Category Archives: evolution

War as a Driver in Tuberculosis Evolution

by Michelle Ziegler

Russia has been all over the news lately. Beyond our recent election, increased Russian activity on the world stage has public health consequences for Europe and farther afield. It has been known for a long time that post-Soviet Russia had and continues to have serious public health problems. One of their particular problems that they have shared with the world is their alarmingly high rate of antibiotic resistant tuberculosis. There is no mystery over the root cause of their antibiotic resistance woes — poor antibiotic stewardship (Garrett, 2000; Bernard et al 2013).

A study by Vegard Eldholm and colleagues that came out this fall sheds light on the origins of particularly virulent tuberculosis strains with high rates of antibiotic resistance that recently entered Europe.  A large outbreak among Afghan refugees and Norwegians in Oslo, Norway, provided a core set of 26 specimens for this study that could be compared with results generated elsewhere in Europe (Eldholm et al, 2010). The Oslo outbreak clearly fits within the Russian clade A group that is concentrated to the east of the Volga River in countries of the former Soviet Union. They name this cluster the Central Asian Clade, noting that it co-localizes with region of origin of migrants carrying the MDR strains of tuberculosis reported in Europe.

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Figure 5. Phylogeny of the Afghan Strain Family (ASF). Colored boxes represent the country of origin: Afghanistan is orange; other countries are gray. (Eldholm et al, 2016)

When the Oslo samples are added to the family tree, phylogeny, of recent tuberculosis isolates from elsewhere in Europe a distinctive pattern emerges. The branches on the family tree are short and dense, suggesting that this is recent diversity, that they calculate to have occurred within approximately the last twenty years (Eldholm et al, 2016).

The Central Asian Clade spread into Afghanistan before drug resistance began to develop, probably during the Soviet-Afghan war (1979-1989) producing the Afghan Strain Diversity clade. Slightly later, the Central Asian Clade still in the former Soviet states begins to accumulate antibiotic resistance as the public health infrastructure crumbles in the wake of the dissolution of the USSR. The invasion of Afghanistan by the US and its allies in 2002 toppled the Afghan state, crippling infrastructure and spurring refugee movements within and out of Afghanistan. The lack of modern public health standards in Afghanistan since their war with the introduction of these strains by the Soviets in the 1980s provided fertile ground for the establishment and diversity of tuberculosis in the country. Instability has been pervasive throughout the entire region sending refugees and economic migrants from both Afghanistan and the former Soviet states into Europe.

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Movements of the Central Asian Clade (CAC) since c. 1960 and the subsequent Afghan Strain Family (ASF). (Eldholm et al, 2016)

Their dating of the last common ancestor for the Central Asian Clade to c. 1961 is significantly younger than the previous dating of 4,415 years before present for the Russian clade A (CC1) of the Beijing lineage of Mycobacteria tuberculosis. They account for this difference by noting differences in their methods of assessing sequence differences and note that their method is in line with other recent evolutionary rates for other tuberculosis clades.  The diagnosis dates and length of the arms on their reconstructed phylogeny suggests that there were multiple, independent introductions of the cases from Afghanistan and the former Soviet republics. This is consistent with a repeated periods of refugee movements from central Asia into Europe.

The rapid proliferation and diversification of the Afghan Strain Family may be explained by a known syndemic between tuberculosis and war (Ostrach & Singer, 2013). Conditions of war everywhere disrupt food systems, destroy critical infrastructures such as electricity and water systems, interrupts medical supplies, and the human public health infrastructure of the country. Malnutrition and stress are known contributors to immune suppression. Many pathogens flourish simultaneously in these conditions increasing the infectious challenges the population must fend off. Diarrheal diseases are the most acute and demanding of rapid attention, allowing longer-term diseases like tuberculosis to slip through the overburdened healthcare system. Afghanistan has experienced nearly forty years of war, political instability, and repeated infrastructure destruction. Thus, they were primed for both the establishment of new tuberculosis strains during the Afghan-Soviet war in the 1980s along with the proliferation and diversification of tuberculosis during the Afghan-American war of the last sixteen years.

