Step aside, ELISA

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I’d like to think that in your typical romantic comedy Elisa is the dreadful girl the leading actor dates before he meets the girl of his dreams. However in this biochemical tale of epitope and antibody ELISA (enzyme-linked immunosorbent assay) isn’t dreadful at all. On the contrary, it serves as a very useful diagnostic tool.my_name_is_elisa_and_i_am_a_ninja_ornament_round

The problem with most infectious diseases lies in the time lapse between accurate diagnosis and onset of treatment. The development of Point-of-Care blood and urine diagnostic kits (based on ELISA) have been crucial in the management of diseases today. Alere Inc is a major service provider for point of care solutions for diseases such as TB, Syphillis and HIV. Introducing *drumroll* Enzymatically enhanced collisions on ultramicroelectrodes (for specific and rapid detection of individual viruses).

We report the specific collision of a single murine cytomegalovirus (MCMV) on a platinum ultramicroelectrode (UME, radius of 1 μm). Antibody directed against the viral surface protein glycoprotein B functionalized with glucose oxidase (GOx) allowed for specific detection of the virus in solution and a biological sample (urine). The oxidation of ferrocene methanol to ferrocenium methanol was carried out at the electrode surface, and the ferrocenium methanol acted as the cosubstrate to GOx to catalyze the oxidation of glucose to gluconolactone. In the presence of glucose, the incident collision of a GOx-covered virus onto the UME while ferrocene methanol was being oxidized produced stepwise increases in current as observed by amperometry. These current increases were observed due to the feedback loop of ferrocene methanol to the surface of the electrode after GOx reduces ferrocenium methanol back to ferrocene. Negative controls (i) without glucose, (ii) with an irrelevant virus (murine gammaherpesvirus 68), and (iii) without either virus do not display these current increases. Stepwise current decreases were observed for the prior two negative controls and no discrete events were observed for the latter. We further apply this method to the detection of MCMV in urine of infected mice. The method provides for a selective, rapid, and sensitive detection technique based on electrochemical collisions.

Read more here: PNAS

How the concept works is simple: An antibody specific to MCMV surface protein glycoprotein B (gB) is bound to glucose oxidase (GOx). The enzyme is responsible for the oxidation of glucose to gluconolactone. It is also responsible for the reduction of ferrocenium methanol (FeMeOH+) to ferrocene methanol (FeMeOH). The oxidation of ferrocene back to ferrocenium occurs at the electrode, and this loop results in an increase in the current response. Naturally, one enzyme is not enough to result in a large enough current response to be detected. The structure of the virus (and its many surface antigens) allows the specific antibody to bind to epitopes across the viral surface, therefore amplifying the signal.

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Anatomy of CMV

The virus generally blocks oxidation of ferrocene by non-specifically binding to the electrode surface, therefore causing a decrease in the current response. In the presence of the GOx bound anti-gB antibody the virus pre-concentrates the enzyme on its surface and when it binds to the electrode feedback is generated resulting in the positive (increase) current response.

The experiment was also performed with Murine Gammaherpesvirus 68 (MHV68), however the virus was unable to pre-concentrate GOx at its surface and no feedback loop was detected, proving the specificity of detection.

Interestingly the study also discussed the ionic strength of urine and how this contributes positively to the number of collisions at the electrode. Plus its less invasive than drawing blood (win-win!)

The researchers believe that this tool can be developed for the rapid diagnosis (as early as five days post-infection) of other viral infections such as Ebola, Zika, HIV and influenza (It’s winter – atishoo anyone?). At this stage factors such as the size of the virus and the accumulative clumping of virus-antibody complexes need to be taken into account, but further research is underway.

Sourced from Popular Science.

Genetic superheroes

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The Resilience project is a major search for individuals that are resilient to genetic disorders, i.e. they have the genotype for the disease, and not the phenotype to prove it. A famous example is that of Stephen Crohn, AKA “The man who can’t catch AIDS” who had a CCR5 -Δ32 mutation that resulted in a non-functional Chemokine Receptor 5 and prevented HIV-1 virus entry. He unfortunately committed suicide in 2013.

