Project suggestion: Flu biology
Lora
http://scienceblogs.com/effectmeasure/2009/02/how_the_flu_virus_performs_cap.php?utm_source=sbhomepage&utm_medium=link&utm_content=channellink
Basically, flu viruses steal pieces of your own RNA in order to mask
their evil intentions. They have a subunit of their polymerase that
does this, the PA unit. The specific epitope of the PA unit that does
this function is an interesting drug target, because if it can be
blocked, we have a nice new antiviral.
Except, drug companies are focusing most of their infectious disease
efforts on vaccines, not antibiotics or antivirals. They buy startup
antibiotics/antivirals when possible, to reduce their own discovery
costs. Most of the easily synthesized antibiotics have already been
made, and now we're scouring the natural products libraries with
little luck.
If you have the flu yourself, you can extract its RNA. Flu is easy to
grow in eggs, that's how the vaccine is made. (No, you're not able to
grow enough flu to start your own personal epidemic in your garage,
try as you might--but you can get enough RNA to experiment with.) Flu
has a segmented genome, so getting the individual pieces of nucleic
acid for each individual protein is as simple as running a gel.
You can use any expression method of choice to grow the individual
proteins, and do shifts or Far Westerns to find binding partners. e.g.
to confirm that it is the PA subunit that steals RNA caps, you could
incubate the various units of the flu protein with RNA extracted from
grocery store chicken, and look for proteins that shift upwards in
size on a gel. Then blot the gel onto nitrocellulose, block with 5%
milk and probe with a labeled version of the original flu RNA
extract.
Pull-downs to find binding partners or potential inhibitors are easy
things that can be done at home. If someone could take the straw
method of electrophoresis and high throughput-ize it, they'd have
their very own drug discovery lab.
Bryan Bishop
> Basically, flu viruses steal pieces of your own RNA in order to mask
> their evil intentions. They have a subunit of their polymerase that
> does this, the PA unit. The specific epitope of the PA unit that does
> this function is an interesting drug target, because if it can be
> blocked, we have a nice new antiviral.
The Replication Activity of Influenza Virus Polymerase Is Linked to
the Capacity of the PA Subunit To Induce Proteolysis
http://jvi.asm.org/cgi/content/abstract/74/3/1307
"The PA subunit of the influenza virus polymerase complex is a
phosphorylated protein that induces a proteolytic process that
decreases its own accumulation levels and those of coexpressed
proteins. The amino-terminal third of the protein is responsible for
the induction of proteolysis. We mutated five potential casein kinase
II phosphorylation sites located in the amino-terminal third of the
protein. Mutations affecting position 157 almost completely abrogated
proteolysis induction, whereas a mutation at position 162 produced a
moderate decrease and mutations at positions 151, 200, and 224 did not
affect proteolysis induction. Reconstitution of the influenza virus
polymerase in vivo with viral model RNA containing the chloramphenicol
acetyltransferase (CAT) gene indicated that the CAT activity obtained
correlated with the capacity of each PA mutant to induce proteolysis.
RNA protection assays of the products obtained with viral polymerase,
reconstituted in vivo with model RNAs, indicated that mutations at
position 157 led to a selective loss of the ability to synthesize cRNA
from the viral RNA template but not to transcribe viral RNA, while a
mutation affecting position 162 showed an intermediate phenotype.
Collectively, these data provide a link between PA-mediated induction
of proteolysis and the replication activity of the polymerase."
So anyway, I did some searching-
influenza genome
http://mirrors.vbi.vt.edu/mirrors/ftp.ncbi.nih.gov/genomes/INFLUENZA/README
(the dot fna file in the parent directory is 120 MB)
search for influenza on this page:
http://www.ncbi.nlm.nih.gov/genomes/genlist.cgi?taxid=10239&type=5&name=Viruses
and in particular get to these 8 sequenced segments of influenzavirus A:
http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Search&dopt=DocSum&term=txid335341%5BOrganism%3Anoexp%5D
and in particular, segment 3 is polymerase PA:
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=CY002069
while segment 2 is polymerase PB1:
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=CY002070
while segment 1 is polymerase PB2:
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=CY002071
Some issues come to mind. First, viruses and any other
self-replicating system is going to generally be subject to natural
selection, so if you make a blocker for the PA subunit, those
influenza viruses that mutate and are able to get around that PA
subunit inhibitor will be able to obviously get past your antiviral,
and using the antiviral would increase the selective pressure on this.
