Thursday, August 8, 2013

Modern applications of evolutionary biology


There are numerous ways to apply evolutionary biology to our needs today, among them:
1.  prolonging the life of drug/chemical resistant compounds
2.  constructing evolutionary trees
3.  pathogen tracking
4.  industrial production of biochemicals and other agents
1. Drug resistance and chemical resistance in microbes, plants, and animals. In the latter half of this century, industry has been exceptionally good at providing compounds to kill viruses, bacteria, insects that eat crops and weeds that grow in crop fields. We even have an abundance of chemotherapy drugs to kill rogue cancer cells. Yet virtually without exception, our attempts to kill these organisms cause them to evolve resistance against the chemicals used to kill them. For example:
AIDS is an example of a virus that evolves to thwart its destruction.
·       Isolates of the AIDS virus with up to 15 different drug-resistance mutations are known, and the latest drugs are becoming ineffective.
·       Some strains of bacteria are resistant to all available antibiotics.
·       For multi-drug resistant tuberculosis, surgery is the only cure because antibiotics don’t work and only 50% of those infected survive.
·       Chemotherapy for cancer often fails because drug-resistant cells evolve during treatment.
·       Pesticide resistance and herbicide resistance is so common now that the financial incentive to make new pesticides and herbicides is break-even or worse.
Evolutionary biology suggests how best to prolong the useful life of drugs/chemicals. The amounts of chemicals used, what combinations of chemicals to use, and when to apply them are all questions that can be assessed from the perspective of preventing or slowing the evolution of resistance. In some cases now, the companies marketing the compounds have a financial interest in maintaining the longevity of their product, and they are funding studies by evolutionary biologists to develop wise use protocols. In other cases, however, economic and emotional forces dictate policies that speed up the evolution of resistance (e.g., patients demand and physicians write prescriptions for antibiotics for viral infections; antibiotics are used in animal feed).
Evolutionary trees help scientists track pathogens that cause disease.
2. Evolutionary trees Perhaps the core of evolutionary theory is that all life forms are connected to each other through common ancestry. Molecular biology has reinforced this view to a far greater level than was deemed possible even 50 years ago. On a short time scale, of course, we observe that this is true — everything alive comes from something else that is both alive and similar. One of the big developments in evolutionary biology over the last 2 decades is a methodology to estimate the underlying patterns of ancestry among living things. These reconstructions of evolutionary history are known as phylogenies, or phylogenetic trees, because they are branched somewhat like trees when drawn from bottom to top. We can use molecular data to estimate the common ancestries of life as far back as we like — for example, between bacteria and our mitochondria (the energy-producing organelles in our cells). But we can also use these methods to estimate much more recent ancestries. And these methods have found many worthy uses in tracking infectious diseases.
3. Molecular epidemiology — pathogen tracking To an epidemiologist studying infectious diseases, it is very useful to know how or where a person became infected with the disease. This information is perhaps the most basic fact we can use in preventing the further spread of a disease. For over a decade now, epidemiologists have been using DNA sequences of viruses to make phylogenetic trees and thereby track the sources of infections. Some of these examples are spectacular.
Law: A case of intentional HIV injection? 
In a highly publicized case in Lafayette, Louisiana in 1998, a woman claimed that her ex-lover (a physician) deliberately injected her with HIV-tainted blood (HIV is the virus that causes AIDS). There were no records of her injection and no witnesses. So how could her story be tested? Evolutionary trees provide the best scientific evidence in a case like this.
A woman’s claim to how she was infected with AIDS was supported by evolution.
·       HIV picks up mutations very fast — even within a single individual.
·       If one person gives the virus to another, there are few differences between the virus in the donor and the virus in the recipient.
·       As the virus goes from person to person, it keeps changing and gets more and more different over time.
·       Thus, the HIV sequences in two individuals who got the virus from two different people will be very different.
·       Thus, if the woman’s story were true, her virus should be very similar to the virus in the person whose blood was drawn but should be very different from viruses taken from other people in Lafayette.
·       That was exactly what the evolutionary trees showed; her virus appeared to have come from the patient’s virus but was unlike the virus taken from other people in town.
·       Since there was no way to explain how she would have gotten that patient’s virus on her own, the evolutionary analysis supported her story. (Incidentally, this case was the first use of phylogenetics in U.S. criminal court.)
Other cases Evolutionary trees have been used in many other cases of infectious disease transmission:
·       the transmission of the AIDS virus by a dentist to his patients
·       deer mice as the source of hantavirus infections in the Four-Corners area
·       the source of rabies viruses in human cases, leading to the discovery of a case in which rabies virus took at least 7 years to kill a person
·       whether recent cases of polio in North America were relict strains from the New World, were vaccine strains, or were introduced from Asia
4. Industrial production of biochemicals and other agents “Directed evolution”, i.e. artificially-induced evolution, has become part of the jargon in biotechnology:
Biotechnology allows us to give direction to evolution.
·       Artificially evolved enzymes and other proteins are soon to become part of household and medical technologies.
·       We will have protein-based drugs that, unlike the proteins inside our bodies, degrade slowly so that we don’t need to take so much of them.
·       Enzymes are being evolved to work in detergents (which they don’t normally do).
·       And as the stuff of futuristic novels, molecules are being developed to bind anthrax spores, ricin molecules, and other potential bioterrorism agents.
All of these developments take advantage of one or more forms of test-tube evolution. Armed with a knowledge of how natural selection works and combined with the right kinds of laboratory technology, people can create molecules to perform seemingly any kind of function. In some of the more spectacular cases, these test tube evolution methods have created enzymes from purely random pools of DNA (or RNA) sequences. Even 10 years ago, it was thought that a DNA enzyme was impossible, yet armed with only an understanding of how to apply test tube evolution, a DNA enzyme can now be created in days.

