Memoirs of a Defective Brain

Blogging Microbes- Communicating Microbiology to Netizens

2014-10-02T12:05:00.003+01:00

There was a conference on Blogging microbes at Nottingham University, with talks from Alan Cann, Shuna Gold, James Gurney and Oscar Rosales, organised by Ivan Lafayette from the Toxicologist Today.
Oh yeah, and I was there as well, talking about how blogging can enhance your scientific career.
We had a lot of fun there, it was crazy meeting other bloggers, and the people who read blogs. It was a really great atmosphere, and it was interesting meeting some of the other people in the community in real life.

It's also time that I confessed. I've been cheating on you guys with another blog. I've started contributing to Sciencemadeeasy.kinja.com. I curate and contribute content there, aimed more towards a general audience than this blog. If you just want to read the stuff I write, then check out Faz-alam.kinja.com.
I have no plans to leave field of science, as there is definitely some content I would like to contribute here, such as the MicroTwJC summaries, which wouldn't really fit for a lay audience. So keep an eye on this space as well.

Microtwjc 49: Discussion and Criticisms

2014-06-09T11:25:00.001+01:00

What is Microbiology About ?

2014-06-01T14:53:00.001+01:00

#MicroTwJC 49 Expanding the Genetic Alphabet

2014-05-25T05:39:00.000+01:00

Recently scientists have achieved a feat the like of which has not been seen in billions of years, they have added new letters to the genetic alphabet of a living organism. What did they do? How did they do it ? Is it too good to be true? All these questions, and more await.

#MicroTwJC 47: Total Synthesis of a "Functional" Designer Yeast Chromosome

2014-04-19T20:34:00.001+01:00

#MicroTwJc 46: FtsZ placement in Bacterial cells

2014-03-31T17:25:00.001+01:00

An In depth look at how bacteria divide in "FtsZ placement in Nucleoid Free Bacteria"!
Link: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0091984

Statistical Websites:
1-way ANOVA
http://www.danielsoper.com/statcalc3/calc.aspx?id=43
Tukey's Honestly Significant Difference
http://faculty.vassar.edu/lowry/hsd.html

Microbiology Twitter Journal Club Website for more details:
http://microtwjc.wordpress.com/

MicroTwJC 45: When Antibiotic Resistant Compete (A video presentation)

2014-03-26T19:46:00.000+00:00

This summary of the latest Microbiology Twitter Journal Club is going to be different in two ways. Firstly, it's late. Secondly, it's a video. I noticed a while ago that my explanations of papers tended to end up longer than the papers themselves, and I decided to experiment. Here are the results

The Post Antibiotic Era will take your job away.

2014-03-16T10:23:00.002+00:00

In 1934, The Los Angeles Milk Commission gave its employees devastating ultimatum- lose their tonsils or lose their jobs. When our antibiotics stop working, your employer may force you to make a similar choice; and they'd be right.

Infectious bacteria are developing resistances to our medicines at alarming rates, and we are entering a Post-Antibiotic Era. If you want to know how people will cope in this new era, the best place to look is in the past. In the era before antibiotics.

In the early 1930's, scarlet fever and strep throat were much more common than they are today, and much more hazardous. In the worst case scenario, a sore throat could develop into full blown sepsis.

So when a report was published in 1931 describing 71 outbreaks of this disease that could be traced to one common factor, people paid attention. That common factor was milk contaminated with bacteria named Streptococcus epidemicus. These bacteria tended to infect the udders of cows, causing mastitis. Since the udders also happen to be where the milk was produced, bacteria inevitably spread to the milk.

Needless to say, these outbreaks needed to be brought under control.

That responsibility fell to the Los Angeles Milk Commission, implemented a number of rules to prevent any more outbreaks occurring.

The first step was to find any dairy cows infected with the disease. Fortunately, there were already rules in place for this. Cows needed to be certified to be free of S. epidemicus, and any infected cows needed to be isolated from the rest of the herd.

Here is the problem. Both humans and cows could carry S.epidemicus. To control any outbreaks, the same restrictions that applied to cows had to apply to the humans who worked with them. Humans could carry S. epidemicus without any symptoms, and appearing perfectly healthy.

Out of a thousand employees tested, fifty were carriers. This was devastating. They could no longer be allowed to work, for the risk of them causing an outbreak was unacceptable.

There were no available antibiotic treatments to allow these carriers to rid themselves of S.epidemicus, but that didn't mean there were no options. It was known at the time that S. epidemicus survived in human tonsils. If the tonsils were removed, then the bacteria would have nowhere to go, and die off.

In a quote from the Director of the milk commission:

"care is taken in each case to impress upon them that the procedure is not compulsory except that otherwise they must retire from employment at certified dairies"

Basically, the procedure was only compulsory if the workers wanted to keep their jobs.

Unsurprisingly, most of the workers chose to go through the operation.

But sixteen of the infected employees either refused to go through with it, or were refused on the basis of underlying health issues that could render such an operation life threatening. These people were forced out of their jobs, and any dairy in the area was given their details should they attempt to apply for another job in the milk industry. Essentially, they were blacklisted from the industry.

You can say it was cruel that employees were forced into this situation, and you're not wrong. But it was an awful dilemma, and one that is destined to repeat. In a world without antibiotics, people who become carriers of diseases may remain carriers for the rest of their lives. Through no fault of their own, these people will pose a threat to the rest of the population, and it will hurt their chances of working in certain jobs. Would you send your kid to a school where a teacher constantly infects their students with life threatening illness ? Or buy groceries from a man with chronic diarrhoea? Allow yourself to get treated by Typhoid Mary?

None of those sixteen workers ever suffered any symptoms. They may spent their life working in the dairy industry, only to be cast aside. They at least had the option of getting surgery to prevent them from being carriers. Not all bacteria are polite enough to live solely within an easily removed organ.

The Post-Antibiotic Era is coming, and it won't just affect "sick" people, it will have wider effects throughout society. If there is one lesson we can learn from this eighty year old paper, it is that you don't even have to be "sick" for bacteria to ruin your life.

*Streptococcus epidemicus is a defunct classification that tends to be refer to what would be called S. zooepidemicus these days, and S. pyogenes.

Bonynge C.W. (1934). Solution of the Streptococcus Carrier Problem *†, American Journal of Public Health and the Nations Health, 24 (10) 1031-1034. DOI: 10.2105/AJPH.24.10.1031

TMI Friday: When you've literrally had your Chips

2014-01-17T15:48:00.001+00:00

The victim of this weeks TMI friday didn't really do anything wrong. In fact, this is less of a TMI Friday than a FML Friday, but it's an odd story that perhaps you'll find interesting, and perhaps learn from.
Our story begins with a 42-year old gym teacher who had hurt her foot. She didn't exactly know what could have caused the pain, but the point was that it was there. She had been enduring it for four weeks, and her doctor had given her painkillers to help her get over the pain.
But it wasn't enough, but there are other things people can do to relieve pain, such as wrapping something very cold in a towel and applying it to the afflicted area. It usually works pretty well.
That was what this lady did. She wrapped a bag of frozen chips in a towel and rested it upon her aching feet. But then she fell asleep.
When she awoke forty minutes later, she found that the pain had subsided, but that her foot had now become swollen and red. The next day, the foot became discoloured, and her doctor prescribed her a course of antibiotics. The day after, she was in the emergency room with what was described in the paper as "third degree frostbite".
Frostbite causes blood vessels to constrict, which helps the body to retain heat, but reduces the blood flow to the extremities. Your extremities need blood, so not getting it can be pretty bad for them. When it gets cold enough for layers of skin to freeze, the real trouble begins, because this can cause the cells to die off, and can lead to blisters. In this particular case, the cold compress froze the nerves and muscles below the skin, causing them to die off.
This is why physicians recommend that you always try to put some layers of fabric between you and the cold surface,and that you should never put on a cold compress for more than 30 minutes. Also, Chips are for eating.

Graham C.A. Frozen chips: an unusual cause of severe frostbite injury, British Journal of Sports Medicine, 34 (5) 382-383. DOI: 10.1136/bjsm.34.5.382

TMI Friday: Frippery Furrows Fanny

2014-01-10T12:00:00.000+00:00

Jewellery, body piercing and other such frippery are usually harmless. A ring here, a piercing there. Humans have been using these accoutrements for millenia without problems. at least, problems they'll admit to.

In this weeks case, we look at a young lady who repeatedly suffered from vaginal bleeding after sex. There was nothing wrong with her menstrual cycles, and there were no signs of bleeding outside of sex.

She had been suffering with this problem for seven months.

Further examination of the lady's delicate area revealed abrasions and lacerations.

She then admitted that her boyfriend wore penile jewellery of the ‘Prince Albert’ type. This information shed light on the aetiology of the post-coital bleeding as well as the bizarre vaginal ﬁndings

With this revealed, the doctors furnished the woman with the rather obvious advice that she should get her boyfriend to remove his penile adornments before the engaged in copulation*.

Esen U.I. & Orife S. (2006). Penile jewellery: a cause of post-coital bleeding, Journal of Obstetrics & Gynaecology, 26 (5) 483-484. DOI: 10.1080/01443610600766884
*I got a thesaurus for Christmas. Apologies.

TMI Friday: How long ?

2014-01-03T22:30:00.000+00:00

This weeks victim/perpetrator showed up to Hirosaki hospital with blood in his urine. The doctors spotted that he had a foreign body that had wormed it's way up into his bladder, but they couldn't quite work out what it was. An x-ray revealed five cm diameter blob, but how could such a large object get up there in the first place ?
The patient revealed the answer. His suffering began when he inserted a long vinyl tube up his urethra.
How long had it been there ?
Two years.
How long was it ?
One and a half metres.
I don't know if the Guinness book of world records recognises the accomplishments of people who stuff things up their own schlongs, but if they did, I suspect the gentleman in this study could be a world record holder.
Just don't try to break his record at home. Or in public.

