Science,Technology,genetics,research on biotechnology

DNA replication

2010-04-02T13:39:00.000-07:00

DNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy fghtheir DNA. This process is "replication" in that each strand of the original double-stranded DNA molecule serves as template for the reproducfgtion of the complemensdftary strand. Hence, following DNA replication, two identical DNA moleculxcves have been produced from a single double-stranded DNA molecule. Cellular proofreading and error toe-checking mechanisms ensure near perfect fidelity for DNA replication.

In a cell, DNA replication begins at specific locations in the genome, called "origins". Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis.

DNA replication can also be performed in vitro (outside a cell). DNA polymerases, isolated from cells, 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 such artificial synthesis in a cyclic manner to amplify a specific target DNA fragment from a pool of DNA..

DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types of nucleotides: adenine, cytosine, guanine, and thymine. A nucleotide is a mono-, di- or triphosphate deoxyribonucleoside; that is, a deoxyribose sugar is attached to one, two or three phosphates . Chemical interaction of these nucleotides forms phosphodiester linkages, creating the phosphate-deoxribose backbone of the DNA double helix with the bases pointing inward. Nucleotides (bases) are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine and cytosine pairs with guanine.

DNA strands have a directionality, and the different ends of a single strand are called the "3' (three-prime) end" and the "5' (five-prime) end." These terms refer to the carbon atom in deoxyribose to which the next phosphate in the chain attaches. In addition to being complementary, the two strands of DNA are antiparallel: they are oriented in opposite directions. This directionality has consequences in DNA synthesis, because DNA polymerase can only synthesize DNA in one direction by adding nucleotides to the 3' end of a DNA strand.

The pairing of bases in DNA through hydrogen bonding means that the information contained within each strand is redundant. The nucleotides on a single strand can be used to reconstruct nucleotides on a newly synthesized partner strand.

DNA polymerases are a family of enzymes that carry out all forms of DNA replication. A DNA polymerase can only extend an existing DNA strand paired with a template strand; it cannot begin the synthesis of a new strand. To begin synthesis of a new strand, a short fragment of DNA or RNA, called a primer, must be created and paired with the template strand before DNA polymerase can synthesize new DNA.

Once a primer pairs with DNA to be replicated, DNA polymerase synthesizes a new strand of DNA by extending the 3' end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from two of the three total phosphates attached to each unincorporated base. (Free bases with their attached phosphate groups are called nucleoside triphosphates.) When a nucleotide is being added to a growing DNA strand, two of the phosphates are removed and the energy produced creates a phosphodiester (chemical) bond that attaches the remaining phosphate to the growing chain. The energetics of this process also help explain the directionality of synthesis - if DNA were synthesized in the 3' to 5' direction, the energy for the process would come from the 5' end of the growing strand rather than from free nucleotides.

DNA polymerases are generally extremely accurate, making less than one error for every 107 nucleotides added. Even so, some DNA polymerases also have proofreading ability; they can remove nucleotides from the end of a strand in order to correct mismatched bases. If the 5' nucleotide needs to be removed during proofreading, the triphosphate end is lost. Hence, the energy source that usually provides energy to add a new nucleotide is also lost.

For a cell to divide, it must first replicate its DNA. This process is initiated at particular points within the DNA, known as "origins", which are targeted by proteins that separate the two strands and initiate DNA synthesis. Origins contain DNA sequences recognized by replication initiator proteins (eg. dnaA in E coli' and the Origin Recognition Complex in yeast). These initiator proteins recruit other proteins to separate the two strands and initiate replication forks.

Initiator proteins recruit other proteins to separate the DNA strands at the origin, forming a bubble. Origins tend to be "AT-rich" (rich in adenine and thymine bases) to assist this process, because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair)—strands rich in these nucleotides are generally easier to separate due the positive relationship between the number of hydrogen bonds and the difficulty of breaking these bonds. Once strands are separated, RNA primers are created on the template strands. More specifically, the leading strand receives one RNA primer per active origin of replication while the lagging strand receives several; these several fragments of RNA primers found on the lagging strand of DNA are called Okazaki fragments, named after their discoverer. DNA polymerase extends the leading strand in one continuous motion and the lagging strand in a discontinuous motion (due to the Okazaki fragments). RNase removes the RNA fragments used to initiate replication by DNA Polymerase, and another DNA Polymerase enters to fill the gaps. When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. Ligase works to fill these nicks in, thus completing the newly replicated DNA molecule.

As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming 2 replication forks. In bacteria, which have a single origin of replication on their circular chromosome, this process eventually creates a "theta structure" (resembling the Greek letter theta: θ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.

When replicating, the original DNA splits in two, forming two "prongs" which resemble a fork (hence the name "replication fork"). DNA has a ladder-like structure; imagine a ladder broken in half vertically, along the steps. Each half of the ladder now requires a new half to match it. Because DNA polymerase can only synthesize a new DNA strand in a 5' to 3' manner, the process of replication goes differently for the two strands comprising the DNA double helix.

Broad-spectrum antibiotic

2009-12-17T10:17:00.001-08:00

The term broad-spectrum antibiotic refers to an antibiotic with activity against a wide range of disease-causing bacteria. It is also means that it acts against both Gram-positive and Gram-negative bacteria. This is in contrast to a narrow-spectrum antibiotic which is effective against only specific families of bacteria. A good example of a commonly used broad-spectrum antibiotic is levofloxacin.

Uses

Broad-spectrum antibiotics are properly used in the following medical situations:

* Empirically prior to identifying the causative bacteria when there is a wide differential and potentially serious illness would result in delay of treatment. This occurs, for example, in meningitis, where the patient can become so ill that he/she could die within hours if broad-spectrum antibiotics are not initiated.

* For drug resistant bacteria that do not respond to other, more narrow-spectrum antibiotics.

* In super-infections where there are multiple types of bacteria causing illness, thus warranting either a broad-spectrum antibiotic or combination antibiotic therapy.

Examples

In medicine:

* amoxycillin
* levofloxacin, gatifloxacin, moxifloxacin

In veterinary medicine, Co-amoxiclav, (in small animals); penicillin & streptomycin and oxytetracycline (in farm animals); penicillin and potentiated sulfonamides (in horses).

Others:

* streptomycin
* tetracycline
* chloramphenicol

Slightly-Broad:

* ampicillin

Drug resistance

2009-12-12T11:01:00.001-08:00

Drug resistance is the reduction in effectiveness of a drug in curing a disease or improving a patient's symptoms. When the drug is not intended to kill or inhibit a pathogen, then the term is equivalent to dosage failure or drug tolerance. More commonly, the term is used in the context of diseases caused by pathogens.

Pathogens are said to be drug-resistant when drugs meant to neutralize them have reduced effect. When an organism is resistant to more than one drug, it is said to be multidrug resistant.

Drug resistance is an example of evolution in microorganisms. Individuals that are not susceptible to the drug effects are capable of surviving drug treatment, and therefore have greater fitness than susceptible individuals. By the process of natural selection, drug resistant traits are selected for in subsequent offspring, resulting in a population that is drug resistant.

Classification

Drug resistance occurs in several classes of pathogens:

* bacteria—antibiotic resistance
* endoparasites
* viruses—resistance to antiviral drugs
* fungi
* cancer cells

Mechanisms

Sometimes the target molecule of the drug evolves so the drugs won't bind as well. Sometimes the target cells or organisms evolve better enzymes to degrade the drug, or evolve better mechanisms to pump the drug out of the target cells.

Metabolic price

Biological cost or metabolic price is a measure of the increased energy metabolism required to achieve a function.

Drug resistance has a high metabolic price, in pathogens for which this concept is relevant (bacteria, endoparasites, and tumor cells.) In viruses, an equivalent "cost" is genomic complexity.

Other Problems

Drug resistance not only causes metabolic problems but also results in issues concerning what more can be done to help the infected people and what better and more effective ways can be used without any further drug resistance. Respiratory infections, HIV/AIDS, diarrhoeal diseases, tuberculosis and malaria are the leading killers among infectious diseases to this date. Resistance to first-line drugs has been observed in all of these diseases. In some cases, the level of resistance has forced a change to more expensive second or third-line agents. When resistance against these drugs also emerges, the world will run out of treatment options until other options emerge.

Antibiotic misuse

2009-12-12T10:57:00.000-08:00

Antibiotic misuse, (sometimes called antibiotic abuse or antibiotic overuse) refers to the misuse and overuse of antibiotics which has serious effects on public health. Antibiotic resistant bacteria is a growing threat and becoming increasingly common. This overuse creates multi-antibiotic resistant life threatening infections by "super bugs", sometimes out of relatively harmless bacteria. Antibiotic abuse also places the patient at unnecessary risk of adverse effects of antibiotics.

Epidemiology

Within a recent study concerning the proper use of this class in the emergency room it was revealed that 99% of these prescriptions were in error. Out of the one hundred total patients studied, eighty one received a fluoroquinolone for an inappropriate indication. Out of these cases, forty three (53%) were judged to be inappropriate because another agent was considered first line, twenty seven (33%) because there was no evidence of a bacterial infection to begin with (based on the documented evaluation) and eleven (14%) because of the need for such therapy was questionable. Out of the nineteen patients who received a fluoroquinolone for an appropriate indication, only one patient out of one hundred received both the correct dose and duration of therapy.

Within a 1994 study it was found that 75% of the fluoroquinolone prescription issued within a long term care setting were judged to be inappropriate by the authors. In more than fifty percent of the cases reviewed, the fluoroquinolone used were not considered to be a first line agent.

Social and economic impact

Increased hospitalizations attributed to adverse drug reactions alone account for billions of dollars each year within the US healthcare system. Severe reactions do occur with antibiotics and can add significantly to the cost of care. Antibacterial adverse effects account for nearly 25% of all adverse drug reactions amongst hospitalized patients. Adverse drug reactions to fluoroquinolones are easily and likely often misdiagnosed as seizure disorder or regular CNS or psychiatric symptoms and the diagnosis of quinolone toxicity or adverse reaction missed. Adverse event reporting in Italy by doctors showed fluoroquinolones among the top 3 prescribed drugs for adverse neurological and psychiatric adverse effects. These neuropsychiatric effects included tremor, confusion, anxiety, insomnia, agitation and in severe cases psychosis. Moxifloxacin came out worst amongst the quinolones for causing CNS toxicity. The central nervous system is an important target for fluoroquinolone mediated neurotoxicity.

Antibiotic resistance

Though antibiotics are considered to be a very important and necessary drugs required to treat severe and life threatening bacterial infections, the associated antibiotic abuse, has contributed to the problem of bacterial resistance. The overuse of antibiotics such as happens with children suffering from otitis media for example has given rise to a breed of super bacteria which are resistant to antibiotics entirely.

The overuse of fluoroquinolone and other antibiotics will eventually result in them becoming useless for treating antibiotic-resistant infections, for which broad-spectrum antibiotics are supposed to be reserved.

The over-prescribing and inappropriate use of antibiotics is fueling antibiotic resistance in bacteria. For example the inapprorpiate wide spread use of fluoroquinolones as first line antibiotics is leading to decreased bacterial sensitivity which has important implications for certain serious bacterial infections such as those associated with cystic fibrosis where quinolones are among the few available antibiotics.

Inappropriate use

Only about 5-10% of bronchitis cases are caused by a bacterial infection. Antibiotics have no effect upon viral infections such as the common head cold. Most cases of bronchitis are caused by a viral infection and are "self-limited" and resolve themselves in a few weeks. The use of antibiotics such as ofloxacin to treat bronchitis is to be considered unnecessary and as such exposes the patient to an unacceptable risk of suffering a severe adverse reaction. Nor does antibiotic treatment help sore throats. Prescribing antibiotics for sore throats encourages increased visits to the doctor. As most cases of sore throats are viral and are self limiting it has been recommended that antibiotic treatment is delayed in most cases. Nevertheless, for severe forms of community-acquired pneumonia the fluoroquinolones seem to be associated with improved treatment rates, but with no differences found in mortality between antibiotic regimens. In spite of this caveat, the use of the fluoroquinolone to treat community acquired pnuemonia (CAP) increased by >50%, from 25% to 39% of all prescriptions. This increase was at the expense of the macrolide class of antimicrobial drugs, the use of which declined 20% during the study period.

As with other fluoroquinolones their use as first line agents is not generally recommended. They are usually reserved for use in patients who are seriously ill and may soon require immediate hospitalization. Though considered to be a very important and necessary drug required to treat severe and life threatening bacterial infections, the associated overprescribing of fluoroquinolones remains unchecked, which has contributed to the problem of bacterial resistance. The overuse of antibiotics such as happens with children suffering from otitis media has given rise to a breed of super bacteria which are resistant to antibiotics entirely. “Fluoroquinolone resistance is an increasing problem not only in the U.S. but also worldwide, potentially due to the widespread misuse of this class of antimicrobials.” For example the use of the fuoroquinolones had increased three-fold in an emergency room environment in the United States between 1995 and 2002, while the use of safer alternatives such as macrolides declined significantly.

Chronic pelvic pain (category IIIB) is often misdiagnosed as chronic prostatitis and needlessly treated with a fluoroquinolone drug. Within a Bulgarian study, where by definition all patients had negative microbiological results, 65% of patients experienced an adverse drug reaction who were treated with a fluoroquinolone in comparison to a 9% rate for the placebo patients. This was combined with a higher cure rate (69% v 53%) found within the placebo group. The authors stated that “The results of our study show that antibiotics have an unacceptably high rate of adverse side effects as well as a statistically insignificant improvement over placebo...” Prostatitis has been termed "the waste basket of clinical ignorance" by prominent Stanford University Urologist Dr. Thomas Stamey. Campbell's Urology, the urologist's most authoritative reference text, identifies only about 5% of all patients with prostatitis as having bacterial prostatitis which can be "cured" at least in the short term by antibiotics. In other words, 95% of men with prostatitis have little hope for a cure with antibiotics alone since they don't actually have any identifiable bacterial infection.

There are limited indications for ciprofloxacin as a first-line therapy within Long Term Care Facilities. Within a 1994 study it was found that 75% of the prescriptions for fluoroquinolones issued within a long term care setting were judged to be inappropriate by the authors. In more than fifty percent of the cases reviewed fluoroquinolones were not considered to be a first line agent.

Xenobiotic metabolism

2009-12-07T05:09:00.000-08:00

Xenobiotic metabolism is the set of metabolic pathways that modify the chemical structure of xenobiotics, which are compounds foreign to an organism's normal biochemistry, such as drugs and poisons. These pathways are a form of biotransformation present in all major groups of organisms, and are considered to be of ancient origin. These reactions often act to detoxify poisonous compounds; however, in some cases, the intermediates in xenobiotic metabolism can themselves be the cause of toxic effects.

Xenobiotic metabolism is divided into three phases. In phase I, enzymes such as cytochrome P450 oxidases introduce reactive or polar groups into xenobiotics. These modified compounds are then conjugated to polar compounds in phase II reactions. These reactions are catalysed by transferase enzymes such as glutathione S-transferases. Finally, in phase III, the conjugated xenobiotics may be further processed, before being recognised by efflux transporters and pumped out of cells.

The reactions in these pathways are of particular interest in medicine as part of drug metabolism and as a factor contributing to multidrug resistance in infectious diseases and cancer chemotherapy. The actions of some drugs as substrates or inhibitors of enzymes involved in xenobiotic metabolism are a common reason for hazardous drug interactions. These pathways are also important in environmental science, with the xenobiotic metabolism of microorganisms determining whether a pollutant will be broken down during bioremediation, or persist in the environment. The enzymes of xenobiotic metabolism, particularly the glutathione S-transferases are also important in agriculture, since they may produce resistance to pesticides and herbicides.

Permeability barriers and detoxification

That the exact compounds an organism is exposed to will be largely unpredictable, and may differ widely over time, is a major characteristic of xenobiotic toxic stress. The major challenge faced by xenobiotic detoxification systems is that they must be able to remove the almost-limitless number of xenobiotic compounds from the complex mixture of chemicals involved in normal metabolism. The solution that has evolved to address this problem is an elegant combination of physical barriers and low-specificity enzymatic systems.

All organisms use cell membranes as hydrophobic permeability barriers to control access to their internal environment. Polar compounds cannot diffuse across these cell membranes, and the uptake of useful molecules is mediated through transport proteins that specifically select substrates from the extracellular mixture. This selective uptake means that most hydrophilic molecules cannot enter cells, since they are not recognised by any specific transporters. In contrast, the diffusion of hydrophobic compounds across these barriers cannot be controlled, and organisms, therefore, cannot exclude lipid-soluble xenobiotics using membrane barriers.

However, the existence of a permeability barrier means that organisms were able to evolve detoxification systems that exploit the hydrophobicity common to membrane-permeable xenobiotics. These systems therefore solve the specificity problem by possessing such broad substrate specificities that they metabolise almost any non-polar compound. Useful metabolites are excluded since they are polar, and in general contain one or more charged groups.

The detoxification of the reactive by-products of normal metabolism cannot be achieved by the systems outlined above, because these species are derived from normal cellular constituents and usually share their polar characteristics. However, since these compounds are few in number, specific enzymes can recognize and remove them. Examples of these specific detoxification systems are the glyoxalase system, which removes the reactive aldehyde methylglyoxal, and the various antioxidant systems that eliminate reactive oxygen species.

Phases of detoxification

The metabolism of xenobiotics is often divided into three phases: modification, conjugation, and excretion. These reactions act in concert to detoxify xenobiotics and remove them from cells.
Phase I - modification

In phase I, a variety of enzymes acts to introduce reactive and polar groups into their substrates. One of the most common modifications is hydroxylation catalysed by the cytochrome P-450-dependent mixed-function oxidase system. These enzyme complexes act to incorporate an atom of oxygen into nonactivated hydrocarbons, which can result in either the introduction of hydroxyl groups or N-, O- and S-dealkylation of substrates. The reaction mechanism of the P-450 oxidases proceeds through the reduction of cytochrome-bound oxygen and the generation of a highly-reactive oxyferryl species, according to the following scheme:

\mbox{NADPH} + \mbox{H}^+ + \mbox{RH} \rightarrow \mbox{NADP}^+ + \mbox{H}_2\mbox{O} +\mbox{ROH} \,
Phase II - conjugation

In subsequent phase II reactions, these activated xenobiotic metabolites are conjugated with charged species such as glutathione (GSH), sulfate, glycine, or glucuronic acid. These reactions are catalysed by a large group of broad-specificity transferases, which in combination can metabolise almost any hydrophobic compound that contains nucleophilic or electrophilic groups. One of the most important of these groups are the glutathione S-transferases (GSTs). The addition of large anionic groups (such as GSH) detoxifies reactive electrophiles and produces more polar metabolites that cannot diffuse across membranes, and may, therefore, be actively transported.
Phase III - further modification and excretion

After phase II reactions, the xenobiotic conjugates may be further metabolised. A common example is the processing of glutathione conjugates to acetylcysteine (mercapturic acid) conjugates. Here, the γ-glutamate and glycine residues in the glutathione molecule are removed by Gamma-glutamyl transpeptidase and dipeptidases. In the final step, the cystine residue in the conjugate is acetylated.

Conjugates and their metabolites can be excreted from cells in phase III of their metabolism, with the anionic groups acting as affinity tags for a variety of membrane transporters of the multidrug resistance protein (MRP) family. These proteins are members of the family of ATP-binding cassette transporters and can catalyse the ATP-dependent transport of a huge variety of hydrophobic anions, and thus act to remove phase II products to the extracellular medium, where they may be further metabolised or excreted.

Endogenous toxins

The detoxification of endogenous reactive metabolites such as peroxides and reactive aldehydes often cannot be achieved by the system described above. This is the result of these species' being derived from normal cellular constituents and usually sharing their polar characteristics. However, since these compounds are few in number, it is possible for enzymatic systems to utilize specific molecular recognition to recognize and remove them. The similarity of these molecules to useful metabolites therefore means that different detoxification enzymes are usually required for the metabolism of each group of endogenous toxins. Examples of these specific detoxification systems are the glyoxalase system, which acts to dispose of the reactive aldehyde methylglyoxal, and the various antioxidant systems that remove reactive oxygen species.

