Chronic inflammation is the common pathological basis for such age-associated diseases as cardiovascular disease, diabetes, cancer and Alzheimer’s disease. A multitude of bodily changes occur with aging that contribute to the initiation and development of inflammation. In particular, the immune system of elderly individuals often exhibits diminished efficiency and fidelity, termed immunosenescence. But, although immune responses to new pathogens and vaccines are impaired, immunosenescence is also characterized by a basal systemic inflammatory state. This alteration in immune system function likely promotes chronic inflammation. Changes in the tissue microenvironment, such as the accumulation of cell debris, and systemic changes in metabolic and hormonal signals, also likely contribute to the development of chronic inflammation. Monocyte/macrophage lineage cells are crucial to these age-associated changes, which culminate in the development of chronic inflammatory diseases. In this review, we will summarize the diverse physiological and pathological roles of macrophages in the chronic inflammation underlying age-associated diseases. Although the chronic inflammation associated with NCDs does not necessarily lead by age-associated changes in the body, the observation that the prevalence of many NCDs increases with advancing age suggests a pathogenic link between inflammaging and age-associated diseases.With advancing age, the immune system undergoes a dynamic change characterized by the coexistence of a smaller immune response to newly encountered pathogens or vaccine antigens, and an elevated systemic inflammatory state made manifest, for example, by elevated levels of proinflammatory cytokines, clotting factors and acute phase reactants.2 Although chronic inflammation may follow acute inflammation, in the most common NCDs of today it likely begins insidiously as a low-grade, smoldering response with no manifestation of the cardinal signs of inflammation (Dolor (pain), Calor (heat), Rubor (redness) and Tumor (swelling)). However, even low-grade inflammation may impair tissue function (Functio laesa). For instance, inflammatory signals interfere with insulin signaling.3 Moreover, the continuous progression of tissue injury and repair promotes tissue remodeling  that may eventually cause irreversible tissue dysfunction.4 In fact, the severity of tissue remodeling determines the prognosis of some NCDs, such as heart failure and chronic kidney disease.5Chronic inflammation is a prolonged condition in which tissue injury and attempts at repair coexist, leading to tissue remodeling and dysfunction.4 In this review, we will summarize the pathological connection between chronic inflammation and age-associated diseases, with a particular focus on what is currently known about the roles played by macrophages.A complex interplay between parenchymal cells within a tissue and the various cells in the stroma, including immune cells, vascular cells and fibroblasts, lead the processes of chronic inflammation under the influence of inputs from both the local microenvironment and the wider system. Of particular interest are monocyte–macrophage lineage cells, which act as major effector cells in chronic inflammatory processes during the pathological development of NCDs.6 The changes in the adaptive immune system are characterized by a decreases in naive T- and B cells, an increase in memory cells and a progressive reduction in the T-cell receptor (TCR) and B-cell receptor (BCR) repertoire.7–9 By contrast, generation of myeloid cells is favored in aging animals.It is often noted that elderly individuals are more vulnerable to infectious diseases. For instance, occult infection with tuberculosis and varicella zoster virus becomes evident, sometimes leading to life-threatening disease. Moreover, vaccines are often ineffective in older adults owing to inability of the adaptive immune system to generate protective immunity. Overall, these changes in the immune system, characterized by declining fidelity and efficiency, are termed immunosenescence. A key feature of immunosenescence is an imbalance between inflammatory and anti-inflammatory networks, leading to a complex presentation of impaired adaptive immune responses with concomitant persistent low-grade inflammation and a greater susceptibility to autoimmune responses.10 Thus, complex mechanisms at multiple levels  appear to contribute to the development of immunosenescence.Immunosenescence involves not only age-related changes intrinsic to immune cells, but also microenvironmental and systemic alterations. Although it is likely that cellular senescence is involved in some of these age-related alterations, many are likely induced independently of cellular senescence pathways. In addition, cellular senescence in non-immune cells may also contribute to immunosenescence.11 as well as changes in non-lymphocyte compartments of the thymus. Elderly HSCs exhibit less potential to produce lymphocytes and an increase in myeloid potential.6 Distinct HSC subsets having lymphoid- or myeloid-biased potential have been identified,12 and while the number of lymphoid-biased HSCs declines with age, myeloid-biased HSCs are increased. This shift toward myelopoiesis is thought to reflect the influence of both intrinsic and extrinsic factors.13 For instance, transforming growth factor (TGF)-β1 promotes proliferation of myeloid-biased HSCs and myeloid progenitors,12 suggesting TGF-β1 may be one of the bone marrow microenvironmental factors altered with age. Decreased production of interleukin (IL)-7 by stromal cells may also contribute to the age-related reduction in B-cell progenitors.14 Age-related microenvironmental changes also impair T-cell development in the thymus. Indeed, age-associated thymal regression resulting in reduced production of naive T cells is now thought to be driven by the epithelial compartment.10 In addition to the local environment, systemic changes that occur with aging, such as decreased levels of growth hormone and IGF-1, may also contribute to the decline in lymphopoiesis.11 Despite the skewing of aged HSCs toward myelopoiesis, however, the basal inflammation seen in older individuals is not associated with increased numbers of myeloid cells, presumably reflecting multiple functional defects in the homing and proliferative responses of aged HSCs.15Multiple causes have been suggested for the age-related functional impairment of lymphocytes, including qualitative changes in hematopoietic stem cells (HSCs) and progenitor cells, resulting in reduced production of T- and B lymphocytes in bone marrow and the thymus,16 And this chronic inflammatory activation, which has been noted in tissues from aged humans and mice,17–19 may have a role in the development of a plethora of age-associated chronic diseases, including cardiovascular and metabolic diseases and cancer. Other factors, including changes in the microenvironment as well as systemic factors, also contribute to chronic inflammation. For instance, senescent cells in tissues may activate inflammatory responses by recruiting immune cells by expressing senescence-associated secretory phenotypes (SASPs).20 Increased cell death or ineffective clearance of dead cells and damaged tissue may also activate immune responses.21Immunosenescence leads not only to impaired immune responses but also to low-grade, chronic, systemic inflammation (inflammaging).With chronic inflammation, innate immune cells act as key effector cells. In the following sections, we will focus on age-related changes in the innate immune system, particularly macrophages, to address how aging paradoxically promotes chronic inflammation.15 age-related dysfunction of neutrophils, natural killer cells, monocytes, macrophages and dendritic cells has been reported.22–25 For instance, the capacity to activate T cells via antigen presentation is impaired in aged dendritic cells, where expression of costimulatory molecules and MHC class II is reportedly downregulated, though the conflicting data have also been reported.26 Dendritic cells from elderly subjects also express lower levels of Toll-like receptors (TLRs) and produce lower levels of cytokines in response to specific TLR ligands, while the baseline cytokine levels in absence of TLR ligand stimulation are increased, suggesting dysregulated cytokine expression.27 Similarly, in blood monocytes from aged human subjects, TLR1/2-induced tumor necrosis factor (TNF)-α and IL-6 production is decreased, suggesting a functional defect in monocyte/macrophage lineage cells  express a variety of pattern-recognition receptors, including TLRs. Upon recognition of a PAMP, innate immune cells are activated to destroy the pathogen and/or pathogen-infected cells. Although PAMPs are exogenous molecules, the damaged or dying cells release endogenous molecules called damage/danger-associated molecular patterns (DAMPs), which also activate the immune system in a manner analogous to PAMPs. A variety of different molecules have now been identified as DAMPS, including high-mobility group box 1 protein (HMGB1), genomic double-stranded DNA and cleaved extracellular matrix proteins.34 Moreover, modified endogenous molecules such as oxidized lipoproteins, cholesterol crystals and uric acid, whose production does not necessarily require cell death, may also serve as DAMPs.36 Those and still other DAMPs, such as damaged macromolecules and cell debris, accumulate with age, leading to activation of the innate immune system. This accumulation of DAMPs may be enhanced by the inadequate phagocytotic activity of aged myeloid cells.1 Thus, a complex reciprocal interplay between immune cells and tissue microenvironmental/non-immune cells very likely contributes to the activation of inflammatory signaling in aged individuals.In addition to the intrinsic dysregulation of innate immune cells, the altered microenvironment within aged tissues and systemic changes in the endocrine and other systems very likely contribute to aberrant activation of innate immune cells. The innate immune system recognizes the repetitive molecular structures of pathogens, known as pathogen-associated molecular patterns (PAMPs), via pattern-recognition receptors.37 Estrogen can activate or inhibit proinflammatory cytokine production, depending on context and dose.38 Obesity associates with activation of chronic low-level inflammation in several tissues, particularly adipose tissue.39 Conversely, calorie restriction without malnutrition suppresses chronic inflammation.40 Malnutrition, on the other hand, leads to immune dysfunction.41 Hormones that control metabolism, such as insulin and leptin, are known to modulate immune cell function.42 Obesity accelerates immunosenescence in T cells,43 and serum cytokines levels are increased in obese subjects.45 Aging associates with an increase in body fat and a shift of fat from subcutaneous to abdominal depots.46 The related systemic metabolic changes likely contribute to the basal activation of inflammation in the elderly. We will discuss later how obesity induces inflammation within visceral adipose tissue and how adipose tissue inflammation not only impacts systemic metabolism but also promotes chronic inflammatory disease.Systemic factors, such as hormonal and metabolic changes, also impact immune responses. For instance, menopause and ovariectomy cause a low-grade systemic inflammatory state with increased plasma chemokine levels that is suppressed by estrogen treatment.4 Acute inflammation is typically self-limiting, and after the offending agent is eliminated the tissue returns to the homeostatic state. The resolution of acute inflammation is an active process