Endoscopy of Large Intestine

Endoscopy means looking inside and typically refers to looking inside the body for medical reasons using an instrument called an endoscope. Endoscopy can also refer to using a borescope in technical situations where direct line-of-sight observation is not feasible.

Endoscopy is a minimally invasive diagnostic medical procedure that is used to assess the interior surfaces of an organ by inserting a tube into the body. The instrument may have a rigid or flexible tube and not only provide an image for visual inspection and photography, but also enable taking biopsies and retrieval of foreign objects. Endoscopy is the vehicle for minimally invasive surgery.

Many endoscopic procedures are considered to be relatively painless and, at worst, associated with mild discomfort; for example, in esophagogastroduodenoscopy, most patients tolerate the procedure with only topical anaesthesia of the oropharynx using lignocaine spray. Complications are not common (only 5% of all operations)but can include perforation of the organ under inspection with the endoscope or biopsy instrument. If that occurs open surgery may be required to repair the injury.

Deciphering the Human Genome

Herceptin: Mechanism of action

Herceptin is a humanized monoclonal antibody that acts on the HER2/neu (erbB2) receptor. Trastuzumab's principal use is as an anti-cancer therapy in breast cancer in patients whose tumors over express (produce more than the usual amount of) this receptor. Trastuzumab is administered either once a week or once every three weeks intravenously for 30 to 90 minutes.

Amplification of HER2/neu (ErbB2) occurs in 25-30% of early-stage breast cancers.[1] It encodes the extracellular domain of her2. Although the signaling pathways induced by the HER2/neu receptor are incompletely characterized, it is thought that activation of the PI3K/Akt pathway is important. This pathway is normally associated with mitogenic signaling involving the MAPK pathway. However in cancer the growth promoting signals from HER2/neu are constitutively transmitted — promoting invasion, survival and angiogenesis of cells.[2] Furthermore overexpression can also confer therapeutic resistance to cancer therapies. The prime mechanism that causes increase in proliferation speed is due to induction of p27Kip1, an inhibitor of cdk2 and of cell proliferation, to remain in the cytoplasm instead of translocation in to the nucleus.[3] This is caused by phosphorylation by Akt.

Herceptin is a humanized monoclonal antibody which binds to the extracellular segment of the HER2/neu receptor. Cells treated with trastuzumab undergo arrest during the G1 phase of the cell cycle so there is reduced proliferation. It has been suggested that trastuzumab induces some of its effect by downregulation of HER2/neu leading to disruption of receptor dimerization and signaling through the downstream PI3K cascade. P27Kip1 is then not phosphorylated and is able to enter the nucleus and inhibit cdk2 activity, causing cell cycle arrest.[3] Also, trastuzumab suppresses angiogenesis by both induction of antiangiogenic factors and repression of proangiogenic factors. It is thought that a contribution to the unregulated growth observed in cancer could be due to proteolytic cleavage of HER2/neu that results in the release of the extracellular domain. Trastuzumab has been shown to inhibit HER2/neu ectodomain cleavage in breast cancer cells.[4] There may be other undiscovered mechanisms by which trastuzumab induces regression in cancer.

T cell lymphocyte

T cells belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and NK cells by the presence of a special receptor on their cell surface called the T cell receptor (TCR). The abbreviation T, in T cell, stands for thymus, since it is the principal organ in the T cell's development.

Several different subsets of T cells have been described, each with a distinct function.

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  • Helper T cells (TH cells) are the "middlemen" of the adaptive immune system. Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or "help" the immune response. Depending on the cytokine signals received, these cells differentiate into TH1, TH2, TH17, or one of other subsets, which secrete different cytokines.
  • Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells, since they express the CD8 glycoprotein at their surface. Through interaction with helper T cells, these cells can be transformed into regulatory T cells, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.
  • Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise two subtypes: central memory T cells (TCM cells) and effector memory T cells (TEM cells). Memory cells may be either CD4+ or CD8+.
  • Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4+ regulatory T cells have been described, including the naturally occurring Treg cells and the adaptive Treg cells. Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus, whereas the adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.
  • Natural Killer T cells (NKT cells) are a special kind of lymphocyte that bridges the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigen presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD1d. Once activated, these cells can perform functions ascribed to both Th and Tc cells (i.e., cytokine production and release of cytolytic/cell killing molecules).
  • γδ T cells represent a small subset of T cells that possess a distinct TCR on their surface. A majority of T cells have a TCR composed of two glycoprotein chains called α- and β- TCR chains. However, in γδ T cells, the TCR is made up of one γ-chain and one δ-chain. This group of T cells is much less common (5% of total T cells) than the αβ T cells, but are found at their highest abundance in the gut mucosa, within a population of lymphocytes known as intraepithelial lymphocytes (IELs). The antigenic molecules that activate γδ T cells are still widely unknown. However, γδ T cells are not MHC restricted and seem to be able to recognise whole proteins rather than requiring peptides to be presented by MHC molecules on antigen presenting cells. Some recognize MHC class IB molecules though. Human Vγ9/Vδ2 T cells, which constitute the major γδ T cell population in peripheral blood, are unique in that they specifically and rapidly respond to a small non-peptidic microbial metabolite, HMB-PP, an isopentenyl pyrophosphate precursor.

