An electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the anode. Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty (they encounter more resistance). After a set amount of time (usually a few hours- though this depends on the voltage applied across the gel; higher voltages run faster but tend to produce somewhat poorer resolution), the proteins will have differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin. Thus proteins may be separated roughly according to size (and therefore, molecular weight). Following electrophoresis, the gel may be stained (most commonly with Coomassie Brilliant Blue or silver stain), allowing visualisation of the separated proteins, or processed further (e.g. Western blot). After staining, different proteins will appear as distinct bands within the gel. It is common to run "marker proteins" of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker. The gel is actually formed because the acrylamide solution contains a small amount, generally about 1 part in 35 of bisacrylamide, which can form cross-links between two polyacrylamide molecules. The ratio of acrylamide to bisacrylamide can be varied for special purposes. The acrylamide concentration of the gel can also be varied, generally in the range from 5% to 25%. Lower percentage gels are better for resolving very high molecular weight proteins, while much higher percentages are needed to resolve smaller proteins. Determining how much of the various solutions to mix together to make gels of particular acrylamide concentration can be done on line
Gel electrophoresis is usually the first choice as an assay of protein purity due to its reliability and ease. The presence of SDS and the denaturing step causes proteins to be separated solely based on size. False negatives and positives are possible. A co migrating contaminant can appear as the same band as the desired protein. This comigration could also cause a protein to run at a different position or to not be able to penetrate the gel. This is why it is important to stain the entire gel including the stacking section. Coomassie Brilliant Blue will also bind with less affinity to glycoproteins and fibrous proteins, which interferes with quantification (Deutscher 1990).

Trinucleotide Repeat Disorders

Trinucleotide repeat disorders (also known as trinucleotide repeat expansion disorders, triplet repeat expansion disorders or codon reiteration disorders) are a set of genetic disorders caused by trinucleotide repeats in certain genes exceeding the normal, stable, threshold, which differs per gene. The mutation is a subset of unstable microsatellite repeats that occur throughout all genomic sequences. If the repeat is present in a healthy gene, a dynamic mutation may increase the repeat count and result in a defective gene.
Trinucleotide repeat disorders are classified as a type of Non-Mendelian inheritance

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Since the early 90’s, a new class of molecular disease has been characterized based upon the presence of unstable and abnormal expansions of DNA-triplets (trinucleotides). The first triplet disease to be identified was fragile X syndrome that has since been mapped to the long arm of the X chromosome. At this point, there are from 230 to 4000 CGG repeats in the gene that causes fragile X syndrome in these patients, as compared with 60 to 230 repeats in carriers and 5 to 54 repeats in normal persons. The chromosomal instability resulting from this trinucleotide expansion presents clinically as mental retardation, distinctive facial features, and macroorchidism in males. The second, related DNA-triplet repeat disease, fragile X-E syndrome, was also identified on the X chromosome, but was found to be the result of an expanded GCC repeat. Identifying trinucleotide repeats as the basis of disease has brought clarity to our understanding of a complex set of inherited neurologic diseases.

