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    Issue Date: October 2016

    Highlights from the 13th Annual IMGC Symposium

    The International Milk Genomics Consortium (IMGC) held its 13th annual conference this past September 27–29, 2016, at the University of California, Davis. With a focus this year on moving “From Milk to Microbes,” the conference included 31 talks, 21 posters, a group dinner at Mulvaney’s B&L in Sacramento, and plenty of networking. Here are some of the top highlights from this year’s IMGC Symposium:

    1) Kicking off the 13th year of the IMGC symposium, Butch Dias, chairman of the California Dairy Research Foundation, welcomed all attendees. Bruce German followed this welcome with a powerfully motivating view ahead to how milk research will soon leave many of today’s most devastating diseases in the past. Lactation and milk is a complex recipe that has been refined for millions of years, and still holds many secrets that will help protect and support infant development. Danielle Lemay presented on the IMGC’s newsletter, “SPLASH! milk science update.” (What’s that? Hint: you’re reading it right now!) SPLASH! contains more than 200 articles and attracts more than 80,000 readers to the IMGC website every year. Danielle also revealed milk science topics that will likely drive future research and discussion in the years to come.

    2) Day one of the symposium focused largely on new insights into the relationship between milk and the gut microbiome in infants. The genus Bifidobacterium claimed center stage; David Mills discussed its unique relationship with milk, feeding on milk glycans and generating metabolites that dampen inflammation and strengthen gut barrier function in the infant. Steve Frese revealed the development of stable replacement microbial communities that can restore a normal microbiome in a clinical setting when given to compromised infants. Jennifer Smilowitz described a clinical trial in which infants were given supplements of a strain of Bifidobacterium and showed that the microbial supplement colonized the gut, was well-tolerated and did not result in any adverse events.

    3) Continuing with the first day’s microbiome theme, Guy Vergères revealed a new strategy for measuring lactic acid fermenting bacteria activity in milk consumers. Nicole Roy presented findings on the differences among cow, goat, sheep, and soy milk when fed to a mouse model of human irritable bowel disease (IBD). Compared with the soy milk, both cow and goat milks reduced inflammation in the gut and altered the gut microbiome. Although individuals diagnosed with IBD are sometimes encouraged to avoid dairy, milk consumption may help reduce intestinal inflammation levels and alleviate symptoms.

    4) Bing Wang, winner of the Most Valuable Presentation award for her talk at 2015 symposium, presented an update on her examination of the influences of milk lactoferrin. In a randomized controlled trial, piglets fed milk supplemented with lactoferrin showed increased activation of genes associated with mental development, learning, and memory. Her ongoing research aims to determine the molecular mechanism that underlies these effects on neural development.

    5) On day 2, the symposium shifted to discussion of complex carbohydrates in milk. Daniela Barile kicked off the day with a great keynote talk on the challenges—and incredible promise—of glycans in human and bovine milk, and how they can be extracted and purified in large quantities. Next, Bethany Henrick discussed human milk oligosaccharides (HMOs) and their bovine analogue, bovine milk oligosaccharides (BMOs), as a powerful new resource for immune modulation. In intestinal epithelial cells, both HMOs and BMOs can influence changes in protein expression and production of cytokines, which play a role in regulating immune response. As well, Michiko Shimoda presented on how HMOs encouraged development of dendritic cells, which play an important role in regulating the adaptive immune response.

    6) Several members of the Barile Lab also shared the results of their work. Valerie Weinborn discussed producing HMO-mimicking molecular products using whey as a starting material, while Randall Robinson shared his success at using mass spectrometry to identify both oligosaccharides in milk and active peptides in different varieties of cheese. Joshua Cohen provided insight into his high-throughput method for isolating glycans and other bioactive molecules from whey.

    7) Elisha Goonatileke presented on Gly-Q, an analytical platform developed for rapid, high-throughput analysis and quantification of carbohydrates. She examined both human and bovine milk using Gly-Q, and demonstrated that the rapidly obtained results did not lead to loss of resolution or sensitivity. This method will offer a new tool to both academia and industry researchers studying milk oligosaccharides.

    8) Christine Leroux extracted and identified microRNA from mammary glands and from milk fat globules, and suggested that these two sources exhibited different miRNAs profiles. Richard Heinz used transgenic mice to demonstrate the important roles of milk miRNAs for milk production; mice that were nursed by mothers with constantly high expression of a particular miRNA in their mammary glands showed a dramatically reduced survival rate.