Nature Biotechnology recently published the results of a systematic study by the Resilience project:

“Genetic studies of human disease have traditionally focused on the detection of disease-causing mutations in afflicted individuals. Here we describe a complementary approach that seeks to identify healthy individuals resilient to highly penetrant forms of genetic childhood disorders. A comprehensive screen of 874 genes in 589,306 genomes led to the identification of 13 adults harboring mutations for 8 severe Mendelian conditions, with no reported clinical manifestation of the indicated disease. Our findings demonstrate the promise of broadening genetic studies to systematically search for well individuals who are buffering the effects of rare, highly penetrant, deleterious mutations. They also indicate that incomplete penetrance for Mendelian diseases is likely more common than previously believed. The identification of resilient individuals may provide a first step toward uncovering protective genetic variants that could help elucidate the mechanisms of Mendelian diseases and new therapeutic strategies.”

Read more: http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3514.html

The study used sequence and genotype data previously submitted to databases such as 23andMe (a saliva-based personal genetic testing service) and 1000 Genomes Project (an effort to create a complete and detailed catalogue of human genetic variations). The pilot study included 589 306 genomes. The aim was to identify healthy individuals that had the genotypes for severe Mendelian childhood disorders (age of onset before 18 years). The mutations studied were completely penetrant, i.e. all individuals should exhibit clinical symptoms.

874 genes were screened for 584 severe childhood disorders and at first 15 597 individuals were found. The researchers were really restrictive and excluded individuals on the basis of low confidence in sequence quality and depth, possible mosaicism (genetic defect affects only some cells in the body), inaccurate database information, environmental factors and repetitive sequence regions. I personally thought that repetitive regions would be a great indicator of gene duplications, however due to a lack of data they did not observe copy number abnormalities, large indels, and genomic rearrangements and focussed on loss of function and nonsense mutations. If the group discussion could not achieve a unified categorization for a candidate, this candidate was rejected from the final candidate table.

The final 13 should have developed one of eight autosomal dominant/recessive diseases: cystic fibrosis, Smith-Lemli-Opitz syndrome, familial dysautonomia, epidermolysis bullosa simplex, Pfeiffer syndrome, autoimmune polyendocrinopathy syndrome, acampomelic campomelic dysplasia and atelosteogenesis.

But they didn’t. They are asymptomatic. They’re called genetic superheroes because they might have some sort of protective mutation that “blocks” the effects of the disease-causing mutation.

Finding and studying these resilient individuals could pave the way to disease prevention and new treatments. An unfortunate occurrence was that the individuals did not submit consent forms to be contacted after the analysis by 23andMe and other companies. Our superheroes remain anonymous.

A follow-up study has been proposed in which test subjects will be required to give consent for further study.

The good news is: superheroes exist – but instead of protecting us from the bad guys out on the streets they’re protecting us from the bad DNA within (i.e. by giving up their DNA for scientific study). Be a superhero!

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Sourced from Scishow.

“Minimal cell raises stakes in race to harness synthetic life”. Really??

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Hahaha! Now to make sure I don’t fall into the “because we can” school of biology =D

ViroBlogy

Genomics entrepreneur Craig Venter has created a synthetic cell that contains the smallest genome of any known, independent organism. Functioning with 473genes, the cell is a milestone in his team?s 20-year quest to reduce life to its bare essentials and, by extension, to design life from scratch.

Venter, who has co-founded a company that seeks to harness synthetic cells for making industrial products, says that the feat heralds the creation of customized cells to make drugs, fuels and other products. But an explosion in powerful ?gene-editing? techniques, which enable relatively easy and selective tinkering with genomes, raises a niggling question: why go to the trouble of making new life when you can simply tweak what already exists?

Thomas Deerinck and Mark Ellisman/NCMIR/UCSD
Each cell of JCVI-syn3.0 contains just 473 genes, fewer than any other independent organism.
Unlike the first synthetic cells made in 20101, in which Venter?s team at the…

View original post 179 more words

The colour belt: mellow yellow

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As not so popularly known, there is currently an outbreak of yellow fever in Angola. A good 3500 km away from where I currently sit the human race is once again at the mercy of the bloodsucking mosquito. As at 15 February 2016, 51 people have died.