T cell Activation

Although the specific mechanisms of activation vary slightly between different types of T cells, the "two-signal model" in CD4+ T cells holds true for most. Activation of CD4+ T cells occurs through the engagement of both the T cell receptor and CD28 on the T cell by the Major histocompatibility complex peptide and B7 family members on the APC, respectively. Both are required for production of an effective immune response; in the absence of CD28 co-stimulation, T cell receptor signalling alone results in anergy. The signalling pathways downstream from both CD28 and the T cell receptor involve many proteins.

The first signal is provided by binding of the T cell receptor to a short peptide presented by the major histocompatibility complex (MHC) on another cell. This ensures that only a T cell with a TCR specific to that peptide is activated. The partner cell is usually a professional antigen presenting cell (APC), usually a dendritic cell in the case of naïve responses, although B cells and macrophages can be important APCs. The peptides presented to CD8+ T cells by MHC class I molecules are 8-9 amino acids in length; the peptides presented to CD4+ cells by MHC class II molecules are longer, as the ends of the binding cleft of the MHC class II molecule are open.

The second signal comes from co-stimulation, in which surface receptors on the APC are induced by a relatively small number of stimuli, usually products of pathogens, but sometimes breakdown products of cells, such as necrotic-bodies or heat-shock proteins. The only co-stimulatory receptor expressed constitutively by naïve T cells is CD28, so co-stimulation for these cells comes from the CD80 and CD86 proteins on the APC. Other receptors are expressed upon activation of the T cell, such as OX40 and ICOS, but these largely depend upon CD28 for their expression. The second signal licenses the T cell to respond to an antigen. Without it, the T cell becomes anergic, and it becomes more difficult for it to activate in future. This mechanism prevents inappropriate responses to self, as self-peptides will not usually be presented with suitable co-stimulation.

The T cell receptor exists as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, which are produced from the independent T cell receptor alpha and beta (TCRα and TCRβ) genes. The other proteins in the complex are the CD3 proteins: CD3εγ and CD3εδ heterodimers and, most important, a CD3ζ homodimer, which has a total of six ITAM motifs. The ITAM motifs on the CD3ζ can be phosphorylated by Lck and in turn recruit ZAP-70. Lck and/or ZAP-70 can also phosphorylate the tyrosines on many other molecules, not least CD28, Trim, LAT and SLP-76, which allows the aggregation of signalling complexes around these proteins.

Phosphorylated LAT recruits SLP-76 to the membrane, where it can then bring in PLCγ, VAV1, Itk and potentially PI3K. Both PLCγ and PI3K act on PI(4,5)P2 on the inner leaflet of the membrane to create the active intermediaries di-acyl glycerol (DAG), inositol-1,4,5-trisphosphate (IP3), and phosphatidlyinositol-3,4,5-trisphosphate (PIP3). DAG binds and activates some PKCs, most important, in T cells PKCθ, a process important for activating the transcription factors NF-κB and AP-1. IP3 is released from the membrane by PLCγ and diffuses rapidly to activate receptors on the ER, which induce the release of calcium. The released calcium then activates calcineurin, and calcineurin activates NFAT, which then translocates to the nucleus. NFAT is a transcription factor, which activates the transcription of a pleiotropic set of genes, most notable, IL-2, a cytokine that promotes long term proliferation of activated T cells.

Pathogen Recognition Receptors

Cells in the immune system like Macrophages and dendritic cells are the first line of defense in recognizing various kinds pathogens.These cells are developed several kind of receptors for recognizing different types of pathogen Associated Molecular patterns known as PAMPs.
There are different classes of these proteins, they recognize different types of PAMPs, Toll-like receptor (TLR) is composed of multiple leucine-rich repeats that are useful for recognizing various PAMPs.Each members of TLR family recognize different kinds of PAMPs,For example TLR5 recognizes flagellin,which is highly conserver constituent of the bacterial flagellum.