    9) Although discussion was serious and focused during the day, researchers and scientists got to lighten up and relax with dinner, music, and even a bit of impromptu dancing at Mulvaney’s B&L in Sacramento. A live band provided some great tunes as attendees shook out some muscles after a long day of presentations.

    10) The third day of the symposium featured new discussion of the proteins and glycoproteins of milk. Carlito Lebrilla gave the keynote talk, discussing new and advanced methods for extracting and profiling milk glycans. Bernd Stahl identified new proteins in the milk extracellular vesicles, while Kasper Hettinga presented on the use of Ultraviolet-C (UVC) light as a sterilization agent for raw milk. UVC light can kill bacteria through DNA damage without requiring the high heat of pasteurization, which can destroy beneficial proteins. Tom Baars and Betty van Esch presented research that showed a difference in the development of allergies between raw and processed milk in a mouse model.

    Over the course of the entire symposium, considerable small group discussion continued to foster the air of collaboration and multi-disciplinary cooperation that has allowed research into milk genomics to be so diverse and successful. Many thanks to the IMGC sustaining members, sponsors, Laurie Jacobson and other staff who ensured that the events ran smoothly, and all of those who attended and contributed to another successful IMGC symposium.

    Photo note: MVP awardee from IMGC 2015, Dr. Bing Wang (left) with Dr. Gonca Pasin (right)

    The Bacterial Diversity in Raw Cow Milk During Its Transport and Storage

    • Identifying the bacteria present in raw milk before processing could help detect sources of spoilage and eventually find ways to get rid of them.
    • In a new study, researchers used high-throughput gene sequencing techniques to conduct a detailed analysis of the bacteria present in raw milk during its transport and storage.
    • The new study found an extremely diverse population of bacteria in raw milk from 899 tanker trucks, and these populations also varied depending on the season.
    • Despite this diversity, there was still a core milk microbiome consisting of 29 different taxa present across all the tanker trucks sampled.
    • The researchers also analyzed the microbiome of raw milk in temporary storage silos and found that milk in some silos had a similar microbiome to that of milk in tanker trucks, but others had a dramatically different microbiome.

     

    Pasteurization helps make raw cow milk safe for human consumption, but it doesn’t get rid of all bacteria. These remaining bacteria can cause spoilage, thus affecting the shelf life and quality of milk products and leading to wastage. Knowing what bacteria are present in milk before and during milk processing could help identify sources of spoilage and find ways to get rid of them.

    Many bacterial species have been found in raw milk or its products [1,2]. But most previous studies have looked either in farms or in processed products, and there’s still relatively little known about the diversity of microbes present in raw milk during its transport and storage [3,4].

    In a new study, Maria Marco and her colleagues at the University of California at Davis used high-throughput gene sequencing techniques to conduct a detailed analysis of the microbes present in raw milk during its transport and storage [5]. “We need to know what bacteria are there, and this first study sets the scene,” says Marco.

    Many previous studies identified raw milk bacteria by seeing what grew on Petri dishes, whereas the new study used high-throughput gene sequencing for the same purpose. “We know we can only cultivate about 1% of bacteria on Earth,” says Marco. “With this new method we can take any kind of dairy sample we want and identify all the bacteria involved within a few days,” she says. “A few years ago we recognized the potential to use this new method to help dairy processors recognize what bacteria are in their milk and dairy processing streams, and to help them get the best dairy possible,” says Marco.

    It’s worth pointing out that raw milk is pasteurized before processing, so all products made from it are safe for human consumption. The milk that the researchers analyzed was also of high quality, so it contained very few bacteria, according to Marco. But milk’s nutrient-rich nature makes it easy for bacteria to grow, and some stick around even after pasteurization.

    For example, bacteria such as Clostridium species that form endospores are resistant to pasteurization and are known to contribute to spoilage and defects [6]. “They’re well known throughout the dairy world, so that’s one group that we were looking for,” says Marco. Other heat-tolerant bacteria or bacterial enzymes can also make it past the pasteurization step. “That’s why we need to understand what’s in there,” she says.

    With funding from the California Dairy Research Foundation, Marco and her colleagues analyzed the microbiome from raw milk in 899 tanker trucks when they arrived at two different dairy processors in the California Central Valley. They found that the raw milk microbiome from the trucks was extremely diverse. “I was surprised at exactly how diverse it is,” says Mary Kable, a postdoc in Marco’s laboratory and first author on the study. In many of the samples, more than half the community consisted of bacteria that formed less than one percent of the total. Other communities were dominated almost entirely by one bacteria. “Lots of things can influence what the bacteria are in milk, and that’s reflected in this huge diversity,” says Kable.

