Using The Power Of Space To Fight Cancer

A man who lives without 90% of his brain is challenging our concept of 'consciousness'

The father of two lives a normal life.

A French man who lives a relatively normal, healthy life - despite missing 90 percent of his brain - is causing scientists to rethink what it is from a biological perspective that makes us conscious.

Despite decades of research, our understanding of consciousness - being aware of one’s existence - is still pretty thin. We know that it’s somehow based in the brain, but then how can someone lose the majority of their neurons and still be aware of themselves and their surroundings?

First described in The Lancet in 2007, the case of the man with the missing brain has been puzzling scientists for almost 10 years.

Read more… 

Anti-microbial Peptides: Proteins that Pack a Punch

Antimicrobial resistance is a growing concern and it is currently estimated that approximately 2 million people are infected annually with serious infections that show antibiotic resistance to some degree. This contributes to the mortality of 23, 000 people with many more suffering severe complications as a direct result of antibiotic resistant infections. The economic burden on the US is thought to exceed $20 billion simply on health care bills alone, and a further $35 billion due to a societal loss in work based productivity (1).

The spread of antibiotic resistance is now widely believed to be a direct result of the anthropogenic release of antibiotics into the biosphere. We are now faced with the dilemma of how to treat these infections. In previous articles, I’ve talked largely about bacteriophages and how they are one possible solution to this complex problem. This article will introduce you to another class of antimicrobial agents, aptly called antimicrobial peptides (AMPs).

What are Antimicrobial Peptides?

Proteins are found ubiquitously throughout all cellular life and are like the mechanical parts of a car, helping your cells carry out a vast array of functions every single day. Peptides are small proteins that contain two or more amino acids joined by peptide bonds. Anyone who is familiar with biochemistry will be aware of the sheer diversity found amongst these versatile molecules. Needless to say, it should not be surprising that there are a large class of proteins involved in offensive cellular warfare. They are found widely in all domains of life and have evolved to give a cell a competitive advantage over its nastier neighbours.

Without getting too bogged down with the biochemistry, AMPs are characterised by their overall properties. AMPs that share common structural features will also have a similar function when targeting a cell. The diversity amongst these proteins can be seen in Figure 1, which shows some examples from the four classes of AMPs. The class I AMPs, the lantibiotics for example, all contain similar motifs which assign them a similar job. AMPs can range from anywhere between 6 to >59 amino acids, but are generally considered to be small proteins (2). They generally have a rather amphipathic nature and feature both positive and negative charges.

These peptides may have a number of rare (Figure 1), modified amino acids. The lanthionines are a class of AMP that contain lanthionine rings made from dehydrated serine and threonine residues connected by thioether cross-links. This happens after the protein leaves the ribosome and gives the protein some very unique properties which will be explained later in the article (3).

Figure 1. The four classes of AMPs, showing common examples in each class. Rare, modified amino acids are indicated by coloured circles with the three letter codes indicating the name of the residue. Thioether cross-links are indicated by an S coordinated by two black lines (3).

Implications for the Pharmaceutical industry

Our antibiotic pipeline is drying up (Figure 2), with few new drugs being approved by the Food and Drug Administration. Identifying novel antibiotics is a tedious process that requires a lot of time and effort from drug companies, which they are not willing to do. The reason for this boils down to economic reasons, as antibiotics are just not worth the investment. Unlike other drugs such as statins, antibiotics are only used for short periods of time by a patient. One course of treatment therefore doesn’t return a massive profit for the company. The second issue antibiotics face is that resistance to them occurs rapidly after they are put into circulation, so the company is not likely to get much use out of the drug. Therefore we need to find a new source for our antimicrobials. This is where the AMPs come in.

Currently, nearly 900 AMPs have been identified and characterised with many more undiscovered (2). They are an untapped source of drug discovery and they exhibit numerous benefits over their antibiotic cousins. As they are proteins, they have a genetic origin, which could provide an amenable platform for further development through random mutagenesis. This could produce a vast library of antimicrobial compounds (4,5), drastically improving our options for therapy.

Figure 2. Graph showing the steady decline in antibiotic development from 1980 to 2012 (1)

Nisin; not so nice if you’re a bacterial cell

AMPs were discovered in the 1930s although their use in the health industry has been fairly limited, resulting from the sheer difficulty and cost of manufacturing and purifying proteins on a large scale. The bacterially produced lantibiotics are by far the most well studied AMPs and have the most potential for the pharmaceutical industry. Nisin (E234) is the most well studied lantibiotic (Refer to Figure 1, Class I) and is produced by the bacterium Lactococcus lactis (6).