Imai A., Suzuki Y., Hashimoto Y., Sasaki A., Saitoh H. & Ohyama C. (2011). A Very Long Foreign Body in the Bladder, Advances in Urology, 2011 1-2. DOI: 10.1155/2011/323197

TMI Friday: The Grinch Syndrome

2013-12-27T16:22:00.001+00:00

Last week, I blogged about the "Santa Claus" Syndrome. Taking inspiration from the "Worst ever Christmas story ever" from the "Gremlins" film, I clawed through the medical case literature to see whether there was any truth behind this story.
After much googling, I tracked down a paper entitled "The Santa Claus Syndrome", which appeared to answer all of my questions, but was locked behind a pay-wall. So I posted the awesome abstract of the paper and left it at that.
But I must have been a good little boy this year, because Santa* left me a present underneath my Christmas tree. A copy of the full paper !
So gather 'round children, you are in for a gruesome tale.

Our story begins with a 17 year old burglar who saw a tempting target of opportunity. It was in the early morning hours, the sun had not risen, and the store was empty. The doors were locked, but our enterprising thief noticed that the owners had left their chimney unguarded. I imagine a Grinch like smile crossing his face, and him rubbing his palms together. For he would not be going down that chimney to give presents, like Santa Claus. The plan was sheer elegance in its simplicity.

The paramedics found him fourteen hours later, jammed in the chimney, his hand caught in a heat vent.
You might be wondering what happens to someone when they get stuck in a chimney. I am here to give you the facts in all of their gory details.
They managed to get the burglar out of the chimney and he was immediately transported to a burns unit. His nostrils and tongue were coated with soot, and he had first and second degree burns all over his chest, abdomen and lower body. But it wasn't just the burns that were the problem for this young gentleman.
He was also covered in pressure sores. These occur at points on the body where bone is close to the surface of the skin (like your elbows, or your knees). When you put pressure on these "bony prominences", the skin, the bone squeezes and tears at the body tissue it's pressing against.
Effectively, the burglar's skin was been worn away, with the chimney acting as the pestle, and his very bones acting as the mortar.
The tissue around the burglars knee had already been severely damaged, and had become "Necrotic", and his hand had become infected and "gangrenous". But these weren't even his biggest problems.
His biggest problems was that there really wasn't much oxygen in that cramped chimney shaft. The oxygen levels in his blood were dangerously low. They intubated him, but his condition just kept getting worse.
He was soon suffering from "Acute respiratory distress", which happens to the cells of the lung that have suffered from so much injury that the immune system kicks into overdrive. In order to get as many immune cells as possible into the lungs, the blood vessels entering the lungs become "leaky". They leak fluid into the lungs as well as inflammatory cells, essentially flooding the lung. It's not unlike drowning.
The physicians prescribed steroids in the hope that they would bring this under control, as well as giving him antibiotics for his infection and intravenous fluid to keep him hydrated.
Yet it still got worse. The burns he had sustained, combined with the severe pressure sores and the gangrene had lead to his muscles breaking down. All muscles contain a compound known as myoglobin, which is like haemoglobin in that it stores oxygen, but only to be used for the muscles. Whilst it is useful, it is also very poisonous to the kidneys if a large amount of it enters the blood stream.
The burglars muscles had been damaged to the point that they were now leaking myoglobin into his blood, and his kidneys were now shutting down. The doctors put him on dialysis to take the pressure off the kidneys.
But his gangrene had now effectively "mummified" the patients hand, leaving it shrivelled, dried out and dead. The surgeons were forced to amputate his whole arm to prevent the spread of the gangrene.
Various parts of his "lower extremities" suffered from the burglar having been stuck so long in an upright position. The blood had flowed down to his legs under the pull of gravity, raising the pressure in his lower limbs, giving him "Compartment Syndrome". The surgeons treated this by cutting the affected areas open so that the pressure can be relieved.
But all of this was for nought, because on the twelfth day after admission, the patient suffered from cardiac arrest and died.

The physicians who had attended this unfortunate ill fated burglar recognised that actually, people get stuck in chimneys more often than you would think. There were a number of cases reported in newspapers of people getting stuck in chimneys during burglaries, or in the process of performing pranks, many of them with fatal consequences. So the researchers devised the "Santa Claus Syndrome". These are series of specific symptoms for other physicians to watch out for if they need to treat someone who had been stuck in a chimney, so that they would have enough preparation to save their patient.
This paper was published in 1994 , and should have been a warning for us all. But housebreakers are still dying from getting stuck in chimneys.
It should be clear now that only Santa Claus is able to enter a chimney with his magic. The poor grinch is probably still stuck in some abandoned chimney in Whoville, legs swollen like melons, skin crackling and popping from the heat, and desperately inhaling lungfuls of soot and ash.

HAPPY HOLIDAYS !

Boglioli L. & Taff M. (1995). The Santa Claus Syndrome' Entrapment in Chimneys, Journal of Forensic Sciences, 40 (3) 499-500. DOI:
*or an interested reader of the blog, who I'm assuming wishes to remain nameless, but I wasn't really very clear on that.

An Unpleasant Pheasant

2013-12-25T18:24:00.000+00:00

Winter is open season for pheasant hunting. Families and friends come together to bond over the hunt. Dogs are released into the underbrush to scare up a few of the birds into the gunsights of the shooters.

But there are occasions when the prey can turn the tables on their predators, to spit their last breath at them in defiance.

Our story regards a man, who was trying to impress his wife with his shooting. She was sat behind him, watching him as he shot at panicked pheasants. Her husband managed to hit one low flying pheasant as it was headed towards them.

Although the pheasant had died, the laws of momentum allowed it to maintain it's trajectory. Had it been alive, it may have been able to manoeuvre out the obstacle that intersected with the aforementioned trajectory. Thus, it hit that object square on at full speed. That object was the man's wife with enough force to burst her spleen.

Enjoy your Christmas meal everybody !

Wilkinson M.C., Klein G., Cornell M. & Rainsbury R.M. (1987). The pheasant's revenge: an unusual zoonotic injury., BMJ, 295 (6613) 1659-1659. DOI: 10.1136/bmj.295.6613.1659

TMI Friday: The Santa Claus Syndrome

2013-12-20T18:32:00.000+00:00

The Journal of Forensic Sciences has one article that I've been attempting to dig out with whatever resources I can muster, but all I could find was the abstract. Thus, I present it to you in it's full form now, until such time as I can find the full journal article and furnish you with the details.

In recent years, there have been sporadic reports in the lay press of individuals stuck in chimneys primarily during burglary attempts. Most of these individuals suffered from suffocation or soot inhalation. Because of the similarities between this form of breaking and entering and Santa Claus' traditional entrance into homes on Christmas Eve, we define the “Santa Claus Syndrome” as postural (positional) asphyxia, inhalational injuries and body burns, and/or complications related to compartment syndrome due to entrapment in chimneys. We report a case of a man who became trapped in a chimney during a burglary attempt and died a delayed death due to postural asphyxia associated with inhalational and burn injuries and anterior compartment syndrome. An analysis of this unusual case is presented. Exhaustional and postural asphyxia, compartment syndromes, and confined space-hypoxia syndrome are also discussed.

Basically, if Santa Claus isn't dead already, he is probably some kind of horribly injured zombie.
G'night kids !

Boglioli L. & Taff M. (1995). The Santa Claus Syndrome' Entrapment in Chimneys, Journal of Forensic Sciences, 40 (3) 499-500. DOI:

TMI Friday: Rectal Repositories !

2013-12-13T12:00:00.000+00:00

On today's TMI Friday, we are once again contemplating colon contents, this time looking at the stories of four people who've used their rectums as repositories for interesting objects. I shall recount those takes to you now.

Case 1. For want of a Wine cabinet

We kick off this series of cases with a 52 year old man who stumbled into the emergency room with a real pain in the ass. He owned up to the emergency room staff that he had a habit of placing objects in his own rectum for sexual gratification. His latest attempt involved a wine glass. Unfortunately the wind glass got turned around in his rectum, making it impossible for him to remove it. After two days, he finally took himself to hospital. All of the doctor attempts at pulling the glass out failed, and eventually he had to go into surgery.

They opened up his abdomen and squeezed out of the colon via a surgical incision.

Case 2. Ball in the buttocks

Our next victim had managed to insert a ball into their anus. At first, the doctors tried giving him a hefty dose of laxatives. When this didn't work they tried to pull the ball out with an endoscope, but the tiny pincers on it couldn't get any purchase on the ball. In the end, they had to put the patient under general anaesthesia and scoop out the ball with two sets of forceps. This was successful, and the patient could go home the next day.

Case 3. Bottling it

The victim (and I guess perpetrator) in this case had a history of inserting objects up his arse for sexual pleasure. The staff couldn't get any purchase on the object just by going through the anus. They had to perform surgery, which revealed that the offending object was an eight centimetre wide bottle.

Case 4. Design Flaw

One must assume that the people who design vibrators try to ensure that the don't get lost in whatever orifice they are inserted in. That would be a key safety feature, unless they thought that their customers first thought after vaginally or anally engorging the vibrator would be to buy another one. Repeat Sales !

In this final case, we have a gentleman who had suffered from that exact problem during sex with his partner. The vibrator was to slippery to grip with endoscopic forceps, and so the patient had to go into surgery to get it out. If only there were easy holds for endoscopic forceps designed into vibrators, it would save on surgery. Think about it vibrator manufacturers !

Sangar P., Henry G. & Sood S. (2013). Foreign bodies in the rectum: report of a case series and review of the literature, Sri Lanka Journal of Surgery, 31 (2) DOI: 10.4038/sljs.v31i2.5960

TMI Friday: The worst way to be caught dead

2013-12-06T12:00:00.000+00:00

There are many things that we all do in our private time when no-one can possibly see us. Things that could potentially embarrass us. Like farting yourself a warm spot in bed. Or rearranging ones testicles to ensure that they don't undergo the dreaded "Testicle Torsion Injury". Or perhaps ordering a whole bucket of Choc-Chip Ice cream to yourself. But no-one would want to be found dead in bed with half a bucket of melted ice cream smeared on ones face whilst having one hand frozen mid scratch and the dreadful aroma of your farts presiding over the affair.

