Baseline Knowledge the “Omes” Contents

Baseline Knowledge The “omes” Contents

1 Microbiota 1 1.1 Introduction ...... 1 1.2 Microbiota by host ...... 2 1.2.1 Humans ...... 2 1.2.2 Non-human animals ...... 3 1.2.3 Plants ...... 4 1.3 Immune system ...... 4 1.4 Co-evolution of microbiota ...... 4 1.5 Research methods ...... 5 1.5.1 Targeted amplicon sequencing ...... 5 1.5.2 Metagenomic sequencing ...... 5 1.5.3 RNA and protein-based approaches ...... 6 1.6 Projects ...... 6 1.7 Privacy ...... 6 1.8 See also ...... 6 1.9 Notes ...... 7 1.10 References ...... 7 1.11 External links ...... 10

2 Human microbiota 11 2.1 Terminology ...... 11 2.2 Relative numbers ...... 12 2.3 Study ...... 12 2.4 Types ...... 13 2.4.1 Bacteria ...... 13 2.4.2 Archaea ...... 13 2.4.3 Fungi ...... 14 2.4.4 Viruses ...... 14 2.5 Anatomical areas ...... 14 2.5.1 Skin ...... 14 2.5.2 Conjunctiva ...... 14 2.5.3 Gut ...... 14 2.5.4 Vagina ...... 15

i ii CONTENTS

2.5.5 Placenta ...... 16 2.5.6 Uterus ...... 16 2.5.7 Oral cavity ...... 16 2.5.8 Lung ...... 16 2.6 Disease ...... 17 2.6.1 Cancer ...... 17 2.7 Research ...... 18 2.8 Environmental health ...... 18 2.9 See also ...... 18 2.10 References ...... 18 2.11 External links ...... 22

3 Genome 23 3.1 Origin of term ...... 23 3.2 Overview ...... 23 3.3 Sequencing and mapping ...... 24 3.4 Genome compositions ...... 24 3.4.1 Genome size ...... 25 3.4.2 Proportion of non-repetitive DNA ...... 25 3.4.3 Proportion of repetitive DNA ...... 25 3.5 Genome evolution ...... 26 3.6 See also ...... 26 3.7 References ...... 27 3.8 Further reading ...... 30 3.9 External links ...... 31

4 Connectome 32 4.1 Origin and usage of the term “connectome” ...... 32 4.2 The connectome at multiple scales ...... 33 4.3 Mapping the connectome at the cellular level ...... 34 4.4 Mapping the connectome at the macro scale ...... 34 4.4.1 Recent advances in connectivity mapping ...... 35 4.4.2 Primary challenge for macroscale connectomics: determining parcellations of the brain ... 35 4.5 Mapping functional connectivity to complement anatomical connectivity ...... 35 4.6 The connectome as a network or graph ...... 35 4.7 See also ...... 36 4.8 References ...... 37 4.9 External links ...... 39

5 Exposome 40 5.1 Background ...... 40 5.2 Research initiatives ...... 40 CONTENTS iii

5.3 Proposed Human Exposome Project (HEP) ...... 41 5.4 Related ﬁelds ...... 41 5.5 See also ...... 41 5.6 References ...... 41

6 Built environment 43 6.1 History ...... 43 6.2 Modern built environment ...... 43 6.3 Public health ...... 43 6.4 Landscape architecture ...... 44 6.5 See also ...... 44 6.6 References ...... 45 6.7 Further reading ...... 45 6.8 External links ...... 46

7 Microbiomes of the built environment 47 7.1 Types of Built Environments For Which Microbiomes Have Been Studied ...... 47 7.2 Results from Studies of the Microbiomes of the Built Environment ...... 48 7.2.1 General Biogeography ...... 48 7.2.2 Human Health and Microbiomes of the Built Environment ...... 48 7.2.3 Components of the Built Environment that Likely Impact Microbiomes ...... 48 7.2.4 Impact of Microbiomes on the Built Environment ...... 48 7.2.5 Possible Uses in Forensics ...... 49 7.2.6 Odor and Microbes in the Built Environment ...... 49 7.3 Methods Used ...... 49 7.4 See also ...... 49 7.5 External links ...... 49 7.5.1 Examples of projects ...... 49 7.5.2 Videos ...... 49 7.5.3 Journals that have a lot of publications on this topic ...... 49 7.5.4 Societies and Organizations ...... 50 7.5.5 News and related coverage ...... 50 7.6 References ...... 50 7.7 Text and image sources, contributors, and licenses ...... 54 7.7.1 Text ...... 54 7.7.2 Images ...... 55 7.7.3 Content license ...... 56 Chapter 1

Microbiota

For other uses, see Microbiota (disambiguation). 1.1 Introduction A microbiota is “the ecological community of Life timeline view • discuss • −4500 — – −4000 — – −3500 — – −3000 — – −2500 — – −2000 — – −1500 — – −1000 — – −500 — – 0 — water Single-celled life photosynthesis Depiction of the human skin and bacteria that predominate Eukaryotes Multicellular life commensal, symbiotic and pathogenic microorganisms Land life that literally share our body space”.[1][2] Joshua Leder- Dinosaurs berg coined the term, emphasising the importance of Mammals microorganisms inhabiting the human body in health Flowers and disease. Many scientiﬁc articles distinguish mi- ← crobiome and microbiota to describe either the collec- Earliest Earth (−4540) tive genomes of the microorganisms that reside in an ← environmental niche or the microorganisms themselves, [3][4][5] Earliest water respectively. However, by the original deﬁnitions, ← these terms are largely synonymous. Earliest life The microbes being discussed generally do not cause dis- (−4100) ease unless they grow abnormally nonpathogenic organ- ← isms; they exist in harmony and symbiotically with their LHB meteorites hosts.[6] The microbiome and host may have emerged as ← a unit by the process of integration.[7] Earliest oxygen

1 2 CHAPTER 1. MICROBIOTA

← but have evolved in the context of complex communities. Atmospheric oxygen A number of advances have driven a change in the per- ← ception of microbiomes, including: Oxygen Crisis ← • the ability to perform genomic and gene expres- Earliest sexual reproduction sion analyses of single cells and even of entire ← microbial communities in the new disciplines of Cambrian explosion metagenomics and metatranscriptomics ← Earliest humans • massive databases making this information accessi- ble to researchers across multiple disciplines

P • methods of mathematical analysis that help re- h searchers to make sense of complex data sets a n Increasingly, biologists have come to appreciate that mi- r crobes make up an important part of an organism’s phe- z notype, far beyond the occasional symbiotic case study.[8] c Pierre-Joseph van Beneden (1809-1894), a Belgian professor at the University of Louvain, developed the con- P cept of commensalism during the nineteenth century. In r his 1875 publication Animal Parasites and Messmates, o Van Beneden presented 264 examples of commensalism. t His conception was widely accepted by his contempo- e raries and commensalism has continued to be used as a r concept right up to the present day: microbiome is clearly o linked to commensalism.[9] z o i c 1.2 Microbiota by host

A There is a strengthening consensus among evolutionary r biologists that one should not separate an organism’s c genes from the context of its resident microbes. h e a 1.2.1 Humans n Main article: Human microbiome H a The human microbiota includes bacteria, fungi, and d archaea. Micro-animals which live on the human body e are excluded. The human microbiome refers to their a genomes.[10] n Humans are colonized by many microorganisms; the traditional estimate was that humans live with ten times Axis scale: millions of years. more non-human cells than human cells; more recent Also see: Human timeline and Nature timeline estimates have lowered that to 3:1 and even to approximately the same number; all the numbers are All plants and animals, from protists to humans, live in estimates.[11][12][13][14] Regardless of the exact number, close association with microbial organisms (see for exam- the microbiota that colonize humans have not merely ple the human microbiome). Up until relatively recently, a commensal (a non-harmful coexistence), but rather a however, biologists have defined the interactions of plants mutualistic relationship with their human hosts.[10]:700[15] and animals with the microbial world mostly in the con- Some of these organisms perform tasks that are known text of disease states and of a relatively small number of to be useful for the human host; for most, the role is not symbiotic case studies. Organisms do not live in isolation, well understood. Those that are expected to be present, 1.2. MICROBIOTA BY HOST 3 and that under normal circumstances do not cause dis- • Leaf-cutter ants form huge underground colonies ease, are deemed normal flora or normal microbiota.[10] with millions of workers, each colony harvesting The Human Microbiome Project took on the project of hundreds of kilograms of leaves each year. Unable sequencing the genome of the human microbiota, focus- to digest the cellulose in the leaves directly, they ing particularly on the microbiota that normally inhabit maintain fungus gardens that are the colony’s pri- the skin, mouth, nose, digestive tract, and vagina.[10] It mary food source. The fungus itself does not digest reached a milestone in 2012 when it published initial cellulose. Instead, a microbial community contain- results.[16] ing a diversity of bacteria is responsible for cellulose digestion. Analysis of the microbial population’s genomic content by community metagenome sequenc- 1.2.2 Non-human animals ing methods revealed the presence of many genes with a role in cellulose digestion. This microbiome’s predicted carbohydrate-degrading enzyme profile is similar to that of the bovine rumen, but the species composition is almost entirely different.[20]

• Mice are the most used models for human disease. As more and more diseases are linked to dysfunc- tional microbiomes, mice have become the most studied organism in this regard. Mostly it is the gut microbiota that have been studied in relation to allergic airway disease, obesity, gastrointestinal diseases and diabetes. Intriguingly, recent work has shown that perinatal shifting of microbiota through A chytrid-infected frog (see Chytridiomycosis) administration of low dose antibiotics can have long- lasting effects on future susceptibility to allergic airway disease.[21][22] These studies showed a re- • A massive, worldwide decline in amphibian pop- markable link between the frequency of certain sub- ulations has been well-publicised. Habitat loss sets of microbes and disease severity. In aggregate and over-exploitation account for part of the prob- these studies suggest that the presence of specific lem, but many other processes seem to be at microbes, early in postnatal life, play an instructive work. The spread of the virulent fungal disease [17] role in the development of future immune responses. chytridiomycosis represents an enigma. The abil- Mechanistically, a recent study done on gnotobiotic ity of some species to coexist with the causative mice described a method in which certain strains agent Batrachochytrium dendrobatidis appears to be of gut bacteria were found to transmit a particu- due to the expression of antimicrobial skin peptides lar phenotype to recipient germ-free mice, iden- along with the presence of symbiotic microbes that tifying an unanticipated range of bacterial strains benefit the host by resisting pathogen colonization that promoted accumulation of colonic regulatory T or inhibiting their growth while being themselves re- cells, as well as strains that modulated mouse adi- sistant to high concentrations of antimicrobial skin [18] posity and cecal metabolite concentrations. Another peptides. study showed that when adult germ-free mice were colonized with the gut flora of obese mice, there • The bovine rumen harbors a complex microbiome was a dramatic weight increase and an observed in- that converts plant cell wall biomass into proteins, creased metabolism of monosaccharides and short- short chain fatty acids, and gases. Multiple species chain fatty acids. Looking at the gut flora compo- are involved in this conversion. Traditional methods sitions between normal and obese mice, obese mice of characterizing the microbial population, based on had less Bacteroidetes than Firmicutes in abundance culture analysis, missed many of the participants in gut flora and it is hypothesized that the micro- in this process. Comparative metagenomic stud- biota of obese mice are more efficient at extracting ies yielded the surprising result that individual steer energy from food.[23] This combinatorial approach had markedly different community structures, pre- enables a systems-level understanding of microbial dicted phenotype, and metabolic potentials,[19] even contributions to human biology.[24] But also other though they were fed identical diets, were housed to- mucoide tissues as lung and vagina have been stud- gether, and were apparently functionally identical in ied in relation to diseases such as asthma, allergy and their utilization of plant cell wall resources. vaginosis [25] 4 CHAPTER 1. MICROBIOTA

1.3 Immune system

The symbiotic relationship between a mammalian host and its microbiota has a significant impact on shaping the host’s immune system.[33] In many animals, the immune system and microbiota engage in “cross-talk”, exchanging chemical signals. This allows the immune system to recognize the types of bacteria that are harmful to the host and combat them, while allowing the helpful bacteria to carry out their functions; in turn, the microbiota influence immune reactivity and targeting.[34] Bacteria can be transferred from mother to child through direct contact Light micrograph of a cross section of a coralloid root of a cycad, and after birth, or through indirect contact through eggs, showing the layer that hosts symbiotic cyanobacteria coprophagy, and several other pathways.[35] As the infant microbiome is established, commensal bacteria quickly populate the gut, prompting a range of immune responses 1.2.3 Plants and “programming” the immune system with long-lasting effects.[34] This early colonization helps to establish the • Plants exhibit a broad range of relationships symbiotic microbiome inside the animal host early in its with symbiotic microorganisms, ranging from life.[33] The bacteria are also able to stimulate lymphoid parasitism, in which the association is disadvan- tissue associated with the gut mucosa. This enables the tageous to the host organism, to mutualism, in tissue to produce antibodies for pathogens that may enter which the association is beneficial to both, to the gut. commensalism, in which the symbiont benefits It has been found that bacteria may also play a role in the while the host is not affected. Exchange of nu- activation of TLRs (toll-like receptors) in the intestines. trients between symbiotic partners is an important TLRs are a type of PRR (pattern recognition receptor) part of the relationship: it may be bidirectional or used by host cells to help repair damage and recognize unidirectional, and it may be context dependent. dangers to the host. This could be important in immune The strategies for nutrient exchange are highly di- tolerance and autoimmune diseases. Pathogens could verse. Oomycetes and fungi have, through conver- influence this symbiotic coexistence leading to immune gent evolution, developed similar morphology and dysregulation and susceptibility to diseases. This could occupy similar ecological niches. They develop hy- provide new direction for managing immunological and phae, filamentous structures that penetrate the host metabolic diseases.[36] cell. In those cases where the association is mutualistic, the plant often exchanges hexose sugars for inorganic phosphate from the fungal symbiont. It is speculated that such associations, which are very 1.4 Co-evolution of microbiota ancient, may have aided plants when they first colonized land.[26][27] Main article: Hologenome theory of evolution Organisms evolve within eco-systems so that the change • A huge range of bacterial symbionts colonize plants. Many of these are pathogenic, but others known as plant-growth promoting bacteria (PGPB) provide the host with essential services such as nitrogen fixation, solubilization of minerals such as phos- phorus, synthesis of plant hormones, direct en- hancement of mineral uptake, and protection from pathogens.[28][29] PGPBs may protect plants from pathogens by competing with the pathogen for an ecological niche or a substrate, producing inhibitory allelochemicals, or inducing systemic resistance in host plants to the pathogen[30]

• Plants are attractive hosts for microorganisms since they provide a variety of nutrients. Microorganisms on plants can be epiphytes (found on the plants) or Bleached branching coral (foreground) and normal branching endophytes (found inside plant tissue).[31][32] coral (background). Keppel Islands, Great Barrier Reef 1.5. RESEARCH METHODS 5

of one organism affects the change of others. Co- 1.5 Research methods evolution (also called “hologenome theory”) proposes that an object of natural selection is not the individual organ- 1.5.1 Targeted amplicon sequencing ism, but the organism together with its associated organisms, including its microbial communities. Targeted amplicon sequencing relies on having some ex- Coral reefs. The hologenome theory originated in stud- pectations about the composition of the community that ies on coral reefs. Coral reefs are the largest structures is being studied. In target amplicon sequencing a phy- created by living organisms, and contain abundant and logenetically informative marker is targeted for sequenc- highly complex microbial communities. Over the past ing. Such a marker should be present in ideally all the several decades, major declines in coral populations have expected organisms. It should also evolve in such a way occurred. Climate change, water pollution and over- that it is conserved enough that primers can target genes fishing are three stress factors that have been described from a wide range of organisms while evolving quickly as leading to disease susceptibility. Over twenty dif- enough to allow for finer resolution at the taxonomic level. ferent coral diseases have been described, but of these, A common marker for human microbiome studies is the only a handful have had their causative agents isolated gene for bacterial 16S rRNA (i.e. “16S rDNA”, the and characterized. Coral bleaching is the most serious sequence of DNA which encodes the ribosomal RNA of these diseases. In the Mediterranean Sea, the bleach- molecule).[41] Since ribosomes are present in all living or- ing of Oculina patagonica was first described in 1994 and ganisms, using 16S rDNA allows for DNA to be ampli- shortly determined to be due to infection by Vibrio shiloi. fied from many more organisms than if another marker From 1994 to 2002, bacterial bleaching of O. patagonica were used. The 16S rDNA gene contains both slowly occurred every summer in the eastern Mediterranean. evolving regions and fast evolving regions; the former can Surprisingly, however, after 2003, O. patagonica in the be used to design broad primers while the latter allow eastern Mediterranean has been resistant to V. shiloi infor finer taxonomic distinction. However, species-level fection, although other diseases still cause bleaching. The resolution is not typically possible using the 16S rDNA. surprise stems from the knowledge that corals are long Primer selection is an important step, as anything that lived, with lifespans on the order of decades,[37] and do cannot be targeted by the primer will not be amplified not have adaptive immune systems. Their innate immune and thus will not be detected. Different sets of primers systems do not produce antibodies, and they should seem- have been shown to amplify different taxonomic groups ingly not be able to respond to new challenges except over due to sequence variation. evolutionary time scales. The puzzle of how corals man- Targeted studies of eukaryotic and viral communities are aged to acquire resistance to a specific pathogen led Eu- limited[42] and subject to the challenge of excluding host gene Rosenberg and Ilana Zilber-Rosenberg to propose DNA from amplification and the reduced eukaryotic and the Coral Probiotic Hypothesis. This hypothesis pro- viral biomass in the human microbiome.[43] poses that a dynamic relationship exists between corals and their symbiotic microbial communities. By altering After the amplicons are sequenced, molecular phyloge- its composition, this holobiont can adapt to changing en- netic methods are used to infer the composition of the vironmental conditions far more rapidly than by genetic microbial community. This is done by clustering the am- mutation and selection alone. Extrapolating this hypoth- plicons into operational taxonomic units (OTUs) and in- esis of adaptation and evolution to other organisms, inferring phylogenetic relationships between the sequences. cluding higher plants and animals, led to the proposal of Due to the complexity of the data, distance measures such the Hologenome Theory of Evolution.[38] as UniFrac distances are usually defined between microbiome samples, and downstream multivariate methods The hologenome theory is still being debated.[39] A major are carried out on the distance matrices. An important criticism has been the claim that V. shiloi was misidenti- point is that the scale of data is extensive, and further fied as the causative agent of coral bleaching, and that approaches must be taken to identify patterns from the its presence in bleached O. patagonica was simply that available information. Tools used to analyze the data in- of opportunistic colonization.[40] If this is true, the basic clude VAMPS,[44] QIIME[45] and mothur.[46] observation leading to the theory would be invalid. Nev- ertheless, the theory has gained significant popularity as a way of explaining rapid changes in adaptation that cannot 1.5.2 Metagenomic sequencing otherwise be explained by traditional mechanisms of natural selection. For those who accept the hologenome the- Main article: Metagenomics ory, the holobiont has become the principal unit of natural selection. On the other hand, it has been stated that the holobiont is the result of other step of integration that it is Metagenomics is also used extensively for studying mi- [47][48][49] also observed at the cell (symbiogenesis, endosymbiosis) crobial communities. In metagenomic sequenc- and genomic levels.[7] ing, DNA is recovered directly from environmental samples in an untargeted manner with the goal of obtain- ing an unbiased sample from all genes of all members 6 CHAPTER 1. MICROBIOTA of the community. Recent studies use shotgun Sanger characterize environments and ecosystems by microbial sequencing or pyrosequencing to recover the sequences composition and interaction. Using these data, new eco- of the reads.[50] The reads can then be assembled into logical and evolutionary theories can be proposed and contigs. To determine the phylogenetic identity of a se- tested.[61] quence, it is compared to available full genome sequences The Brazilian Microbiome Project (BMP) aims to assem- using methods such as BLAST. One drawback of this ap- ble a Brazilian Microbiome Consortium/Database. At proach is that many members of microbial communities [41] present, many metagenomic projects underway in Brazil do not have a representative sequenced genome. are widely known. It’s goal is to co-ordinate and standard- Despite the fact that metagenomics is limited by the avail- ize these, together with future projects. This is the first ability of reference sequences, one significant advantage attempt to collect and collate information about Brazilian of metagenomics over targeted amplicon sequencing is microbial genetic and functional diversity in a systematic that metagenomics data can elucidate the functional po- and holistic manner. New sequence data have been gener- tential of the community DNA.[51][52] Targeted gene sur- ated from samples collected in all Brazilian regions, how- veys cannot do this as they only reveal the phylogenetic ever the success of the BMP depends on a massive col- relationship between the same gene from different organ- laborative effort of both the Brazilian and international isms. Functional analysis is done by comparing the recov- scientific communities. ered sequences to databases of metagenomic annotations such as KEGG. The metabolic pathways that these genes are involved in can then be predicted with tools such as MG-RAST,[53] CAMERA[54] and IMG/M.[55] 1.7 Privacy