It shows broad spectrum activity on a large number of Gram-positive bacteria including other lactic acid bacteria, which has made it a coveted preservative in food processing. Currently it is added to cheeses, meats and beverages to extend shelf life and prevent the growth of spoilage organisms including spore forming bacteria such as Clostridium botulinum (6). The lantibiotics have also proven their capabilities for treating the clinically relevant pathogens methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci (7). They are also seen to have similar levels of activity as antibiotics and express low levels of toxicity to mammalian cells. Nisin exhibits poor oral availability making it more appealing as a topical agent or for intravenous application but there are also intentions to use it as a sterilising agent for catheters and medical equipment to help reduce the risk of infection (3).

So could lantibiotics like nisin be a good solution to antimicrobial resistance? Well more compelling evidence for nisin is that resistance has not been thoroughly documented. Nisin has been applied in sub-therapeutic concentrations in the food industry since the 1960s but still mostly retains its bactericidal ability. Resistance has been achieved artificially in the lab (discussed later in the article), but due to the mechanism of some lantibiotics including nisin, resistance is thought to be unlikely (6).

The mechanism behind nisin’s potency

Unlike animal cells, generally bacterial membranes have an overall negative charge and lack cholesterol (8). Nisin contains a high proportion of the positively charged (basic) amino acids lysine and arginine. These positive charges allow the protein to interact with the negative charges commonly associated with bacterial cell membranes (2). Nisin is good at aligning against Gram-positive bacterial membranes, where they multimerise to form short-lived pores (Figure 3). Hydrophobic regions help the protein to insert into the membrane and stabilise the pore (2), which allows the transport of ATP, ions and amino acids, eliminating the cellular membrane potential (9).

Nisin has a second trick up its sleeve. Its C-terminal, the portion of the protein containing the lanthionine ring motifs, allows it to latch onto the important membrane component lipid II (Figure 3). Lipid II is a precursor for peptidoglycan; the cell wall strengthening polymer found in both Gram-positive and Gram-negative bacteria. It is a common target for antibiotics including penicillin and vancomycin, which both target different stages of its synthesis. It helps to maintain the cell structure and prevents it from bursting under high osmotic pressure. When nisin binds to lipid II, it sequesters this molecule from the enzymes that catalyse its addition to growing peptidoglycan chains. Binding lipid II also helps to stabilise the transmembrane pores, further damaging the cell. As a result, not only is the cell wall weakened, but the cell loses its metabolic capabilities, through the loss of charged molecules.

The dual targeting system of nisin is thought to be the reason why resistance to nisin has not be well documented (10). The two processes are completely physiologically separate, and therefore to develop resistance, the bacteria would have to develop two unrelated mutations to counteract the effects of nisin.

Figure 3. Diagram showing the mechanism of several lantibiotics including nisin. AMPs are represented by lines made with clear circles. Phospholipids represented by green circles with tails. Lipid II is represented by orange hexagons (3).

What do we know about resistance towards nisin?

There are several proposed means by which an organism can be resistance to a toxin. Firstly, an organism may have innate immunity to a toxin simply because of its physiology. We see this largely in the Gram-negative bacteria towards nisin. The lipopolysaccharide (LPS) layer found on the outside of their cell wall provides protection against nisin and it has been shown that the oligosaccharides found within the core region of this structure greatly improve protection against nisin. It is believed that this is because metal ions are sequestered within this layer, adding additional positive charges to the site. Such charges would help to prevent nisin from aligning with the cell membrane (11). Removing these metal ions by sequestering them sensitises Gram-negative bacteria to nisin.

Emergent resistance is the type of resistance that should concern us the most, as it is the reason why we are now faced with the problem of antimicrobial resistance. It involves the acquisition of mutations or DNA that help confer tolerance to stress resulting from the action of a toxin (12). Although currently only produced in the laboratory, experiments carried out on the tolerance of clinically relevant bacteria towards nisin are crucial in highlighting the future of implementing an antimicrobial.

Resistance mechanisms have been documented in several bacteria including the causative agent of listeriosis, Listeria monocytogenes. Although not fully understood, changes in membrane composition have been attributed for the decreased susceptibility in resistant strains. In resistant strains, the bacterial membrane is composed of less negatively charged phospholipids. Similarly to sequestering metal ions near the membrane, this alters the overall net charge, helping to repel nisin.

The number of long chain fatty acids within its membrane is increased helping to reducing fluidity. This is believed to play a role in preventing nisin from inserting itself into the membrane. Studies show that nisin resistant strains were also less susceptible to cell wall acting components such as lysozyme and cell wall acting antibiotics. They did not identify the phenotypic change that gave additional protection, but this does indicate that a number of defence mechanisms are involved in defending cells against environmental stress from nisin (13).