Today's TMI Friday was simply asking for this fate.

There is a sexual practice known alliteratively as "autoerotic asphyxiation". The idea behind this practice is that suffocating at the point of climax is meant to somehow improve the experience. But it can be dangerous, especially if the person engaging in autoerotic asphyxiation is doing it alone, and as a result we will no doubt encounter this practice in the future.

There are a number of ways to induce asphyxiation. The subject of today's topic decided to use a plastic bag to help improve his masturbatory experience. He set up a complex series of luggage rack straps to control the degree of asphyxiation he experienced, but some how it all went wrong.

But it get's worse. Here is the full description of how he was found.

A case is reported of a 36-year-old male, found dead in his locked room, lying on a bed, dressed in his mother's clothes, with a plastic bag over his head, hands tied and with a barrel wooden cork in his rectum. Two pornographic magazines were found on a chair near the bed, so that the deceased could see them well.

Atanasijević T., Jovanović A.A., Nikolić S., Popović V. & Jasović-Gasić M. (2009). Accidental death due to complete autoerotic asphyxia associated with transvestic fetishism and anal self-stimulation - case report., Psychiatria Danubina, PMID: 19556957

TMI Friday: Telephone support

2013-11-29T12:30:00.000+00:00

When things go wrong, often we turn to the humble telephone to help us out. Whether we turn to it to call for services found in the yellow pages, family, friends and the authorities when an emergency strikes. So when the subject of this weeks TMI Friday was faced with a problem he could not solve, he too turned to the telephone.

He had unfortunately "Lost his erections" after suffering a heart attack. Instead of opting for the usual impotence medications, he discovered another way of coping. He used his telephone to find his erections again.

He threaded telephone wire up his urethra to give himself a solid enough erection to allow him to masturbate. He did this for three years before running into any problems. One day, the telephone wire just got stuck. When he tried to pull it out, all he got was blood, and soon he lost control of his bladder.

When he eventually got to hospital, a X ray revealed that the telephone wire had coiled inside his bladder. The doctors managed to successfully pull it out with much difficulty and a heavy dose of local anaesthetic.

Trehan R.K., Haroon A., Memon S. & Turner D. (2007). Successful removal of a telephone cable, a foreign body through the urethra into the bladder: a case report, Journal of Medical Case Reports, 1 (1) 153. DOI: 10.1186/1752-1947-1-153

TMI Friday: A Shocking Discovery

2013-11-22T12:00:00.000+00:00

You should always be careful with electricity, a fact that is perfectly illustrated in this weeks TMI Friday. Today we find yet another story of a poor soul who recklessly endangered their life during the course of masturbation.

He lived in a country mining town in Australia, and had last been seen drinking at a bar. No-one knew he'd be dead within three days. We don't exactly know the circumstances of his death, save from what could be deduced from the state of his corpse.

It seems that before he died, he decided to do some experimentation with electricity.

He took the cord from one of his electrical appliances, and cut away the protective rubber around them to expose the wires. In any plug cord (In most countries) there are three wires. The live wire, the neutral wire and the earth wire. The live wire delivers the electricity, the neutral wire carries it away, and the earth wire diverts electricity to a safe place in the event of a fault.

The man had no use for the earth wire, so he tucked it back inside its sheath. He linked the neutral wire to his chest. He hooked the live wire up to a chain and wrapped it around his dick. With everything to his satisfaction, he plugged the cord in. Sparks flew.

It is thought that after he had separated from his wife, he had taken some time to travel overseas where he discovered how to use electricity for autoerotic stimulation. But when he tried to repeat the practice in his home town, he fell foul of one major difference between Australia and other countries.

The electric voltage overseas was less than half that of the mining town (110 V as compared with 240 V throughout Australia)

Cooke C.T., Cadden G.A. & Margolius K.A. (1994). Autoerotic Deaths: Four cases, Pathology, 26 (3) 276-280. DOI: 10.1080/00313029400169631

History of Bacteriology: Miasmas and Contagions

2013-11-19T16:56:00.000+00:00

Have you ever found yourself walking down a street, and be confronted with a pungent turd blocking your path?

You don't know where it came from, but whether it be a dog or a tramp, you know to avoid it. It's a natural instinct. Toiletry behaviour can be found all over the natural world. Even tree sloths will make the effort to climb down to defecate far away from where they feed. The disgusting odours we associate with putrefying meat, faeces and death are ones we naturally try to avoid, and when that isn't possible we try to cleanse ourselves thoroughly so that we don't have to experience their stench.

If you were to ask me where the Miasma theory came from, I would point not to any one individual, but to that instinct of disgust. It was it's link to this natural response that also made it so difficult for people to dismiss miasmas even when confronted with overwhelming evidence that they didn't exist.

Many peoples across the world developed their own culture of hygiene and cleanliness, which usually involved some form of cleaning ritual and disposal of waste.
Often, these traditions would propose a specific association between bad smells and disgusting objects with disease. Across the world, incense and pungent smelling products tended to be used for treatments. Getting rid of the smell equated to getting rid of the disease.
Hippocrates advised people to stay away from places with "bad air" in many of his writings, and expounded the earliest iteration of the Miasma theory. His work was translated into multiple languages and passed through the ancient world, forming the basis of medical practice for centuries after his death. The concept of "Bad air" being the cause of diseases became well established. But it was not the only theory of disease people used.
In 1546, Girolamo Fracastoro published "De Contagione et Contagiosis Morbis", where he wrote about the "Contagion" theory of disease. He drew together various observations on disease that he had accumulated over his forty year long career as a physician. He proposed that diseases could be caused by the transfer of some imperceptible, yet corrupt matter between the sick and the well. He called this corrupt matter "the seeds of contagion". He proposed that these seeds could spread in three distinct ways. Through direct contact with infected people, through contact with inanimate objects that had been in contact with sick people (he named these "Fomites") and at a distance. He connected these seeds to "putrefaction", in that they could cause it in their hosts, explaining diseases such as gangrene. His explanations generally attempted to reconcile contagion with Hippocratic theories that all diseases were caused by imbalances between four mysterious "humours" found within the body. The types of disease would be determined by which particular humour interacted with specific seeds.
His work sparked off as debate throughout Europe. Whilst the idea of contagion became more widely known, there was much debate over its theoretical causes and his interpretation of the work of Galen and Aristotle and objection over creating a whole new form of life, these seeds, out of thin air. Fracastoro's most fierce critic was one of the first converts to contagion, Giambattista da Monte. The two would continually clash over the differences in their ideas, a hostile relationship that would eventually develop into one of mutual respect. The students who were privy to these lectures came from all over Europe, and when they returned to their home countries, they would bring the theory of contagion with them.
When proponents of "Bad Air" theory heard of the contagion theory, sparks most conspicuously did not fly. There were arguments, but they were no less vicious than the ones going on between the people who accepted contagion as a theory. Contagion theory still accepted that diseases could be caused by "Bad Air", but that the reason for this was that the air was filled with the "seeds of disease". "Bad Air" Theory held that sufferers of disease were primary sources of "Bad air", and people could catch disease from close contact with the sick.
However, the "Bad Air" theory got a boost in the 17th century when the Hippocratic corpus began to be re-evaluated by a number physicians. I've already talked about a gentleman named Thomas Sydenham who was key for bringing the Hippocratic corpus into the English language and kick starting a new era in evidence based medicine. He lived in an era where doctors applied many treatments based on theoretical ideas, such as bloodletting, sweating and forcing patients to vomit in order to "balance the humors". Sydenham however recognised that many of these treatments did more harm than good, and that the only way to truly divine a theory of medicine would be by a patients bedside.
Unfortunately, he did not have access to the fabulous microscopes of Anton Leeuwenhoek (No-one did, Leeuwenhoek was incredibly protective of his work) so he had no way of determining an evidence basis for contagion. In the end, he tended to favour Hippocratic theories of "Bad air" being the cause of disease, as he had no evidence available to prove otherwise.
In a treatise about malaria* written by Giovanni Maria Lancisi called "On the Noxious Effluvium of Marshes"he connected the disease with the noxious smells emanating from marshes and the mosquitoes that live within them. He gave a name to these noxious smells, he was the first to call them "Miasmas".
The Contagion theory and the Miasma theory were no longer seen as compatible by a growing number of physicians.
His writings went global, and were of particular influence in the United States on medical professionals such as Noah Webster and Benjamin Rush. During an outbreak of Yellow Fever in Philadelphia, many suspected that it had arrived from a contaminated consignment of Coffee from a ship with a crew riddled with the disease. However, Benjamin Rush disagreed with this vehemently, suspecting it had come from the marshy miasma's he believed surrounded the city. Even though Rush believed that contagion played a role in these early cases, that belief eventually waned. By 1799 he actively protested against quarantine procedures for sailors entering the city. By 1805 he began writing treatises to dismiss the idea of contagionism.
Maritime Quarantine procedures were a problem the world over, and cut into the business of many growing companies. It was in the interests of these companies to fuel the backlash against Contagionism.
When quarantine procedures** failed to prevent the Liverpool Cholera epidemic, a major blow was struck to the theory. The Cholera Riots demonstrated the true chaos which could follow a disease epidemic, and spurred people to enact new precautions against these outbreaks. These precautions would be based on the theory of Miasma.
Edwin Chadwick was a major supporter of the Miasma theory, and a vehement anti-contagionist. . He was aware that often, Cholera was associated with unsanitary conditions, and he realised that if the Miasma theory was true, then the greatest threat to peoples health was the presence of open sewers within cities. Through his efforts, open sewers were eliminated, and the cleaning of the streets began to be seen as an issue of public health. These massive clean-up efforts were incredibly effective, and would fuel further support for Miasma theory.
As we now know, Miasma's do not exist, at least not in the form that most people believed in at the time. There are solid reason why humans developed an aversion to particular bad smells. Often the sources of these smells would contain harmful diseases. Clearing out sewage from cities helped people because it meant that flies that fed on faeces could not then transfer microbes from those faeces onto food. It removed opportunities for bacteria such as typhoid from spreading through the streets in shit.
Whilst Chadwick's reforms produced results, they still had problems that are a lot more obvious to modern readers. Whilst the sewers were covered, they still discharged into the water supply, which caused regular outbreaks of cholera and typhus within the city.
Nevertheless, the Miasma Theory had risen to prominence by the 1830's, and its supporters would often define themselves as "anti-contagionist".
But the picture, as always, is more complex. Contagionism, whilst under attack, was far from dead.