The DNA of the microbes that inhabit a person’s human 1.5.3 RNA and protein-based approaches body can uniquely identify the person. A risk to violating a person’s privacy may exist, if the person anonymously Metatranscriptomics studies have been performed to donated microbe DNA data, and the data could be used study the gene expression of microbial communities to identify the person and their medical condition, and if through methods such as the pyrosequencing of extracted the person’s identity were revealed.[62][63][64][65] RNA.[56] Structure based studies have also identiﬁed non-coding RNAs (ncRNAs) such as ribozymes from microbiota.[57] Metaproteomics is a new approach that studies the proteins expressed by microbiota, giving in- 1.8 See also sight into its functional potential.[58] • Anagenesis

1.6 Projects • Biome

• The Human Microbiome Project (HMP) was a United Human microbiome States National Institutes of Health initiative with the • goal of identifying and characterizing the microorgan- Human microbiome project isms which are found in association with both healthy and diseased humans (their microbial flora).[59] Launched in • Human virome 2008, it was a five-year project, best characterized as a feasibility study, with a total budget of $115 million. The • List of bacterial vaginosis microbiota ultimate goal of this and similar NIH-sponsored microbiome projects is to test how changes in the human mi- • Microbiota of the lower reproductive tract of women crobiome are associated with human health or disease.[59] • The Earth Microbiome Project (EMP) is an initiative to Metagenomics collect natural samples and analyze the microbial com- • munity around the globe. Microbes are highly abundant, Multigenomic organism diverse and have an important role in the ecological system. Yet as of 2010, it was estimated that the total global • Psychobiotic environmental DNA sequencing effort had produced less than 1 percent of the total DNA found in a liter of seawa- • Skin flora ter or a gram of soil,[60] and the specific interactions between microbes are largely unknown. The EMP aims to • Vaginal flora process as many as 200,000 samples in different biomes, generating a complete database of microbes on earth to • Vaginal microbiota in pregnancy 1.10. REFERENCES 7

1.9 Notes [13] Alison Abbott for Nature News. Jan 8 2016 Scientists bust myth that our bodies have more bacteria than human cells

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1.11 External links Chapter 2

Human microbiota

human hosts via the metabolites that they produce, like trimethylamine.[7][8] Certain microbiota perform tasks that are known to be useful for the human host; for most, the role is not well understood. Those that are expected to be present, and that under normal circumstances do not cause disease, are deemed normal ﬂora or normal microbiota.[1] The Human Microbiome Project took on the project of sequencing the genome of the human microbiota, focusing particularly on the microbiota that normally inhabit the skin, mouth, nose, digestive tract, and vagina.[1] It reached a milestone in 2012 when it published initial results.[9]

2.1 Terminology

Life timeline view • discuss • −4500 — – −4000 — Graphic depicting the human skin microbiota, with relative – prevalences of various classes of bacteria. −3500 — – −3000 — The human microbiota is the aggregate of – microorganisms, a microbiome that resides on or −2500 — within a number of tissues and bioﬂuids, including the – skin, mammary glands, placenta, seminal ﬂuid, uterus, −2000 — ovarian follicles, lung, saliva, oral mucosa, conjunctiva, – and gastrointestinal tracts. They include bacteria, fungi, −1500 — and archaea. Micro-animals which live on the human – body are excluded. The human microbiome refers to − [1] 1000 — their genomes. – Humans are colonized by many microorganisms; the tra- −500 — ditional estimate was that humans live with ten times – more non-human cells than human cells; more recent esti- 0 — mates have lowered that to 3:1 and even to approximately water the same number; all the numbers are estimates.[2][3][4][5] Single-celled Some microbiota that colonize humans have not merely life a commensal (a non-harmful coexistence), but rather a photosynthesis mutualistic relationship with their human hosts.[1]:700[6] Eukaryotes Conversely, some non-pathogenic microbiota can harm Multicellular

11 12 CHAPTER 2. HUMAN MICROBIOTA life d Land life e Dinosaurs a Mammals n Flowers ← Axis scale: millions of years. Earliest Earth (−4540) Also see: Human timeline and Nature timeline ← Earliest water ← Though widely known as flora or microflora, this is a Earliest life misnomer in technical terms, since the word root flora (−4100) pertains to plants, and biota refers to the total collection of ← organisms in a particular ecosystem. Recently, the more LHB meteorites appropriate term microbiota is applied, though its use has ← not eclipsed the entrenched use and recognition of flora Earliest oxygen with regard to bacteria and other microorganisms. Both ← terms are being used in different literature.[6] Atmospheric oxygen ← Oxygen Crisis 2.2 Relative numbers ← Earliest sexual reproduction ← As of 2014, it was often reported in popular media and Cambrian explosion in the scientific literature that there are about 10 times as ← many microbial cells in the human body than there are Earliest humans human cells; this figure was based on estimates that the human microbiome includes around 100 trillion bacterial cells and an adult human typically has around 10 trillion [2] P human cells. In 2014 the American Academy of Micro- h biology published an FAQ that emphasized that the num- a ber of microbial cells and the number of human cells are n both estimates, and noted that recent research had arrived r at a new estimate of the number of human cells at around z 37 trillion cells, meaning that the ratio of microbial to [2][3] c human cells is probably about 3:1. In 2016 another group published a new estimate of ratio as being roughly 1:1 (1.3:1, with “an uncertainty of 25% and a variation of P 53% over the population of standard 70 kg males”).[4][5] r o t e 2.3 Study r o Main article: Human Microbiome Project z The problem of elucidating the human microbiome is es- o sentially identifying the members of a microbial commu- i nity which includes bacteria, eukaryotes, and viruses.[10] c This is done primarily using DNA-based studies, though RNA, protein and metabolite based studies are also A performed.[10][11] DNA-based microbiome studies typi- r cally can be categorized as either targeted amplicon stud- c ies or more recently shotgun metagenomic studies. The h former focuses on specific known marker genes and is pri- e marily informative taxonomically, while the latter is an a entire metagenomic approach which can also be used to n study the functional potential of the community.[10] One of the challenges that is present in human microbiome H studies but not in other metagenomic studies is to avoid a including the host DNA in the study.[12] 2.4. TYPES 13

2.4 Types

2.4.1 Bacteria

Populations of microbes (such as bacteria and yeasts) inhabit the skin and mucosal surfaces in various parts of the body. Their role forms part of normal, healthy human physiology, however if microbe numbers grow beyond their typical ranges (often due to a compromised immune system) or if microbes populate (such as through poor hygiene or injury) areas of the body normally not colonized or sterile (such as the blood, or the lower respiratory tract, or the abdominal cavity), disease can result (causing, respectively, bacteremia/sepsis, pneumonia, and peritoni- tis). The Human Microbiome Project found that individuals host thousands of bacterial types, different body sites having their own distinctive communities. Skin and vagi- Flowchart illustrating how the human microbiome is studied on the DNA level. nal sites showed smaller diversity than the mouth and gut, these showing the greatest richness. The bacterial makeup for a given site on a body varies from person to person, not only in type, but also in abundance. Bacte- ria of the same species found throughout the mouth are of multiple subtypes, preferring to inhabit distinctly dif- Aside from simply elucidating the composition of the hu- ferent locations in the mouth. Even the enterotypes in man microbiome, one of the major questions involving the human gut, previously thought to be well-understood, the human microbiome is whether there is a “core”, that are from a broad spectrum of communities with blurred is, whether there is a subset of the community that is taxon boundaries.[17][18] [13][14] shared among most humans. If there is a core, then It is estimated that 500 to 1,000 species of bacte- it would be possible to associate certain community com- ria live in the human gut but belong to just a few positions with disease states, which is one of the goals of phyla: Firmicutes and Bacteroidetes dominate but there the Human Microbiome Project. It is known that the hu- are also Proteobacteria, Verrumicrobia, Actinobacteria, man microbiome is highly variable both within a single Fusobacteria and Cyanobacteria.[19] subject and between different individuals. For example, the gut microbiota of humans is markedly dissimilar be- A number of types of bacteria, such as Actinomyces vis- tween individuals, a phenomenon which is also observed cosus and A. naeslundii, live in the mouth, where they are in mice.[6] part of a sticky substance called plaque. If this is not re- moved by brushing, it hardens into calculus (also called On 13 June 2012, a major milestone of the Human Mi- tartar). The same bacteria also secrete acids that dissolve crobiome Project (HMP) was announced by the NIH di- tooth enamel, causing tooth decay. rector Francis Collins.[9] The announcement was accom- panied with a series of coordinated articles published in The vaginal microflora consist mostly of various Nature[15][16] and several journals in the Public Library lactobacillus species. It was long thought that the most of Science (PLoS) on the same day. By mapping the nor- common of these species was Lactobacillus acidophilus, mal microbial make-up of healthy humans using genome but it has later been shown that L. iners is in fact most sequencing techniques, the researchers of the HMP have common, followed by L. crispatus. Other lactobacilli created a reference database and the boundaries of nor- found in the vagina are L. jensenii, L. delbruekii and mal microbial variation in humans. From 242 healthy L. gasseri. Disturbance of the vaginal flora can lead U.S. volunteers, more than 5,000 samples were collected to infections such as bacterial vaginosis or candidiasis from tissues from 15 (men) to 18 (women) body sites (“yeast infection”). such as mouth, nose, skin, lower intestine (stool), and vagina. All the DNA, human and microbial, were an- alyzed with DNA sequencing machines. The microbial 2.4.2 Archaea genome data were extracted by identifying the bacterial specific ribosomal RNA, 16S rRNA. The researchers cal- Archaea are present in the human gut, but, in contrast culated that more than 10,000 microbial species occupy to the enormous variety of bacteria in this organ, the the human ecosystem and they have identified 81 – 99% numbers of archaeal species are much more limited.[20] of the genera. The dominant group are the methanogens, particularly 14 CHAPTER 2. HUMAN MICROBIOTA

Methanobrevibacter smithii and Methanosphaera stadt- The skin acts as a barrier to deter the invasion of manae.[21] However, colonization by methanogens is pathogenic microbes. The human skin contains microbes variable, and only about 50% of humans have easily de- that reside either in or on the skin and can be residen- tectable populations of these organisms.[22] tial or transient. Resident microorganism types vary in As of 2007, no clear examples of archaeal pathogens were relation to skin type on the human body. A majority of known,[23][24] although a relationship has been proposed microbes reside on superﬁcial cells on the skin or pre- between the presence of some methanogens and human fer to associate with glands. These glands such as oil periodontal disease.[25] or sweat glands provide the microbes with water, amino acids, and fatty acids. In addition, resident bacteria that associated with oil glands are often Gram-positive and [1] 2.4.3 Fungi can be pathogenic.

See also: Human virome

A small number of bacteria and fungi are normally Viruses, especially bacterial viruses (bacteriophages), present in the conjunctiva.[26][40] Classes of bacteria colonize various body sites. These colonized sites in- include Gram-positive cocci (e.g., Staphylococcus and clude the skin,[32] gut,[33] lungs,[34] and oral cavity.[35] Streptococcus) and Gram-negative rods and cocci (e.g., Virus communities have been associated with some Haemophilus and Neisseria) are present.[40] Fungal gen- diseases, and do not simply reflect the bacterial era include Candida, Aspergillus, and Penicillium.[26] The communities.[36][37][38] lachrymal glands continuously secrete, keeping the conjunctiva moist, while intermittent blinking lubricates the conjunctiva and washes away foreign material. Tears con- 2.5 Anatomical areas tain bactericides such as lysozyme, so that microorganisms have difficulty in surviving the lysozyme and settling Main article: List of human flora on the epithelial surfaces.

2.5.1 Skin

Main article: Skin ﬂora

A study of twenty skin sites on each of ten healthy humans found 205 identified genera in nineteen bacterial phyla, with most sequences assigned to four phyla: Actinobacteria (51.8%), Firmicutes (24.4%), Proteobacteria (16.5%), and Bacteroidetes (6.3%).[39] A large number of fungal genera are present on healthy hu- 2.5.3 Gut man skin, with some variability by region of the body; however, during pathological conditions, certain genera tend to dominate in the affected region.[26] For example, Malassezia is dominant in atopic dermatitis and Tryptophan metabolism by human gastrointestinal Acremonium is dominant on dandruff-afflicted scalps.[26] microbiota () 2.5. ANATOMICAL AREAS 15

The gut flora has the largest numbers of bacteria and the greatest number of species compared to other areas of the body.[6] In humans the gut flora is established at one to two years after birth, and by that time the intestinal epithelium and the intestinal mucosal barrier that it secretes have co-developed in a way that is tolerant to, and even supportive of, the gut flora and that also provides a barrier to pathogenic organisms.[19][45] The relationship between some gut flora and humans is not merely commensal (a non-harmful coexistence), but rather a mutualistic relationship.[1] Some human gut microorganisms benefit the host by fermentating dietary fiber into short-chain fatty acids (SCFAs), such as acetic acid and butyric acid, which are then absorbed by the host.[6][46] Intestinal bacteria also play a role in synthesiz- ing vitamin B and vitamin K as well as metabolizing bile acids, sterols, and xenobiotics.[1][46] The systemic importance of the SCFAs and other compounds they produce are like hormones and the gut flora itself appears to function like an endocrine organ,[46] and dysregulation of the gut flora has been correlated with a host of inflammatory and autoimmune conditions.[6][47] The composition of human gut flora changes over time, when the diet changes, and as overall health changes.[6][47] A systematic review of 15 human randomized con- trolled trials from July 2016 found that certain com- mercially available strains of probiotic bacteria from the Bifidobacterium and Lactobacillus genera (B. longum, B. This diagram shows the biosynthesis of bioactive breve, B. infantis, L. helveticus, L. rhamnosus, L. plan- compounds (indole and certain derivatives) from tarum, and L. casei), when taken by mouth in daily doses [41] tryptophan by bacteria in the gut. Indole is pro- of 109–1010 colony forming units (CFU) for 1–2 months, duced from tryptophan by bacteria that express possess treatment efficacy (i.e., improved behavioral out- [41] tryptophanase. Clostridium sporogenes metabolizes comes) in certain central nervous system disorders – in- [42] indole into 3-indolepropionic acid (IPA), a highly cluding anxiety, depression, autism spectrum disorder, potent neuroprotective antioxidant that scavenges and obsessive–compulsive disorder – and improved cer- [41][43][44] hydroxyl radicals. In the intestine, IPA binds tain aspects of memory.[48] to pregnane X receptors (PXR) in intestinal cells, thereby facilitating mucosal homeostasis and barrier function.[41] Following absorption from the intestine 2.5.4 Vagina and distribution to the brain, IPA confers a neuroprotective effect against cerebral ischemia and Alzheimer’s Main article: Vaginal flora disease.[41] Lactobacillus species metabolize tryptophan See also: List of microbiota species of the lower re- into indole-3-aldehyde (I3A) which acts on the aryl productive tract of women, List of bacterial vaginosis hydrocarbon receptor (AhR) in intestinal immune cells, microbiota, and Vaginal microbiota in pregnancy in turn increasing interleukin-22 (IL-22) production.[41] Indole itself acts as a glucagon-like peptide-1 (GLP-1) Vaginal microbiota refers to those species and genera secretagogue in intestinal L cells and as a ligand for that colonize the lower reproductive tract of women. AhR.[41] Indole can also be metabolized by the liver These organisms play an important role in protecting into indoxyl sulfate, a compound that is toxic in high against infections and maintaining vaginal health.[49] The concentrations and associated with vascular disease and most abundant vaginal microorganisms found in pre- renal dysfunction.[41] AST-120 (activated charcoal), an menopausal women are from the genus Lactobacillus, intestinal sorbent that is taken by mouth, adsorbs indole, which suppress pathogens by producing hydrogen per- in turn decreasing the concentration of indoxyl sulfate in oxide and lactic acid.[28][49][50] Bacterial species com- blood plasma.[41] position and ratios vary depending on the stage of the Main article: Gut flora menstrual cycle.[51][52] Ethnicity also influences vaginal See also: Gut–brain axis flora. The occurrence of hydrogen peroxide-producing 16 CHAPTER 2. HUMAN MICROBIOTA

lactobacilli is lower in African American women and Candida, Cladosporium, Aspergillus, Fusarium, Glomus, vaginal pH is higher.[53] Other influential factors such Alternaria, Penicillium, and Cryptococcus, among as sexual intercourse and antibiotics have been linked to others.[26] Bacteria accumulate on both the hard and soft the loss of lactobacilli.[50] Moreover, studies have found oral tissues in biofilms. Bacterial adhesion is particularly that sexual intercourse with a condom does appear to important for oral bacteria. change lactobacilli levels, and does increase the level of [50] Oral bacteria have evolved mechanisms to sense their en- Escherichia coli within the vaginal flora. Changes in vironment and evade or modify the host. Bacteria oc- the normal, healthy vaginal microbiota is an indication of cupy the ecological niche provided by both the tooth sur- infections, such as candidiasis or bacterial vaginosis.[50] face and gingival epithelium. However, a highly efficient Candida albicans inhibits the growth of Lactobacillus innate host defense system constantly monitors the bac- species, while Lactobacillus species which produce hy- terial colonization and prevents bacterial invasion of lo- drogen peroxide inhibit the growth and virulence of Can- cal tissues. A dynamic equilibrium exists between dental dida albicans in both the vagina and the gut.[26][28][29] plaque bacteria and the innate host defense system.[59] Fungal genera that have been detected in the vagina Of particular interest is the role of oral microorgan- include Candida, Pichia, Eurotium, Alternaria, isms in the two major dental diseases: dental caries and Rhodotorula, and Cladosporium, among others.[26] periodontal disease.[59] Additionally, research has correlated poor oral heath and the resulting ability of the oral microbiota to invade the body to affect cardiac health as 2.5.5 Placenta well as cognitive function.[60]