    Despite this huge diversity and even though the milk was collected at different geographic locations and across multiple seasons, every sample also had a core microbiome consisting of 29 different taxa, including Streptococcus. “That’s a little surprising too, because of how robust this core microbiome was,” says Kable. “We looked at close to 900 samples, so it’s a pretty big deal to find it in every single one,” she says. Kable points out that this particular core microbiome might be specific to California, and more studies will be needed at other locations to figure out if that’s the case.

    The researchers collected milk samples in fall, spring, and summer, and found that the bacteria in raw milk also varied depending on the season. That’s not surprising, as bacteria are known to vary based on differences in temperature and moisture, and there may also be seasonal differences in what cattle are fed or how they’re housed. But this seasonal variation might be something for dairy processors to keep in mind. “It would be interesting to see if there is some seasonality to any product issues or defects they’re having,” says Marco.

    Marco and her colleagues also sampled raw milk in large-volume storage silos, where it is temporarily kept before being processed. “It seemed like there were two defined groups of bacterial communities among the five silos we sampled,” says Kable. The microbiome in some silos was drastically different from that in the tanker trucks, while in others it was fairly similar. “It seems like there may be an intrinsic community in some silos despite robust cleaning protocols,” says Kable. “Maybe that’s a good first place for the dairy community to look when looking at sporadic spoilage,” she says.

    Marco and her colleagues are currently working on analyzing the microbiome at later stages of milk processing, including in the final processed product. “It goes back to the idea of what we want to do, which is to identify milk as it moves through the processor, and identify early on if this milk has a higher likelihood of bacteria that would cause product defects,” she says. “This is just the tip of the iceberg.”

    References

    1. Quigley L., O’Sullivan O., Beresford T.P., Ross R.P., Fitzgerald G.F., Cotter P.D. High-throughput sequencing for detection of subpopulations of bacteria not previously associated with artisanal cheeses. Appl Environ Microbiol. 2012 Aug;78(16):5717-23. doi: 10.1128/AEM.00918-12.
    2. Vacheyrou M., Normand A.C., Guyot P., Cassagne C., Piarroux R., Bouton Y. Cultivable microbial communities in raw cow milk and potential transfers from stables of sixteen French farms. Int J Food Microbiol. 2011 Apr;146(3):253-62. doi: 10.1016/j.ijfoodmicro.2011.02.033.
    3. Pantoja J.C, Reinemann D.J., Ruegg P.L. Associations among milk quality indicators in raw bulk milk. J Dairy Sci. 2009 Oct;92(10):4978-87. doi: 10.3168/jds.2009-2329.
    4. Pantoja J.C, Reinemann D.J, Ruegg P.L. Factors associated with coliform count in unpasteurized bulk milk. J Dairy Sci. 2011 Jun;94(6):2680-91. doi: 10.3168/jds.2010-3721.
    5. Kable M.E., Srisengfa Y., Laird M., Zaragoza J., McLeod J., Heidenreich J., Marco M.L. The core and seasonal microbiota of raw bovine milk in tanker trucks and the impact of transfer to a milk processing facility. mBio. 2016 Aug;7(4):e00836-16. doi: 10.1128/mBio.00836-16.
    6. Klijn N., Nieuwenhof F.F., Hoolwerf J.D., van der Waals C.B., Weerkamp A.H. Identification of Clostridium tyrobutyricum as the causative agent of late blowing in cheese by species-specific PCR amplification. Appl Environ Microbiol. 1995 Aug;61(8):2919-24.

    Human Milk Makes People Smarter at Age 30

    • Studies claiming a link between breastfeeding and intelligence rarely test whether IQ differences persist into adulthood.
    • Research from Brazil finds a difference of four IQ points between 30-year-olds who received human milk for at least their first year of life and people of the same age who received it for less than a month.
    • This difference was associated with a substantially higher monthly income among those breastfed for a longer time.
    • The study is important because it was not confounded by the developed-world tendency of socioeconomically advantaged women to breastfeed for longer.

