The circadian clock orchestrates daily rhythms in many physiological, behavioral and molecular processes, providing means to anticipate, and adapt to environmental changes. A specific role of the circadian clock is to coordinate functions of the immune system both at steady-state and in response to infectious threats. Hence, time-of-day dependent variables are found in the physiology of immune cells, host-parasite interactions, inflammatory processes, or adaptive immune responses. Interestingly, the molecular clock coordinates transcriptional-translational feedback loops which orchestrate daily oscillations in expression of many genes involved in cellular functions. This clock function is assisted by tightly controlled transitions in the chromatin fiber involving epigenetic mechanisms which determine how a when transcriptional oscillations occur. Immune cells are no exception, as they also present a functional clock dictating transcriptional rhythms. Hereby, the molecular clock and the chromatin regulators controlling rhythmicity represent a unique scaffold mediating the crosstalk between the circadian and the immune systems. Certain epigenetic regulators are shared between both systems and uncovering them and characterizing their dynamics can provide clues to design effective chronotherapeutic strategies for modulation of the immune system. The immune response is perhaps the most evident organismal response against infection, and mounting research depicts the circadian system as a critical regulator of immune defense. Circadian rhythms are endogenous processes with an oscillatory pattern following a daily cycle, mostly controlled by the circadian system. Circadian clocks are hierarchically organized, with the central pacemaker located in the suprachiasmatic nucleus (SCN) in the brain, and subordinate clocks in almost all peripheral tissues  and Cryptochrome Cry1-2 genes  themselves, such as the PAR (proline and acidic amino acid–rich) basic leucine zipper (bZIP) TF family including DBP (D-box binding protein), TEF (thyrotroph embryonic factor) and HLF (hepatic leukemia factor), which impose circadian transcription to subordinate genes. Remarkably, the coordinated action between all these transcriptional regulators orchestrates a tissue or cell type-specific circadian transcriptome, and constitutes the molecular basis of circadian rhythmicity . The core components of this machinery are the clock proteins, mostly transcriptional regulators. Within the positive loop, the heterodimer CLOCK:BMAL1 rhythmically binds to e-boxes at regulatory regions in the genome, hereby inducing architectural changes in chromatin states to promote transcription of CCGs and the clock repressors -2 genes . PER and+)- dependent histone deacetylase (HDAC) SIRT1 (Sirtuin1)  to their promoters, such as the H3K27 methyltransferases EZH2 and SUZ12 and their partner EED  and their progenitors also circulate in the bloodstream in physiological conditions, showing marked circadian fluctuations in their trafficking (>2-fold), both in mice and humans  or T helper 17 cells (Th17), which participate in defense mechanisms on mucous membranes  and regulatory T-cell (Treg) differentiation  locus and a metabolic switch toward oxidative reactions - dependent SIRT1 HDAC activity is circadian by the fluctuating levels of NAD+ due to the circadian expression of the enzyme NAMPT, the rate-limiting enzyme on the NAD+ biosynthesis  histone deacetylation is crucial for differentiation (Nencioni et al., Responses to infection require big amounts of energy and resources to adequately fight threats, however these responses need to be balanced to avoid compromising critical organism functions. The circadian clock regulates daily rhythms in immune functions in normal conditions; however, during infection circadian regulation becomes critical to temporally organize the immune response and make it more effective and compatible with the host vital functions. The clock machinery are mostly transcriptional regulators which act in the chromatin fiber, and many epigenetic mechanisms are shared between the circadian and the immune systems. The circadian TFs BMAL1 and RevErbα regulate immune cell trafficking by directing the expression of chemokines involved in mobilization, and to do so they cooperate with chromatin modifiers such as members of the Polycomb family, or regulate active enhancers. Histone acetylation also plays a prominent role in orchestrating a circadian transcriptome in regulatory cell types for the immune response, while inflammatory processes are coordinated by multiple interactions between NF-κB and the clock TFs. At this regard, it will be necessary to further dissect the interactions between metabolism and the epigenome during the circadian cycle in immune cells, as it will provide means to understand how metabolic cues regulate immunity. Finally, it is important to note that much of our understanding in the circadian aspects of immunity has been studied in rodents, thus advancing this knowledge in humans will be essential for opening possibilities of translation and, specifically, to design adequate strategies in circadian medicine (Munch and Kramer, RO-S and LA-A wrote the paper.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.