Established syndemics between tuberculosis and war have been made retrospectively following the Vietnam war and the Persian Gulf war of 1991 (Ostrach & Singer, 2013). In Vietnam, prolonged malnutrition caused an eruption of tuberculosis along with malaria, leprosy, typhoid, cholera, plague, and parasitic diseases.  A WHO survey in 1976 found that Vietnam had twice the incidence of tuberculosis over all of its neighboring countries (Ostrach & Singer, 2013). When the military intentionally targets water infrastructure as it did in Vietnam and Iraq, the production of civilian infectious disease is a tactic of war. In both Vietnam and post-Gulf war Iraq, more civilians died of malnutrition and infectious disease than enemy soldiers died of all causes (Ostrach & Singler, 2013).

It seems likely that this is just one of the first studies to establish a link between serious infectious disease developments and the Afghan wars. The current war zones throughout central Asia and the Middle East already have ramifications for the public health of the entire world that walls along borders will not be able to stop. Most of the cases in the Oslo outbreak were Norwegians, not Afghan immigrants. Diseases will spread beyond the migrants so country of origin screening will be of little use before long.


Reference

Eldholm, V., Pettersson, J. H. O., Brynildsrud, O. B., Kitchen, A., Rasmussen, E. M., Lillebaek, T., et al. (2016). Armed conflict and population displacement as drivers of the evolution and dispersal of Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America, 201611283–16. http://doi.org/10.1073/pnas.1611283113

Ostrach, B., & Singer, M. C. (2013). Syndemics of War: Malnutrition-Infectious Disease Interactions and the Unintended Health Consequences of Intentional War Policies. Annals of Anthropological Practice, 36(2), 257–273. http://doi.org/10.1111/napa.12003

Bernard, C., Brossier, F., Sougakoff, W., Veziris, N., Frechet-Jachym, M., Metivier, N., et al. (2013). A surge of MDR and XDR tuberculosis in France among patients born in the Former Soviet Union. Euro Surveillance: Bulletin Européen Sur Les Maladies Transmissibles = European Communicable Disease Bulletin, 18(33), 20555.

Evolutionary Clues in 17th-Century Smallpox Genome

By Michelle Ziegler

Smallpox is one of those diseases long believed to have an ancient pedigree, the suspected culprit of legendary epidemics. Yet, so far, smallpox hasn’t made a big impression in ancient DNA surveys. If it was truly endemic throughout the Old World before 1492, so much so that it pops up in the New World almost immediately after contact, it’s odd that it has not been more prominent in ancient DNA surveys. Be ready for a smallpox paradigm shift and reexamination of its reputed history.

In December, Ana Duggan, Maria Perdomo, and the McMaster Ancient DNA Centre team announced the first full ancient smallpox genome isolated from a mummified 17th-century child in Vilnius, Lithuania. Radiocarbon dates of the child place him or her in the mid 17th century (est. c. 1654) in the midst of dated smallpox epidemics from all over Europe.

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Fig 1 (Duggan et al, 2016): Left: Distribution of smallpox records in Europe. Right upper: Dominican Church of the Holy Spirit, Vilnius. Lithuania. Right lower: Crypt containing the child’s remains.

Their finding was unexpected. They were not looking for smallpox at all; the child had no observable lesions. They were hoping to find JC polyomavirus, of particular interest to one of the co-authors, and so they first enriched the specimen for this virus (McKenna 2016). After detecting variola virus (VARV), smallpox, instead they then enriched for VARV to confirm the initial signal.

duggan_cbsmallpox_finalMore than just confirming the signal, they were able to reconstruct the entire genome producing the entire sequence at an average depth of 18X. The surprising child had more revelations in his or her viral sequence. The sequence is ancestral to all existing reference strains. This is consistent with short stretches of aDNA amplified from 300-year-old frozen Siberian remains (Biagini et al, 2012). Unfortunately, the sequences from 2012 were not distinctive enough from the new Lithuanian sequence to give phylogicial resolution between them. Oddly, the frozen Siberian remains also lacked smallpox skin lesions with one showing signs of pulmonary hemorrhages.

Its ancestral position in the phylogeny suggests that a severe bottleneck occurred before c. 1654. As Duggan et al (2016) remark, vaccination would cause a very strong bottleneck, but this occurs after 1654 and there is new diversity among the descendent reference specimens producing two major clades. Yet to be determined is the evolutionary effect of extensive variolation practices in the early modern period. In contrast to vaccination, variolation is a form of intentional smallpox transmission that sometimes went horribly wrong.