    Experts generally agree that consuming human milk as opposed to formula during infancy has a beneficial effect on brain development, and consequently, a beneficial effect on intelligence. Although there are swathes of studies on this topic, often the methods available to researchers are criticized—and imperfect methods make drawing firm conclusions risky. Furthermore, almost all studies in this field test intelligence during childhood and teenage years only, even though we know that cognitive functions continue to develop well into adulthood. Without data from older study participants, the field cannot be certain that people who do not receive human milk in early life don’t catch up on cognitive development later.

    For the above-cited reasons, a recently published paper about a population in Brazil is especially rare and intriguing [1]. First, the researchers studied the intellectual performance of people who, on average, had already hit the age of 30 years. This is even older than the participants in a 2002 Danish study of human milk consumption during infancy and subsequent IQ and had a mean participant age of 27 years [2]. The Brazilian study also had a much bigger sample size.

    Second—and in contrast to the Danish study—the work from Brazil is immune to a methodological problem that plagues all research on this topic from developed countries. In Denmark, as in the United States, wealthier women tend to breastfeed for longer, and also provide more cognitive stimulation to their offspring. Unless such socioeconomic differences can be accurately measured and properly included as potential confounders in statistical models, studies conducted in developed countries tend to overestimate the influence of human milk on IQ. But in Brazil, breastfeeding showed no social patterning (by maternal education nor family income), at least during the years when the study participants were infants.

    The Brazilian participants were all born in 1982, in one of the five maternity hospitals in Pelotas—a city in the very south of the country. Back then, academics visited these hospitals on a daily basis and asked new mothers if they (and their babies) wanted to be involved in the research. Less than 1% said no, meaning that almost everyone born in Pelotas at that time was enrolled.

    In 2012, the two main universities in Pelotas started to collaborate to re-assess the original participants’ IQ. Researchers evaluated almost three-and-a-half thousand 30-year-old individuals for whom breastfeeding information was available, which was equivalent to 68% of those enrolled as babies back in 1982. Crucially, this 68% was representative of the full sample by the vast majority of measures—with the exception that those tested at 30 years of age were more likely to be female, which is probably because men in Brazil are more likely to die young. Four psychologists who did not know the participants’ breastfeeding history conducted a battery of IQ tests, and also recorded each participant’s number of completed years of schooling and their income for the previous month.

    Breastfeeding was very common in the whole cohort, so the researchers focused on the period of time for which it continued. Participants who were breastfed for a year or more had an IQ about four points higher than those who were breastfed for less than a month. The differences didn’t end there—and because breastfeeding was not linked to socioeconomic status, the additional differences are more likely to be directly due to IQ levels than in studies from richer countries. Participants who were breastfed for more than a year had almost a year’s more education at age 30 than those breastfed for less than a month—and their income was about a third higher. To drill down on this point, the researchers used a technique called mediation analysis, which showed that adult IQ levels accounted for 72% of the effect of breastfeeding on income. (In other words, other differences—perhaps physical health benefits afforded by human milk consumption—played a much smaller role.)

    That is fairly convincing evidence for the link between human milk consumption and intelligence. The conclusions are put into context in a review of the field by two researchers involved in the study, Bernardo Horta and Cesar Victora, of the Federal University of Pelotas, which was published by the World Health Organization in 2013 [3]. In this report, Horta and Victora conducted a pooled analysis of all studies in the field that met various criteria for methodological quality. Their pooled analysis included a well-known study by Der et al. [4] that had claimed that the IQ benefits of breastfeeding are, in fact, the result of differences in cognitive stimulation growing up, and of maternal (i.e. inherited) IQ.

    Overall, Horta and Victora concluded that maternal IQ is an important confounder, but when taken into account across studies, it only alters the estimate of higher IQ performance by breastfed individuals slightly. Their pooled analysis of all studies on the topic suggests that breastfeeding adds 3.5 IQ points on average; the same analysis on only the studies that statistically removed the influence of maternal IQ suggests that breastfeeding adds 2.19 IQ points—slightly less, but still a gain that could perhaps make the marginal difference between getting the grade you need for a job or higher education course, rather than the grade below.

    Many participants of the original Pelotas study will today have gone on to have kids of their own. The lifestyle that these kids enjoy now may in part due to breastfeeding by their grandmothers.