History

Studies on how people transform the substances that they ingest began in the mid-nineteenth century, with chemists discovering that organic chemicals such as benzaldehyde could be oxidized and conjugated to amino acids in the human body. During the remainder of the nineteenth century, several other basic detoxification reactions were discovered, such as methylation, acetylation, and sulfonation.

In the early twentieth century, work moved on to the investigation of the enzymes and pathways that were responsible for the production of these metabolites. This field became defined as a separate area of study with the publication by Richard Williams of the book Detoxication mechanisms in 1947. This modern biochemical research resulted in the identification of glutathione S-transferases in 1961, followed by the discovery of cytochrome P450s in 1962, and the realization of their central role in xenobiotic metabolism in 1963

Antibiotic resistance

2009-12-07T05:07:00.000-08:00

Antibiotic resistance is a specific type of drug resistance when a microorganism has the ability of withstanding the effects of antibiotics. Antibiotic resistance evolves via natural selection acting upon random mutation, but it can also be engineered by applying an evolutionary stress on a population. Once such a gene is generated, bacteria can then transfer the genetic information in a horizontal fashion (between individuals) by plasmid exchange. If a bacterium carries several resistance genes, it is called multiresistant or, informally, a superbug. The term antimicrobial resistance is sometimes used to explicitly encompass organisms other than bacteria.

Antibiotic resistance can also be introduced artificially into a microorganism through transformation protocols. This can aid in implanting artificial genes into the microorganism. If the resistance gene is linked with the gene to be implanted, the antibiotic can be used to kill off organisms that lack the new gene.

Causes

The widespread use of antibiotics both inside and outside of medicine is playing a significant role in the emergence of resistant bacteria. They are often used in animals but also in other industries which at least in the case of agricultural use lead to the spread of resistant strains to human populations. In some countries antibiotics are sold over the counter without a prescription which compounds the problem. In human medicine the major problem of the emergence of resistant bacteria is due to misuse and overuse of antibiotics by doctors as well as patients. Other practices contributing towards resistance include the addition of antibiotics to the feed of livestock. Household use of antibacterials in soaps and other products, although not clearly contributing to resistance, is also discouraged (as not being effective at infection control). Also unsound practices in the pharmaceutical manufacturing industry can contribute towards the likelihood of creating antibiotic resistant strains.

Certain antibiotic classes are highly associated with colonisation with superbugs compared to other antibiotic classes. The risk for colonisation increases if there is a lack of sensitivity (resistance) of the superbugs to the antibiotic used and high tissue penetration as well as broad spectrum activity against "good bacteria". In the case of MRSA, increased rates of MRSA infections are seen with glycopeptides, cephalosporins and especially quinolones. In the case of colonisation with C difficile the high risk antibiotics include cephalosporins and in particular quinolones and clindamycin.
In medicine

The volume of antibiotic prescribed is the major factor in increasing rates or bacterial resistance rather than compliance with antibiotics. Inappropriate prescribing of antibiotics has been attributed to a number of causes including: people who insist on antibiotics, physicians simply prescribe them as they feel they do not have time to explain why they are not necessary, physicians who do not know when to prescribe antibiotics or else are overly cautious for medical legal reasons. A third of people for example believe that antibiotics are effective for the common cold and 22% of people do not finish a course of antibiotics primarily due to that fact that they feel better (varying from 10% to 44% depending on the country). Compliance with once daily antibiotics is better than with twice daily antibiotics. Sub optimum antibiotic concentrations in critically ill people increase the frequency of antibiotic resistance organisms. While taking antibiotics doses less than those recommended may increase rates of resistance, shortening the course of antibiotics may actually decrease rates of resistance.

Poor hand hygiene by hospital staff has been associated with the spread of resistant organisms and an increase in hand washing compliance results in decreased rates of these organisms.
Role of other animals

Drugs are used in animals that are used as human food, such as cows, pigs, chickens, fish, etc, and these drugs can affect the safety of the meat, milk, and eggs produced from those animals and can be the source of superbugs. For example, farm animals, particularly pigs, are believed to be able to infect people with MRSA. The resistant bacteria in animals due to antibiotic exposure can be transmitted to humans via three pathways, those being through the consumption of meat, from close or direct contact with animals, or through the environment.

The World Health Organization concluded that antibiotics as growth promoters in animal feeds should be prohibited (in the absence of risk assessments). In 1998, European Union health ministers voted to ban four antibiotics widely used to promote animal growth (despite their scientific panel's recommendations). Regulation banning the use of antibiotics in European feed, with the exception of two antibiotics in poultry feeds, became effective in 2006. In Scandinavia, there's evidence that the ban has led to a lower prevalence of antimicrobial resistance in (non-hazardous) animal bacterial populations. In the USA federal agencies do not collect data on antibiotic use in animals but animal to human spread of drug resistant organisms has been demonstrated in research studies. Antibiotics are still used in U.S. animal feed—along with other ingredients which have safety concerns.

Growing U.S. consumer concern about using antibiotics in animal feed has led to a niche market of "antibiotic-free" animal products, but this small market is unlikely to change entrenched industry-wide practices.

In 2001, the Union of Concerned Scientists estimated that greater than 70% of the antibiotics used in the US are given to food animals (e.g. chickens, pigs and cattle) in the absence of disease. In 2000 the US Food and Drug Administration (FDA) announced their intention to revoke approval of fluoroquinolone use in poultry production because of substantial evidence linking it to the emergence of fluoroquinolone resistant campylobacter infections in humans. The final decision to ban fluoroquinolones from use in poultry production was not made until five years later because of challenges from the food animal and pharmaceutical industries. Today, there are two federal bills (S. 549 and H.R. 962) aimed at phasing out "non-therapeutic" antibiotics in US food animal production.
Mechanisms
Schematic representation of how antibiotic resistance evolves via natural selection. The top section represents a population of bacteria before exposure to an antibiotic. The middle section shows the population directly after exposure, the phase in which selection took place. The last section shows the distribution of resistance in a new generation of bacteria. The legend indicates the resistance levels of individuals.

Researchers have recently demonstrated the bacterial protein LexA may play a key role in the acquisition of bacterial mutations.

Antibiotic resistance can be a result of horizontal gene transfer, and also of unlinked point mutations in the pathogen genome and a rate of about 1 in 108 per chromosomal replication. The antibiotic action against the pathogen can be seen as an environmental pressure; those bacteria which have a mutation allowing them to survive will live on to reproduce. They will then pass this trait to their offspring, which will result in a fully resistant colony.

The four main mechanisms by which microorganisms exhibit resistance to antimicrobials are:

1. Drug inactivation or modification: e.g. enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of β-lactamases.
2. Alteration of target site: e.g. alteration of PBP—the binding target site of penicillins—in MRSA and other penicillin-resistant bacteria.
3. Alteration of metabolic pathway: e.g. some sulfonamide-resistant bacteria do not require para-aminobenzoic acid (PABA), an important precursor for the synthesis of folic acid and nucleic acids in bacteria inhibited by sulfonamides. Instead, like mammalian cells, they turn to utilizing preformed folic acid.
4. Reduced drug accumulation: by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface.

There are three known mechanisms of fluoroquinolone resistance. Some types of efflux pumps can act to decrease intracellular quinolone concentration. In gram-negative bacteria, plasmid-mediated resistance genes produce proteins that can bind to DNA gyrase, protecting it from the action of quinolones. Finally, mutations at key sites in DNA gyrase or Topoisomerase IV can decrease their binding affinity to quinolones, decreasing the drug's effectiveness.
Resistant pathogens
Staphylococcus aureus

Staphylococcus aureus (colloquially known as "Staph aureus" or a Staph infection) is one of the major resistant pathogens. Found on the mucous membranes and the skin of around a third of the population, it is extremely adaptable to antibiotic pressure. It was the first bacterium in which penicillin resistance was found—in 1947, just four years after the drug started being mass-produced. Methicillin was then the antibiotic of choice, but has since been replaced by oxacillin due to significant kidney toxicity. MRSA (methicillin-resistant Staphylococcus aureus) was first detected in Britain in 1961 and is now "quite common" in hospitals. MRSA was responsible for 37% of fatal cases of blood poisoning in the UK in 1999, up from 4% in 1991. Half of all S. aureus infections in the US are resistant to penicillin, methicillin, tetracycline and erythromycin.

This left vancomycin as the only effective agent available at the time. However, strains with intermediate (4-8 ug/ml) levels of resistance, termed GISA (glycopeptide intermediate Staphylococcus aureus) or VISA (vancomycin intermediate Staphylococcus aureus), began appearing in the late 1990s. The first identified case was in Japan in 1996, and strains have since been found in hospitals in England, France and the US. The first documented strain with complete (>16 ug/ml) resistance to vancomycin, termed VRSA (Vancomycin-resistant Staphylococcus aureus) appeared in the United States in 2002.

A new class of antibiotics, oxazolidinones, became available in the 1990s, and the first commercially available oxazolidinone, linezolid, is comparable to vancomycin in effectiveness against MRSA. Linezolid-resistance in Staphylococcus aureus was reported in 2003.

CA-MRSA (Community-acquired MRSA) has now emerged as an epidemic that is responsible for rapidly progressive, fatal diseases including necrotizing pneumonia, severe sepsis and necrotizing fasciitis. Methicillin-resistant Staphylococcus aureus (MRSA) is the most frequently identified antimicrobial drug-resistant pathogen in US hospitals. The epidemiology of infections caused by MRSA is rapidly changing. In the past 10 years, infections caused by this organism have emerged in the community. The 2 MRSA clones in the United States most closely associated with community outbreaks, USA400 (MW2 strain, ST1 lineage) and USA300, often contain Panton-Valentine leukocidin (PVL) genes and, more frequently, have been associated with skin and soft tissue infections. Outbreaks of community-associated (CA)-MRSA infections have been reported in correctional facilities, among athletic teams, among military recruits, in newborn nurseries, and among men who engage in frequent homosexual activities. CA-MRSA infections now appear to be endemic in many urban regions and cause most CA-S. aureus infections.
Streptococcus and Enterococcus

Streptococcus pyogenes (Group A Streptococcus: GAS) infections can usually be treated with many different antibiotics. Early treatment may reduce the risk of death from invasive group A streptococcal disease. However, even the best medical care does not prevent death in every case. For those with very severe illness, supportive care in an intensive care unit may be needed. For persons with necrotizing fasciitis, surgery often is needed to remove damaged tissue. Strains of S. pyogenes resistant to macrolide antibiotics have emerged, however all strains remain uniformly sensitive to penicillin.

Resistance of Streptococcus pneumoniae to penicillin and other beta-lactams is increasing worldwide. The major mechanism of resistance involves the introduction of mutations in genes encoding penicillin-binding proteins. Selective pressure is thought to play an important role, and use of beta-lactam antibiotics has been implicated as a risk factor for infection and colonization. Streptococcus pneumoniae is responsible for pneumonia, bacteremia, otitis media, meningitis, sinusitis, peritonitis and arthritis.

Penicillin-resistant pneumonia caused by Streptococcus pneumoniae (commonly known as pneumococcus), was first detected in 1967, as was penicillin-resistant gonorrhea. Resistance to penicillin substitutes is also known as beyond S. aureus. By 1993 Escherichia coli was resistant to five fluoroquinolone variants. Mycobacterium tuberculosis is commonly resistant to isoniazid and rifampin and sometimes universally resistant to the common treatments. Other pathogens showing some resistance include Salmonella, Campylobacter, and Streptococci.

Enterococcus faecium is another superbug found in hospitals. Penicillin-Resistant Enterococcus was seen in 1983, vancomycin-resistant enterococcus (VRE) in 1987, and Linezolid-Resistant Enterococcus (LRE) in the late 1990s.
Pseudomonas aeruginosa

Pseudomonas aeruginosa is a highly prevalent opportunistic pathogen. One of the most worrisome characteristics of P. aeruginosa consists in its low antibiotic susceptibility. This low susceptibility is attributable to a concerted action of multidrug efflux pumps with chromosomally-encoded antibiotic resistance genes (e.g. mexAB-oprM, mexXY etc) and the low permeability of the bacterial cellular envelopes. Besides intrinsic resistance, P. aeruginosa easily develop acquired resistance either by mutation in chromosomally-encoded genes, or by the horizontal gene transfer of antibiotic resistance determinants. Development of multidrug resistance by P. aeruginosa isolates requires several different genetic events that include acquisition of different mutations and/or horizontal transfer of antibiotic resistance genes. Hypermutation favours the selection of mutation-driven antibiotic resistance in P. aeruginosa strains producing chronic infections, whereas the clustering of several different antibiotic resistance genes in integrons favours the concerted acquisition of antibiotic resistance determinants. Some recent studies have shown that phenotypic resistance associated to biofilm formation or to the emergence of small-colony-variants may be important in the response of P. aeruginosa populations to antibiotics treatment.
Clostridium difficile

Clostridium difficile is a nosocomial pathogen that causes diarrheal disease in hospitals world wide. Clindamycin-resistant C. difficile was reported as the causative agent of large outbreaks of diarrheal disease in hospitals in New York, Arizona, Florida and Massachusetts between 1989 and 1992. Geographically dispersed outbreaks of C. difficile strains resistant to fluoroquinolone antibiotics, such as Cipro (ciprofloxacin) and Levaquin (levofloxacin), were also reported in North America in 2005.
Salmonella and E. coli

E. coli and Salmonella come directly from contaminated food. Of the meat that is contaminated with E. coli, eighty percent of the bacteria are resistant to one or more drugs made; it causes bladder infections that are resistant to antibiotics (“HSUS Fact Sheet”). Salmonella was first found in humans in the 1970s and in some cases is resistant to as many as nine different antibiotics (“HSUS Fact Sheet”). When both bacterium are spread, serious health conditions arise. Many people are hospitalized each year after becoming infected, and some die as a result.
Acinetobacter baumannii

On November 5, 2004, the Centers for Disease Control and Prevention (CDC) reported an increasing number of Acinetobacter baumannii bloodstream infections in patients at military medical facilities in which service members injured in the Iraq/Kuwait region during Operation Iraqi Freedom and in Afghanistan during Operation Enduring Freedom were treated. Most of these showed multidrug resistance (MRAB), with a few isolates resistant to all drugs tested.
Alternatives
Prevention

Rational use of antibiotics may reduce the chances of development of opportunistic infection by antibiotic-resistant bacteria due to dysbacteriosis. In one study the use of fluoroquinolones are clearly associated with Clostridium difficile infection, which is a leading cause of nosocomial diarrhea in the United States, and a major cause of death, worldwide.

There is clinical evidence that topical dermatological preparations containing tea tree oil and thyme oil may be effective in preventing transmittal of CA-MRSA.

Vaccines do not suffer the problem of resistance because a vaccine enhances the body's natural defenses, while an antibiotic operates separately from the body's normal defenses. Nevertheless, new strains may evolve that escape immunity induced by vaccines.

While theoretically promising, anti-staphylococcal vaccines have shown limited efficacy, because of immunological variation between Staphylococcus species, and the limited duration of effectiveness of the antibodies produced. Development and testing of more effective vaccines is under way.

The Australian Commonwealth Scientific and Industrial Research Organization (CSIRO), realizing the need for the reduction of antibiotic use, has been working on two alternatives. One alternative is to prevent diseases by adding cytokines instead of antibiotics to animal feed. These proteins are made in the animal body "naturally" after a disease and are not antibiotics so they do not contribute to the antibiotic resistance problem. Furthermore, studies on using cytokines have shown that they also enhance the growth of animals like the antibiotics now used, but without the drawbacks of non-therapeutic antibiotic use. Cytokines have the potential to achieve the animal growth rates traditionally sought by the use of antibiotics without the contribution of antibiotic resistance associated with the widespread non-therapeutic uses of antibiotics currently utilized in the food animal production industries. Additionally, CSIRO is working on vaccines for diseases.
Phage therapy

Phage therapy, an approach that has been extensively researched and utilized as a therapeutic agent for over 60 years, especially in the Soviet Union, is an alternative that might help with the problem of resistance. Phage therapy was widely used in the United States until the discovery of antibiotics, in the early 1940s. Bacteriophages or "phages" are viruses that invade bacterial cells and, in the case of lytic phages, disrupt bacterial metabolism and cause the bacterium to lyse. Phage therapy is the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections.

Bacteriophage therapy is an important alternative to antibiotics in the current era of multidrug resistant pathogens. A review of studies that dealt with the therapeutic use of phages from 1966–1996 and few latest ongoing phage therapy projects via internet showed: phages were used topically, orally or systemically in Polish and Soviet studies. The success rate found in these studies was 80–95% with few gastrointestinal or allergic side effects. British studies also demonstrated significant efficacy of phages against Escherichia coli, Acinetobacter spp., Pseudomonas spp and Staphylococcus aureus. US studies dealt with improving the bioavailability of phage. Phage therapy may prove as an important alternative to antibiotics for treating multidrug resistant pathogens.
Research
New medications

Until recently, research and development (R&D) efforts have provided new drugs in time to treat bacteria that became resistant to older antibiotics. That is no longer the case. The potential crisis at hand is the result of a marked decrease in industry R&D, and the increasing prevalence of resistant bacteria. Infectious disease physicians are alarmed by the prospect that effective antibiotics may not be available to treat seriously ill patients in the near future.

The pipeline of new antibiotics is drying up. Major pharmaceutical companies are losing interest in the antibiotics market because these drugs may not be as profitable as drugs that treat chronic (long-term) conditions and lifestyle issues.

The resistance problem demands that a renewed effort be made to seek antibacterial agents effective against pathogenic bacteria resistant to current antibiotics. One of the possible strategies towards this objective is the rational localization of bioactive phytochemicals. Plants have an almost limitless ability to synthesize aromatic substances, most of which are phenols or their oxygen-substituted derivatives such as tannins. Most are secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be less than 10% of the total. In many cases, these substances serve as plant defense mechanisms against predation by microorganisms, insects, and herbivores. Many of the herbs and spices used by humans to season food yield useful medicinal compounds including those having antibacterial activity.

Traditional healers have long used plants to prevent or cure infectious conditions. Many of these plants have been investigated scientifically for antimicrobial activity and a large number of plant products have been shown to inhibit growth of pathogenic bacteria. A number of these agents appear to have structures and modes of action that are distinct from those of the antibiotics in current use, suggesting that cross-resistance with agents already in use may be minimal. For example the combination of 5'-methoxyhydnocarpine and berberine in herbs like Hydrastis canadensis and Berberis vulgaris can block the MDR-pumps that cause multidrug resistance. This has been shown for Staphylococcus aureus.

Archaeocins is the name given to a new class of potentially useful antibiotics that are derived from the Archaea group of organisms. Eight archaeocins have been partially or fully characterized, but hundreds of archaeocins are believed to exist, especially within the haloarchaea. The prevalence of archaeocins is unknown simply because no one has looked for them. The discovery of new archaeocins hinges on recovery and cultivation of archaeal organisms from the environment. For example, samples from a novel hypersaline field site, Wilson Hot Springs, recovered 350 halophilic organisms; preliminary analysis of 75 isolates showed that 48 were archaeal and 27 were bacterial.

In research published on October 17, 2008 in Cell, a team of scientists pinpointed the place on bacteria where the antibiotic myxopyronin launches its attack, and why that attack is successful. The myxopyronin binds to and inhibits the crucial bacterial enzyme, RNA polymerase. The myxopyronin changes the structure of the switch-2 segment of the enzyme, inhibiting its function of reading and transmitting DNA code. This prevents RNA polymerase from delivering genetic information to the ribosomes, causing the bacteria to die.

One of the major causes of antibiotic resistance is the decrease of effective drug concentrations at their target place, due to the increased action of ABC transporters. Since ABC transporter blockers can be used in combination with current drugs to increase their effective intracellular concentration, the possible impact of ABC transporter inhibitors is of great clinical interest. ABC transporter blockers that may be useful to increase the efficacy of current drugs have entered clinical trials and are available to be used in therapeutic regimes.
Applications

Antibiotic resistance is an important tool for genetic engineering. By constructing a plasmid which contains an antibiotic resistance gene as well as the gene being engineered or expressed, a researcher can ensure that when bacteria replicate, only the copies which carry along the plasmid survive. This ensures that the gene being manipulated passes along when the bacteria replicates.