in which anti-inflammatory signals suppress inflammation, clear immune cells and promote healing, leading to the restoration of normal tissue function.47 Because the resolution involves dynamic processes, dysfunctional resolution prolongs and perpetuates inflammation. Many mechanisms can cause impaired resolution and sustained inflammation—failure to eliminate the offending agent, for instance. Insufficient clearance of proinflammatory cells may also hamper resolution, and functional alteration of immune cells may affect resolution-related signaling and processes. It can be inferred from this that in aged tissue, accumulation of DAMPs due to their increased production and/or impaired clearance may predispose one to chronic inflammation.Acute inflammation is a protective response to injury or infection.48 In a mouse self-resolving peritonitis model, resolution of acute inflammation was delayed in aged mice.49 Numbers of granulocytes and levels of proinflammatory cytokines were higher in aged mice than young mice 24 h after peritonitis was triggered. Moreover, although the numbers of monocytes/macrophages in peritoneal exudates were increased in aged mice, bone marrow-derived macrophages from aged mice showed less capacity to take up apoptotic granulocytes, suggesting diminished macrophage activity may contribute to failed resolution of inflammation. Impaired phagocytosis of apoptotic cells has also been noted in other in vivo mouse models and in human monocyte-derived dendritic cells in vitro.51 One study also suggests that aging impairs the phagocytotic capacity of microglia.52 In addition, macrophages from obese ob/ob mice exhibit a diminished ability to phagocytose apoptotic cells,53 suggesting that systemic metabolic dysfunction, which is often seen in the elderly, may further impair phagocytotic capacity. This may mean that diminished macrophage phagocytotic activity results in failed resolution and accumulation of DAMPs in aged animals. However, that notion needs to be directly tested in future studies. It will also be important to determine mechanistically how aging causes loss of phagocytotic functionality. A previous observation that apoptotic clearance by thioglycollate-elicited peritoneal macrophages from young mice was impaired after the cells were exposed to serum from aged mice suggests exogenous factors contribute to the age-related decline in phagocytotic activity.50 On the other hand, the finding that dendritic cells generated in vitro from human monocytes exhibited age-associated phagocytotic dysfunction with impairment of the PI3K/AKT pathway51 suggests a cell-autonomous mechanism. Presumably, both intrinsic and extrinsic mechanisms drive the functional alterations in macrophages and reflect the cells’ highly plastic gene expression, as we will discuss in the following sections.A primary function of macrophages during inflammatory resolution is the clearance of effete cells. Particularly important is their phagocytosis of apoptotic granulocytes.54 It has been suggested that accumulation of senescent cells within tissues contributes to age-related organ dysfunction and pathology. Baker et al.51 showed that removing cells expressing the senescence-marker gene Cdkn2a (p16) in a progeroid context (BubR1H/H mice) delays the onset of age-related phenotypes, including sarcopenia, cataracts and loss of adipose tissue, which highlights the importance of proper clearance of senescent cells. Very recently, the same group showed that partial ablation of p16+ cells extended lifespan and healthspan in a non-progenoid context as well, and this was accompanied by reduced expression of inflammation markers in various tissues.56Macrophages are also essential for clearing senescent cells.57 SASP factors recruit immune cells, including macrophages, neutrophils, natural killer cells and T cells, and promote inflammation  produced by senescent cells are involved in chronic inflammation and tissue dysfunction  in age-associated diseases, it has been suggested that cellular senescence contributes to the development of these diseases by inducing SASP expression. Further studies will be needed to determine the extent to which SASPs contribute to the initiation and development of chronic inflammation in the elderly and to clarify the causal link between cellular senescence, SASPs and age-related pathologies.Senescent cells produce proinflammatory mediators and proteases in what is collectively termed the SASP.ammation . This in60 If, however, apoptotic cells are not promptly cleared, they undergo secondary necrosis, leading to the leakage of molecules that elicit inflammatory responses by acting as DAMPs and autoantigens.It would also be expected that insufficient clearance of senescent cells would prolong inflammatory processes owing to the production of SASP factors and accumulation of DAMPs. Other types of cell death may also contribute to the prolongation of inflammatory processes. For instance, improper clearance of apoptotic cells may contribute to the pathogenesis of age-associated diseases as well as autoimmune diseases. Apoptotic cells are normally rapidly phagocytosed, preventing the leakage of their immunogenic and cytotoxic intracellular contents.61 Secondary necrosis of the foam cells leads to the expansion of a necrotic core that consists primarily of debris from dead macrophages.62 This necrotic debris also promotes inflammation and destabilization of the plaque, which can lead to plaque rupture.63 It is currently unclear whether macrophage apoptosis is accelerated by aging, but peritoneal macrophages from aged mice are more susceptible to endoplasmic reticulum stress-induced apoptosis.64 The age-related changes in macrophage function may thus contribute to the progression of atherosclerosis. Cellular senescence of endothelial cells and smooth muscle cells also likely contribute to the inability to resolve inflammation in atherosclerosis.65Atherosclerosis is a chronic inflammatory disease in which unresolved inflammation promotes vessel wall remodeling and destabilization of atherosclerotic plaque. Improper clearance of apoptotic cells by macrophages (efferocytosis) is one of the mechanisms thought to underlie the unresolved inflammation in atherosclerosis. Within atherosclerotic plaque, macrophages take up modified lipoproteins and become foam cells. As the plaque progresses, foam cell apoptosis overwhelms the remaining macrophages’ capacity for phagocytotic clearance.Monocyte–macrophage lineage cells are multifunctional and found in nearly all tissues throughout the body. In addition to their essential roles in host defense, they are pivotally involved in the maintenance of tissue homeostasis. As we have discussed, macrophages are essential for proper remodeling and healing after tissue injury. Phagocytosis by macrophages is also indispensable for tissue remodeling during organ development and ontogeny.66 Although this dichotomy is widely used, it is important to keep in mind that the M1/M2 dichotomy is clearly not sufficient to encompass the diverse phenotypes and functions of macrophages, and that classification of macrophages into these two groups is often not straightforward and relies on different sets of markers in different tissues. Exposure to TLR ligand or Th1 cytokines, such as TNF-α and IFN-γ, polarizes macrophages into the proinflammatory, M1 phenotype. M1 activation increases expression of proinflammatory cytokines and production of reactive oxygen species. By contrast, Th2 cytokines, such as IL-4 and IL-13, induce the M2 phenotype, though other factors may be involved.67 M2 macrophages are known to be essential for parasite clearance and have also been shown to promote resolution of inflammation and fibrosis.Macrophages are often divided into two subgroups: M1 and M2.69 Later, however, they preferentially assume the M2 phenotype and promote fibrosis. Similar transitions of macrophage phenotypes occur after skeletal muscle injury or myocardial infarction.71 In these models M2 macrophages are increased through recruitment of monocytes and/or in situ proliferation of resident macrophages.72The different functions of M1 and M2 macrophages have been demonstrated in various tissues relevant to NCDs. For instance, following renal injury the initially recruited monocytes preferentially differentiate into M1 macrophages and promote inflammation.74 SASP factors secreted from hepatic stellate cells shift macrophage polarization from M2 toward M1.75 Accordingly, it is likely that exogenous changes in the microenvironment  in combination with the systemic low-grade, inflammatory state modulate macrophage activation programs and their responses to stimuli.Aging may modulate M1/M2 activation and polarization.76Monocyte–macrophage lineage cells constitutively localized in peripheral tissues are called tissue-resident macrophages and exhibit highly diverse and plastic phenotypes that depend on the tissue type. Recent studies have shown that in addition to monocyte-derived macrophages, tissue-resident macrophages crucially contribute to age-related pathology. For instance, a recent study found that an age-dependent increase in reactive oxygen species in mitochondria and the associated NLRP3 inflammasome activation contributed to the pathogenesis of fibrosis in aged mice.As we will discuss in the following section, the origins of tissue-resident macrophages may differ among tissues and may be affected by aging. As such, different origins add another level of complexity to macrophage diversity.77 On the other hand, inflammatory activated Kupffer cells secrete proinflammatory cytokines, likely promoting insulin resistance.79 High-fat diet-induced obesity induces chronic, low-grade inflammation in the hypothalamus accompanied by activation of microglia, the resident macrophages in the central nervous system.80 This hypothalamic inflammation increases appetite by modulating the balance between anorexic and orexigenic neuron activities. Within pancreatic islets, resident macrophages are important for β-cell development, and M2-type islet macrophages have been shown to have a protective role in pancreatitis models.81 On the other hand M1 macrophages promote β-cell dysfunction.82Macrophages variously contribute to the physiology and pathology of metabolic tissues . For exa83Macrophages also have diverse and pivotal roles within adipose tissue. Visceral obesity has been shown to promote both cardiovascular and metabolic disease. Inflammation within visceral adipose tissue both modulates the function of adipose tissue locally and promotes systemic metabolic dysfunction. For example, when secreted by immune cells within adipose tissue, inflammatory cytokines such as TNF-α not only alter local adipocyte function but they also act systemically to induce insulin resistance. In addition, adipose tissue-derived proinflammatory cytokines may promote atherosclerosis and cancer by activating inflammatory processes in responsive tissues.84 These macrophages on one hand promote adipose tissue inflammation that has a strong impact on systemic metabolism, but on the other hand they are important for maintaining lipid metabolic homeostasis, clearing dead adipocytes and generating new adipocytes (adipogenesis).85 Macrophages are thus an essential component of normal adipose tissue function. Within healthy adipose tissue in lean animals, macrophages mainly exhibit the M2 phenotype.86 Obesity greatly increases numbers of M1 macrophages in visceral adipose tissue. M1 macrophages impair adipose tissue function in part by promoting insulin resistance and lipolysis. In addition, other subpopulations and/or activation states have been proposed for some