*The very name "Malaria" refers to the bad air theory, with "Mal" meaning bad and "Aria" referring to the air. Although it didn't necessarily just refer to our modern interpretation of the disease. Many sweating sicknesses similar to what we understand as malaria would be referred to by its name as well.
** No-one knew of the waterborne nature of the disease, and nor was it thought that fomites could have played a role.

References

Curtis V.A. (2007). Dirt, disgust and disease: a natural history of hygiene, Journal of Epidemiology & Community Health, 61 (8) 660-664. DOI: 10.1136/jech.2007.062380

Nutton V. (1990). The Reception of Fracastoro's Theory of Contagion: The Seed That Fell among Thorns?, Osiris, 6 (1) 196. DOI: 10.1086/368701

Ayliffe G.A.J, English M.P. (2003) Hospital Infection: From Miasmas to MRSA, Cambridge University Press. Link

TMI Friday: Friction Burns

2013-11-15T12:00:00.000+00:00

When engaging in Slicking the Willie, Molesting the monkey and Throttling the turkey, a thought may occur to the male masturbator. "If I do this too much, will it fall off ?"
So we come to the subject of today's topic. This gentleman showed up to the emergency room with a heavy fever, vomiting and muscle pain.
The patient had developed "Fournier's Gangrene" on his member. To those of you who don't know what Fournier's Gangrene is, it is a flesh eating disease which afflicts male genitalia. I probably don't have to tell you how awful it is, but you can always google image it.
How did this individual get Founrier's Gangrene ?
The doctors asked the patient.

Upon further questioning, he also endorsed severe scrotal pain and swelling and frequent masturbation with soap as a lubricant. He reported that past episodes of masturbation often resulted in recurrent penile erythema and abrasions

I am not exactly sure what the authors mean when they say that the patient "Endorsed" severe scrotal pain, but the frequent masturbation whilst using soap may have dried out his skin, and caused friction burns. These burns then got infected , and caused gangrene.
Unfortunately, the paper does not relate exactly how frequently the man masturbated before he gave himself a flesh eating disease.
But it turns out that if you do masturbate enough to give yourself friction burns, it could drop off. So ease up on the joystick jacking.

Heiner J.D., Eng K.D., Bialowas T.A. & Devita D. (2012). Fournier's Gangrene due to Masturbation in an Otherwise Healthy Male, Case Reports in Emergency Medicine, 2012 1-3. DOI: 10.1155/2012/154025

#MicroTwJC: Dicing Up Virus Genomes Part 3: The Controversy

2013-11-11T19:46:00.003+00:00

In this weeks Microbiology Twitter Journal Club we are going to be discussing two papers published a month ago in Science. These papers purport to provide evidence for RNA Silencing as an antiviral mechanism in mammals.

For those of you who don't know what RNA silencing is, or have no idea about these papers, feel free to check out my first two posts for this weeks MicroTwJC.

Basically an enzyme known as DICER spots double stranded RNA within cells, and chops it up into short 22 base pair sequences. These sequences are then grasped by the RNA Silencing complex. The RNA Silencing complex uses these short sequences to recognise full length RNA floating within the cell. Once the RNA Silencing complex recognises these full length RNAs, they break them down. Through this, they can "silence" the expression of genes.

For a long time, its been known that many viruses use RNA to make their genomes, and when they reproduce their genomes they can form double stranded RNA, which is a prime target for DICER and the RNA Silencing Complex.

Can organisms use RNA Silencing to fight against viruses ?

The answer to that question up till this point has been a Yes, with an important caveat. There has been definite evidence for RNA silencing playing a role in resisting viruses in plants and in invertebrates.... but not mammals.

Whilst mammals are known to have active RNA silencing systems, the evidence for them playing a role in combating viruses rather than just regulating cellular RNA has been hard to discover.

Interfering Interferon

The main problem with proving that RNA Silencing is an antiviral response in mammalian cells is that mammals have evolved a much stronger ways of responding to viral double stranded RNA.

For a start, there are Pathogen recognition receptor system, which acts like an early warning system for a cell when its being attacked by a pathogen. These receptors can detect specific bacterial or viral components, and send out an alarm to the rest of the cell so that it can take appropriate action.

Toll-like Receptor 3 is perhaps one of the most important ones for recognising viruses, as it can specifically recognise double stranded RNA from viruses. It is usually found in Dendritic cells, which are immune cells that tend to mop up debris from damaged areas of the human body, so if a virus is causing damage, it's likely the'll be around to detect it.

Once they do recognise it, they produce Interferon, which warns cells that viruses may be in the area. The cells then upregulate production of anti-RNA effectors, such as RNA activated Protein Kinase and Latent Ribonucleases. These slow down protein production in response to double stranded RNA and break down any double stranded RNA in the cell. In extreme cases, they can push the cell self destruct button (known as Apoptosis) and kill the cell, taking the virus with it.

These antiviral measures tend to swamp any effects that small interfering RNA's may exert. Remember, the main effect of RNA interference is a decrease in the number of viral genomes floating around a cell, a number that would already be reduced by Ribonucleases.

Viruses do have some defences against this response. If they protect their dsRNA from attack, they can avoid being detected by TLR3 and RNA-activated Protein Kinases, and being broken down by ribonucleases. In a funny/frustrating coincidence, the proteins that enable this defence also enable protection against DICER and the RNA-silencing complex.

This makes it very difficult to obtain unequivocal evidence that RNA silencing actually occurs in mammalian cells.

What evidence needs to be gained ?

In 2006, Bryan Cullen threw down the gauntlet, and came up with three predictions of what you would see if the RNA silencing complex genuinely broke down viral RNAs. I will quote directly from the article.

"viral infection should result in the production of siRNA of viral origin"
"inhibition of the RNAi response should enhance virus replication"
"as an adaptive response to that antiviral mechanism, many viruses should have evolved gene products that specifically inhibit RNAi"

Do these papers show that the RNA Silencing complex can break down viral RNA into siRNA's ?

Both of these papers fulfil the first prediction. They detected siRNA fragments of a viral origin in mouse embryonic stem cells, in hamster kidney cells, and during infection of full grown mice.

Is there evidence that inhibition of the RNAi response enhances viral replication ?

Let's take a look at the kinds of evidence researchers present to prove that inhibition on the RNAi response improves replication.

There are a number of ways which the researchers investigated this. The first was to look at what happens when we remove the activity of viral suppressors of RNA interference.

Li et al focussed their study on Nodamura virus with or without expression of B2. In one study they infected the B2 deficient mutant into cells that were engineered to produce RNA interference suppressors, either B2 or VP35. When these cells were infected with wild type viruses, they tended to accumulate more viral RNA than when the wild type virus infected normal cells. The over expression of these RNA suppressors appeared to enhance replication.

At least it would if the researchers had used the mysterious science of statistics to analyse this data. There could be a trend there, but we can't be sure.

Both of them however demonstrate that the removal of B2 reduces the abundance of the RNA genome within a cell.

But as I noted before, B2 can potentially protect against all of the other antiviral mechanisms within a cell. So this evidence alone isn't necessarily enough. They needed to disrupt the DICER system, and show that removing that improves the survival of viral RNA's.

Neither study showed this in their published papers, although Maillard et al came the closest, as they actually produced a DICER knockout model. In their paper, they showed that without DICER, virus derived siRNAs didn't show up.

However, if you dig into the supplementary section of the paper, and do some hunting, you will find a really interesting figure. In the DICER knockout, we find that there is a higher accumulation of VP3 RNA, indicating that knocking out DICER improves viral survival.

This seems to strongly suggest that in embryonic stem cells at least, the ECM virus infection is impaired by RNA silencing.

Do these papers show evidence that the virus response evolved to specifically hamper RNA interference in mammals?

This is the hardest of the predictions to prove, because as I mentioned earlier, you can argue that any protein that protects RNA could hamper both the TLR response and the RNA interference response.

It's not enough that viruses have evolved genes like B2 and VP35, you need to prove that these genes actually have a functional effect that is not related to inhibiting the interferon response.

In embryonic stem cells, the interferon response doesn't generally happen. In fact, the researchers showed that when the interferon response rolled into action, RNA interference ceased to be important in infection.

Things get a lot weirder when we get to the mouse experiments, where researchers took samples of RNA from mice during infection and used that to check whether there were any differences in the expression of immune genes. All of that data was stuffed into a supplementary table that I did not have the energy to get through yesterday. Essentially, the proteins that regulate interferon are expressed in different patterns, and there is a slight up-regulation in the production of interferon alpha, but the relevance is debatable

My main problem with this data is that yet again we don't get any idea of the variance of the data, which is very important.

Even more importantly, we don't get any idea of when in the infection these mice are being sampled. If the infection has resolved in one group of mice and not the other, then it is effectively the same as comparing infected mice with uninfected mice. Any differences due to the infection of a mutated virus could have completely been resolved by the time point they are examining. The inflammatory changes could simply be due to the infection resolving. The researchers needed to communicate what day of infection they were examining these genes on, and also provide the reader with an idea of the viral abundance at this point.

All I can say for this is that it looks like RNA interference is important for embryonic stem cells, and not unconditionally proven for the rest of the other models.

