Using Molecular Approaches to Combat Illegal Wildlife Trade by Emily Ford

The illegal trade of wildlife (plants, animals, and their byproducts) is the second largest threat facing endangered species today, outranked only by habitat destruction. Frequently trafficked species span all ecosystems in every part of the world, making the $20 billion/year market incredibly difficult for law enforcement to handle. Most trade occurs along existing drug smuggling routes, and once wildlife is seized, it’s often tough to pinpoint where exactly these species came from– that’s where molecular genetics comes in.

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Turtles are one of the most threatened species worldwide, with 51% of their species extinct, endangered, or vulnerable of becoming endangered. (Image source)

In the past, identifying trafficked organisms was based primarily on recognizing physical traits unique to each species. Unfortunately, this requires the plant or animal to be mostly intact, and is almost useless in identifying the origins of byproducts often seized (such as ivory, cooked/dried meats, feathers, etc.); however, molecular approaches require only a small sample of DNA, making them the preferred method of species identification in these instances.

Each cell contains multiple copies of mitochondrial DNA (mtDNA) because there are several mitochondrion inside. Plant and animal cells only contain one nucleus a piece (generally), meaning that mtDNA is more likely to be found in a sample (like decomposing tissue) than nuclear DNA (nDNA). Segments of mtDNA can be copied over and over again using a process known as the polymerase chain reaction (PCR), which allows genes (like Cyt b or CO1) to be identified easily. The differences in DNA patterns within these genes is what makes each species unique– that’s how they’re able to be identified. heraa-hashmi-atcaveheraa-mitochondria-is-the-powerhouse-of-the-cell-false-mitochondria-are-the-powerhouses-of-the-cell-mitochondria-is-plural-the-singular-is-mitochondrion-if-thats-the-o

Having multiple mitochondrion in each cell makes mtDNA the preferred genetic marker for identifying a degraded sample’s species of origin. (Image Source)

In terms of conservation efforts, simply identifying a species isn’t enough– it’s important to determine where these organisms are starting their trafficked journey. “Phylogeography” refers to the relationship of very similar genetic patterns (like those found in mtDNA) and their location across the globe. A species’ region of origin can be determined by comparing it’s mtDNA sequences to what we already know about the mtDNA of similar species living in different parts of the world.

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Phylogeographic methods were used to compare seahorse species Hippocampus barbouri, H. spinosissimus (pictured here), H. trimaculatus, and H. ingens to pinpoint the birthplace of seahorses listed for sale in Californian traditional medicine shops. (Image Source)

Not only is the trafficking of species cruel to individual organisms, it disrupts their home ecosystems by removing key players from their natural balance, while simultaneously exposing species to foreign diseases they have no immunity against. The methods described above, in part with harsher legal consequences for traffickers, can help decrease illegal wildlife trade, therefore improving the overall health of species everywhere.

Source: Alacs, et al. (2009). DNA detective: a review of molecular approaches to wildlife forensics. Forensic science, medicine, and pathology. DOI: 10.1007/s12024-009-9131-7.

 

 

 

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Segregated Human Pedigree Analysis by DNA Fingerprints By J. Munoz

Human pedigree analysis, also known as the “genetic family tree,” shows certain genetic traits that have been through generations between families. Determining a carrier for a genetic disease is one of the ways these pedigrees are useful. The use of DNA fingerprinting, in a study of Jeffreys et al., extended their research in a co-segregated research with human pedigrees. To help with the pedigree, they first used RFLPs (Restriction Fragment Length Polymorphisms). On how that works, DNA is digested with a restriction enzyme that cuts DNA at a specific site making hypervariable minisatellite fragments. 

Everyone’s DNA is specific on the way that its cut. The cut fragments are gel electrophoresed with two different probes (33.6 and 33.15) to obtain different sets of fragments and allow a better scoring of hypervariable minisatellites. It will display different patterns of segments revealing the DNA fingerprints that are individual-specific. This starts the linkage analysis in man.RFLP genotyping

Example of how RFLPs can associate with pedigrees. *(This picture is not related to the study of Jeffrey et al.)*

The picture above helps one understand which siblings has the same specific DNA cuts from each parent. One parent’s DNA can be unavailable, but it can still manage to construct the pedigree with the help of RFLP in identifying parental minisatellite fragments present in some siblings but absent from the father. 

Using this analysis of DNA fingerprints the study was able to become a pedigree of Gujarati Asians. Jeffreys et al. were able to form 2-four generations with the determination of hypervariable fragments. This co-segregated study was able to determine the transmission in hereditary persistence of fetal hemoglobin in the sibship (siblings from the same two parents) and other generations as well.