Conclusion:

So could AMPs like nisin possibly serve as a replacement to our current armamentarium of antibiotics? AMPs are a largely untapped source of antimicrobials with many more still to be identified. AMPs may therefore serve as a new source of antimicrobials to help relieve the stress exerted on microorganisms by antibiotics. We have seen that nisin is an effective antimicrobial against a wide range of Gram-positive bacteria including spore forming bacteria. The dual-action of nisin challenges bacterial cells making it difficult for them to develop resistance. However, lab-based experiments have shown that it is possible to generate resistant strains showing the tenacity of bacteria to adapt to such potent environmental stresses. To learn from our previous mistakes with antibiotics, more responsible practices would need to be applied. Using combination therapy or rotating drug usage, as done with pesticides, could help further prevent resistance. Where they are likely to be applied in high concentration (in medical settings and agriculture), combination therapies should be used to further reduce the likelihood of resistance.

1. CDC. Antibiotic resistance threats. US Dep Healh Hum Serv. 2013;22–50.

2. Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol [Internet]. 2005;3(3):238–50. Available from: http://www.nature.com/doifinder/10.1038/nrmicro1098

3. Dischinger J, Basi Chipalu S, Bierbaum G. Lantibiotics: Promising candidates for future applications in health care. Int J Med Microbiol [Internet]. Elsevier GmbH.; 2014;304(1):51–62. Available from: http://dx.doi.org/10.1016/j.ijmm.2013.09.003

4. Field D, Begley M, O’Connor PM, Daly KM, Hugenholtz F, Cotter PD, et al. Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens. PLoS One. 2012;7(10).

5. Hilpert K, Volkmer-Engert R, Walter T, Hancock REW. High-throughput generation of small antibacterial peptides with improved activity. Nat Biotechnol. 2005;23(8):1008–12.

6. van Heel AJ, Montalban-Lopez M, Kuipers OP. Evaluating the feasibility of lantibiotics as an alternative therapy against bacterial infections in humans. Expert Opin Drug Metab Toxicol. 2011;7(6):675–80.

7. Barbosa J, Caetano T, Mendo S. Class I and Class II Lanthipeptides Produced by Bacillus spp. J Nat Prod [Internet]. 2015;151008121848005. Available from: http://pubsdc3.acs.org/doi/10.1021/np500424y

8. Neumann A, Berends ETM, Nerlich A, Molhoek EM, Gallo RL, Meerloo T, et al. The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps. Biochem J [Internet]. 2014 Nov 15 [cited 2014 Oct 28];464(1):3–11. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25181554

9. Kordel M, Schuller F, Sahl HG. Interaction of the pore forming-peptide antibiotics Pep 5, nisin and subtilin with non-energized liposomes. FEBS Lett. 1989;244(1):99–102.

10. Islam MR, Nagao J, Zendo T, Sonomoto K. Antimicrobial mechanism of lantibiotics. Biochem Soc Trans [Internet]. 2012;40(6):1528–33. Available from: http://www.biochemsoctrans.org/bst/040/bst0401528.htm

11. Stevens K a., Sheldon BW, Klapes N a., Klaenhammer TR. Nisin treatment for inactivation of Salmonella species and other gram- negative bacteria. Appl Environ Microbiol. 1991;57(12):3613–5.

12. Kaur G, Malik RK, Mishra SK, Singh TP, Bhardwaj A, Singroha G, et al. Nisin and class IIa bacteriocin resistance among Listeria and other Foodborne pathogens and spoilage bacteria. Microb Drug Resist. 2011;17(2).

13. Crandall AD, Montville TJ. Nisin resistance in Listeria monocytogenes ATCC 700302 is a complex phenotype. Appl Environ Microbiol. 1998

Molecule of the Day: Limonene

d-Limonene (C10H16) is a naturally occurring, chiral cyclic hydrocarbon with an orangey scent, and is found in citrus peels and citrus essential oils. It is a colourless liquid that is immiscible with water at room temperature and pressure. 

Limonene undergoes reactions typical of an alkene, such as electrophilic addition and oxidative cleavage. It is biosynthesised from geranyl pyrophosphate in plants, and is classified as a terpene.

While limonene can be synthesised in the lab, as shown below, it is produced industrially from the steam distillation of citrus peels due to its natural abundance. Furthermore, citrus peels are a by-product of orange juice manufacturing, which makes it environmentally-friendly.

Limonene is commonly used in perfumes, soaps, and foods due to its fresh, citrus-like scent, and can also act as a pesticide. It is gaining prominence as an environmentally-friendly solvent and paint stripper. However, it is also a skin sensitiser, as it can dissolve the oils and fat underneath the skin!

Links:

Extraction of limonene from orange peels - YouTube

Dissolving styrofoam using limonene - YouTube

Leopard shark makes world-first switch from sexual to asexual reproduction

A leopard shark in an Australian aquarium has reproduced asexually after being separated from her mate.

It is the first reported case of a shark switching from sexual to asexual or parthenogenetic reproduction and only the third reported case among all vertebrate species.