Bacterial genomes contain methylated CPG oligonucleotide motifs, which are recognized by TLR9, once the genome is degraded in the lysosome.
TLR6 and TLR2 are dimers,that recognize diacyllipopeptide.
TLR1 and TLR2 are dimers that recognize triacyllipopetide and TLR4 recognize lipopolysaccharide (LPS) a component of Gram Negative bacteria.
Like TLR9 TLR3 and TLR7 are located endocylic vesciles and recognize double stranded RNA and single stranded RNA respectively.
When any TLRs activated, it sends the signal to nucleus by activating transcription factors.
Some pathogens such as viruses exists and replicate in cytosol.there are at least two classes receptors that an detect pathogens in cytosol and signal their presence to the immune system.One class of such receptors are members of nuclear oligomerization domain family or NOD proteins.
For example NOD 2 protein, which is located in the cytosol, can detect bacterioproteoglycans of intracellular bacteria.When NOD2 protein recognizes its ligands the muramyl dipeptide, it sends the signal to nucleus to activate transcription.
Finally there is a class intracellular receptor protein that can contain a RNA helicase domain and two caspase recruitment domains, one member of this family RIG-I recognizes double stranded RNAs that are component of life cycle of many RNA virus,
This class of proteins also sends the signal to nucleus, but unlike TLRs it activates the production type 1 interferons. In all the toll like receptors, NOR proteins and RNA helicase domain family provide the innate immune system with the ability to detect both extra cellular and intracellular pathogens and to activate immune response

What is Hemolysis

Hemolysis is the breakdown of red blood cells. The ability of bacterial colonies to induce hemolysis when grown on blood agar is used to classify certain microorganisms. This is a particularly useful in classifying streptococcal species. A substance that causes hemolysis is a hemolysin.
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Types of hemolysis Alpha
When Alpha hemolysis (α-hemolysis) is present the agar under the colonies is dark and greenish. Streptococcus pneumoniae and a group of oral streptococci (Streptococcus viridans or viridans streptococci) display alpha hemolysis. This is sometimes called green hemolysis because of the color change in the agar. Other synonymous terms are incomplete hemolysis and partial hemolysis. Alpha hemolysis is generally caused by peroxides produced by the bacterium.
The bacterium Staphylococcus aureus, a common cause of infection in humans, secretes a toxin known as alpha-hemolysin that kills human cells by forming holes in their membranes, through which chemicals essential to survival leak out. The movie illustrates the structure of the hole, which is created when identical proteins released by the bacterium (in pink, yellow, gold, red, grey, green, and white) assemble in groups of seven in the cell's membrane (in cyan).
Beta hemolysis (β-hemolysis), sometimes called complete hemolysis, is a complete lysis of red cells in the media around and under the colonies: the area appears lightened and transparent. Streptococcus pyogenes, or Group A beta-hemolytic Strep (GAS), displays beta hemolysis.
Some weakly beta-hemolytic species cause intense beta hemolysis when grown together with a strain of Staphylococcus. This is called the CAMP test1. Streptococcus agalactiae displays this property. Clostridium perfringens can be identified presumptively with this test.

If an organism does not induce hemolysis, it is said to display gamma hemolysis (γ-hemolysis): the agar under and around the colony is unchanged (this is also called non-hemolytic). Enterococcus faecalis (formerly called Group D Strep) displays gamma hemolysis.

Rh Factor

Individuals either have, or do not have, the Rhesus factor (or Rh D antigen) on the surface of their red blood cells. This is usually indicated by 'RhD positive' (does have the RhD antigen) or 'RhD negative' (does not have the antigen) suffix to the ABO blood type. Unlike the ABO antigens, the only ways antibodies are developed against the Rh factor are through placental sensitization or translation. That is, if a person who is RhD-negative has never been exposed to the RhD antigen, they do not possess the RhD antibody. The 'RhD-' suffix is often shortened to 'D pos'/'D neg', 'RhD pos'/RhD neg', or +/-. The latter is generally not preferred in research or medical situations, because it can be altered or obscured accidentally.

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There may be prenatal danger to the fetus when a pregnant woman is RhD-negative and the biological father is RhD-positive. But, as discussed below, the situation is considerably more complex than that.

Rh factor, protein substance present in the red blood cells of most people, capable of inducing intense antigenic reactions. The Rh, or rhesus, factor was discovered in 1940 by K. Landsteiner and A. S. Wiener, when they observed that an injection of blood from a rhesus monkey into rabbits caused an antigenic reaction in the serum component of rabbit blood (see immunity). When blood from humans was tested with the rabbit serum, the red blood cells of 85% of the humans tested agglutinated (clumped together). The red blood cells of the 85% (later found to be 85% of the white population and a larger percentage of blacks and Asians) contained the same factor present in rhesus monkey blood; such blood was typed Rh positive. The blood of the remaining 15% lacked the factor and was typed Rh negative. Under ordinary circumstances, the presence or lack of the Rh factor has no bearing on life or health. It is only when the two blood types are mingled in an Rh-negative individual that the difficulty arises,