adipose-resident macrophages, as their surface phenotypes and gene expression profiles do not fit the typical M1/M2 types.87–89Visceral adipose tissue contains large populations of immune cells and their interactions with adipocytes and other cells have a strong impact on adipose tissue function. Within visceral adipose tissue, macrophage numbers increase as the body gains weight.90 Aging also increases the numbers of both CD4+ and CD8+ T cells in visceral fat. Proinflammatory cytokine levels are higher in aged visceral fat, and adipose tissue macrophages from aged mice express higher levels of proinflammatory cytokines.91 These changes in macrophages may promote adipose tissue inflammation, which may in turn have systemic impact on basal activation of inflammation in the elderly.Aging alters the balance of visceral adipose tissue macrophages toward the proinflammatory M1 phenotype in mice.93 Moreover, differentiation of preadipocytes is impaired, and the capacity of subcutaneous adipose tissue to store lipids is decreased. Consequently, fat is redistributed to visceral depots and extra-adipose sites, including bone marrow, muscle and liver.94 Because visceral fat is more prone to inflammation than subcutaneous fat, and accumulation of fat in ectopic tissues  promotes inflammation in the affected tissues,95 the redistribution of fat may contribute to age-related activation of inflammation. Evidence suggests adipose tissue macrophages promote the generation of new adipocytes (adipogenesis).96 It will be important to assess whether the functional alteration of macrophages with age contributes to the diminished adipogenesis seen in the elderly.The metabolic function of adipose tissue changes with increasing age. Aged adipose tissue becomes less sensitive to insulin, lipolytic stimulation and fatty acids.97–99 However, a recent series of studies demonstrated that many tissue-resident macrophages are established during embryonic development and, in the healthy steady state, persist into the adulthood independently of blood monocytes.100–105 Nonetheless, aging may promote replacement of embryo-derived tissue macrophages with monocyte-derived ones.106 And while monocyte-derived macrophages are highly plastic and responsive to environmental changes, this shift in the origin of tissue-resident macrophages may still affect the function of macrophages residing within aged tissues. This will need to be directly addressed in future.For years, it was believed that all tissue-resident macrophages originate from circulating adult blood monocytes, despite evidence that some tissue-resident macrophages are independent of circulating monocytes.108 In particular, bone marrow monocytes and fetal macrophages acquire an epigenome and functionality that are very similar to those of the native resident macrophages in colonized tissues.107–109 This plasticity suggests age-related changes in the microenvironment alter macrophage function within tissues.75 What is more, nearly all microRNAs involved in immune regulation are functionally modulated during aging.110 Clearly, a greater understanding of the changes in macrophage origin and the epigenetic changes that occur with aging is needed. For instance, macrophages in the elderly exhibit age-related reductions in phagocytosis and antigen-presenting capacity.29 It will be important to determine whether changes endogenous to macrophages induce this dysfunction. A population of senescent-associated CD4+ T cells exhibits features of cellular senescence and a unique transcriptomic signature partly driven by C/EBPα.112 It is possible that the age-related functional alterations in macrophages are driven by similar cell-autonomous mechanisms. It will also be important to determine whether aging induces epigenetic changes in monocyte/macrophage progenitors and/or tissue macrophages that maintain macrophage populations through self-renewal, and to test whether such epigenetic alterations modulate macrophage function. Because macrophages are so susceptible to environmental cues, it will be important to examine the interplay between cell autonomous and externally driven mechanisms to elucidate age-associated changes in macrophages. In future studies, it will also be important to assess the causative link between age-related alterations in the function of macrophages and the onset and progression of chronic inflammatory diseases.The epigenomes and transcriptomes of macrophages are highly plastic, and the tissue microenvironment within certain tissues can transform the enhancer landscape of exogenous macrophages into one mimicking that of resident macrophages.As we have summarized here, age-related changes in immunity are characterized by both impairment of adaptive immunity and activation of low-grade chronic inflammation. The paradoxical activation of chronic inflammatory states very likely contributes to the progression of age-associated diseases. Macrophages not only promote inflammation and tissue dysfunction but also are essential for resolution and healing of inflammation, as well as maintenance of tissue homeostasis. Accordingly, macrophages appear to contribute crucially to the paradoxical activation of basal chronic inflammatory states in the elderly and to the progression of age-associated diseases.21 (3) Systemic factors, such as metabolic and hormonal signals and inflammation in distant tissues . Proinflammatory cytokines and free fatty acids secreted from inflamed adipose tissue can activate inflammation in distant tissues.114 Age-related changes in adiposity and hormones  may also have a systemic impact on inflammation. (4) Changes in macrophage subtype and origin. Macrophages can assume diverse phenotypes and their epigenomes are highly plastic. Aging may shift the balance among macrophages. Moreover, age may promote replacement of embryo-derived tissue macrophages with monocyte-derived ones. Many other factors, including nutrients, gut microbiota and tissue remodeling are also likely involved.4 For instance, improper tissue remodeling  may impair tissue function and result in the generation of endogenous proinflammatory stimuli such as extracellular matrix molecules and their degradation products (matrikines).4 Macrophages are crucially involved in all these processes, which impair the proper resolution of inflammation. As such, a better understanding of the changes in macrophage function, phenotype and epigenome that occur with aging will not only shed a new light on inflammaging, but also lead to identification of new therapeutic targets for the treatment of age-associated ailments.Many factors are likely involved in the activation of chronic inflammation in the elderly, as we have seen. These include (1) intrinsic dysregulation of immune cell responses to inflammatory stimuli—i.e., macrophages may be activated at baseline and overreact to certain stimuli. (2) Age-related alterations to the microenvironment. For instance, macrophages may be activated in response to the accumulation of DAMPs brought about by insufficient clearance of dead cells. In some cases, this may reflect the macrophages’ diminished phagocytotic capacity. However, increased cell death induced by cellular senescence and stress  may also overwhelm the capacity of the phagocytotic system.


We highlight that switching OTU picking to a closed reference approach greatly enhances performance. Finally, recommendations are given on how to perform ITS based mycobiota analysis with the currently available measures.Fungi constitute an important part of the human microbiota and they play a significant role for health and disease development. Advancements made in the culture-independent analysis of microbial communities have broadened our understanding of the mycobiota, however, microbiota analysis tools have been mainly developed for bacteria  and they often fall short if applied to fungal marker-gene based investigations . In the current paper we discuss all major steps of a fungal amplicon analysis starting with DNA extraction from specimens up to bioinformatics analyses of next-generation sequencing data. Specific points are discussed at each step and special emphasis is placed on the bioinformatics challenges emerging during operational taxonomic unit (OTU) picking, a critical step in mycobiota analysis. By using an  STAT1, CARD9, etc.) or Il-17 and Il-22 signaling pathways lead to severe fungal syndromatic infections, such as chronic mucocutaneous candidiasis or the APECED  syndrome -, urogenital-, and respiratory tract  also other resources for mycobiota investigations exist. Formalin-fixed paraffin-embedded (FFPE) tissue samples play an important role in the clinical context. Biopsies or surgically removed tissues are typically fixed in formalin (10%) immediately after they are collected from the patient, thus they represent a well-preserved resource for the analysis of biomolecules including nucleic acids  analytical process of mycobiota studies mandatory. To highlight the influence of pre-analytics on ITS based mycobiota investigations we assessed the performance of DNA extraction from human skin FFPE samples  protein  pre-processing, (ii) OTU picking, (iii) taxonomic classification, and (iv) visualization and statistical analysis Figure . So far Current pre-processing recommendations include rigorous length filtering of reads, noise reduction , quality filtering , chimera removal , as well as removal of singletons/doubletons  closed reference-, and (iii) open reference-based clustering. Briefly, a closed reference approach calculates for each input sequence the best pairwise alignment to a pre-defined reference database collection. Sequences with the same best match are binned into the same cluster . In contrast, de novo based strategies cluster sequences within a pre-defined distance (commonly 3%). For each of these clusters a representative sequence is selected and taxonomically classified. Open-reference OTU picking is a mixture of both. Reads are first clustered using a closed reference approach and all reads which fail in this first step are subsequently clustered using a de novo strategy  as the optimal method for clustering 16S rRNA gene sequences into OTUs  database for ITS fragments represents a curated full-length ITS sequence repository devoid of ambiguous sequences . A SH represents an operational taxonomic unit at approximately species level . These counts give already an idea about the “completeness” of the current fungal reference databases , which behaved poor (classifying only 33.53% of sequences correctly at phylum level and 0.07% at species level). A high number of singletons emerged by using all three de novo approaches, leading to OTU inflation, and wrongly clustered OTUs. Importantly, changing the default OTU picking approach of QIIME (de novo) to a closed reference approach increased the amount of correctly classified species to 71.62% , as well as taxonomic turnover  called beta-diversity, which can be calculated with different distance measurements . Principle coordinates analysis (PCoA) plots based on these distance matrices enable simplified visualization of the structural resemblance of mycobiota profiles. Statistical identification of differential abundant taxa between groups could be achieved using tools such as LEfSe . Based on our experience, pre-processing, and quality filtering of ITS sequencing data, as well as chimera filtering could be done with standard 16S rRNA gene based procedures. We use the default workflow of mothur for ITS data pre-processing, assembling of paired reads, length-, quality-, and chimera filtering, as well as noise reduction as described in the MiSeq 16S SOP of Kuczynski et al. .