How we used CRISPR to narrow in on a possible antidote to box jellyfish venom

File 20190430 136800 u6bay2.jpg?ixlib=rb 1.1 — Venom from box jellyfish causes extreme pain and tissue damage. Massive exposure can cause death.
from www.shutterstock.com

Warm Australian waters are home to the box jellyfish (Chironex fleckeri), which is considered to be one of the most venomous animals on the planet.

Box jellyfish stings lead to excruciating pain lasting days, tissue death and scarring at the site of the sting, and with significant exposure, death within minutes. While most jellyfish stings do not lead to death, pain and scarring is quite common.

Despite its potent ability to cause pain and death, to date we’ve had very little understanding of how this deadly venom works. This makes it very difficult to understand how it can cause so much pain – and how to develop medicines to block venom actions.

Published today, our new research has uncovered a potential antidote for box jellyfish venom. By working with humans cells and the gene-editing tool CRISPR, we identified a common, cheap drug that is already on the market and which could be a candidate for treating box jellyfish stings.

Flipping all the switches

This work began in 2012, when we set out to determine what it was about box jellyfish venom molecules that made them so effective in causing pain and damage.

The venom didn’t seem to work through the known pathways that cause cell death. So we used CRISPR genome editing technologies in human cells grown in the laboratory. This let us systematically turn off each gene in the human genome, and test to see which of these is needed for the jellyfish venom to kill the cells.

It’s kind of like flipping all the switches in a house, trying to figure out which one turns off the kitchen lights, but at the whole genome level. We actually didn’t even know if it would be possible to find single genes that when turned off could block the venom action.

But luckily, we were successful. While normal human cells exposed to venom die in the laboratory within five minutes, we identified gene-edited cells that could last for two weeks continually exposed to venom.

Putting the evidence together

Then using new DNA sequencing technologies (that allow us to identify CRISPR guide RNAs targeting specific genes), we identified which human genes had been switched off in our genome editing experiments.

By putting the evidence together, we worked out which genes the box jellyfish venom needs to target in order to kill human cells in the lab.

One we identified is a calcium transporter molecule called ATP2B1, and is present on the surface of cells.

We tested a drug that we know targets this gene. If we added the drug before the venom, we could block cell death, but if we added the drug after the venom, it didn’t have any effect.

So this helped us understand more about how the venom works – and maybe even how it causes pain. We are still looking at this particular pathway in more detail, but at the moment it doesn’t seem promising for a therapy.

Stopping cell death

Next we looked at the pathways involved in how box jellyfish venom kills cells.

We found four of the top ten genes required for venom action were all part of a pathway that makes cholesterol in cells.

Since cholesterol has been heavily studied over the last 30 years, there are already drugs available that target lots of different steps in cholesterol regulation. We focused on drugs that could bind to cholesterol and remove it quickly, basically acting like a cholesterol sponge.

We found these drugs could completely block the box jelly fish venom’s ability to kill human cells in the lab if added before venom exposure. We also found there is a 15-minute window after venom exposure where if we add this cholesterol sponge, it still blocks venom action.

This was exciting, as the capacity to have effect after the venom means the drug could work as a treatment in the case of being stung by a box jellyfish.

So far our additional studies show that these same drugs can block pain, tissue death and scarring associated with a mouse model of box jellyfish stings.

Moving towards a human treatment

The really cool thing about this work is that the potential box jellyfish antidote we found is in a family of drugs called cyclodextrins. These are known to be safe for us in humans, and are cheap and stable.

So now we are trying to work with the state or national government, or first responders, to see if we can move this venom antidote forward for human use.

As well as developing a topical application at the site of a sting, we also aim to develop this idea as a potential treatment for cardiac injection in the emergency room in the case of very severe box jellyfish sting cases.

Greg Neely, Associate Professor , University of Sydney

This article is republished from The Conversation under a Creative Commons license. Read the original article.

Citizen scientists count nearly 2 million birds and reveal a possible kookaburra decline

Kerryn Herman, Deakin University

The fourth Aussie Backyard Bird Count has just ended, with nearly 2 million birds from 635 species submitted to the BirdLife Australia app. The count, which is in its fourth year, has created a national database of birds found in our backyards.

We don’t know yet exactly how many people participated this year, but more than 60,000 people submitted checklists in 2016. Participants span the whole country, though participation is highest in our urban areas. By surveying our backyards (rather than “good” bird spots), these citizen scientists provide ecologists – like me – with information from urban areas we would not otherwise sample.

This includes data on a range of common bird species that are not frequently analysed because these species are believed to be secure. One of the most surprising results is a decline in the frequency of occurrence of the laughing kookaburra across southeast Australia.

Counting birds

Everyone has a bird story – and fortunately for ecologists, everyone is willing to share them. With 85% of Australia’s population living in cities and towns, birds are an important connection to our natural environment.

But birds are also good environmental indices. They’re generally easy to measure, they respond quickly to environmental change and we know a reasonable amount about the ecology of most species.