Since the Rh factor acts as an antigen in Rh-negative persons, causing the production of antibodies. Besides the Rh factor, human red blood cells contain a large number of additional antigenic substances that have been classified into many blood group systems (see blood groups); however, the Rh system is the only one, aside from the ABO system, that is of major importance in blood transfusions. If Rh-positive blood is transfused into an Rh-negative person, the latter will gradually develop antibodies called anti-Rh agglutinins, that attach to the Rh-positive red blood cells, causing them to agglutinate. Destruction of the cells (hemolysis) eventually results. If the Rh-negative recipient is given additional transfusions of Rh-positive blood, the concentration of anti-Rh agglutinins may become high enough to cause a serious or fatal reaction. The same type of immune reaction occurs in the blood of an Rh-negative mother who is carrying an Rh-positive fetus. (The probability of this situation occurring is high if the father is Rh positive.) Some of the infant's blood may enter the maternal circulation, causing the formation of agglutinins against the fetal red blood cells. The first baby is usually not harmed. But, if the mother's agglutinins pass into the circulation of subsequent fetuses, they may destroy the fetal red blood cells, causing the severe hemolytic disease of newborns known as erythroblastosis fetalis

HIV Drug groups - Protease inhibitors

Protease inhibitors (PIs) are a class of medications used to treat or prevent infection by viruses, including HIV and Hepatitis C. PIs prevent viral replication by inhibiting the activity of HIV-1 protease, an enzyme used by the viruses to cleave nascent proteins for final assembly of new virons.
Protease inhibitors have been developed or are presently undergoing testing for treating various viruses:
HIV/AIDS: antiretroviral protease inhibitors such assaquinavir, ritonavir, indinavir, nelfinavir etc.

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Saquinavir is an antiretroviral drug used in HIV therapy. It falls in the protease inhibitor class. Two formulations have been marketed:
a hard-gel capsule formulation of the mesylate, with trade name Invirase®, which requires combination with ritonavir to increase the saquinavir bioavailability; a soft-gel capsule formulation of saquinavir, with trade name Fortovase®. Both formulations are generally used as a component of highly active antiretroviral therapy (HAART). Ritonavir
Ritonavir, with trade name Norvir® (Abbott Laboratories), is an antiretroviral drug from the protease inhibitor class used to treat HIV infection and AIDS.
Ritonavir is frequently prescribed with HAART, not for its antiviral action, but as it inhibits the same host enzyme that metabolizes other protease inhibitors. This inhibition leads to higher plasma concentrations of these latter drugs, allowing the clinician to lower their dose and frequency and improving their clinical efficacy.

Indinavir (IDV; trade name Crixivan, manufactured by Merck) is a protease inhibitor used as a component of highly active antiretroviral therapy (HAART) to treat HIV infection and AIDS.
Nelfinavir (Viracept®) is an antiretroviral drug used in the treatment of the human immunodeficiency virus (HIV). Nelfinavir belongs to the class of drugs known as protease inhibitors (PIs) and like other PIs is generally used in combination with other antiretroviral drugs. Nelfinavir is presented as the mesilate (mesylate) ester prodrug.
Nelfinavir mesylate (Viracept, formally AG1343) is a potent and orally bioavailable human immunodeficiency virus HIV-1 protease inhibitor (Ki=2nM) and is being widely prescribed in combination with HIV reverse transcriptase inhibitors for the treatment of HIV infection. Nelfinavir mesylate contains the Castor oil derivative Cremophor EL

Protein Folding

Protein folding is the physical process by which a polypeptide folds into its characteristic three-dimensional structure.

Each protein begins as a polypeptide, translated from a sequence of mRNA as a linear chain of amino acids. This polypeptide lacks any developed three-dimensional structure (the left hand side of the neighboring figure). However each amino acid in the chain can be thought of having certain 'gross' chemical features. These may be hydrophobic, hydrophilic, or electrically charged, for example. These interact with each other and their surroundings in the cell to produce a well-defined, three dimensional shape, the folded protein (the right hand side of the figure), known as the native state. The resulting three-dimensional structure is determined by the sequence of the amino acids. The mechanism of protein folding is not completely understood.

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Experimentally determining the three dimensional structure of a protein is often very difficult and expensive. However the sequence of that protein is often known. Therefore scientists have tried to use different biophysical techniques to manually fold a protein. That is, to predict the structure of the complete protein from the sequence of the protein.

For many proteins the correct three dimensional structure is essential to function. Failure to fold into the intended shape usually produces inactive proteins with different properties (details found under prion). Several neurodegenerative and other diseases are believed to result from the accumulation of misfolded (incorrectly folded) proteins.
A simulation of protein folding in the HP lattice model from a random state to ground state with maximum number of HH contacts. The red balls are Hydrophobic amino acids.
The relationship between folding and amino acid sequence

The amino-acid sequence (or primary structure) of a protein predisposes it towards its native conformation or conformations. It will fold spontaneously during or after synthesis. While these macromolecules may be regarded as "folding themselves", the mechanism depends equally on the characteristics of the cytosol, including the nature of the primary solvent (water or lipid), the concentration of salts, the temperature, and molecular chaperones.

Most folded proteins have a hydrophobic core in which side chain packing stabilizes the folded state, and charged or polar side chains on the solvent-exposed surface where they interact with surrounding water molecules. It is generally accepted that minimizing the number of hydrophobic sidechains exposed to water is the principal driving force behind the folding process,although a recent theory has been proposed which reassesses the contributions made by hydrogen bonding.