Is RNA interference a relevant antiviral response for whole organisms ?

These studies have demonstrated that in embryonic stem cells, RNA interference does suppress the activity of the ECM virus and the Nodamura virus. I will concede that there is enough evidence for that.

I can also say that based on the evidence presented, viral RNA's from the Nodamura virus and the ECMV get processed into short RNAs during the course of infections, and that B2 can halt this process.

The main problem comes from that damn interfering interferon response. The changes in viral genome abundance could alternatively be explained as a result of B2 flipping off the interferon response.

Whilst Li et al tried to account for it, they haven't presented their data in enough detail for me to conclusively say that B2 had no effect on the interferon response. Key details are missing, like how long after infection did they collect their samples? Where exactly from the mouse's anatomy did those samples come from ?

Conclusions

These papers have enough in them to convince me that RNA interference of viruses can occur in mammalian cells, and that they could be particularly important in embryonic stem cells. This is an evolutionarily ancient antiviral response, so it make sense that it could be important for the most basal cells we know of. But just as the Interferon response superseded these responses evolutionarily, they superseded them developmentally during the lifespan of an organism.

Verdict

This is a really difficult one for me. These papers have provided quite compelling evidence that RNA interference does affect viruses in certain situations, but not enough evidence to fully support RNA interference as a key part of the mammalian immune response.

These papers have pushed the goal posts of this debate towards debating whether siRNA's are important in viral infection rather than whether they actually exist.

But the controversy is alive and kicking.

Join in the discussion this Tuesday at 8pm GMT. Follow the #microtwjc hashtag and be a part of Microbiology Twitter Journal Club !

Cullen B., Cherry S. & tenOever B. (2013). Is RNA Interference a Physiologically Relevant Innate Antiviral Immune Response in Mammals?, Cell Host & Microbe, 14 (4) 374-378. DOI: 10.1016/j.chom.2013.09.011

Cullen B.R. (2006). Is RNA interference involved in intrinsic antiviral immunity in mammals?, Nature Immunology, 7 (6) 563-567. DOI: 10.1038/ni1352

MicroTwJC: Dicing up Viral Genomes Part 2

2013-11-10T20:34:00.000+00:00

Good news everybody ! I managed to source a copy of the second paper for this weeks' Microbiology Twitter Journal club, after not an insignificant expenditure of time and energy.

In the last blog post, I went through the basics of how siRNA mediated silencing works, and how it could potentially be used to attack the RNA genomes of viruses. The paper I reviewed for that post focused on what happened when viruses infected embryonic stem cells, and looked at how these cells chopped up the viral genome into siRNA's.

The paper we will be discussing today will extend that work, focussing on a virus we had just been introduced to in the last #microtwjc post. The authors use Nodamura virus in their studies. Nodamura virus has a positive stranded RNA genome. It produces a protein called B2, which protects viral DNA from DICER and the RNA induced silencing complex.

B2 is a viral suppressor of RNA interference.

The researchers used a variant of the Nodamura virus with a mutation in B2, so that they could see what happens to the virus without B2 there to protect it.

So without further ado, let us get to the first experiment.

Infecting Hamster Kidney cells

The researchers took a cell culture, derived from the Hamster Kidney, and infected it with either the normal Nodamura virus, or the Nodamura virus with the B2 gene knocked out. They then measured the presence of small virus derived RNA's between 18 and 28 nucleotides in length from the positive strand (in red) and from the negative strand (in blue). They looked at these at 2 days post-infection (dpi) and 3 days post infection.

It is very important that you look at the scales of these graphs, because otherwise you may not see the main differences between the groups.

The Wild Nodamura virus (with functioning B2) doesn't really have many short RNA fragments from the negative strand, and the positive strand experiences a lot of natural degradation anyway. There are usually a lot more positive RNA fragments hanging around in a cell, so this is usually why they have more degradation products than from the negative strands.

When we take B2 away, we suddenly see a spike in the presence of 21-23 nucleotide long RNA's from both the positive and the negative strand of RNA. Look at the scales, and you realise that there are generally much less viral RNA products in cells infected with this strain of virus. You could take this to show that the Silencing complex is having an effect on the abundance of viral RNA.

You can take this speculation further, if you observe that there appear to be more small RNAs recovered from the wild Nodamura virus on day 3 post infection compared to day 2, and the numbers don't appear to change for it's B2 deficient counterpart.

Deep sequencing of the Small RNAs

Alright, so the researchers showed that under certain circumstances, the Nodamura virus RNA can be chopped up into smaller pieces approximately 22 nucleotides in length. But are these short nucleotides really small interfering RNA's ?

The researchers needed to prove that these RNA's fit the structure of small interfering RNA's. The question is whether they have special 3' overhangs on both sides of the double stranded RNA.

So they took a look at the sequences of the small viral RNA's they had discovered previously to see whether they had an abundance of RNA's that can neatly bind to eachother, leaving an overhang on either side of the molecule.

So the researchers took the sequences they had obtained from both the positive and the negative strands, and using special software, experimented whether they would match up or not. Exact matches would produce a spike at zero, matches that were offset by a certain number of nucleotides would produce a spike at however many nucleotides they were offset (in this case, we are looking for a 2 nucleotide offset).

But there is a wrinkle to this. If the nucleotides are chopped up into neat little 22 nucleotide segments that are just slightly offset with the other strand, these short nucleotides will also be able to match up with the next segment of nucleotides (depicted at the bottom of the figure below).

We can see no real discernible pattern in the short RNA pairings for the wild type viral RNA strands, but for those strands not protected by B2, the outcome is much different. We can see peaks at the -2 offset, and the 20 offset. The -2 offset confirms that these strands have 3' overhangs. The 20 offset confirms that these strands are chopped up neatly all along the RNA strand with no gaps in between them. The evidence points to these being small interfering RNAs.

Is Fighting of DICER and the RNA silencing system essential for Nodamura virus infection ?

So we've shown that B2 is essential for Nodamura virus infection, but is it because it stops the RNA silencing system ?

They engineered some of the hamster kidney cells to produce the B2 protein on their own. They also took another protein called VP35 ,derived from an Ebola virus, that can also repress the RNA silencing system, and engineered that into the hamster kidney cells as well.

They then infected these with the wild Nodamura virus and it's B2 deficient counterpart. They then counted the genomic RNA of the Nodamura virus over the next 72 hours using real-time PCR.

The cells with the lowest amount of viral RNA were the normal cells infected with the B2 mutants (Deep blue line).

However when the B2 mutants were infected into cells that produced either their own version of B2 or VP35, the RNA was protected, and could be detected in higher numbers throughout infection. In fact, it appears to compensate for the loss of B2 in this strain, such that its numbers are comparable to the wild -type strain infecting a normal cell.

But it gets more interesting when the wild type infects cells which already produce B2 and VP35, because apparently this increases their numbers more. However, it should be noted that RNA silencing plays an important function in the growth of any healthy cell, and knocking it down could have many unforeseen effects that could potentially aid viral infection.

We next see a northern blot, to test directly for viral RNA in these cultures to further back up the evidence gathered from RT-PCR.

Here we see the direct accumulation of RNA's from the virus infection. Effectively, these blots say the same thing as the previous figure. The important column is the middle one,and the band you should be focussing on is the top one. When the RNA silencing system is suppressed by B2 or VP35, we can see big black bands indicating the RNA is still there, but without the there, the genomic RNA is pretty much gone. Destroyed. Kaput.

Nodamura virus Infection in Mice

The researchers next wanted to see what would happen in whole organisms. So they infected mice with the wild virus and the mutant to compare how well they could spread over the course of a week long experiment. The researchers infected newborn mice (I don't know why they used newborn mice, other than because it was what everyone's been doing since it was first discovered.). The virus tends to be very lethal to these mice, producing paralysis over the course of a week, with death occurring soon after.

For this study they managed to acquire a strain of nodamura virus which produced a mutated B2 protein, named Nov-mB2, as opposed to ^B2 which produces no protein whatsoever, and the wild type (NoV).

At specific time points the researchers took samples of the hind limbs and the fore limbs to check them for viral RNA. The wild type virus had the highest accumulation of RNA (measured in Fold change) compared to the other two strains.

So let's bring out the northern blots, because sometimes you just need to see the black smudges for yourself to truly believe.

The first Northern blot shows the accumulation of viral RNA in mice infected with the Nodamura virus that produces no B2. in either the Hindlimb (H) or the Forelimb (F) over the course of the seven day infection.

The big smudge on the other end is from the wild type virus control, demonstrating that whilst the B2-deficient virus can be detected, it is present at a much lower level than it would if it had functioning B2.

What's more, mice infected with this attenuated virus all survived the experiment, as opposed to the ones infected with the Nodamura virus. The researchers put in a picture to demonstrate that there was no difference between a survivor of infection and a normal mouse, and they don't show an infected mouse for comparison.

Frankly this picture is an utterly meaningless addition to this paper. If they had some standardised method for measuring the paralysis, or perhaps showed the weight changes of the mice over the time course, y'know, ACTUAL DATA, I would be able to be convinced about the effects on mouse welfare. But frankly just giving me a photograph of two mice for a disease that has no symptoms that won't even show up on a still image is insulting.

Anyway, the researchers next looked out for virus derived short RNAs in mice infected with either the mB2 strain or the ^B2 strain.

Turns out that they could detect short RNA's for both of these mutants.

Nov ^B2

The mutantB2 had a signal on days three and four post infection, but the strain that produced no B2 showed the consistent presence of viral small RNAs from day 3 post infection right up to day 7. The authors say some stuff about there being some heterogeneity in the sizes of these bands, but frankly with blots there is always a chance of a photographic artefact from overexposure.

Can you see what's missing ? I didn't at first, and I desperately scrabbled through the supplementary data for it. Heck, there was one moment where an apparent mis-spelling in the text made me hope for it being present somewhere, but for nought. There was no wild-type control for this. It may be a small nitpick, but frankly I think that even if it is a blank gel, it is worth having a control for comparison.