Now, I suppose I can imagine a scenario where you are able to
simultaneously get the rates down for the flu so low in that these
selective pressures might not "have as much time to kick into action"
(somehow overcoming the natural rate of mutation of influenza, and
whatever benefit that natural rate of mutation may or may not have).
See also:
The PB1 subunit alone can catalyze cRNA synthesis, and the PA subunit
in addition to the PB1 subunit is required for viral RNA synthesis in
replication of the influenza virus genome.J. Virol. 1996 Sep;
70(9):6390-6394
Crystal structure of the polymerase PAC–PB1N complex from an avian
influenza H5N1 virus
http://heybryan.org/~bbishop/docs/Crystal%20structure%20of%20the%20polymerase%20PAC%3fPB1N%20complex%20from%20an%20avian%20influenza%20H5N1%20virus.pdf
Figure 2c shows locations of known mutations to PAc. "The recent
emergence of highly pathogenic avian influenza A virus strains with
subtype H5N1 pose a global threat to human health1. Elucidation of the
underlying mechanisms of viral replication is critical for development
of anti-influenza virus drugs2. The influenza RNA-dependent RNA
polymerase (RdRp) heterotrimer has crucial roles in viral RNA
replication and transcription. It contains three proteins: PA, PB1 and
PB2. PB1 harbours polymerase and endonuclease activities and PB2 is
responsible for cap binding3, 4; PA is implicated in RNA replication5,
6, 7, 8, 9, 10 and proteolytic activity11, 12, 13, 14, although its
function is less clearly defined. Here we report the 2.9 ångström
structure of avian H5N1 influenza A virus PA (PAC, residues 257–716)
in complex with the PA-binding region of PB1 (PB1N, residues 1–25).
PAC has a fold resembling a dragon's head with PB1N clamped into its
open 'jaws'. PB1N is a known inhibitor that blocks assembly of the
polymerase heterotrimer and abolishes viral replication. Our structure
provides details for the binding of PB1N to PAC at the atomic level,
demonstrating a potential target for novel anti-influenza
therapeutics. We also discuss a potential nucleotide binding site and
the roles of some known residues involved in polymerase activity.
Furthermore, to explore the role of PA in viral replication and
transcription, we propose a model for the influenza RdRp heterotrimer
by comparing PAC with the 3 reovirus polymerase structure, and docking
the PAC structure into an available low resolution electron microscopy
map."
The dragon head:
http://www.edu.cn/cheng_guo_zhan_shi_1085/20080831/W020080831298498757881.jpg
> If you have the flu yourself, you can extract its RNA. Flu is easy to
> grow in eggs, that's how the vaccine is made. (No, you're not able to
Can you describe this some more please, or provide some references?
What type of eggs?
> grow enough flu to start your own personal epidemic in your garage,
> try as you might--but you can get enough RNA to experiment with.) Flu
> has a segmented genome, so getting the individual pieces of nucleic
> acid for each individual protein is as simple as running a gel.
I wonder if just ingesting PB1N ("residues 1-25") would be enough
here- or at least somehow getting into the body and on the scene. In
which case, that would be:
MDVNPTLLFLKVPAQNAISTTFPYT
> You can use any expression method of choice to grow the individual
> proteins, and do shifts or Far Westerns to find binding partners. e.g.
> to confirm that it is the PA subunit that steals RNA caps, you could
> incubate the various units of the flu protein with RNA extracted from
> grocery store chicken, and look for proteins that shift upwards in
> size on a gel. Then blot the gel onto nitrocellulose, block with 5%
> milk and probe with a labeled version of the original flu RNA
> extract.
Right. Sounds like the above study on the replication activity of the
subunits would be a good starting point on that type of home
experimentation.
> Pull-downs to find binding partners or potential inhibitors are easy
> things that can be done at home. If someone could take the straw
> method of electrophoresis and high throughput-ize it, they'd have
> their very own drug discovery lab.
So, the experimental design would be to do some large scale automation
or high throughput work to figure out which mutations cause the virus
replication activity to drop like a rock, from looking at the gel
results. What about actually getting the PAc subunit inhibitor though?
I suppose that one terrible way to do it would be to separate the
genome by mass via gel electrophoresis, cut out the PB1N proteins, and
make a giant library and keep detailed records of where each one came
from for each experiment, trying to find a natural mutation from your
many rounds of replicating the virus' proteins. But maybe there's a
more direct method that might involve, say, ordering some oligos over
the net?