The process of folding in vivo often begins co-translationally, so that the N-terminus of the protein begins to fold while the C-terminal portion of the protein is still being synthesized by the ribosome. Specialized proteins called chaperones assist in the folding of other proteins.A well studied example is the bacterial GroEL system, which assists in the folding of globular proteins. In eukaryotic organisms chaperones are known as heat shock proteins. Although most globular proteins are able to assume their native state unassisted, chaperone-assisted folding is often necessary in the crowded intracellular environment to prevent aggregation; chaperones are also used to prevent misfolding and aggregation which may occur as a consequence of exposure to heat or other changes in the cellular environment.

For the most part, scientists have been able to study many identical molecules folding together en masse. At the coarsest level, it appears that in transitioning to the native state, a given amino acid sequence takes on roughly the same route and proceeds through roughly the same intermediates and transition states. Often folding involves first the establishment of regular secondary and supersecondary structures, particularly alpha helices and beta sheets, and afterwards tertiary structure. Formation of quaternary structure usually involves the "assembly" or "coassembly" of subunits that have already folded. The regular alpha helix and beta sheet structures fold rapidly because they are stabilized by intramolecular hydrogen bonds, as was first characterized by Linus Pauling. Protein folding may involve covalent bonding in the form of disulfide bridges formed between two cysteine residues or the formation of metal clusters. Shortly before settling into their more energetically favourable native conformation, molecules may pass through an intermediate "molten globule" state.

The essential fact of folding, however, remains that the amino acid sequence of each protein contains the information that specifies both the native structure and the pathway to attain that state. This is not to say that identical amino acid sequences always fold similarly. Conformations differ based on environmental factors as well; similar proteins fold differently based on where they are found. Folding is a spontaneous process independent of energy inputs from nucleoside triphosphates. The passage of the folded state is mainly guided by hydrophobic interactions, formation of intramolecular hydrogen bonds, and van der Waals forces, and it is opposed by conformational entropy, which must be overcome by extrinsic factors such as chaperones.

Disruption of the native state
In certain solutions and under some conditions proteins will not fold into their biochemically functional forms. Temperatures above (and sometimes those below) the range that cells tend to live in will cause proteins to unfold or "denature" (this is why boiling makes an egg white turn opaque). High concentrations of solutes, extremes of pH, mechanical forces, and the presence of chemical denaturants can do the same. A fully denatured protein lacks both tertiary and secondary structure, and exists as a so-called random coil. Under certain conditions some proteins can refold; however, in many cases denaturation is irreversible. Cells sometimes protect their proteins against the denaturing influence of heat with enzymes known as chaperones or heat shock proteins, which assist other proteins both in folding and in remaining folded. Some proteins never fold in cells at all except with the assistance of chaperone molecules, which either isolate individual proteins so that their folding is not interrupted by interactions with other proteins or help to unfold misfolded proteins, giving them a second chance to refold properly. This function is crucial to prevent the risk of precipitation into insoluble amorphous aggregates.

Incorrect protein folding and neurodegenerative disease
Misfolded proteins are responsible for prion-related illnesses such as Creutzfeldt-Jakob disease, bovine spongiform encephalopathy (mad cow disease), amyloid-related illnesses such as Alzheimer's Disease, and a number of other forms of proteopathy such as cystic fibrosis. These diseases are associated with the multimerization of misfolded proteins into insoluble, extracellular aggregates and/or intracellular inclusions; it is not clear whether the plaques are the cause or merely a symptom of illness.

Kinetics and the Levinthal Paradox
The entire duration of the folding process varies dramatically depending on the protein of interest. The slowest folding proteins require many minutes or hours to fold, primarily due to proline isomerizations or wrong disulfide bond formations, and must pass through a number of intermediate states, like checkpoints, before the process is complete. On the other hand, very small single-domain proteins with lengths of up to a hundred amino acids typically fold in a single step. Time scales of milliseconds are the norm and the very fastest known protein folding reactions are complete within a few microseconds.

The Levinthal paradox observes that if a protein were to fold by sequentially sampling all possible conformations, it would take an astronomical amount of time to do so, even if the conformations were sampled at a rapid rate (on the nanosecond or picosecond scale). Based upon the observation that proteins fold much faster than this, Levinthal then proposed that a random conformational search does not occur in folding, and the protein must, therefore, fold by a directed process.

Techniques for studying protein folding
Modern studies of folding with high time resolution

The study of protein folding has been greatly advanced in recent years by the development of fast, time-resolved techniques. These are experimental methods for rapidly triggering the folding of a sample of unfolded protein, and then observing the resulting dynamics. Fast techniques in widespread use include ultrafast mixing of solutions, photochemical methods, and laser temperature jump spectroscopy. Among the many scientists who have contributed to the development of these techniques are Heinrich Roder, Harry Gray, Martin Gruebele, Brian Dyer, William Eaton, Sheena Radford, Chris Dobson, Sir Alan R. Fersht and Bengt Nölting.