Deep Sequencing Viral siRNA's from an infection

They took samples from mice, and sequenced the RNA's in the way we saw in figure 1 at the top of the post.

They took samples from mice infected with B2 deficient virus on days 1 ad 2 post infection, and compared them to samples taken from mice infected with the wild virus on day 4 post infection.

In the left column we have the read counts for positive and negative strands showing a slight trend on day 1 of showing viral siRNA's, which are more pronounced on day 2 post infection, but not present in the wild type strain of day 4 post infection.

In the right hand column we have the sRNA pairing graphs, which show offsets at -2 and 20, just like in the previous figure, indicating that we are looking at siRNA's. The wild type trace of this does not show seem to show a similar pattern.

The researchers then tried to work out which specific parts of the viral genome were the primary targets for being chopped up into siRNA. They compared them based on data from their mouse experiments and their cell culture experiments.

The latter end of the region encoding the RNA-dependent RNA polymerase appears to be a major target, as well as (to a smaller extent) the starting regions in the genome. These results are somewhat different to those obtained from embryonic stem cells from the other paper we're looking at for MicroTwJC, mainly because the 5' end clearly isn't getting attacked as much. However the latter end of the genome is still being attacked by DICER in both.

Summary

This paper quite quickly and cleanly demonstrates that viral RNA can get chopped up into siRNA's by a mammalian cell, and in whole organisms. The researchers demonstrated that if you remove a virus's defences against the RNA silencing complex and DICER, you hamper it's ability to reproduce within a host.

Criticisms

Firstly, the authors really missed an opportunity to demonstrate that the removal of B2 function could aid host survival during an infection. All I have are pictures of two mice, no data on health or their recovery. Is there lasting damage from the paralysis induced by Nodamura virus ? Do the mice infected with the mutants experience less severe symptoms ? Nope, got no idea. It's not like this data is difficult to get, most places actively require you to keep a running tally of a mouse's weight throughout an experiment, as it allows a you to keep an eye on its health. If they are running a tight ship, then they will no doubt have a record of those weights lying around somewhere.

Opinion

I wish that this was the article on open access, because it is so much more readable than the other available paper. It is well written (aside from a few typos) and the data flows as a cogent story.

But the thing about this weeks #MicroTwJC is that we are genuinely going to discuss a contentious issue. I am going to attempt to dive into for my next post. Wish me luck !

Li Y., Lu J., Han Y., Fan X. & Ding S.W. (2013). RNA Interference Functions as an Antiviral Immunity Mechanism in Mammals, Science, 342 (6155) 231-234. DOI: 10.1126/science.1241911

#MicroTwJC: Dicing up Viral Genomes

2013-11-08T17:02:00.001+00:00

In this week's Microbiology Twitter Journal Club, we have been challenged to dissect not one, but two papers.

Fortunately, the University of California San Francisco have made one of the papers widely available, so you can access it here, and the ever important supplemental material can be found here.

The title of the paper we'll be going through is "Antiviral RNA Interference in Mammalian Cells".

So let's quickly refresh ourselves on how viruses cause infections. They attach to the surface to a host cell, inject their genetic material into said cell (which can be in the form of either DNA or RNA). The Viruses genetic material does all of the hard work, finding ways to trick the host into replicating it and create new viruses, and possibly killing the host cell in the process.

There are ways in which we can disrupt the replication cycle of viruses, and I've talked about some of the new drugs being developed in previous blog posts.

A key part of any viruses replication cycle is the need for it to use RNA RNA is the tool cells use to deliver the genetic code instructions to Ribosomes, which use that code to make proteins. Viruses take advantage of this system by delivering their own RNA to the Ribosomes and tricking into making viral proteins. This paper focuses on one way that cells could potentially resist infection.

Many organisms produce a protein in their cells known as "DICER". This protein can recognise double stranded RNA, and when it does, it cuts it up into much smaller RNA strands, at around 20-25 bases long.

But the fun simply does not stop here, because those small RNA fragments created by the DICER will play an important role. They get taken up by the RNA-induced Silencing Complex (RISC), a complex set of molecular machinery which uses these short RNA fragments to recognise complementary sequences in complete RNA strands. Once the RISC recognises these RNA's, it breaks them down.

As I explain in the simplified diagram below, which in no way was affected by the Doctor Who season 6 marathon I just finished.

The Silence, I mean ..er.. the RNA Induced Silencing Complex (RISC) plays a key role in regulating the cellular RNAs within a Cell. Can this system also be used to fight against viruses ?

It's known that if we artificially introduce short synthetic RNA fragments into mammalian cells that match to viral RNA, the cells primed with this RNA can resist infection. In fact, it is know that some invertebrates and plants can use this system to fight viruses without any outside assistance.

Can mammalian cells do it as well ?

How would we even go about investigating this question ?

The Experimental Set Up

Firstly, we need a experimental set up that will allow us to pull apart all of the individual parts that contribute to the creation of siRNA's, and allow those siRNA's to regulate degradation of mRNAs. We can then knock out or alter some of the genes that regulate key steps in the production of siRNAs and their implementation in the RISC. The researchers decided to use Mouse Embryonic Stem cells because these cells can survive without a fully functioning siRNA system.

The next thing we need to get straight is what kind of virus we want to use for this investigation. They selected the Encephalomyocarditis virus (ECMV). This virus stores its genome in the form of single stranded RNA, and is known to make its host cells reproduce a lot of double stranded RNA during its infection cycle.

The researchers made a recombinant form of the virus genome using PCR, and then amplified it up in a special mammalian cell type called BHK to create a pure form of the virus, which then transferred off to another culture to allow it to go through a full infection cycle to increase the numbers of virus that the researchers could use for an infection.

There are a number of different lineages of embryonic stem cells that the researcher could potentially use, but they wanted to make sure to select one which would produce loads of virus. The more viral RNA going through a system, the more likely that they will detect even a weak host effect acting against them.

They tested 3 lineages of stem cells, named E14, PGK and HM1.

After they infected the cells, they waited until either 3 or 6 hours post infection (hpi) to take samples from the cells, and measured the amounts of specific proteins within the cells (which are indicated by the black smudges).

They measured the dose of virus by adding samples to a standard tissue culture at different dilutions, and then determining the amount of virus needed to kill of 50% of the cells in the tissue culture. The less you need to dilute a sample to get to this point, the more virus there actually is in the original sample, so I'm guessing the lower numbers mean there is more virus.

In this figure, we want to find the cells that produce the highest levels of the viral protein VP1. The researchers also measured the abundance of Actin, a normal component of the cell which doesn't generally vary that much, as a control, to show that they aren't cheating by including more cells in the prep. The E14 lineage wins this hands down, so this would be used for further studies.

They next took samples of cells 3 hours and 6 hours after being infected, and extracted all of the RNA from these cells. The Y-axis of the graph indicates "read counts" which essentially describe how much of an RNA transcript there is. Of the total RNA within a cell, around 0.7% belonged to the virus after 6 hours.

Figure 1B shows how the amounts of virus RNA vary over the course of an infection. The RNA's are classed based on the size of the RNA transcripts. At 3 hours post inoculation, there isn't much viral RNA hanging around the cell, but at 6 hours (the grey bars) we can see a sharp spike in the amount of transcripts.

There is an interesting spike for transcripts side between 21, 22 and 23 nucleotides long, which intriguingly is the sie that DICER tends to chop RNA molecules into.

But at this point in the paper, we don't know whether these are all the natural sizes of the virus transcripts.

So lets drill down deeper. Where are these viral transcripts coming from ?

We know that ECMV creates a double stranded RNA molecule, and these RNA's could come from either strand. So the researchers looked at the code for each strand, and checked which part of the HCMV genome these small RNA strands came from.

These are shown in the graph below, with the top graph indicating where the RNA's come from on the positive strand, and the bottom graph shows the numbers of RNA's coming from the bottom strand.

The blue bars indicate strands in the 21-23 range, i.e. the kinds of RNA we'd expect to see accumulate if DICER is chopping these strands up. The 24-44 nucleotide length RNA's are shown up by the grey bars.

You should be able to see that the top graph has most of the bars, and the higher bars. Most of these short RNAs are coming from the positive strand of the ECMV genome. The Positive strand has pretty much all of the grey bars, and the authors suggest that these grey bars represent natural breakdown produces for the RNA. The Blue bars are found on both strands, and are primarily found within the first 200 nucleotides of the genome, which is known as the 5' untranslated region. There is a much smaller region shared by both strands on the opposite end of the genome in the 3' untranslated region. There are also a ton of different regions on the positive strand. But the problem with interpreting most of the data from the positive strand is that breakdown products will be mixed in with those that have been genuinely broken up by DICER.

These two regions of the Virus genome play an important role in allowing the genome to duplicate during an infection. They can lasso in important transcription enzymes onto itself to start the replication process, or start transcribing its genetic code into viral proteins.

In the next figure, the authors zoom into the first 300 base pairs of the genome from the last figure to show what's happening in more detail. Each of the bars are now numbered as well, You may notice that the bars 1&2 and 3&4 tend to mirror each-other. The sequences for these bars are shown above, and they are complementary. They form duplexes with slight overhangs, such as might be created if they had been chopped up by DICER.

You may be wondering what those circular graphs mean. They describe the the Phase of the register of an RNA transcript. The "Register" of the an RNA transcript describes where the reading frame the sequence operates in, i.e. where it starts. We are assuming that the actual read length here is 22, and that there are 22 registers. These are represented by the 22 arms of these plots. The actual data is represented by the jagged black line, which represents the percentage of reads within a specific register.

The top radar plot shows the registers for the postive strand, and the bottom one shows the registers for the negative strand (although the labelling is really ambiguous, and could suggest that they are taking the registers of RNA strands that are 21 base pairs (top) or 23 base pairs (bottom))

The top radar plot shows that most of its transcripts fall within the second register. The 23 base pair log strands tend to start in the 22nd register. So the genome at this end is being parcelled up into registers that are off by two base pairs. Those base pairs likely are left dangling off to the side in different directions, leaving overhangs.