Any thoughts?
Jeswin John
What type of eggs?"
http://www.rkm.com.au/VIRUS/Influenza/influenza-vaccines.html
*-----------------------------------------------------------*
Join the Revolution
http://diybio.org/
http://homebrewbioscience.blogspot.com/
*------------------------------------------------------------*
Lora
unfertilized eggs, actually, I don't have any other kind. Pretty much,
you have a filtered inoculum (in this case, derived from your
nose ;-) ), you sterilize a needle, wipe the eggshell w/ alcohol and
stick the needle in the inoculum and then through the eggshell. Put a
bandaid on the eggshell if you like to keep out bacteria while it
grows. Incubate as usual @ 37C. Crack egg about a week later & harvest
RNA as usual protocol for getting RNA out of blood or tissue.
> Some issues come to mind. First, viruses and any other
> self-replicating system is going to generally be subject to natural
> selection, so if you make a blocker for the PA subunit, those
> influenza viruses that mutate and are able to get around that PA
> subunit inhibitor will be able to obviously get past your antiviral,
> and using the antiviral would increase the selective pressure on this.
most antibiotics sooner or later, too, but we keep looking for new
antibiotics, you know? Ideally you'd want 100% vaccination, but that
ain't ever going to happen.
> I wonder if just ingesting PB1N ("residues 1-25") would be enough
> here- or at least somehow getting into the body and on the scene. In
> which case, that would be:
>
> MDVNPTLLFLKVPAQNAISTTFPYT
what else.
> So, the experimental design would be to do some large scale automation
> or high throughput work to figure out which mutations cause the virus
> replication activity to drop like a rock, from looking at the gel
> results.
structures, similar to how Joseph Schlessinger @ Yale did a lot of
estrogen analog work. It depends on what you're interested in and
where your spatial reasoning talents lie.
>But maybe there's a
> more direct method that might involve, say, ordering some oligos over
> the net?
Douglas Ridgway
> If you have the flu yourself, you can extract its RNA. Flu is easy to
> grow in eggs, that's how the vaccine is made.
Uh, can't the flu virus cause disease in healthy humans? Doesn't that
mean it's at least BSL 2?
Jeswin John
It would be real helpful if DIYers post their lab location e.g. corporation, university, home lab, etc when you describe your method/experiment. That way we know if you do crazy experiments in your house which is highly not recommended.
Douglas Ridgway
cultivating a human pathogen at home, which seems like a bad idea to
me.
Daniel C.
> I'm sure she does. But it almost sounded like a suggestion to try
> cultivating a human pathogen at home, which seems like a bad idea to
> me.
Education time. If you're already sick with the flu, how is
cultivating it going to hurt you more?
-Dan
Brandon Ason
One concern would be that the strain you isolate could potentially become more pathogenic in culture overtime.
> Date: Sat, 14 Feb 2009 17:01:13 -0700
> Subject: Re: Project suggestion: Flu biology
> From: dcroo...@gmail.com
> To: diy...@googlegroups.com
Nick Taylor
>> cultivating a human pathogen at home, which seems like a bad idea to
>> me.
>
> Education time. If you're already sick with the flu, how is cultivating it going to hurt you more?
There's a completely unrelated flu epidemic that kills about 50 million people like the one in 1918, then someone reads this mailing list looking for someone to blame, and sees that you once said "If you're already sick with the flu, how is cultivating it going to hurt you more?"
And then they all come round to your house with flaming torches and pitchforks.
Or there isn't, but for some reason there's a massive panic because someone's created the rumour that this year's flu was actually created in a DIYbio lab by mistake... so loads of people come round to your house with flaming torches and pitchforks.
Or you could actually create a variant that kills 50 million people. Or just one person.
And so on. Use your imagination, because sooner or later someone wanting to sell newspapers is going to use theirs.
Nick
Lora
On Feb 14, 7:50 pm, Brandon Ason <brandon_a...@hotmail.com> wrote:
> One concern would be that the strain you isolate could potentially become more pathogenic in culture overtime.
flu *more* virulent in a deliberate way--in other words, you could not
decide, "Aha, today I shall create a horrible flu pandemic!" and then
go about doing that, because too much is unknown about flu biology.