Energy landscape theory of protein folding
The protein folding phenomenon was largely an experimental endeavor until the formulation of energy landscape theory by Joseph Bryngelson and Peter Wolynes in the late 1980s and early 1990s. This approach introduced the principle of minimal frustration, which asserts that evolution has selected the amino acid sequences of natural proteins so that interactions between side chains largely favor the molecule's acquisition of the folded state. Interactions that do not favor folding are selected against, although some residual frustration is expected to exist. A consequence of these evolutionarily selected sequences is that proteins are generally thought to have globally "funneled energy landscapes" (coined by José Onuchic) that are largely directed towards the native state. This "folding funnel" landscape allows the protein to fold to the native state through any of a large number of pathways and intermediates, rather than being restricted to a single mechanism. The theory is supported by both computational simulations of model proteins and numerous experimental studies, and it has been used to improve methods for protein structure prediction and design.

Computational prediction of protein tertiary structure
De novo or ab initio techniques for computational protein structure prediction is related to, but strictly distinct from, studies involving protein folding. Molecular Dynamics (MD) is an important tool for studying protein folding and dynamics in silico. Because of computational cost, ab initio MD folding simulations with explicit water are limited to peptides and very small proteins. MD simulations of larger proteins remain restricted to dynamics of the experimental structure or its high-temperature unfolding. In order to simulate long time folding processes (beyond about 1 microsecond), like folding of small-size proteins (about 50 residues) or larger, some approximations or simplifications in protein models need to be introduced. An approach using reduced protein representation (pseudo-atoms representing groups of atoms are defined) and statistical potential is not only useful in protein structure prediction, but is also capable of reproducing the folding pathways.

Because of the many possible ways of folding, there can be many possible structures. A peptide consisting of just five amino acids can fold into over 100 billion possible structures.

Techniques for determination of protein structure

The determination of the folded structure of a protein is a lengthy and complicated process, involving methods like X-ray crystallography and NMR. One of the major areas of interest is the prediction of native structure from amino-acid sequences alone using bioinformatics and computational simulation methods.

There are distibuted computing projects which use idle CPU time of personal computers to solve problems such as protein folding or prediction of protein structure. People can run these programs on their computer or PlayStation 3 to support them. See links below (for example Folding@Home) to get information about how to participate in these projects.

Intracellular Infection by Salmonella

Many bacterial pathogens produce virulence factors that alter the host cell cytoskeleton to promote infection. Salmonella strains target cellular actin in a carefully orchestrated series of interactions that promote bacterial uptake into host cells and the subsequent proliferation and intercellular spread of the organisms. The Salmonella Pathogenicity Island 1 (SPI1) locus encodes a type III protein secretion system (TTSS) that translocates effector proteins into epithelial cells to promote bacterial invasion through actin cytoskeletal rearrangements.

SPI1 effectors interact directly with actin and also alter the cytoskeleton through activation of the regulatory proteins, Cdc42 and Rac, to produce membrane ruffles that engulf the bacteria. SPI1 also restores normal cellular actin dynamics through the action of another effector, SptP. A second TTSS, Salmonella Pathogenecity Island 2 (SPI2), translocates effectors that promote intracellular survival and growth, accompanied by focal actin polymerization around the Salmonella-containing vacuole (SCV). A number of Salmonella strains also carry the spv virulence locus, encoding an ADP-ribosyl transferase, the SpvB protein, which acts later during intracellular infection to depolymerize the actin cytoskeleton. SpvB produces a cytotoxic effect on infected host cells leading to apoptosis. The SpvB effect appears to promote intracellular infection and may facilitate cell-to-cell spread of the organism, thereby enhancing virulence

CYT387 Potent Dual Inhibitor of JAK 12 in Phase III Clinical Development

CYT387 is a novel oral JAK1/JAK2 inhibitor initially targeting the treatment of a group of hematological disorders known as myeloproliferative neoplasms (MPNs), also known as myeloproliferative disorders. Additional potential treatment indications for CYT387 include other MPNs, cancer (solid and liquid tumors), graft-vs-host disease, and inflammatory conditions.

CYT387 is a potent, selective JAK1/JAK2 inhibitor designed to suppress the over-activity of the mutant JAK2. The compound possesses an excellent selectivity and safety profile with minimal off-target activities, favorable pharmacokinetic properties, a clean toxicological profile and the prospect of limited drug/drug interactions. Preliminary data using samples derived from MPN patients have shown promising activity in suppressing the over-activity of the JAK2V617F mutant enzyme.

DNA sequencing

DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. The sequence of DNA constitutes the heritable genetic information in nuclei, plasmids, mitochondria, and chloroplasts that forms the basis for the developmental programs of all living organisms. Determining the DNA sequence is therefore useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic or forensic research. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attainable with modern DNA sequencing technology has been instrumental in the large-scale sequencing of the human genome, in the Human Genome Project. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences of many animal, plant, and microbial genome.