Aedes aegypti is a vector for the yellow fever virus, which causes an acute haemorrphagic disease that has no treatment. Interestingly, the same vector is also responsible for the transmission of the infamous Zika virus. And let’s not forget about dengue and malaria. Plainly said, mosquito vectors kill more humans than any animal on earth. BmjVoEqIcAAikrH

And we can’t kill all of them because “every organism has an important role to play in its habitat”. Hank Green covers it better on SciShow: What If We Killed All the Mosquitoes?

Honestly speaking, the outbreak in Angola isn’t too much of a shocker. Angola falls within the yellow fever belt: 40 countries where there is a realistic chance of contracting yellow fever. This has massive implications for travel (vaccination certificates need to be presented at border posts and airports).yellow efevr belt

The first observations of the cause of the virus were made in the 1793 Philadelphia yellow fever epidemic in which Dr Benjamin Rush reported that ”Mosquitoes were uncommonly numerous..” giving other researchers the idea of the mosquito vector. Later Dr Jesse William Lazear allowed an infected mosquito to bite him in order to study the disease. He died 17 days later. Talk about sacrificial science! Thankfully, it was not in vain. His notes led to the development of the 17-D vaccine (yay!) and strains of the live, attenuated vaccine is still used today. This means that the vaccine is a weakened form of the viable virus with reduced virulence (as opposed to an inactivated vaccine where the virus is killed).

Unfortunately, there is still no cure for yellow fever. Health care facilities provide supportive care to treat the symptoms, i.e. dehydration, respiratory failure and fever. However with new advances in scientific techniques, there is hope in the form of mosquito population control (see here). Developments in studies of Zika will hopefully prove useful as well.

News report from Reuters.

CRISPR-Cas: milk and honey for scientists?

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Since the first demonstration that CRISPR–Cas systems provide
bacteria and archaea with adaptive immunity against phages and plasmids,
numerous studies have yielded key insights into the molecular mechanisms
governing how these systems attack and degrade foreign DNA. However, the
molecular mechanisms underlying the adaptation stage, in which new
immunological memory is formed, have until recently represented a major
unresolved question. In this Progress article, we discuss recent discoveries that
have shown both how foreign DNA is identified by the CRISPR–Cas adaptation
machinery and the molecular basis for its integration into the chromosome to
form an immunological memory. Furthermore, we describe the roles of each of
the specific CRISPR–Cas components that are involved in memory formation,
and consider current models for their evolutionary origin.

Read more: http://www.nature.com/nrmicro/journal/v14/n2/abs/nrmicro.2015.14.html

From: Nature Reviews Microbiology

CRISPR-Cas is a genome editing tool that originally was used by bacteria to defend themselves against bacteriophages and other invader DNA that might prove to be dangerous to them. Since its discovery the CRISPR-Cas complex has been studied extensively and researchers in the field are said to be favourites for a Nobel Prize in Chemistry.

All in all, it is great news. Insights into the mechanism of action bring about loads of opportunity for graduate studies in the field (yay us!), and open up new avenues for bioentrepreneurship (yay economy!)

Brief overview as to how CRISPR works:

  1. Cas proteins 1 and 2 recognize foreign DNA
  2. Cas proteins cut out a piece of foreign DNA to be integrated into CRISPR
  3. CRISPR transcribed, RNA binds to Cas 9 (and other proteins)
  4. Complex destroys foreign DNA through complementary base pairing and nuclease activity

Further studies are now considering how to improve the precision of the Cas proteins and the specificity of the DNA cut out. Scientists at MIT have knocked out a couple of genes in Plasmodium falciparum in order to understand the malaria parasite.

The applications appear to be endless. We could turn off antibiotic resistance, increase pseudovirion production (yes, I’ve been looking into the project at BRU), erase genetic mutations, and do gene function studies. Pigs are already set to serve as human organ donors (see http://www.nature.com/news/new-life-for-pig-to-human-transplants-1.18768).

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There is even talk of the storage of DNA data and what that means for biocomputing. And yes, biocomputing opens doors for biohacking.

What is biohacking? Stay tuned to find out.