As more repeat expansion diseases have been discovered, several categories have been established to group them based upon similar characteristics. Category 1 includes Huntington’s disease (HD) and the spinocerebellar ataxias that are caused by a CAG repeat expansion in a protein-coding portion of specific genes. Category 2 expansions tend to be more phenotypically diverse with heterogeneous expansions that are generally small in magnitude, but also found in the exons of genes. Category 3 includes fragile X syndrome, myotonic dystrophy, two of the spinocerebellar ataxias, juvenile myoclonic epilepsy, and Friedreich’s ataxia. These diseases are characterized by typically much larger repeat expansions than the first two groups, and the repeats are located outside of the protein-coding regions of the genes.
Currently, ten neurologic disorders are known to be caused by an increased number of CAG repeats that encode an expanded series of glutamine residues in otherwise unrelated proteins. During protein synthesis, the expanded CAG repeats are translated into a series of uninterrupted glutamine residues forming what is known as a polyglutamine tract. These disorders are characterized by autosomal dominant mode of inheritance (with the exception of spino-bulbar muscular atrophy which shows X-linked inheritance), midlife onset, a progressive course, and a correlation of the number of CAG repeats with the severity of disease and the age at onset. Family studies have also suggested that these diseases are associated with anticipation, the tendency for progressively earlier or more severe expression of the disease in successive generations. Although the causative genes are widely expressed in all of the known polyglutamine diseases, each disease displays an extremely selective pattern of neurodegeneration.
A common symptom of Polyq diseases is characterized by a progressive degeneration of nerve cells usually affecting people later in life. Although these diseases share the same repeated codon (CAG) and some symptoms, the repeats for the different polyglutamine diseases occur on different chromosomes.
Trinucleotide repeat disorders generally show genetic anticipation, where their severity increases with each successive generation that inherits them.
Trinucleotide repeat disorders are the result of extensive duplication of a single codon. In fact, the cause is trinucleotide expansion up to a repeat number above a certain threshold level.
Why three nucleotides?
An interesting question is why three nucleotides are expanded, rather than two or four or some other number. Dinucleotide repeats are a common feature of the genome in general, as are larger repeats (e.g. VNTRs - Variable Number Tandem Repeats). One possibility is that repeats that are not a multiple of three would not be viable. Trinucleotide repeat expansions tend to be near coding regions of the genome, and therefore repeats that are not multiples of three could cause frameshift mutations that would be deadly
The non-Polyq diseases do not share any specific symptoms and are unlike the Polyq diseases. Trinucleotide repeat expansion
Trinucleotide repeat expansion, also known as triplet repeat expansion, is the DNA mutation responsible for causing any type of disorder categorized as a trinucleotide repeat disorder. Robert I. Richards and Grant R. Sutherland called these phenomena, in the framework of dynamical genetics, dynamic mutations.
Triplet expansion is caused by slippage during DNA replication. Due to the repetitive nature of the DNA sequence in these regions, 'loop out' structures may form during DNA replication while maintaining complementary base paring between the parent strand and daughter strand being synthesized. If the loop out structure is formed from sequence on the daughter strand this will result in an increase in the number of repeats. However if the loop out structure is formed on the parent strand a decrease in the number of repeats occurs. It appears that expansion of these repeats is more common than reduction. Generally the larger the expansion the more likely they are to cause disease or increase the severity of disease. This property results in the characteristic of anticipation seen in trinucleotide repeat disorders. Anticipation describes the tendency of age of onset to decrease and severity of symptoms to increase through successive generations of an affected family due to the expansion of these repeats.
In 2007, a team of scientists led by Ehud Shapiro at the Weizmann Institute of Science in Rehovot, Israel, proposed a new disease model to explain the progression of Huntington's Disease and similar trinucleotide repeat disorders. The team's computer simulations accurately predict age of onset and the way the disease will progress in an individual, based on the number of repeats of a genetic mutation.

DNA Replication Lecture

DNA replication is the process of copying a double-stranded DNA molecule to form two double-stranded molecules. The process of DNA replication is a fundamental process used by all living organisms as it is the basis for biological inheritance. As each DNA strand holds the same genetic information, both strands can serve as templates for the reproduction of the opposite strand. The template strand is preserved in its entirety and the new strand is assembled from nucleotides — this process is called "semiconservative replication". The resulting double-stranded DNA molecules are identical; proofreading and error-checking mechanisms exist to ensure near perfect fidelity.
DNA Replication DNA Replication 2
Dna replication 3
In a cell, DNA replication must happen before cell division can occur. DNA synthesis begins at specific locations in the genome, called "origins", where the two strands of DNA are separated. RNA primers attach to single stranded DNA and DNA polymerase extends from the primers to form new strands of DNA, adding nucleotides matched to the template strand. The unwinding of DNA and synthesis of new strands forms a replication fork. In addition to DNA polymerase, a number of enzymes are associated with the fork and assist in the initiation and continuation of DNA synthesis.
DNA replication can also be performed artificially, using the same enzymes used within the cell. DNA polymerases and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs artificial synthesis in a cyclic manner to rapidly and specifically amplify a target DNA fragment from a pool of DNA.