Main article: Placental microbiome

Until recently the placenta was considered to be a sterile organ but commensal, nonpathogenic bacterial species and genera have been identiﬁed that reside in the placental tissue.[54][55][56] 2.5.8 Lung

2.5.6 Uterus Main article: Lung microbiome Main article: Uterine microbiome Much like the oral cavity, the upper and lower respiratory system possess mechanical deterrents to remove mi- Until recently, the upper reproductive tract of women crobes. Goblet cells produce mucous which traps mi- was considered to be a sterile environment. A variety crobes and moves them out of the respiratory system via of microorganisms inhabit the uterus of healthy, asymp- continuously moving ciliated epithelial cells.[1] In addi- tomatic women of reproductive age. The microbiome of tion, a bactericidal effect is generated by nasal mucus the uterus differs significantly from that of the vagina and which contains the enzyme lysozyme.[1] gastrointestinal tract.[57] Nonetheless, the upper and lower respiratory tract appears to have their own set of microbiota. Pulmonary 2.5.7 Oral cavity bacterial microbiota belong to 9 major bacterial genera: Prevotella, Sphingomonas, Pseudomonas, Acineto- Main article: Oral microbiology bacter, Fusobacterium, Megasphaera, Veillonella, Staphy- lococcus, and Streptococcus. Some of the bacteria consid- The environment present in the human mouth allows the ered “normal biota” in the respiratory tract can cause se- growth of characteristic microorganisms found there. It rious disease especially in immunocompromised individ- provides a source of water and nutrients, as well as a mod- uals; these include Streptococcus pyogenes, Haemophilus erate temperature.[1] Resident microbes of the mouth ad- influenzae, Streptococcus pneumoniae, Neisseria meningi- here to the teeth and gums to resist mechanical flushing tidis, and Staphylococcus aureus. from the mouth to stomach where acid-sensitive microbes Fungal genera that compose the pulmonary myco- are destroyed by hydrochloric acid.[1][28] biome include Candida, Malassezia, Neosartorya, Sac- [26] Anaerobic bacteria in the oral cavity include: charomyces, and Aspergillus, among others. Actinomyces, Arachnia, Bacteroides, Bifidobacterium, Unusual distributions of bacterial and fungal genera in the Eubacterium, Fusobacterium, Lactobacillus, Leptotrichia, respiratory tract is observed in people with cystic fibro- Peptococcus, Peptostreptococcus, Propionibacterium, sis.[26][61] Their bacterial flora often contains antibiotic- Selenomonas, Treponema, and Veillonella.[58] Genera resistant and slow-growing bacteria, and the frequency of of fungi that are frequently found in the mouth include these pathogens changes in relation to age.[61] 2.6. DISEASE 17

2.6 Disease human colorectal cancers and the ability of colibactin- expressing E. coli to potentiate intestinal tumorigenesis Communities of microflora have been shown to change in mice. Data also support a role for enterotoxigenic their behavior in diseased individuals.[62] B. fragilis in both human and animal models of colon tumors. Both colibactin and CDT can cause double- stranded DNA damage in mammalian cells. In contrast, 2.6.1 Cancer Bft acts indirectly by eliciting high levels of reactive oxygen species (ROS), which in turn damage host DNA. Chronically high ROS levels can outpace DNA repair Although cancer is generally a disease of host genet- [63] ics and environmental factors, microorganisms are im- mechanisms, leading to DNA damage and mutations. plicated in ~20% of human malignancies. Mucosal microbes can become part of the tumor microenvironment β-catenin Several microbes possess proteins that en- (TME) of aerodigestive tract malignancies. Intratumoral gage host pathways involved in carcinogenesis. The microbes can affect cancer growth and spread. Gut mi- Wnt/β-catenin signaling pathway, which regulates cells’ crobiota also detoxify dietary components, reducing in- polarity, growth and differentiation, is one example and is flammation and balancing host cell growth and prolifera- altered in many malignancies. Multiple cancer-associated tion. Coley’s toxins were one of the earliest forms of can- bacteria can influence β-catenin signaling. Oncogenic cer bacteriotherapy. Synthetic biology employs designer type 1 strains of Helicobacter pylori express CagA, which microbes and microbiota transplants against tumors.[63] is injected directly into the cytoplasm of host cells and Microbes and the microbiota affect carcinogenesis in modulates β-catenin to drive gastric cancer. This mod- three broad ways: (i) altering the balance of tumor ulation leads to up-regulation of cellular proliferation, cell proliferation and death, (ii) regulating immune sys- survival and migration genes, as well as angiogenesis— tem function and (iii) influencing metabolism of host- all processes central to carcinogenesis. Oral microbiota produced factors, foods and pharmaceuticals.[63] Fusobacterium nucleatum is associated with human colorectal adenomas and adenocarcinomas and amplified intestinal tumorigenesis in mice. F. nucleatum expresses Modes of action FadA, a bacterial cell surface adhesion component that binds host E-cadherin, activating β-catenin. Enterotoxi- Ten microbes are designated by the International Agency genic B. fragilis, which is enriched in some human col- for Research on Cancer (IARC) as human carcinogens. orectal cancers, can stimulate E-cadherin cleavage via Most of these microbes colonize large percentages of the Btf, leading to β-catenin activation. Salmonella Typhi human population, although only genetically susceptible strains that maintain chronic infections secrete AvrA, individuals develop cancer. Tumors arising at bound- which can activate epithelial β-catenin signaling and are ary surfaces, such as the skin, oropharynx and respira- associated with hepatobiliary cancers.[63] tory, digestive and urogenital tracts, harbor a microbiota, Several of these bacteria are normal microbiota con- which complicates cancer-microbe causality. Substan- stituents. The presence of these cancer-potentiating mi- tial microbe presence at a tumor site does not establish crobes and their access to E-cadherin in evolving tumors association or causal links. Instead, microbes may find demonstrate that a loss of appropriate boundaries and the tumor’s oxygen tension or nutrient profile supportive. barrier maintenance between host and microbe is a criti- Decreased populations of specific microbes may also in- cal step in the development of some tumors.[63] crease risks.[63] Human oncoviruses can drive carcinogenesis by integrating oncogenes into host genomes. Human papillo- Inflammation maviruses (HPV) express oncoproteins such as E6 and E7. Viral integration selectively amplifies host genes in Mucosal surface barriers are subject to environmental pathways with established cancer roles.[63] insult and must rapidly repair to maintain homeostasis. Microbes affect genomic stability, resistance to cell death Compromised host or microbiota resiliency also reduce and proliferative signaling. Many bacteria can damage resistance to malignancy. Cancer and inflammatory dis- DNA, to kill competitors/survive. These defense fac- orders can then arise. Once barriers are breached, microbes can elicit proinflammatory or immunosuppressive tors can lead to mutational events that contribute to car- [63] cinogenesis. Examples include colibactin encoded by programs. the pks locus (expressed by B2 group Escherichia coli as Inflammation, whether high-grade as in inflammatory well as by other Enterobacteriaceae), Bacteroides fragilis disorders or low-grade as in malignancies and obesity, toxin (Bft) produced by enterotoxigenic B. fragilis and drive a tumor-permissive milieu. Pro-inflammatory fac- cytolethal distending toxin (CDT) produced by several ε- tors such as reactive oxygen and nitrogen species, cy- and γ-proteobacteria. Colibactin is of interest in colorec- tokines and chemokines can also drive tumor growth tal carcinogenesis, given the detection of pks+ E. coli in and spread. Tumors can up-regulate and activate pat- 18 CHAPTER 2. HUMAN MICROBIOTA

tern recognition receptors (e.g. toll-like receptors), driv- • Microbiome ing feedforward loops of activation of cancer-associated • inﬂammation regulator NF-κΒ. Cancer-associated mi- Microorganism crobes appear to activate NF-κΒ signaling within the • Human Microbiome Project TME. The activation of NF-κΒ by F. nucleatum may be the result of pattern recognition receptor engagement or • Human virome FadA engagement of E-cadherin. Other pattern recog- • Initial acquisition of microbiota nition receptors, such as nucleotide-binding oligomeriza- tion domain–like receptor (NLR) family members NOD- • List of bacterial vaginosis microbiota 2, NLRP3, NLRP6 and NLRP12, may play a role in me- • diating colorectal cancer.[63] uBiome Immune system TME engagement is not restricted to the innate immune system. Once the innate immune sys- 2.10 References tem is activated, adaptive immune responses ensue, often with tumor progression. The interleukin-23 (IL-23)–IL- [1] Sherwood, Linda; Willey, Joanne; Woolverton, Christo- 17 axis, tumor necrosis factor–α (TNF-α)–TNF recep- pher (2013). Prescott’s Microbiology (9th ed.). New York: tor signaling, IL-6–IL-6 family member signaling, and McGraw Hill. pp. 713–721. ISBN 9780073402406. STAT3 activation all represent innate and adaptive path- OCLC 886600661. ways contributing to tumor progression and growth.[63] [2] American Academy of Microbiology FAQ: Human Mi- The microbiota adapts to host changes such as inﬂam- crobiome January 2014 mation. Adaptation shift microbiota to a vulnerable tissue site. Genotoxin azoxymethane and colon barrier– [3] Judah L. Rosner for Microbe Magazine, Feb 2014. Ten disrupting agent dextran sodium sulfate independently re- Times More Microbial Cells than Body Cells in Humans? sult in colon tumors in susceptible mouse strains; combin- [4] Alison Abbott for Nature News. Jan 8 2016 Scientists bust ing them accelerates tumorigenesis.[63] myth that our bodies have more bacteria than human cells

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Indole-3-propionate (IPA), a deamination product of tryp- as biomarkers and agents that can promote various as- tophan formed by symbiotic bacteria in the gastrointesti- pects of vaginal health”. Frontiers in Physiology. 6. nal tract of mammals and birds. 3-Indolepropionic acid doi:10.3389/fphys.2015.00081. ISSN 1664-042X. has been shown to prevent oxidative stress and death of primary neurons and neuroblastoma cells exposed to [50] Witkin, S. S.; Linhares, I. M.; Giraldo, P. (2007). the amyloid beta-protein in the form of amyloid fibrils, “Bacterial flora of the female genital tract: Function one of the most prominent neuropathologic features of and immune regulation”. Best Practice & Research Alzheimer’s disease. 3-Indolepropionic acid also shows Clinical Obstetrics & Gynaecology. 21 (3): 347–354. a strong level of neuroprotection in two other paradigms doi:10.1016/j.bpobgyn.2006.12.004. PMID 17215167. of oxidative stress. (PMID 10419516 ) [51] Todar, Kenneth (2012). “The Normal Bacterial Flora Origin: • Endogenous • Microbial of Humans”. Todar’s Online Textbook of Bacteriology. Madison, WI: Kenneth Todar. Retrieved 2012-04-06. [44] Chyan YJ, Poeggeler B, Omar RA, Chain DG, Fran- gione B, Ghiso J, Pappolla MA (July 1999). “Potent [52] Onderdonk, A. B.; Zamarchi, G. R.; Walsh, J. A.; Mellor, neuroprotective properties against the Alzheimer beta- R. D.; Muñoz, A.; Kass, E. H. (1986). “Methods for quan- amyloid by an endogenous melatonin-related indole struc- titative and qualitative evaluation of vaginal microflora ture, indole-3-propionic acid”. J. Biol. Chem. 274 (31): during menstruation”. Applied and Environmental Mi- 21937–21942. doi:10.1074/jbc.274.31.21937. PMID crobiology. 51 (2): 333–339. PMC 238869 . PMID 10419516. [Indole-3-propionic acid (IPA)] has previ- 3954346. ously been identified in the plasma and cerebrospinal fluid of humans, but its functions are not known. ... In ki- [53] Antonio, M. A. D.; Hawes, S. E.; Hillier, S. L. (1999). netic competition experiments using free radical-trapping “The Identification of VaginalLactobacillusSpecies and agents, the capacity of IPA to scavenge hydroxyl radicals the Demographic and Microbiologic Characteristics of exceeded that of melatonin, an indoleamine considered to Women Colonized by These Species”. The Jour- be the most potent naturally occurring scavenger of free nal of Infectious Diseases. 180 (6): 1950–1956. radicals. In contrast with other antioxidants, IPA was not doi:10.1086/315109. PMID 10558952. converted to reactive intermediates with pro-oxidant activity. [54] Fox, Chelsea; Eichelberger, Kacey (2015). “Ma- ternal microbiome and pregnancy outcomes”. [45] Faderl, M; et al. (Apr 2015). “Keeping bugs in check: Fertility and Sterility. 104 (6): 1358–1363. The mucus layer as a critical component in maintaining doi:10.1016/j.fertnstert.2015.09.037. ISSN 0015- intestinal homeostasis”. IUBMB Life. 67 (4): 275–85. 0282. PMID 26493119. doi:10.1002/iub.1374. PMID 25914114. [55] Wassenaar, T.M.; Panigrahi, P. (2014). “Is a foetus devel- [46] Clarke, G; et al. (Aug 2014). “Minireview: Gut micro- oping in a sterile environment?". Letters in Applied Micro- biota: the neglected endocrine organ”. Mol Endocrinol. biology. 59 (6): 572–579. doi:10.1111/lam.12334. ISSN 28 (8): 1221–38. doi:10.1210/me.2014-1108. PMID 0266-8254. PMID 25273890. 24892638. [56] Schwiertz, Andreas (2016). Microbiota of the human body : implications in health and disease. Switzerland: [47] Shen S, Wong CH. “Bugging inflammation: role of the gut Springer. p. 1. ISBN 978-3-319-31248-4. microbiota. Clin Transl Immunology. 2016 Apr;5(4):e72. Review. doi:10.1038/cti.2016.12 PMID 27195115 PDF [57] Franasiak, Jason M.; Scott, Richard T. (2015). “Repro- ductive tract microbiome in assisted reproductive tech- [48] Wang H, Lee IS, Braun C, Enck P (July 2016). “Effect of nologies”. Fertility and Sterility. 104 (6): 1364–1371. probiotics on central nervous system functions in animals doi:10.1016/j.fertnstert.2015.10.012. ISSN 0015-0282. and humans - a systematic review”. J. Neurogastroen- PMID 26597628. terol Motil. doi:10.5056/jnm16018. PMID 27413138. These probiotics showed efficacy in improving psychiatric [58] Sutter, V. L. (1984). “Anaerobes as normal oral disorder-related behaviors including anxiety, depression, flora”. Reviews of infectious diseases. 6 Suppl 1: S62– autism spectrum disorder (ASD), obsessive-compulsive S66. doi:10.1093/clinids/6.Supplement_1.S62. PMID disorder, and memory abilities, including spatial and non- 6372039. spatial memory. Because many of the basic science stud- [59] Rogers A H (editor). (2008). Molecular Oral Microbiol- ies showed some efficacy of probiotics on central nervous ogy. Caister Academic Press. ISBN 978-1-904455-24-0. system function, this background may guide and promote further preclinical and clinical studies. ... According to [60] Noble JM, Scarmeas N, Papapanou PN (2013). “Poor the qualitative analyses of current studies, we can provi- oral health as a chronic, potentially modifiable dementia sionally draw the conclusion that B. longum, B. breve, B. risk factor: review of the literature.”. Curr Neurol Neu- infantis, L. helveticus, L. rhamnosus, L. plantarum, and L. rosci Rep. 13 (10): 384. doi:10.1007/s11910-013-0384- casei were most effective in improving CNS function, in- x. PMID 23963608. cluding psychiatric disease-associated functions (anxiety, depression, mood, stress response) and memory abilities [61] Beringer, P M; M D Appleman (November 2000). “Unusual respiratory bacterial flora in cystic fibro- [49] Petrova, Mariya I.; Lievens, Elke; Malik, Shweta; Imholz, sis: microbiologic and clinical features” (PDF). Cur- Nicole; Lebeer, Sarah (2015). “Lactobacillus species rent Opinion in Pulmonary Medicine. 6 (6): 545–550. 22 CHAPTER 2. HUMAN MICROBIOTA

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[62] “Mouth bacteria can change its diet, supercomputers reveal”. medicalxpress.com.

[63] Garrett, Wendy S. (3 April 2015). “Cancer and the microbiota”. Science Magazine. doi:10.1126/science.aaa4972. Retrieved 2015-06-29.

[64] Lavigne, Jean-Philippe; Sotto, Albert; Dunyach-Remy, Catherine; Lipsky, Benjamin A. (2015). “New Molec- ular Techniques to Study the Skin Microbiota of Diabetic Foot Ulcers”. Advances in Wound Care. 4 (1): 38–49. doi:10.1089/wound.2014.0532. ISSN 2162-1918. PMC 4281861 . PMID 25566413.

[65] Katherine Harmon (16 December 2009). “Bugs Inside: What Happens When the Microbes That Keep Us Healthy Disappear?". Scientiﬁc American. Retrieved 27 Decem- ber 2008.