The most commonly used antibiotics in genetic engineering are generally "older" antibiotics which have largely fallen out of use in clinical practice. These include:

* ampicillin
* kanamycin
* tetracycline
* chloramphenicol

Industrially the use of antibiotic resistance is disfavored since maintaining bacterial cultures would require feeding them large quantities of antibiotics. Instead, the use of auxotrophic bacterial strains (and function-replacement plasmids) is preferred.

Antibiotic

2009-11-01T23:05:00.000-08:00

The term "antibiotic" was coined by Selman Waksman in 1942 to describe any substance produced by a microorganism that is antagonistic to the growth of other microorganisms in high dilution. This original definition excluded naturally occurring substances that kill bacteria but are not produced by microorganisms (such as gastric juice and hydrogen peroxide) and also excluded synthetic antibacterial compounds such as the sulfonamides. Many antibiotics are relatively small molecules with a molecular weight less than 2000 Da.

With advances in medicinal chemistry, most antibiotics are now semisynthetic—modified chemically from original compounds found in nature, as is the case with beta-lactams (which include the penicillins, produced by fungi in the genus Penicillium, the cephalosporins, and the carbapenems). Some antibiotics are still produced and isolated from living organisms, such as the aminoglycosides, and others have been created through purely synthetic means: the sulfonamides, the quinolones, and the oxazolidinones. In addition to this origin-based classification into natural, semisynthetic, and synthetic, antibiotics may be divided into two broad groups according to their effect on microorganisms: those that kill bacteria are bactericidal agents, while those that only impair bacterial growth are known as bacteriostatic agents.

History of antibiotics

Many cures for infectious diseases prior to the beginning of the twentieth century were based on medicinal folklore. Cures for infection in ancient Chinese medicine using plants with antibiotic-like properties began to be described over 2,500 years ago. Many other ancient cultures, including the ancient Egyptians, ancient Greeks and medieval Arabs already used molds and plants to treat infections. Cinchona bark was a widely effective treatment of malaria in the 17th century, the disease caused by protozoan parasites of the genus Plasmodium. Scientific endeavours to understand the science behind what caused these diseases, the development of synthetic antibiotic chemotherapy, the isolation of the natural antibiotics marked milestones in antibiotic development.

Originally known as antiobiosis, antibiotics were drugs that had actions against bacteria. The term antibiosis which means ‘against life’ was introduced by the French bacteriologist Vuillemin as a descriptive name of the phenomenon exhibited by these drugs. (Antibiosis was first described in 1877 in bacteria when Louis Pasteur and Robert Koch observed that an airborne bacillus could inhibit the growth of Bacillus anthracis.). These drugs were later renamed antibiotics by Selman Wakeman, an American microbiologist in 1942.

Synthetic antibiotic chemotherapy as a science and the story of antibiotic development began in Germany with Paul Ehrlich, a German medical scientist in the late 1880s. Dr. Ehrlich noted that certain dyes would bind to and color human, animal or bacterial cells, while others did not. He then extended the idea that it might be possible to make certain dyes or chemicals that would act as a magic bullet or selective drug that would bind to and kill bacteria while not harming the human host. After much experimentation, screening hundreds of dyes against various organisms, he discovered the first medicinally useful drug, the man-made antibiotic, Salvarsan.However, the adverse side-effect profile of salvarsan, coupled with the later discovery of the antibiotic penicillin, superseded its use as an antibiotic. The work of Ehrlich, which marked the birth of the antibiotic revolution, was followed by the discovery of Prontosil by Domagk in 1932. Prontosil, the first commercially available antibacterial antibiotic was developed by a research team led by Gerhard Domagk (who received the 1939 Nobel Prize for Medicine for his efforts) at the Bayer Laboratories of the IG Farben conglomerate in Germany. Prontosil had a relatively broad effect against Gram-positive cocci but not against enterobacteria. The discovery and development of this first sulfonamide drug opened the era of antibiotics.

The discovery of natural antibiotics produced by microorganisms stemmed from earlier work on the observation of antibiosis between micro-organisms. Pasteur observed that "if we could intervene in the antagonism observed between some bacteria, it would offer ‘perhaps the greatest hopes for therapeutics’". Bacterial antagonism of Penicillium sp. were first described in England by John Tyndall in 1875. However, his work went by without much notice from the scientific community until Alexander Fleming's discovery of Penicillin in 1928. Even then the therapeutic potential of penicillin was not pursued. More than ten years later, Ernst Chain and Howard Florey became interested in Fleming's work, and came up with the purified form of penicillin. The purified antibiotic displayed antibacterial activity against a wide range of bacteria. It also had low toxicity and could be taken without causing adverse effects. Furthermore its activity was not inhibited by biological constituents such as pus, unlike the sulfonamides. At the time, no-one had discovered a compound equalling this activity. The discovery of penicillin led to renewed interest in the search for antibiotic compounds with similar capabilities. Because of their discovery of penicillin Ernst Chain, Howard Florey and Alexander Fleming shared the 1945 Nobel Prize in Medicine. In 1939, Rene Dubos isolated gramicidin, one of the first commercially manufactured antibiotics in use during World War II to prove highly effective in treating wounds and ulcers. Florey credited Dubos for reviving his research in penicillin.

Antimicrobial pharmacodynamics

The environment of individual antibiotics varies with the location of an infection, the ability of the antibiotic to reach the infection site, and the ability of the microbe to inactivate or excrete the antibiotic. At the highest level, antibiotics can be classified as either bactericidal or bacteriostatic. Bactericidals kill bacteria directly where bacteriostatics prevent cell division. However, these classifications are based on laboratory behavior; in practice, both of these are capable of ending a bacterial infection. The bactericidal activity of antibiotics may be growth phase dependent and in most but not all cases the action of many bactericidal antibiotics requires ongoing cell activity and cell division for the drugs' killing activity. The minimum inhibitory concentration and minimum bactericidal concentration are used to measure in vitro activity of an antimicrobial and are excellent indicators of antimicrobial potency. However, in clinical practice, these measurements alone are insufficient to predict clinical outcome. By combining the pharmacokinetic profile of an antibiotic with the antimicrobial activity, several pharmacological parameters appear to be significant markers of drug efficacy. The activity of antibiotics may be concentration-dependent and their characteristic antimicrobial activity increases with progressively higher antibiotic concentrations. They may also be time-dependent, where their antimicrobial activity does not increase with increasing antibiotic concentrations; however, it is critical that a minimum inhibitory serum concentration is maintained for a certain length of time.

Administration

Oral antibiotics are simply ingested, while intravenous antibiotics are used in more serious cases, such as deep-seated systemic infections. Antibiotics may also sometimes be administered topically, as with eye drops or ointments.

Antibiotic classes

Unlike many previous treatments for infections, which often consisted of administering chemical compounds such as strychnine and arsenic, which also have high toxicity against mammals, most antibiotics from microbes have fewer side-effects and high effective target activity. Most anti-bacterial antibiotics do not have activity against viruses, fungi, or other microbes. Anti-bacterial antibiotics can be categorized based on their target specificity: "narrow-spectrum" antibiotics target particular types of bacteria, such as Gram-negative or Gram-positive bacteria, while broad-spectrum antibiotics affect a wide range of bacteria.

Antibiotics which target the bacterial cell wall (penicillins, cephalosporins), or cell membrane (polymixins), or interfere with essential bacterial enzymes (quinolones, sulfonamides) usually are bactericidal in nature. Those which target protein synthesis, such as the aminoglycosides, macrolides and tetracyclines, are usually bacteriostatic.

In the last few years three new classes of antibiotics have been brought into clinical use. This follows a 40-year hiatus in discovering new classes of antibiotic compounds. These new antibiotics are of the following three classes: cyclic lipopeptides (daptomycin), glycylcyclines (tigecycline), and oxazolidinones (linezolid). Tigecycline is a broad-spectrum antibiotic, while the two others are used for Gram-positive infections. These developments show promise as a means to counteract the bacterial resistance to existing antibiotics.

Production

Since the first pioneering efforts of Florey and Chain in 1939, the importance of antibiotics to medicine has led to much research into discovering and producing them. The process of production usually involves the screening of wide ranges of microorganisms, and their testing and modification. Production is carried out using fermentation, usually in strongly aerobic form.

Side effects

Although antibiotics are generally considered safe and well tolerated, they have been associated with a wide range of adverse effects. Side effects are many, varied and can be very serious depending on the antibiotics used and the microbial organisms targeted. The safety profiles of newer medications may not be as well established as those that have been in use for many years. Adverse effects can range from fever and nausea to major allergic reactions including photodermatitis. One of the more common side effects is diarrhea, sometimes caused by the anaerobic bacterium Clostridium difficile, which results from the antibiotic disrupting the normal balance of the intestinal flora, Such overgrowth of pathogenic bacteria may be alleviated by ingesting probiotics during a course of antibiotics.. An antibiotic-induced disruption of the population of the bacteria normally present as constituents of the normal vaginal flora may also occur, and may lead to overgrowth of yeast species of the genus Candida in the vulvo-vaginal area. Other side effects can result from interaction with other drugs, such as elevated risk of tendon damage from administration of a quinolone antibiotic with a systemic corticosteroid.

Drug-Drug interactions

Contraceptive pill

Hypothetically, interference of some antibiotics with the efficiency of birth control pills is thought to occur in two ways. Modification of the intestinal gut flora resulting in the reduced absorption of the estrogens and induction of hepatic liver enzymes which metabolise the pills active ingredients faster may affect the pill's usefulness. However, the majority of studies indicate that antibiotics do not interfere with contraception, even though a small percentage of women may experience decreased effectiveness of birth control pills while taking an antibiotic the failure rate is comparible to those taking the pill.Moreover, there have been no studies that have conclusively demonstrated that disruption of the gut flora affects contraception. Interaction with the combined oral contraceptive pill through induction of hepatic enzymes by the antifungal medication griseofulvin and the broad-spectrum antibiotic rifampicin has been shown to occur. It is recommended that extra contraceptive measures are applied during antimicrobial therapy using these antimicrobials.

Alcohol

Alcohol can interfere with the activity or metabolization of antibiotics. It may affect the activity of liver enzymes, which break down the antibiotics. Moreover, certain antibiotics, including metronidazole, tinidazole, co-trimoxazole, cephamandole, ketoconazole, latamoxef, cefoperazone, amoxicillin, cefmenoxime, and furazolidone, chemically react with alcohol, leading to serious side effects, which include severe vomiting, nausea, and shortness of breath. Alcohol consumption while taking such antibiotics is therefore not recommended. Additionally, serum levels of doxycycline and erythromycin succinate may, in certain circumstances, be significantly reduced by alcohol consumption.

Antibiotic resistance

The emergence of antibiotic resistance is an evolutionary process that is based on selection for organisms that have enhanced ability to survive doses of antibiotics that would have previously been lethal. Antibiotics like Penicillin and Erythromycin which used to be one-time miracle cures are now less effective because bacteria have become more resistant. Antibiotics themselves act as a selective pressure which allows the growth of resistant bacteria within a population and inhibits susceptible bacteria. Antibiotic selection of pre-existing antibiotic resistant mutants within bacterial populations was demonstrated in 1943 by the Luria-Delbrück experiment. Survival of bacteria often results from an inheritable resistance. Any antibiotic resistance may impose a biological cost and the spread of antibiotic resistant bacteria may be hampered by the reduced fitness associated with the resistance which proves disadvantageous for survival of the bacteria when antibiotic is not present. Additional mutations, however, may compensate for this fitness cost and aids the survival of these bacteria.

The underlying molecular mechanisms leading to antibiotic resistance can vary. Intrinsic resistance may naturally occur as a result of the bacteria's genetic makeup. The bacterial chromosome may fail to encode a protein which the antibiotic targets. Acquired resistance results from a mutation in the bacterial chromosome or the acquisition of extra-chromosomal DNA. Antibiotic-producing bacteria have evolved resistance mechanisms which have been shown to be similar to and may have been transferred to antibiotic resistant strains. The spread of antibiotic resistance mechanisms occurs through vertical transmission of inherited mutations from previous generations and genetic recombination of DNA by horizontal genetic exchange. Antibiotic resistance exchanged between different bacteria by plasmids that carry genes which encode antibiotic resistance which may result in co-resistance to multiple antibiotics. These plasmids can carry different genes with diverse resistance mechanisms to unrelated antibiotics but because they are located on the same plasmid multiple antibiotic resistance to more than one antibiotic is transferred. Alternatively, cross-resistance to other antibiotics within the bacteria results when the same resistance mechanism is responsible for resistance to more than one antibiotic is selected for.

Antibiotic misuse

The first rule of antibiotics is try not to use them, and the second rule is try not to use too many of them.
—Paul L. Marino, The ICU Book

Inappropriate antibiotic treatment and overuse of antibiotics have been a contributing factor to the emergence of resistant bacteria. The problem is further exacerbated by self-prescribing of antibiotics by individuals without the guidelines of a qualified clinician and the non-therapeutic use of antibiotics as growth promoters in agriculture. Antibiotics are frequently prescribed for indications in which their use is not warranted, an incorrect or sub-optimal antibiotic is prescribed or in some cases for infections likely to resolve without treatment.

Several organizations concerned with antimicrobial resistance are lobbying to improve the regulatory climate. Approaches to tackling the issues of misuse and overuse of antibiotics by the establishment of the U.S. Interagency Task Force on Antimicrobial Resistance which aims actively address the problem antimicrobial resistance are being organised and coordinated by the US Centers for Disease Control and Prevention, the Food and Drug Administration (FDA), and the National Institutes of Health (NIH) and also includes several other federal agencies. An NGO campaign group is Keep Antibiotics Working. In France, an "Antibiotics are not automatic" government campaign starting in 2002 led to a marked reduction of unnecessary antibiotic prescriptions, especially in children.

The overuse of antibiotics like penicillin and erythromycin which used to be one-time miracle cures were associated with emerging resistance since the 1950s. Therapeutic usage of antibiotics in hospitals has been seen to be associated with increases in multi-antibiotic resistant bacteria

Common forms of antibiotic misuse include failure to take into account the patient's weight and history of prior antibiotic use when prescribing, since both can strongly affect the efficacy of an antibiotic prescription, failure to take the entire prescribed course of the antibiotic, failure to prescribe or take the course of treatment at fairly precise correct daily intervals (e.g. "every 8 hours" rather than merely "3x per day"), or failure to rest for sufficient recovery to allow clearance of the infecting organism. These practices may facilitate the development of bacterial populations with antibiotic resistance. Inappropriate antibiotic treatment is another common form of antibiotic misuse. A common example is the prescription and use of antibiotics to treat viral infections such as the common cold that have no effect.

In agriculture, associated antibiotic resistance with the non-therapeutic use of antibiotics as growth promoters in animals resulted in their restricted use in the UK in the 1970 (Swann report 1969). Currently there is a EU wide ban on the non-therapeutic use of antibiotics as growth promoters. It is estimated that greater than 70% of the antibiotics used in U.S. are given to feed animals (e.g. chickens, pigs and cattle) in the absence of disease. Antibiotic use in food animal production has been associated with the emergence of antibiotic-resistant strains of bacteria including Salmonella spp., Campylobacter spp., Escherichia coli, and Enterococcus spp. Evidence from some US and European studies suggest that these resistant bacteria cause infections in humans that do not respond to commonly prescribed antibiotics. In response to these practices and attendant problems, several organizations (e.g. The American Society for Microbiology (ASM), American Public Health Association (APHA) and the American Medical Association (AMA)) have called for restrictions on antibiotic use in food animal production and an end to all non-therapeutic uses.However, delays in regulatory and legislative actions to limit the use of antibiotics are common, and may include resistance to these changes by industries using or selling antibiotics, as well as time spent on research to establish causal links between antibiotic use and emergence of untreatable bacterial diseases. Two federal bills (S.742 and H.R. 2562) aimed at phasing out non-therapeutic antibiotics in US food animal production were proposed but not passed. These bills were endorsed by public health and medical organizations including the American Holistic Nurses’ Association, the American Medical Association, and the American Public Health Association (APHA). The EU has banned the use of antibiotics as growth promotional agents since 2003.

One study on respiratory tract infections found "physicians were more likely to prescribe antibiotics to patients who they believed expected them, although they correctly identified only about 1 in 4 of those patients". Multifactorial interventions aimed at both physicians and patients can reduce inappropriate prescribing of antibiotics. Delaying antibiotics for 48 hours while observing for spontaneous resolution of respiratory tract infections may reduce antibiotic usage; however, this strategy may reduce patient satisfaction.

Excessive use of prophylactic antibiotics in travelers may also be classified as misuse.

In the United Kingdom, there are NHS posters in many doctors surgeries indicating that 'unfortunately, no amount of antibiotics will get rid of your cold', following on from many patients specifically requesting antibiotics from their doctor inappropriately, believing they will help treat viral infections.

Resistance modifying agents

One solution to combat resistance currently being researched is the development of pharmaceutical compounds that would revert multiple antibiotic resistance. These so called resistance modifying agents may target and inhibit MDR mechanisms, rendering the bacteria susceptible to antibiotics to which they were previously resistant. These compounds targets include among others

* Efflux inhibition(Phe-Arg-β-naphthylamide)
* Beta Lactamase inhibitors - Including Clavulanic acid and Sulbactam

Beyond antibiotics

The comparative ease of identifying compounds which safely cured bacterial infections was more difficult to duplicate in treatments of fungal and viral infections. Antibiotic research led to great strides in the knowledge of biochemistry, establishing large differences between the cellular and molecular physiology of the bacterial cell and that of the mammalian cell. This explained the observation that many compounds that are toxic to bacteria are non-toxic to human cells. In contrast, the basic biochemistries of the fungal cell and the mammalian cell are much more similar. This restricts the development and use of therapeutic compounds that attack a fungal cell, while not harming mammalian cells. Similar problems exist in antibiotic treatments of viral diseases. Human viral metabolic biochemistry is very closely similar to human biochemistry, and the possible targets of antiviral compounds are restricted to very few components unique to a mammalian virus.

Research into bacteriophages for use as antibiotics is presently ongoing. Several types of bacteriophage appear to exist that are specific for each bacterial taxonomic group or species. Research into bacteriophages for medicinal use is just beginning, but has led to advances in microscopic imaging. While bacteriophages provide a possible solution to the problem of antibiotic resistance, there is no clinical evidence yet that they can be deployed as therapeutic agents to cure disease.

Phage therapy, the use of particular viruses to attack bacteria, has been used in the past on humans in the US and Europe during the 1920s and 1930s, but these treatments had mixed results. With the discovery of penicillin in the 1940s, Europe and the US changed therapeutic strategies to using antibiotics. However, in the former Soviet Union phage therapies continued to be studied. In the Republic of Georgia, the Eliava Institute of Bacteriophage, Microbiology & Virology continues to research the use of phage therapy. Various companies and foundations in North America and Europe are currently researching phage therapies.However, phage are living and reproducing; concerns about genetic engineering in freely released viruses currently limit certain aspects of phage therapy.

Bacteriocins are also a growing alternative to the classic small-molecule antibiotics . Different classes of bacteriocins have different potential as therapeutic agents. Small molecule bacteriocins (microcins, for example, and lantibiotics) may be similar to the classic antibiotics; colicin-like bacteriocins are more likely to be narrow-spectrum, demanding new molecular diagnostics prior to therapy but also not raising the spectre of resistance to the same degree. One drawback to the large molecule antibiotics is that they will have relative difficulty crossing membranes and travelling systemically throughout the body. For this reason, they are most often proposed for application topically or gastrointestinally. Because bacteriocins are peptides, they are more readily engineered than small molecules. This may permit the generation of cocktails and dynamically improved antibiotics that are modified to overcome resistance.

Probiotics are another alternative that goes beyond traditional antibiotics by employing a live culture which may in theory establish itself as a symbiont, competing, inhibiting, or simply interfering with colonization by pathogens.