The different probes allowed to obtain about 34 isolated minisatellite for hypervariable loci to determine loci that is closely linked to a given disease loci to help in distinguishing a disease gene. This approach is very helpful in linkage data to locate minisatellites in other human genomes to detect affected families in future generations.

 

Article Source: Jeffreys, A J et al. “DNA ‘Fingerprints’ and Segregation Analysis of Multiple Markers in Human Pedigrees.” American Journal of Human Genetics 39.1 (1986): 11–24. Print.

Picture Source: https://www.ncbi.nlm.nih.gov/probe/docs/techrflp/

The Use of DNA Fingerprinting in Identifying Diseases by Seth Browning

Individuals can be identified as part of a group in a number of ways.  Besides identifying someone based on physical appearance, a person can be labeled through the use of genetic testing.  One of these mechanisms is the usage of DNA fingerprinting.  DNA fingerprinting is the use of polymerase chain reaction (PCR) and gel electrophoresis to determine the genetic makeup of a DNA sample.  With these two techniques, we can distinguish DNA samples between individuals.  PCR makes multiple copies of DNA and amplifies the specific sequence that researchers want to observe.  Gel electrophoresis allows the researcher to match the DNA being tested with the original DNA.  The similarities of the DNA are based upon the number of short tandem repeats within the genetic sequence.  In a study completed in 1994, DNA fingerprinting was used to determine which tuberculosis samples were latent infections and which were transmitted within the community.

http://thetimesweekly.com/news/2015/nov/19/tb-cases-rise-will-county-health-officials-say/
                            Tuberculosis

Tuberculosis (TB) is caused by Mycobacterium tuberculosis, and mostly colonizes the lungs.  The signs and symptoms of TB include a persistent cough which can be accompanied by the presence of blood or phlegm, chest pain, fatigue, weight loss, absence of appetite, chills, fever, and night sweats.  There has been an increase of TB cases since the 1980s.  New York City has contributed to 14% of these cases, and researchers believe that most individuals with TB are homeless or part of the minority population.  To distinguish between the different TB strains in New York City, researchers used DNA fingerprinting.  After gathering 104 patients that tested positive for TB, the DNA from the bacteria were extracted.  Enzymes used to digest the extracted DNA included PvuII and AluI.  The samples then underwent PCR and gel electrophoresis.  After examining the fingerprints, the researchers divided the samples into two groups.  Group 1 contained nonclustered isolates that produced a unique fingerprint and Group 2 which contained clustered isolates.  Of the 104 samples, 62.5% consisted of Group 1 and the other 37.5% consisted of Group 2 strains.  This study concluded that the transmissible strain of TB caused more cases than TB strains that reactivated during adulthood.  DNA fingerprinting allowed scientists to distinguish between different strains of M. tuberculosis.  This has proven that DNA fingerprinting is a promising tool.  This instrument can be also used to predict future strains of bacteria that are responsible for outbreaks when looking at a large population.

 

Sources:

https://www.cdc.gov/tb/topic/basics/signsandsymptoms.htm

Alland D, et al. 1994. Transmission of Tuberculosis in New York City: An Analysis by DNA Fingerprinting and Conventional Epidemiologic Methods. NEJM. 330:1710-1716.

 

Picture Source:

http://thetimesweekly.com/news/2015/nov/19/tb-cases-rise-will-county-health-officials-say/

The Woes of DNA Fingerprinting by K. Gleason

DNA fingerprinting has more uses than solving crimes, like murder; it is also employed in paternal cases to identify a child’s father. DNA fingerprinting, or DNA profiling, is a technique that is used to determine a genetic match between people and samples that are found at crime scenes. The genetic material is retrieved from a variety of cells, like semen, blood, skin cells, and saliva. Everybody has their own unique DNA fingerprint, unless they are identical twins. Like all innovative sciences and technologies, there are some controversies surrounding DNA fingerprinting.

https://sanfrancisco.cbslocal.com/photo-galleries/2011/10/24/famous-identical-twins/

One of the most prominent controversies is the reference population that forensic scientists use. The reference population is a general template of what someone’s DNA would look like from a certain population (Caucasian, African American, etc). The biggest concern with the use of reference populations is that there are subpopulations (German, Irish, etc) within these ethnicities. It was assumed for a long time that people only married and had children with others in their subpopulation, but with the modernization of relationships, people are starting to marry outside of their subpopulations and even ethnicities. These populations are expressing increased variability, which means the chances of having two people with matching DNA is slim to none, especially in the US, which is considered a “melting pot” of cultures and genetics. To remedy this outdated reference population system, new databases are being made. These new databases will ignore the subpopulations and look only at ethnicity unless some populations are incredibly different, like the different tribes of Native Americans, which is when the databases are separated by region in case there is small variability.