    References

    1. Victora, C. G., Horta, B. L., de Mola, C. L., Quevedo, L., Pinheiro, R. T., Gigante, D .P. et al. 2015. Association between breastfeeding and intelligence, educational attainment, and income at 30 years of age: a prospective birth cohort study from Brazil. The Lancet 3(4), e199-e205.
    2. Mortensen, E. L., Michaelsen, K. F., Sanders, S. A., & Reinisch, J. M. 2002. The association between breastfeeding and adult intelligence. JAMA 287 (18), 2365-2946.
    3. Horta, B. L. & Victora, C. G. 2013. Long-term effects of breastfeeding. A systematic review. The World Health Organization. ISBN 978 92 4 150530 7.
    4. Der, G., Batty, G. D., & Deary, I. J. 2006. Effect of breastfeeding on intelligence in children: prospective study, sibling pairs analysis, and meta-analysis. BMJ, 333(7575), 945.

    The First Farmers: Where Did They Come From and Where Did They Go?

    • Agriculture began in the Fertile Crescent more than 11,000 years ago.
    • DNA samples from ancient farmers reveal their relationship to present day humans.
    • The first farmers made an enormous genetic contribution to diverse European, Asian, and African populations.
    • Domestication of cattle and the subsequent advent of cow’s milk as a source of nutrition was integral to the growth and expansion of human societies.

    Farming was a transformational technology that began the expansion of human populations and created settlements leading to the emergence of civilization. The origin of farming can be traced to the region known as the Fertile Crescent, which covered the area from modern Egypt around the eastern Mediterranean to Anatolia, the southern Caucasus mountains in the north, and the Euphrates and Tigris valleys in the east. Archaeologists have discovered evidence of early crop production from before 11,000 years ago and have traced the spread of agriculture in all directions from this region. One of the remaining questions is whether local hunter-gatherer populations across Europe and southern Asia learned about farming from afar and began their own farming culture, or whether Neolithic farmers migrated and brought agriculture and settlement with them. So, who were these ancient farmers, where did they come from, and where did their descendants emigrate?

    The advent of modern genomics and an increasing number of fully sequenced animal and plant genomes have given rise to the field of paleogenomics, which has provided new insights into the genetics of human populations. Detailed analysis of ancient DNA is providing clarity on the genetic lineages of prehistoric humans and identifying patterns of migration. Some recent research analyzed DNA from Neolithic farmers and defined the origins and spread of agriculture in ancient times [1-3].

    The introduction of agriculture had an enormous social and genetic impact. Farming led directly to a shift from nomadic to sedentary populations, and eventually gave rise to early civilizations where food security enabled social activities that were free from the daily imperative to find food. Population expansion and the demand for resources provided the most likely driver for migration, but determining whether the spread of agricultural technology was coincident with the migratory patterns has been a difficult question to address. Did the idea of farming, and/ or the technological knowledge underlying Neolithic agriculture, propagate human expansion or was it the migration of farmers? What happened when migratory farmers met hunter-gatherer populations? They possibly pushed out hunter-gathers, or mixed with the local people, or did hunter-gatherers adopt the technology and form their own settlements ahead of the migrating farmer peoples? These are the issues where paleogenomics is providing new insights.

    Ancient DNA is the term used to describe samples of DNA derived from prehistoric sites. Scientists have developed the techniques to painstakingly extract very small amounts of DNA from the most incredible sources, such as the inner ear of a human buried more than 10,000 years before the present (B.P.). Preserving these precious specimens and maintaining their purity, in an environment that is potentially contaminated with remnants from microbes, plants, animals, and even other humans, is a highly specialized skill. These scientists are truly the sleuths of the DNA research world.

    Archaeological evidence provides a picture of the Neolithic farmer who cultivated wheat or grass, fed grain to livestock and harvested manure to fertilize crop production [4]. From some of these archaeological sites, ancient DNA has been isolated from the remnants of individual farmers. Just six samples, from an area that straddled the Aegean Sea between Greece and Turkey established a continuous genetic relationship between the populations and linked the earliest European farmers directly to those of the Near East [2]. Paleogeneticists were able to do this because of the power in having genomic sequence data from modern populations to compare with the data obtained from ancient DNA from specific regions. Using a similar approach, Brousaki et al. [1] compared DNA from Neolithic farmers found in the Zagros region of Iran with DNA of modern-day populations, and they found that there was a distinct migration from that region towards the east. There was no evidence that these farmers contributed to the European spread of farming, but there was very strong evidence that they were ancestors of populations living in modern-day Pakistan and Afghanistan [1]. A third study isolated ancient DNA samples from 44 individuals that lived in different sites from the Southern Levant (in the region of modern day Israel and Jordan) to the Zagros Mountains, and ranged in age from about 14,000–3,400 B.P. [3]. They merged these data with existing data to generate information on 238 ancient people and compared it to the data with that from over 2,500 modern day individuals. They found that the farmers from the northern site were distinct from those in the south, and showed that their ancestors were the hunter-gather people of their respective regions. When comparing the DNA from ancient peoples with that of modern populations, they concluded that there were distinct migratory patterns to the north and east from the Zagros region towards Europe from Anatolia, and towards East Africa from the Southern Levant.