2.11 External links

• The Secret World Inside You Exhibit 2015-2016, American Museum of Natural History • FAQ: Human Microbiome, January 2014 American Society For Microbiology Chapter 3

Genome

For a non-technical introduction to the topic, see netics, in a sexually reproducing organism (typically Introduction to genetics. For other uses, see Genome eukarya) the gamete has half the number of chromo- (disambiguation). somes of the somatic cell and the genome is a full set of In modern molecular biology and genetics, a genome is chromosomes in a diploid cell. The halving of the genetic material in gametes is accomplished by the segregation of homologous chromosomes during meiosis.[6] In haploid organisms, including cells of bacteria, archaea, and in organelles including mitochondria and chloroplasts, or viruses, that similarly contain genes, the single or set of circular or linear chains of DNA (or RNA for some viruses), likewise constitute the genome. The term genome can be applied speciﬁcally to mean what is stored on a complete set of nuclear DNA (i.e., the "nuclear genome") but can also be applied to what is stored within organelles that contain their own DNA, as with the "mitochondrial genome" or the "chloroplast genome”. Additionally, the genome can comprise non- chromosomal genetic elements such as viruses, plasmids, and transposable elements.[7]

An image of the 46 chromosomes making up the diploid genome Typically, when it is said that the genome of a sexually of a human male. (The mitochondrial chromosome is not reproducing species has been "sequenced", it refers to a shown.) determination of the sequences of one set of autosomes and one of each type of sex chromosome, which together the genetic material of an organism. It consists of DNA represent both of the possible sexes. Even in species that (or RNA in RNA viruses). The genome includes both exist in only one sex, what is described as a “genome se- the genes, (the coding regions), the noncoding DNA[1] quence” may be a composite read from the chromosomes and the genomes of the mitochondria[2] and chloroplasts. of various individuals. Colloquially, the phrase “genetic makeup” is sometimes used to signify the genome of a particular individual or organism. The study of the 3.1 Origin of term global properties of genomes of related organisms is usually referred to as genomics, which distinguishes it from genetics which generally studies the properties of single The term genome was created in 1920 by Hans Win- genes or groups of genes. kler,[3] professor of botany at the University of Hamburg, Germany. The Oxford Dictionary suggests the name is a Both the number of base pairs and the number of genes blend of the words gene and chromosome.[4] However, see vary widely from one species to another, and there is only omics for a more thorough discussion. A few related -ome a rough correlation between the two (an observation is words already existed—such as biome, rhizome, forming known as the C-value paradox). At present, the high- a vocabulary into which genome ﬁts systematically.[5] est known number of genes is around 60,000, for the protozoan causing trichomoniasis (see List of sequenced eukaryotic genomes), almost three times as many as in 3.2 Overview the human genome. An analogy to the human genome stored on DNA is that Some organisms have multiple copies of chromosomes: of instructions stored in a book: diploid, triploid, tetraploid and so on. In classical ge-

23 24 CHAPTER 3. GENOME

• The book (genome) would contain 23 chapters Methanococcus jannaschii, was completed in 1996, again (chromosomes); by The Institute for Genomic Research. The development of new technologies has made it dra- • Each chapter contains 48 to 250 million letters matically easier and cheaper to do sequencing, and (A,C,G,T) without spaces; the number of complete genome sequences is growing • Hence, the book contains over 3.2 billion letters to- rapidly. The US National Institutes of Health main- tal; tains one of several comprehensive databases of genomic information.[9] Among the thousands of completed • The book fits into a cell nucleus the size of a pin- genome sequencing projects include those for rice, a point; mouse, the plant Arabidopsis thaliana, the puffer fish, and the bacteria E. coli. In December 2013, scientists first • At least one copy of the book (all 23 chapters) is con- sequenced the entire genome of a Neanderthal, an extinct tained in most cells of our body. The only exception species of humans. The genome was extracted from the in humans is found in mature red blood cells which toe bone of a 130,000-year-old Neanderthal found in a become enucleated during development and there- Siberian cave.[10][11] fore lack a genome. New sequencing technologies, such as massive parallel sequencing have also opened up the prospect of personal genome sequencing as a diagnostic tool, as pioneered by 3.3 Sequencing and mapping Manteia Predictive Medicine. A major step toward that goal was the completion in 2007 of the full genome of James D. Watson, one of the co-discoverers of the struc- For more details on this topic, see Genome project. [12] In 1976, Walter Fiers at the University of Ghent (Bel- ture of DNA. Whereas a genome sequence lists the order of every DNA base in a genome, a genome map identifies the landmarks. A genome map is less detailed than a genome sequence and aids in navigating around the genome. The Human Genome Project was organized to map and to sequence the human genome. A fundamental step in the project was the release of a detailed genomic map by Jean Weis- senbach and his team at the Genoscope in Paris.[13][14] Reference genome sequences and maps continue to be updated, removing errors and clarifying regions of high allelic complexity.[15] The decreasing cost of genomic mapping has permitted genealogical sites to offer it as a service,[16] to the extent that one may submit one’s genome to crowd sourced scientific endeavours such as DNA.land at the New York Genome Center, an example both of the economies of scale and of citizen science.[17]

Part of DNA sequence - prototypification of complete genome of 3.4 Genome compositions virus gium) was the first to establish the complete nucleotide Genome composition is used to describe the make up sequence of a viral RNA-genome (Bacteriophage MS2). of contents of a haploid genome, which should include The next year Fred Sanger completed the first DNA- genome size, proportions of non-repetitive DNA and genome sequence: Phage Φ-X174, of 5386 base pairs.[8] repetitive DNA in details. By comparing the genome The first complete genome sequences among all three do- compositions between genomes, scientists can better un- mains of life were released within a short period dur- derstand the evolutionary history of a given genome. ing the mid-1990s: The first bacterial genome to be se- When talking about genome composition, one should quenced was that of Haemophilus influenzae, completed distinguish between prokaryotes and eukaryotes as there by a team at The Institute for Genomic Research in are significant differences with contents structure. In 1995. A few months later, the first eukaryotic genome prokaryotes, most of the genome (85–90%) is non- was completed, with sequences of the 16 chromosomes repetitive DNA, which means coding DNA mainly forms of budding yeast Saccharomyces cerevisiae published as it, while non-coding regions only take a small part.[18] the result of a European-led effort begun in the mid- On the contrary, eukaryotes have the feature of exon- 1980s. The first genome sequence for an archaeon, intron organization of protein coding genes; the varia- 3.4. GENOME COMPOSITIONS 25 tion of repetitive DNA content in eukaryotes is also ex- minimum and still have the organism in question sur- tremely high. In mammals and plants, the major part of vive. There is experimental work being done on mini- the genome is composed of repetitive DNA.[19] mal genomes for single cell organisms as well as minimal genomes for multi-cellular organisms (see Developmental Most biological entities that are more complex than a [23][24] virus sometimes or always carry additional genetic ma- biology). The work is both in vivo and in silico. terial besides that which resides in their chromosomes. Here is a table of some significant or representative In some contexts, such as sequencing the genome of a genomes. See #See also for lists of sequenced genomes. pathogenic microbe, “genome” is meant to include information stored on this auxiliary material, which is carried in plasmids. In such circumstances then, “genome” de- 3.4.2 Proportion of non-repetitive DNA scribes all of the genes and information on non-coding DNA that have the potential to be present. The proportion of non-repetitive DNA is calculated by using the length of non-repetitive DNA divided by In eukaryotes such as plants, protozoa and animals, how- genome size. Protein-coding genes and RNA-coding ever, “genome” carries the typical connotation of only in- genes are generally non-repetitive DNA.[66] A bigger formation on chromosomal DNA. So although these or- genome does not mean more genes, and the proportion ganisms contain chloroplasts or mitochondria that have of non-repetitive DNA decreases along with increasing their own DNA, the genetic information contained in genome size in higher eukaryotes.[19] DNA within these organelles is not considered part of the genome. In fact, mitochondria are sometimes said to have It had been found that the proportion of non-repetitive their own genome often referred to as the "mitochondrial DNA can vary a lot between species. Some E. coli as genome". The DNA found within the chloroplast may be prokaryotes only have non-repetitive DNA, lower eukary- referred to as the "plastome". otes such as C. elegans and fruit fly, still possess more non-repetitive DNA than repetitive DNA.[19][67] Higher eukaryotes tend to have more repetitive DNA than non- 3.4.1 Genome size repetitive ones. In some plants and amphibians, the proportion of non-repetitive DNA is no more than 20%, be- coming a minority component.[19]

3.4.3 Proportion of repetitive DNA

The proportion of repetitive DNA is calculated by using length of repetitive DNA divide by genome size. There are two categories of repetitive DNA in genome: tandem repeats and interspersed repeats.[68]

Tandem repeats

Tandem repeats are usually caused by slippage Log-log plot of the total number of annotated proteins in genomes during replication, unequal crossing-over and gene submitted to GenBank as a function of genome size.[20] conversion,[69] satellite DNA and microsatellites are forms of tandem repeats in the genome.[70] Although Genome size is the total number of DNA base pairs tandem repeats count for a significant proportion in in one copy of a haploid genome. In humans, the nu- genome, the largest proportion in mammalian is the clear genome comprises approximately 3.2 billion nu- other type, interspersed repeats. cleotides of DNA, divided into 24 linear molecules, the shortest 50 000 000 nucleotides in length and the longest Interspersed repeats 260 000 000 nucleotides, each contained in a different [21] chromosome. The genome size is positively correlated Interspersed repeats mainly come from transposable el- with the morphological complexity among prokaryotes ements (TEs), but they also include some protein cod- and lower eukaryotes; however, after mollusks and all ing gene families and pseudogenes. Transposable el- the other higher eukaryotes above, this correlation is no [19][22] ements are able to integrate into the genome at an- longer effective. This phenomenon also indicates other site within the cell.[18][71] It is believed that TEs the mighty influence coming from repetitive DNA act on are an important driving force on genome evolution of the genomes. higher eukaryotes.[72] TEs can be classified into two cat- Since genomes are very complex, one research strategy is egories, Class 1 (retrotransposons) and Class 2 (DNA to reduce the number of genes in a genome to the bare transposons).[71] 26 CHAPTER 3. GENOME

Retrotransposons Retrotransposons can be tran- Duplications play a major role in shaping the genome. scribed into RNA, which are then duplicated at another Duplication may range from extension of short tandem site into the genome.[73] Retrotransposons can be divided repeats, to duplication of a cluster of genes, and all the into Long terminal repeats (LTRs) and Non-Long Termi- way to duplication of entire chromosomes or even entire nal Repeats (Non-LTR).[72] genomes. Such duplications are probably fundamental to the creation of genetic novelty. Long terminal repeats (LTRs) similar to retro- Horizontal gene transfer is invoked to explain how there viruses, which have both gag and pol genes to is often an extreme similarity between small portions of make cDNA from RNA and proteins to insert into the genomes of two organisms that are otherwise very dis- genome, but LTRs can only act within the cell as tantly related. Horizontal gene transfer seems to be com- [71] they lack the env gene in retroviruses. It has been mon among many microbes. Also, eukaryotic cells seem reported that LTRs consist of the largest fraction in to have experienced a transfer of some genetic material most plant genome and might account for the huge from their chloroplast and mitochondrial genomes to their [74] variation in genome size. nuclear chromosomes.

Non-long terminal repeats (Non-LTRs) can be divided into long interspersed elements (LINEs), short interspersed elements (SINEs) and Penelope- 3.6 See also like elements. In Dictyostelium discoideum, there is another DIRS-like elements belong to Non- • Bacterial genome size LTRs. Non-LTRs are widely spread in eukaryotic • Cryoconservation of animal genetic resources genomes.[75] • Genome Browser Long interspersed elements (LINEs) are able to en- code two Open Reading Frames (ORFs) to gener- • Genome Compiler ate transcriptase and endonuclease, which are essen- • tial in retrotransposition. The human genome has Genome topology around 500,000 LINEs, taking around 17% of the • Genome-wide association study genome.[76] • List of sequenced animal genomes Short interspersed elements (SINEs) are usually less than 500 base pairs and need to co-opt with the • List of sequenced archaeal genomes LINEs machinery to function as nonautonomous • List of sequenced bacterial genomes retrotransposons.[77] The Alu element is the most common SINEs found in primates, it has a length • List of sequenced eukaryotic genomes of about 350 base pairs and takes about 11% of the human genome with around 1,500,000 copies.[72] • List of sequenced fungi genomes

• List of sequenced plastomes DNA transposons DNA transposons generally move by “cut and paste” in the genome, but duplication has also • List of sequenced protist genomes been observed. Class 2 TEs do not use RNA as interme- • diate and are popular in bacteria, in metazoan it has also Metagenomics been found.[72] • Microbiome

• Molecular epidemiology 3.5 Genome evolution • Molecular pathological epidemiology

Genomes are more than the sum of an organism’s genes • Molecular pathology and have traits that may be measured and studied without reference to the details of any particular genes and their • Nucleic acid sequence products. Researchers compare traits such as karyotype • (chromosome number), genome size, gene order, codon Pan-genome usage bias, and GC-content to determine what mecha- • Precision medicine nisms could have produced the great variety of genomes that exist today (for recent overviews, see Brown 2002; • Sequenceome Saccone and Pesole 2003; Benfey and Protopapas 2004; Gibson and Muse 2004; Reese 2004; Gregory 2005). • Whole genome sequencing 3.7. REFERENCES 27

3.7 References [15] Genome Reference Consortium. “Assembling the Genome”. Retrieved 23 August 2016. [1] Brosius, J (2009), “The Fragmented Gene”, Annals of [16] Kaplan, Sarah (2016-04-17). “How do your 20,000 genes the New York Academy of Sciences, 1178: 186–193, determine so many wildly diﬀerent traits? They multi- doi:10.1111/j.1749-6632.2009.05004.x task.”. The Washington Post. Retrieved 2016-08-27. [2] Ridley, M. (2006), Genome: the autobiography of a species in 23 chapters (PDF), New York: Harper Peren- [17] Zimmer, Carl. “Game of Genomes, Episode 13: Answers nial, ISBN 0-06-019497-9 and Questions”. STAT. Retrieved 2016-08-27.

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[9] “Genome Home”. 2010-12-08. Retrieved 27 January [23] Glass JI; Assad-Garcia N; Alperovich N; Yooseph 2011. S; Lewis MR; Maruf M; Hutchison CA 3rd; Smith HO; Venter JC (2006). “Essential genes of a min- [10] Zimmer, Carl (December 18, 2013). “Toe Fossil Provides imal bacterium”. Proc Natl Acad Sci USA. 103 Complete Neanderthal Genome”. New York Times. Re- (2): 425–30. Bibcode:2006PNAS..103..425G. trieved 18 December 2013. doi:10.1073/pnas.0510013103. PMC 1324956 . [11] Prüfer, Kay; Racimo, Fernando; Patterson, Nick; Jay, PMID 16407165. Flora; Sankararaman, Sriram; Sawyer, Susanna; Heinze, [24] Forster AC; Church GM (2006). “Towards synthe- Anja; Renaud, Gabriel; Sudmant, Peter H.; De Fil- sis of a minimal cell”. Mol Syst Biol. 2 (1): 45. ippo, Cesare; Li, Heng; Mallick, Swapan; Dannemann, Michael; Fu, Qiaomei; Kircher, Martin; Kuhlwilm, doi:10.1038/msb4100090. PMC 1681520 . PMID Martin; Lachmann, Michael; Meyer, Matthias; Ongy- 16924266. erth, Matthias; Siebauer, Michael; Theunert, Christoph; Tandon, Arti; Moorjani, Priya; Pickrell, Joseph; Mul- [25] Mankertz P (2008). “Molecular Biology of Porcine Cir- likin, James C.; Vohr, Samuel H.; Green, Richard E.; coviruses”. Animal Viruses: Molecular Biology. Caister Hellmann, Ines; Johnson, Philip L. F.; et al. (De- Academic Press. ISBN 978-1-904455-22-6. cember 18, 2013). “The complete genome sequence of a Neanderthal from the Altai Mountains”. Nature. [26] Fiers W; Contreras, R.; Duerinck, F.; Haegeman, 505 (7481): 43–49. Bibcode:2014Natur.505...43P. G.; Iserentant, D.; Merregaert, J.; Min Jou, W.; Molemans, F.; Raeymaekers, A.; Van Den Berghe, doi:10.1038/nature12886. PMC 4031459 . PMID A.; Volckaert, G.; Ysebaert, M. (1976). “Complete 24352235. Retrieved 18 December 2013. nucleotide-sequence of bacteriophage MS2-RNA – pri- [12] Wade, Nicholas (2007-05-31). “Genome of DNA Pio- mary and secondary structure of replicase gene”. Nature. neer Is Deciphered”. The New York Times. Retrieved 2 260 (5551): 500–507. Bibcode:1976Natur.260..500F. April 2010. doi:10.1038/260500a0. PMID 1264203.

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• Saccone, Cecilia; Pesole, Graziano (2003). Hand- book of Comparative Genomics. Chichester: John Wiley & Sons. ISBN 0-471-39128-X.

• Werner, E. (2003). “In silico multicellular systems biology and minimal genomes”. Drug Discov Today. 8 (24): 1121–1127. doi:10.1016/S1359- 6446(03)02918-0. PMID 14678738.

3.9 External links

• UCSC Genome Browser – view the genome and annotations for more than 80 organisms. • genomecenter.howard.edu

• Build a DNA Molecule • Some comparative genome sizes

• DNA Interactive: The History of DNA Science • DNA From The Beginning

• All About The Human Genome Project—from Genome.gov

• Animal genome size database • Plant genome size database

• GOLD:Genomes OnLine Database • The Genome News Network

• NCBI Entrez Genome Project database • NCBI Genome Primer

• GeneCards—an integrated database of human genes • Visualization of nucleotide sequence - prototypiﬁca- tion of complete genome of virus, sequence of 5418 nucleotides • BBC News – Final genome 'chapter' published

• IMG (The Integrated Microbial Genomes system)— for genome analysis by the DOE-JGI

• GeKnome Technologies Next-Gen Sequencing Data Analysis—next-generation sequencing data analysis for Illumina and 454 Service from GeKnome Tech- nologies. Chapter 4

Connectome

Rendering of a group connectome based on 20 subjects. Anatom- ical ﬁbers that constitute the white matter architecture of the human brain are visulized color-coded by traversing direction (xyz- White matter tracts within a human brain, as visualized by MRI directions mapping to rgb colors respectively). Visualization of [1] tractography ﬁbers was done using TrackVis software.