Evolution continues unabated. The molecular clock is consistent among the 20th-century specimens and the latest aDNA from the Lithuanian child. The two clades of smallpox collected from 20th-century specimens diverged from each other sometime around the mid-17th century after vaccination began. Interestingly, the less virulent Variola minor strain is not predicted to have emerged from Variola major clade P-II until the mid-19th century.

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Evolutionary history of Variola (Duggan et al, 2016)

It’s not entirely surprising that smallpox, a highly transmissive human-only virus, has a relatively recent last common ancestor; other viruses like measles do as well. Measles last common ancestor is probably in the early 20th century (Furuse, Suzuki & Oshitani, 2010). The dominance of the 1918 influenza strain in recent influenza phylogeny is another example; incomplete because influenza swaps genes with influenza viruses that are circulating primarily in birds, but also in swine and earlier in equines (Taubenberger & Morens, 2005). Improved transmission strains are likely to out-compete strains with a lower transmission rate if they achieve a global spread. For some viruses, though not necessarily all, improved transmissibility and virulence go hand in hand. So, in the end, the relatively recent last common ancestor says more about its global transmissibility than anything else.

The antiquity of the virus needs two components to estimate. The molecular clock must be steady, and it is so far (though this could change with more ancient specimens), and a near relative ‘out group’, related strains outside the Variola clade (a branch of the larger genetic tree). One potential problem here is that as transmissibility improves the clock may speed up. The speed of the clock is determined by the reproduction rate. The relatively steady clock back to this 17th-century specimen suggests that the transmission rate was pretty steady — after the evolutionary/transmission leap that swept aside other Variola strains. The inactivation of several orthopoxvirus genes in smallpox that are functional in vaccinia (used in smallpox vaccines), camelpox, and taterapox  (the ‘out groups’ used) may suggest that one or more of these genes had been protective. When the genes were inactivated, smallpox probably became a much more dangerous virus to humans.

Historical epidemiology suggests that there was once more variation in the virulence of smallpox epidemics.  Securely identifying smallpox epidemics in the historical record is much harder than is generally assumed, and it is harder yet to make a claim for a significant demographic impact prior to the Renaissance (Carmichael & Silverman, 1987). This is the problem with theories that smallpox was the cause of the second-century Antonine plague and then failed to cause an epidemic with a major demographic effect for many centuries. I find this very hard to believe. Additionally, the infamous smallpox epidemics in the New World are now also be reevaluated in ways that diminish smallpox’s toll and add in a wide variety of contributing factors to produce a colonization syndemic. This has most recently been summarized in essays collected in Beyond Germs: Native Depopulation in North America (2015).

One other observation from these studies: All ancient smallpox DNA to date has been extracted from mummy tissue, not a tooth or bone. This may point toward one of the limitations of ancient DNA pathogen surveys that currently use primarily teeth. Since neither mummy had visible smallpox lesions, smallpox should be considered a possibility in any mummy.


References

Duggan, A. T., Perdomo, M. F., Piombino-Mascali, D., Marciniak, S., Poinar, D., Emery, M. V., et al. (2016). 17th Century Variola Virus Reveals the Recent History of Smallpox. Current Biology, 1–7. http://doi.org/10.1016/j.cub.2016.10.061

Biagini, P., Thèves, C., Balaresque, P., Géraut, A., Cannet, C., Keyser, C., et al. (2012). Variola virus in a 300-year-old Siberian mummy. The New England Journal of Medicine, 367(21), 2057–2059. http://doi.org/10.1056/NEJMc1208124

McKenna, Maryn (8 Dec 2016) Child Mummy Found with Oldest Known Smallpox Virus. National Geographic. (online)

Carmichael, A. G., & Silverstein, A. M. (1987). Smallpox in Europe before the seventeenth century: virulent killer or benign disease? Journal of the History of Medicine and Allied Sciences, 42(2), 147–168.