Between 1998 and 2014, BirdLife Australia volunteers collected a significant amount of data. This was used to develop a terrestrial bird index in 2015 – a bird “Dow Jones” to track our biodiversity. It was here that the decline in kookaburras was first identified.

The data were drawn from BirdLife Australia’s ongoing atlas project, now called Birdata. However, there are biases in this data set, as people obviously like to go birdwatching where they will see more birds. This may inflate the frequency of encountering some species and decrease the chances of encountering others – particularly rare and cryptic species.

For the last four years, we’ve asked volunteers to add to this data by counting birds around their home for a week in October, when many birds are highly active and visible. These counts complement the data already available in Birdata by allowing access to backyards across Australia, which are generally poorly represented in the larger data set.

While there are still limitations in the Backyard Bird Count data, such as the risk of mis-identification, for common species like the laughing kookaburra we can generally be confident that the identification is correct. Even if the same bird is counted multiple times, our models report only a species’ presence or absence, so inflated numbers don’t affect the trend.

Are kookaburras really declining?

The below figures show modelled trends for the kookaburra across metropolitan Melbourne and Sydney. These figures are derived from the volunteer-collected Birdata, much of which comes from green spaces and remnant vegetation in these landscapes.

I wondered whether these declines are true changes in the populations, or reflect a change in the way kookaburras are using the landscape, possibly moving into the matrix of urban backyards that just don’t get surveyed. Looking solely at the backyard count data, I found similar trends in the reporting rates of kookaburras as those in the models, supporting that this decline is at the population level. What started out in 2014 as a way of engaging the broader community with their birds is now collecting useful ecological data.

Further exploration of the ABBC data across other capital cities found some interesting things. In both Perth and Hobart, where the kookaburra is considered an introduced species, the birds are recorded more frequently than in Melbourne and across the ACT. In Perth, increases in 2016 compared to previous years suggest an increase in the species there.

Modelled trends for Kookaburras from 1998-2014. The figure on the left is for Melbourne, and on the right is for Sydney. The reporting rate shows the percentage of surveys where kookaburras were recorded as present. The thick black line is the modelled trend (with confidence intervals in dashed line), the pink line shows the statistically significant linear trend, the thin black line shows the monthly calculated reporting rate, and the green spots show acceleration (or a favourable change) in the trend. Note that due to modelling method, the ends of figures tend to blow out as there is no data from which to predict trend.
Unpublished models/K. Herman/BirdLife Australia, Author provided

While three years does not make a trend, Aussie Backyard Bird Count data from heavily urbanised areas suggest we are seeing a decline in this iconic species in the eastern capitals. Likely reasons for this are the loss of nesting hollows and possibly reductions in the availability of prey as we increasingly modify our urban landscapes. We don’t really know as this is not an area that has been researched.

We need citizen scientists

Collecting enough data (especially from the backyards of towns and cities) to detect these kinds of changes can be an overwhelming task. This is where citizen science programs like the Aussie Backyard Bird Count can help.

As well as helping ecologists track large-scale biodiversity trends, it also gives people the chance to connect with their natural environment and gain a greater appreciation of our unique fauna.

As with all citizen science projects, there are limitations in the data being collected. However, the Backyard Bird app has been designed to make counting as simple and standardised as possible, providing confidence in the tally of common and “iconic” species, and filling in the gaps found in other data sets.

The good old kookaburra is neither rare or cryptic. If anything, if people are seeking out “good” bird habitat to survey we would expect that kookaburras would be one of those species subject to inflated reporting. But this is not what we encountered.

If we are starting to see declines in species that we have traditionally considered secure, what does this mean for those that are already at risk? Once all the data from the Aussie Backyard Bird Count have been collated and vetted we will continue to explore the developing trends in Australia’s urban birds. Increasing engagement and awareness in our communities can help ensure our backyard birds are still around to count next year.

Kerryn Herman, Research Ecologist, Deakin University

This article was originally published on The Conversation. Read the original article.

Arctic: Catastrophic Methane Release Coming

The link below is to an article reporting on the possible release of massive volumes of methane when the permafrost of the Arctic melts due to climate change and global warming.

For more visit:
http://inhabitat.com/new-report-finds-arctic-ice-melt-could-cost-70-trillion/

Climate Change: Temperatures to Rise

The link below is to an article that reports on possible temperature rises associated with climate change over the next century.

For more visit:
http://news.mongabay.com/2013/0530-hance-temperature-range.html

Biofuel from CO2 Possible?

The link below is to an interesting article reporting on the discovery of a way to possibly transform CO2 in the atmosphere into biofuel.

For more visit:
http://scitechdaily.com/discovery-may-lead-to-the-creation-of-biofuel-from-co2-in-the-atmosphere/

Tasmanian Devil: Vaccine Near?

The link below is to an article reporting on the Tasmanian Devil and a possible vaccine in the near future for the deadly facial tumour disease that is wiping out the species.

For more visit:
http://www.australiangeographic.com.au/journal/one-step-closer-to-tassie-devil-vaccine.htm