Project, sponsored by the National Institutes of Health, A connectome (/kəˈnɛktoʊm/) is a comprehensive map whose focus is to build a network map of the human brain of neural connections in the brain, and may be thought in healthy, living adults. of as its "wiring diagram". More broadly, a connectome would include the mapping of all neural connections within an organism's nervous system. 4.1 Origin and usage of the term The production and study of connectomes, known as connectomics, may range in scale from a detailed map “connectome” of the full set of neurons and synapses within part or all of the nervous system of an organism to a macro scale In 2005, Dr. Olaf Sporns at Indiana University and Dr. description of the functional and structural connectivity Patric Hagmann at Lausanne University Hospital inde- between all cortical areas and subcortical structures. The pendently and simultaneously suggested the term “con- term “connectome” is used primarily in scientific efforts nectome” to refer to a map of the neural connections to capture, map, and understand the organization of neu- within the brain. This term was directly inspired by the ral interactions within the brain. ongoing effort to sequence the human genetic code—to build a genome. Research has successfully constructed the full connectome of one animal: the roundworm C. elegans (White “Connectomics” (Hagmann, 2005) has been defined as et al., 1986,[2] Varshney et al., 2011[3]). Partial con- the science concerned with assembling and analyzing nectomes of a mouse retina[4] and mouse primary visual connectome data sets.[6] [5] cortex have also been successfully constructed. Bock et In their 2005 paper, “The Human Connectome, a struc- al.'s complete 12 TB data set is publicly available at Open tural description of the human brain”, Sporns et al. wrote: Connectome Project. The ultimate goal of connectomics is to map the human To understand the functioning of a network, brain. This effort is pursued by the Human Connectome one must know its elements and their inter-

32 4.2. THE CONNECTOME AT MULTIPLE SCALES 33

connections. The purpose of this article is However, structure-function relationships in the brain are to discuss research strategies aimed at a com- unlikely to reduce to simple one-to-one mappings. In prehensive structural description of the net- fact, the connectome can evidently support a great num- work of elements and connections forming ber of variable dynamic states, depending on current the human brain. We propose to call this sensory inputs, global brain state, learning and develop- dataset the human “connectome,” and we argue ment. Some changes in functional state may involve rapid that it is fundamentally important in cognitive changes of structural connectivity at the synaptic level, as neuroscience and neuropsychology. The con- has been elucidated by two-photon imaging experiments nectome will significantly increase our under- showing the rapid appearance and disappearance of den- standing of how functional brain states emerge dritic spines (Bonhoeffer and Yuste, 2002).[11] from their underlying structural substrate, and Despite such complex and variable structure-function will provide new mechanistic insights into how mappings, the connectome is an indispensable basis for brain function is affected if this structural sub- [7] the mechanistic interpretation of dynamic brain data, strate is disrupted. from single-cell recordings to functional neuroimaging. The term “connectome” was more recently popularized In his 2005 Ph.D. thesis, From diffusion MRI to brain con- by Sebastian Seung's “I am my Connectome” speech nectomics, Hagmann wrote: given at the 2010 TED conference, which discusses the high-level goals of mapping the human connectome, as It is clear that, like the genome, which is much well as ongoing efforts to build a three-dimensional neural more than just a juxtaposition of genes, the map of brain tissue at the microscale.[12] In 2012, Seung set of all neuronal connections in the brain published the book Connectome: How the Brain’s Wiring is much more than the sum of their individ- Makes Us Who We Are. ual components. The genome is an entity it-self, as it is from the subtle gene interaction that [life] emerges. In a similar manner, one could consider the brain connectome, 4.2 The connectome at multiple set of all neuronal connections, as one sin- scales gle entity, thus emphasizing the fact that the huge brain neuronal communication capacity Brain networks can be defined at different levels of scale, and computational power critically relies on corresponding to levels of spatial resolution in brain imag- this subtle and incredibly complex connectiv- [13][14] [6] ing (Kötter, 2007, Sporns, 2010). These scales ity architecture. can be roughly categorized as microscale, mesoscale and macroscale. Ultimately, it may be possible to join con- Pathways through cerebral white matter can be charted nectomic maps obtained at different scales into a single by histological dissection and staining, by degeneration hierarchical map of the neural organization of a given methods, and by axonal tracing. Axonal tracing meth- species that ranges from single neurons to populations of ods form the primary basis for the systematic charting neurons to larger systems like cortical areas. Given the of long-distance pathways into extensive, species-specific methodological uncertainties involved in inferring con- anatomical connection matrices between gray matter re- nectivity from the primary experimental data, and given gions. Landmark studies have included the areas and con- that there are likely to be large differences in the connec- nections of the visual cortex of the macaque (Felleman tomes of different individuals, any unified map will likely and Van Essen, 1991)[8] and the thalamo-cortical system rely on probabilistic representations of connectivity data in the feline brain (Scannell et al., 1999).[9] The develop- (Sporns et al., 2005).[7] ment of neuroinformatics databases for anatomical con- Mapping the connectome at the “microscale” (microm- nectivity allow for continual updating and refinement of eter resolution) means building a complete map of the such anatomical connection maps. The online macaque [10] neural systems, neuron-by-neuron. The challenge of do- cortex connectivity tool CoCoMac (Kötter, 2004) is a ing this becomes obvious: the number of neurons com- prominent example of such a database. prising the brain easily ranges into the billions in more In the human brain, the significance of the connectome highly evolved organisms. The human cerebral cortex stems from the realization that the structure and function alone contains on the order of 1010 neurons linked by of the human brain are intricately linked, through mul- 1014 synaptic connections.[15] By comparison, the num- tiple levels and modes of brain connectivity. There are ber of base-pairs in a human genome is 3×109. A few of strong natural constraints on which neurons or neural pop- the main challenges of building a human connectome at ulations can interact, or how strong or direct their inter- the microscale today include: (1) data collection would actions are. Indeed, the foundation of human cognition take years given current technology; (2) machine vision lies in the pattern of dynamic interactions shaped by the tools to annotate the data remain in their infancy, and connectome. are inadequate; and (3) neither theory nor algorithms are 34 CHAPTER 4. CONNECTOME

readily available for the analysis of the resulting brain- cell populations and single axonal pathways. EM recon- graphs. To address the data collection issues, several struction was successfully used for the compilation of groups are building high-throughput serial electron mi- the C. elegans connectome (White et al., 1986).[2] How- croscopes (Kasthuri et al., 2009; Bock et al. 2011). To ever, applications to larger tissue blocks of entire nervous address the machine-vision and image-processing issues, systems have traditionally had difficulty with projections the Open Connectome Project is alg-sourcing (algorithm that span longer distances. outsourcing) this hurdle. Finally, statistical graph the- Recent advances in mapping neural connectivity at the ory is an emerging discipline which is developing sophis- cellular level offer significant new hope for overcoming ticated pattern recognition and inference tools to parse the limitations of classical techniques and for compiling these brain-graphs (Goldenberg et al., 2009). cellular connectome data sets (Livet et al., 2007; Licht- A “mesoscale” connectome corresponds to a spatial res- man et al., 2008).[18][19][20] Using Brainbow, a combi- olution of hundreds of micrometers. Rather than at- natorial color labeling method based on the stochastic tempt to map each individual neuron, a connectome at the expression of several fluorescent proteins, Lichtman and mesoscale would attempt to capture anatomically and/or colleagues were able to mark individual neurons with one functionally distinct neuronal populations, formed by lo- of over 100 distinct colors. The labeling of individual cal circuits (e.g. cortical columns) that link hundreds or neurons with a distinguishable hue then allows the trac- thousands of individual neurons. This scale still presents ing and reconstruction of their cellular structure including a very ambitious technical challenge at this time and can long processes within a block of tissue. only be probed on a small scale with invasive techniques In March 2011, the journal Nature published a pair of or very high field MRI on a local scale. articles on micro-connectomes: Bock et al.[5] and Brig- A connectome at the macroscale (millimeter resolution) gman et al.[4] In both articles, the authors first character- attempts to capture large brain systems that can be par- ized the functional properties of a small subset of cells, cellated into anatomically distinct modules (areas, parcels and then manually traced a subset of the processes em- or nodes), each having a distinct pattern of connectivity. anating from those cells to obtain a partial subgraph. In Connectomic databases at the mesoscale and macroscale alignment with the principles of open-science, the authors may be significantly more compact than those at cellular of Bock et al. (2011) have released their data for pub- resolution, but they require effective strategies for accu- lic access. The full resolution 12TB dataset from Bock rate anatomical or functional parcellation of the neural et al. is available at the Open Connectome Project. In volume into network nodes (for complexities see, e.g., 2012, a Citizen science project called EyeWire began at- Wallace et al., 2004).[16] tempting to crowdsource the mapping of the connectome through an interactive game.[21] Independently, important topologies of functional interactions among several 4.3 Mapping the connectome at the hundred cells are also gradually going to be declared (Shi- mono and Beggs, 2014).[22] Scaling up ultrastructural cir- cellular level cuit mapping to the whole mouse brain is currently underway (Mikula, 2012).[23] An alternative approach to Current non-invasive imaging techniques cannot capture mapping connectivity was recently proposed by Zador [17] the brain’s activity on a neuron-by-neuron level. Map- and colleagues (Zador et al., 2012). Zador’s technique, ping the connectome at the cellular level in vertebrates called BOINC (barcoding of individual neuronal connec- currently requires post-mortem microscopic analysis of tions) uses high-throughput sequencing to map neural cir- limited portions of brain tissue. Non-optical techniques cuits. Briefly, the approach consists of (1) labelling each that rely on high-throughput DNA sequencing have been neuron with a unique DNA barcode; (2) transferring bar- proposed recently by Anthony Zador (CSHL).[17] codes between synaptically coupled neurons (for example using PRV); and (3) fusion of barcodes to represent Traditional histological circuit-mapping approaches rely a synaptic pair. This approach has the potential to be on imaging and include light-microscopic techniques for cheap, fast, and extremely high-throughput. cell staining, injection of labeling agents for tract tracing, or chemical brain preservation, staining and reconstruction of serially sectioned tissue blocks via electron microscopy (EM). Each of these classical approaches has 4.4 Mapping the connectome at the specific drawbacks when it comes to deployment for connectomics. The staining of single cells, e.g. with the macro scale Golgi stain, to trace cellular processes and connectivity suffers from the limited resolution of light-microscopy Established methods of brain research, such as axonal as well as difficulties in capturing long-range projections. tracing, provided early avenues for building connectome Tract tracing, often described as the “gold standard” of data sets. However, more recent advances in living sub- neuroanatomy for detecting long-range pathways across jects has been made by the use of non-invasive imaging the brain, generally only allows the tracing of fairly large technologies such as diffusion magnetic resonance imag- 4.5. MAPPING FUNCTIONAL CONNECTIVITY TO COMPLEMENT ANATOMICAL CONNECTIVITY 35 ing and functional magnetic resonance imaging (fMRI). 4.4.2 Primary challenge for macroscale The first, when combined with tractography allows recon- connectomics: determining parcella- struction of the major fiber bundles in the brain. The sec- tions of the brain ond allows the researcher to capture the brain’s network activity (either at rest or while performing directed tasks), The initial explorations in macroscale human connec- enabling the identification of structurally and anatomi- tomics were done using either equally sized regions or cally distinct areas of the brain that are functionally con- anatomical regions with unclear relationship to the un- nected. derlying functional organization of the brain (e.g. gyral Notably, the goal of the Human Connectome Project, led and sulcal-based regions). While much can be learned by the WU-Minn consortium, is to build a structural and from these approaches, it is highly desirable to parcel- functional map of the healthy human brain at the macro late the brain into functionally distinct parcels: brain re- scale, using a combination of multiple imaging technolo- gions with distinct architectonics, connectivity, function, gies and resolutions. and/or topography (Felleman and Van Essen, 1991).[25] Accurate parcellation allows each node in the macroscale connectome to be more informative by associating it with a distinct connectivity pattern and functional profile. Parcellation of localized areas of cortex have been 4.4.1 Recent advances in connectivity accomplished using diffusion tractography (Beckmann et mapping al. 2009)[26] and functional connectivity (Nelson et al. 2010)[27] to non-invasively measure connectivity patterns and define cortical areas based on distinct connectivity patterns. Such analyses may best be done on a whole brain scale and by integrating non-invasive modalities. Accurate whole brain parcellation may lead to more accurate macroscale connectomes for the normal brain, which can then be compared to disease states.

4.5 Mapping functional connectivity to complement anatomical connectivity

Using functional MRI (fMRI) in the resting state and during tasks, functions of the connectome circuits are being studied.[28] Just as detailed road maps of the earth’s surface do not tell us much about the kind of vehicles that travel those roads or what cargo they are hauling, to understand how neural structures result in specific functional behavior such as consciousness, it is neces- sary to build theories that relate functions to anatomical connectivity.[29] However, the bond between struc- Tractographic reconstruction of neural connections via DTI tural and functional connectivity is not straightforward. Computational models of whole-brain network dynamics Over the past few years, several investigators have at- are valuable tools to investigate the role of the anatomi- [30][31] tempted to map the large-scale structural architecture of cal network in shaping functional connectivity. In the human cerebral cortex. One attempt exploited cross- particular, computational models can be used to predict [32][33] correlations in cortical thickness or volume across indi- the dynamic effect of lesions in the connectome. viduals (He et al., 2007).[24] Such gray-matter thickness correlations have been postulated as indicators for the presence of structural connections. A drawback of the 4.6 The connectome as a network approach is that it provides highly indirect information about cortical connection patterns and requires data from or graph large numbers of individuals to derive a single connection data set across a subject group. Other investigators have The connectome can be studied as a network by means attempted to build whole-brain connection matrices from of network science and graph theory. In case of a micro- diffusion imaging data. scale connectome, the nodes of this network (or graph) 36 CHAPTER 4. CONNECTOME are the neurons, and the edges correspond to the synapses of the brain graphs of multiple subjects. It is possible between those neurons. For the macro-scale connectome, to build a consensus graph such the Budapest Reference the nodes correspond to the ROIs (regions of interest), Connectome by allowing only edges that are present in at while the edges of the graph are derived from the ax- least k connectomes, for a selectable k parameter. The ons interconnecting those areas. Thus connectomes are Budapest Reference Connectome has led the researchers sometimes referred to as brain graphs, as they are indeed to the discovery of the Consensus Connectome Dynam- graphs in a mathematical sense which describe the con- ics of the human brain graphs. The edges appeared in nections in the brain (or, in a broader sense, the whole all of the brain graphs form a connected subgraph around nervous system). the brainstem. By allowing gradually less frequent edges, One group of researchers (Iturria-Medina et al., 2008)[34] this core subgraph grows continuously, as a shrub. The growth dynamics may reflect the individual brain devel- has constructed connectome data sets using diffusion tensor imaging (DTI)[35][36] followed by the derivation of opment and provide an opportunity to direct some edges of the human consensus brain graph.[46] average connection probabilities between 70-90 cortical and basal brain gray matter areas. All networks were The possible causes of the difference between individual found to have small-world attributes and “broad-scale” connectomes were also investigated. It has been found degree distributions. An analysis of betweenness central- that the macro-scale connectomes of women contain sig- ity in these networks demonstrated high centrality for the nificantly more edges than those of men, and a larger precuneus, the insula, the superior parietal and the supe- portion of the edges in the connectomes of women run rior frontal cortex. Another group (Gong et al. 2008)[37] between the two hemispheres.[47][48] In addition, con- has applied DTI to map a network of anatomical connec- nectomes generally exhibit a small-world character, with tions between 78 cortical regions. This study also identi- overall cortical connectivity decreasing with age.[49] The fied several hub regions in the human brain, including the aim of the as of 2015 ongoing HCP Lifespan Pilot Project precuneus and the superior frontal gyrus. is to identify connectome differences between 6 age Hagmann et al. (2007)[38] constructed a connection groups (4–6, 8–9, 14–15, 25–35, 45–55, 65–75). matrix from fiber densities measured between homoge- More recently, connectograms have been used to visu- neously distributed and equal-sized regions of interest alize full-brain data by placing cortical areas around a (ROIs) numbering between 500 and 4000. A quanti- circle, organized by lobe.[50][51] Inner circles then depict tative analysis of connection matrices obtained for ap- cortical metrics on a color scale. White matter fiber con- proximately 1000 ROIs and approximately 50,000 fiber nections in DTI data are then drawn between these cor- pathways from two subjects demonstrated an exponential tical regions and weighted by fractional anisotropy and (one-scale) degree distribution as well as robust small- strength of the connection. Such graphs have even been world attributes for the network. The data sets were de- used to analyze the damage done to the famous traumatic rived from diffusion spectrum imaging (DSI) (Wedeen, brain injury patient Phineas Gage.[52] [39] [40][41] 2005), a variant of diffusion-weighted imaging Statistical graph theory is an emerging discipline which that is sensitive to intra-voxel heterogeneities in diffusion is developing sophisticated pattern recognition and infer- directions caused by crossing fiber tracts and thus allows ence tools to parse these brain graphs (Goldenberg et al., more accurate mapping of axonal trajectories than other 2009). diffusion imaging approaches (Wedeen, 2008).[42] The combination of whole-head DSI datasets acquired and processed according to the approach developed by Hag- mann et al. (2007)[38] with the graph analysis tools con- 4.7 See also ceived initially for animal tracing studies (Sporns, 2006; Sporns, 2007)[43][44] allow a detailed study of the network structure of human cortical connectivity (Hagmann et al., • Outline of brain mapping 2008).[45] The human brain network was characterized using a broad array of network analysis methods includ- • Outline of the human brain ing core decomposition, modularity analysis, hub classifi- cation and centrality. Hagmann et al. presented evidence • Human Connectome Project for the existence of a structural core of highly and mu- tually interconnected brain regions, located primarily in posterior medial and parietal cortex. The core comprises • Drosophila connectome portions of the posterior cingulate cortex, the precuneus, the cuneus, the paracentral lobule, the isthmus of the cin- • List of animals by number of neurons gulate, the banks of the superior temporal sulcus, and the inferior and superior parietal cortex, all located in both • Interactome cerebral hemispheres.

A subﬁeld of connectomics deals with the comparison • Budapest Reference Connectome 4.8. REFERENCES 37

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ONE. 2 (10): e1049. Bibcode:2007PLoSO...2.1049S. 4.9 External links doi:10.1371/journal.pone.0001049. PMC 2013941 . PMID 17940613. • Open Connectome Project • [45] Hagmann, Patric; Cammoun, Leila; Gigandet, Xavier; The Connectome Project at Harvard Meuli, Reto; Honey, Christopher J.; Wedeen, Van J.; • The official site for the NIH-sponsored Human Con- Sporns, Olaf (2008). Friston, Karl J., ed. “Mapping the nectome Project Structural Core of Human Cerebral Cortex”. PLoS Bi- ology. 6 (7): e159. doi:10.1371/journal.pbio.0060159. • The NITRC Human Connectome Project Site PMC 2443193 . PMID 18597554. • Connectome Research by EPFL/CHUV, Lausanne, [46] Kerepesi, Csaba; et al. (2016). “How to Direct the Switzerland Edges of the Connectomes: Dynamics of the Consensus • Connectomes and the Development of the Connections Database of hundreds of braingraphs with different in the Human Brain”. PLOS ONE. 11 (6): e0158680. resolutions and weight functions at braingraph.org doi:10.1371/journal.pone.0158680. PMC 4928947 . • The NIH Blueprint for Neuroscience Research PMID 27362431. • The Budapest Reference Connectome Server [47] Ingalhalikar, M.; Smith, A.; Parker, D.; Satterthwaite, T. D.; Elliott, M. A.; Ruparel, K.; Hakonarson, H.; • Connectome Research led by Dr. Shawn Mikula Gur, R. E.; Gur, R. C.; Verma, R. (2013). “Sex • differences in the structural connectome of the human TED talk by Sebastian Seung: I am my connectome brain”. Proceedings of the National Academy of Sciences. • EyeWire, a citizen science game to map the retinal 111 (2): 823–828. Bibcode:2014PNAS..111..823I. connectome doi:10.1073/pnas.1316909110. ISSN 0027-8424. PMC 3896179 . PMID 24297904. • Connectome on Scholarpedia • [48] Szalkai, Balázs; Varga, Bálint; Grolmusz, Vince (2015). MITK Diffusion: Free software for the processing “Graph Theoretical Analysis Reveals: Women’s Brains of diffusion-weighted MR data including connec- Are Better Connected than Men’s”. PLOS ONE. 10 (7): tomics e0130045. doi:10.1371/journal.pone.0130045. ISSN 1932-6203. PMC 4488527 . PMID 26132764.