Microarray Method for Genetic Testing

Genetic testing allows the genetic diagnosis of vulnerabilities to inherited diseases, and can also be used to determine a person's ancestry. Normally, every person carries two copies of every gene, one inherited from their mother, one inherited from their father. The human genome is believed to contain around 20,000 - 25,000 genes. In addition to studying chromosomes to the level of individual genes, genetic testing in a broader sense includes biochemical tests for the possible presence of genetic diseases, or mutant forms of genes associated with increased risk of developing genetic disorders. Genetic testing identifies changes in chromosomes, genes, or proteins. Most of the time, testing is used to find changes that are associated with inherited disorders. The results of a genetic test can confirm or rule out a suspected genetic condition or help determine a person's chance developing or passing on a genetic disorder. Several hundred genetic tests are currently in use, and more are being developed.

Chromosomal Crossing Over

Chromosomal crossover (or crossing over) is the process by which two chromosomes pair up and exchange sections of their DNA. This often occurs during prophase 1 of meiosis in a process called synapsis. Synapsis begins before the synaptonemal complex develops, and is not completed until near the end of prophase 1. Crossover usually occurs when matching regions on matching chromosomes break and then reconnect to the other chromosome. The result of this process is an exchange of genes, called genetic recombination. Chromosomal crossovers also occur in asexual organisms and in somatic cells, since they are important in some forms of DNA repair.

 A double crossing over Recombination involves the breakage and rejoining of parental chromosomes Crossing over was first described, in theory, by Thomas Hunt Morgan. The physical basis of crossing over was first demonstrated by Harriet Creighton and Barbara McClintock in 1931
Meiotic recombination initiates with double-stranded breaks that are introduced into the DNA by the Spo11 protein. One or more exonucleases then digest the 5’ ends generated by the double-stranded breaks to produce 3’ single-stranded DNA tails. The meiosis-specific recombinase Dmc1 and the general recombinase Rad51 coat the single-stranded DNA to form nucleoprotein filaments.The recombinases catalyze invasion of the opposite chromatid by the single-stranded DNA from one end of the break. Next, the 3’ end of the invading DNA primes DNA synthesis, causing displacement of the complementary strand, which subsequently anneals to the single-stranded DNA generated from the other end of the initial double-stranded break. The structure that results is a cross-strand exchange that is known as a Holliday junction. Another word for crossing-over is chiasmata which refers to the contact between two chromatids that will soon undergo crossing-over. The Holliday junction is a tetrahedral structure which can be 'pulled' by other recombinases, moving it along the four-stranded structure.

DNA Sequencers

DNA sequencers have become more important due to large genomics projects and the need to increase productivity.
Modern automated DNA sequencing instruments (called DNA sequencers) are able to sequence as many as 384 fluoresecently labeled samples in a batch (run) and perform as many as 24 runs a day. These perform only the size separation and peak reading; the actual sequencing reaction(s), cleanup and resuspension in a suitable buffer must be performed separately.
The magnitude of the fluorescent signal is related to the number of strands of DNA that are in the reaction. If the initial amount of DNA is small, the signals will be weak. However, the properties of PCR allow one to increase the signal by increasing the number of cycles in the PCR program.
A simple DNA sequencer will have one or more lasers that emit at a wavelength that is absorbed by the fluorescent dye that has been attached to the DNA strand of interest. It will then have one or more optical detectors that can detect at the wavelength that the dye fluoresces at. The presence or absence of a strand of DNA is then detected by monitoring the output of the detector. Since shorter strands of DNA move through the gel matrix faster they are detected sooner and there is then a direct correlation between length of DNA strand and time at the detector. This relationship is then used to determine the actual DNA sequence.

How Ear Works

The ear is the sense organ that detects sounds. The vertebrate ear shows a common biology from fish to humans, with variations in structure according to order and species. It not only acts as a receiver for sound, but plays a major role in the sense of balance and body position. The ear is part of the auditory system.
The word "ear" may be used correctly to describe the entire organ or just the visible portion. In most animals, the visible ear is a flap of tissue that is also called the pinna. The pinna may be all that shows of the ear, but it serves only the first of many steps in hearing and plays no role in the sense of balance. In people, the pinna is often called the auricle. Vertebrates have a pair of ears, placed symmetrically on opposite sides of the head. This arrangement aids in the ability to localize sound sources.