Furuse Y, Suzuki A, & Oshitani H (2010). Origin of measles virus: divergence from rinderpest virus between the 11th and 12th centuries. Virology journal, 7 PMID: 20202190

Taubenberger, J. K., & Morens, D. M. (2005). 1918 Influenza: the mother of all pandemics. Emerging Infectious Diseases, 12(1), 15–22. http://doi.org/10.3201/eid1201.050979

Beyond Germs: Native Depopulation in North America. Edited by Catherine Cameron, Paul Kelton, and Alan Swedlund. University of Arizona Press, 2015.

Plague Dialogues: Monica Green and Boris Schmid on Plague Phylogeny (I)

In keeping with this blog’s goal to be a meeting ground for interdisciplinary discussions, I’ll be hosting a series of dialogues between scholars in the humanities and sciences. If you would like to be involved in one of these dialogues, please use the contact form on the about page.

On behalf of today’s participants, I invite you to post comments on this dialogue or on twitter.


Monica H. Green (monica.green@asu.edu, @MonicaMedHist ) is a historian of medieval medicine. An elected Fellow of the Medieval Academy of America, she teaches both global history and the global history of health. She was the editor in 2014 of Pandemic Disease in the Medieval World: Rethinking the Black Death, the inaugural issue of a new journal, The Medieval Globe.

Boris Schmid (@BorisVSchmid) is a theoretical biologist at the University of Oslo, Norway, and specializes in disease ecology and epidemiology. He recently described a link between climate fluctuations in medieval Central Asia and what looks like repeated introductions of plague into Europe’s harbors, a hypothesis that can be tested by the analysis of ancient DNA samples of Y. pestis. He works in a multidisciplinary team of theoreticians, archeologists, microbiologists and historians, led by Nils Chr. Stenseth.


“Tiny Changes with Huge Implications: Counting SNPs in Plague’s History”

Monica:

I’d like to raise the question of SNPs and why understanding them is so important for those keeping an eye on the “plague aDNA debates.” SNPs – single nucleotide polymorphisms, one of the smallest levels of genetic change you can document on a genome — are critical to the evolutionary, and historical, story of Yersinia pestis (the causative agent of plague), because Y. pestis in fact has been proven to change so little over time. Nucleotide changes are a normal part of evolution. They just happen. But they don’t necessarily happen at the same rate in all organisms, or even necessarily at the same rate in the same species in all circumstances. On top of that, sudden proliferation of an organism — say, in the context of an epidemic in the case of pathogenic organisms — may increase the rapidity with which a random evolutionary change gets fixed in a population. (See Cui et al. 2013)

In the case of Y. pestis, it’s actually been shown to be “genetically monomorphic.” That’s another way of saying, it doesn’t change very much or very often. Within its core genome (the common set of genes shared by all Y. pestis bacteria), most any Y. pestis cell you find is going to look pretty much like every other one across much of its ±3.5 million nucleotides, differentiated only by a few dozen or a few hundred different SNPs (Achtman 2012). So when a ‘C’ (cytosine) in one position on the genome changes to a ‘G’ (guanine) or ’T’ (thymine) or ‘A’ (adenine), that can actually be a historically significant change. And when that new SNP suddenly shows up in a whole population, that’s a sign that something major has happened. That’s what Cui et al. 2013 talked about in the “Big Bang” they posited for the later medieval period: the old Branch 0 of Y. pestis (see fig. 1) suddenly split into four new branches, each characterized by a different SNP signature. And the sequencing in 2011 of the first three Y. pestis genomes from the London Black Death cemetery (Bos et al. 2011; I’ll want to talk about the 4th genome, London 6330, later), made clear that that split did indeed happen quite suddenly. The split happens (presumably somewhere in western China or central Eurasia) and then, “two SNPs later,” Y. pestis shows up in London. Given the distances involved in transmitting this single-celled organism — several thousands of kilometers —  and the fact that there are no airplanes in the 14th century to account for rapid dissemination, that’s quite significant.

Cui et al 2013 fig S3B Minimum spanning tree, with SNP counts (post-polytomy branches only)
Fig. 1: Cui et al 2013 fig S3B Minimum spanning tree of relationships between 133 core genomes of Y. pestis based on 2298 SNPs. Detail to show post-polytomy branches (the “Big Bang”), at the middle of the tree. Branch 0 is the vertical stem below that. New aDNA work in 2016 has added additional sub branches just after the polytomy on Branch 1 (upper left).