SARS Virus Genetically Engineered

2009-10-23T07:03:00.000-07:00

The SARS epidemic started in the weeks that the ‘allied forces’ were waging war on Iraq to hunt down Saddam Hussein and his still elusive ‘weapons of mass destruction’.

SARS – Severe Acute Respiratory Syndrome – is a completely new infectious disease spread by human contact. By 20 June 2003, World Health Organisation figures registered 8461 cases in 31 countries worldwide, and 804 deaths. The overall death rate is nearly 10% and could be 20% or higher.

Although there are signs that the disease is under control, there are also fears that it may return.
Mystery of the SARS virus

The World Health Organisation, which played the key role in coordinating the research of a dozen laboratories, formally announced on 16 April that a new pathogen, a member of the coronavirus family never before seen in humans, is the cause of SARS, though lingering doubt has remained. The virus cannot be identified all patients diagnosed with SARS, and it can only be isolated from cultured green monkey kidney cells.

Known coronaviruses are placed in three groups based on similarities in their genomes. Group 1 contains the porcine epidemic diarrhoea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), canine coronavirus (CCV), feline infectious peritonitis virus (FIPV) and human coronovirus 229E (HuCV229E); Group 2 contains the avian infectious bronchitis virus (AIBV) and turkey coronavirus; while Group 3 contains the murine hepatitis virus (MHV) bovine coronavirus (BCV), human coronavirus (HuOC43) and others.

The molecular phylogenies published 10 April in the New England Journal of Medicine, based on small fragments of the polymerase gene, have placed the SARS virus in a separate group somewhere between groups 2 and 3.

More detailed analysis, subsequently published in the New England Journal of Medicine, Science and the Lancet indicate that the new virus is not closely related to any known virus at all, human, mouse, bovine, cat, pig, bird, notwithstanding. It is neither a mutant that switched host, nor a recombinant from existing coronaviruses. It is more complicated than that.
SARS virus - a product of genetic engineering?

Two scientists who have genetic engineered coronaviruses in their laboratories, Holmes and Enjuanes, suggested in a commentary in the journal Science that the SARS virus probably "evolved separately from an ancestor of the known coronavirus, and infected an unidentified animal, bird, or reptile host for a very long time before infecting humans and starting the SARS epidemic." (p.1377)

Following soon afterwards, there was a claim that the SARS virus came from the masked civet cat in south China. But that claim could not be substantiated. An alternative hypothesis entertained in the mainstream journals was that the virus came from outerspace.

There are very unusual features to the SARS virus. Its sequence most closely matches that of mouse hepatitis virus (MHV) and Bovine corona virus (BCV), both in group 3. The match is quite good in the middle third of the genome that’s nearly 30 000nt long, and not good at all for the first third or last third of the sequence.

But, antibodies to the SARS virus cross react with FIPV, HuCV229E and TGEV, all in Group 1. And the SARS virus can grow in Vero green monkey kidney cells, which no other coronavirus can, with the exception of PEDV, another virus in Group 1.

Could the SARS virus have come from genetic engineering? This is a question that Ho and Cummins have put to the scientific community. So far, we have not had a proper reply.

Holmes and Enjuanes stated in their commentary, "SARS-CoV is also unlikely to have been created from known coronaviruses by genetic engineering, because at present it would be impossible to modify 50% of a coronavirus genome without abrogating viral infectivity."

This is a quite a feeble response. The whole point to genetic engineering is that it greatly increases the scope of recombination, and provides selective tools to find the most unlikely recombinants that are still infectious.

Coronaviruses have been subjected to increasing genetic manipulation since the latter half of the 1990s, when P.S. Masters in Wadworth Center, New York State Department of Health and New York State University at Albany, used RNA recombination to introduce extensive changes into the genome of mouse hepatitis virus (MHV). In a review published in 1999, he wrote, "targeted recombination could be used to create extensive substitutions to the cornavirus genome, generating recombinants that could not be made otherwise between two viruses separated by a species barrier." (p.254)

‘Defective interfering RNAs’ – sequences of the viral genome with large deletions as well as mutations and substitutions or insertions - were used as donor sequences to introduce major substitutions and point mutations into the genome of the viruses by RNA recombination.

In the course of such work, researchers have even isolated a recombinant of cororanvirus with the green fluorescent protein (GFP) gene, presumably from cells in which coronaviruses have been cultured, which has become inserted into the spike protein gene. The GFP gene, originally from a jelly-fish, is extensively used in genetic engineering as a marker gene because it makes the cells that have taken up the foreign genes give off a green glow under uv light. The GFP-coronavirus recombinant could only have come about as an unintended by-product of genetic engineering.

In the same review, P.S. Masters showed that both point mutations and large substitutions can readily be transferred to the last third of the genome of MHV and other coronaviruses. He further indicated that similar strategies could be used to mutate and substitute the first third of the genome, though not for the middle third. "A comprehensive genetic study of the highly complex gene for the RNA polymerase and all of its associated activities [encoded by the middle third of the genome] will likely await either the construction of an infectious full-length clone or the development of an innovative scheme for mutant selection." (p.259)

Is that why the middle third of SARS virus genome has retained good homology to MHV and BCV, which were the first coronaviruses to be engineered in this manner, while the other parts are much more different?

Another feature of the SARS virus is that the spike protein, which determines host range, is unlike the spike protein of any known coronavirus. Instead, it appears to have homologies to segments of the human chromosome 7, according to sequence analysis performed by Howard Urnovitz.

Urnovitz believes that the spike protein of the SARS virus is the result of genetic rearrangements provoked by environmental genotoxic agents, much like those he and his colleagues have detected in Gulf War I veterans suffering from Gulf War Syndrome.

But how did the virus get to south China? A possible answer was provided by Urnovitz: Migratory birds that frequent gene-swapping hot spots like southeast China could have carried the SARS virus there.

Urnovitz himself doesn’t think the SARS virus is the real cause of SARS. Instead, it is the piece of reshuffled human chromosome 7 that others are referring to as the spike protein gene of the SARS virus. That alone is sufficient to trigger serious autoimmune responses in people.

Hence, to create vaccines against that ‘spike’ protein is also tantamount to vaccinating people against their own genes (see "Dynamic genomics")

Adult Bone Marrow Cells Mend Heart without Transplant

2009-10-23T07:02:00.000-07:00

Sudden blockages of a major artery to the heart cuts off blood supply and lead to rapid death of the muscle cells and blood vessels in the heart. This condition, myocardial infarction, is a common form of heart disease. Despite the demonstration that some of the heart muscle cells can multiply and new vessels formed, regeneration is restricted to the living part of the heart wall. The ‘infarcted’ or dead area is irreversible, and in time, scar tissue is formed. Attempts to replace the dead tissue by transplanting heart muscle cells or skeletal muscle cells have failed to mend the damaged part properly.

In previous experiments on mice, researchers in New York Medical College and the National Institute of Health injected bone marrow cells along the border of the damaged area of the heart, and found that the cells did differentiate into muscle and blood vessels. But this surgical intervention killed a high number of the mice and the grafting success was only 40%. This prompted them to consider a ‘non-invasive’ method, which involved stimulating the mice to overproduce bone marrow cells before and after myocardial infarction was induced .

For the purpose, the mice were given daily injections of two cytokines (small molecules that influence the activities of cells), stem cell factor (SCF) and granulocyte-colony-stimulating factor (G-CSF), which increased the number of circulating stem cells two to three hundred fold.

Mice given cytokines had a survival rate of 73% after the operation, compared with 20% in controls not given cytokines. There were clear signs of repair in the damaged area of the heart in the cytokine-injected group, both new heart muscle and blood vessels were formed, whereas only scar-tissue was found in controls. The hearts of the cytokine-injected group also performed significantly better than the controls.

The experimental results looked impressive enough even though the protocol of inducing myocardial infarction in such large numbers of animals is debatable. In addition, there was an unaccountably small number of experimental animals, only 15 compared to 52 in the group of controls. This may be because the researchers excluded mice that died within 48h of the operation, "to minimize the influence of the surgical trauma". But could it be that the mice died from stress of overproduction of bone marrow cells caused by the cytokines injected? There are certainly more ways to be invasive; and much more effort should be devoted to reducing unnecessary and stressful interventions, both physical and chemical.

Scrambled Genomes in Human Gene Therapy and Transgenic Plants

2009-10-22T03:07:00.001-07:00

Human gene therapy is usually considered separate and distinct from genetic modification (GM) of crops, but this is misleading.

Adeno-associated virus vectors (AAV) are most commonly used in clinical gene-therapy trials. The wild-type adenovirus has terminal repeats that enable it to integrate into human chromosome 19 at a specific site. The AAV vector, however, has no specific integration site. It often integrates into chromosome 19, though not at the integration site of the wild-type virus, and it may also integrate into any of the other human chromosomes. AAV vector is preferred for most gene therapy experiments because the chromosome insertion is more stable and the AAV vector transforms both dividing and non-dividing cells.

Crop GM is achieved using Agrobacterium transformation or direct plasmid transfer using biolistic transformation (gene gun) methods. The Agrobacterium T –DNA vector is flanked by 25 base-pair direct repeats that facilitate integration of plasmid sequences into the plant chromosome.

The common features of gene therapy in human cells and crop GM is the presence of integrating vector sequences flanking transgene(s) each equipped with a promoter to drive expression.

While many studies have been carried out on transgenic DNA in plants, there have been relatively few that analyze host genome at the site of insertion. In a recent issue of Nature Genetics, researchers in the Department of Medicine, University of Washington Seattle, report that integrated AAV are associated with chromosomal deletions and other rearrangements and are frequently located on chromosome 19 (although not at the wildtype AAV integration site).

The researchers analysed the chromosomal DNA flanking the site of vector insertion. By searching the human genome sequence databases, the junctions were located to 6 different chromosomes. Four integrated into genes. Four of nine inserts went into a relatively large, 22-Mb (millions of base pairs) region of chromosome 10, but not the wild type site, which only spans 1kb.

Chromosomal deletions and additions were found, as well as translocations of parts of one chromosome to another. There were also unexpected vector sequences at integration sites.

The study is reminiscent of the recent finding of unexpected sequences and genome scrambling associated with transgenes in GM crops such as soybean (see "Scrambled genome of Roundup Ready Soya", ISIS News 9/10 www.i-sis.org.uk). Further analysis is likely to show that scrambled and unexpected sequences are commonplace in GM crops. We have questioned the legality and safety of approving crops that contain unknown, uncharacterised DNA sequences. If scrambled and unexpected sequences are found even in the most widely distributed and established commercial GM crop, the problems are likely to be worse with newer transgenic crops of corn, cotton or canola, which have yet to be analysed. Certainly, government regulators and their academic satellites seem passive and submissive in dealing with important findings that question the safety of GM crops.

The observation that gene scrambling occurs in both human gene therapy and in GM crops suggest that there is a fundamental flaw in both genetic engineering technology and in the auditing of molecular properties of the modified humans or crops.

The corporate audit of molecular characteristics in gene therapy vectors and those of GM crops may be analogous to the Enron audits of the Arthur Anderson Accounting Firm. Those outside of direct involvement in gene therapy and crop genetic modification should be well enough informed to require full and truthful molecular audits of gene therapy vectors and GM crops.

Stem cell

2009-10-03T21:09:00.000-07:00

Stem cells are cells found in most, if not all, multi-cellular organisms. They are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. Research in the stem cell field grew out of findings by Canadian scientists Ernest A. McCulloch and James E. Till in the 1960s. The two broad types of mammalian stem cells are: embryonic stem cells that are isolated from the inner cell mass of blastocysts, and adult stem cells that are found in adult tissues. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin or intestinal tissues.

Stem cells can now be grown and transformed into specialized cells with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture. Highly plastic adult stem cells from a variety of sources, including umbilical cord blood and bone marrow, are routinely used in medical therapies. Embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have also been proposed as promising candidates for future therapies.

Properties

The classical definition of a stem cell requires that it possess two properties:

* Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated state.
* Potency - the capacity to differentiate into specialized cell types. In the strictest sense, this requires stem cells to be either totipotent or pluripotent - to be able to give rise to any mature cell type, although multipotent or unipotent progenitor cells are sometimes referred to as stem cells.

Potency definitions
Pluripotent, embryonic stem cells originate as inner mass cells within a blastocyst. The stem cells can become any tissue in the body, excluding a placenta. Only the morula's cells are totipotent, able to become all tissues and a placenta.
Human embryonic stem cells
A: Cell colonies that are not yet differentiated.
B: Nerve cell

Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell.

* Totipotent (a.k.a omnipotent) stem cells can differentiate into embryonic and extraembryonic cell types. Such cells can construct a complete, viable, organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.
* Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells, i.e. cells derived from any of the three germ layers.
* Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of cells.
* Oligopotent stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.
* Unipotent cells can produce only one cell type, their own, but have the property of self-renewal which distinguishes them from non-stem cells (e.g. muscle stem cells).

Identification

The practical definition of a stem cell is the functional definition - a cell that has the potential to regenerate tissue over a lifetime. For example, the gold standard test for a bone marrow or hematopoietic stem cell (HSC) is the ability to transplant one cell and save an individual without HSCs. In this case, a stem cell must be able to produce new blood cells and immune cells over a long term, demonstrating potency. It should also be possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into another individual without HSCs, demonstrating that the stem cell was able to self-renew.

Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, where single cells are characterized by their ability to differentiate and self-renew. As well, stem cells can be isolated based on a distinctive set of cell surface markers. However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells will behave in a similar manner in vivo. Considerable debate exists whether some proposed adult cell populations are truly stem cells.

Embryonic

Embryonic stem cell lines (ES cell lines) are cultures of cells derived from the epiblast tissue of the inner cell mass (ICM) of a blastocyst or earlier morula stage embryos. A blastocyst is an early stage embryo—approximately four to five days old in humans and consisting of 50–150 cells. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta.

Nearly all research to date has taken place using mouse embryonic stem cells (mES) or human embryonic stem cells (hES). Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin and require the presence of Leukemia Inhibitory Factor (LIF). Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic Fibroblast Growth Factor (bFGF or FGF-2). Without optimal culture conditions or genetic manipulation, embryonic stem cells will rapidly differentiate.

A human embryonic stem cell is also defined by the presence of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency. The cell surface antigens most commonly used to identify hES cells are the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81. The molecular definition of a stem cell includes many more proteins and continues to be a topic of research.

After nearly ten years of research, there are no approved treatments using embryonic stem cells. The first human trial was approved by the US Food & Drug Administration in January 2009. ES cells, being pluripotent cells, require specific signals for correct differentiation - if injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma. Differentiating ES cells into usable cells while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell researchers still face. Many nations currently have moratoria on either ES cell research or the production of new ES cell lines. Because of their combined abilities of unlimited expansion and pluripotency, embryonic stem cells remain a theoretically potential source for regenerative medicine and tissue replacement after injury or disease.

Molecular Genetic Engineers in Junk DNA

2009-10-02T11:58:00.000-07:00

Perhaps only 1% of the human genome codes for genes, and that’s what the human genome map contains. The rest is mainly repetitive DNA, commonly known as ‘junk DNA’.

However, evidence has been emerging that lurking within junk DNA are armies of transposons (mobile genetic elements) that play an indispensable role in ‘natural genetic engineering’ the genome. They make up nearly half of the human genome, and serve as ‘recombination hotspots’ for cutting and splicing, and hence reshuffling the genome. They are also a source of ready to use motifs for gene expression, as well as new protein-coding sequences.

These important transposons are scattered throughout the genome. There are two main categories: Long Interspersed Elements (LINEs) about 6.7 kilobasepairs in length and Short Interspersed Elements (SINEs) of several hundred basepairs.

The most abundant SINEs are Alu elements, of which 1.4 million copies exist, comprising 10% of the human genome, and are apparently only found in primates.

Most LINEs are dormant, except for LINE1. But both LINE 1 and other LINEs are abundant in areas of the genome where the bases A and T predominate, which do not have many genes. Alu elements, however, are more common in ‘GC-rich’regions that are also gene-rich. This is quite baffling as Alu itself cannot move, but depends on enzymes encoded by LINE1 in order to insert itself.

Alu elements, like LINE1 are retrotransposons that move and multiply by being transcribed into RNA, then reverse transcribed into DNA copies that jump into new sites.

New research is suggesting that Alu elements may help create new proteins from existing ones. The reasons the human genome contains so few genes – the latest count is just under 25 000, is that more than half the genes are interrupted and subject to alternative splicing.

In other words, the coding sequence of the gene is broken up into segments (exons) interrupted by non-coding segments (introns) that are spliced out from the RNA transcript before it is translated into protein. But there are alternative splicing pathways that join different exons together, with the result that many different proteins can be made from a single gene.

It appears that about 5% of alternatively spliced internal exons in the human genome originate in an Alu sequence. It suggests that Alu elements can actually jump into genes and, instead of destroying that gene, actually contributes a new coding sequence to it.

There are two ways in which this could be done, either by jumping into an existing gene so that the gene gains a new exon and increases its repertoire of possible proteins by alternative splicing, or else, the gene can become duplicated first, with one copy remaining intact, while the other is crafted by the Alu element. Currently, it is estimated that 238 000 Alu elements are located within introns of protein-coding genes and each one can actually or potentially become an exon.

Gil Ast, head of a group in Tel Aviv University, Israel, which has made some of the most significant discoveries about Alu, is understandably pleased. "We believe that Alus allowed the shuffling of genetic information that may have led to the evolution of primates," said Ast. "They may contribute to a lot of disorders we don’t even know about yet. But they have also created genetic diversity."

Alternative splicing is quite precisely regulated, and all other things being equal, depends on the spacing of potential splice sites consisting of the dinucleotide, AG, as well as neighbouring nucleotides. A single base change can destroy alternative splicing, and this can cause a disease, as in the case of Alport syndrome.

Also, gaining an exon is not always a good thing. There are numerous ‘insertion mutations’ associated with the inappropriate insertion of transposons into genes. And even Alu sequences that are already in introns can cause problems. More than a decade ago, it was discovered that a point mutation in an Alu element residing in the third intron of the ornithine aminotransferase gene activated a cryptic splice site, and led to part of the Alu element becoming incorporated into the coding sequence. Unfortunately, it carried a stop codon, which cut the protein short, leading to ornithine aminotransferase deficiency.

There is increasing evidence that physical and chemical stresses to the cell, such as heat shock, chemical poisons and viral infections, tend to activate Alu elements. The resultant gene reshuffling may be responsible for a variety of chronic diseases.

source:- i-sis.org.uk

Endogenous Viruses and Chronic Disease

2009-10-02T11:55:00.000-07:00

Retroviruses are RNA viruses found in all vertebrates. The retrovirus reproduces by reverse transcribing (copying) its RNA into DNA that is then integrated into the host-cell genome to be transcribed into viruses. Many retroviral sequences have become permanently integrated into the human genome as human endogenous retroviruses, or HERVs. The human genome (indeed all genomes) also contains retrovirus-like retrotransposons, mobile elements that multiply by making RNA copies that are reverse transcribed into DNA and integrated into new sites in the genome. The main difference between a retrovirus and a retrotransposon is that the latter lacks an envelope.

Most HERVs and retrotransposons are defective, having lost one or more gene functions; but can nevertheless multiply and move with the help of other elements or infecting viruses.

Which came first, retrovirus or retrotransposon? As viruses depend on cells to reproduce, it seems reasonable to suppose that retroviruses are simply retrotransposons that have gained an envelop, which helps in entering other cells. That idea was first put forward by Howard Temin, who discovered reverse transcriptase, the enzyme that reverse transcribes, encoded by the retrovirus and retrotransposon.

Retrotransposons play an important role in turning genes on or off, and in rearranging and amplifying genes during development and cell differentiation. And evidence has been accumulating that they are the natural molecular genetic engineers of the fluid genome, which are necessary for the survival of the organism .