Audition is the scientific name for the perception of sound. Sound is a form of energy that moves through air, water, and other matter, in waves of pressure. Sound is the means of auditory communication, including frog calls, bird songs and spoken language. Although the ear is the vertebrate sense organ that recognizes sound, it is the brain and central nervous system that "hears". Sound waves are perceived by the brain through the firing of nerve cells in the auditory portion of the central nervous system. The ear changes sound pressure waves from the outside world into a signal of nerve impulses sent to the brain.

The outer part of the ear collects sound. That sound pressure is amplified through the middle portion of the ear and, in land animals, passed from the medium of air into a liquid medium. The change from air to liquid occurs because air surrounds the head and is contained in the ear canal and middle ear, but not in the inner ear. The inner ear is hollow, embedded in the temporal bone, the densest bone of the body. The hollow channels of the inner ear are filled with liquid, and contain a sensory epithelium that is studded with hair cells. The microscopic "hairs" of these cells are structural protein filaments that project out into the fluid. The hair cells are mechanoreceptors that release a chemical neurotransmitter when stimulated. Sound waves moving through fluid push the filaments; if the filaments bend over enough it causes the hair cells to fire. In this way sound waves are transformed into nerve impulses. In vision, the rods and cones of the retina play a similar role with light as the hair cells do with sound. The nerve impulses travel from the left and right ears through the eighth cranial nerve to both sides of the brain stem and up to the portion of the cerebral cortex dedicated to sound. This auditory part of the cerebral cortex is in the temporal lobe.

The part of the ear that is dedicated to sensing balance and position also sends impulses through the eighth cranial nerve, the VIIIth nerve's Vestibular Portion. Those impulses are sent to the vestibular portion of the central nervous system. The human ear can generally hear sounds with frequencies between 20 Hz and 20 kHz (the audio range). Although the sensation of hearing requires an intact and functioning auditory portion of the central nervous system as well as a working ear, human deafness (extreme insensitivity to sound) most commonly occurs because of abnormalities of the inner ear, rather than the nerves or tracts of the central auditory system.

The pinna collects the sound.The ear drum is a muscle thet vibrates.The small bones pass the vibrations to the cochlea.The cochlea turns vibrations into electricty.The electricity moves through the auditory nervet to the brain.

Animals can hear many different sounds these are same order of how their hearing is:Human Grandparent,Elephant,Human Parent, Yound Human,Dog,Cat,Bat and Dolphin.This order is worst to best hearing.

Chromosomal translocation

Chromosome translocation is a chromosome abnormality caused by rearrangement of parts between nonhomologous chromosomes. It is detected on cytogenetics or a karyotype of affected cells. There are two main types, reciprocal (also known as non-Robertsonian) and Robertsonian. Also, translocations can be balanced (in an even exchange of material with no genetic information extra or missing, and ideally full functionality) or unbalanced (where the exchange of chromosome material is unequal resulting in extra or missing genes).

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Reciprocal (non-Robertsonian) translocations

Reciprocal translocations are usually an exchange of material between nonhomologous chromosomes. They are found in about 1 in 600 human newborns. Such translocations are usually harmless and may be found through prenatal diagnosis. However, carriers of balanced reciprocal translocations have increased risks of creating gametes with unbalanced chromosome translocations leading to miscarriages or children with abnormalities. Genetic counseling and genetic testing is often offered to families that may carry a translocation.

Robertsonian translocations

This type of rearrangement involves two acrocentric chromosomes that fuse near the centromere region with loss of the short arms. The resulting karyotype in humans leaves only 45 chromosomes since two chromosomes have fused together. Robertsonian translocations have been seen involving all combinations of acrocentric chromosomes. The most common translocation in human involves chromosomes 13 and 14 and is seen in about 1 in 1300 persons. Like other translocations, carriers of Robertsonian translocations are phenotypically normal, but there is a risk of unbalanced gametes which lead to miscarriages or abnormal offspring. For example, carriers of Robertsonian translocations involving chromosome 21 have a higher chance to have a child with Down syndrome.