The mechanics of doing so are still a mystery. But in general, unless
you have a sizable flock of ducks and a pig farm in your backyard,
you're not going to be able to get significant recombinations that
would be more hazardous. I'm thinking your neighbors would *notice*
200 sneezing ducks and a couple dozen pigs in your yard, you know? The
places where we KNOW the weird mutations and pandemics DO arise, is
where people don't really notice that sort of a thing. (Hence my
opposition to factory farming, but that's a rant for another day.)
> > On Sat, Feb 14, 2009 at 2:19 PM, Douglas Ridgway <ridg...@dridgway.com> wrote:
> > > I'm sure she does.
doors. You shed nastier pathogens into the sewer every day. Think of
everyone who gets Salmonella from the peanut butter / spinach / recall
of the day: They're mainly at home, spewing all those cooties all over
their bathrooms, not in a hospital, as are just about everyone with
infectious diseases, including flu. Back in The Olden Days, when I was
a wee lass of a student, we used to teach the nursing students'
microbiology labs using what are now considered BSL2s and even 3s. We
routinely used all the enteric bacteria, complete with nasty toxins,
the STDs, the toxin-producing staph, the vector-borne pathogens,
Bacillus spp, some of that stuff is now considered select agents. The
point being that you learn to handle things safely and the techniques
are truly no different from safe food handling: Wipe your workspace
down with disinfectant before & after work. Wash your hands thoroughly
w/ disinfectant soap before and after work. Practice good aseptic
technique. Wear gloves, safety glasses and a lab coat, and when you
are done for the day, wash your lab coat with a bit of bleach, wipe
down your glasses with an alcohol wipe, and throw your gloves away.
Put used culture materials in a container and cook them in a pressure
cooker (30 min, 15 psig, 120C) to sterilize. Same rules you would
follow when working with transgenic bacteria, really.
You ARE using BSL2 precautions when working with transgenic bacteria,
right? Right? I mean, antibiotic-resistant E. coli isn't exactly a
healthy thing to drink...
If you're very very worried, or very much into this stuff, you can
even get a biosafety cabinet at home; some hobby mycologists have them
for airflow control during the cultivation of particularly nitpicky
fungi (e.g. morels). Then everything is neatly contained in your
cabinet, which makes things a wee bit easier for cleanup and allows
your aseptic technique to be a little more lax (you can leave
containers open for longer, do pass-overs if you need to, don't worry
about contamination from the air column).
However, as Bryan pointed out, you can just order the DNA on the
internet if you want--the sequences are already published. One protein
does not a virus make. Then you're working only with DNA and store-
bought chicken, which is safe as houses.
Now that I think about it, safe as...um...something really safe...
Lora
to teach the undergrads in Medical Microbio 301: Disease Surveillance.
One of my fellow students even got a couple of nice publications for
discovering new bacteria and new gene cassettes and so forth, so could
be worth doing at home.
Take several blood, BHI or chocolate agar plates and sterile swabs.
Swab everything you think is probably dirty, including the dreaded
Public Toilet Seat, a local body of water, industrial waste
discharges, ballast water from ship docks and at least one person's
nose. Also, sample things you would devoutly hope are clean, like your
drinking water. Streak onto plates and let grow. ID per Bergey's
Manual of Det. Microbio, confirm by PCR of 16s RNA. Repeat at
different times of year, as the winter bugs are likely to be somewhat
different from the summer bugs. Once ID'ed, look for antibiotic and
heavy metals resistance genes.
Fellow student discovered a new species of Enterobacter, which was
resistant to all classes of antibiotics invented at that time and
heavy metals too. This was discovered in a local body of water used
for fishing and recreation, and it turned out to be the direct result
of raw sewage being discharged by a municipality that had so little
tax revenue it could no longer maintain its own sewage services. The
kicker was, no one ever did anything about the sewage system, and it's
still oozing filth into the river to this day, creating heaven knows
what recombinations.
Bryan Bishop
> Take several blood, BHI or chocolate agar plates and sterile swabs.
> Swab everything you think is probably dirty, including the dreaded
> Public Toilet Seat, a local body of water, industrial waste
> discharges, ballast water from ship docks and at least one person's
> nose. Also, sample things you would devoutly hope are clean, like your
> drinking water. Streak onto plates and let grow. ID per Bergey's
> Manual of Det. Microbio, confirm by PCR of 16s RNA. Repeat at
> different times of year, as the winter bugs are likely to be somewhat
> different from the summer bugs. Once ID'ed, look for antibiotic and
> heavy metals resistance genes.