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hfe+/+ and Hfe−/− mice in combination with oral iron overload in a model of Salmonella enterica serovar Typhimurium infection, we found animals of either genotype to induce hepcidin antimicrobial peptide expression and hypoferremia following systemic infection in an Hfe-independent manner. As predicted, Hfe−/− mice, a model of hereditary hemochromatosis, displayed reduced spleen iron content, which translated into improved control of Salmonella replication. Salmonella adapted to the iron-poor microenvironment in the spleens of Hfe−/− mice by inducing the expression of its siderophore iron-uptake machinery. Dietary iron loading resulted in higher bacterial numbers in both WT and Hfe−/− mice, although Hfe deficiency still resulted in better pathogen control and improved survival. This suggests that Hfe deficiency may exert protective effects in addition to the control of iron availability for intracellular bacteria. Our data show that a dynamic adaptation of iron metabolism in both immune cells and microbes shapes the host-pathogen interaction in the setting of systemic Salmonella infection. Moreover, Hfe-associated iron overload and dietary iron excess result in different outcomes in infection, indicating that tissue and cellular iron distribution determines the susceptibility to infection with specific pathogens.Genetic and dietary forms of iron overload have distinctive clinical and pathophysiological features. HFE-associated hereditary hemochromatosis is characterized by overwhelming intestinal iron absorption, parenchymal iron deposition, and macrophage iron depletion. In contrast, excessive dietary iron intake results in iron deposition in macrophages. However, the functional consequences of genetic and dietary iron overload for the control of microbes are incompletely understood. Using  HFE encodes an atypical MHC class I molecule which plays a major role in the regulation of iron homeostasis under basal conditions  hereditary hemochromatosis (HH)  impair this iron-sensing mechanism, resulting in the insufficient generation of Hamp and increased iron absorption : Hfe fw: 5′-GAATTAACAGGCCGTTTCTAAAG-3′, Hfe rev: 5′-CTTGGAGTAGTGGCTCACACT-3′, Hfe neo: 5′-GAGATCAGCAGCCTCTGTTCC-3′.All animal experiments described were performed in accordance with Austrian legal requirements. Design of the animal experiments was approved by the Austrian Federal Ministry of Science and Research . Mice were maintained at the central animal facilities of the Medical University of Innsbruck and given free access to water and food. in vivo infection experiments .Serum iron was measured using the QuantiChrom Iron Assay kit (BioAssay Systems) according to the manufacturer's instructions. Serum FT was measured by a specific ELISA kit (LifeSpan BioSciences) according to the manufacturer's protocol  according to a standard protocol  of 10.Thioglycolate-elicited primary peritoneal macrophages were harvested as described  or loaded onto human apo-transferrin , cells were incubated for an additional 8 h. Intracellular bacilli were harvested according to a modified protocol as described  statistic was used. Non-parametric variables (CFU and serum FT) were log-transformed prior to testing. P < 0.05 was used to determine statistical significance, 0.05 ≤ P < 1.0 was considered a statistical trend and depicted.Statistical analysis was carried out using a SPSS statistical package. Calculations for statistical differences between various groups were carried out by ANOVA and Tukey's correction for multiple tests. Otherwise, a two-tailed unpaired Student's Salmonella infection. Wildtype (WT) C57BL/6 (Hfe+/+) and congenic Hfe/−− mice were fed either a standard rodent diet with adequate iron content (IA) or an iron-enriched (IE) diet for 3 weeks prior to and during infection. Congenic WT and Hfe−/− mice were then systemically infected with 500 colony-forming units (CFU) of Salmonella enterica serovar Typhimurium ATCC14028  via intraperitoneal (i.p.) injection  diet showed elevated serum iron and serum ferritin (FT) levels as compared to Hfe+/+ controls. Both Hfe+/+ and Hfe−/− responded to systemic infection with a reduction of serum iron concentrations (hypoferremia)  Figure . Of intel Figure .Hfe−/− mice tended to have lower hepatic Hamp mRNA expression as compared to congenic WT mice, the induced Hamp expression in response to infection or dietary iron overload remained intact in Hfe−/− mice compared to WT littermates  expression was significantly reduced following Salmonella infection  diet succumbed 1–2 days earlier  or iron-enriched (IE) diet (3 of 9 mice in each group) survived the infection beyond day 10 of the observation period. Hepatic and splenic microbial loads of randomly selected animals were quantified on days 2 and 4 of infection. Dietary iron overload significantly increased the bacterial load in both organs in WT mice as well as in Hfe−/− animals 2 days post-infection  were not affected by the Hfe-genotype Figures . IncreasSalmonella-infected mice on day 2 post-infection. We found that multiple genes involved in iron uptake were expressed at higher levels in the spleens of Hfe−/− mice on an IA diet as compared to the spleens of WT mice receiving the same diet. These genes encoded outer membrane siderophore receptors IroN, FepA, and CirA, as well as the siderophore exporter IroC  through a combined limitation of intestinal iron absorption and macrophage iron recycling. Hamp and its receptor Fpn1 are primary mediators of the hypoferremia, and thereby influence Salmonella-host interactions  has long been known to influence the course of infection with Salmonella-infected mice and that Hfe deficiency reduces the bacterial load. Of note, oral iron overload in the setting of Hfe deficiency resulted in intermediate pathogen numbers in spleen and liver on days 2 and 4 of infection. In contrast, the survival of Hfe−/− mice over a 10 day period was not affected by dietary iron. Hfe−/− mice with dietary iron overload remain more resistant to Salmonella infection than WT mice receiving the same diet, with reduced organ loads and increased survival  (Berger et al., Hfe−/− mice, it is possible that one of the siderophore-independent effects of Lcn2 may play a role. Lcn2 is a chemoattractant for neutrophils, but this is unlikely to account for a survival difference beyond day 3 of infection (Schroll et al., Hfe−/− and Hfe+/+ mice (Nairz et al., Salmonella infection is directly or indirectly influenced by the intestinal microbiome, which is modulated by Lcn2 (Raffatellu et al., Hfe−/− mice on an IE on IA diet may involve an Lcn2-independent mechanism beyond innate immunity. For instance, Hfe deficiency may have beneficial effects on apoptosis, ferroptosis, autophagy, or the oxidative stress response within or outside of the myeloid compartment that is independent of dietary iron. If a vital organ system were to be involved, an effect on host survival would be a plausible. An unbiased approach such as RNA-sequencing may be required to identify such a mechanism.The iron content of macrophages is influenced by several mechanisms including iron levels in the extracellular microenvironment, expression of iron importers, and exporters, and the rates of erythrophagocytosis and heme-iron recycling (Canonne-Hergaux et al., l Figure  despite l Figure . Albeit S. Typhimurium expressed genes required for siderophore-mediated iron uptake in vivo in the iron-poor spleens of Hfe−/− mice. This induction was specifically abrogated by dietary iron overload. This observation raises the question whether virulence factors other than siderophore genes may have been repressed in the setting of dietary iron overload to enhance the survival of Hfe−/− mice. This possibility is supported by the known cross-regulation of bacterial iron homeostasis and virulence gene expression (Zaharik et al., Neisseria gonorrhoeae residing within human monocytes (Zughaier et al., A central and novel finding of our study is that both the host and the microbe adapt their iron metabolism during infection. Hfe mutation alters the iron content of macrophages, which renders the host more resistant to infections with the intracellular pathogen S. Typhimurium. The selective pressure imposed by intracellular pathogens may have contributed to the evolutionary conservation of the HFE C282Y mutation, accounting for its high allelic frequency in Caucasians (Datz et al., In summary, the present study highlights the central role of macrophage iron homeostasis in the outcome of infections with iron-dependent intracellular microbes and the differential effects of genetic and dietary iron overload. We also demonstrate that Hfe is not required for the induction of hypoferremia in infected animals on an iron-replete diet. Nevertheless, the MN planned and performed experiments, analyzed the data and wrote the manuscript. AS, DH, SD, PT, ED, and PM performed experiments. HH, FF, and IT interpreted results and edited the manuscript. GW conceived the study, analyzed the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This encouraging result indicates that the allogeneic option is viable and should be tested in future clinical trials if the donor and recipient are HLA matched.Age-related macular degeneration (AMD) is a medical condition of the eye that results in blurred vision and/or a loss of central vision. The wet form of AMD – caused by abnormal blood vessel growth – is often treated using anti-VEGF medication. This approach can slow deterioration but is unable to treat the cause, or reverse the effects of, retinal pigment epithelium (RPE) atrophy. A regenerative medicine strategy currently being explored is the possibility that healthy RPE tissue could replace defective RPE. Masayo Takahashi from the RIKEN Center for Developmental Biology  presented data on the creation and utilization of RPE cells from inducible pluripotent stem cells (iPSCs) to treat wet AMD. In 2014, Takahashi led a pilot study of the first transplant of human iPSC-derived tissue into a human being. Skin cells taken from a patient suffering from wet-AMD were converted into iPSCs, differentiated into RPE cells and transplanted back into the patient's eye. Importantly, transplanted cells were not rejected and resulted in the stabilization of visual acuity. However, using autologous cells is a costly option and is of limited use to patients with acute ocular diseases. To address these points, Takahashi has explored the therapeutic use of RPE cells from iPSCs derived from human leukocyte antigen (HLA)-matched homozygote donors. When tested in a monkey model, no rejection signs were observed in monkey iPSC-RPE allografts of MHC-matched animal models, whereas immune attacks around the graft were detected in the MHC-mismatched situation. Using an 2Myocardial infarction (MI) occurs when blood flow to the heart is prevented, usually as a result of a blood clot. This can cause damage to the heart muscle, resulting in death of cardiac tissue which is replaced with collagen scar tissue. Scar tissue is not contractile and does not possess the electrical conductivity of cardiac tissue, putting the patient at risk of heart failure and arrhythmias. Michael Laflamme  presented preclinical data on the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to improve the function of infarcted hearts. Using a guinea pig model, when hESC-CM cells were grafted into injured hearts they were able to integrate into host heart muscle. Shared gap junctions between these tissues were observed along with an improved mechanical function in treated animals. To test host-graft electrical coupling, grafted tissue was labelled with a genetically-encoded