    Cultivation of grasses and crops were the first signs of farming, but domestication of animals was also evident in the early Neolithic period. The mobility of farming animals provided an impetus to move in order to expand or secure pastures and had the capacity to enable migration by providing a source of food. This was evident in the spread from the Zagros region towards the steppes where farming of animals flourished. The earliest animals to be domesticated were probably goats and sheep, perhaps as long ago as 11000 B.P. Cattle (Bos taurus) were first domesticated in the Taurus mountain region of Anatolia around the same period [5,6]. Milk use for nutritional purposes was probably coincident with the domestication of the cattle [7]. The use of milk to generate dairy products, particularly cheese, was evident in the Near East from at least 9000 B.P. [7]. This technology spread with evidence in Eastern Europe 8000 B.P., Britain in 6000 B.P., and 7000 B.P. in North Africa [8,9]. By 5000 B.P., farming had reached the furthest western point in Europe—Ireland—and recent evidence suggests even the early farmers of Ireland had a direct genetic link to the farmers of the Near East [10].

    The agricultural revolution of the Neolithic period was the most significant advance in human pre-history, and set the direction for the genetic diversity and population expansion of Europe. Cattle herding and the early adoption of dairy were an integral part of its success. The value of milk in the human diet is emphasized by the spread of lactase persistence, which expanded after beneficial mutations arose in a central European population around 7,500 B.P. [11–13]. The rapid spread and relatively high frequency of the allelic variants in European populations suggest an evolutionary advantage from the capacity to digest lactose in adulthood. The most plausible explanation is the high nutritional value of milk and its potential contribution to successful survival during conditions that may have otherwise led to starvation.

    References

    1. Broushaki F., Thomas M.G., Link V., Lopez S., van Dorp L., et al. 2016. Early Neolithic genomes from the eastern Fertile Crescent. Science 353: 499-503.
    2. Hofmanova Z., Kreutzer S., Hellenthal G., Sell C., Diekmann Y., et al. 2016. Early farmers from across Europe directly descended from Neolithic Aegeans. Proc Natl Acad Sci U S A 113: 6886-6891.
    3. Lazaridis I., Nadel D., Rollefson G., Merrett D.C., Rohland N., et al. 2016. Genomic insights into the origin of farming in the ancient Near East. Nature 536: 419-424.
    4. Bogaard A., Fraser R., Heaton T.H., Wallace M., Vaiglova P., et al. 2013. Crop manuring and intensive land management by Europe’s first farmers. Proc Natl Acad Sci U S A 110: 12589-12594.
    5. Bollongino R., Burger J., Powell A., Mashkour M., Vigne J.D., et al. 2012. Modern taurine cattle descended from small number of near-eastern founders. Mol Biol Evol 29: 2101-2104.
    6. Scheu A., Powell A., Bollongino R., Vigne J.D., Tresset A., et al. 2015. The genetic prehistory of domesticated cattle from their origin to the spread across Europe. BMC Genet 16: 54.
    7. Evershed R.P., Payne S., Sherratt A.G., Copley M.S., Coolidge J., et al. 2008. Earliest date for milk use in the Near East and southeastern Europe linked to cattle herding. Nature 455: 528-531.
    8. Dunne J., Evershed R.P., Salque M., Cramp L., Bruni S., et al. 2012. First dairying in green Saharan Africa in the fifth millennium BC. Nature 486: 390-394.
    9. Salque M., Bogucki P.I., Pyzel J., Sobkowiak-Tabaka I., Grygiel R., et al. 2013. Earliest evidence for cheese making in the sixth millennium BC in northern Europe. Nature 493: 522-525.
    10. Cassidy L.M., Martiniano R., Murphy E.M., Teasdale M.D., Mallory J., et al. 2016. Neolithic and Bronze Age migration to Ireland and establishment of the insular Atlantic genome. Proc Natl Acad Sci U S A 113: 368-373.

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