Archive for April, 2012

Silencing is golden!

18 April, 2012

An excellent journal club article by Mark Whitehead:

Pavan Kumar, Sagar Subhash Pandit, Ian T. Baldwin. Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference. PLoS ONE, 2012; 7 (2): e31347 DOI: 10.1371/journal.pone.0031347

Specific Insect Gene Silencing achieved by ingestion of plant produced dsRNA, via a transient viral vector platform.

RNAi- mediated gene silencing is an endogenous mechanism and has been utilised in reverse genetics in a number of organisms and it has the potential to be used as a tool for pest control.

The diagram below gives a good summary on the standard RNAi process. Briefly,  dsRNA produced in the nucleus is transported to the cytoplasm; alternatively, exogenous dsRNA can be taken up by cells with the help of a cell surface protein. In the cytoplasm, dsRNA is cleaved by RNaseIII type enzymes (dicers) to produce approximately 22 bp fragments, called small interfering RNAs (siRNAs). One strand of the siRNA (guide strand) is incorporated into the RNA-induced silencing complex (RISC) with the perfectly complementary site in a target mRNA to form a guide strand-target mRNA duplex. The target mRNA is then sliced by the Argonaute protein of RISC.

(With permission from http://www.RNAiweb.com.)

Plants have RNA-dependant RNA polymerases (RdRPs) that accentuate the process as they extend the bound guide strand to create more dsRNA that can then re-enter the RNAi cycle. dsRNA delivered to insects by various routes has been seen to induce RNAi, however insects lack RdRPs and therefore require a large constant supply of siRNAs for sustained gene silencing. Herbivorous insects feeding on stably transformed transgenic host plants have been seen to take up the produced dsRNA molecules into their gut cells, causing post transcriptional gene silencing. Generation of these stable transgenic plant lines is a time consuming task, while transient plant transformation offers a faster and more versatile approach, allowing for a number of dsRNA products to be created as a quicker screening method.

Larvae of the tobacco hornworm Manduca sexta contain genes that encode for nicotine-catabolising enzymes, rendering them resistant to the toxic nicotine alkaloid produced by their host plant Nicotiana attenuata. It was previously seen that some cytochrome P450 (CYP) genes were up-regulated in the larval gut in response to nicotine ingestion (CYP4M1 and CYP4M3 genes) and CYP6B46 was down-regulated when fed on nicotine suppressed plants.

In this paper the tobacco rattle virus (TRV) was used to transiently produce dsRNAs in Nicotiana attenuata – this approach was termed plant-virus based dsRNA producing system (VDPS) – in comparison to stably transformed plants – termed plant mediated RNAi (PMRi) for the silencing of these lepidopteran genes.

They initially checked to see if M. sexta could indeed take up the dsRNA and cause PMRi. It was observed that when the larvae were fed on a transgenic plant expressing dsRNA for the CYP6B46 gene, there was CYP6B46 smRNA found in the midgut and a reduction in the CYP6B46 transcript levels was observed, effectively causing silencing of the gene. It was very specific as the transcript levels of a similar gene (CYP6B45 – 80% similarity) was not affected. The VDPS was tested and compared to the PMRi for the same target and produced comparable silencing, that was also highly specific and did not cause any “off-target” effects.

Since the VDPS is a more rapid technique and was seen to be comparable to the PMRi, it was therefore used to screen the other gene targets – CYP4M1 and CYP4M3. Again the smRNAs for each were seen to be present in the midgut of the larvae when fed on the plants and the transcript levels were reduced with high specificity. The reduction of the CYP4M3 transcription levels also caused larval growth to decrease, indicating that this gene may a central role in nicotine tolerance.

The length of the dsRNA is known to have an effect on RNAi experiments and it would be ideal if the lengths were standardised. It is possible that the lepidopteran dicers that function in extremely alkaline environments of the midgut are specialized and possess different dicing properties than the plant dicers; consequently, insect-dicer diced smRNA might be more effective than the plant-dicer diced smRNA in gene silencing in insects.

Plant Dicers (DCLs) are involved in the biogenesis of smRNA by cleaving longer dsRNA. Four different types of DCLs are reported in higher plants. Their function has been found to overlap in plants, suggesting that one DCL can contribute to and/or compensate for the function of the others. Hence, more than one DCL might be involved in processing long dsRNA.

To address this they then silenced different combinations of the four N. attenuata’s Dicer genes in the transgenic PMRi lines producing CYP6B46 dsRNA. Long CYP6B46 transcript levels in the plants was found to be increased more than 50 fold when the DCL 1,3,4 or DCL 2,3,4 were co-silenced. These then lead to an enhanced silencing effect in the larvae midgut, indicating that there could be a preference for insect diced smRNAs or simply that the larger dsRNAs were more stable and the higher concentration enhanced the silencing effect. It also suggests that the plant and insect RNAi machinery respond differently to the dsRNA.

In conclusion PMRi can be a specific and robust system of gene silencing in M. sexta. PMRi would be the method of choice for crop protection in countries which allow the growth of transgenic crops. While retaining all the virtues of PMRi, VDPS promises to be a rapid and high throughput alternative, suitable for ecological research.

This article has been a short review of the journal article stated below. For more in depth information on this research, follow the link and download the freely available journal article.

From herd immunity and complacency to group panic: How vaccine scares unfold

17 April, 2012

Via Scoop.itVirology News

A new study, published in PLoS Computational Biology, shows how worries over vaccine risks can allow preventable contagious diseases, such as measles and whooping cough, to make a comeback.