calcium sensor. Host ECG could be correlated to calcium flux in donor tissue, demonstrating a certain degree of 1:1 host-graft coupling in damaged hearts. To determine if this approach could be translated into larger animal models where many more cells would be needed, a non-human primate model of myocardial ischemia-reperfusion was utilized. Macaques with MI were treated with 1 billion hESC-CMs, delivered to and around the infarct region. Autopsy showed significant remuscularization of the infarcted heart, with reciprocal gap junctions and integration of host vasculature into donor tissue. However, in contrast to small animal models, small non-fatal arrhythmias were observed in engrafted macaques. These results suggest that hESC-CM grafts are able to improve the function of infarcted hearts, but the challenge is to improve electrical properties of the graft to minimize arrhythmic complications. The next step is to refine this promising approach in a pig MI model which is more faithful to the human situation.3NCT02808442). This trial is currently recruiting participants and hopes to prove the effectiveness of UCART19 cells in larger groups of patients. Similar studies are needed to test if this therapeutic approach can be expanded to treat other aggressive forms of chemotherapy-resistant cancer.Chemotherapy has been used with great success to treat patients with leukemia. Unfortunately, this approach is not always effective, especially when cancerous cells become refractory through resistance mechanisms. Until recently, palliative care has been the only option left for patients suffering from aggressive drug-resistant leukemia. However, developments in the field of immunology are revolutionizing the way hematological cancers are tackled. Waseem Qasim  presented one such strategy that is being used with great success to treat CD19-positive B-cell Acute Lymphoblastic Leukemia (B-ALL). Essentially, T cells derived from healthy donors were molecularly tweaked using genome editing technology. These so-called UCART19 cells express chimeric antigen receptors (CARs) that target surface CD19 proteins found on leukemic cells, and kill them. CART19 cells were initially tested in two extremely sick children with relapsed B-ALL. Using a special license allowing application of unlicensed medical products, CART19 cells were administered. This was a landmark experiment in which gene-edited therapeutic T cells were being tested in humans for the first time. Both patients responded remarkably well to the treatment, showing robust abolition of leukemic cells. This pioneering study informed the dosing and schedule of a Phase 1 trial to evaluate the safety and ability to induce molecular remission in pediatric patients with relapsed/refractory CD19-positive B-ALL, taking place at Great Ormond Street hospital , discussed the generation of kidney organoids from hPSCs and posed the question: where to from here? Approximately 10% of Australian's suffer from chronic kidney disease, driving the need to better model diseases in the laboratory to dissect etiological mechanisms, screen therapeutic agents and explore the possibility of regenerative applications. Little presented a protocol to differentiate hPSCs into all four kidney progenitor populations , that could be further directed to form self-organized kidney organoids. Detailed analysis of these 3D organoids identified at least nine different kidney cell types, and transcriptional profiling indicated similarity to that of a first trimester fetal kidney. Functional testing demonstrated that these kidney organoids exhibit some of the actions of an intact kidney, including formation of a brush border within the proximal tubules that can absorb nutrients. These structures also express multiple drug transporters that respond to nephrotoxins, making them appropriate for drug screening assays. However, fundamental processes such as blood filtration and urine production are not present and introducing additional steps or bioengineering methods to refine the protocol might expand organoid functionality. Nevertheless, creating kidney organoids from patient-derived iPSCs provides a means to understand and validate novel mutations. One third of kidney diseases are inherited, so recapitulating renal kidney disease in the dish will enable the development of mutation-specific therapies and even correct the mutation using gene editing technologies. Perhaps in the future, corrected kidney organoids could be transplanted back into the patient to replace defective kidney tissue. Organoids recapitulating many different body parts have now been created, providing an 5Streptococcus pyogenes Cas9 (SpCas9), to abolish off-target effects while preserving on-target activity. The crystal structure of SpCas9:DNA indicated that positively charged amino acids were found in the cleft of SpCas9 that holds the negatively charged non-target DNA strand. These residues could be systematically substituted for neutral amino acids to reduce this binding and, in theory, necessitate more stringent base pairing between the RNA guide and target DNA strand to improved specificity. Indeed, a number of mutants were identified that were able to reduce off-target activity by at least 10-fold compared to wildtype SpCas9, while maintaining on-target cleavage activity. The second topic discussed was how bacterial diversity could be explored to identify new CRISPR-Cas systems. To date, only two Class 2 nucleases – those with a single effector subunit – have been described, Cas9 and Cpf1. This runs counter to the extreme diversity shown by microbes that are likely to have evolved additional Class 2 variants. Using a computational prediction approach across microbial genome sequences, new candidate Class 2 CRISPR-Cas9 loci were identified. A total of 53 loci were discovered, that were divided into three distinct groups based on the nature of their effector proteins, denoted C2c1–3 (Class 2 candidate 1–3). The functionality of these Class 2 variants are currently being tested, and will perhaps offer novel mechanisms of action to expand the CRISPR-Cas toolkit.Feng Zhang from The Broad Institute MIT  gave an inspiring talk on the prospects and challenges of genome engineering. The presentation covered two main topics. First, strategies to improve the specificity of genome editing were discussed. A propensity for RNA-guided nucleases to cut at non-specific sites, resulting in unintended mutagenesis, has hampered the use of CRISPR-Cas in situations requiring high levels of specificity. Zhang's approach to tackle this issue was to use structure–guided protein engineering of 6NCT01541579). The primary endpoint was met with half of the patients treated with a single injection of Cx601 achieving remission at week 24. Cx601 was well tolerated in the study population and no immune reaction or specific treatment emergent adverse events were detected. Follow-up analysis at 52 weeks confirmed the sustained efficacy and safety profile of Cx601. As a result of these encouraging results, TiGenix has submitted a Marketing Authorization Application to the EMA and is preparing to develop Cx601 in the United States after having reached an agreement with the FDA. Other ASC products are in the clinical pipeline at TiGenix to treat a range of inflammatory diseases that are treatment-refractory, underscoring the clinical utility of ASCs as a therapeutic entity.In a session dedicated to MSC Gene & Cell Therapy, Wilfried Daelemans from TiGenix , presented data on the use of expanded allogeneic Adipose-derived Mesenchymal Stem Cells (ASCs) to treat inflammatory disorders. The rationale for using ASCs is that they possess anti-inflammatory and immunomodulatory potential, whilst being well tolerated. ASCs are separated from liposuction-derived adipose fat, characterized, expanded and stored in a cell bank until required. The use of ASCs in a variety of preclinical mouse models of inflammation were presented. Administration of ASCs resulted in decreasing the severity of established arthritis in a collagen-induced mouse model for rheumatoid arthritis, primarily by restoring the Treg compartment in treated animals. Similarly encouraging results were seen in a dextran sulfate sodium-induced model of colitis, and in both lipopolysaccharide and cecal ligation and puncture models of sepsis ASC-treated mice showed decreased mortality compared to non-treated controls. To illustrate the success of this approach in humans, results from a recently completed clinical trial were highlighted. This Phase 3 randomized, multicenter trial aimed to evaluate the safety and efficacy of allogeneic ASCs (Cx601) for treatment of treatment-refractory complex perianal fistulas in Crohn's disease patients (


The development of accurate, non-invasive urinary assays for bladder cancer would greatly facilitate the detection and management of a disease that has a high rate of recurrence and progression. In this study, we employed a discovery and validation strategy to identify microRNA signatures that can perform as a non-invasive bladder cancer diagnostic assay. Expression profiling of 754 human microRNAs (TaqMan low density arrays) was performed on naturally voided urine samples from a cohort of 85 subjects of known bladder disease status (27 with active BCa). A panel of 46 microRNAs significantly associated with bladder cancer were subsequently monitored in an independent cohort of 121 subjects (61 with active BCa) using quantitative real-time PCR (RT-PCR). Multivariable modeling identified a 25-target diagnostic signature that predicted the presence of BCa with an estimated sensitivity of 87% at a specificity of 100% (AUC 0.982). With additional validation, the monitoring of a urinary microRNA biomarker panel could facilitate the non-invasive evaluation of patients under investigation for BCa. The development of accurate assays that can detect and monitor bladder cancer (BCa) non-invasively through urinalysis would benefit both patients and healthcare systems. A number of tests have been developed to detect bladder tumor-associated urinary biomarkers, but due to inadequate sensitivity and poor overall accuracy, none of these assays have sufficient predictive power to be applied to the management of individual patients . A shiftIn previous studies, we demonstrated the feasibility of profiling the mRNA transcriptome of urothelia obtained from naturally micturated urine and developed an analytical approach to identify cancer-associated gene signatures. Genome-wide expression profiling and validation of selected candidate biomarkers in an independent cohort of subjects identified multiplex mRNA signatures that achieved promising diagnostic performance –5. In thFrom the profiling of 754 human miRNAs in a cohort of 85 subjects, 46 miRNAs were identified as being significantly associated with the presence of BCa, and these were monitored in an independent validation cohort of 121 subjects using quantitative real-time PCR (RT-PCR). Multivariable modeling identified multiplex miRNA signatures that achieved encouraging diagnostic performance values. This phase II biomarker development study [Urine samples were collected consecutively from a total of 206 subjects, of which, 88 subjects had biopsy-proven, active BCa. Samples from the first 85 subjects collected (discovery-cohort 1) were profiled to identify candidate miRNA markers, and samples from the subsequent 121 subjects  were used to assess the potential utility of the selected markers in an independent analysis. Demographic and clinicopathological details of cases and controls for each cohort are provided in Table P < 0.05) with BCa status. A set of 46 miRNAs . Of the 