The leopard shark, Leonie, was captured in the wild in 1999 and introduced to a male shark at the Reef HQ aquarium in Townsville, Queensland, in 2006. Leopard sharks are also known as zebra sharks.

One of the baby sharks born to leopard sharks at Reef HQ aquarium in Townsville, who have produced live young through asexual reproduction. Photograph: Tourism and Events Queensland

Leonie, the world’s first shark known to have switched from sexual to asexual reproduction, at Reef HQ aquarium in Townsville. Photograph: Tourism and Events Queensland

For #WorldBeeDay, here’s a look at the chemistry behind the honey some bees produce: https://ift.tt/2GV5qtq https://ift.tt/2LJpsIe

Plantibodies and Plant-Derived Edible Vaccines

Throughout history, humans have used plants in the treatment of disease. This includes more traditional methods involving direct consumption with minimal preparation involved and the extraction of compounds for use in modern pharmaceuticals. One of the more recent methods of using plants in medicine involves the synthesis and application of plantibodies and plant produced antigens. These are recombinant antibodies and antigens respectively, which have been produced by a genetically modified plant (1, 2).        

Antibodies are a diverse set of proteins which serve the purpose of aiding the body in eliminating foreign pathogens. They are secreted by effector B lymphocytes which are a type of white blood cell that circulate throughout the body. An antigen is a molecule or a component of a molecule, such as a protein or carbohydrate, which can stimulate an immune response. The human body is capable of producing around 1012  different types of antibodies, each of which can bind to a specific antigen or a small group of related motifs (3). When an antibody encounters the antigen of a foreign pathogen to which it has high affinity, it binds to it which can disable it or alert the immune system for its destruction (4).

Figure 1: Each type of antibody has the ability to bind to a specific antigen or group of antigens with high affinity.

Plants do not normally produce antibodies and thus must be genetically modified to produce plantibodies as well as foreign protein antigens. Plantibodies produced in this manner function the same way as the antibodies native to the human body (1). The main ways to do this are to stably integrate foreign DNA into a host cell and place it into a plant embryo resulting in a permanent change of the nuclear genome, or to induce transient gene expression of the specified protein (5). In both cases, the genetic material introduced to the plant codes for the protein of choice. Several of the methods used to induce permanent transgene expression include agrobacterium-mediated transformation, particle bombardment using a gene gun, or the transformation of organelles such as chloroplasts. Transient transgene expression can be done using plant viruses as viral vectors or agroinfiltration (2). Once the genetic material has been inserted, the specified protein is produced via the plant endomembrane and secretory systems, after which it can be recovered through purification of the plant tissue to be used for injection (1). The production of these proteins can also be directed to specific organs of the plant such as the seeds using targeting signals (2). Stable integration techniques are generally used for more large scale production and when the gene in question has a high level of expression, while transient techniques are used to produce a greater yield in the short term (5).

Figure 2: A gene gun being used to introduce genetic material into the leaves of a plant.

Now how can plantibodies and plant produced antigens help us as humans? The primary purpose of producing plantibodies is for the treatment of disease via immunotherapy. Immunotherapy is a method of treatment in which one’s immune response to a particular disease is enhanced. Specific plantibodies can be produced in order to target a particular disease and then be applied to patients via injection as a means of treatment (6). Doing so provides a boost to the number of antibodies against the targeted disease in the patient’s body which helps to enhance their immune system response against it. An example of this is CaroRx, the first clinically tested plantibody which has the ability to bind to Streptococcus mutans. CaroRx has been shown to be effective in the treatment of tooth decay caused by this species of bacteria (1). More recently, a plantibody known as ZMapp has shown potential in the treatment of Ebola. A study by Qiu et al showed that when administered up to 5 days after the onset of the disease, 100% of rhesus macaques that were administered the drug were shown to have recovered from its effects while all of the control group animals perished as a result of the disease (7). In addition, it has been experimentally administered to some humans who later recovered from the disease, although its role in their recovery was not fully ascertained (8).

Plant produced antigens on the other hand can be used to produce oral vaccines (9). Vaccines are typically biological mixtures containing a weakened pathogen and its antigens. Injection of this results in priming of the body’s adaptive immune system against the particular pathogen so that it can more easily recognize and respond to the threat in the future (4). By producing the antigens of targeted pathogens in plants through transgenic expression, edible vaccines can be created if the plant used is safe to eat. Tobacco, potato, and tomato plants have typically been used in past attempts to create them, showing success in both animal studies and a number of human trials. The advantages of using an oral vaccine include ease of administration and lower costs since specialised personel are not required for administration (9). In addition, oral vaccines are more effective in providing immunity against pathogens at mucosal surfaces as they can be directly applied to the gastrointestinal tract (1). The primary issue with the usage of oral vaccines is that protein antigens must avoid degradation in the stomach and intestines before they can reach the targeted sites in the body. Several solutions to this dilemma include using other biological structures such as liposomes and proteasomes as a means of delivery. This helps to prevent the proteins from being degraded by digestive enzymes and the acidic environment of the stomach before they can reach their destination (1, 9).