Monday, June 24, 2013

ELISA/Western blot tests for HIV


HIV ELISA/Western blot is a set of blood tests used to diagnose chronic infection with human immunodeficiency virus (HIV).

How the Test is Performed
A blood sample is needed. For information on how this is done, see: Venipuncture.

How to Prepare for the Test
No preparation is necessary.

How the Test Will Feel
When the needle is inserted to draw blood, some people feel moderate pain, while others feel only a prick or stinging sensation. Afterward, there may be some throbbing.

Why the Test is Performed
Testing for HIV infection is done for many reasons, including:
  • Screening people who want to be tested
  • Screening people in high-risk groups (men who have sex with men, injection drug users and their sexual partners, and commercial sex workers)
  • Screening people with certain conditions and infections (such as Kaposi's sarcoma or Pneumocystis jirovecii pneumonia)
  • Screening pregnant women to help prevent them from passing the virus to the baby
  • When a patient has an unusual infection

Normal Results
A negative test result is normal. However, people with early HIV infection (termed acute HIV infection or primary HIV infection) often have a negative test result.

What Abnormal Results Mean
A positive result on the ELISA screening test does not necessarily mean that the person has HIV infection. Certain conditions may lead to a false positive result, such as Lyme diseasesyphilis, and lupus.
A positive ELISA test is always followed by a Western blot test. A positive Western blot confirms an HIV infection. A negative Western blot test means the ELISA test was a false positive test. The Western blot test can also be unclear, in which case more testing is done.
Negative tests do not rule out HIV infection. There is a period of time (called the "window period") between HIV infection and the appearance of anti-HIV antibodies that can be measured.
If a person might have acute or primary HIV infection, and is in the "window period," a negative HIV ELISA and Western blot will not rule out HIV infection. More tests for HIV will need to be done.

Risks
Veins and arteries vary in size from one patient to another and from one side of the body to the other. Obtaining a blood sample from some people may be more difficult than from others.
Other risks associated with having blood drawn are slight but may include:
  • Excessive bleeding
  • Fainting or feeling light-headed
  • Hematoma (blood accumulating under the skin)
  • Infection (a slight risk any time the skin is broken)
Considerations
People who are at high risk (men who have sex with men, injection drug users and their sexual partners, commercial sex workers) should be regularly tested for HIV.
If the health care provider suspects early acute HIV infection, other tests (such as HIV viral load) will be needed to confirm this diagnosis, because the HIV ELISA/Western blot test will often be negative during this window period.

Alternative Names
HIV testing

References
Dewar R, Goldstein D, Maldarelli F. Diagnosis of human immunodeficiency virus infection. In: Mandell GL, Bennett GE, Dolin R, eds. Principles and Practice of Infectious Diseases. 7th ed. Philadelphia, Pa: Elsevier Churchill Livingstone; 2009:chap 119.
Sax PE, Walker BD. Immunopathogenesis of human immunodeficiency infection. In: Goldman L, Ausiello D, eds. Cecil Medicine. 23rd ed. Philadelphia, PA: Saunders Elsevier; 2007:chap 408.