 

Boris:

The mutation rates of bacteria is indeed a topic with quite some nuance, and one that is worth delving into in order to understand the slew of aDNA plague papers that are currently coming online.

The rate at which plague accumulates new point mutations has been calculated from the plague outbreaks in Madagascar (Morelli 2010) to be on average of 1 mutation for every 100 million nucleotides copied (Cui 2013). While that sounds like a very uncommon event, it really is not, given the length of its genome – a single bacterium that is replicating itself would after 4 generations (so roughly 7 hours within a human (Chauvaux 2007)) be a small colony of 16 bacteria, with a total of 105 million nucleotides of core genome copied, and therefore most likely already with 1 SNP among them. But here we are comparing two different things – the mutation rate used by Morelli and Cui is valid for the amount of variation you’d expect to accumulate in each surviving lineage of plague during an epidemic; many of the mutations that were generated are lost again simply because those bacteria were not the ones that continued the epidemic.

Bos (2011) notices with surprise that the plague at the time of the Black Death differed just 97 SNPs from CO92 (a sample collected from a human patient in 1992 in Colorado, and the plague strain that by convention is used as a baseline against which to compare other plague strains). So that is 1 accumulated mutation every 6.6 years, counting from 1348 to 1992. That compares pretty well to the mutation rate in a plague focus we in Oslo are currently studying, where we see an accumulation of mutations along each lineage of about 1 mutation every 8 years. Continuing with our back-of-the-envelope calculation, this “two SNPs later” since the sudden divergence of plague into 4 lineages puts the date of the “Big Bang” at just 10-20 years prior to the arrival of the Black Death in Europe in 1347. There are some caveats that come with this analysis, notably that the rate at which mutations are accumulated can be quite slower in some branches of plague (up to 6-fold, Cui 2013), but for now let us continue with a simplistic 1 new SNP being fixed into a plague lineage approximately every 7 years.

With that 1 SNP per 7 years rate in mind, let’s look at the stylized version of the evolution of Yersinia pestis, recently presented by Johannes Krause (fig. 2 below), with the recently published medieval plague sequences (and now the new publication from Spyrou et al. 2016), and see what it tells us in terms of the history of plague.

last-tree-plague-krause
Fig. 2: A simplified diagram of the “Big Bang” (polytomy) proposed by Cui et al. 2013, where plague split up into its 4 branches in the 13th or 14th century. Branch 1 is the lineage responsible for both the Second (medieval) Pandemic in western Eurasia and Africa and the Third (modern, global) Pandemic. Moving along Branch 1 from the initial split, two SNPs after the “Big Bang,” we see the introduction of the Black Death into Europe (the red-darkred circle). In our discussion, we call the branch leading to Marseille (the grey circle) “Branch 1A”; the branch leading to the rest of Branch 1 we call “Branch 1B.” (Source: Krause 2016.)

 

Monica:

Thanks, Boris, for laying out these calculations. So, we’ve established that, even though Y. pestis is genetically monomorphic—that it doesn’t have sex and doesn’t even often switch genetic material laterally—that it does have a tendency to develop random mutations over time. Par for the course for basic biology. I’d like to return now to a point I already made, which is more historical: “And when that new SNP suddenly shows up in a whole population, that’s a sign that something major has happened.” The big question we’re looking at with the Black Death strain and the “founding” of Branch 1 of the Y. pestis phylogenetic tree is: how did one—just one—Y. pestis cell get its SNP profile to be the founder of the whole lineage that caused millions of deaths in the first wave of the Black Death? This seems to be a classic “founder effect” scenario, the Mitochondrial Eve of this new phase of plague history. (And yes, I know that Y. pestis doesn’t have mitochondria.) Granted, we only have fully sequenced aDNA from two Black Death sites: London and Barcelona. But Spyrou et al. report that the Barcelona sample is identical in every respect with the three London Black Death genomes that were sequenced in 2011.