Figure 3: An overview of one method of producing an edible vaccine using a potato plant. A gene coding for the protein of a human pathogen is used in agrobacterium-mediated transformation to produce a transgenic potato plant. The potatoes from this plant can then serve as an edible vaccine against pathogen from which the protein originated.

There are a number of advantages to using these plant based pharmaceuticals. First of all, they can be produced on a large scale at a relatively low cost through agriculture and are convenient for long-term storage due to the resiliency and size of plant seeds (5). There is also a low risk of contamination by mammalian viruses, blood borne pathogens, and oncogenes which can remove the need for expensive removal steps (1). In addition, purification steps can be skipped if the plants used are edible and ethical problems that come with animal production can be avoided (5). The disadvantages include the potential for allergic reactions to plant antigens and contamination by pesticides and herbicides. There is also the possibility of outcrossing of transgenic pollen to weeds or related crops which would lead to non-target crops also expressing the pharmaceutical.This could lead to public concern along with the potential that other species which ingest these plants may be negatively affected (9).  While plantibodies and plant produced antigens have not yet been extensively tested in clinical trials, going forward they represent a new treatment option with great promise.

References

1. Jain P, Pandey P, Jain D, Dwivedi P. Plantibody: An overview. Asian journal of Pharmacy and Life Science. 2011 Jan;1(1):87-94.

2. Stoger E, Sack M, Fischer R, Christou P. Plantibodies: applications, advantages and bottlenecks. Current Opinion in Biotechnology. 2002 Apr 1;13(2):161-166.

3. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell. 4th Edition. New York: Garland Science; 2002.

4. Parham P. The immune system. 4th Edition. New York: Garland Science; 2014.

5. Ferrante E, Simpson D. A review of the progression of transgenic plants used to produce plantibodies for human usage. J. Young Invest. 2001;4:1-0.

6. Smith MD. Antibody production in plants. Biotechnology advances. 1996 Dec 31;14(3):267-81.

7. Qiu X, Wong G, Audet J, Bello A, Fernando L, Alimonti JB, Fausther-Bovendo H, Wei H, Aviles J, Hiatt E, Johnson A. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature. 2014 Aug 29.

8. Sneed A. Know the Jargon. Scientific american. 2014 Dec 1;311(6):24-24.

9. Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends in plant science. 2001 May 1;6(5):219-26.

Virus carrying DNA of black widow spider toxin discovered

A tiny virus that may sting like a black widow spider.

That is one of the surprise discoveries made by a pair of Vanderbilt biologists when they sequenced the genome of a virus that attacks Wolbachia, a bacterial parasite that has successfully infected not only black widow spiders but more than half of all arthropod species, which include insects, spiders and crustaceans.

“Discovering DNA related to the black widow spider toxin gene came as a total surprise because it is the first time that a phage – a virus that infects bacteria – has been found carrying animal-like DNA,” said Associate Professor of Biological Sciences Seth Bordenstein. He and Senior Research Specialist Sarah Bordenstein reported the results of their study in a paper titled “Eukaryotic association module in phage WO genomes from Wolbachia” published Oct. 11 in the journal Nature Communications.

Sarah R. Bordenstein, Seth R. Bordenstein. Eukaryotic association module in phage WO genomes from Wolbachia. Nature Communications, 2016; 7: 13155 DOI: 10.1038/ncomms13155

DNA related to black widow spider toxin has been found in a bacterial virus. (iStock)

The oval shape in this electron microphotograph is a Wolbachia bacterium that has infected a Nasonia wasp. The small dots in the bacterium are WO phage particles. The inset shows them at a higher magnification. The white arrows in the inset point to the phage tails. The scale bar in the image is 200 nm and the bar in he inset is 100 nm. (Bordenstein Lab / Vanderbilt)

The Red Hot Debate about Transmissible Alzheimer’s

In the 25 years that John Collinge has studied neurology, he has seen hundreds of human brains. But the ones he was looking at under the microscope in January 2015 were like nothing he had seen before.

He and his team of pathologists were examining the autopsied brains of four people who had once received injections of growth hormone derived from human cadavers. It turned out that some of the preparations were contaminated with a misfolded protein—a prion—that causes a rare and deadly condition called Creutzfeldt–Jakob disease (CJD), and all four had died in their 40s or 50s as a result. But for Collinge, the reason that these brains looked extraordinary was not the damage wrought by prion disease; it was that they were scarred in another way. “It was very clear that something was there beyond what you’d expect,” he says. The brains were spotted with the whitish plaques typical of people with Alzheimer’s disease. They looked, in other words, like young people with an old person’s disease.