To confirm that most of these transcripts were 22 base pairs long, the extracted all of the RNA from an infected cell and ran it out on a northern blot specifically geared to spot low nucleotide products.

They showed that most of the transcripts are about 22 base pairs long. It's shown alongside some Arabidopsis thaliana RNAs for size comparison, a Not infected (NI) control, and with the ubiquitous U6 RNA transcript to show that the cells in both samples were basically alive.

We have so far learned that the 5' edge of the virus genome appears to be heavily attacked by DICER.

But this is all circumstantial evidence. We don't know that DICER is involved in this at all. The only way to show it would be to remove DICER from the equation completely.

In these next set of experiments, the researchers tried infecting Embryonic stem cells that could not produce DICER, to see whether infections with ECMV would produce the same outcome.

They used Embronic stem cells that had been genetically modified with "flox" tags around the DICER genes. When activated, these tags cause the entire DICER gene to be removed.

They then infected these stem cells with ECMV, and checked to see whether the 22bp RNA's were still detectable when DICER wasn't present.

Essentially this figure is the same deal as the last one, with one little extra. We get to look at miR-16 in the control embryonic stem cells and the DICER knockout cells. Cells with DICER present can make miR-16, and those without cannot.

The control cells have detectable 21bp transcripts, and the DICER knockout cells do not. The transcripts probably were genuine viral siRNAs.

To further highlight this, they looked at whether the siRNAs could be incorporated into the RISC. The RISC consists of multiple proteins, and one of the most important ones is the Argonaute protein. It is this protein which directly binds to the siRNA and brings it into the RISC.

They used embryonic stem cells that expressed a specially tagged version of the Argonaute protein (AGO2). During an infection, the siRNAs would be bound by the Argonaute protein. At the end of the infection, the researchers harvested all of the Argonaute protein, and got it to spit out the siRNA sequence so that they could run it on a northern blot.

The Argonaute specifically works by picking up siRNA's, and the researchers use it here to confirm that the EMCV short RNA's are bound by the Argonaute protein just like real siRNA's.

So what would happen if we were to take these siRNA's gathered by the argonaute protein and find out where they fit on the ECMV genome ?

We can see that viral siRNA's bound by Argonaute come primarily from the positive strand of the ECMV genome, and from a number of hotspots, some of which may be crucial to the function of the viral genomic RNA.

But it should be noted that the total make-up of the RNA's bound to the argonaute protein fundamentally differ from the small transcripts that were freely extracted from the medium in the previous experiments. This suggests something else may be going on, that some unknown process may be causing this difference.

The authors of this paper then decided that it would be interesting to look at what would happen if they infected the embryonic stem cells at different developmental stages. At day 10, embryonic stem cells stop producing a protein called OCT4 as a sign of their new relative maturity.

The point here is that after a certain stage of maturity, we simply don't see the siRNA's anymore, even though infected cells ar both maturities were still producing viral protein.

When the researchers looked at the RNA transcripts, they found again that the more mature cells had lower levels of short transcripts, especially those in the 21-23 nucleotide range i.e. the siRNAs

If we look at the 5' portion of the genome again and the numbers of transcripts produced from them in the less mature and more mature cells, we get the graph below:

Whilst siRNA transcripts are still detectable at day 10, they are so much lower than the transcripts from the younger cells. Good to know.

But the question remains as to whether this whole siRNA system actually helps protect cells from viruses. The problem here is that many viruses have Viral Suppressors of RNA interference (VSR's) which act to directly confound RNA interference. Whilst these proteins are active, it would be nigh impossible to tell whether siRNA's in mammals were having an effect on them. They have to be removed if we really want to get answers over whether siRNA systems have effects on viral infection.

Unfortunately, whilst ECMV is purported to produce a VSR, it is not yet known. So they switched their work to focus on a different positive strand RNA virus with a known VSR called Nodamura virus. It produces a B2 protein which binds to and protects double stranded viral RNA.

The researchers used a mutant of Nodamura virus which didn't have B2, and used that to infect embryonic stem cells along with its wild type counterpart.

The researchers then extracted the RNA from the infected cells, and pulled out the genomic viral RNA for each of the virus, and ran them out on a gel.

As can be seen the genome, consisting of RNA1 and sub-genomic RNA produce much weaker bands when B2 is not present, suggesting that the B2 mutant may not be present in high numbers within an infected cell.

The researchers then looked even more directly at the sizes of the RNA transcripts. The Nodamura virus without B2 had a spike in the amount of small RNA transcripts between 21-23 nucleotides long.

This could suggest that the unprotected Nodamura virus's genome is being chopped up into small RNA fragments.

Now let us take a look at where in the Nodamura viruses genome these RNA transcripts come from. We're looking at both the wild type and the mutant versions of the virus.

Again most of the reads come from the positive strand of the viral genome. But the interesting ting here is that without B2, nearly all of the viral short transcripts are now 21-23 base pairs long. Furthermore, we yet again see that they tend to come from the 5' portion of the virus genome. We can zoom in on the 5' untranslated region again, to see how much of a difference the lack of protective B2 has on the amount of short RNAs.

Were you missing the Phase Register radial graphs ? We have more of those for you in the next figure.

These ones are at least more clearly labelled.

We are looking at the wild type Nodamuravirus in the first two plots. There is no distinct phase register for the short RNA's on either of these strands. This makes sense, considering that the virus stops DICER from working. The RNA doesn't get broken down into neat 21-23 base pair chunks, all we are seeing in these graphs are the natural degradation products of the RNA, distributed across random registers.

But when we look at the mutant, which is vulnerable to DICER, we see the that the short RNA's on the positive strand and negative strand all fall into just one of the 22 registers. They are two nucleotides out of phase with eachother, again suggesting that these are siRNA's with nucleotide overhangs.

In the next figure, the researchers show the sequences of the the siRNA's from the first 180 nucleotides of the 5' end of the genome.
The Black Bold numbers indicate the numbers of each transcript, the normal numbers indicate the position of the transcript along the viruses genome. The transcripts marked XXX are ones that were not detected. The blue coloured siRNA sequences are ones that were also shown to come up in a different study which used newborn mice instead of embryonic stem cells, and the overhangs are in red.

This shows that these effects in embryonic stem cells are also mirrored in mouse models, although since I don't have access to the other article, I can't give you the full story on this figure. I don't know how the researchers in the other paper came to their data, I don't know how the transcript numbers could compare. I have to back away from this one.
So for the next part of their work, they decided to knock out the Argonaute protein. They used the Flox system previously used to knockout DICER to knockout the Argonaute gene. This is triggered through the addition of the hormone Tamoxifen. So the next graph shows that the Argonaute protein is knocked out when Tamoxifen is added, even when the cells are infected with Nodamura virus with or without active B2.

In the next figure they try to look at the survival of the embryonic stem cells after they've been infected with Nodamura virus, but they use a really odd method of measuring the effects of removing the effect of Argonaute.
They infected embryonic stem cells that have active Argonaute protein and inactive Argonaute protein with both mutants of the Nodamura virus. What is odd is how they represent the data.
They take the ratio the Nodamura virus genome abundance in cells without Argonaute protein (E7 cells with tamoxifen added) compared to cells with active Argonaute protein.
So in both cases there appears to be more Nodavirus RNA present when the Argonaute protein is knocked out, which makes sense because the whole RNA induced Silencing Complex pretty much falls apart without it. In the virus that doesn't produce B2, the ratio is a whole lot higher, because in the wild-type E7 the genome is more vulnerable to being chewed up by the RNA Silencing Complex.

But they show the actual data in the Northern blots shown below. The NI and NoV columns are pretty much controls. The important one to pay attention to is the NoV^B2 , which shows what happens when you add the mutant Nodamuravirus to cells with and without active Argonaute protein. When it's there, and the RNA Silencing Complex is active, we can see that there is less of the Nodamuravirus RNA hanging around, and when it isn't there there is more of it.

So this is the evidence they present to show that mammalian cells can use siRNAs to break down viral dsRNA.

Summary

The first set of figures suggest that ECMV is broken down into short interfering RNAs. In the next set of Figures, they looked at what happened when they knocked out DICER, although apparently missed the opportunity to catalogue the siRNA present during infection in as much detail as they had in the previous figure. A missed opportunity ?
They then focussed on the argonaute protein, the protein which draws the siRNA's into the RNA silencing complex. When they extracted the Argonaute proteins from their stem cells and got them to spit out whatver siRNA they had bound, they found somehting interesting. They found that the content of these siRNA's differed from the total siRNA content of the cells. They suggest that some other process may be delivering siRNA's to these argonaute proteins, processes that don't require DICER.
Hey, wouldn't it be cool if they had embryonic stem cells without DICER which they could do deep sequencing on and get an idea about the viral siRNA generated by other processes within mammalian cells ?
The researchers then showed that more mature embryonic stem cells don't produce siRNA's even though the virus is still there.
We then go through this whole dance again with Nodamura virus, so that we can see what happens when we remove a viruses defences against siRNA silencing. It looks like the B2 virus suppressor of siRNA works very well, and when it's removed there is less intact viral RNA and more viral derived siRNA.

Criticisms
It's hard to properly critique a paper when you have very little idea about how the experiments were designed. I have no idea about the sizes of the embryonic stem cell populations we are dealing with in each figure. I don't have any idea of how variable the viral siRNA content is from infected cell to infected cell. Most of the results are presented in transcript numbers ( Except for one of the figures where they inexplicably switch to reads per million) , but I don't know what that relates to. Is it transcripts per cell, or for each millilitre of culture ?
They don't use any statistics that I'm really familiar with, and whilst I'm fascinated by the stuff they did do, I don't have enough time to learn it all.
If there is anyone who can give me an explanation of how single spectrum analysis works, and what the researchers use it for in this paper's supplementary section, I would be most indebted.