To guarantee that no other controversy could arise about the uniqueness of a person’s DNA, scientists studied how many genotypes could be observed. They found that the number of possible genotypes far exceeded the population of the US and the chance of two people with the same genotype (excluding identical twins) was 10 raised to the negative 12, which means that it is negligible.

Since we are always shedding skin cells and hair, our DNA is everywhere. DNA fingerprinting is used to place a suspect at a crime scene or to get some basic information about the people that were there, like their gender and ethnicity. While most of us know that DNA fingerprinting is used to place a suspect at the scene, it can also be used to exonerate suspects. A boy that was a suspect of raping and murdering two females in England requested a blood test to prove that he didn’t do it. The blood test showed that he was indeed innocent of the crime, preventing an innocent person from being imprisoned. If there had been a lab error, his case could have ended differently. Lab errors can occur when the same sample is tested twice, there is a high number of different DNA profiles analyzed, and chain of custody problems. There aren’t really any solutions to these concerns other than the lab being careful and proper protocol being followed. As science advances, these concerns could vanish or open the door for even greater concerns to emerge.

References:

Roeder, K. (1994). DNA Fingerprinting: A Review of the Controversy. Statistical Science, 9(2), 222-278. doi:198.51.92.3

 

 

AraC Regulators by Daniel Achee

Bacteria need different sets of enzymes to digest different types of food, or substrates. It would be energetically inefficient for the bacteria to produce all possible sets of enzymes in the absence of substrates to digest. Because of this, many bacterial enzymes are only made, or the genes are only expressed, in presence of the particular food that they are able to help digest. One example of this is the 5-carbon sugar arabinose. All of the enzymes needed to consume this sugar are coded for in a series of genes that are activated together and share a promoter. This set of connected genes is referred to as the arabinose operon. The activation of this operon is governed by the AraC regulator, a type of positive gene regulator. Positive gene regulation is whenever the presence of the regulator  activates transcription, which is the making of the protein. When there is a low level of arabinose and a high level of glucose, arabinose will bind to two seperate spots on the operon, ara02 and aral, and bring them together, forming a dimer and creating DNA loop. This loop structure prevents transcription of the enzymes as well as the AraC protein.

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*AraC protein binding to ara02 and aral causing a DNA loop*

However, whenever arabinose is present and glucose concentration is low, the AraC protein will bind to arabinose and undergo a conformational change. This change in shape allows for the AraC protein to become an activator, opening the DNA loop and binding to the activation site. Once binded, AraC will cooperate with CAP-cAMP to facilitate the transcription of the operon.

Gene Wilder

*Whenever arabinose is bonded to the AraC protein, the gene becomes wilder. Whenever it is unbonded the AraC protein will cause a loop and cause the gene to be calmer.*

Despite having a much larger family of proteins, AraC regulators.in particular are the most extensive group of transcriptional regulator proteins in bacteria. The list of microbes that have AraC regulators includes Citrobacter freundii, Escherichia coli, Erwinia chrysanthemi, and Salmonella typhimurium. This particular regulator was of incredible importance because it was the first of its kind to be discovered. The AraC regulator of the L-arabinose operon in E. coli was the first found example of positive gene regulation, this opened up a new area of research and allowed for further understanding of how genes can be expressed. 

 

References:

thumbpress.com

Gallegos, M T et al. “Arac/XylS Family of Transcriptional Regulators.” Microbiology and Molecular Biology Reviews 61.4 (1997): 393–410. Print.

A New Method of Screening for Tuberculosis Drug Candidates by Testing With a Luciferase-Expressing Microbe : Jonah Albares

Tuberculosis (TB) is an infectious microbial disease caused by Mycobacterium tuberculosis that targets the lungs. Tuberculosis is an incredibly lethal disease, claiming the lives of around 1.5 million of the 9 million patients diagnosed in 2013. Tuberculosis is also incredibly contagious, and infected patients have to be isolated in a biosafety facility. TB has also become resistant to a multitude of drugs, making it even more difficult to treat. Due to these factors, it is imperative that tuberculosis is diagnosed and treated with extreme haste. Unfortunately, tuberculosis replicates at an incredibly low rate, multiplying as little as once per day. Tuberculosis’ slow replication rate makes traditional TB tests impractical, as they take longer than three weeks to provide results. Developing more efficient TB screening methods and vaccines could be the key to saving millions of lives.