For Collinge, this led to a worrying conclusion: that the plaques might have been transmitted, alongside the prions, in the injections of growth hormone—the first evidence that Alzheimer’s could be transmitted from one person to another. If true, that could have far-reaching implications: the possibility that ‘seeds’ of the amyloid-β protein involved in Alzheimer’s could be transferred during other procedures in which fluid or tissues from one person are introduced into another, such as blood transfusions, organ transplants and other common medical procedures.

Collinge felt a duty to inform the public quickly. And that’s what he did, publishing the study inNature in September, to headlines around the world. “Can you CATCH Alzheimer’s?” asked Britain’s Daily Mail, about the “potentially explosive new study”. Collinge has been careful to temper the alarm. “Our study does not mean that Alzheimer’s is actually contagious,” he stresses. Carers won’t catch it on the job, nor family members, however close. “But it raises concern that some medical procedures could be inadvertently transferring amyloid-β seeds.”

Since then, the headlines have died away, but the academic work and discussion have taken off. Could seeds of amyloid-β proteins really be transmitted and, if so, are they harmless or do they cause disease? And could seeds of other related diseases involving misfolded proteins be transmitted in a similar way? In the past decade or so, evidence has been mounting for a controversial theory that rogue proteins, known collectively as amyloids and associated with diverse neurodegenerative diseases—from Alzheimer’s to Parkinson’s and Huntington's—might share some properties of prions, including their transmissibility. Collinge’s data bolstered that theory.

Urgent though these questions are, it could take years to find answers. The paper by Collinge and his colleagues has sparked a worldwide hunt for similar amyloid pathology in autopsied brains, and a small study published in January 2016 revealed a handful of related cases. Researchers are also trying to work out what the putative amyloid seeds look like, and whether different 'strains’ of amyloids exist that are particularly damaging.

Some researchers say that it is much too early to be alarmed. They point out that the number of patients in Collinge’s study was tiny, that none had displayed symptoms of Alzheimer’s disease before their death and that another toxic protein called tau also seems to be required to cause the condition. “We have to remember that there is no conclusive evidence that seeds of amyloids can transmit actual disease or that amyloids spread in the brain in a prion-like way,” says Pierluigi Nicotera, scientific director of the German Centre for Neurodegenerative Diseases in Bonn. “There may be other biological explanations.”

Right now, there are few solid answers, but plenty of concerns. The sceptics worry that they might one day find themselves working under tight biosecurity regulations to handle proteins that they view as relatively innocuous. Others feel that the dangers may have been underestimated, and that scientists have a duty to investigate this as quickly as they can. “In my opinion, all amyloids should be considered dangerous until proven safe,” says prion and amyloid researcher Adriano Aguzzi at the University Hospital Zurich in Switzerland.

DANGEROUS FOLDS

A few decades ago, it was almost inconceivable that a protein, which has no genetic material or any other obvious way to self-replicate, could cause infectious disease. But that changed in 1982, when Stanley Prusiner, now at the University of California, San Francisco, introduced evidence for disease-causing prions, coining the term from the words 'proteinacious’ and 'infectious’. Prusiner showed that prion proteins (PrP) exist in a normal cellular form, and in a misfolded infectious form. The misfolded form causes the normal protein to also misfold, creating a cascade that overwhelms and kills cells. It cause animal brains to turn into a spongy mess in scrapie, a disease of sheep, and in bovine spongiform encephalopathy (BSE or 'mad cow disease’), as well as in human prion diseases such as CJD.

Prusiner and others also investigated how prions could spread. They showed that injecting brain extracts containing infectious prions into healthy animals seeds disease. These prions can be so aggressive that in some cases, simply eating infected brains is sufficient to transmit disease. For example, many cases of variant CJD (vCJD) are now thought to have arisen in the United Kingdom in the 1990s after people ate meat from cattle that were infected with BSE.

Since then, scientists have come to appreciate that many proteins associated with neurodegenerative diseases—including amyloid-β and tau in Alzheimer’s disease and α-synuclein in Parkinson’s disease—misfold catastrophically. Structural biologists call the entire family of misfolded proteins (including PrP) amyloids. Amyloid-β clumps into whitish plaques, tau forms ribbons called tangles and α-synuclein creates fibrous deposits called inclusions.

A decade ago, these similarities prompted neuroscientist Mathias Jucker at the University of Tübingen in Germany to test whether injecting brain extracts containing misfolded amyloid-β into mice could seed an abnormal build-up of amyloid in the animals’ brains. He found that it could, and that it also worked if he injected amyloids into the muscles. “We saw no reason not to believe that if amyloid seeds entered the human brain, they would also cause amyloid pathology in the same way,” says Jucker.