[49] Gong, G.; Rosa-Neto, P.; Carbonell, F.; Chen, Z. J.; He, Y.; Evans, A. C. (2009). “Age- and Gender- Related Diﬀerences in the Cortical Anatomical Net- work”. Journal of Neuroscience. 29 (50): 15684–15693. doi:10.1523/JNEUROSCI.2308-09.2009. ISSN 0270- 6474.

[50] Irimia A, Chambers MC, Torgerson CM, Van Horn JD (April 2012). “Circular representation of human cortical networks for subject and population-level connectomic visualization”. NeuroImage. 60 (2): 1340– 51. doi:10.1016/j.neuroimage.2012.01.107. PMC 3594415 . PMID 22305988.

[51] Irimia A, Chambers MC, Torgerson CM, et al. (2012). “Patient-tailored connectomics visualization for the assessment of white matter atrophy in traumatic brain injury”. Frontiers in Neurology. 3: 10. doi:10.3389/fneur.2012.00010. PMC 3275792 . PMID 22363313.

[52] Van Horn JD, Irimia A, Torgerson CM, Chambers MC, Kikinis R, Toga AW (2012). “Mapping connectivity damage in the case of Phineas Gage”. PLOS ONE. 7 (5): e37454. Bibcode:2012PLoSO...737454V. doi:10.1371/journal.pone.0037454. PMC 3353935 . PMID 22616011. Chapter 5

Exposome

The exposome encompasses the totality of human en- More recently, G.W. Miller and D. P. Jones proposed vironmental (i.e. non-genetic) exposures from con- a revised definition of the exposome that explicitly in- ception onwards, complementing the genome. It was corporates the body’s response to environmental influ- first proposed by Dr. Christopher Wild, a cancer epi- ences and also includes the endogenous metabolic pro- demiologist, in a 2005 article entitled “Complement- cesses that can alter or process the chemicals to which ing the Genome with an “Exposome”: The Outstanding humans are exposed.[17] This definition is explained in Challenge of Environmental Exposure Measurement in greater depth in the new book by G.W. Miller entitled Molecular Epidemiology”.[1] The concept of the expo- “The Exposome: A Primer” published by Elsevier in late some and how to assess it has led to lively discussions 2013.[18] This introductory text is the first book on the with varied views.[2][3][4][5][6][7][8][9][10][11] Although at exposome and explores the gene versus environmental this stage it may not be possible to measure or model argument.[19] the full exposome, some recent European projects such as For complex disorders specific genetic causes appear to HELIX,[8][12] EXPOsOMICS,[13][14] and HEALS[15] and [16] only account for 10-30% of the disease incidence, al- the American initiative HERCULES have started to though as genomic approaches improve this percentage make first attempts. could increase. Environmental influences contribute to human disease, but unlike with genetics, there is no standard or systematic way to measure the influence of en- 5.1 Background vironmental exposures. Some studies, such as those by C. J. Patel et al into the interaction of genetic and en- In his 2005 article Wild stated, “At its most complete, the vironmental factors in the incidence of diabetes have exposome encompasses life-course environmental expo- demonstrated that environmental-wide association stud- sures (including lifestyle factors), from the prenatal pe- ies (EWAS, or exposome-wide association studies) may riod onwards.” The concept was first proposed to draw be feasible.[20][21] However, it is not clear what data sets attention to the need for better and more complete envi- are most appropriate to represent the value of “E”.[22] ronmental exposure data for etiologic research, in order to balance the investment, tools and knowledge in genetics. Wild also stated that even incomplete versions of the 5.2 Research initiatives exposome could be of great use to field of epidemiology. Wild published a follow-up paper in 2012 where he outlines methods, including personal sensors, biomarkers In 2012, the European Commission awarded two large- [23] and 'omics' technologies, to better define the exposome.[4] grants to pursue exposome-related research. The HE- He has described three overlapping domains within the LIX project at the Barcelona-based Centre for Research exposome: in Environmental Epidemiology will attempt to develop an early life exposome, noting that the first exposures occur during development.[8][12] It will build upon six ex- 1. a general external environment including the urban isting birth cohorts across Europe and “measure the ex- environment, education, climate factors, social cap- posome” at key prenatal and early childhood time points, ital, stress, through the use of GIS, personal sensors, biomarkers and 2. a specific external environment with specific omics platforms. The second project, Exposomics, led contaminants, radiation, infections, lifestyle factors by Paolo Vineis, is a consortium based at Imperial Col- [24] (e.g. tobacco, alcohol), diet, physical activity, etc. lege London. This project will use smartphones that utilize GPS and environmental sensors to assess expo- 3. an internal environment to include internal biologi- sures. Another major initiative that started in late 2013 cal factors such as metabolic factors, hormones, gut is the Health and Environment-Wide Associations based microflora, inflammation, oxidative stress. on Large Scale population Surveys or HEALS. Touted

40 5.5. SEE ALSO 41 as the largest environmental health-related study in Eu- ness of the exposome and its unique influence on molec- rope, HEALS proposes to reverse the paradigm of “na- ular pathologic processes (including alterations in the ture versus nurture” and adopt one defined by complex interactome) in each individual.[34] This principle was and dynamic interactions between DNA sequence, epi- first described in neoplastic diseases as “the unique tu- genetic DNA modifications, gene expression and envi- mor principle”.[35] Based on this unique disease princi- ronmental factors that all combine to influence disease ple, interdisciplinary field of molecular pathological epi- phenotypes.[25] demiology (MPE) emerged as an integration of molecular [36] In the US, the National Academy of Sciences hosted a pathology and epidemiology. Because heterogeneity of exposome, disease etiology, and pathogenesis is a ubiq- meeting in December 2011 entitled “Emerging Tech- nologies for Measuring Individual Exposomes.”[26] A uitous phenomenon, the MPE paradigm will become in- herent in epidemiology and population health science. Centers for Disease Control and Prevention overview “Exposome and Exposomics” outlines the three pri- ority areas for researching the occupational exposome as identified by the National Institute for Occupa- 5.5 See also tional Safety and Health.[10] The National Institutes of Health (NIH) has made investments in technolo- • Envirome gies that support exposome-related research, including • biosensors, and supports research on gene-environment Epidemiology [27][28] interactions. In May, 2013, the National Institute • Human Toxome Project[37] of Environmental Health Sciences (NIEHS) awarded a Core Center Grant to Emory University that is focused • Molecular epidemiology on the exposome.[29] • Molecular pathological epidemiology • Public health 5.3 Proposed Human Exposome Project (HEP) 5.6 References The idea of a Human Exposome Project, analogous to [1] Wild, CP (Aug 2005). “Complementing the genome with the Human Genome Project, has been proposed and an “exposome": the outstanding challenge of environmen- discussed in numerous scientific meetings, but no such tal exposure measurement in molecular epidemiology.”. project exists at this time. Several investigators have ar- Cancer Epidemiology, Biomarkers & Prevention. 14 (8): gued that such an initiative should occur, but given the 1847–50. doi:10.1158/1055-9965.EPI-05-0456. PMID lack of clarity on how science would go about pursuing 16103423. such a project, support has been lacking.[30] Reports on the issue include: [2] Rappaport SM, Smith MT (2010). “Epidemiology. En- vironment and disease risks”. Science. 330 (6003): 460– 461. doi:10.1126/science.1192603. • A 2011 review on the exposome and exposure science by Paul Lioy and Stephen Rappaport, “Expo- [3] Rappaport SM (2011). “Implications of the exposome for sure science and the exposome: an opportunity for exposure science”. J Expo Sci Environ Epidemiol. 21 (1): coherence in the environmental health sciences” was 5–9. doi:10.1038/jes.2010.50. published in the journal Environmental Health Per- [31] [4] Wild, CP (Feb 2012). “The exposome: from concept to spectives. utility”. International Journal of Epidemiology. 41 (1): • “Exposure Science in the 21st Century: A Vi- 24–32. doi:10.1093/ije/dyr236. PMID 22296988. sion and A Strategy”, an official report from [5] Peters A, Hoek G, Katsouyanni K (2012). “Understand- the United States National Research Council pub- ing the link between environmental exposures and health: lished in September 2012 outlines the challenges does the exposome promise too much?". Epidemiol Com- in implementing systematic evaluation of the munity Health. 66: 103–105. doi:10.1136/jech-2011- exposome.[32][33] 200643. [6] Buck Louis GM, Sundaram R (2012). “Exposome: time for transformative research”. Stat Med. 31 (22): 2569–75. 5.4 Related fields doi:10.1002/sim.5496.

[7] Buck Louis G. M.; Yeung E.; Sundaram R.; Laughon S. The concept of exposome contributes to a new paradigm K.; Zhang C. (2013). “The Exposome – Exciting Oppor- in disease phenotype. Essentially, an individual has a tunities for Discoveries in Reproductive and Perinatal Epi- unique disease process diﬀerent from any other individ- demiology”. Paediatric and Perinatal Epidemiology. 27: ual (“the unique disease principle”), considering unique- 229–236. doi:10.1111/ppe.12040. 42 CHAPTER 5. EXPOSOME

[8] Vrijheid M, Slama R, Robinson O, Chatzi L, Coen M, [27] “NIEHS Gene-Environment studies”. Retrieved 21 Jan- et al. “The Human Early-Life Exposome (HELIX): uary 2013. Project Rationale and Design”. Environ Health Perspect. doi:10.1289/ehp.1307204. [28] “Genes and Environment Initiative”. Retrieved 21 Jan- uary 2013. [9] Miller Gary W.; Jones Dean P (2014). “The Na- ture of Nurture: Reﬁning the Deﬁnition of the Ex- [29] “Emory HERCULES Exposome Center”. Retrieved 15 posome”. Toxicological Sciences. 137 (1): 1–2. January 2014. doi:10.1093/toxsci/kft251. PMID 24213143. [30] Arnaud, Celia Henry (16 August 2010). “Exposing The Exposome”. Chemical & Engineering News, Vol. 88, No. [10] Centers for Disease Control and Prevention (2012). 33, pp. 42-44. American Chemical Society. Retrieved 5 “Exposome and Exposomics”. Retrieved 5 March 2013. March 2013. [11] Porta M, editor. Greenland S, Hernán M, dos Santos Silva [31] Lioy, PJ; Rappaport, SM (Nov 2011). “Exposure science I, Last JM, associate editors (2014). A dictionary of epi- and the exposome: an opportunity for coherence in the en- demiology, 6th. edition. New York: Oxford University vironmental health sciences”. Environmental Health Per- Press. ISBN 9780199976737 spectives. 119 (11): A466–7. doi:10.1289/ehp.1104387. [12] Home - HELIX | Building the early life exposome PMC 3226514 . PMID 22171373.

[13] Callaway E (2012) Daily dose of toxics to be tracked. Na- [32] “NRC report supports NIEHS vision of the exposome”. ture.vol 491. 19 November 2012 Retrieved 21 January 2013.

[14] About Exposomics | Exposome [33] “Exposure Science in the 21st Century: A Vision and a Strategy”. Retrieved 21 January 2013. [15] HEALS [34] Ogino S, Lochhead P, Chan AT, Nishihara R, Cho E, [16] Hercules Exposome Research Center | Emory University Wolpin BM, Meyerhardt AJ, Meissner A, Schernhammer ES, Fuchs CS, Giovannucci E (2013). “Molecular patho- [17] Miller Gary W.; Jones Dean P. (January 2014). “The logical epidemiology of epigenetics: emerging integrative Nature of Nurture: Reﬁning the Deﬁnition of the science to analyze environment, host, and disease”. Mod Exposome”. Toxicological Sciences. 137 (1): 1–2. Pathol. 26: 465–484. doi:10.1038/modpathol.2012.214. doi:10.1093/toxsci/kft251. PMID 24213143. PMC 3637979 . PMID 23307060.

[18] “The Exposome: A Primer by Elsevier”. Retrieved 16 [35] Ogino S, Fuchs CS, Giovannucci E (2012). “How many January 2014. molecular subtypes? Implications of the unique tumor principle in personalized medicine”. Expert Rev Mol [19] “G x E = ?". Retrieved 16 January 2014. Diagn. 12: 621–628. doi:10.1586/erm.12.46. PMC [20] Patel, CJ; Bhattacharya, J; Butte, AJ (May 20, 2010). 3492839 . PMID 22845482. “An Environment-Wide Association Study (EWAS) on type 2 diabetes mellitus.”. PLoS ONE. 5 (5): e10746. [36] Ogino S, Stampfer M (2010). “Lifestyle factors and microsatellite instability in colorectal cancer: the evolving doi:10.1371/journal.pone.0010746. PMC 2873978 . field of molecular pathological epidemiology”. J Natl PMID 20505766. Cancer Inst. 102: 365–367. doi:10.1093/jnci/djq031. [21] Patel, CJ; Chen, R; Kodama, K; Ioannidis, JP; Butte, AJ PMC 2841039 . PMID 20208016. (Jan 20, 2013). “Systematic identification of interaction [37] “Human Toxome Project”. Retrieved 21 June 2013. effects between genome- and environment-wide associations in type 2 diabetes mellitus” (PDF). Human Genet- ics. 132 (5): 495–508. doi:10.1007/s00439-012-1258-z. PMID 23334806. Retrieved 4 March 2015.

[22] Smith Martyn T.; Rappaport Stephen M. (August 2009). “Building Exposure Biology Centers to Put the E into “G × E” Interaction Studies”. Environmental Health Per- spectives. 117 (8): A334–A335. doi:10.1289/ehp.12812. PMC 2721881 . PMID 19672377.

[23] Callaway, Ewen (27 November 2012). “Daily dose of toxics to be tracked”. Nature. Retrieved 4 March 2013.

[24] “Imperial College News.”. Retrieved 21 January 2013.

[25] “HEALS-EU.”. Retrieved 16 January 2014.

[26] “National Academy of Sciences meeting”. Retrieved 21 January 2013. Chapter 6

Built environment

beautification process included parks and architectural design.[5] By mid-century modernist “indifferent” design influenced the character of work and public spaces, followed by what Alexander describes as a late twentieth century “revival of interest relating to the concept of place (including the built environment), and its relevance to mental health and other fields of study.”[6]

6.2 Modern built environment

Currently built environments are typically used to describe the interdisciplinary field that addresses the design, construction, management, and use of these man-made Part of the built environment: suburban tract housing in Colorado Springs, Colorado. surroundings as an interrelated whole as well as their relationship to human activities over time (rather than a particular element in isolation or at a single moment in time). In social science, the term built environment refers to The field is generally not regarded as a traditional profes- the man-made surroundings that provide the setting for sion or academic discipline in its own right, instead draw- human activity, ranging in scale from buildings and parks. ing upon areas such as economics, law, public policy, pub- It has been defined as “the humanitarian-made space in lic health, management, geography, design, engineering, which people live, work, and recreate on a day-to-day technology, and environmental sustainability. Within the basis.”[1] The “built environment encompasses places and field of public health, built environments are referred to spaces created or modified by people including buildings, as building or renovating areas in an effort to improve the parks, and transportation systems.” In recent years, public community’s well-being through construction of “aesthet- health research has expanded the definition of “built envi- ically, health improved, and environmentally improved ronment” to include healthy food access, community gar- landscapes and living structures”.[7] For example; com- dens,mental health,[2] "walkability" and "bikeability.”[3] munity forest user group in Nepal is multidimensional in- stitution, which serves goods and services to the communities through natural resource management. 6.1 History

Early concepts of built environment date to Classical An- 6.3 Public health tiquity: Hippodamus of Miletos, known as the “father of urban planning,” developed Greek cities from 498 BC to 408 BC that created order by using grid plans that In public health, built environment refers to physical en- mapped the city. These early city plans eventually gave vironments that are designed with health and wellness as way to the City Beautiful movement in the late 1800s integral parts of the communities. Research has indi- and early 1900s, inspired by Daniel Hudson Burnham, cated that the way neighborhoods are created can affect both the physical activity and mental health of the com- a reformist for the Progressivism movement who actively [8] promoted “a reform of the landscape in tandem with po- munities’ residents. Studies have shown that built envi- litical change.”[4] The effort was in partnership with oth- ronments that were expressly designed to improve physical activity are linked to higher rates of physical activity, ers who believed that beautifying American cities would [9] improve the moral compass of the cities and encour- which in turn, positively affects health. age the upper class to spend their money in cities. This Neighborhoods with more walkability had lower rates of

43 44 CHAPTER 6. BUILT ENVIRONMENT

stores has been associated with obesity in children.[13] In contrast, improved access to community supermar- kets and farmer’s markets is correlated with lower overweight status.[14] Specifically in low income neighborhoods, the presence of a local grocery store is correlated with lower BMI/overweight risk.[15] Community gardens are also considered a part of the built environment, and have been shown to increase fruit and vegetable intake among gardeners.[16] Scholars say that community gardens have also been shown to have positive social and psychological impacts that lead to lower levels of stress, hypertension, and an improved sense of wellness, affect- ing the overall health of the individual and the commu- A separated bike lane in New York City. nity. The intersection of public health with other disciplines is obesity as well as increased physical activity among its evident in the design process of built environments which residents. They also had lower rates of depression, higher includes environmental planning, policy development and social capital, and less alcohol abuse. Walkability fea- land-use planning.[1] Research suggests that people are tures in these neighborhoods include safety, sidewalk con- more active in mixed-use communities or those that in- struction, as well as destinations in which to walk.[8] In corporate retail and residential and densely populated ar- addition, the perception of a walkable neighborhood, one eas as well as those with good street connectivity.[17] that is perceived to have good sidewalks and connectivity, Those who preferred to walk and live in walkable en- is correlated with higher rates of physical activity.[9] vironments often have lower obesity rates and drive less over those who preferred living in auto-dependent Assessments of walkability have been completed through environments.[18] The strength of the evidence for reduc- the use of GIS programs. One such program, Street ing obesity through environment has been highlighted by Smart Walk Score, is a walkability assessment tool which the Center for Disease Control in its Common Commu- determines distances to grocery stores and other ameni- nity Measures for Obesity Prevention Project, which in- ties, as well as connectivity and intersection frequency cludes measures of healthy food access and physical ac- using specific addresses.[10] Assessments such as Street tivity environments.[19] Smart Walk Score can be utilized by city and country planning departments to improve existing walkability of communities. 6.4 Landscape architecture

In landscape architecture, the built environment is understood to mean a human-made landscape, as distinguished from the natural environment; for example, a city park is a built environment.