754 miRNAs included on the Taqman low density arrays (Human MicroRNA Set v3.0), 267 were detected in at least one urothelial cell sample. Comparative group (cases vs. controls) analysis identified 108 miRNAs that were significantly associated  were significantly associated with gender  while maintaining optimal specificity (100%). These values compare very favorably with those of cystoscopy and VUC, which both rely on high specificity for overall accuracy. Restriction to reduced target prediction models resulted in an expected reduction in performance values, but even a 10-miRNA model achieved sensitivity of 84% and specificity of 87% (AUC 0.902). The model performance values also compare well with existing urinary tests for BCa detection. To date, there are four urine tests that have received FDA approval for diagnostic clinical use , and a couple of others with approval restricted to post-treatment monitoring . In a meet al. reported that the ratio of miR-126:miR-152 in urine was indicative of BCa [et al. monitored 15 miRNAs in a cohort of 121 subjects [et al. described levels of the miR-200 family plus miR-192 and miR-155 associated with Bca [A number of studies have investigated the expressed miRNA repertoire in excised bladder tumor tissues and identified specific miRNAs that inform tumor stratification, predict outcome –21 or ine of BCa , Miah etwith Bca , but theet al. monitored over 750 miRNAs in urine samples obtained from 40 subjects, and identified a 6-miRNA diagnostic biomarker panel that achieved 85% sensitivity and 86% specificity for BCa detection in an independent cohort [et al. profiled 730 miRNAs in pooled samples from 130 subjects to identify a 4-miRNA panel that had a diagnostic sensitivity of 88% and specificity of 78%, but the panel was not tested in an independent cohort [As miRNA characterization and reagent availability has improved, a number of more comprehensive urinary profiling studies have been performed. In a study conducted in Spain, Mengual t cohort . In a USt cohort . Most reIn our study, the optimal miRNA signature was comprised of 32 targets, and with improving quantitative PCR technologies the monitoring of multiplex RNA panels is not a limiting factor. The limiting of prediction models to 15 and 10 miRNAs expectedly resulted in some loss of performance, but it also revealed several miRNAs  that consistently contributed to all models. Given the relatively small number of characterized miRNAs , there iWe recognize that the study has a number of limitations. Although both cases and controls were collected consecutively, molecular analyses were only initiated when balance of samples in each group was achieved. Firstly, disease prevalence is typically considerably lower in urologic practice, so evaluation of the validation study cohort is likely to provide an overly optimistic assessment of the assay predictive value. Secondly, as samples were collected prior to clinical evaluation for bladder cancer, other neoplastic urological conditions  are under-represented in our control study cohort. It is also understood that biomarker performance values derived from limited validation studies can over-estimate potential value with respect to utility in larger, more diverse independent cohorts. Continued recruitment of patients in ongoing in order to perform larger studies that will better represent urological disease prevalence, and evaluate potential confounding comorbidities. As we expand the cohorts to include the breadth of urology clinic visitors, we will also endeavor to derive predictive models that might perform well for specific clinical scenarios, including first-event diagnosis or disease recurrence monitoring. If a highly sensitive microRNA test can be established, there is potential to reduce unnecessary, invasive cystoscopy procedures. Furthermore, beyond VUC, it will be of interest to directly compare the performance of a miRNA-based diagnostic signature with existing urine tests in the same samples. While there are issues with the measurement of labile RNA analytes, a number of RNA-based assays are being translated into tests that meet clinical laboratory standards .The development of accurate assays that can detect and monitor bladder cancer non-invasively through urinalysis would be a major advance, benefiting both patients and healthcare systems, but this remains a challenge. In this study, we have identified urinary miRNA signatures that achieve encouraging diagnostic performance values. Additional, prospective studies are underway that will incorporate enough samples to stringently test the robustness of biomarkers and models , 15 and Under IRB approval and informed consent, urine samples and associated clinical information were consecutively collected from subjects visiting the urology clinic at MD Anderson Cancer Center at Orlando, FL between 2010 and 2013. The discovery cohort consisted of 58 individuals with no evidence of active urothelial cell carcinoma (controls) and 27 individuals with primary urothelial carcinoma (cases). The validation cohort consisted of 60 individuals with no evidence of active urothelial cell carcinoma (controls) and 61 individuals with newly diagnosed primary urothelial carcinoma (cases). All subjects underwent standard clinical work-up, including office cystoscopy, and the majority also had axial imaging of the abdomen and pelvis. For the bladder cancer case group, histological confirmation of urothelial carcinoma, including grade and stage was defined from excised tissue. A summary of clinical data for both cohorts is given in Table ® Array Human MicroRNA A+B Cards Set v3.0 (Applied Biosystems Cat# 4444913). The Taqman low density array (TLDA) format is a 384-well system that uses standard TaqMan assays and enables automated loading and high-throughput analyses [P-value) and fold-change, including 4 targets negatively associated with BCa. The complete list of targets selected for validation and Applied Biosystems TaqMan assay ID details are listed in Profiling of 754 human miRNAs was performed using TaqMananalyses . Details-DDCT. Differential miRNA expression between bladder cancer cases and controls was determined using Student's t-statistic. For the validation study analyses (cohort 2), differences in clinical covariates between bladder cancer cases and non-malignant controls were evaluated via Chi-squared test and Wilcoxon Rank Sum test, as appropriate. For each miRNA, the percentage of samples that were censored  was calculated for cases and controls separately  values from duplicate data were calculated by normalization with the endogenous reference Mamm U6 miRNA and the fold-change between bladder cancer and control samples was calculated as log 2 parately . To avoi


Hospital Exposures and Long-Term Health), part of the DINE  cohort of the ECHO  program. Seventy-six percent of eligible infants enrolled in the study. Sixty-four of 81 infants survived and are included in this analysis. 164 urine specimens were analyzed for phthalate metabolites using high-performance liquid chromatography/tandem mass spectrometry. The NICU Network Neurobehavioral Scale (NNNS) was performed prior to NICU discharge. Linear and weighted quantile sum regression quantified associations between phthalate biomarkers and NNNS performance, and between phthalate biomarkers and intensity of medical intervention. The sum of di(2-ethylhexyl) phthalate metabolites (∑DEHP) was associated with improved performance on the Attention and Regulation scales. Specific mixtures of phthalate biomarkers were also associated with improved NNNS performance. More intense medical intervention was associated with higher ∑DEHP exposure. NICU-based exposure to phthalates mixtures was associated with improved attention and social response. This suggests that the impact of phthalate exposure on neurodevelopment may follow a non-linear trajectory, perhaps accelerating the development of certain neural networks. The long-term neurodevelopmental impact of NICU-based phthalate exposure needs to be evaluated.Every year in the United States, more than 300,000 infants are admitted to neonatal intensive care units (NICU) where they are exposed to a chemical-intensive hospital environment during a developmentally vulnerable period. The neurodevelopmental impact of environmental exposure to phthalates during the NICU stay is unknown. As phthalate exposure during the third trimester developmental window has been implicated in neurobehavioral deficits in term-born children that are strikingly similar to a phenotype of neurobehavioral morbidity common among children born premature, the role of early-life phthalate exposure on the neurodevelopmental trajectory of premature infants may be clinically important. In this study, premature newborns with birth weight <1500g were recruited to participate in a prospective environmental health cohort study, NICU-HEALTH ( Every year in the United States, more than 300,000 infants are admitted to neonatal intensive care units (NICUs) where, in addition to experiencing life-saving treatments, they are exposed to a chemical-intensive hospital environment during a developmentally vulnerable period.–5 PrematPhthalates are a family of industrial chemicals non-covalently bonded to plastic materials to enhance flexibility and durability. Particulin utero exposure to phthalate mixtures has been associated with poorer infant executive function, attention, and motor reflexes.– 1)/(SG[specimen]– 1) where SG[reference] was the median specific gravity for all specimens analyzed. The geometric mean per participant of biomarker levels from all specimens collected during the hospitalization was used for regression analyses of association with neurodevelopmental performance. Individual specimen biomarker levels were used in analyses aimed at medical equipment exposure classification.Each measured biomarker level was adjusted for urinary dilution with urine specific gravity using the following adjustment: We created a directed acyclic graph  and NNNS summary scales adjusted for covariates. We expressed DEHP molar sum results in ng/mL for convenience of interpretation, using the molecular weight of MEHP (278g/mol) as a molecular weight estimate per Teitelbaum and WolfAs the complex NICU environment conveys exposure to mixtures of phthalates rather than exposure to single phthalate diesters in isolation, WQS regression was perfet al.,[et al. raises the possibility of misclassification between the medium and high exposure groups, we chose to use these established groupings to allow comparison to the existing, if sparse, literature on NICU-based phthalate exposure.From enrollment until NICU discharge, trained study staff kept daily inventory of medical equipment exposure for each infant. Following Green et al., we categEighty-one premature infants were enrolled in phase I of NICU-HEALTH. The 64 who survived, had at least one urine specimen analyzed for phthalate monoesters, and had NNNS performed prior to NICU discharge are the cohort evaluated in this study. Pertinent demographic characteristics are presented in One hundred sixty four urine specimens were analyzed for 15 phthalate monoester metabolites. Five monoesters, MiPP, MCHP, MnPP, MiNP, and MOP, were measured above the LOD in < 15% of specimens and were not included in statistical analyses. Specific gravity-adjusted urinary phthalate concentrations are presented in in utero drug exposure are known to be more easily excitable and with a baseline higher level of arousal than non-withdrawing populations.Assessment with the NNNS was completed prior to NICU discharge. Descriptive statistics of our cohort’s performance alongside available