Let us assume then, for the moment, that our historical sources are correct that the famous outbreak of plague in Caffa in 1346 (the bodies being hurled over the walls) caused a single chain of events that carried our one strain of Y. pestis all the way to Barcelona and London within a two-year period. Presumably, if we had samples from all the other places plague struck in those brutal first years as it spread from the Black Sea to the Mediterranean—Alexandria, Aleppo, Almeria, etc., etc.—they would all have the same SNP signature that Haensch et al. 2010 reported from sample SNP typing for Hereford (UK) and Saint-Laurent-de-la-Cabrerisse (France), and that Seifert et al. 2016 reported, also from sample SNP typing, for Manching-Pichl (Bavaria). None of those sites are as precisely dated as the first three genomes sequenced from the East Smithfield cemetery in London (which is dated from documentary records to late 1348 to 1350). But for the sake of argument let’s assume that they are, in fact, all first outbreak burials. So all those human deaths are caused by an identical organism, moving into new vulnerable hosts at the same time.

But now we have to ask: what happened after the Black Death? What was Y. pestis’s next stage of evolution? The samples we have thus far, whether whole genome sequences or just SNP profiles, are all taken from human bodies. But humans are not natural carriers of plague. Every aDNA sample found thus far is probably a dead end, literally: the end of the line for the unique SNP profile (if it had one) of the Y. pestis that infected that individual. It died with them. If we’re going to look for what sustained plague, what allowed it to focalize and continue replicating for centuries thereafter, then we need to look at what plague strains survived in rodent populations.

Boris:

The question of where plague’s reservoir was in medieval Eurasia is indeed the question that, as a biologist, I was eager to work on, Monica. Validation of any theory of how plague has moved across Eurasia in the past would have to come from the analysis of the SNPs that plague gathered along the way. However, the interpretation of those medieval SNPs turns out to less clear-cut than most of us had anticipated, as we will discuss in the next blog post.

References:

Achtman, M. (2012). Insights from genomic comparisons of genetically monomorphic bacterial pathogens. Philosophical Transactions of the Royal Society B: Biological Sciences, 367(1590), 860–867. http://doi.org/10.1098/rstb.2011.0303

Bos, K. I., Schuenemann, V. J., Golding, G. B., Burbano, H. A., Waglechner, N., Coombes, B. K., et al. (2011). A draft genome of Yersinia pestis from victims of the Black Death. Nature, 478(7370), 506–510. http://doi.org/10.1038/nature10549

Chauvaux, Sylvie, Marie-Laure Rosso, Lionel Frangeul, Céline Lacroix, Laurent Labarre, Angèle Schiavo, Michaël Marceau, et al. 2007. “Transcriptome Analysis of Yersinia Pestis in Human Plasma: An Approach for Discovering Bacterial Genes Involved in Septicaemic Plague.” Microbiology 153 (Pt 9): 3112–3124

Cui, Y., Yu, C., Yan, Y., Li, D., Li, Y., Jombart, T., et al. (2012). Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis. Proceedings of the National Academy of Sciences, 110(2), 577–582. http://doi.org/10.1073/pnas.1205750110/-/DCSupplemental/sd01.xls

Haensch, S., Bianucci, R., Signoli, M., Rajerison, M., Schultz, M., Kacki, S., et al. (2010). Distinct Clones of Yersinia pestis Caused the Black Death. PLoS Pathogens, 6(10), e1001134. http://doi.org/10.1371/journal.ppat.1001134.t001

Krause, Johannes (4-12-2016)  Oral Presentation #S577:  Ancient pathogen genomics: what we learn from historic pandemics. European Congress  of Clinical Microbiology and Infectious Diseases http://eccmidlive.org/#resources/ancient-pathogen-genomics-what-we-learn-from-historic-pandemics

Seifert, L., Wiechmann, I., Harbeck, M., Thomas, A., Grupe, G., Projahn, M., et al. (2016). Genotyping Yersinia pestis in Historical Plague: Evidence for Long-Term Persistence of Y. pestis in Europe from the 14th to the 17th Century. PLoS ONE, 11(1), e0145194–8. http://doi.org/10.1371/journal.pone.0145194

Spyrou, M. A., Tukhbatova, R. I., Feldman, M., Drath, J., Kacki, S., de Heredia, J. B., et al. (2016). Historical Y. pestis Genomes Reveal the European Black Death as the Source of Ancient and Modern Plague Pandemics. Cell Host and Microbe, 19(6), 874–881. http://doi.org/10.1016/j.chom.2016.05.012