Some human diseases caused by translocations are:

* Cancer: several forms of cancer are caused by translocations; this has been described mainly in leukemia (acute myelogenous leukemia and chronic myelogenous leukemia).
* Infertility: one of the would-be parents carries a balanced translocation, where the parent is asymptomatic but conceived fetuses are not viable.
* Down syndrome is caused in a minority (5% or less) of cases by a Robertsonian translocation of about a third of chromosome 21 onto chromosome 14.

Coronary Artery Bypass Graft (CABG )

Coronary artery bypass grafting (CABG) is a type of surgery called revascularization (re-VAS-kyu-lar-i-ZA-shun), used to improve blood flow to the heart in people with severe coronary artery disease (CAD).

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CAD occurs when the arteries that supply blood to the heart muscle (the coronary arteries) become blocked due to the buildup of a material called plaque (plak) on the inside of the blood vessels. If the blockage is severe, chest pain (also called angina), shortness of breath, and, in some cases, heart attack can occur.

CABG is one treatment for CAD. During CABG, a healthy artery or vein from another part of the body is connected, or grafted, to the blocked coronary artery. The grafted artery or vein bypasses (that is, it goes around) the blocked portion of the coronary artery. This new passage routes oxygen-rich blood around the blockage to the heart muscle. As many as four major blocked coronary arteries can be bypassed during one surgery.

CABG is the most common type of open-heart surgery in the United States, with more than 500,000 surgeries performed each year. Doctors called cardiothoracic (KAR-de-o-tho-RAS-ik) surgeons perform this surgery.

CABG isn’t used for everyone with CAD. Many people with CAD can be treated by other means, such as lifestyle changes, medicines, and another revascularization procedure called angioplasty.

CABG may be an option if you have severe blockages in the large coronary arteries that supply a major part of the heart muscle with blood—especially if the heart’s pumping action has already been weakened.

CABG may also be an option if you have blockages in the heart that can’t be treated with angioplasty. In these situations, CABG is considered more effective than other types of treatment.

If you’re a candidate for CABG, the goals of having the surgery are to:

  • Improve your quality of life and decrease angina and other symptoms of CAD
  • Resume a more active lifestyle
  • Improve the pumping action of the heart if it has been damaged by a heart attack
  • Lower the chances of a heart attack (in some patients, such as those with diabetes)
  • Improve your chance of survival
Repeat surgery may be needed if grafted arteries or veins become blocked, or if new blockages develop in arteries that weren’t blocked before. Taking medicines as prescribed and making lifestyle changes that your doctor recommends can lower the chance of a graft becoming blocked.

In people who are candidates for the surgery, the results are usually excellent, with 85 percent of people having significantly reduced symptoms, less risk for future heart attacks, and a decreased chance of dying within 10 years following the surgery.

During the last three decades careful studies have clearly shown that coronary artery bypass surgery relieves angina pectoris and other symptoms caused by coronary artery disease and, for some patients, prolongs their lives. However, coronary artery bypass surgery alone does not remove the metabolic causes of coronary artery disease and even after successful operation the occurrence of new obstructions may cause problems as the years ago by. These new obstructions may develop either in the patient’s own coronary arteries (progression of native coronary artery disease) or in bypass grafts, particularly in saphenous vein grafts.

Saphenous vein graft

Within a decade of the development of bypass surgery it became apparent that obstructions could develop in saphenous vein to coronary bypass grafts and that the likelihood of obstructions developing was related to time. Within 5 years of surgery approximately 20% of saphenous vein grafts developed partial or total obstructions, and between 5 and 10 years after operation these processes continued to progress such that by 10 years after operation almost half of saphenous vein grafts were either totally obstructed or showed some angiographic evidence of pathologic changes .

Since those early days of bypass surgery progress has been made in the treatment of patients with vein grafts that decreases the rate of vein graft failure. Taking aspirin early after operation increases the percentage of grafts that are functioning well a year after surgery and, more recently, treatment with HMG-CoA reductase inhibitors, also known as "statin" type drugs, has been shown to have long term benefit. However, the failure of vein grafts over the long term remains a significant problem effecting outcomes after bypass surgery and it is the single greatest cause of the need for repeat surgery for bypass grafting .