This didn’t cause alarm at the time, because it wasn’t clear how an amyloid seed from the brain of someone with Alzheimer’s could be transferred into another person’s body and find its way to their brain. To investigate that, what was needed was a group of people who had been injected with material from another person, and the opportunity to examine their brains in great detail, preferably when they were still relatively young and before they might have spontaneously developed early signs of Alzheimer’s.

The CJD brains provided just that opportunity. Between 1958 and 1985, around 30,000 people worldwide received injections of growth hormone derived from the adrenal glands of cadavers to treat growth problems. Some of the preparations were contaminated with the prion that causes CJD. Like all prion diseases, CJD has a very long incubation period, but once it gets going it rages through the brain, destroying all tissue in its wake and typically killing people from their late 40s onwards. According to 2012 statistics, 226 people around the world have died from CJD as a result of prion-contaminated growth-hormone preparations.

Collinge had not set out to find a link with Alzheimer's—it emerged as part of routine work at the National Prion Clinic in London, which he heads, and where around 70% of all people in the United Kingdom who die from prion-related causes are now autopsied. The clinic routinely looks for signs of all amyloid proteins in these brains to distinguish prion disease from other conditions. It was thanks to this routine work that the cluster of unusual cases emerged of people who had clearly died of CJD, but who also had obvious signs of amyloid pathology in their grey matter and cerebral blood vessels.

As soon as he saw these brains, Collinge knew that he could get into stormy waters. Keen to strike a balance between warning of a possible public-health risk and causing unwarranted panic, he sketched a carefully worded press release that would go out from the National Prion Centre and set up hotlines for people who had been treated with growth hormone in the past. But no panic occurred: apart from one or two overwrought headlines, the news stories were fairly measured, he says. Only around ten people called the hotlines.

For scientists, however, the paper was a red flag. “As soon as the paper came out we realized the health implications and started collecting slides and paraffin blocks from patients,” says Jiri Safar, director of the National Prion Disease Pathology Surveillance Center at Case Western Reserve University in Cleveland, Ohio. Like other pathologists in countries where people had died of CJD associated with medical procedures, he rushed to check the centre’s archives of autopsied brains to see if any of them contained the ominous amyloid deposits.

The answers are not yet in. Safar says that it has not proved easy to trace brain samples in the United States, but that he is working to do so with the National Institutes of Health and the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia. Charles Duyckaerts at the Pitié-Salpêtrière Hospital in Paris, France, has now examined brain tissues from around 24 patients and is likely to report the results later this year.

A further 228 cases of CJD were caused by transplantation of prion-contaminated dura mater—the membrane surrounding the brain and spinal cord—prepared from cadavers around the world. Dura-mater preparations were regularly used in brain surgery as repair patches until the late 1990s. For the study published in January, Herbert Budka at the National Prion Diseases Reference Center at University Hospital Zurich and his colleagues examined the brains of seven such patients from Switzerland and Austria, and found that five had amyloid deposits in grey matter and blood vessels. In Japan, dementia researcher Masahito Yamada at Kanazawa University is making his way through a large number of such autopsy specimens and says that the 16 brains he has examined so far show signs of unusually high levels of amyloid deposition in cerebral blood vessels.

Yet such case studies can only ever provide circumstantial evidence that seeds of amyloid-β were transferred during the treatments. And they cannot entirely rule out the possibility that the treatments themselves—or the patients’ original medical conditions—caused the amyloid pathology. More-conclusive evidence would come from checking whether the original growth hormone and dura-mater preparations contained infectious amyloid seeds, by injecting them into animals and seeing whether this triggers disease. Most of these preparations, however, have long since disappeared. Collinge has access to some original samples of growth hormone stored by the UK Department of Health, and he is planning to analyse them for the presence of amyloid seeds and then inject them into mice. That work will take a couple of years to complete, he says.

SEEDS OF DOUBT

There is another hitch: no one knows for sure what size and shape the amyloid seeds might be. Jucker is hunting for them in an unusual source of human brain tissue that has nothing to do with CJD. A team in Bonn has collected frozen samples from more than 700 people with epilepsy who were operated on over the past 25 years to remove tissue that was driving their seizures. “It is the best source of fresh human brain tissue available at the moment,” says Jucker, who plans to scrutinize it carefully under the microscope for anything that might resemble tiny clumps or seeds of amyloid-β. The team also has records of the patients’ cognitive skills, such as language and memory skills, before and at regular intervals after the operations. This should allow Jucker’s team to correlate the presence of any amyloid-β seeds it finds with changes in the cognitive function of individual patients over time.

Scientists have shown that tau and α-synuclein can also seed pathological features in mice. In two studies, from 2012, scientists injected fibrils of α-synuclein into the brains of mice already engineered to develop some of the characteristics of Parkinson’s disease. This triggered the early onset of some of the signs and symptoms of Parkinson’s, and eventually killed the animals. A third study showed that similar injections into normal mice caused some of the neurodegeneration typical of Parkinson’s disease and the mice became less agile. In humans, α-synuclein would not necessarily turn out to be equally aggressive—mouse models of neurodegenerative diseases do not mimic human disease very closely—but scientists are taking the possibility seriously.