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chain-termination or Sanger method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into four separate sequencing reactions, containing the four standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). These dideoxynucleotides are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides during DNA strand elongation. Incorporation of a dideoxynucleotide into the nascent (elongating) DNA strand therefore terminates DNA strand extension, resulting in various DNA fragments of varying length. The dideoxynucleotides are added at lower concentration than the standard deoxynucleotides to allow strand elongation sufficient for sequence analysis.

The newly synthesized and labeled DNA fragments are heat denatured, and separated by size (with a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel. Each of the four DNA synthesis reactions is run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The terminal nucleotide base can be identified according to which dideoxynucleotide was added in the reaction giving that band. The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence as indicated.
There are some technical variations of chain-termination sequencing. In one method, the DNA fragments are tagged with nucleotides containing radioactive phosphorus for radiolabelling. Alternatively, a primer labeled at the 5’ end with a fluorescent dye is used for the tagging. Four separate reactions are still required, but DNA fragments with dye labels can be read using an optical system, facilitating faster and more economical analysis and automation. This approach is known as 'dye-primer sequencing'. The later development by L Hood and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.
The different chain-termination methods have greatly simplified the amount of work and planning needed for DNA sequencing. For example, the chain-termination-based "Sequenase" kit from USB Biochemicals contains most of the reagents needed for sequencing, prealiquoted and ready to use. Some sequencing problems can occur with the Sanger Method, such as non-specific binding of the primer to the DNA, affecting accurate read out of the DNA sequence. In addition, secondary structures within the DNA template, or contaminating RNA randomly priming at the DNA template can also affect the fidelity of the obtained sequence. Other contaminants affecting the reaction may consist of extraneous DNA or inhibitors of the DNA polymerase.
Dye-terminator sequencing
An alternative to primer labelling is labelling of the chain terminators, a method commonly called 'dye-terminator sequencing'. The major advantage of this method is that the sequencing can be performed in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with a different fluorescent dye, each fluorescing at a different wavelength. This method is attractive because of its greater expediency and speed and is now the mainstay in automated sequencing with computer-controlled sequence analyzers (see below). Its potential limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis (see figure to the right). This problem has largely been overcome with the introduction of new DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs", caused by certain chemical characteristics of the dyes that can result in artifacts in DNA sequence traces. The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects, as it is both easier to perform and lower in cost than most previous sequencing methods.

Action Potential

Action potential (also known as a nerve impulse or spike) is a pulse-like wave of voltage that travels along several types of cell membranes. The best-understood example is generated on the membrane of the axon of a neuron, but also appears in other types of excitable cells, such as cardiac muscle cells, and even plant cells. The resting voltage across the axonal membrane is typically −70 millivolts (mV), with the inside being more negative than the outside. As an action potential passes through a point, this voltage rises to roughly +40 mV in one millisecond, then returns to −70 mV. The action potential moves rapidly down the axon, with a conduction velocity as high as 100 meters/second (225 miles per hour). Because of this high speed, action potentials are used to transmit information, with this being particularly important in neurons, as these cells can be more than a meter long.

An action potential is provoked on a patch of membrane when the membrane is depolarized sufficiently strongly, i.e., when the voltage of the cell's interior relative to the cell's exterior is raised above a threshold. Such a depolarization opens voltage-sensitive channels, which allow positive current to flow into the axon, further depolarizing the membrane. This will cause the membrane to "fire", initiating a positive feedback loop that suddenly and rapidly causes the voltage inside the axon to become more positive. After this rapid rise, the membrane voltage is restored to its resting value by a combination of effects: the channels responsible for the initial inward current are inactivated, while the raised voltage opens other voltage-sensitive channels that allow a compensating outward current. Because of the positive feedback, an action potential is all-or-none; there are no partial action potentials. In neurons, a typical action potential lasts for just a few thousandths of a second at any given point along their length. The passage of an action potential can leave the ion channels in a non-equilibrium state, making them more difficult to open, and thus inhibiting another action potential at the same spot: such an axon is said to be refractory.