comparative populations are provimproved performance. After Holm-Bonferroni correction, the association between ∑DEHP and Attention remained significant.Adjusted multivariable linear regression analysis demonstrated small but significant associations between ∑DEHP exposure and multiple summary scales of the NNNS . ∑DEHP wimprovement in summary scale performance in all subscales except habituation. Habituation summary scale assessments were only available for 18/64 (28%) infants due to the specific resting state requirements of habituation maneuvers. Other NNNS summary scales were based on the full cohort. Certain phthalate monoesters—notably MEOHP—were implicated as clinically important to performance on multiple summary scales.WQS regression revealed significant associations between specific mixtures of phthalate biomarkers and performance on the NNNS summary scales of Attention, Handling, Non-Optimal Reflexes, Regulation, Excitability, and Habituation . After HBased on daily assessments of clinical equipment exposure and the known timing of urine specimen collection, we were able to link 158 individual urine specimens with a distinct medical exposure category . Exposurimproved performance on specific NNNS summary scales was unexpected, but consistent across analyses focused only on DEHP metabolites and on agnostically derived phthalate mixtures.This study is the first to relate NICU-based phthalate exposure to neurobehavioral outcome. As a biomarker-based prospective study designed from its inception as an environmental health cohort of premature infants, it is unique in its population and approach. The primary finding, that NICU-based phthalate exposure was associated with in utero exposure associated with adverse neurodevelopmental outcomes in recent term birth cohorts.[Phthalate metabolite levels in the first phase of NICU-HEALTH were lower than those previously reported in NICU patients., 28 This cohorts., 45, 70in utero phthalate exposure in term children and behavioral outcomes in middle childhood,[better on multiple NNNS summary scales.Based on studies of hildhood,, 42–45 whildhood,–45 but ahildhood,, 28, 71 development, not a measure of static cognitive ability or behavior. As NNNS performance improves with maturity, improved summary scale scores can be interpreted as either improved ability compared to PMA-matched peers, or attainment of neurodevelopmental milestones earlier than expected. Commonly proposed mechanisms of action of phthalate neurotoxicity involve endocrine disruption via interference with androgen synthesis and thyroid function—two pathways known to be critically important to neurodevelopment. Additionally, phthalate exposure is linked to epigenetic modification which could alter neurodevelopmental velocity during times of neuronal plasticity.[Although unexpected, our findings are biologically plausible and may provide insight into the sometimes inconsistent data on the relationship between phthalate exposure during early development and neurodevelopmental outcomes. Our outcome measure, the NNNS, is a measure of neurobehavioral asticity.–74 If phasticity.et al.[well on neurobehavioral testing early in life. It was hypothesized that the stressful in utero environment provoked rapid maturation as a protective mechanism for an anticipated stressful postnatal environment. When followed into middle childhood, these children transition to a phenotype of inattention and hyperactivity, thus demonstrating the widely recognized association between prenatal exposure to stress and poor behavioral outcome in childhood.[Epidemiological studies in other fields have demonstrated the relationship between environmental exposures during the third trimester neurodevelopmental window and more rapid behavioral maturation. Specifically, Posner et al. found thhildhood.–80 The “improved NNNS performance reduced concerns about confounding by indication.Our study has a number of strengths. It was designed to address limitations of the few other biomarker-based studies of NICU-based phthalate exposure in the literature,–28 whichOur study does have some limitations. Our patient population, very low birth weight infants cared for at a level IV academic regional perinatal center, may not be representative of the entire NICU population. As data on “typical” community-based exposure to phthalates in early infancy in non-NICU and non-preterm populations is not available , comparisons to a relevant non-NICU group is not possible. The half-life of phthalate diester metabolism in preterm infants is not well characterized, but is known to vary between 4 and 24 hours in adults.–84 This Although many NICUs—including ours—are increasingly labeled “phthalate-free” to signify the use of DEHP-free equipment whenever available, replacements for DEHP are often other phthalates or non-pimproved attention and social response on the NICU Network Neurobehavioral Scale. The long-term impact of this association between phthalate exposure and neurobehavior needs to be evaluated as our preterm cohort ages.In this first phase of a prospective cohort study of the neurodevelopmental impact of NICU-based phthalate exposure on very low birth weight infants, specific mixtures of phthalate biomarkers were associated with S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 FigCovariates included in the model were unique elements of the CRIB-II score, gestational age at birth, a variable representing medical illness in the NICU, and gender.(TIF)Click here for additional data file.


The population of mixed ethnicity individuals in the UK is growing. Despite this demographic trend, little is known about mixed ethnicity children and their problem behaviours. We examine trajectories of behavioural problems among non-mixed and mixed ethnicity children from early to middle childhood using nationally representative cohort data in the UK.Data from 16 330 children from the Millennium Cohort Study with total difficulties scores were analysed. We estimated trajectories of behavioural problems by mixed ethnicity using growth curve models.White mixed , Indian mixed (7.7), Pakistani mixed (8.9) and Bangladeshi mixed (7.2) children had fewer problem behaviours than their non-mixed counterparts at age 3 . White mixed, Pakistani mixed and Bangladeshi mixed children had growth trajectories in problem behaviours significantly different from that of their non-mixed counterparts.Using a detailed mixed ethnic classification revealed diverging trajectories between some non-mixed and mixed children across the early life course. Future studies should investigate the mechanisms, which may influence increasing behavioural problems in mixed ethnicity children. Research has documented ethnic inequalities in health across the early life course.There is a paucity of longitudinal research investigating mixed ethnic differences in children's behavioural problems using a comprehensive mixed ethnic classification.We find White mixed, Indian mixed, Pakistani mixed and Bangladeshi mixed 3-year-old children to have fewer problem behaviours than their non-mixed counterparts.Growth curve models demonstrated that White mixed, Pakistani mixed and Bangladeshi mixed children experienced increases in problem behaviours compared with their non-mixed counterparts, particularly after age 7.Existing evidence shows ethnic variation for a variety of markers of health across the early life course.Only one study investigated mixed ethnic differences in behavioural problems during early childhood and reported no link between mixed ethnicity and behavioural problems among 3-year-old children.Research has called for more robust evidence on mixed ethnicity children's behavioural problems.The Millennium Cohort Study (MCS) is a nationally representative longitudinal study of 18 552 infants born in the UK between September 2000 and January 2002.Problem behaviours were assessed using the Strengths and Difficulties Questionnaire (SDQ),Ethnic categories were constructed using mother's reports of the cohort child's ethnicity and were based on census categories. We drew upon father's ethnicity to facilitate the ethnic classification of the cohort child. A cohort child's ethnicity is categorised as mixed if the main respondent  chose a mixed category or if the ethnic categories for the child's parents were different. If the main respondent chose a ‘mixed’ ethnic category for the child, but the categories of the child’s natural parents were the same, we reclassified the child according to the parents’ ethnicity. Ethnic categories used for analysis are white; Indian; Pakistani; Bangladeshi; black Caribbean; black African; other and their ‘mixed’ counterparts. The white mixed group is made up of children whose parents are from different white backgrounds, for example, a white mixed child might have a white European mother and a white British father.Our analyses included cohort member's gender, age, sweep at assessment as a measure of time and a quadratic term for time. The linear but not the quadratic term varied by ethnicity. We explored the impact of equivalised household income in quintiles, measured as a time-varying variable (middle-income quintile is reference)Behavioural outcomes are moderated by multiple births,We estimated growth curve models for children's behavioural problems trajectories for the analytic sample.10.1136/archdischild-2015-309701.supp1Supplementary tablesThe patterns in problem behaviours are less consistent at age 11. White and Indian mixed children have fewer problem behaviours compared with their non-mixed counterparts. Pakistani, Bangladeshi and Black Caribbean mixed children have more problem behaviours than their non-mixed counterparts. Black African mixed children have fewer behavioural problems than their non-mixed counterparts. In order to closely scrutinise the increase and decrease in problem behaviours over time, we turn to growth curve models to more accurately distinguish differences between mixed ethnicity children and their counterparts.In sensitivity analyses, adjustments for equivalised household income decreased the mean differences in intercepts of child behaviour between mixed and non-mixed ethnic minorities. However, the magnitude and significance of trajectories were not sensitive to adjustments for income (see In this paper, we extend our understanding of mixed ethnicity differences in behavioural problems by examining children's trajectories beginning in early childhood and including a comprehensive mixed ethnic classification using nationally representative data. At age 3, we find white mixed, Indian mixed, Pakistani mixed and Bangladeshi mixed children to have fewer problem behaviours than their non-mixed counterparts. This finding is in stark contrast to previous research finding no differences in problem behaviours between a catch-all mixed group and non-mixed 3-year-old children.Our findings suggest that by age 11 some mixed children have more problem behaviours than their non-mixed counterparts. There are two possible explanations for this result. One, mixed ethnicity as a category may reflect not only social stratification processes but also shifts in identity and potential confusions between personal and social identities.The aim of this short report was to describe, for the first time, trajectories in problem behaviours for non-mixed and mixed ethnicity children in a nationally representative sample. White mixed, Pakistani mixed and Bangladeshi mixed children had significantly different growth trajectories compared with their non-mixed counterparts. Future research might examine the mechanisms, such as school, psychosocial and socio-demographic factors, behind these differences.