Opinion
The problem with this paper is that it makes me paranoid. I've got a nasty feeling that if I could better understand the data, and was more fully up to date with Virology and deep sequencing techniques that my opinion may be different.
The researchers do show an effect, but they are also quite (rightly) modest about it, suggesting it is only really relevant in the embryonic stages of a mammalian cell.
Unfortunately, I don't have access to the second paper for this journal club, meaning that I'll have to hitch a ride to the British Library over the week-end to get it. I'll have to save my thoughts on the wider applicability of this research for after I've read it, and the associated commentary pieces.

Join in the Microbiology Twitter Journal Club discussion, 8pm GMT next Tuesday, follow the #microtwjc hashtag.
I apologise for yet again producing a review that is almost as long as the paper.

Maillard P.V., Ciaudo C., Marchais A., Li Y., Jay F., Ding S.W. & Voinnet O. (2013). Antiviral RNA Interference in Mammalian Cells, Science, 342 (6155) 235-238. DOI: 10.1126/science.1241930

TMI Friday: Eat your Vegetables

2013-11-08T12:00:00.000+00:00

There are a number of odd things that people do in pursuit of a sexual high, but today's story may just be the most bizarre event I've written about for TMI Friday.

It was a peaceful afternoon, when a middle aged couple heard a frantic tapping, a frantic rapping at their door. It was their neighbour, and he was in quite a lot of trouble. He was a 29 year old man, of normal build, dressed only in trousers with the waist button unfastened and desperately attempting to talk before dropping dead in front of them.
What happened to him, and why did he die ?
Perhaps an autopsy could answer these questions.

Removal of the trousers showed the penis to be semi-erect; around the base of the penis was a rubber band. On the lower abdomen and in the groin was dried, white coloured material, subsequently identified as semen.

Okay, so now we can draw a few conclusions as to what the young man was doing before he showed up at his neighbours door. It rhymes with "Rasta-Dating". But that still doesn't explain what killed him.

However, when they opened up his throat cavity, the revealed it. The man had inhaled a whole zucchini, and suffocated as a result. His cries for help were stymied because the zucchini had blocked his airways.

Further enquiry revealed that the deceased had been married for 2 yrs at the time of his death. He was casually employed but, unlike his wife, was not working on that day. The couple grew zucchinis in their backyard garden.

Cooke C.T., Cadden G.A. & Margolius K.A. (1994). Autoerotic Deaths: Four cases, Pathology, 26 (3) 276-280. DOI: 10.1080/00313029400169631

History of Bacteriology: The Cholera Riots

2013-11-07T00:38:00.001+00:00

Murmurs of murder rippled through the crowd as it accumulated outside the entrance of the building, cursing the people who entered and exited it. They had watched helpless woman stretchered into the building, knowing she would soon join of the hundreds of people who had died within its walls. Whole city of Liverpool was in uproar, and had endured enough.

History does not record who threw the first stone, but soon the air was thick with them. They thudded against the buildings wall's, breaking the windows and scattering the people within. The men and women escaping the building were chased and beaten.

The building was Toxteth Park Hospital, the people being chased were Doctors and nurses, and this was the beginning of the Liverpool Cholera Riots.

It was the age of the Industrial Revolution, Empire and Mass migration. Irish immigrants formed a major part of this migration, travelling to America to avoid the depredations back home. The primary intersection between the British Isles and America was a Liverpool. Immigrants awaiting passage to the new world would often find themselves stuck in the overcrowded city of Liverpool.

Like many cities of this era, Liverpool was transforming into a haven of squalor and disease. Urine and faeces were flung freely into the streets where they flowed into the rivers from which people drank. Tuberculosis and Typhoid ravaged the poor.

The industrialization of Europe had meant that transport links had become much quicker, and trade had improved, but brought with it diseases. Rags from continental hospitals sold to farmers in Yorkshire to help manure hops also carried with them a disease that had not been seen in England before. It was known as "Asiatic Cholera" * at the time, and it frightened the rich and poor alike. Before its appearence, "Cholera" had only referred to seasonal stomach bugs and diarrhoea, and didn't relate to the deadly bacterium which we now refer to as Vibrio cholerae.

The month after the infected rags had been imported into Hull, the first cases of Cholera began to be recorded. Patients suffered from diarrhoea, severe cramps, followed by severe dehydration and then death, with the final symptom being the patients turning blue. It could turn a healthy person into a corpse within twenty four hours.

In 1831, an epidemic devastated Sunderland, killing over 20,000 people. The doctors could not contain the outbreak, their treatments consisting of brandy, bleeding and opium. Fear of this disease was high when it reached Liverpool in 1832.

A veteran medic who had experienced Cholera first hand whilst stationed in India tried his best to calm the situation. After the first two cases reported in Liverpool, he publicly stated that this "was not the case of an epidemic" like people may have heard about in Europe or Dublin. Not long after this, Cholera broke out on a vessel named the Brutus, claiming eighty-one deaths. Liverpool's Board of Health were slow to act, at first apparently denying the news of the outbreak within their city. At the boards very outset it was criticised as being filled with "a few fat-bellied magistrates" who had obtained their position through patronage rather than any medical expertise. Their sluggish reaction to this epidemic did not help that public perception.

The hysteria surrounding this disease was only rivalled by the scandal surrounding the whole medical profession. In the early half of this century, medical schools suffered from a dearth of human cadavers for students to practice on, and had begun to pay quite handsomely for them. In Edinburgh, two enterprising gentleman by the names of Burke and Hare decided to capitalise on this need by making a few corpses of their own, killing 16 people and making approximately £8K in today's money. The complicity of the medical establishment in this case combined with widespread reports of grave-robbing and the publics general distaste for dissection stained the medical establishment. People were now well aware of the high prices doctors would pay for a good corpse. A patient walking into a doctors surgery may have worried that they could be worth more dead than alive.

When Cholera began to spread through Liverpool people began to refer to doctors as "Burkers", invoking the more notorious of the murderers and implying that doctors were profiting from the deaths of their patients.

The medical board in the meantime were doing their best to contain the disease, setting up new hospitals for patients to go to, and arranging carts to carry sufferers to these hospitals. The doctors and nurses worked hard to help their patients, but were severely hampered by the fact that none of their treatments appeared to work. In fact, it is likely that treatments like bloodletting made the disease a lot more dangerous.

Things however came to a head when Mr Clarke and his wife fell ill from Cholera. The doctors were jeered at by the mob when they brought the woman into the building. At this point the Liverpool Chronicle picks up the story.

“Stones and brickbats were thrown at the premises, several windows were broken, even in the room where the woman, now in a dying state, was lying, and the medical gentleman who was attending her was obliged to seek safety in flight. Several individuals were pursued and attacked by the mob and some hurt."

The next few days saw the protests escalate. Mobs prevented doctors from carrying away their patients by any means necessary. They would halt the palanquins that were used to carry patients away, and when that didn't work they started to smash them to pieces. In one incident, people opted to hide a patient away from a surgeon tasked with treating her, and upon confronting them is chased across town to take refuge in a shop. Nightly gatherings surrounded the hospital in Toxteth Park. The police were often called in to hold back the worst excesses of the violence, but were simply overwhelmed.

But it wasn't just the fear of the doctors that motivated people. Cholera hospitals were rapidly being set up, bringing sick people to places of business. Some of those in the crowd wanted the doctors to take their grisly business elsewhere. Conspiracy theories abounded about how doctors were perpetuating the epidemic for a £10 "cholera fee" paid out by local bureaucrats. In some cities, Grocers believed doctors were advising people from staying away from certain food, leading them to be pelted with fruit.

The riots in Liverpool were solved when a threatening letter was sent to the mayor of the city. In the content of the letter, the author promised to do "wicked things" to any doctors who attempted to treat their patients. The author signed the letter off simply as "An Irishman". It was this last part of the message that suggested an alternative solution to the violence. Most of the cholera victims were the Irish Catholics crammed together within cramped underbelly of the city, and they were the loudest voices speaking out against doctors.

The Board of Health invited the Catholic clergy into a meeting to discuss solutions to the violence, and the clergy were given a message to deliver to their congregations. The speech addressed people fears about the cholera outbreak, and more importantly announced in no uncertain terms that the people who were dying were not being dissected. Furthermore, they declared that people had the right to go into the hospitals to see this for themselves, and to see the untouched bodies of their deceased before burial.

This was supported by an article published in the Liverpool journal by Dr James Collins , who also made a point of talking to people during church meetings.

Soon, the streets of Liverpool were once again relatively quiet.

These riots occurred before anybody had a real handle on how infectious diseases spread. It was an era where the doctors had little idea of how to control a cholera outbreak, nor even what truly caused it. But the massive death toll and the incredible civil unrest spurred the government into action nonetheless. At this point, people had started to make the connection between overcrowding and poor sewerage to the spread of disease. The government would soon take steps to solving these problems, but in the process promote a troublesome theory that nearly strangled the nascent science of microbiology in its crib.

References

Burrell S. & Gill G. The Liverpool cholera epidemic of 1832 and anatomical dissection--medical mistrust and civil unrest., Journal of the history of medicine and allied sciences, PMID: 16144959

Puntis J. 1832 cholera riots., Lancet, PMID: 11597715

Gill G., Burrell S. & Brown J. Fear and frustration--the Liverpool cholera riots of 1832., Lancet, PMID: 11476860
Howie W.B. (1981). Stephen T. Anning, The history of medicine in Leeds, Leeds, W. S. Maney, 1980, 8vo, pp. ix, 218, illus., [no price stated], (paperback)., Medical History, 25 (04) 442-443. DOI: 10.1017/S002572730003502X

Further Reading

The First Spasmodic Cholera Epidemic in York, 1832, Issues 37-46 By Michael Durey

* The only doctors who had observed it were those who had been serving in the armed forces in the Empire when this disease swept through India during the Kumbh Mela, hence why it is known as Asiatic Cholera. A second pandemic had been working its way across Europe.