That sounds like it would be a fantastically fun kit to build,
distribute and use. The instructions are fairly simple- even PCR could
be made to have a simple protocol and instructions- and then the
antibiotics and resistance tests might have to be purchased from the
pharmacy or drug store down the street (that sort of thing), but other
than that, a package of cotton swabs, some plastic plates with tops,
and some instructions might make for an interesting project. And if
I'm not mistaken, this is what the bioweathermaps project is about-
except getting some deals for individuals to send in DNA samples for
sequencing and subsequent identification (which, by the way, should
probably be confirmed visually by looking at the cultures and culture
behavior).
Btw, I see two books for Bergey: Bergey's Manual of Systematic
Bacteriology, and Bergey's Manual of Determinitive Bacteriology; which
one are you referring to, or is there a book specifically on micro
that I'm not seeing?
Douglas Ridgway
There's no blanket requirement that transgenic bacteria be handled at
BSL 2. There *is* such a requirement for culturing human pathogens. As
I think you know, probably much better than me, a BSL 2 laboratory
isn't compatible with a home kitchen. Were someone to follow your
suggestion, they could get in serious trouble, or even risk hurting
somebody.
Lora
habitats--nice to have, but expensive and not needed for this
project.
> 1 512 203 0507
Lora
I understand your concern, really I do, I heard many undergrads with
the same concerns...But there is no magic wand that makes a BSL2 lab
special. There is a sort of belief that BSL2 labs have special
oversight and management and auditing that somehow goes on by The
Authorities to ensure that everything is safe and no one is doing
anything especially dangerous, but honestly I do not know where this
belief comes from. **In no microbiology lab I have worked in over the
past 15 years has anyone, ever, cared one whit about the safety of the
BSL2 infectious cooties I was working with, including doing gov't
work.** They were worried more about whether or not my pipettes were
calibrated than about whether I was adequately cleaning up after my
Salmonella spp. Visit any university and watch how many random people
go in and out of the microbiology labs with nary a "hey, excuse me,
what are you doing?" BSL2s are not a big deal. Again, to re-emphasize:
I'm talking about something that is *already* circulating in the
environment, things to which your entire household has already been
exposed. NOT a novel organism.
BSL3 is very different. As soon as you work with BSL3s, then the Very
Serious Regulations kick in. I would never advocate anyone working
with BSL3s. Those things are nasty critters, and those WILL get you in
hot water for playing with. Don't go near the dead squirrels or
prairie dogs...
I know you're not saying that adding antibiotic resistance genes to
gut bacteria is completely non-hazardous, right? The fact is that just
about everything in a lab is conceivably a hazard: electricity,
antibiotic resistant bugs, caustic reagents, flammable solvents,
allergy-inducing proteases, autoclaves/pressure cookers, these are all
nasty things. But, just like any of the ordinary household hazards,
you learn to handle them safely.
On Feb 15, 11:24 pm, Douglas Ridgway <ridg...@dridgway.com> wrote:
> Lora,
>
> There's no blanket requirement that transgenic bacteria be handled at
> BSL 2. There *is* such a requirement for culturing human pathogens. As
> I think you know, probably much better than me, a BSL 2 laboratory
> isn't compatible with a home kitchen. Were someone to follow your
> suggestion, they could get in serious trouble, or even risk hurting
> somebody.
>
ndpm
There are areas of science where DIY can make a contribution and/or
the individual can get a lot out of it personally. This isn't one of
them.
Comparing the risks associated with operating a pressure cooker full
of agar plates with the risks associated with influenza virus is
illogical.
There are plenty of relatively safe, challenging, important, and fun
things to work on. Leave the relatively unsafe, challenging,
important, and fun things to others.
Phil...
**********************
Douglas Ridgway
No, there's no magic in being safe with pathogens (aka BSL2), just a
nearly trivial amount of facilities and equipment, plus the training
and supervision so that, as you put it, "you learn to handle them
safely". You obviously not only have that training, but also
substantial experience providing that training and supervision to
others. If you took the food out of your kitchen, installed an
autoclave and any other equipment needed, and hung a sign on the door,
I think you could reasonably do that work in the room formerly known
as your kitchen. You'd have to find someplace else to cook and eat,
what with your kitchen now being a BSL 2 lab, but you could do it.