HERVs are flanked by ‘long terminal repeats’ that contain strong promoters for gene expression. Promoters are stretches of DNA with binding sites for transcription factors of the host cell that boosts transcription, effectively saying to the cell, "make many copies of the message following". HERVs and retrotransposons are regulated by the cell, and ultimately, by the organism as a whole, which stops most of them from being expressed; though they can be activated under a variety of conditions (see below).

Many endogenous viruses show xenotropism, i.e., they are not active in the host cell but can become infectious to cells of non-host species. Xenotropism is one of the major health hazards of xenotransplantation, the transplant of organs and tissues between species, as from pig to humans.

Defective or dormant HERVs, like defective retrotransposons, can become expressed when missing gene-functions are provided by a ‘helper’ virus that happens to infect the cell, and that includes ‘attenuated’ viruses in vaccines. Like retrotransposons, HERVs can also be induced: by X-rays or various chemical agents and drugs, such as inhibitors of protein synthesis, organophosphates and other pesticides, inflammatory cytokines (hormone-like factors that influence cells of the immune system) or steroid hormones, and retinoic acid.

In a comprehensive review published in 1996, virologists Howard Urnovitz and William Murphy raised the possibility that many chronic debilitating diseases may be linked to HERVs. These include leukaemia and other cancers, B-cell immunoglobulin diseases, inflammatory diseases of the nervous system, autoimmune rheumatic and connective tissue disease and chronic fatigue syndrome.

There are several mechanisms linking HERVs with chronic diseases, though it is not at all clear which mechanism comes into effect under different circumstances.

One way in which endogenous viruses can cause disease is for them to move and insert itself next to certain genes, that, when over-expressed, results in uncontrolled cell division, or cancer. This mechanism may be involved in mouse and human leukaemia, breast cancer and teratocarcinoma. This is also the mechanism that causes cancer in gene therapy, when viral vectors integrate next to these same genes.

Another possibility is for an HERV to recombine with an infecting virus to generate new viruses. One theory for the origin of the AIDS virus it that it may have come from early preparations of polioviruses used for vaccines that were propagated in rhesus and African green monkey kidney cells. At least 26 monkey viruses, including adenoviruses, cosackievirus, herpesvirus, echovirus, and possibly other groups of viruses were found as contaminants in such preparations. Current vaccines are presumably free of such contamination, though that does not necessarily make them safe (see below).

Urnovitz and Murphy suggested that human immunodeficiency virus type 1 (HIV-1) may be a chimera between one of the simian (monkey) viruses (simian immunodeficiency virus) and HERV sequences. Simian immunodeficiency virus capable of causing simian AIDS, appears to occur exclusively in African monkeys, particularly in the African green monkey. And DNA sequences related to the highly conserved domain of the HIV reverse transcriptase and glycoprotein gp41 (part of the gp160 polyprotein of the HIV that’s cleaved into gp120 and gp41) have been found in the human genome.

Another way in which disease may arise is when HERV encoded proteins are expressed. This provokes antibodies against the body’s own cells, giving rise to autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, Sjögren’s syndrome, mixed connective tissue diseases and inflammatory neurologic disease. The inflammatory response could be the most important trigger for the development of autoimmune disease, as infecting viruses can strongly activate HERVs to express, resulting in production of HERV protein antigens.

In this context, vaccines came in for special criticism in Unovitz’ testimony to the United States House of Representatives in 1999.

In his view, "there appears to be a limit on how much foreign material to which the human body can be exposed before some level of genetic damage occurs and a chronic disease initiates". Gulf War I veterans (GWIVs) were given large numbers of vaccines (see "Gulf War Syndrome and vaccinations", this series), and vaccine overload is a significant factor in Gulf War Syndrome.

Urnovitz described a case of a woman who died from a mysterious case of AIDS. Over several years, laboratory tests failed to reveal HIV-1 antibodies in her blood. However, she was subsequently tested HIV-1 positive in her urine. The virus was eventually isolated and sequenced; and came to be known as HIV-1 Group O. Analyses of the viral genetic material suggest that the virus originated, in part, from genetic reshuffling of human chromosomal material.

Vaccination against HIV-1 is particularly dangerous, if as Urnovitz and Murphy have suggested, HIV arose from recombination between the simian aids virus and HERV sequences (see also "AIDS vaccines worse than useless?" this series).

"To put it simply, are we embarking on a course that will vaccinate people against their own genes?" Dr. Urnovitz asked.

A second example is the intensive effort directed to creating a vaccine for the hepatitis C virus. Unfortunately, there is no hard evidence in the scientific literature that hepatitis C virus exists. Urnovitz continued, "As a scientist I am compelled to ask, how can we vaccinate people against a disease-causing agent that has not been fully characterised?"

Finally, he drew attention to the contaminated polio vaccines that is now being increasingly implicated in cancer,

"Had my mother and father known that the poliovirus vaccines of the 1950s were heavily contaminated with more than 26 monkey viruses, including the cancer virus SV40, I can say with certainty that they would not have allowed their children and themselves to take those vaccines. Both of my parents might not have developed cancers suspected of being vaccine-related, and might even be alive today."

But even uncontaminated Polio vaccines are of doubtful efficacy in protecting against viral infections and chronic disease. Enteroviruses have been shown to be a major factor in Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome by John Richardson who studied more than 4000 patients over a 50 year period. Vaccination against polio provides protection against only 3 strains of polio leaving no protection against the other 70 or so enteroviruses- coxsackie, echo and others. These viruses have a range of pathological effects on the central nervous system, the cardiovascular system, and endocrine and exocrine glands.

source:- i-sis.org.uk

Dynamic Genomics

2009-10-02T11:52:00.000-07:00

During the Persian Gulf War, some 700 000 individuals were exposed to a whole range of environmental hazards, including low-level chemical warfare agents, investigational drugs (inclucing pyridostigmine bromide, used as a prophylactic against nerve agents), organophosphate, carbamate, and other pesticides and insect repellents, low levels of nuclear and electromagnetic radiation, toxic combustion products from oilwell fires, diesel exhaust products and airborne particles, all collectively known to be genotoxic, or capable of causing harm through effects on the genetic material. The veterans were also exposed to multiple vaccinations, also of questionable safety. A significant proportion of the veterans developed a pattern of symptoms that have been referred to as Persian Gulf War-Related Illnesses, or Gulf War Syndrome (GWS): rash, fatigue, muscle and joint pain, headache, irritability, depression, unrefreshing sleep, gastrointestinal and respiratory disorders and cognitive defects. These were eventually defined as a clinical entity in 1998.

Most Gulf War I veterans (GWIVs) received oral poliovirus vaccine before deployment. Persistent enterovirus infection has been implicated in the chronic fatigue syndrome, one of the major disorders of GWIVs. There has already been a report that enterovirus-specific RNA was found in the sera of patients with chronic fatigue syndrome. For these reasons, Howard Urnovitz, Scientific Director of the Chronic Illness Research Foundation, and his colleagues decided to search for virus-specific nucleic acids in the sera of the GWIVs by using virus-specific primers to amplify RNA sequence. The sera from 24 GWIV with GWS deployed approximately 5 years previously were compared with serum samples from 50 controls, for the most part matched by age, sex and race.

When the amplified RNAs were separated according to size by running the mixtures through an agarose gel in an electric field, a striking difference between the GWIVs and controls was seen. Controls typically gave no more than three faint RNA bands, all less than 350 nucleotides (nt) in length. The sera from GWIVs, in contrast, contained numerous bright bands of very large RNAs, most of them longer than 750nt and especially longer than 2 000 nt. Most of the bands, moreover, did not belong to either the poliovirus or enterovirus. Both viral RNAs tended to be found more frequently in the sera of GWIVs, but the differences from controls were not significant.

The team sequenced two of the many bands that were found only in GWIVs, one 414nt and the other, 759nt, from three different samples. They were 99% identical between samples, but unrelated to each other, and were not homologous (similar) to any sequence found the public DNA database GenBank. However, short stretches of 14 to 15nt were homologous to segments in a region on the short arm of chromosome 22, 22q11.2. It is as though something had chopped up that region into pieces, shuffled them, and joined them up together again.

Thus, 3 sequences of 15nt and 8 of 14nt in the 759nt RNA had 100% homology to short segments of chromosome 22q11.2. Five of these segments occur only on chromosome 22q11.2. For the 414nt RNA, there were 2 sequences of 15nt and 4 of 14nt with 100% homology to the 22q11.2 region, but these segments also occur on other chromosomes, so it cannot be excluded that other chromosome regions were also involved in this gene shuffling exercise. Another important feature is that 6 of the segments in the 759nt RNA and 2 of those in the 414nt RNA occur near, between, or in Alu elements ("Molecular genetic engineers in junk DNA?", this series) that are capable of multiplying and jumping around the genome, and are hence thought to be involved in genetic recombination or gene shuffling.

This is a surprising finding. After all, GWS is generally considered to be a ‘multifactorial’ disease, ie, a disease due to multiple causes, possibly one for each of the symptoms. And yet, for the first time, Urnovitz and his colleagues have demonstrated that there could be a common molecular marker for the disease.

Not only that, using the same techniques, Urnovitz and colleagues were able to identify another unique RNA molecular marker in patients with multiple myeloma (malignant transformation of blood plasma precursor cells) and related disorders. They analysed 65 patients with multiple myeoloma (MM) 3 with Waldenstrom’s macroglobulinemia (WM), 2 with monoclonal gammopathy of undetermined significance (MGUS), and 50 healthy controls.

A 713nt plasma RNA occurred in 16/18 of MM patients in relapse, 5/8 MM patients who were untreated, 2/3 WM patients and ½ MGUS patients. None of the MM patients in remission, nor the 50 healthy controls was positive. The homology of the 713nt RNA between four samples was > 99.7% and matched (99.6%) a 704ng sequence of the flanking region of the peroxisome proliferator activator receptor gene, located in the same genome region, chromosome 22q11.2. A 255nt sequence within the 713nt RNA had a 90.2% homology with an Alu consensus sequence.

There is reasonable evidence that multiple myeloma is associated with exposure to industrial chemicals, pesticides or other environmental insults, as in the case of GWS.

This raises key questions: what is the origin of these RNAs? What is the possible role of these RNAs and of chromosome 22q11.2 in these diseases? Have environmental genotoxins played a role in causing disease? And finally, could the RNA molecular markers offer diagnostic tools for the diseases?

Chromosome 22q11.2 has been identified as a region full of hotspots for genetic deletions and translocations correlated with multiple myelomas and related disorders, as well as with rearrangements of the immunoglobulin lambda light chains in the normal immune response. Chromosome 22 appears to be involved in the so-called Goldenhar complex, a birth defect possibly associated with GWS.

That region is full of Alu sequences, previously thought to be nothing but junk DNA. But it is becoming increasingl clear that they have important regulatory functions. Alu expression is induced when cells are stressed by heat shock, or genotoxic agents, and may be part of the detoxification response. Alu sequences are known to be involved in genetic recombination or gene shuffling. Alu-Alu rcombinants are generated by both extrachromosomal and chromosomal genetic mechanisms.

Thus, it seems reasonable to conclude that exposure to toxic substances had activated retrotransposable Alu elements, possibly in specific parts of the genome, which results in gene shuffling to produce the unique sequences of RNAs circulating in the serum.

These circulating RNAs appear to be derived from white blood cells that have died, and are enclosed in proteolipid vesicles that protect them from being broken down. There is evidence that such plasma RNAs account for at least some of the illnesses. They are capable of transforming the blood cells of healthy animals in a mouse model, and are associated with immune suppression, making them more susceptible to infections.

At a conference celebrating the Centennial of the University of Michigan Department of Microbiology and Immunology in May 2003, Urnovitz, presented the new concept of "the dynamic genome", the idea that the genome contains "an operating system that instructs the organism how to both use and adapt genomic elements to the constant challenges of a dynamic environment."

This concept led to a practical breakthough, surrogate marker blood tests for yet another condition, mad cow disease, which can be performed on live animals. And, he also mentioned potential public health application for understanding the role of the genome in epidemics ranging from influenza-like pandemics (SARS) to "Gulf War syndrome, chronic fatigue syndrome, and AIDS".

What led him to the idea of the dynamic genome is the discovery that blood borne particles, or "microvesicles" contain "non-blueprint" RNA. In the past, they were assumed to be foreign, and hence mistaken as viruses.

He rejects the theory that a coronavirus is the cause of SARS. The virus was isolated from lab cultures that showed sick and dying cells. "Transmissible factors don’t have to kill a cell to be part of the disease," Urnovitz says, "they could just dysregulate cell function without killing the host cell."

He has carried out his own analysis on the so-called SARS-related coronavirus gene sequence. "Frankly, I do not see a virus. I see a unique and complete rearrangement of genomic elements. For example, when I look at what is believed to be the gene sequence coding for the spike protein of this coronavirus, I see a complicated gene rearrangement of a region of human chromosome 7." As with the Gulf War Syndrome, gene rearrangements like this immediately says to him, "search for an associated catastrophic environmental event that could have caused such genomic rearrangement."

He sees a correlation between nuclear and chemical weapons deployment over the last 100 years and the associated occurrence of flu-like pandemics. He postulates that when animals are exposed to nuclear or chemical weapons, entirely new regulatory gene set are expressed and packaged into non-viral RNA regulatory microvesicles. The risk of turning an epidemic into a pandemic is increased when the exposed animals are migratory birds that frequent gene-swapping hot spots like southeast China. He says, "The recent sightings in eastern China and Hong Kong of rare migratory birds – white cranes, grey cranes, and swans – that spend significant time feeding in the radioactive-contaminated regions of Siberia suggest that international efforts should be focussed on not only hunting for weapons of mass destruction but also on cleaning up the ones that have already been released into the environment."

He rejects the common belief that vaccines are the key to stopping epidemics: "While the current dogma states that vaccines stop viral epidemics, the historical data do not support that claim. From smallpox to polio to HIV, all vaccine attempts have been ineffective or hazardous to the vaccinee."

His company, Chronix Biomedical, develop screening and diagnostic tests based on the detection of non-viral RNA regulatory microvesicles for both veterinary and human diseases. Is it making a profit? "Not yet," he answered.

The blood test for mad cow disease, or bovine spongiform encephalitis (BSE) —the first that can be performed on live animals—is under development in the laboratory of Professor Bertram Brenig, Director of the Institute of Veterinary Medicine, Georg-August University, Göttingen, Germany. Urnovitz’s collaboration with Brenig’s laboratory has resulted in the detection of a specific RNA unique to cows at risk for developing, or that have confirmed cases of BSE.

Urnovitz claims that the BSE blood test is 100% sensitive on all 6 BSE cows confirmed with a licensed prion test, and 100% specific on all 46 animals from known healthy herds. They found that 3.5% of cohort animals (two animals out of 57) showed a positive response in the surrogate blood marker for BSE. Cohorts are animals born and/or raised in the same herd as a confirmed BSE case within approximately 12 months before and after the date of birth of the BSE case. Positive cohort cases may represent animals at risk for developing BSE.
If Urnovitz is right, we have to seriously rethink environmental health.

source:- i-sis.org.uk

Xenotransplantation

2009-09-18T12:00:00.001-07:00

Xenotransplantation (xeno- from the Greek meaning "foreign") is the transplantation of living cells, tissues or organs from one species to another such as from pigs to humans (see Medical grafting). Such cells, tissues or organs are called xenografts or xenotransplants. The term allotransplantation refers to a same-species transplant. Human xenotransplantation offers a potential treatment for end-stage organ failure, a significant health problem in parts of the industrialized world. It also raises many novel medical, legal and ethical issues. A continuing concern is that pigs have different lifespans than humans and their tissues age at a different rate. Disease transmission (xenozoonosis) and permanent alteration to the genetic code of animals are also a cause for concern.

Because there is a worldwide shortage of organs for clinical implantation, about 60% of patients awaiting replacement organs die on the waiting list. Recent advances in understanding the mechanisms of transplant organ rejection have brought science to a stage where it is reasonable to consider that organs from other species, probably pigs, may soon be engineered to minimize the risk of serious rejection and used as an alternative to human tissues, possibly ending organ shortages.

Other procedures, some of which are being carefully investigated in early clinical trials, aim to use cells or tissues from other species to treat life-threatening and debilitating illnesses such as cancer, diabetes, liver failure and Parkinson's disease. If vitrification can be perfected it could allow for long-term storage of xenogenic cells, tissues and organs so they would be more readily available for transplant.

There are only a few published successful xenotransplant procedures.

Xenotransplants are transplants of organs from one species to another, such as an animal organ to humans. They are cutting edge of medical science and could save thousands of people’s lives who are waiting for an organ donation. The animal organ, probably from a pig or baboon could be genetically altered with human genes to trick a patient’s immune system into accepting it as apart of its own body. They have re-emerged because of the lack of organs available and the constant battle to keep immune systems from rejecting the organs. Xenotransplants are thus hopefully able to provide a way of transplants which are safe and effective.

Acceptance

Xenografts have been a controversial procedure since they were first attempted. Many, including animal rights groups, strongly oppose killing animals in order to harvest their organs for human use. Medical concerns exist about possible disease transfer between animals and humans, such as the porcine endogenous retrovirus found in pig tissues. Religious beliefs, such as the Jewish and Muslim prohibition against eating pork, have been sometimes thought to be a problem, however according to a Council of Europe documentation both religions agree that this rule is overridden by the preservation of human life.

In general, however, the use of pig and cow tissue in humans has been met with little resistance, save some religious beliefs.

Some of the main and biggest biological/human health problems involved with Xenotransplants are that of transmitting animal diseases to humans, the unknown certainty of an outbreak of infectious diseases and the probability of rejection of donor organs. Baboons and pigs carry myriad transmittable agents which are harmless in their natural host but extremely toxic and deadly in humans. HIV is an example of a disease which was believed to be transmitted to humans by monkeys. Scientists and researchers also do not know if an outbreak of infectious diseases could occur and if they could contain the outbreak even though they have measures for control. Another obstacle for Xenotransplants is that of the body’s rejection of foreign objects by it immune system. These antigens (foreign objects) are often treated with powerful drugs which may in turn make the patient vulnerable to other infections and actually aid the disease trying to be cured. This is the reason the organs would have to be altered to fit with the patients DNA. In 2005, the Australian National Health and Medical Research Council declared a eighteen-year moratorium on all animal-to-human transplantation, concluding that the risks of transmission of animal viruses to patients and the wider community have not yet been resolved . The main ethical issues associated with Xenotransplants are that the animals which would be commonly used for their organs, such as pigs and baboons are killed or sacrificed. Baboons are very similar to humans with human-like hands, faces and a developed social structure. For this reason pigs could be used more as their anatomies are similar to humans and are a lot easier to breed than baboons that only produce one offspring at a time. Pigs are also a lot healthier and carry less disease than primates as well. There are less moral objections to the killing of pigs as they are already killed for food and are already being produced.

Transcriptome

2009-09-18T11:59:00.001-07:00

The transcriptome is the set of all messenger RNA (mRNA) molecules, or "transcripts," produced in one or a population of cells. The term can be applied to the total set of transcripts in a given organism, or to the specific subset of transcripts present in a particular cell type. Unlike the genome, which is roughly fixed for a given cell line (excluding mutations), the transcriptome can vary with external environmental conditions. Because it includes all mRNA transcripts in the cell, the transcriptome reflects the genes that are being actively expressed at any given time, with the exception of mRNA degradation phenomena such as transcriptional attenuation. The study of transcriptomics, also referred to as Expression Profiling, examines the expression level of mRNAs in a given cell population, often using high-throughput techniques based on DNA microarray technology. The use of next-generation sequencing technology to study the transcriptome at the nucleotide level is known as RNA-Seq .

Transcriptomics is the branch of chemistry that deals with the study of messenger RNA molecules produced in an individual or population of a particular cell type.