If the transmissibility hypothesis proves true, the implications could be severe. Amyloids stick like glue to metal surgical instruments, and normal sterilization does not remove them, so amyloid seeds might possibly be transferred during surgery. The seeds might sit in the body for years or decades before spreading into plaques, and perhaps enabling the other pathological changes needed to induce Alzheimer’s disease. Having amyloid plaques in cerebral blood vessels could be dangerous in another way, because they increase the risk that the vessel walls might break, leading to small strokes.

But if common medical procedures really increased the risk of neurodegenerative disorders, then wouldn’t that already have been detected? Not necessarily, says epidemiologist Roy Anderson at Imperial College London. “The proper epidemiological studies have not been done yet,” he says. They require very large and carefully curated databases of people with Alzheimer’s disease, which include information about the development of symptoms and autopsy data. He and his team are now studying the handful of reliable databases that exist to tease out a signal that might associate medical procedures with Alzheimer’s progression. The number of patients currently available may turn out to be too small to draw conclusions, he says, but a more definitive answer could emerge as the databases grow.

Faced with so much uncertainty, some researchers and public-health agencies have adopted a wait-and-see approach. “We are right at the beginning of this story,” says Nicotera, “and if there is one message to come out right now it is that we need more work to see if this is a relevant mechanism.” The CDC and the European Centre for Disease Prevention and Control in Solna, Sweden, say that they are keeping a cautious eye on the issue.

If further research does confirm that common neurodegenerative diseases are transmissible, what then? One immediate priority would be rigorous sterilization procedures for medical and surgical instruments that would destroy amyloids, in the way that extremely high temperatures and harsh chemicals destroy prions. Aguzzi says that funding agencies should put out calls now to researchers to develop cheap and simple sterilization methods. “It’s not very sexy science, but it is urgently needed,” he says. He also worries about the safety of researchers working with amyloids—particularly α-synuclein. “I have nightmares that someone in my lab may catch Parkinson’s,” he says. “While the story is in flux, our first duty is to protect lab workers.”

STRAIN SEEKERS

The similarities between prions and other amyloids is throwing open other avenues of research. Prions can exist as distinct strains—proteins that have the same sequence of amino acids but misfold in different ways and have distinct biological behaviours, much as different strains of a pathogenic virus can be aggressive or weak. The outbreak of vCJD in the United Kingdom in the 1990s was traced to BSE-contaminated meat because the prion strain was the same in both.

Over the past few years, research in animals has shown that different strains of amyloid-β and α-synuclein exist. And a landmark paper in 2013 reported that strains of amyloid-β with different 3D structures were associated with different disease progression in two people with Alzheimer’s. Structural biologist Robert Tycko, who led the work at the National Institute of Diabetes and Digestive and Kidney Diseases in Bethesda, Maryland, is now looking at many more brain samples from such patients.

Knowing the structures of pathological forms of amyloid seeds should help to design small molecules that bind to them and stop them doing damage, says biophysicist Ronald Melki at the Paris-Saclay Institute of Neuroscience, who works on α-synuclein strains. His lab is designing small peptides that target the seeds and mimic regions of 'chaperone’ molecules, which usually bind to proteins and help them to fold correctly. Melki’s small peptides mimic these binding regions, sticking to the amyloid proteins to stop them from aggregating further.

In the research community, much of the agitation in response to Collinge’s paper boils down to semantics. Some scientists do not like to use the word 'prion’ in connection with the amyloids associated with common neurodegenerative diseases, or to describe any of their properties as 'prion-like'—because of its connotation of infectious, deadly disease. “The public has this perception of the word 'prion’,” says Alzheimer’s researcher Brad Hyman at Harvard Medical School in Boston, Massachusetts, and this matters, even if their ideas are wrong. “One of my patients told me that she wasn’t getting any hugs any more from her husband who had read about the case in the media—that made me sad,” he says.

Others, however, feel that it is helpful to consider prions and other amyloids as being part of a single spectrum of conditions involving proteins that misfold and misbehave. It means that researchers studying prion diseases and neurodegenerative diseases, who until recently had considered their disciplines to be separate, now find themselves tackling shared questions.

Both fields are wary of raising premature alarm, even though they wonder what the future will bring. Jucker, only half-jokingly, says he could imagine a future in which people would go into hospital every ten years or so and get the amyloid seeds cleared out of their brains with antibodies. “You’d be good then to go for another decade.”

Image 1 Credit: ©iStock.com

Image 2 Credit:  Juan Gaertner/Shutterstock

Source: Scientific American (By Alison Abbott, Nature magazine)

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