Via www.eurekalert.org

How old human tissue can help unlock secrets of the past

17 April, 2012

Via Scoop.itVirology News

Tissue samples have been used to reconstruct the 1918 influenza epidemic and to illuminate the proliferation of the AIDS virus.

Preserved human tissue can help shed light on why diseases — caused by microbes, environmental exposures and lifestyle changes — emerged when they did. It might even help scientists predict disease trends or outbreaks. Michael Worobey, an evolutionary biologist at the University of Arizona, is among the top researchers responsible for establishing the most credible date for when HIV entered the human population. He was able to do so in part thanks to analysis of tissue samples such as the ones pictured, from Congo, where some of the earliest HIV cases were discovered. The tissues are stored in blocks of wax.

 

A good archive is a wonderful thing – as I have discovered, when I have found that my first-ever clone is just a bit of dried-up agar in a cracked Petri dish….

Via www.washingtonpost.com

On Second Thought, Flu Papers Get Go-Ahead

17 April, 2012

Via Scoop.itVirology News

“The end of an impassioned and often strident global debate over the proper balance between scientific openness and security began with 2 hours of mandatory, studious silence in a room protected by an armed guard.

When members of a U.S. government advisory panel gathered last week on the campus of the U.S. National Institutes of Health (NIH) near Washington, D.C., to reconsider their controversial December 2011 recommendation that two groups of scientists redact key details from papers describing how they made the H5N1 avian influenza virus more transmissible between mammals, one of the first items on the agenda was to read revised versions of the manuscripts.”

 

Well, I would hope so – and then stop being silly.

Image courtesy Russell Kightley Media

Via www.sciencemag.org

Biotechnology science fiction on your Kindle this weekend …

17 April, 2012

Via Scoop.itVirology News

PROJECT GENESIS: Triumph, Tragedy, & Romance in the Futuristic Realm of Biotechnology [Kindle Edition] Markus Fredericks (Author). “Our story begins in year 2055 A.D. Felix and Monique are two attractive bioengineers …

 

I said “and other things”.  This is one of them.

Via cbt20.wordpress.com

NMAH | Polio: Two Vaccines

13 April, 2012

Via Scoop.itVirology News

This site explores the history of polio, the science and philanthropy behind the vaccines, the experiences of people who contracted polio and their influence on American culture, and current global efforts at stopping transmission of the poliovirus.

 

Vaccine denialists really, really need to go and have a look at this…15 000 kids paralysed and 1000 died per year in teh USA in the mid-1950s.  And now parents don’t want to vaccinate.

Via americanhistory.si.edu

The Birth of Polio Eradication: The Salk Vaccine Turns 57

13 April, 2012

Via Scoop.itVirology News

On April 12, 1955, scientists and reporters gathered in Ann Arbor, Michigan, for a momentous event. Millions of Americans huddled around radios and televisions that day to learn whether the world’s first polio vaccine, developed by Dr. Jonas Salk at the University of Pittsburgh, could prevent a devastating disease that killed and paralyzed thousands upon thousands of people, mainly children.

It’s hard to overstate the terror of polio back then. It would arrive each summer, like clockwork, leaving behind vivid reminders for all to see: wheelchairs, crutches, leg braces, iron lungs, deformed limbs. When Dr. Salk’s injectable vaccine was declared “safe, effective, and potent” that remarkable day in Ann Arbor, a nation celebrated. In churches, department stores, and coffee shops people wept openly with relief. President Eisenhower invited Dr. Salk to the White House where, in a trembling voice, he thanked the young researcher for saving children everywhere.

Via www.impatientoptimists.org

Can stem cells cure HIV?

13 April, 2012

Via Scoop.itVirology News

Because they are rich in stem cells, the cord blood of babies is being stored in blood banks, fueling a growing industry. Human stem cells, after all, have been credited with vast medical powers.

But can stem cells be used to cure one of the major scourges of the modern age: HIV-AIDS?

A series of studies conducted by researchers at the University of California in Los Angeles (UCLA) suggest that this might indeed be possible. In the most recent study, published on April 12 in the journal PLoS Pathogens, the researchers demonstrated that these stem cells can actually attack HIV-infected cells in a living organism.

 

Nice account of an interesting topic.

Via www.rappler.com

Vaccine Scares Could Become More Common, Experts Say | MyHealthNewsDaily.com

7 April, 2012

Via Scoop.itVirology News

“Vaccine scares that lead portions of the population to forgo vaccination could become more common as more diseases become eradicated.”

 

This is becoming a major problem for developed countries – that is to say, high GDP countries where people mostly have jobs and houses and electricity, and TV – and of course, that bringer of disinformation about vaccines, the internet.

 

Seriously: there are some 300 000 ANTI-vaccine sites out there, according to contacts in the USA, and some of them are so rabid they would make neo-nazis look respectable.

 

As I have written elsewhere – in a comment on an article in The Scientist – it would be ironic if developing countries started instituting stricter vaccination controls for travellers from the more affluent world.  It is coming, though: the first importations of diseases like measles from Europe to the Americas have already occurred, and I am sure the incidence of these reports will rise as vaccine paranoia grows.

Via www.myhealthnewsdaily.com

Sex and oral cancer: What is the connection? – DentistryIQ

7 April, 2012

Via Scoop.itVirology News

Are sexual relations closely intertwined with oral health? Jo-Anne Jones, RDH, addresses that question by sharing some of the latest statistics regarding a possible connection between the human papillomavirus (HPV) and oral cancer.

Via www.dentistryiq.com