Applications and analysis

The transcriptomes of stem cells and cancer cells are of particular interest to researchers who seek to understand the processes of cellular differentiation and carcinogenesis. A number of organism-specific transcriptome databases have been constructed and annotated to aid in the identification of genes that are differentially expressed in distinct cell populations or subtypes; however, the analysis of relative mRNA expression levels can be complicated by the fact that relatively small changes in mRNA expression can produce large changes in the total amount of the corresponding protein present in the cell. One analysis method, known as Gene Set Enrichment Analysis, identifies coregulated gene networks rather than individual genes that are up- or down-regulated in different cell populations.

mRNA regulation

Although microarray studies can reveal the relative amounts of different mRNAs in the cell, levels of mRNA are not directly proportional to the expression level of the proteins they code for. The number of protein molecules synthesized using a given mRNA molecule as a template is highly dependent on translation-initiation features of the mRNA sequence; in particular, the ability of the translation initiation sequence is a key determinant in the recruiting of ribosomes for protein translation. The complete protein complement of a cell or organism is known as the proteome.

A study of 158,807 mouse transcripts revealed that 4520 of these transcripts form antisense partners that are base pair complementary to the exons of genes. These results raise the possibility that significant numbers of "antisense RNA-coding genes" might participate in the regulation of the levels of expression of protein-coding mRNAs.

Human Insulin

2009-09-18T11:58:00.000-07:00

Amongst the earliest uses of biotechnology in pharmaceutical manufacturing is the use of recombinant DNA technology to modify escherichia coli bacteria to produce human insulin, which was performed at Genentech in 1978. Prior to the development of this technique, insulin was extracted from the pancreas glands of cattle, pigs, and other farm animals. While generally efficacious in the treatment of diabetes, animal-derived insulin is not indistinguishable from human insulin, and may therefore produce allergic reactions. Genentech researchers produced artificial genes for each of the two protein chains that comprise the insulin molecule. The artificial genes were "then inserted... into plasmids... among a group of genes that" are activated by lactose. Thus, the insulin producing genes were also activated by lactose. The recombinant plasmids were inserted into Escherichia coli bacteria, which were "induced to produce 100,000 molecules of either chain A or chain B human insulin." The two protein chains were then combined to produce insulin molecules.

Human Growth Hormone

Prior to the use of recombinant DNA technology to modify bacteria to produce human growth hormone, the hormone was manufactured by extraction from the pituitary glands of cadavers, as animal growth hormones have no therapeutic value in humans. Production of a single year's supply of human growth hormone required up to fifty pituitary glands, creating significant shortages of the hormone. In 1979, scientists at Genentech produced human growth hormone by inserting DNA coding for human growth hormone into a plasmid that was implanted in escherichia coli bacteria. The gene that was inserted into the plasmid was created by reverse transcription of the mRNA found in pituitary glands to complementary DNA. HaeIII, a type of restriction enzyme which acts at restriction sites "in the 3' noncoding region" and at the 23rd codon in complementary DNA for human growth hormone, was used to produce "a DNA fragment of 551 base pairs which includes coding sequences for amino acids 24 - 191 of HGH." Then "a chemically synthesized DNA 'adaptor' fragment containing an ATG initiation codon..." was produced with the codons for the first through 23rd amino acids in human growth hormone. The "two DNA fragments... [were] combined to form a synthetic-natural 'hybrid' gene." The use of entirely synthetic methods of DNA production to produce a gene that would be translated to human growth hormone in escherichia coli would have been exceedingly laborious due to the significant length of the amino acid sequence in human growth hormone. However, if the cDNA reverse transcribed from the mRNA for human growth hormone were inserted directly into the plasmid inserted into the escherichia coli, the bacteria would translate regions of the gene that are not translated in humans, thereby producing a "pre-hormone containing an extra 26 amino acids" which might be difficult to remove.

Human Blood Clotting Factors

Prior to the development and FDA approval of a means to produce human blood clotting factors using recombinant DNA technologies, human blood clotting factors were produced from donated blood that was inadequately screened for HIV. Thus, HIV infection posed a significant danger to patients with hemophilia who received human blood clotting factors:

Most reports indicate that 60 to 80 percent of patients with hemophilia who were exposed to factor VIII concentrates between 1979 and 1984 are seropositive for HIV by [the] Western blot assay. As of May 1988, more than 659 patients with hemophilia had AIDS...

The first human blood clotting factor to be produced in significant quantities using recombinant DNA technology was Factor IX, which was produced using transgenic Chinese hamster ovary cells in 1986. Lacking a map of the human genome, researchers obtained a known sequence of the RNA for Factor IX by examining the amino acids in Factor IX:

Microsequencing of highly purified... [Factor IX] yielded sufficient amino acid sequence to construct oligonucleotide probes.

The known sequence of Factor IX RNA was then used to search for the gene coding for Factor IX in a library of the DNA found in the human liver, since it was known that blood clotting factors are produced by the human liver:

A unique oligonucleotide... homologous to Factor IX mRNA... was synthesized and labeled... The resultant probe was used to screen a human liver double-stranded cDNA library... Complete two-stranded DNA sequences of the... [relevant] cDNA... contained all of the coding sequence COOH-terminal of the eleventh codon and the entire 3'-untranslated sequence.

This sequence of cDNA was used to find the remaining DNA sequences comprising the Factor IX gene by searching the DNA in the X chromosome:

A genomic library from a human XXXX chromosome was prepared... and screen[ed] with a Factor IX cDNA probe. Hybridizing recombinant phage were isolated, plaque-purified, and the DNA isolated. Restriction mapping, Southern analysis, and DNA sequencing permitted identification of five recombinant phage-containing inserts which, when overlapped at common sequences, coded the entire 35kb Factor IX gene.

Plasmids containing the Factor IX gene, along with plasmids with a gene that codes for resistance to methotrexate, were inserted into Chinese hamster ovary cells via transfection. Transfection involves the insertion of DNA into a eukaryotic cell. Unlike the analogous process of transformation in bacteria, transfected DNA is not ordinarily integrated into the cell's genome, and is therefore not usually passed on to subsequent generations via cell division. Thus, in order to obtain a "stable" transfection, a gene which confers a significant survival advantage must also be transfected, causing the few cells that did integrate the transfected DNA into their genomes to increase their population as cells that did not integrate the DNA are eliminated. In the case of this study, "grow[th] in increasing concentrations of methotrexate" promoted the survival of stably transfected cells, and diminished the survival of other cells.

The Chinese hamster ovary cells that were stably transfected produced significant quantities of Factor IX, which was shown to have substantial coagulant properties, though of a lesser degree than Factor IX produced from human blood:

The specific activity of the recombinant Factor IX was measured on the basis of direct measurement of the coagulant activity... The specific activity of recombinant Factor IX was 75 units/mg... compared to 150 units/mg measured for plasma-derived Factor IX...

In 1992, the FDA approved Factor VIII produced using transgenic Chinese hamster ovary cells, the first such blood clotting factor produced using recombinant DNA technology to be approved.

AnaSpec

2009-08-18T21:27:00.001-07:00

AnaSpec Inc. is a biotechnology company headquartered in San Jose, California. Located in the Silicon Valley, it is a provider of custom and catalog research peptides, antibodies, dyes, assay kits, and synthesis reagents. AnaSpec focuses on three main technologies: peptides, detection reagents (including dyes and antibodies), and combinatorial chemistry.

History

AnaSpec acquired HiLyte Biosciences, a manufacturer of fluorescent dyes, in 2003.

In 2008 AnaSpec established a new dedicated GMP manufacturing facility.

In 2009 AnaSpec launched its new facilities in Fremont, CA.

Genetic use restriction technology

2009-08-18T21:26:00.000-07:00

Genetic use restriction technology (GURT), colloquially known as terminator technology, is the name given to proposed methods for restricting the use of genetically modified plants by causing second generation seeds to be sterile. The technology was developed under a cooperative research and development agreement between the Agricultural Research Service of the USDA and Delta and Pine Land company in the 1990s, but it is not yet commercially available. Because some stakeholders expressed concerns that this technology might lead to dependence for poor smallholder farmers, Monsanto Company, an agricultural products company and the world's biggest seed supplier, pledged not to commercialize the technology in 1999. Late in 2006, it acquired Delta and Pine Land company.

The technology was discussed during the 8th Conference of the Parties to the UN's Convention on Biological Diversity in Curitiba, Brazil, March 20-31, 2006.

Variants

There are conceptually two types of GURT:

1. V-GURT: This type of GURT produces sterile seeds meaning that a farmer that had purchased seeds containing v-GURT technology could not save the seed from this crop for future planting. This would not have an immediate impact on the large number of primarily western farmers who use hybrid seeds, as they do not produce their own planting seeds, and instead buy specialized hybrid seeds from seed production companies. However, currently around 80 percent of farmers in both Brazil and Pakistan grow crops based on saved seeds from previous harvests. Consequentially, resistance to the introduction of GURT technology into developing countries is strong. The technology is restricted at the plant variety level - hence the term V-GURT. Manufacturers of genetically enhanced crops would use this technology to protect their products from unauthorised use.
2. T-GURT: A second type of GURT modifies a crop in such a way that the genetic enhancement engineered into the crop does not function until the crop plant is treated with a chemical that is sold by the biotechnology company. Farmers can save seeds for use each year. However, they do not get to use the enhanced trait in the crop unless they purchase the activator compound. The technology is restricted at the trait level - hence the term T-GURT.

Possible advantages

Where effective intellectual property protection systems don't exist or are not enforced, GURTs could be an alternative to stimulate plant developing activities by biotech firms.

Non-viable seeds produced on V-GURT plants will reduce the propagation of volunteer plants. Volunteer plants can become an economic problem for larger-scale mechanized farming systems that incorporate crop rotation.

Under warm, wet harvest conditions non V-GURT grain can sprout, which lowers the quality of grain produced. It is speculated[weasel words] that this problem would not occur with the use of V-GURT grain varieties.

Use of V-GURT technology could prevent escape of transgenes into wild relatives and prevent any impact on biodiversity. Crops modified to produce non-food products could be armed with GURT technology to prevent accidental transmission of these traits into crops destined for foods.

Possible disadvantages

There is a concern that V-GURT plants could cross-pollinate with non-genetically modified plants, either in the wild or on the fields of farmers who do not adopt the technology. Though the V-GURT plants are supposed to produce sterile seeds, there is concern that this trait will not be expressed in the first generation of a small percentage of these plants, but be expressed in later generations. This does not seem to be much of a problem in the wild, as a sterile plant would naturally be selected out of a population within one generation of trait expression.

As with all genetically modified crops, the food safety of GURT technology would need to be assessed if a commercial release of a GURT containing crop were proposed.

Initially developed by the US Department of Agriculture and multinational seed companies, “suicide seeds” have not been commercialized anywhere in the world due to an avalanche of opposition from farmers, indigenous peoples, civil society, and some governments[which?]. In 2000, the United Nations Convention on Biological Diversity recommended a de facto moratorium on field-testing and commercial sale of Terminator seeds; the moratorium was re-affirmed in 2006. India and Brazil have already passed national laws to prohibit the technology

Bionic architecture

2009-08-18T21:25:00.002-07:00

Bionic architecture is a movement for the design and construction of expressive buildings whose layout and lines borrow from natural (i.e. biological) forms. The movement began to mature in the early 21st century, and thus in early designs research was stressed over practicality. Bionic architecture sets itself in opposition to traditional rectangular layouts and design schemes by using curved forms and surfaces reminiscent of structures in biology and fractal mathematics. One of the tasks set themselves by the movement's early pioneers was the development of aesthetic and economic justifications for their approach to architecture.

Amgen

2009-08-18T21:25:00.001-07:00

Amgen Inc. (NASDAQ: AMGN, SEHK: 4332) is an international biotechnology company headquartered in Thousand Oaks, California. Located in the Conejo Valley, Amgen is the largest independent biotech firm. The company employs approximately 14,000 staff members including the 125 Allied-Barton Security staff and A-post personnel in 2007. Its products include Epogen, Aranesp, Enbrel, Kineret, Neulasta, Neupogen, Sensipar / Mimpara and Nplate. Epogen and Neupogen (the company's first products on the market) were the two most successful biopharmaceutical products at the time of their respective releases.

BusinessWeek ranked Amgen fourth on the S&P 500 for being the most "future-oriented" of those five hundred corporations. BusinessWeek ostensibly calculated the ratio of research and development spending, combined with capital spending, to total outlays; Amgen had the fourth highest ratio, at 506:1000.

Amgen is the largest employer in Thousand Oaks and second only to the United States Navy in terms of number of people employed in Ventura County. Amgen is also a member of the Pennsylvania Bio commerce organization.

With plans to expand into a new campus under construction in South San Francisco, Amgen abruptly halted construction on the plans and instead put the 365,000 square feet (33,900 m2) of new space on the sublease market.

In 2006, Amgen began sponsoring the Tour of California, one of only three major Union Cycliste Internationale events in the United States.

History

The word AMGen is a portmanteau of the company's original name, Applied Molecular Genetics, which became the official name of the company in 1983 (three years after incorporation and coincident with its initial public offering). The company's first chief executive officer, from 1980, was George B. Rathmann, followed by Gordon M. Binder in 1988, followed by Kevin W. Sharer in 2000. The company has made at least five major corporate acquisitions.

Agrobacterium

2009-08-18T21:23:00.000-07:00

Agrobacterium is a genus of Gram-negative bacteria that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus. Agrobacterium is well known for its ability to transfer DNA between itself and plants, and for this reason it has become an important tool for plant improvement by genetic engineering.

The Agrobacterium genus is quite heterogeneous. Recent taxonomic studies have reclassified all of the Agrobacterium species into new genera, such as Ruegeria, Pseudorhodobacter and Stappia, but most species have been reclassified as Rhizobium species.

Plant pathogen

A. tumefaciens causes crown-gall disease in plants. The disease is characterised by a tumour-like growth or gall on the infected plant, often at the junction between the root and the shoot. Tumors are incited by the conjugative transfer of a DNA segment (T-DNA) from the bacterial tumour-inducing (Ti) plasmid. The closely related species, A. rhizogenes, induces root tumors, and carries the distinct Ri (root-inducing) plasmid. Although the taxonomy of Agrobacterium is currently under revision it can be generalised that 3 biovars exist within the genus, A. tumefaciens or biovar 1, A. rhizogenes or biovar 2, and A. vitis or biovar 3. Strains within biovars 1 and 2 are known to be able to harbour either a Ti or Ri-plasmid, whilst strains of biovar 3, generally restricted to grapevines, can harbour a Ti-plasmid. Non-Agrobacterium strains have been isolated from environmental samples which harbour a Ri-plasmid whilst laboratory studies have shown that non-Agrobacterium strains can also harbour a Ti-plasmid. Many environmental strains of Agrobacterium do not possess either a Ti or Ri-plasmid. These strains are avirulent.

The plasmid T-DNA is integrated semi-randomly into the genome of the host cell (Francis and Spiker, 2005. Plant Journal. 41(3): 464.), and the virulence (vir) genes on the T-DNA are expressed, causing the formation of a gall. The T-DNA carries genes for the biosynthetic enzymes for the production of unusual amino acids, typically octopine or nopaline. It also carries genes for the biosynthesis of the plant hormones, auxin and cytokinins. By altering the hormone balance in the plant cell, the division of those cells cannot be controlled by the plant, and tumors form. The ratio of auxin to cytokinin produced by the tumor genes determines the morphology of the tumor (root-like, disorganized or shoot-like).

Agrobacterium in humans

Although generally seen as an infection in plants, Agrobacterium can be responsible for opportunistic infections in humans with weakened immune systems, but has not been shown to be a primary pathogen in otherwise healthy individuals. A 2000 study published by the National Academy of Sciences suggested that Agrobacterium attaches to and genetically transforms several types of human cells by integrating its T-DNA into the human cell genome. The study was conducted under laboratory conditions and states that it does not draw any conclusions regarding related biological activity in nature.

There is a conjectured connection with Morgellons syndrome. Dr. Stricker, along with Dr. Citovsky, MRF board member from the State University of New York at Stony Brook and an expert on plant pathogens, reported in January, 2007, that Morgellons skin fibers appear to contain cellulose. Five skin samples of Morgellons patients contained evidence of DNA from Agrobacterium.

Uses in biotechnology

The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement. A modified Ti or Ri plasmid can be used. The plasmid is 'disarmed' by deletion of the tumor inducing genes; the only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation. Marc Van Montagu and Jozef Schell at the University of Ghent (Belgium) discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter Agrobacterium into an efficient delivery system for gene engineering in plants. A team of researchers led by Dr Mary-Dell Chilton were the first to demonstrate that the virulence genes could be removed without adversely affecting the ability of Agrobacterium to insert its own DNA into the plant genome (1983).

The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die. An alternative method is agroinfiltration.
Plant (S. chacoense) transformed using Agrobacterium. Transformed cells start forming calluses on the side the leaf pieces

Transformation with Agrobacterium can be achieved in two ways. Protoplasts, or leaf-discs can be incubated with the Agrobacterium and whole plants regenerated using plant tissue culture. A common transformation protocol for Arabidopsis is the floral-dip method: the flowers are dipped in an Agrobacterium culture, and the bacterium transforms the germline cells that make the female gametes. The seeds can then be screened for antibiotic resistance (or another marker of interest), and plants that have not integrated the plasmid DNA will die.

Agrobacterium does not infect all plant species, but there are several other effective techniques for plant transformation including the gene gun.

Agrobacterium is listed as being the original source of genetic material that was transferred to these USA GMO foods :

* Soybean
* Cotton
* Corn
* Sugar Beet
* Alfalfa
* Wheat
* Rapeseed Oil (Canola)
* Creeping bentgrass (for animal feed)

Genomics

The sequencing of the genomes of several species of Agrobacterium has permitted the study of the evolutionary history of these organisms and has provided information on the genes and systems involved in pathogenesis, biological control and symbiosis. One important finding is the possibility that chromosomes are evolving from plasmids in many of these bacteria. Another discovery is that the diverse chromosomal structures in this group appear to be capable of supporting both symbiotic and pathogenic lifestyles. The availability of the genome sequences of Agrobacterium species will continue to increase, resulting in substantial insights into the function and evolutionary history of this group of plant-associated microbes.

Agricultural Biotechnology

2009-08-18T21:20:00.000-07:00

With increase in population and concern about the quality of food, the bio-agriculture has gained focus in the recent past in India. The farmers in India are looking at the GM seeds, biofertilizers, biopesticides from which they can expect more return on their investments and also increase productivity. The farmers are now getting a premium for the organic produce. With this now they are able to export their produce at much higher price. Lot of research initiatives are also being undertaken in the field to explore new technologies and new tie ups and joint ventures are being set up and are getting approvals from government for exploiting the technologies for the betterment of farmers and obtaining decent returns

GM Seeds
The challenge of producing more food grains to feed the ever increasing population of India that has already crossed one billion mark with less resources has bought companies like Mahyco, Monsanto, Syngenta, ProAgro, Advanta to invest in GM crops. It was in 2002 a joint venture between Mahyco and Monsanto called Mahyco- Monsanto Biotech Ltd got the green signal from the Government of India for the commercial production and sale of Bt cotton (Bollgard) in six southern states of India.

A lot of awareness campaigns have to be conducted to reach out to the farmers to brief them about the benefits of using seeds that are resistant to pests, diseases, herbicides, and crops which are tolerant to drought, cold, salinity and other harsh environments. This will bring in confidence among the farmers as well as the industry people.

Biofertilizers, biopesticides
In addition to GM seeds, the farmers are also looking at biofertilizers, biopesticides to get more benefits. Now the farmers are using formulations based on Bt, viruses like NPV, and GV, as well as neem-based pesticides. To meet the increasing demand, the industry has to scale up investments in biofertilizers and biopesticides. Conservative estimate shows that the 10 percent saving through the use of biofertilizers will result in an annual saving of 1.094 million tons of nitrogenous fertilizers costing around Rs 550 crore.

Biofuel
Looking at the opportunity in biofuel sector, the central government has taken an initiative to promote this sector in a big way. The total consumption of ethanol-blended petrol is expected to be 4.6 million tons per year. This sector not only helps sugarcane farmers, as cane is used as raw material for production of ethanol, but also helps in building up the oil security apart from benefiting the environment. It can save foreign exchange to the tune of Rs 80,000 crore, as India imports about 70 percent of its requirement of crude oil.