Model Organisms' Resource


9      Mesoplasma florum:Genomic DNA      
14    Yeast PCR Protocols   
15    Yeast Genetics    
17    Yeast Culture and Storage       
18    Yeast Cellular Biology       
20    Arabidopsis Protocols        

Monday, June 10, 2013

Separation of normal CD34+ cells from fresh pheresis of mobilized stem cells


1.      Resuspend sample up to 100 mL of MACS buffer (see recipe below). Aliquot to two 50-mL conical tubes. Centrifuge for 10 minutes at 1000 rpm to "soft spin" the pellet. A soft spin keeps the platelets, which are concentrated in pheresis samples, in the supernatant. Platelets cause major problems with the staining of the sample as well as the running of the sample through the magnetic column. The supernantant must be aspirated, not poured off, since the pellet is loose.
2.      If the supernatant from the soft spin is still relatively cloudy, the soft spin may be repeated.
3.      ACD-A changes the density of the cells in the pheresis sample so that granulocytes will stay at the interphase of the Ficoll. Although pheresis samples have a high concentration of granulocytes, it is more important to remove the platelets before they activate. An alternative protocol may be that the sample is initially suspended in buffer without ACD-A and then Ficolled. The mononuclear layer may then be resuspended in buffer with ACD-A, and the soft spin performed. We have found it best to just leave the granulocytes and adjust concentration of the antibody (follows).
4.      Count the total number of cells. Combine sample into one 50-mL conical tube. Miltenyi lists antibody amount according to the total number of cells; however, this may be adjusted according to the estimate of number of cells positive for the sorting parameter. Since pheresis has between 1-10% CD34+ cells, we usually use 50% of the recommended amount of antibody. If a Ficoll is not performed, this may be reduced to ? the amount of antibody, but the buffer should not go below ?, and the incubation time should be extended to 30 minutes. Add ? the amount of buffer recommended by Miltenyi. Add ? the amount of reagent A1, shake gently. Add ? the recommended amount of reagent A2, shake gently. Incubate in the refrigerator for 15 minutes, gently shaking the sample periodically.
5.      Wash the sample two times with 50 mL of MACS buffer.
6.      Resuspend the sample in _ the recommended amount of buffer, and _ the amount of reagent B, shake gently. Incubate in the refrigerator for 15 minutes, gently shaking the sample periodically.
7.      Wash the sample one time with 50 mL of MACS buffer.
8.      Resuspend the sample in at least 10 mL degassed (see Note) MACS buffer for 1 x 109 total cells, or up to 20 mL for 2 x 109 total cells. Run sample over a VS positive selection column.
9.      Wash 2X with 3 mL of degassed buffer.
10.   Attach stop cock and syringe to bottom of VS column. Remove column from magnet. Backflush column with 6 mL of degassed buffer. Replace column in magnet.
11.   Remove stop cock. Allow buffer to flow through column. Wash 2X with 3 mL of buffer.
12.   Remove column from magnet. Add 6 mL buffer to column and allow to run through. Add 6 mL buffer to column and plunge the column.
13.   Count the total number of cells collected from each fraction to calculate the recovery of the separation.
14.   Perform flow cytometry on the collected fractions to assess sample purity with CD45-FITC and CD34-PE (Becton Dickinson).

MACS buffer
Hank's Balanced Saline Solution (HBSS) -Ca+2, -Mg+2
0.5% BSA
0.6% Anticoagulant Citrate Dextrose- Formula A (ACDA) (Baxter)
Filter sterilize and store at 4°C

Notes: It is very important when running the magnetic column that only degassed MACS buffer be used. To degas the buffer, place 100 mL of buffer in a 150-mL bottle. Place a rubber stopper attached to a vacuum line over the mouth of the bottle. Turn vacuum on. Allow the buffer to degas at room temperature for at least 30 minutes. Replace cap on bottle and refrigerate buffer until cold. Use buffer as directed.

Mouse Tumor Biology (MTB) Database

The Mouse Tumor Biology (MTB) Database supports the use of the mouse as a model system of hereditary cancer by providing electronic access to:

l  Information on endogenous spontaneous and induced tumors in mice, including tumor frequency & latency data,
l  Information on genetically defined mice (inbred, hybrid, mutant, and genetically engineered strains of mice) in which tumors arise,
l  Information on genetic factors associated with tumor susceptibility in mice and somatic genetic-mutations observed in the tumors,
l  Tumor pathology reports and images,
l  References, supporting MTB data
l  Links to other online resources for cancer

http://tumor.informatics.jax.org/mtbwi/index.do