I, however, could not. I don't have the skills for handling pathogens,
and reading a few tips from you on email doesn't give them to me. Lab
skills are taught hands-on: you'd have to show me the first time, be
available as I asked questions, and keep half an eye on me until you
felt confident I wasn't going to do something stupid, endangering
myself or the data. Just like you were taught, just like you'd teach
one of your undergrads.
Fine, so that's safety. But in addition to plain safety, ie the direct
health risks to the workers and others of becoming infected with the
lab culture, there is also regulatory risk. A DIYer (and isn't that
what we're talking about here?), fairly or unfairly, will be treated
differently from a recognized institution. Screw up in an academic
lab, as eg the incident where a UCLA tech was killed a couple months
ago, and the Authorities will probably just give the institution a
fine, and the institution will make the parties at fault sit through a
lot of meetings. The assumption is that the institution was acting in
good faith, but may need to change practices so as to be more safe in
the future. Personal basement labs, on the other hand, can get an
assumption of bad faith. Google Victor Deeb, or Steven Kurtz, or Lewis
Casey, for examples of what I'm talking about. One criminal
indictment, no matter how unjustified, can put a serious cramp on your
life plans.
Now, there's no absolute defense against being raided or charged by an
ignorant or corrupt prosecutor. They can always find something.
(Heating broth in an Erlenmeyer in Texas? Unregistered glassware, they
gotcha. Use a pickle jar instead? "Ghetto glassware", and it'll be
introduced as evidence of illicit intent. Culturing RG2 pathogens in
Canada without a license? Under C-11, that'll be a federal offense and
a prison term.) The question is, what evidence are you going to give
your defense attorney to work with? I'd think a reasonable minimum
would be to document that you understand and are abiding by the rules
that would govern that work being done in any other place. Being
casual about the well established and accepted distinctions between
different risk groups of organisms is not the way to go.
Tom Knight
> DIYbio folks working on live influenza viruses is a bad idea. Period.
> There are areas of science where DIY can make a contribution and/or
> the individual can get a lot out of it personally. This isn't one of
> them.
>
> Comparing the risks associated with operating a pressure cooker full
> of agar plates with the risks associated with influenza virus is
> illogical.
>
> There are plenty of relatively safe, challenging, important, and fun
> things to work on. Leave the relatively unsafe, challenging,
> important, and fun things to others.
Just so, and well said. Don't confuse two issues:
* Being safe
* Giving others confidence of your work being safe
These are definitely not the same, and you can confuse
yourself (in either direction) if you mix them up.
You need to both be safe and provide others assurance
that you are being safe. Doing BL-2 work at home, while
perhaps safe for some people, does not give the appearance
of being safe. You (and our community!) will suffer.
sgt york
well, so I do know what it entails. Our Health & Safety readiness
entailed that I (1) had access to an autoclave and (2) had plenty of
"BIOHAZARD-BSL2" scarystickers to put around the lab. Today, I ate
lunch not 2 meters from where I prep about 4 different BSL2 organisms
on a daily basis, and I know there's nothing wrong with it. It's not
even against regulations here. We have a 3 feet rule.
But you should hear people freak out when I tell them, or worse, they
see me working with them. Some of my equipment looks like it belongs
on the set of Outbreak. In reality, it's to protect the bugs from me;
not the other way around. But I can't tell anyone that and have them
think I'm not just placating them.
There is a perception issue, and a perception like this could be very
harmful to this community.
Nick Taylor
> Just so, and well said. Don't confuse two issues:
>
> * Being safe
> * Giving others confidence of your work being safe
1) corporate/private ownership of our life-support systems.
2) prohibition (and all the damage that that causes)
3) ecosystemic inbalance
4) superbugs
To a greater or lesser degree we already have most of these, and aren't dealing with them now... 1) and 2) are intimately intertwined, 3) and 4) are intimately intertwined.
And 1) and 2) are bound up with perceptions of safety... which isn't to say that perceived safety is more important than actual safety... it's just that in my opinion, the consequences of ignorance are greater than the consequences of an accident... in fact ignorance makes the accidents so much worse.
I actually find Lora pretty convincing... in fact things that she says have formed a cornerstone of how I think we should respond/adapt etc... but getting these ideas across in a soundbite may not be so simple... particularly given the whole Persistence of Myth thing - the biproduct of which is that the more you talk about safety and security, the less safe and secure people think they are.