Affymetrix

2009-08-18T21:19:00.000-07:00

Affymetrix (NASDAQ: AFFX) is a manufacturer of DNA microarrays, based in Santa Clara, California, United States. The company was co-founded by Dr. Stephen Fodor in 1992. The company was begun as a unit in Affymax N.V. in 1991 by Fodor's group, which had in the late 1980s developed methods for fabricating DNA microarrays, called "GeneChips" according to the Affymetrix trademark, using semiconductor manufacturing techniques. The company's first product, an HIV genotyping GeneChip, was introduced in 1994 and the company went public in 1996. As a result of their pioneering work and the ensuing popularity of microarray products, Affymetrix derives significant benefit from its patent portfolio in this area.

Acquisitions have included Genetic MicroSystems for slide-based microarrays and scanners, Neomorphic for bioinformatics, ParAllele Bioscience for custom SNP genotyping, USB/Anatrace for biochemical reagents, and Panomics and True Materials to expand its offering of low to mid-plex applications. In 2000 Perlegen Sciences was spun out to focus on wafer-scale genomics for massive data creation and collection required for characterizing population variance of genomic markers and expression for the drug discovery process.

Description of product

Affymetrix makes quartz chips for analysis of DNA Microarrays. These chips are sold under the trademarked name GeneChip. Affymetrix's GeneChips assist researchers in quickly scanning for the presence of particular genes in a biological sample. Within this area, Affymetrix is focused on oligonucleotide microarrays. These microarrays are used to determine which genes exist in a sample by detecting specific pieces of mRNA. A single chip can be used to do thousands of experiments in parallel. Chips can be used only once.

Affymetrix sells both mass produced GeneChips intended to match scientifically important parts of human and other animal genomes. Affymetrix manufactures its GeneChips using photolithography. The company also manufactures machinery for high speed analysis of biological samples.

Affymetrix GeneChip Operating Software is a software system for managing Affymetrix microarray data.

Competitors in the DNA Microarray business include Illumina, GE Healthcare, Applied Biosystems, Beckman Coulter, Eppendorf Biochip Systems, and Agilent. There are also various inexpensive plastic-based technologies under development in small companies and laboratories around the world. It has been widely speculated that mass-produced plastic chips can be produced at lower prices than Affymetrix's quartz chips.

Affymetrix has established a licensing program to make its intellectual property accessible to stimulate the broad commercialization of genome analysis technologies. They have several collaboration relationships with other companies that utilize their patented GeneChip technology.

Future Innovations & Direction

It is unknown whether or not Affymetrix is currently focusing any effort on full genome sequencing, which is thought to be the next major breakthrough in genetic technology and could significantly disrupt the array markets. It's competitor, Illumina, as well as a number of private companies, such as Pacific Biosciences and Complete Genomics have been heavily invested in the race to commercialize full genome sequencing for a number of years. Complete Genomics has stated that they will be able to provide a full genome sequencing service for $5,000 by the summer of 2009. In June 2009, Illumina, Affymetrix's primary competitor, announced that they were launching their own Personal Full Genome Sequencing Service at a depth of 30X for $48,000 per genome. This is still too expensive for true commercialization but the price will most likely decrease substantially over the next few years as they realize economies of scale and given the competition with other companies such as Complete Genomics.

Variants of Biotechnology

2009-08-12T00:33:00.002-07:00

Biotechnology is the use of biological processes, organisms, or systems to manufacture products intended to improve the quality of human life. The earliest biotechnologists were farmers who developed improved species of plants and animals by cross pollenization or cross breeding. In recent years, biotechnology has expanded in sophistication, scope, and applicability in various sectors of modern economy.

The science of biotechnology can be broken down into different sub-disciplines called red, white, green, and blue.

Red biotechnology involves medical processes such as getting organisms to produce new drugs, or using stem cells to regenerate damaged human tissues and perhaps re-grow entire organs, designing of organisms to produce antibiotics, and the engineering of genetic cures through genomic manipulation, DNA fingerprinting to achieve advancements in understanding human evolution and origin of various diseases, tissue culture for detection of diseases etc. are examples of red biotechnology.

White biotechnology, also known as grey biotechnology, is biotechnology applied to industrial processes. An example is the designing of an organism to produce a useful chemical. White biotechnology tends to consume less in resources than traditional processes used to produce industrial goods. Fermentation process which is used for making bread and other eatables is an example of white biotechnology.

Green biotechnology is biotechnology applied to agricultural processes. An example is the designing of transgenic plants to grow under specific environmental conditions or in the presence (or absence) of certain agricultural chemicals. One hope is that green biotechnology might produce more environmentally friendly solutions than traditional industrial agriculture. An example of this is the engineering of a plant to express a pesticide, thereby eliminating the need for external application of pesticides. An example of this would be Bt corn. Green revolution in India is a successful example of green biotechnology, resulting in increased yield and productivity and self sufficiency in food production. Introduction of various biofertilsers and biopesticides are other examples of green biotechnology.

Bioinformatics is an interdisciplinary field which addresses biological problems using computational techniques. The field is also often referred to as computational biology. It plays a key role in various areas, such as functional genomics, structural genomics, and proteomics, and forms a key component in the biotechnology and pharmaceutical sector. This is the sunrise industry in the field with growth of IT industry.

Plant Production Biotechnology

2009-08-12T00:33:00.001-07:00

There is scarcely any aspect of plant production that will not undergo profound changes as a result of the application of biotechnology. Commercial applications of plant genetic engineering have not yet occurred. At the present time, more traditional aspects of biotechnology such as tissue culture have had an important impact, especially in the acceleration of the breeding process for new varieties and in the multiplication of disease-free seed material.

Provision of seeds: Plant breeding has been enhanced considerably by in vitro development of improved varieties which are better adapted to a specific environment. The application of tissue culture has several advantages, including rapid reproduction and multiplication, availability of seed material throughout the year etc. Since the application of tissue culture does not require very expensive equipment, this technology can be applied easily in developing countries and can help to improve local varieties of food-crops. For example, using traditional methods for propagating potatoes.

Reduced use of agrochemicals: Biotechnology can help reduce the need for agrochemicals which small farmers in developing countries often cannot afford. A reduction in the use of agrochemicals implies fewer residues in the final product. This is expected to enhance the productivity and land fertility as well as reduction in toxic elements in crops.

Increased production: Biotechnology can be used in many ways to achieve higher yields; for example by improving flowering capacity and increasing photosynthesis or the intake of nutritive elements. Productivity increases may lead to lower prices¸ which is a vital policy objective in many a developing nation.

Improved harvesting: The cloning of plants can help to reduce the work necessary for harvesting. When individual plants show more uniform characteristics, grow at the same speed and ripen at the same time, harvesting will be less laborious. A reduction in the workload is not only an objective in highly industrialized countries, it can also be very important for small farmers in developing countries.

Improved storage: Food shortages would not exist in many countries if the problem of post-harvest losses could be solved. In the future, genetic engineering may be used to remove plant components that cause early deterioration of the harvest. For instance, a technique to reduce the presence of a normal tomato enzyme involved in the softening of ripe tomato fruit has been patented and would be found very useful for enhancing the shelf life of crops of various varieties.

Molecular Biotechnology

2009-08-12T00:31:00.000-07:00

Perhaps the most potent use of biotechnology for development of human well being has been in the field of molecular biotechnology. The powerful revolution in medicine during the past decade has been in the field of genomic research that has completely transformed conventional medicine into molecular medicine. The remarkable unveiling of virtually complete Human Genome in June, 2000, and release of the genetic code in February, 2001, has been instrumental in unraveling path breaking research and wonder drugs and application for scientific community. While it has created jubilation amongst medical professionals across the globe, but at the same time, the intended applications raised several ethical, legal and issues social too (ELSI).

Advances in genomic medicine research in India in the field of cancer genomics, vaccinology, microbial genomics, pharmacogenomics, vector genomics, neurogenetics and molecular basis of diseases have resulted in development of new drugs and finding new cures for ailments which were considered to be fatal in the past.

While new developments are taking place in the field, the need to further upgrade the proven technologies such as diagnostics and vaccines and make them use for application is also emerging. It is here that the Indian Industry will have to put extra efforts to benefit out of biotechnology revolution. Thus it is seen that while the Indian industry is strong in product development and marketing for commercial benefits, biotech in India still lacks the infrastructure required for R&D in molecular modelling, protein engineering, drug designing and immunological studies. This issue needs to be addressed immediately to gain out of research initiatives.

Another aspect worth considering is that various technologies in the field of molecular biotechnologies have the potential to improve productivity and increase the number and quality of new drugs by validating more genomically diverse and higher quality drug targets and speeding-up clinical development by designing better trials that clearly show better safety, efficacy and compliance. As per an estimate, by improvising medical outcomes by use of well developed drugs and diagnostics, pharmaceutical companies could benefit to the order of US$ 200-500 million in extra revenue for each drug. Apex scientific bodies in India e.g. CSIR, ICMR, DBT have launched country-wide programmes to identify and characterize new drug targets, especially in the area of tuberculosis, malaria, leishmania etc., besides new drug targets for diabetes, cardiovascular and neurological disorders. In addition, there is also in the pipeline a proposal to undertake single nucleotide polymorphism (SNP) mapping in over 500 genes to identify and characterize in Indian population, the genes linked to susceptibility to malaria, TB, diabetes and some cardiovascular and neurological disorders, which are more common in Indian context.

Nanobiotechnology

2009-01-07T04:49:00.001-08:00

Nanobiotechnology is the branch of nanotechnology with biological and biochemical applications or uses. Nanobiotechnology often studies existing elements of nature in order to fabricate new devices.

The term bionanotechnology is often used interchangeably with nanobiotechnology, though a distinction is sometimes drawn between the two. If the two are distinguished, nanobiotechnology usually refers to the use of nanotechnology to further the goals of biotechnology, while bionanotechnology might refer to any overlap between biology and nanotechnology, including the use of biomolecules as part of or as an inspiration for nanotechnological devices.

Examples

One example of current nanobiotechnological research involves nanospheres coated with fluorescent polymers. Researchers are seeking to design polymers whose fluorescence is quenched when they encounter specific molecules. Different polymers would detect different metabolites. The polymer-coated spheres could become part of new biological assays, and the technology might someday lead to particles that could be introduced into the human body to track down metabolites associated with tumors and other health problems.

Antibody-Nanoparticle Computational Modeling

The conjugation of antibodies and nanoparticles with high affinity & specificity through receptor-ligand recognition modes is of paramount importance in the development of vehicles which can be used for diagnosis, treatment of cancer and various other diseases, application of immudiagnostic nano-biosensors etc. The bio-nanocomplex formed by an artificial nanomaterial (nanoliposomes , nanoparticles ) and a biological entity such as an antibody is brought about by the formation of covalent bonds based on their specific chemical and structural properties such as water solubility, biocompatibility, and biodegradability. There is a requirement of a comprehensive understanding of the relationship of the thermodynamic & kinetic aspects of antibody-membrane association, translational , rotational mobilities of membrane bound antibodies, interactions with the diverse cell surface , circulating molecules and various artificial nanomolecules as well as the conformation. These details are of great importance in the development, application of various nanoscale immunodiagnostic devices. The association of antibodies with cell surfaces is a key molecular event in antibody-mediated immune mechanisms such as phagocytosis, antibody mediated immune dependent cell-mediated cytotoxicity.

The interfacial properties, especially the dynamic, thermodynamic, and mechanical properties, at different spatial and temporal resolutions of these bio-nano systems can be readily investigated with the aid of computer simulations, which consist of studies of interactions of the proteins as well as those of various nanomaterials with organic biological molecules such as proteins, nucleic acids, membrane lipids, and water and of significance importance is the study of the interactions of nanoparticles in the protein binding sites and optimization of the same for improved bio-nano recognition. Recently it has been noted that there exists certain natural proteins, antibodies, that can recognize specific nanoparticles . For example, a specific antibody from the mouse immune system can specifically recognize derivatized C60 fullerenes with a binding affinity of about 25 nM . From the studies carried out by Noon et al, it is hypothized that the fullerene-binding site is formed at the interface of the light and heavy chains lined with a cluster of shape-complementary hydrophobic amino acid residues. As the covalent modifications of the functionalized fullerenes, occupy only a small fraction of the particle surface area , the largely unoccupied surface would be free to interact with the antibody. Therefore, in order to gain in-depth understanding of the detailed interactions of the nps and the antibody, molecular dynamics simulation is carried out using molecular dynamics simulation; the purpose of our theoretical modeling studies is to be able to identify the energetically favorable binding modes.

For the modeling study, the initial coordinates of the antibody can be made available from the Protein Data Bank (PDB). The coordinates of the nanoparticle in this case , would be obtained from the AFM, TEM studies carried out at the AMERI and Nano-biotechnology laboratory, FIU, Miami. The CHARMm (Chemistry at HARvard Macromolecular Mechanics) an Unix-based commercialized software using Fortran 77 source codes uses set of force fields for molecular dynamics for simulation and analysis.

The basic assumptions, as a first approximation, during the modeling study would be that the hydrophilic derivatizations do not play a critical role in the predominantly hydrophobic nanomaterial-antibody interactions and that the electronic structure remains undisturbed during the conjugation. The nanoparticle is docked into a suggested binding site from the previously done literature studies. Polar-hydrogen potential function (PARAM19) and a modified TIP3P water solvent model for the protein is used.

The simulation involves approximately about 300 steps of minimization, using the Steepest Descent and the Newton Raphson method. To reduce the necessary simulation time, a highly efficient method for simulating the localized interactions in the active site of a protein, the stochastic boundary molecular dynamics (SBMD) is used. The reference point for partitioning the system in SBMD was chosen to be near the center of the nanomaterials, which is assumed to be an uniform sphere. The complex nano-bio system can be assumed to be separated into spherical reservoir and reaction zones; the latter is further sub-divided into a reaction region and a buffer region. The atoms in the reaction region are propagated by molecular dynamics, whereas those in the buffer region involve Langevin dynamics are retained using harmonic restoring forces.

Agriculture (Biotechnology)

2009-01-07T04:48:00.001-08:00

Responsible biotechnology is not the enemy; starvation is. Without adequate food supplies at affordable prices, we cannot expect world health or peace.
—Jimmy Carter, Former President of the United States, 11 Jul 1997,

Improve yield from crops

Using the techniques of modern biotechnology, one or two genes may be transferred to a highly developed crop variety to impart a new character that would increase its yield. However, while increases in crop yield are the most obvious applications of modern biotechnology in agriculture, it is also the most difficult one. Current genetic engineering techniques work best for effects that are controlled by a single gene. Many of the genetic characteristics associated with yield (e.g., enhanced growth) are controlled by a large number of genes, each of which has a minimal effect on the overall yield. There is, therefore, much scientific work to be done in this area.

Reduced vulnerability of crops to environmental stresses

Crops containing genes that will enable them to withstand biotic and abiotic stresses may be developed. For example, drought and excessively salty soil are two important limiting factors in crop productivity. Biotechnologists are studying plants that can cope with these extreme conditions in the hope of finding the genes that enable them to do so and eventually transferring these genes to the more desirable crops. One of the latest developments is the identification of a plant gene, At-DBF2, from thale cress, a tiny weed that is often used for plant research because it is very easy to grow and its genetic code is well mapped out. When this gene was inserted into tomato and tobacco cells (see RNA interference), the cells were able to withstand environmental stresses like salt, drought, cold and heat, far more than ordinary cells. If these preliminary results prove successful in larger trials, then At-DBF2 genes can help in engineering crops that can better withstand harsh environments. Researchers have also created transgenic rice plants that are resistant to rice yellow mottle virus (RYMV). In Africa, this virus destroys majority of the rice crops and makes the surviving plants more susceptible to fungal infections.

Increased nutritional qualities &quantity of food crops

Proteins in foods may be modified to increase their nutritional qualities. Proteins in legumes and cereals may be transformed to provide the amino acids needed by human beings for a balanced diet. A good example is the work of Professors Ingo Potrykus and Peter Beyer on the so-called Golden rice (discussed below).

Improved taste, texture or appearance of food

Modern biotechnology can be used to slow down the process of spoilage so that fruit can ripen longer on the plant and then be transported to the consumer with a still reasonable shelf life. This improves the taste, texture and appearance of the fruit. More importantly, it could expand the market for farmers in developing countries due to the reduction in spoilage.

The first genetically modified food product was a tomato which was transformed to delay its ripening. Researchers in Indonesia, Malaysia, Thailand, Philippines and Vietnam are currently working on delayed-ripening papaya in collaboration with the University of Nottingham and Zeneca.

Biotechnology in cheese production: enzymes produced by micro-organisms provide an alternative to animal rennet – a cheese coagulant - and an alternative supply for cheese makers. This also eliminates possible public concerns with animal-derived material, although there is currently no plans to develop synthetic milk, thus making this argument less compelling. Enzymes offer an animal-friendly alternative to animal rennet. While providing comparable quality, they are theoretically also less expensive.

About 85 million tons of wheat flour is used every year to bake bread. By adding an enzyme called maltogenic amylase to the flour, bread stays fresher longer. Assuming that 10-15% of bread is thrown away, if it could just stay fresh another 5–7 days then 2 million tons of flour per year would be saved. That corresponds to 40% of the bread consumed in a country such as the USA. This means more bread becomes available with no increase in input. In combination with other enzymes, bread can also be made bigger, more appetizing and better in a range of ways.

Reduced dependence on fertilizers, pesticides and other agrochemicals

Most of the current commercial applications of modern biotechnology in agriculture are on reducing the dependence of farmers on agrochemicals. For example, Bacillus thuringiensis (Bt) is a soil bacterium that produces a protein with insecticidal qualities. Traditionally, a fermentation process has been used to produce an insecticidal spray from these bacteria. In this form, the Bt toxin occurs as an inactive protoxin, which requires digestion by an insect to be effective. There are several Bt toxins and each one is specific to certain target insects. Crop plants have now been engineered to contain and express the genes for Bt toxin, which they produce in its active form. When a susceptible insect ingests the transgenic crop cultivar expressing the Bt protein, it stops feeding and soon thereafter dies as a result of the Bt toxin binding to its gut wall. Bt corn is now commercially available in a number of countries to control corn borer (a lepidopteran insect), which is otherwise controlled by spraying (a more difficult process).

Crops have also been genetically engineered to acquire tolerance to broad-spectrum herbicide. The lack of cost-effective herbicides with broad-spectrum activity and no crop injury was a consistent limitation in crop weed management. Multiple applications of numerous herbicides were routinely used to control a wide range of weed species detrimental to agronomic crops. Weed management tended to rely on preemergence — that is, herbicide applications were sprayed in response to expected weed infestations rather than in response to actual weeds present. Mechanical cultivation and hand weeding were often necessary to control weeds not controlled by herbicide applications. The introduction of herbicide tolerant crops has the potential of reducing the number of herbicide active ingredients used for weed management, reducing the number of herbicide applications made during a season, and increasing yield due to improved weed management and less crop injury. Transgenic crops that express tolerance to glyphosate, glufosinate and bromoxynil have been developed. These herbicides can now be sprayed on transgenic crops without inflicting damage on the crops while killing nearby weeds.

From 1996 to 2001, herbicide tolerance was the most dominant trait introduced to commercially available transgenic crops, followed by insect resistance. In 2001, herbicide tolerance deployed in soybean, corn and cotton accounted for 77% of the 626,000 square kilometres planted to transgenic crops; Bt crops accounted for 15%; and "stacked genes" for herbicide tolerance and insect resistance used in both cotton and corn accounted for 8%.