6.5 See also

• Center for the Built Environment • City planning

A community garden located in Montreal, Canada. • Environmental psychology • Environmental sustainability Public health also addresses additional components of built environments including “bikeability” and healthy • International Association of People-Environment food access such as proximity to grocery stores and Studies community gardens. Bikeability refers to the access that • National Building Museum an area has granted to safe biking through multiple bike paths and bike lanes.[11] Both walkability and bikeability • Natural environment have been cited as determinants of physical activity.[12] • Public health Access to healthy food is also an important component of the built environment. A higher density of convenience • Social environment 6.7. FURTHER READING 45

• Urbanism [15] Zick, C; Smith, K; Fan, J; Brown, B; Yamada, I; Kowaleski-Jones, L (2009). “Running to the store? The • Urban planning relationship between neighborhood environments and the risk of obesity”. Soc Sci Med. 69: 1493–500. • Vernacular architecture • [16] Litt, J; Soobader, M; Turbin, M; Hale, J; Buchenau, M; Microbiomes of the built environment Marshall, J (2011). “The inﬂuence of social involvement, neighborhood aesthetics, and community garden partici- pation on fruit and vegetable consumption”. Am J Public 6.6 References Health. 101: 1466–73. doi:10.2105/ajph.2010.300111. PMID 21680931.

[1] Roof, K; Oleru N. (2008). “Public Health: Seattle and [17] Heath, G; Brownson, R; Kruger, J; et al. (2006). “The King County’s Push for the Built Environment.”. J Envi- eﬀectiveness of urban design and land use and transport ron Health. 71: 24–27. policies and practices to increase physical activity: a systematic review”. J Phys Act Health. 3: S55–S76. [2] Assari, A Birashk, B Nik, M Mousavi Naghdbishi, R (2016). “IMPACT OF BUILT ENVIRONMENT ON [18] Frank, L; Saelens, B; Powell, K; Chapmen, J (2007). MENTAL HEALTH: REVIEW OF TEHRAN CITY IN “Stepping towards causation: Do built environments or IRAN” (PDF). International Journal on Technical and neighborhood and travel preferences explain physical ac- Physical Problems of Engineering. 8(26): 81–87 – via tivity, driving, and obesity?". Social Science & Medicine. IJTPE. 65: 1898–1914. doi:10.1016/j.socscimed.2007.05.053.

[3] Lee, V; Mikkelsen, L; Srikantharajah, J; Cohen, L. [19] Kahn, LK; Sobush K; Keener D; et al. (2009). “Recom- “Strategies for Enhancing the Built Environment to Sup- mended community strategies and measurements to pre- port Healthy Eating and Active Living”. Prevention Insti- vention obesity in the United States”. MMWR Recomm tute. Retrieved 29 April 2012. Rep. 58: 1–26. [4] “The City Beautiful Movement”. Retrieved 26 April 2012. 6.7 Further reading [5] “Architecture: The City Beautiful Movement”. Retrieved 22 April 2012. • Jackson, Richard J.; Dannenberg, Andrew L.; [6] Alexander, Donald (2008). “Physical determinism, mod- Frumkin, Howard (2013). “Health and the Built ernism and mental health”. Environments (35 no.3). Environment: 10 Years After”. American Jour- [7] “The Built Environment and Health: 11 Proﬁles of Neigh- nal of Public Health. 103 (9): 1542–1544. borhood Transformation”. Retrieved 12 April 2012. doi:10.2105/ajph.2013.301482.

[8] Renalds, A; Smith, T; Hale, P (2010). “A Sys- • Leyden, Kevin M (2003). “Social Capital tematic Review of Built Environment and Health”. and the Built Environment: The Importance Family and Community Health. 33: 68–78. of Walkable Neighborhoods” (PDF). American doi:10.1097/fch.0b013e3181c4e2e5. Journal of Public Health. 93: 1546–1551. doi:10.2105/ajph.93.9.1546. [9] Carlson, C; Aytur, S; Gardner, K; Rogers, S (2012). “Complexity in Built Environment, Health, and Destina- • Jeb Brugmann, Welcome to the urban revolution: tion Walking: A Neighborhood-Scale Analysis”. J Urban how cities are changing the world, Bloomsbury Health. 89: 270–84. doi:10.1007/s11524-011-9652-8. Press, 2009 [10] “Walk Score Methodology” (PDF). Retrieved 30 March • Jane Jacobs, The Death and Life of Great American 2012. Cities, Random House, New York, 1961 [11] Horacek, TM; White AA; Greene GW; et al. (2012). • “Sneakers and spokes: an assessment of the walkability Andrew Knight & Les Ruddock, Advanced Re- and bikeability of U.S. postsecondary institutions”. J En- search Methods in the Built Environment, Wiley- viron Health. 74: 8–15. Blackwell 2008

[12] Cochrane, T; Davey, R (2008). “Increasing uptake of • Paul Chynoweth, The Built Environment Interdis- physical activity: A social ecological approach”. J R Soc cipline: A Theoretical Model for Decision Makers Promot Health. 128: 31–40. in Research and Teaching, Proceedings of the CIB Working Commission (W089) Building Education [13] Grafova, I (2008). “Overweight Children: Assessing The Contribution Of The Built Environment.”. Prev Med. 47: and Research Conference, Kowloon Sangri-La Ho- 304–308. doi:10.1016/j.ypmed.2008.04.012. tel, Hong Kong, 10 - 13 April 2006.

[14] Rahman, T; Cushing RA; Jackson RJ (2011). “Contribu- • Richard J. Jackson with Stacy Sinclair, Designing tions of built environment to childhood obesity”. Mt Sinai Healthy Communities, Jossey-Bass, San Francisco, J Med. 78: 49–57. doi:10.1002/msj.20235. 2012 46 CHAPTER 6. BUILT ENVIRONMENT

• Russell P. Lopez, The Built Environment and Public Health, Jossey-Bass, San Francisco, 2012

6.8 External links

• Australian Sustainable Built Environment Council (ASBEC)

• Faculty of Built Environment, UTM, Skudai, Johor, Malaysia

• Designing Healthy Communities, link to nonproﬁt organization and public television documentary of same name • The Built Environment and Health: 11 Proﬁles of Neighborhood Transformation Chapter 7

Microbiomes of the built environment

Microbiomes of the built environment [1][2] is a ﬁeld of • “Indoor bacterial communities often originate from inquiry focusing on the study of the communities of mi- indoor sources.” croorganisms found in human constructed environments (i.e., the built environment). It is also sometimes referred • “Humans are also major sources of bacteria to in- to as “microbiology of the built environment”. door air”

This field encompasses studies of any kind of microor- • “Building design and operation can influence indoor ganism (e.g. bacteria, archaea, viruses, various microbial microbial communities.” eukaryotes including yeasts, and others sometimes generally referred to as protists) and studies of any kind of built environment such as buildings, vehicles, and water The microbiomes of the built environment are being stud- systems. ied for multiple reasons including how they may impact the health of humans and other organisms occupying the Some key highlights emphasizing the growing importance built environment but also some non health reasons such of the field include: as diagnostics of building properties, for forensic application, impact on food production, impact on built envi- • The field has accelerated somewhat in recent years, ronment function, and more. with significant funding from the Alfred P. Sloan Foundation.[3] and with the increase attention being given to microbiomes and communities of microbes 7.1 Types of Built Environments generally. For Which Microbiomes Have • The National Academies of Sciences, Engineering, and Medicine of the USA is conducting a study of Been Studied the this field with the study entitled "Microbiomes of the Built Environment: From Research to Appli- Extensive research has been conducted on individual mi- cation". crobes found in the built environment. More recently there has been a significant expansion in the number of • The American Association for the Advancement of studies that are examining the communities of microbes [4] Science ran a symposium on the topic in 2014. (i.e., microbiomes) found in the built environment. Such studies of microbial communities in the built environ- • The American Academy of Microbiology had ment have covered a wide range of types of built envi- a colloquium on this topic in September 2015 ronments including those listed below. and published a report “Microbiology of Built Environments”.[5] Buildings. Examples include homes,[7][8][9] dormitories,[10] offices,[11][12] hospitals,[13][14][15] [16][17][18] [19] [20][21] A 2016 paper by Brent Stephens [6] highlights some of operating rooms, NICUs, classrooms, the key findings of studies of “microbiomes of the indoor transportation facilities such as train and subway stations,[22][23] food production facilities [24] (e.g. environment”. These key findings include those listed be- [25] [26][27] low: dairies, wineries, cheesemaking facilities, sake breweries [28] and beer breweries [29]), aquaria,[30] libraries,[31] cleanrooms,[32][33] zoos, animal shelters, • “Culture-independent methods reveal vastly greater farms, and hicken coops and housing.[34] microbial diversity compared to culture-based [35] methods” Vehicles. Examples include airplanes, ships, tains,[23] automobiles [36] and space vehicles includ- • “Indoor spaces often harbor unique microbial coming International Space Station,[37] MIR,[38] the Mars munities” Odyssey,[39] the Herschel Spacecraft.[40]

47 48 CHAPTER 7. MICROBIOMES OF THE BUILT ENVIRONMENT

Water Systems. Examples include shower heads,[41] chil- Mental health. In a 2015 review Hoisington et al. dis- dren’s paddling pools,[42] municipal water systems,[43] cuss possible connections between the microbiology of drinking water and premise plumbing systems the built environment and human health.[62] The concept [44][45][46][47] and saunas.[48] presented in this paper is that more and more evidence is Other. Examples include art and cultural heritage accumulating that the human microbiome has some im- items,[49] clothing,[50] and household appliances such as pact on the brain and thus if the built environment either dishwashers [51] and washing machines.[52] directly or indirectly impacts the human microbiome, this in turn could have impacts on human mental health. Pathogen transmission. Many pathogens are transmit- 7.2 Results from Studies of the Mi- ted in the built environment and may also reside in the built environment for some period of time.[63] Good crobiomes of the Built Environ- examples include influenza, Norovirus, Legionella, and ment MRSA. The study of the transmission and survival of these pathogens is a component of studies of microbiomes of the built environment. 7.2.1 General Biogeography Indoor Air Quality The study of Indoor air quality and the health impact of such air quality is linked at least in Overall the many studies that have been conducted on part to microbes in the built environment since they can the microbiomes of the built environment have started to impact directly or indirectly indoor air quality. identify some general patterns regarding the microbes are found in various places. For example, Adams et al., in a comparative analysis of ribosomal RNA based studies in the built environment found that geography and building type had strong associations with the types of microbes 7.2.3 Components of the Built Environ- seen in the built environment.[53] Pakpour et al. in 2016 ment that Likely Impact Micro- reviewed the patterns relating to the presence of archaea biomes in indoor environments (based on analysis of rRNA gene [54] sequence data. A major component of studies of Microbiomes of the Built Environment involves determining how components of the built environment impact these microbes 7.2.2 Human Health and Microbiomes of and microbial communities. Factors that are thought the Built Environment to be important include humidity, pH, chemical exposures, temperature, filtration, surface materials, and air Many studies have documented possible human health flow.[64] There has been an effort to develop standards implications of the microbiomes of the built environment for what built environment “metadata” to collect asso- (e.g.,[55] ). Examples include those below. ciated with studies of the microbial communities in the [65] Newborn colonization. The microbes that colonize new- built environment. A 2014 paper reviews the tools borns come in part from the built environment (e.g., hos- that are available to improve the built environment data [66] pital rooms). This appears to be especially true for babies that is collected associated with such studies. Exam- born by C-section (see for example Shin et al. 2016 [56]) ples of types of built environment data covered in this and also babies that spend time in a NICU.[19] review include building characteristics and environmental conditions, HVAC system characteristics and ventila- Risk of allergy and asthma. The risk of allergy and tion rates, human occupancy and activity measurements, asthma is correlated to differences in the built environ- surface characterizations and air sampling and aerosol dy- ment microbiome. Some experimental tests (e.g., in namics. mice) have suggested that these correlations may actually be causal (i.e., the differences in the microbiomes may actually lead to differences in risk of allergy or asthma). Review papers on this topic include Casas et al. 2016 [57] 7.2.4 Impact of Microbiomes on the Built and Fujimura and Lynch 2015.[58] Studies of dust in various homes has shown that the microbiome found in the Environment dust is correlated to the risk of children in those homes developing allergy, asthma, or phenotypes connected to Just as the built environment has an impact on the mi- these ailments.[59][60][61] The impact of the microbiome crobiomes found therein, the microbial communities of of the built environment on the risk of allergy and asthma the built environment can impact the built environment and other inflammatory or immune conditions is a possi- itself. Examples include degradation of building mate- ble mechanism underlying what is known as the hygiene rials, altering fluid and airflow, generating volatiles, and hypothesis. more. 7.5. EXTERNAL LINKS 49

7.2.5 Possible Uses in Forensics 7.5.1 Examples of projects

The microbiome of the built environment has some po- There are a growing number of research projects and tential for being used as a feature for forensic studies. groups focusing directly or indirectly on microbiomes Most of these applications are still in the early research of the built environment. Some of these are targeted phase. For example, it has been shown that people leave towards speciﬁc environments (e.g., homes, hospitals, behind a somewhat diagnostic microbial signature when showerheads) and others are broader (e.g., BIMERC, the they type on computer keyboards,[67] use phones [68] or BioBE Center). More detail about some of these projects occupy a room.[10] is below.

• BIMERC - the Berkeley Indoor Microbial Ecology 7.2.6 Odor and Microbes in the Built En- Research Consortium. This group is focused on vironment “understanding the microbial components of indoor air, including the identification of the source pop- There has been a significant amount of research on the ulations and illuminating the processes that suspend role that microbes play in various odors in the built envi- and disseminate microbes and microbial products in ronment. For example, Diekmann et al. examined the buildings.” connection between volatile organic emissions in automobile air conditioning units.[69] They reported that the • The BioBE Center - Biology and the Built Environ- types of microbes found were correlated to the bad odors ment Center found. Park and Kim examined which microbes found in an automobile air conditioner could produce bad smelling • The Wildlife of Your Homes is a Citizen science volatile compounds and identified candidate taxa produc- project focusing on "the diversity of bacterial coming some such compounds.[70] munities found in nine distinct locations within our homes.”

• Baby Associated Built Environment Microbiome 7.3 Methods Used Project

• Many methods are used to study microbes in built en- Showerhead microbiome project vironment. A review of such methods are some of • the challenges in using them was published by NIST: Hospital Microbiome Project Challenges in Microbial Sampling in the Indoor Envi- • ronment. Hoisington et al. in 2015 reviewed methods Home Microbiome that could be used by building professionals to study the • PRoBE – Pathogen Research in the Built Environ- microbiology of the built environment.[71] Methods used ment in the study of microbes in the built environment include culturing (with subsequent studies of the cultured microbes), microscopy, air, water and surface sampling, chemical analyses, and culture independent DNA studies 7.5.2 Videos such as ribosomal RNA gene PCR and metagenomics. • Jessica Green TED talk Are We Filtering the Wrong Microbes

7.4 See also • Jessica Green TED talk We're covered in germs. Let’s design for that. • Building science • Rob Knight TED Talk How our microbes make us who we are • Microbial biogeography

• Microbial ecology 7.5.3 Journals that have a lot of publica- • Indoor air quality tions on this topic

• Indoor Air Journal

7.5 External links • Microbiology of the Built Environment paper collection at Biomed Central 50 CHAPTER 7. MICROBIOMES OF THE BUILT ENVIRONMENT

7.5.4 Societies and Organizations [9] Dunn, Robert R.; Fierer, Noah; Henley, Jessica B.; Leff, Jonathan W.; Menninger, Holly L. (2013). “Home • ISIAQ - International Society of Indoor Air Quality Life: Factors Structuring the Bacterial Diversity Found and Climate within and between Homes”. PLoS ONE. 8 (5): e64133. doi:10.1371/journal.pone.0064133. ISSN 1932-6203. • Alfred P. Sloan Foundation program in Microbiol- ogy of the Built Environment [10] Luongo, Julia C; Barberán, Albert; Hacker-Cary, Robin; Morgan, Emily E.; Miller, Shelly L; Fierer, Noah (2016). “Microbial analyses of airborne dust collected from dor- 7.5.5 News and related coverage mitory rooms predict the sex of occupants”. Indoor Air: n/a–n/a. doi:10.1111/ina.12302. ISSN 0905-6947. • Nova Next: Mapping the Microbiome [11] Kembel, Steven W.; Meadow, James F.; O’Connor, • NPR: Your Invisible Neighbors: Each City Has Timothy K.; Mhuireach, Gwynne; Northcutt, Dale; Unique Microbes Kline, Jeff; Moriyama, Maxwell; Brown, G. Z.; Bohan- nan, Brendan J. M.; Green, Jessica L. (2014). “Ar- • The Scientists: Your Office Has a Distinct Micro- chitectural Design Drives the Biogeography of Indoor biome Bacterial Communities”. PLoS ONE. 9 (1): e87093. doi:10.1371/journal.pone.0087093. ISSN 1932-6203. • The Conversation article by Erica Hartmann Scientists at work: studying indoor microbial [12] Chase, John; Fouquier, Jennifer; Zare, Mahnaz; Son- ecology means sampling in public restrooms deregger, Derek L.; Knight, Rob; Kelley, Scott T.; Siegel, Jeffrey; Caporaso, J. Gregory; Gilbert, Jack A. (2016). • TIME Magazine Your Home is Covered in Microbes “Geography and Location Are the Primary Drivers of Office Microbiome Composition”. mSystems. 1 (2): e00022–16. doi:10.1128/mSystems.00022-16. ISSN 7.6 References 2379-5077. [13] Smith, Daniel; Alverdy, John; An, Gary; Coleman, Mau- [1] Konya, Theodore; Scott, James A. 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7.7 Text and image sources, contributors, and licenses