The principal ions involved in an action potential are sodium and potassium cations; sodium ions enter the cell, and potassium ions leave, restoring equilibrium. Relatively few ions need to cross the membrane for the membrane voltage to change drastically. The ions exchanged during an action potential, therefore, make a negligible change in the interior and exterior ionic concentrations. The few ions that do cross are pumped out again by the continual action of the sodium–potassium pump, which, with other ion transporters, maintains the normal ratio of ion concentrations across the membrane. Calcium cations and chloride anions are involved in a few types of action potentials, such as the cardiac action potential and the action potential in the single-celled alga Acetabularia, respectively..
The action potential "travels" along the axon without fading out because the signal is regenerated at each patch of membrane. This happens because an action potential at one patch raises the voltage at nearby patches, depolarizing them and provoking a new action potential there. In unmyelinated neurons, the patches are adjacent, but in myelinated neurons, the action potential "hops" between distant patches, making the process both faster and more efficient. The axons of neurons generally branch, and an action potential often travels along both forks from a branch point. The action potential stops at the end of these branches, but usually causes the secretion of neurotransmitters at the synapses that are found there. These neurotransmitters bind to receptors on adjacent cells. These receptors are themselves ion channels, although—in contrast to the axonal channels—they are generally opened by the presence of a neurotransmitter, rather than by changes in voltage. The opening of these receptor channels can help to depolarize the membrane of the new cell (an excitatory channel) or work against its depolarization (an inhibitory channel). If these depolarizations are sufficiently strong, they can provoke another action potential in the new cell.

Development of Fetus

A fetus (or foetus or fœtus) is a developing mammal or other viviparous vertebrate, after the embryonic stage and before birth. The plural is fetuses, or sometimes feti.
In humans, the fetal stage of prenatal development begins about eight weeks after fertilization, when the major structures and organ systems have formed, until birth.

The fetal stage begins eight weeks after fertilization. Miscarriage is much less likely at the beginning of the fetal stage.The fetus is not as sensitive to damage from environmental exposures as the embryo was, though toxic exposures can often cause physiological abnormalities or minor congenital malformation. Fetal growth can be terminated by various factors, including miscarriage, feticide committed by a third party, or induced abortion.

Development The following timeline describes some of the specific changes in fetal anatomy and physiology by fertilization age (i.e. the time elapsed since fertilization). Obstetricians often use "gestational age" which, by convention, is measured from 2 weeks earlier than fertilization. For purposes of this article, age is measured from fertilization, except as noted.

8 to 15 weeks The fetal stage commences at eight weeks when the fetus is typically about 30 mm (1.2 inches) in length from crown to rump and the head makes up nearly half of the fetus' size.. The fetus cannot feel pain, is not yet sentient, and moves involuntarily as tissues, organs and pathways begin to develop. The movements include motor patterns, and localized movement of the arms and legs, hiccups, stretches and yawns, sideward bendings of the head, and generalized movements that involve the whole body. These movements are involuntary, and the parts of the fetal brain that control movement will not fully form until late in the second trimester, and the first part of the third trimester. At this stage, the heart is beating but not functional.The hands, feet, head, brain, and other organs are present, but not yet functional. The breathing-like movement of the fetus is necessary for stimulation of lung development, rather than for obtaining oxygen. At nine weeks the fetus' involuntary movements include curling toes to move away from an object, and fingers are structurally able to bend. During weeks 9-12, the face is “well-formed,” though the fetal head is only one to three inches long. From weeks 9 to 12, the fetal eyelids close and remain closed for several months, and the appearance of the genitals in males and females becomes more apparent. Tooth buds appear, the limbs are long and thin, and red blood cells are produced in the liver, however the majority of red blood cells will be made later in gestation (at 21 weeks) by bone marrow. A fine hair called lanugo develops on the head. The gastrointestinal tract, still forming, starts to collect sloughed skin and lanugo, as well as hepatic products, forming meconium (stool). Fetal skin is almost transparent. More muscle tissue and bones have developed, and the bones become harder. The first measurable signs of EEG movement occur in the 12th week. By the end of this stage, the fetus has reached about 15 cm (6 inches). 16 to 25 weeks The lanugo covers the entire body. Eyebrows, eyelashes, fingernails, and toenails appear. The fetus has increased muscle development. Alveoli (air sacs) are forming in lungs. The nervous system develops enough to control some body functions. The cochlea are now developed, though the myelin sheaths in the neural portion of the auditory system will continue to develop until 18 months after birth. The respiratory system has developed to the point where gas exchange is possible. The quickening, the first maternally discernable fetal movements, are often felt during this period. A woman pregnant for the first time (i.e. a primiparous woman) typically feels fetal movements at about 18-19 weeks, whereas a woman who has already given birth at least two times (i.e. a multiparous woman) will typically feel movements around 16 weeks.[22] By the end of the fifth month, the fetus is about 20 cm (8 inches). 26 to 38 weeks The amount of body fat rapidly increases. Lungs are not fully mature. Thalamic brain connections, which mediate sensory input, form. Bones are fully developed, but are still soft and pliable. Iron, calcium, and phosphorus become more abundant. Fingernails reach the end of the fingertips. The lanugo begins to disappear, until it is gone except on the upper arms and shoulders. Small breast buds are present on both sexes. Head hair becomes coarse and thicker. Birth is imminent and occurs around the 38th week. The fetus is considered full-term between weeks 35 and 40,[23] which means that the fetus is considered sufficiently developed for life outside the uterus.[24] It may be 48 to 53 cm (19 to 21 inches) in length, when born.