Most of these variants reside in non-coding regions of the genome potentially affecting transcriptional and post-transcriptional gene regulation. Although a few hallmark examples of driver mutations in non-coding regions have been reported, the functional role of the vast majority of somatic non-coding variants remains to be determined. This is because the few driver variants in each sample must be distinguished from the thousands of passenger variants and because the logic of regulatory element function has not yet been fully elucidated. Thus, variants prioritized based on mutational burden and location within regulatory elements need to be validated experimentally. This is generally achieved by combining assays that measure physical binding, such as chromatin immunoprecipitation, with those that determine regulatory activity, such as luciferase reporter assays. Here, we present an overview of  Cancer initiation, progression, maintenance, and metastasis originate from somatic single nucleotide variants (SNVs), small insertions and deletions, structural variants, and epigenetic alterations . In partFigure 1)  . This inigure 1) .Despite recent advances in the understanding of the downstream consequences of non-coding SNVs, it remains a challenge to identify non-coding driver mutations and the mechanisms through which they effect biological functions. First, as stated above, non-coding SNVs can affect multiple regulatory functions including transcriptional and post-transcriptional regulation. Second, non-coding regions present higher mutations rates than coding regions, due to weaker selective pressure . As a reEarly studies that identified non-coding driver SNVs compared the sequence of regulatory regions of candidate cancer-related genes between tumor and non-tumor samples in order to determine whether these mutations disrupt or create TF binding sites. For example, SNVs were identified in the GTAAC sequence within the first intron of MYC in samples from multiple patients with Burkitt lymphomas . These mMore recently, whole-genome sequencing of matched tumor and normal samples has enabled the identification of millions of SNVs. However, the identity of the SNVs responsible for driving cancer and those that constitute passenger mutations remains to be determined. Two pioneering studies showed that mutations present in the telomerase reverse transcriptase (TERT) promoter in tumor samples of patients with melanoma lead to increased TERT mRNA expression . These sFigure 2).Computational approaches to predict functional SNVs within regulatory regions share a common general pipeline, including the identification of somatic SNVs, comparison with common germline variants, constraining the analysis to regulatory regions , identification of mutational hotspots, and determining altered TF binding sites , which needs to be distinguished from errors derived from whole-genome sequencing and genome alignment pipelines . Thus, mTable 1). While the first type of methods models sequencing errors based on statistical parameters from the sequencing reads or from non-matched normal samples, the second type of methods compare matched tumor-normal samples to distinguish true mutations from sequencing errors. Even though these algorithms have been used as stand-alone methods to call SNVs, some studies have used a combination of methods for a “wisdom of the crowd” approach with the goal of increasing the confidence in the SNVs detected  those that separately call SNVs in tumor and normal samples and then identify tumor-specific SNVs by comparison, such as GATK , GATKcandetected .Among the millions of non-coding somatic SNVs identified in different cancers, only a small number are expected to be drivers. Given that it is not currently possible to experimentally test most of the SNVs identified, methods have been developed to prioritize which SNVs are more likely to be functional. A common approach to prioritize somatic SNVs is to determine genomic regions with high mutation frequency across different cancer samples. Given the billions of bases in the human genome, the thousands of mutations per cancer sample, and that we only have sequencing data for a few thousand tumors, the chances of detecting a significantly enriched mutation across cancers after multiple hypothesis testing correction is almost null.Figure 2)  or histone marks such as H3K4me and H4K27ac (igure 2) . Furtherigure 2) . OverallFigure 2). This reduces dimensionality and increases the frequency of SNVs within each DNA window leading to increased statistical power. The identification of these mutational hotspots across cancers involves comparing the SNV frequency within a DNA window to a background distribution of SNV frequencies. These methods can be divided into local and global models, comparing the SNV frequencies to other windows in neighboring genomic regions or to functionally similar regions , respectively. The window size selection can vary widely between analysis, ranging from 50 bp . Other computational frameworks have also been used to also include distal elements in the analyses, including LARVA that incorporates background models for non-coding regions by integrating SNVs with a comprehensive set of non-coding functional elements based on DHSs and histone marks (Table 1). In addition, LARVA uses regional genomic features like replication timing allowing to better estimate local mutation rates and mutational hotspots.Although studies using low tumor sample numbers may be underpowered to identify hotspot regions, large samples sizes can also be challenging to analyze. This is because large sample sizes frequently lead to larger lists of potentially significant genes which in many cases do not have cancer-related functions, suggestive of a high false positive prediction rate . This stne marks  (Table 1Table 1). The variety of co-existing computational approaches, background models, and covariates included in those models, highlights the challenges currently faced in identifying mutational hotspots associated with cancer.Further covariates can be included while modeling mutation frequencies. For instance, recent studies have shown that some breast tumors have mutations mediated by the alipoprotein B messenger RNA-editing enzyme catalytic (APOBEC) which have been found to occur in dense hypermutated regions in the genome (kataegis) . These mFigure 2). These TF binding differences between SNV alleles can be predicted based on DNA specificities determined by protein-binding microarrays, SELEX, bacterial one-hybrid assays, or chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-seq) (Table 1). Differences in TF binding between SNV alleles can be predicted using position weight matrices (PWMs), probabilistic representations of DNA binding specificities, and motif prediction algorithms such as FIMO (Table 1). For example, MotifLocator, a tool to score how mutations affect wild-type TF binding sites, led to the identification of gain of binding sites for RB1, E2F1 and ETS to multiple promoter regions in tumor samples from TCGA  . CurrenthIP-seq)  . These approaches include RegulomeDB (Table 1).In addition, motif analyses can integrate functional annotations of regulatory sequences  and TF expression levels such as those provided by the ENCODE, Roadmap Epigenomics, FANTOM, and GTEx Projects to constrain the analyses to TFs expressed and regulatory elements active in the tissues of interest  . ChIP has been successfully used to study differential TF binding between non-coding SNV alleles in vivo . For example, several studies have identified mutations in the TERT promoter, such as G228A, that lead to the creation of de novo bind site for ETS factors , and enhanced yeast one-hybrid (eY1H) assays  (A recent study using enhanced yeast one-hybrid (eY1H) assays, a method that tests protein-DNA interactions in the milieu of the yeast nucleus, has increased the screening throughput for TF binding differences between SNV alleles by testing > 1,000 TFs in parallel, without the need for antibodies or patient samples (gure 3C) . AlthougFigure 4A)  . In addiFigure 4B)  .Recent studies using massively parallel reporter assays (MPRAs), a high-throughput technology based on barcodes and next generation sequencing, have made progress in determining whether germline SNVs associated with genetic disorders affect transcriptional regulation (gure 4B) . In partgure 4B) . Thus, tgure 4C) . These sCompared to promoters, dissecting the functional effects of mutations in distal regulatory elements such as enhancers is a more complex task as it is not trivial to determine which of these elements are functional in different cells/conditions nor the identity of the target gene involved. This, and the fact that including distal elements in hotspot analyses increases the search space and reduces statistical power are the main reasons why most studies characterizing germline and somatic non-coding SNVs have focused on promoter regions .LMO1 oncogene generated a de novo binding site for the MYB TF in patients with T-cell acute lymphoblastic leukemia   . For exablastoma , while mficiency . In addi1 levels . As with 3′ UTRs .BRAF induces the expression of a splice variant that confers resistance to vemurafenib treatment in melanoma (Mutations in the exon–intron boundaries, introns, and coding regions can affect splicing and lead to the upregulation oncogenic isoforms or the downregulation of tumor suppressor isoforms. Various cancer tumor suppressor genes such as TP53, ARID1A, PTEN, CHD1, MLL2, and PTCH1 were found to carry mutations in the exon–intron boundaries which led to intron retention . For examelanoma . These amelanoma .in silico parameters that can accurately predict differential TF binding between alleles.Recent studies have identified a handful of somatic SNVs in regulatory regions that affect TF binding and target gene expression. However, the number of functional non-coding SNVs associated with cancer is expected to be much higher given the low overlap between those reported in different studies, and given that non-coding SNVs seem to play an important role in disease based on the hundreds of functional non-coding SNVs identified in genome-wide association and genetic studies . AdvanceAnother source of underestimation of non-coding driver SNVs stems from the hotspot analysis itself as it assumes that driver mutations in a particular regulatory region should be present in multiple patients. Given the hundreds of thousands of regulatory elements in the human genome we may be far from having a sample size sufficiently large to detect most functional SNVs. An alternative approach would be to lower the stringency in the statistical pipelines and directly test thousands of “moderate-confidence” SNVs using MPRAs to identify functional variants. Ultimately, a combination of computational and experimental methods along with new technical innovations will increase our ability to identify and characterize the mechanisms by which non-coding SNV drive cancer.KG, SCP, JS, and JFB participated in the writing, reviewing, and critical analysis of the manuscript. JFB prepared the illustrations and coordinated the manuscript. All authors agreed and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.