7.7.1 Text

• Microbiota Source: https://en.wikipedia.org/wiki/Microbiota?oldid=758237294 Contributors: Deb, Fred Bauder, Darrell Greenwood, Alan Liefting, Gzuckier, Thorwald, Syp, Mwanner, Pol098, GregorB, Drbogdan, Rjwilmsi, Jongbhak, Bgwhite, Wavelength, Number 57, Epipelagic, Kkmurray, Arthur Rubin, Chris the speller, Rijkbenik, Headbomb, DadaNeem, Kellydcarter, Steel1943, TXiKiBoT, Bear- ian, NinjaRobotPirate, TheAlmightyEgg, Bfpage, Alexbrn, Zifra, Genes2007, Dodger67, Denisarona, Niceguyedc, Peteruetz, Eredrian, Gciriani, Rui Gabriel Correia, Qwfp, Koumz, Jytdog, Richard-of-Earth, Addbot, Fgnievinski, Quercus solaris, Ludovika26, Luckas-bot, Yobot, Nallimbot, EnBob08, AnomieBOT, Jim1138, Citation bot, GrouchoBot, Zefr, Jatlas, Jonesey95, Jandalhandler, Trappist the monk, Mindmonk, Woodlot, Yaxy2k, RjwilmsiBot, John of Reading, Ashton 29, Dcirovic, ZéroBot, SemanticMantis, ClueBot NG, Kmcnagny, Drlectin, Bibcode Bot, BG19bot, Cervantescid, MrBill3, Ginger Maine Coon, Estevezj, Matt.g.bakker, Csample, Stigmatella aurantiaca, ChrisGualtieri, Axtian, Projectphobos, Dexbot, CuriousMind01, FallingGravity, Wuerzele, Jamie Hlusko, AioftheStorm, Emibacter, Fprael, Coreyemotela, InsightSeeker, Vpylro, Monkbot, Asucur, Archiloc, Bhagiraj46, Ahonnecke, Panchoorlando, Zlfediay, FourVio- las, Jerryi Abalyi, Kevo Strevin, Hausmannmd, Pre-pro-page, JulesDarwin, Vonjo, DrFattyAcids, Toothcollector, McortNGHH, Barbara (WVS), Patriciapr12, ECE2509, Julia Lindén (SLU), Niklasgrassl-1 and Anonymous: 61 • Human microbiota Source: https://en.wikipedia.org/wiki/Human_microbiota?oldid=753071224 Contributors: AxelBoldt, Bryan Derk- sen, Manning Bartlett, Heron, Someone else, Shyamal, Ahoerstemeier, Ronz, Mark Foskey, Tristanb, DJ Clayworth, Profdhw, Robbot, Bkell, Fuelbottle, Alan Liefting, Christopher Parham, Nunh-huh, Jfdwolff, Peter Ellis, Beland, Wilagobler, Gscshoyru, Thorwald, Rich Farmbrough, Bender235, CanisRufus, Atcack, Madler, Passw0rd, Hu, Dirac1933, SteinbDJ, Gene Nygaard, Jeffrey O. 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Flyer22 Reborn, Twinsday, ClueBot, The Thing That Should Not Be, CounterVandalismBot, Niceguyedc, G-turbozone153, Rprpr, DragonBot, Gciriani, Jack-A-Roe, BOTarate, Thingg, XLinkBot, Jytdog, Memstone, Ost316, Richard-of-Earth, Addbot, DOI bot, Belmond, Kgh69, Luckas-bot, Yobot, The Earwig, Drgao, AnomieBOT, Dwot, JackieBot, Materialscientist, Pepo13, Citation bot, Marshallsumter, Xqbot, Magicxcian, GrouchoBot, Zefr, Nagualdesign, FrescoBot, TimonyCrickets, Citation bot 1, Pinethicket, I dream of horses, Abductive, Nightsmaiden, RedBot, Lester- Freamon00, Rickhev1, RjwilmsiBot, Androstachys, Helium4, Dcirovic, AManWithNoPlan, KittykatLA, ClueBot NG, Drlectin, Curb Chain, Plantdrew, BG19bot, Hank.stevens, Torreano61, Enderskye, Lhb15, AdventurousSquirrel, NotWith, Kmxrmach, BattyBot, Starry- Grandma, Stigmatella aurantiaca, Cyberbot II, JYBot, Dexbot, CuriousMind01, Joeinwiki, Wuerzele, Pasteurella, BallenaBlanca, Seppi333, Ajpolino, Jpgs33, Coreyemotela, Anrnusna, Ljstudent17, Gurtljb, 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ambitious, Xmteam, RjwilmsiBot, TjBot, Aircorn, EmausBot, WikitanvirBot, Immunize, GoingBatty, Tareqiu, Thecheesykid, Felipe Sobreira Abrahão, DelianDiver, John Mackenzie Burke, Wayne Slam, Sahimrobot, Orange Suede Sofa, ChuispastonBot, Woodsrock, Will Beback Auto, ClueBot NG, Jack Greenmaven, Carandraug, Amigoswiki, Jbkgiants, Zakarps, Cntras, Matt11111111, A.cristianlucian, Helpful Pixie Bot, TrenSur, Bibcode Bot, Gitana127, BG19bot, Waerfeles, Northamerica1000, Californiadreams, MusikAnimal, Silvrous, Cita- tionCleanerBot, Manchester123456789~enwiki, NotWith, Estevezj, BattyBot, Indianpjn, Mkh388, Wenfuli, Dexbot, Sminthopsis84, Mo- 7.7. TEXT AND IMAGE SOURCES, CONTRIBUTORS, AND LICENSES 55

gism, Lugia2453, Sampsonjk, Alglascock, YaguchiA, Haywardlc, Genome biologist KS, Bradleysp1, Sumukal, Vasi31, CsDix, FamAD123, Evolution and evolvability, The Herald, Seppi333, Askpat13, Cyborg1981, Newsrihari, Quigend, Skr15081997, Monkbot, ShawntheGod, Bobsaw8081, Kaytopi, Χρυσάνθη Λυκούση, ASDFghJKL:2255, M.Jormungand, GenomeEditor, Iwilsonp, MoreTomorrow, CV9933, 1115crocodileov, KasparBot, Delxy, CLCStudent, InternetArchiveBot, Sarush soti, GreenC bot, Vegethermar, Southardemily, Abhigyan Chakraborty and Anonymous: 299 • Connectome Source: https://en.wikipedia.org/wiki/Connectome?oldid=755483650 Contributors: Kku, Anders Feder, Phil Boswell, Pas- cal666, Thorwald, Syp, Robert P. O'Shea, Giraffedata, Robert K S, BD2412, Rjwilmsi, Fragglet, Chris Capoccia, Daniel Mietchen, Rjlabs, Arthur Rubin, DoriSmith, SmackBot, Snori, George Church, Stevage, Frap, LouScheffer, Matstuff, MrPMonday, Pwjb, Joshuav, Was a bee, CopperKettle, The Transhumanist, Greensburger, Yakushima, Sbump, Nono64, DadaNeem, Trondarild, Medlat, Markdask, Abhishikt, Marine-Blue, Alfnie, Superbatfish, ImageRemovalBot, Ingenuity Arts, Wkboonec, Addbot, Luckas-bot, Yobot, Pahagman, AnomieBOT, Citation bot, LilHelpa, Frebule, Kortikal, Thehelpfulbot, FrescoBot, Unidesigner, Piscosour00, Trappist the monk, Felis do- mestica, 564dude, RjwilmsiBot, Gcastellanos, Yevrah342, Dcirovic, Serketan, ZéroBot, Brainiac now, BWP1234, R.prentki, Hazard-Bot, ChuispastonBot, Spicemix, Willat600series, MelbourneStar, Bibcode Bot, BG19bot, Bdvj1son, Brian Tomasik, BattyBot, ChrisGualtieri, Iztwoz, NEuRDo, Loniucla, Comp.arch, Kokapellimt, Schmiani, Paul2520, Hypercyclic, Monkbot, UConnectome, Andreashorn, Bender the Bot, LessusMoore and Anonymous: 26 • Exposome Source: https://en.wikipedia.org/wiki/Exposome?oldid=754834316 Contributors: Kku, Beland, Rjwilmsi, Bgwhite, Kkmurray, Voceditenore, GordiasAchos, Mblumber, Headbomb, Ktr101, Vael Victus, Jytdog, Hersfold non-admin, Yobot, Citation bot, FrescoBot, Gcastellanos, GoingBatty, Dcirovic, BG19bot, CitationCleanerBot, ArticlesForCreationBot, Cyberbot II, Me, Myself, and I are Here, Mark viking, Gwmiller23, Stamptrader, Monkbot, RCrucial, Kkdenni, Michaela Dijmarescu and Anonymous: 6 • Built environment Source: https://en.wikipedia.org/wiki/Built_environment?oldid=759829095 Contributors: Kku, Ronz, Astudent, BobCMU76, Editor B, Wetman, Merovingian, Alan Liefting, Christiaan, Andycjp, Mzajac, Bender235, Lankiveil, Hayabusa future, Mis- creant, Erauch, Remuel, La goutte de pluie, Rajah, Alansohn, Ringbang, Woohookitty, BD2412, Mendaliv, Solace098, Rjwilmsi, Lockley, Vegaswikian, RobertG, Alex Sims, Gurch, Dmittleman, Wavelength, Manop, Jpbowen, Arthur Rubin, Mdwyer, Jeff Silvers, DVD R W, SmackBot, McGeddon, Gilliam, Mairibot, Amazins490, Esy~enwiki, Germanomaniac, Fryeb, CMG, Islescape, Nick Number, Escarbot, AntiVandalBot, AubreyEllenShomo, MER-C, Nyttend, Gabriel Kielland, User A1, CommonsDelinker, Tgeairn, Hasanisawi, Hans Dunkel- berg, DASonnenfeld, Funandtrvl, PercyWaldram, Coolestkitty, K. Annoyomous, Hseneff, Bentogoa, RW Marloe, ClueBot, PipepBot, Bmartens, John loza2, KKoolstra, Addbot, Knight of Truth, Quantumobserver, AnomieBOT, Galoubet, Roux-HG, CyeCenter, FrescoBot, Haeinous, Erin Inglish, Pinethicket, Tawnysea, Roopi04, Trappist the monk, Buddy23Lee, Darigan, Brianatkin, Onel5969, Rjwilmsi- Bot, Dcirovic, BeGenderNeutral, Azreen09, Echidna18, B william r, Rcsprinter123, Arman Cagle, BuiltEnvirons, Ellisun, ClueBot NG, SpikeTorontoRCP, Phylogenomics, Wbm1058, Gob Lofa, Ramaksoud2000, BG19bot, Northamerica1000, ISTB351, NGC 2736, SD5bot, Dexbot, Envhealthstudent, Ali.assari, Budhathokyp, Mrm7171, Prokaryotes, Inaaaa, Mmi7593, Monkbot, CMSnow14, CTS1996, Hustle- cat, InternetArchiveBot, McFarlandDana, Tonystudentnot, Tikvah17, GoFarGoWell and Anonymous: 83 • Microbiomes of the built environment Source: https://en.wikipedia.org/wiki/Microbiomes_of_the_built_environment?oldid= 757929255 Contributors: Ubiquity, Feanor~enwiki, Rjwilmsi, Bckirkup, Yobot, John of Reading, Phylogenomics, CitationCleanerBot and Anonymous: 6

7.7.2 Images

• File:Chytridiomycosis.jpg Source: https://upload.wikimedia.org/wikipedia/commons/0/01/Chytridiomycosis.jpg License: CC BY 2.5 Contributors: From Riders of a Modern-Day Ark. Gewin V. PLoS Biology Vol. 6, No. 1, e24 doi:10.1371/journal.pbio.0060024. Original artist: Photo credit: Forrest Brem • File:Commons-logo.svg Source: https://upload.wikimedia.org/wikipedia/en/4/4a/Commons-logo.svg License: PD Contributors: ? Origi- nal artist: ? • File:Community_garden.jpg Source: https://upload.wikimedia.org/wikipedia/commons/d/da/Community_garden.jpg License: Public domain Contributors: Own work Original artist: Klest • File:Cycas_coralloid_root_XS_high.jpg Source: https://upload.wikimedia.org/wikipedia/commons/6/62/Cycas_coralloid_root_XS_ high.jpg License: CC BY-SA 3.0 Contributors: Own work Original artist: Curtis Clark • File:DTI-sagittal-fibers.jpg Source: https://upload.wikimedia.org/wikipedia/commons/8/82/DTI-sagittal-fibers.jpg License: CC-BY- SA-3.0 Contributors: Own work Original artist: Thomas Schultz • File:Edit-clear.svg Source: https://upload.wikimedia.org/wikipedia/en/f/f2/Edit-clear.svg License: Public domain Contributors: The Tango! Desktop Project. Original artist: The people from the Tango! project. And according to the meta-data in the ﬁle, speciﬁcally: “Andreas Nilsson, and Jakub Steiner (although minimally).” • File:Esculaap4.svg Source: https://upload.wikimedia.org/wikipedia/commons/6/66/Esculaap4.svg License: GFDL Contributors: self- made, SVG-version of Image:Esculaap3.png by Evanherk, GFDL Original artist: .Koen • File:Folder_Hexagonal_Icon.svg Source: https://upload.wikimedia.org/wikipedia/en/4/48/Folder_Hexagonal_Icon.svg License: Cc-by- sa-3.0 Contributors: ? Original artist: ? • File:Genome_size_vs_number_of_genes.svg Source: https://upload.wikimedia.org/wikipedia/commons/0/0d/Genome_size_vs_ number_of_genes.svg License: CC BY-SA 3.0 Contributors: Own work Original artist: Estevezj • File:Hybridogenesis_in_water_frogs_gametes.gif Source: https://upload.wikimedia.org/wikipedia/commons/7/7f/Hybridogenesis_ in_water_frogs_gametes.gif License: CC BY-SA 4.0 Contributors: Own work Original artist: Darekk2 • File:Issoria_lathonia.jpg Source: https://upload.wikimedia.org/wikipedia/commons/2/2d/Issoria_lathonia.jpg License: CC-BY-SA-3.0 Contributors: ? Original artist: ? • File:Keppelbleaching.jpg Source: https://upload.wikimedia.org/wikipedia/en/9/90/Keppelbleaching.jpg License: CC-BY-3.0 Contribu- tors: ? Original artist: ? 56 CHAPTER 7. MICROBIOMES OF THE BUILT ENVIRONMENT

• File:Lock-green.svg Source: https://upload.wikimedia.org/wikipedia/commons/6/65/Lock-green.svg License: CC0 Contributors: en:File: Free-to-read_lock_75.svg Original artist: User:Trappist the monk • File:Microbiome_analysis_flowchart.png Source: https://upload.wikimedia.org/wikipedia/commons/a/ac/Microbiome_analysis_ flowchart.png License: CC BY-SA 3.0 Contributors: Own work Original artist: Axtian • File:Microbiota-derived_3-Indolepropionic_acid.jpg Source: https://upload.wikimedia.org/wikipedia/commons/7/7d/ Microbiota-derived_3-Indolepropionic_acid.jpg License: CC BY 4.0 Contributors: (April 2016). "Microbial metabolism of dietary components to bioactive metabolites: opportunities for new therapeutic interventions". Genome Med 8 (1): 46. DOI:10.1186/s13073-016- 0296-x. PMID 27102537. PMC: 4840492. Figure 1: Molecular mechanisms of action of indole and its metabolites on host physiology and disease Original artist: Zhang LS, Davies SS • File:Open_Access_logo_PLoS_transparent.svg Source: https://upload.wikimedia.org/wikipedia/commons/7/77/Open_Access_logo_ PLoS_transparent.svg License: CC0 Contributors: http://www.plos.org/ Original artist: art designer at PLoS, modified by Wikipedia users Nina, Beao, and JakobVoss • File:Part_of_DNA_sequence_prototypification_of_complete_genome_of_virus_5418_nucleotides.gif Source: https: //upload.wikimedia.org/wikipedia/commons/6/63/Part_of_DNA_sequence_prototypification_of_complete_genome_of_virus_5418_ nucleotides.gif License: CC BY-SA 4.0 Contributors: http://studia.scienceontheweb.net/visualization.php Original artist: Gregory Podgorniak • File:Portal-puzzle.svg Source: https://upload.wikimedia.org/wikipedia/en/f/fd/Portal-puzzle.svg License: Public domain Contributors: ? Original artist: ? • File:Skin_Microbiome20169-300.jpg Source: https://upload.wikimedia.org/wikipedia/commons/0/0a/Skin_Microbiome20169-300. jpg License: Public domain Contributors: http://www.genome.gov/dmd/img.cfm?node=Photos/Graphics&id=85320 Original artist: Darryl Leja, NHGRI • File:Suburbia_by_David_Shankbone.jpg Source: https://upload.wikimedia.org/wikipedia/commons/3/37/Suburbia_by_David_ Shankbone.jpg License: CC-BY-SA-3.0 Contributors: David Shankbone Original artist: David Shankbone • File:Sustainable_development.svg Source: https://upload.wikimedia.org/wikipedia/commons/7/70/Sustainable_development.svg Li- cense: CC-BY-SA-3.0 Contributors: • Inspired from Developpement durable.jpg Original artist: • original: Johann Dréo (talk · contribs) • File:Symbol_template_class.svg Source: https://upload.wikimedia.org/wikipedia/en/5/5c/Symbol_template_class.svg License: Public domain Contributors: ? Original artist: ? • File:The_Human_Connectome.png Source: https://upload.wikimedia.org/wikipedia/commons/c/cd/The_Human_Connectome.png Li- cense: CC BY-SA 4.0 Contributors: Own work Original artist: Andreashorn • File:UCSC_human_chromosome_colours.png Source: https://upload.wikimedia.org/wikipedia/commons/d/db/UCSC_human_ chromosome_colours.png License: CC0 Contributors: This file was derived from: NHGRI human male karyotype.png Original artist: HYanWong • File:WTM3_Gnarly_0028.jpg Source: https://upload.wikimedia.org/wikipedia/commons/e/e1/WTM3_Gnarly_0028.jpg License: CC BY-SA 3.0 Contributors: Uploaded from Wikis Take Manhattan 2009 Original artist: Gnarly (Wikis Take Manhattan 2009 participant) • File:White_Matter_Connections_Obtained_with_MRI_Tractography.png Source: https://upload.wikimedia.org/wikipedia/ commons/f/f2/White_Matter_Connections_Obtained_with_MRI_Tractography.png License: CC BY 2.5 Contributors: Gigandet X, Hagmann P, Kurant M, Cammoun L, Meuli R, et al. (2008) Estimating the Confidence Level of White Matter Connections Obtained with MRI Tractography. PLoS ONE 3(12): e4006. doi:10.1371/journal.pone.0004006 Original artist: Xavier Gigandet et. al. • File:Wiki_letter_w_cropped.svg Source: https://upload.wikimedia.org/wikipedia/commons/1/1c/Wiki_letter_w_cropped.svg License: CC-BY-SA-3.0 Contributors: This file was derived from Wiki letter w.svg: Original artist: Derivative work by Thumperward • File:Wikiquote-logo.svg Source: https://upload.wikimedia.org/wikipedia/commons/f/fa/Wikiquote-logo.svg License: Public domain Contributors: Own work Original artist: Rei-artur • File:Wiktionary-logo-v2.svg Source: https://upload.wikimedia.org/wikipedia/commons/0/06/Wiktionary-logo-v2.svg License: CC BY- SA 4.0 Contributors: Own work Original artist: Dan Polansky based on work currently attributed to Wikimedia Foundation but originally created by Smurrayinchester

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