Internal thoracic artery (ITA, also called mammary artery) graft
(bypass graft using left and/or right internal thoracic artery from the chest wall)

Distal end of ITA attached to LAD
Fortunately there have been other bypass grafts available that are resistant to a late failure - internal thoracic (mammary) artery grafts. Internal thoracic artery (ITA) grafts were used from the beginning of bypass surgery although at relatively few centers during the early years.

Most commonly the left ITA was left attached at its origin from the left subclavian artery and the distal end was dissected away from the chest wall, swung over, and its distal end was attached with sutures to the side of the left anterior descending (LAD) coronary artery.

Multiple vein bypass graft

In the most common situation the left ITA was used as a graft to the LAD coronary artery and saphenous vein grafts were used from the aorta to the other coronary vessels. Studies of angiograms performed after bypass surgery have shown that not only did the LITA to LAD graft have a more than 90% chance of functioning well early after operation, but that these grafts continued to function well for many years and that even 20 years after operation the development of obstructions in these grafts is extremely uncommon (Ref 1).

Long-term follow-up studies done at The Cleveland Clinic Foundation during the 1980s show that not only is the LITA-LAD graft likely to stay functioning over the years but also, that graft has an important long-term effect on clinical outcomes. Over time, patients with a LITA-LAD graft are less likely to die or to need a reoperation when compared with patients who received only vein grafts (Ref. 3). Since these studies have been completed the LITA-LAD graft has become a standard part of operations for coronary bypass grafting.

There are two internal thoracic arteries, one on either side of the sternum (breast bone) and more extensive use of ITA grafts can be accomplished by using the right ITA as an in situ graft (left attached to the right subclavian artery), as a "free" graft from the aorta to the coronary artery, or attached to the left ITA as a composite graft.

ITA as in situ graft ITA as "free" graft ITA as composite graft

Despite the logic that more extensive ITA grafting would be an advantage over the use of only one ITA graft it has only recently been shown by long-term follow-up studies from The Cleveland Clinic Foundation that bilateral ITA grafts further decrease the long-term risks of death and reoperation when compared to patients receiving only one ITA graft (Ref 4). The use of both ITAs as bypass grafts is a more complicated operation and there are some patients where this strategy is not appropriate. However, two ITA grafts do produce better outcomes than just one ITA graft for many patients.

Gastroepiploic graft

Because of the success of ITA grafts, surgeons have search for other arterial bypass grafts. The gastroepiploic artery (GEA) is a branch of the blood supply to the stomach (an organ with a very rich blood supply) that has been used as a bypass graft usually to the right coronary artery. This is a technically difficult operation to perform and it has not become a popular bypass graft but it has a high likelihood of good long-term functioning when used in the proper situation and in some patients represents a significant advantage over vein grafts.

Radial artery graft
(bypass graft using artery from inner forearm)

Radial artery graft

The radial artery was used as a bypass graft in the early years of coronary surgery but its use was abandoned for a number of years because of the occurrence of graft occlusions. In the past few years, its use was revived because of the hope that new methods of preparation and drug treatment with antispasm agents might improve the long-term results. The advantage of radial artery grafts is that they are easy to prepare. The hope is that they will be resistant to the development of atherosclerosis, a problem that has plagued vein grafts. However, the long-term (more than 10 years) of outcomes of radial artery grafts are as yet unknown.

Our data indicates that the long-term results of radial artery grafts are not as good as those for ITA grafts, in particular we have seen more early graft failures. In fact, radial artery graft patency was not better than for saphenous vein grafts. We continue to recommend and use radial artery grafts, particularly for young patients with hyperlipidemia (high cholesterol or triglycerides) who have a relatively high risk of vein graft failure because of the occurrence of vein graft atherosclerosis. In patients who are 70 years or older we use radial artery grafts more cautiously, mainly when alternative grafts are not available. In addition, a radial graft needs a severe blockage or stenosis in the native artery to be grafted, to have a better chance to be promoted and to stay open.(Ref.5)

Total revascularization
It is very clear that the internal thoracic arteries are the best bypass grafts that we have. Because not all patients can be completed treated with just the internal thoracic arteries, the search continues to go on for other arterial bypass conduits and/or total arterial revascularization.