Production of novel substances in crop plants

Biotechnology is being applied for novel uses other than food. For example, oilseed can be modified to produce fatty acids for detergents, substitute fuels and petrochemicals.[citation needed] Potatoes, tomatos, rice, tobacco, lettuce, safflowers, and other plants have been genetically-engineered to produce insulin[citation needed] and certain vaccines. If future clinical trials prove successful, the advantages of edible vaccines would be enormous, especially for developing countries. The transgenic plants may be grown locally and cheaply. Homegrown vaccines would also avoid logistical and economic problems posed by having to transport traditional preparations over long distances and keeping them cold while in transit. And since they are edible, they will not need syringes, which are not only an additional expense in the traditional vaccine preparations but also a source of infections if contaminated. In the case of insulin grown in transgenic plants, it is well-established that the gastrointestinal system breaks the protein down therefore this could not currently be administered as an edible protein. However, it might be produced at significantly lower cost than insulin produced in costly, bioreactors. For example, Calgary, Canada-based SemBioSys Genetics, Inc. reports that its safflower-produced insulin will reduce unit costs by over 25% or more and reduce the capital costs associated with building a commercial-scale insulin manufacturing facility by approximately over $100 million compared to traditional biomanufacturing facilities.

Gene therapy (Biotechnology)

2009-01-07T04:47:00.001-08:00

Gene therapy using an Adenovirus vector. A new gene is inserted into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein.

Gene therapy may be used for treating, or even curing, genetic and acquired diseases like cancer and AIDS by using normal genes to supplement or replace defective genes or to bolster a normal function such as immunity. It can be used to target somatic (i.e., body) or germ (i.e., egg and sperm) cells. In somatic gene therapy, the genome of the recipient is changed, but this change is not passed along to the next generation. In contrast, in germline gene therapy, the egg and sperm cells of the parents are changed for the purpose of passing on the changes to their offspring.

There are basically two ways of implementing a gene therapy treatment:

1. Ex vivo, which means “outside the body” – Cells from the patient’s blood or bone marrow are removed and grown in the laboratory. They are then exposed to a virus carrying the desired gene. The virus enters the cells, and the desired gene becomes part of the DNA of the cells. The cells are allowed to grow in the laboratory before being returned to the patient by injection into a vein.
2. In vivo, which means “inside the body” – No cells are removed from the patient’s body. Instead, vectors are used to deliver the desired gene to cells in the patient’s body.

Currently, the use of gene therapy is limited. Somatic gene therapy is primarily at the experimental stage. Germline therapy is the subject of much discussion but it is not being actively investigated in larger animals and human beings.

As of June 2001, more than 500 clinical gene-therapy trials involving about 3,500 patients have been identified worldwide. Around 78% of these are in the United States, with Europe having 18%. These trials focus on various types of cancer, although other multigenic diseases are being studied as well. Recently, two children born with severe combined immunodeficiency disorder (“SCID”) were reported to have been cured after being given genetically engineered cells.

Gene therapy faces many obstacles before it can become a practical approach for treating disease. At least four of these obstacles are as follows:

1. Gene delivery tools. Genes are inserted into the body using gene carriers called vectors. The most common vectors now are viruses, which have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists manipulate the genome of the virus by removing the disease-causing genes and inserting the therapeutic genes. However, while viruses are effective, they can introduce problems like toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, in order for gene therapy to provide permanent therapeutic effects, the introduced gene needs to be integrated within the host cell's genome. Some viral vectors effect this in a random fashion, which can introduce other problems such as disruption of an endogenous host gene.
2. High costs. Since gene therapy is relatively new and at an experimental stage, it is an expensive treatment to undertake. This explains why current studies are focused on illnesses commonly found in developed countries, where more people can afford to pay for treatment. It may take decades before developing countries can take advantage of this technology.
3. Limited knowledge of the functions of genes. Scientists currently know the functions of only a few genes. Hence, gene therapy can address only some genes that cause a particular disease. Worse, it is not known exactly whether genes have more than one function, which creates uncertainty as to whether replacing such genes is indeed desirable.
4. Multigene disorders and effect of environment. Most genetic disorders involve more than one gene. Moreover, most diseases involve the interaction of several genes and the environment. For example, many people with cancer not only inherit the disease gene for the disorder, but may have also failed to inherit specific tumor suppressor genes. Diet, exercise, smoking and other environmental factors may have also contributed to their disease.

Human Genome Project

The Human Genome Project is an initiative of the U.S. Department of Energy (“DOE”) that aims to generate a high-quality reference sequence for the entire human genome and identify all the human genes.

The DOE and its predecessor agencies were assigned by the U.S. Congress to develop new energy resources and technologies and to pursue a deeper understanding of potential health and environmental risks posed by their production and use. In 1986, the DOE announced its Human Genome Initiative. Shortly thereafter, the DOE and National Institutes of Health developed a plan for a joint Human Genome Project (“HGP”), which officially began in 1990.

The HGP was originally planned to last 15 years. However, rapid technological advances and worldwide participation accelerated the completion date to 2003 (making it a 13 year project). Already it has enabled gene hunters to pinpoint genes associated with more than 30 disorders.

Cloning

Cloning involves the removal of the nucleus from one cell and its placement in an unfertilized egg cell whose nucleus has either been deactivated or removed.

There are two types of cloning:

1. Reproductive cloning. After a few divisions, the egg cell is placed into a uterus where it is allowed to develop into a fetus that is genetically identical to the donor of the original nucleus.
2. Therapeutic cloning. The egg is placed into a Petri dish where it develops into embryonic stem cells, which have shown potentials for treating several ailments.

In February 1997, cloning became the focus of media attention when Ian Wilmut and his colleagues at the Roslin Institute announced the successful cloning of a sheep, named Dolly, from the mammary glands of an adult female. The cloning of Dolly made it apparent to many that the techniques used to produce her could someday be used to clone human beings. This stirred a lot of controversy because of its ethical implications.

Genetic testing (Biotechnology)

2009-01-07T04:46:00.000-08:00

There are two major types of gene tests. In the first type, a researcher may design short pieces of DNA (“probes”) whose sequences are complementary to the mutated sequences. These probes will seek their complement among the base pairs of an individual’s genome. If the mutated sequence is present in the patient’s genome, the probe will bind to it and flag the mutation. In the second type, a researcher may conduct the gene test by comparing the sequence of DNA bases in a patient’s gene to disease in healthy individuals or their progeny.

Genetic testing is now used for:

* Carrier screening, or the identification of unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to manifest;
* Confirmational diagnosis of symptomatic individuals;
* Determining sex;
* Forensic/identity testing;
* Newborn screening;
* Prenatal diagnostic screening;
* Presymptomatic testing for estimating the risk of developing adult-onset cancers;
* Presymptomatic testing for predicting adult-onset disorders.

Some genetic tests are already available, although most of them are used in developed countries. The tests currently available can detect mutations associated with rare genetic disorders like cystic fibrosis, sickle cell anemia, and Huntington’s disease. Recently, tests have been developed to detect mutation for a handful of more complex conditions such as breast, ovarian, and colon cancers. However, gene tests may not detect every mutation associated with a particular condition because many are as yet undiscovered, and the ones they do detect may present different risks to different people and populations.

Controversial questions

Several issues have been raised regarding the use of genetic testing:

1. Absence of cure. There is still a lack of effective treatment or preventive measures for many diseases and conditions now being diagnosed or predicted using gene tests. Thus, revealing information about risk of a future disease that has no existing cure presents an ethical dilemma for medical practitioners.
2. Ownership and control of genetic information. Who will own and control genetic information, or information about genes, gene products, or inherited characteristics derived from an individual or a group of people like indigenous communities? At the macro level, there is a possibility of a genetic divide, with developing countries that do not have access to medical applications of biotechnology being deprived of benefits accruing from products derived from genes obtained from their own people. Moreover, genetic information can pose a risk for minority population groups as it can lead to group stigmatization.

At the individual level, the absence of privacy and anti-discrimination legal protections in most countries can lead to discrimination in employment or insurance or other misuse of personal genetic information. This raises questions such as whether genetic privacy is different from medical privacy.

1. Reproductive issues. These include the use of genetic information in reproductive decision-making and the possibility of genetically altering reproductive cells that may be passed on to future generations. For example, germline therapy forever changes the genetic make-up of an individual’s descendants. Thus, any error in technology or judgment may have far-reaching consequences. Ethical issues like designer babies and human cloning have also given rise to controversies between and among scientists and bioethicists, especially in the light of past abuses with eugenics.
2. Clinical issues. These center on the capabilities and limitations of doctors and other health-service providers, people identified with genetic conditions, and the general public in dealing with genetic information.
3. Effects on social institutions. Genetic tests reveal information about individuals and their families. Thus, test results can affect the dynamics within social institutions, particularly the family.
4. Conceptual and philosophical implications regarding human responsibility, free will vis-à-vis genetic determinism, and the concepts of health and disease.

Medicine (a biotechnological invention),drug production,pharmacogenomics,gene therapy,genetic testing

2009-01-07T04:43:00.000-08:00

In medicine, modern biotechnology finds promising applications in such areas as

* drug production;
* pharmacogenomics;
* gene therapy; and
* genetic testing;

Pharmacogenomics

Pharmacogenomics is the study of how the genetic inheritance of an individual affects his/her body’s response to drugs. It is a coined word derived from the words “pharmacology” and “genomics”. It is hence the study of the relationship between pharmaceuticals and genetics. The vision of pharmacogenomics is to be able to design and produce drugs that are adapted to each person’s genetic makeup.

Pharmacogenomics results in the following benefits:

1. Development of tailor-made medicines. Using pharmacogenomics, pharmaceutical companies can create drugs based on the proteins, enzymes and RNA molecules that are associated with specific genes and diseases. These tailor-made drugs promise not only to maximize therapeutic effects but also to decrease damage to nearby healthy cells.
2. More accurate methods of determining appropriate drug dosages. Knowing a patient’s genetics will enable doctors to determine how well his/ her body can process and metabolize a medicine. This will maximize the value of the medicine and decrease the likelihood of overdose.
3. Improvements in the drug discovery and approval process. The discovery of potential therapies will be made easier using genome targets. Genes have been associated with numerous diseases and disorders. With modern biotechnology, these genes can be used as targets for the development of effective new therapies, which could significantly shorten the drug discovery process.
4. Better vaccines. Safer vaccines can be designed and produced by organisms transformed by means of genetic engineering. These vaccines will elicit the immune response without the attendant risks of infection. They will be inexpensive, stable, easy to store, and capable of being engineered to carry several strains of pathogen at once.

Pharmaceutical products

Most traditional pharmaceutical drugs are relatively simple molecules that have been found primarily through trial and error to treat the symptoms of a disease or illness. Biopharmaceuticals are large biological molecules known as proteins and these usually target the underlying mechanisms and pathways of a malady (but not always, as is the case with using insulin to treat type 1 diabetes mellitus, as that treatment merely addresses the symptoms of the disease, not the underlying cause which is autoimmunity); it is a relatively young industry. They can deal with targets in humans that may not be accessible with traditional medicines. A patient typically is dosed with a small molecule via a tablet while a large molecule is typically injected.

Small molecules are manufactured by chemistry but larger molecules are created by living cells such as those found in the human body: for example, bacteria cells, yeast cells, animal or plant cells.

Modern biotechnology is often associated with the use of genetically altered microorganisms such as E. coli or yeast for the production of substances like synthetic insulin or antibiotics. It can also refer to transgenic animals or transgenic plants, such as Bt corn. Genetically altered mammalian cells, such as Chinese Hamster Ovary (CHO) cells, are also used to manufacture certain pharmaceuticals. Another promising new biotechnology application is the development of plant-made pharmaceuticals.

Biotechnology is also commonly associated with landmark breakthroughs in new medical therapies to treat hepatitis B, hepatitis C, cancers, arthritis, haemophilia, bone fractures, multiple sclerosis, and cardiovascular disorders. The biotechnology industry has also been instrumental in developing molecular diagnostic devices than can be used to define the target patient population for a given biopharmaceutical. Herceptin, for example, was the first drug approved for use with a matching diagnostic test and is used to treat breast cancer in women whose cancer cells express the protein HER2.

Modern biotechnology can be used to manufacture existing medicines relatively easily and cheaply. The first genetically engineered products were medicines designed to treat human diseases. To cite one example, in 1978 Genentech developed synthetic humanized insulin by joining its gene with a plasmid vector inserted into the bacterium Escherichia coli. Insulin, widely used for the treatment of diabetes, was previously extracted from the pancreas of abattoir animals (cattle and/or pigs). The resulting genetically engineered bacterium enabled the production of vast quantities of synthetic human insulin at relatively low cost[9], although the cost savings was used to increase profits for manufacturers, not passed on to consumers or their healthcare providers. According to a 2003 study undertaken by the International Diabetes Federation (IDF) on the access to and availability of insulin in its member countries, synthetic 'human' insulin is considerably more expensive in most countries where both synthetic 'human' and animal insulin are commercially available: e.g. within European countries the average price of synthetic 'human' insulin was twice as high as the price of pork insulin. Yet in its position statement, the IDF writes that "there is no overwhelming evidence to prefer one species of insulin over another" and "[modern, highly-purified] animal insulins remain a perfectly acceptable alternative.

Modern biotechnology has evolved, making it possible to produce more easily and relatively cheaply human growth hormone, clotting factors for hemophiliacs, fertility drugs, erythropoietin and other drugs. Most drugs today are based on about 500 molecular targets. Genomic knowledge of the genes involved in diseases, disease pathways, and drug-response sites are expected to lead to the discovery of thousands more new targets.

Applications of biotechnology

2009-01-07T04:42:00.001-08:00

Biotechnology has applications in four major industrial areas, including health care (medical), crop production and agriculture, non food (industrial) uses of crops and other products (e.g. biodegradable plastics, vegetable oil, biofuels), and environmental uses.

For example, one application of biotechnology is the directed use of organisms for the manufacture of organic products (examples include beer and milk products). Another example is using naturally present bacteria by the mining industry in bioleaching. Biotechnology is also used to recycle, treat waste, clean up sites contaminated by industrial activities (bioremediation), and also to produce biological weapons.

A series of derived terms have been coined to identify several branches of biotechnology, for example:

* Bioinformatics is an interdisciplinary field which addresses biological problems using computational techniques, and makes the rapid organization and analysis of biological data possible. The field may also be referred to as computational biology, and can be defined as, "conceptualizing biology in terms of molecules and then applying informatics techniques to understand and organize the information associated with these molecules, on a large scale." Bioinformatics plays a key role in various areas, such as functional genomics, structural genomics, and proteomics, and forms a key component in the biotechnology and pharmaceutical sector.
* Blue biotechnology is a term that has been used to describe the marine and aquatic applications of biotechnology, but its use is relatively rare.
* Green biotechnology is biotechnology applied to agricultural processes. An example would be the selection and domestication of plants via micropropagation. Another example is the designing of transgenic plants to grow under specific environmental conditions or in the presence (or absence) of certain agricultural chemicals. One hope is that green biotechnology might produce more environmentally friendly solutions than traditional industrial agriculture. An example of this is the engineering of a plant to express a pesticide, thereby eliminating the need for external application of pesticides. An example of this would be Bt corn. Whether or not green biotechnology products such as this are ultimately more environmentally friendly is a topic of considerable debate.
* Red biotechnology is applied to medical processes. Some examples are the designing of organisms to produce antibiotics, and the engineering of genetic cures through genomic manipulation.
* White biotechnology, also known as industrial biotechnology, is biotechnology applied to industrial processes. An example is the designing of an organism to produce a useful chemical. Another example is the using of enzymes as industrial catalysts to either produce valuable chemicals or destroy hazardous/polluting chemicals. White biotechnology tends to consume less in resources than traditional processes used to produce industrial goods.
* The investments and economic output of all of these types of applied biotechnologies form what has been described as the bioeconomy.

History of biotechnology

2009-01-07T04:41:00.001-08:00

The most practical use of biotechnology, which is still present today, is the cultivation of plants to produce food suitable to humans. Agriculture has been theorized to have become the dominant way of producing food since the Neolithic Revolution. The processes and methods of agriculture have been refined by other mechanical and biological sciences since its inception. Through early biotechnology, farmers were able to select the best suited and highest-yield crops to produce enough food to support a growing population. Other uses of biotechnology were required as crops and fields became increasingly large and difficult to maintain. Specific organisms and organism by-products were used to fertilize, restore nitrogen, and control pests. Throughout the use of agriculture, farmers have inadvertently altered the genetics of their crops through introducing them to new environments and breeding them with other plants--one of the first forms of biotechnology. Cultures such as those in Mesopotamia, Egypt, and Pakistan developed the process of brewing beer. It is still done by the same basic method of using malted grains (containing enzymes) to convert starch from grains into sugar and then adding specific yeasts to produce beer. In this process the carbohydrates in the grains were broken down into alcohols such as ethanol. Ancient Indians also used the juices of the plant Ephedra vulgaris and used to call it Soma. Later other cultures produced the process of Lactic acid fermentation which allowed the fermentation and preservation of other forms of food. Fermentation was also used in this time period to produce leavened bread. Although the process of fermentation was not fully understood until Louis Pasteur’s work in 1857, it is still the first use of biotechnology to convert a food source into another form.

Combinations of plants and other organisms were used as medications in many early civilizations. Since as early as 200 BC, people began to use disabled or minute amounts of infectious agents to immunize themselves against infections. These and similar processes have been refined in modern medicine and have led to many developments such as antibiotics, vaccines, and other methods of fighting sickness.

In the early twentieth century scientists gained a greater understanding of microbiology and explored ways of manufacturing specific products. In 1917, Chaim Weizmann first used a pure microbiological culture in an industrial process, that of manufacturing corn starch using Clostridium acetobutylicum, to produce acetone, which the United Kingdom desperately needed to manufacture explosives during World War I.

The field of modern biotechnology is thought to have largely begun on June 16, 1980, when the United States Supreme Court ruled that a genetically-modified microorganism could be patented in the case of Diamond v. Chakrabarty. Indian-born Ananda Chakrabarty, working for General Electric, had developed a bacterium (derived from the Pseudomonas genus) capable of breaking down crude oil, which he proposed to use in treating oil spills.

Revenue in the industry is expected to grow by 12.9% in 2008. Another factor influencing the biotechnology sector's success is improved intellectual property rights legislation -- and enforcement -- worldwide, as well as strengthened demand for medical and pharmaceutical products to cope with an ageing, and ailing, U.S. population.

Rising demand for biofuels is expected to be good news for the biotechnology sector, with the Department of Energy estimating ethanol usage could reduce U.S. petroleum-derived fuel consumption by up to 30% by 2030. The biotechnology sector has allowed the U.S. farming industry to rapidly increase its supply of corn and soybeans -- the main inputs into biofuels -- by developing genetically-modified seeds which are resistant to pests and drought. By boosting farm productivity, biotechnology plays a crucial role in ensuring that biofuel production targets are met.

Biotechnology (Introduction)

2009-01-07T04:35:00.000-08:00

Biotechnology is technology based on biology, especially when used in agriculture, food science, and medicine. The United Nations Convention on Biological Diversity defines biotechnology as:

Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.

Biotechnology is often used to refer to genetic engineering technology of the 21st century, however the term encompasses a wider range and history of procedures for modifying biological organisms according to the needs of humanity, going back to the initial modifications of native plants into improved food crops through artificial selection and hybridization. Bioengineering is the science upon which all biotechnological applications are based. With the development of new approaches and modern techniques, traditional biotechnology industries are also acquiring new horizons enabling them to improve the quality of their products and increase the productivity of their systems.

Before 1971, the term, biotechnology, was primarily used in the food processing and agriculture industries. Since the 1970s, it began to be used by the Western scientific establishment to refer to laboratory-based techniques being developed in biological research, such as recombinant DNA or tissue culture-based processes, or horizontal gene transfer in living plants, using vectors such as the Agrobacterium bacteria to transfer DNA into a host organism. In fact, the term should be used in a much broader sense to describe the whole range of methods, both ancient and modern, used to manipulate organic materials to reach the demands of food production. So the term could be defined as, "The application of indigenous and/or scientific knowledge to the management of (parts of) microorganisms, or of cells and tissues of higher organisms, so that these supply goods and services of use to the food industry and its consumers.

Biotechnology combines disciplines like genetics, molecular biology, biochemistry, embryology and cell biology, which are in turn linked to practical disciplines like chemical engineering, information technology, and biorobotics. Patho-biotechnology describes the exploitation of pathogens or pathogen derived compounds for beneficial effect.

biotechnology

2008-12-25T19:58:00.000-08:00