Multi-drug resistant tuberculosis (Mtb) has drastically affected the way we treat tuberculosis. Fortunately, we have found ways to effectively test TB vaccines. Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is a strain of bacteria that is genetically similar to TB. BCG responds to drugs in a similar way to tuberculosis. A lot of the TB vaccinations have been effective towards BCG. Using this information, scientists have been able to make more effective TB drugs by testing with BCG!

Now that we can more effectively treat tuberculosis, it is important that we find better methods of diagnosing it. Luciferase is an enzyme that is known for producing photons. This essentially means that Luciferase is bioluminescent, or that it naturally glows. This enzyme has recently been used to detect tuberculosis in as early as four days. Luciferase  hydrolyzes ATP that is found abundantly in living cells. This means that even with few TB cells present, Luciferase will hydrolyze their ATP, releasing photons that can be examined by a sensitive light detecting system. This lets doctors know there are viable TB cells in the sample!

Though they may seem like simple discoveries, these strides in treating and detecting tuberculosis are great discoveries in the medical field. It makes testing for more viable methods to treat TB much safer and more efficient now that scientists can test on BCG. This will hopefully lead to the development of better treatments of the disease. Also, being able to detect TB in a sample more than 5 times faster could lead to quicker diagnosis and treatment, ultimately helping doctors save lives!

 

References:

1.) Ozeki Y, Igarashi M, Doe M, Tamaru A, Kinoshita N, Ogura Y, et al. (2015) A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Gueren. PLoS ONE 10(11): e0141658. doi:10,1371/journal.pone.0141658

 

Image Sources:

1.)  www.funnycaptions.com/img/230327/

 

 

 

 

Use of High-Throughput Luciferase-Based Assay in Aiding Malaria Prevention: Karoline Ellsworth

Malaria is a parasitic disease that stems back as long as mankind. The infection is caused by Plasmodium parasites being transferred into the blood through the bite of an Anopheles mosquito. These parasites can evade the body’s immune response by their ability to change surface proteins rapidly and make themselves unrecognizable. In later stages of infection, the host’s red blood cells become a vessel where the parasites can multiply, this causes the cells to undergo substantial morphological and structural changes. As the red blood cells’ physical properties become dramatically altered, the body’s circulation becomes impaired due to rigidity of the cell shape. The change in cell shape causes adhering to the lining of the blood vessels and also to other cell types. Malaria victims can then develop issues concerning anemia, hypoglycemia, cerebral malaria, and other serious health risks.

                        Mosquito (Clyde) from Southpark

The Plasmodium parasite has evolved and spread alongside humans as long ago as human expansion out of Africa around 70,000 years ago. With malaria having a history as old ours, it is unsurprising to find out that the disease has begun adapting to our current methods of eradication. A majority of our antimalarial medications only treat for the erythrocytic stages of the parasite, where the red blood cells are impacted and the host begins to feel symptoms. Prior to these erythrocytic stages, are the exoerythrocytic stages, where the parasite lives dormant in host liver cells. Scientists are now focusing on inhibiting development of the parasite during this pre-symptomatic phase.

                               A depiction of the malaria lifecycle in a human

In order to obtain new information, many small molecules will need to be examined while being located among a diverse range of chemicals. A new method of high-throughput screening has shown potential to be a catalyst for our discovery of improved antimalarial medication. High-throughput screening allows scientists to conduct trials using hundreds of wells at a time and this new method is a luciferase-based assay, meaning it utilizes the class of oxidative enzymes called luciferases. It is found in several species and allows the organisms that express them to emit light.

                Luciferase enzyme aides in the oxidation of luciferin and produces light!

Nearly all the energy put into the reaction is rapidly converted to light, making the reaction highly energy efficient. The rapid conversion into light results in a high sensitivity that allows identification of even small changes during transcription. These observations during gene expression can aide scientists in chemically characterizing the early developmental stages of Plasmodium parasites. With this knowledge we can inhibit parasitic development and potentially save hundreds of thousands of people from malaria.

References:

  1. Lobo, I. A. (2015) How the Malaria Parasite Remodels and Takes Over a Human Host Cell. Nature Education 8(3):8
  2. Cell Press. “How The Malaria Parasite Hijacks Human Red Blood Cells.” ScienceDaily. ScienceDaily, 10 July 2008.
  3. Biotechnology and Biological Sciences Research Council. “Malaria threat is as old as humanity, new research shows.” ScienceDaily. ScienceDaily, 18 June 2010.
  4. Swann, J., Corey, V., Scherer, C. A., Kato, N., Comer, E., Maetani, M., … Meister, S. (2016). High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria. ACS Infectious Diseases
  5. Khan, F. (2013) The Luciferase Reporter Assay: How it works. Bitesize Bio.

Image Sources:

Fractured But Whole

https://www.cdc.gov

https://bitesizebio.com