Purpose: Quantifying stenosis in cardiac computed tomography angiography (CTA) images remains a difficult task, as image noise and cardiac motion can degrade image quality and distort underlying anatomic information. The purpose of this study was to develop a computational framework to objectively assess the precision of quantifying coronary stenosis in cardiac CTA.Approach: The framework used models of coronary vessels and plaques, asymmetric motion point spread functions, CT image blur (task-based modulation transfer functions) and noise (noise-power spectrums), and an automated maximum-likelihood estimator implemented as a matched template squared-difference operator. These factors were integrated into an estimability index (Results: The Conclusions: A computational framework to objectively quantify stenosis estimation task performance was successfully implemented and was reflective of clinical results in the context of a prominent clinical trial with diverse sites, readers, scanners, acquisition protocols, and patients. It also demonstrated the potential for prospective optimization of imaging protocols toward targeted precision and measurement consistency in cardiac CT images. Many of the advancements have been focused on the technical capabilities of the CT scanners themselves. In terms of hardware, faster rotation times, dual source–detector geometries, and multisegment acquisition and reconstruction methods have been aimed at improving the effective temporal resolution of the scanner to reduce the impact of coronary vessel motion on image quality. In terms of software, metrics and algorithms have been developed to better quantify, mitigate, or correct motion artifacts at both the data acquisition and image reconstruction stages.For a metric of image quality to add clinical utility and predictive power in the context of efficient CTA utilization and optimization, one can postulate that it must (1) be reflective of an actual clinical task as opposed to the generic aspects of image quality such as noise or blur, (2) account for the attributes of the imaging systems and techniques as they render the patient information, (3) incorporate all influencing factors into an estimator of stenosis accuracy, (4) be relatable to and validated against image quality performance as experienced clinically, and (5) be applicable to provide guidance for optimizing CTA. In line with these objectives, the purpose of this work was to develop a computational framework to objectively quantify the task performance of estimating stenosis in CTA. The proposed framework’s figure-of-merit, the estimability index We first developed a library of idealized task functions, utilizing virtual coronary vessels and plaques, which incorporated the most significant elements of the designated clinical task.(2)We mathematically modeled the attributes of the complete imaging chain, including the blurring and distortion due to cardiac motion for a variety of modern, clinically employed CT systems.(3)A model observer framework based on the ideal maximum-likelihood estimator (MLE) was developed to quantify the stenosis estimation performance ((4)The framework was validated against a retrospective cohort of 132 patient cases from the Prospective Multicenter Imaging Study for Evaluation of Chest Pain (PROMISE)(5)Finally, the framework was applied to ascertain the imaging parameters for optimum cardiovascular imaging as a part of the CT angiography biomarker initiative2.1To address the first requirement for developing a task-specific metric of image quality, we developed a library of idealized task functions based on the morphology explicit to our targeted indications, i.e., coronary plaques and stenoses. These functions utilize virtual coronary artery and plaque models that provide a spectrum of morphology, composition , and degree of stenosis (15% to 85%) typically seen in patient populations. The task functions serve as the basis of the ideal estimator (described below) to ascertain the likelihood of assessing the degree of stenosis within a lumen.,The disease-free coronary artery was modeled as a uniform cylinder with 0.5 mm of concentric vessel wall material. The inner luminal radius was then defined according to the sex and involved segment of a given patient case with the added assumption of a right dominant coronary arterial system.A plaque, either noncalcified, calcified, or mixed, was included to narrow the artery. Our model assigned specific material densities and corresponding Hounsfield Unit (HU) values, according to the three typical plaque material classes: noncalcified: ,As coronary vessels contract and relax per heart motion, our library of idealized task functions included the temporal dimension. A given vessel segment at a given phase in the cardiac cycle may have both in-plane , and the involved vessel segment with the added assumption that the phase of acquisition corresponds to the period of relative least motion (quiescence or diastasis) for the given patient-vessel combination.2.2To address the second requirement, in terms of modeling the deterministic spatial and stochastic noise attributes of CTA, we incorporated empirically measured task-based modulation transfer functions (TTF) and noise-power spectrums (NPS) from commercial CT systems. “Task-based” refers to the measurements being made relevant to a specific plaque material  targeted task with corresponding local contrast, noise, and patient attenuation size. Task-based measures of resolution and noise are a necessary component of modeling nonlinear reconstruction effects and local spectral degradations like beam hardening and calcium blooming present in cardiac CTA images.The TTF and NPS, specific to the applied scanner model, reconstruction kernel, plaque material, tube-potential (kV), and radiation dose from a wide array of clinical scanners, were applied to our idealized hyper-resolution vessel and plaque models to “image” them according to the associated imaging conditions. The deployed NPS and TTF quantities had been previously measured from images of the American College of Radiology and Mercury Phantoms scanned on machines within our clinical operation using a method described by Solomon et al.We included the image-domain asymmetric motion point spread functions (mPSF) induced by the complex interaction of the scanner temporal resolution, half-scan (180 deg) projection data, and coronary vessel motion vectors. Coronary vessel motion during CT acquisition results in asymmetric blurring of the vessel region within the reconstructed image due to spatial inconsistencies in the acquired projection data. Although this vessel motion is negligible in the source-to-detector direction (motion directly toward or away from the detector results in negligible magnification differences), motion in the detector channel direction (perpendicular to the source-to-detector direction) is significant relative to the detector channel size and does result in spatial inconsistencies in the projection data and subsequent asymmetric blurring. Furthermore, due to the acquisition geometry of modern third generation CT scanners, the channel direction is constantly changing as the x-ray source rotates about the object in a uniform circular motion. For a clockwise source rotation as seen from an object at the center of the scanner field of view, the channel direction can be described by the vector To form a single instance of a two-dimensional (2D) image-domain asymmetric mPSF, the vessel motion vector of known magnitude and direction was projected onto the channel direction vector for every projection angle used to reconstruct the image. The mPSF was then calculated as the channel direction displacement path weighted by the respective projection angle reconstruction weights and rotation time of the specific CT scanner model. For dual source acquisition geometries and multisegment acquisition techniques, the mPSF was weighted and summed according to each subset of projection angles and their effective temporal resolution. This process was stochastically repeated for different uniformly random relative vessel motion directions and projection angles to form a set of mPSF instances. With each mPSF instance resulting in a unique degree of image degradation and visualized vessel deformation, the complete set of all possible mPSFs stochastically represents the mathematical imaging operator for the motion blur.Finally, to include the stochastic noise properties of the simulated imaging chain, the filtered image was downsampled to the target clinical resolution and correlated noise was added by filtering a zero-mean white Gaussian noise field , there may exist multiple parameter values for a given object that achieve the same minimum squared difference. The ideal estimate may correspond to multiple template values. Second, because the vessel motion in cardiac CTA results in stochastic asymmetric motion degradation, the residuals for each squared difference operation may not be “white,” meaning there may exist spatial correlations within the residuals that do not satisfy the requirement for Gaussian distributed residuals. Although these caveats do partially disqualify this operator from setting the upper bound on achievable estimation performance, it still provides a representative measure of estimation performance under realistic operating conditions. Taking these limitations into account, the ensemble of ideal estimates may include both bias and variability with respect to the ground truth value and may not be well described by the normal distribution.The ideal estimator assumed that a given patient–scanner-acquisition case, as imaged, may be stochastically represented by a specific noise and motion condition. 2300 such stochastic instances of noise and motion were synthesized for a case being modeled. Each instance was then individually compared to a bank of precomputed template images to find the “ideal estimate”  for that instance. The templates provided a systematic representation of influencing factors including vessel diameter (1.5 to 5.0 mm), stenosis (15% to 85%), vessel velocity  were identified to fall within these two divergent cohorts. The associated patient and scanner characteristics from this cohort are tabulated in For each case, In calculating For a small subset of patients–scanner-protocol combinations , there was missing or conflicting information, which was replaced by concurrent values from similar patient or scanner attributes, case averages, and published scanner details. For patients with multiple image series with differing acquisition and reconstruction parameters, the single-image series or protocol with the highest The receiver operating curve (ROC) and its associated summary metrics were calculated for the task of classifying the high-quality versus low-quality classes for the 132 PROMISE datasets. Furthermore, we employed both a fourfold cross-validation approach and a Monte Carlo bootstrapping method using within-class case replacement, which maintained the original dataset size and class ratio to calculate confidence intervals (CI) on the class separation threshold and ROC performance.2.5An estimability index for coronary stenosis can have multiple applications including characterization and comparison of coronary imaging hardware and software, harmonization of data across clinical practices, and optimization of the imaging protocols toward targeted precision or consistency. As a demonstration, we implemented the metrology to ascertain the imaging parameters for optimum cardiovascular imaging as a part of the CT Angiography Biomarker initiative3The estimability index , and hardware-based controllable factors . A given factor combination and its associated interdependencies with the clinical task (stenosis estimation) determines the upper-bound on achievable estimation precision. This upper-bound is applicable to both expert readers and advanced computer segmentation algorithms. The problem we aimed to address was uncertainty in the effectiveness of coronary CTA in certain clinical settings that result in a higher than usual number of nondiagnostic CT scans and subsequently an inefficient use of high-cost imaging technology. The developed task-based metric of image quality In this study, we devised a framework to account for the prominent degradation factors that affect the characterization of cardiac stenosis. The innovative and necessary inclusion of analytically derived mPSFs and their treatment as a stochastic image degradation factor was critical in the proper application of ideal estimation theory in the context of cardiac CT.Our gold standard of quality for the validation of In this work, we validated This Practical use of the Although our developed platform does incorporate, and is sensitive to, the primary factors that influence image quality and diagnostic performance, it did not isolate the sources of image degradation separately . Further, there were other factors that were not included in the model. For example, we did not explicitly include spectral effects, such as calcium blooming and local beam hardening that can negatively impact image quality and make image interpretation near impossible in high calcium burden patients. Although patients with a prescan probability of calcium or very high coronary artery calcium score on noncontrast CT may not be the candidates for stenosis evaluation through CTA, these factors are important. Likewise, our motion model focused primarily on motion within the more influential axial plane and not in the craniocaudal direction, even though the influence is likely less prominent. Future studies may explicitly include these aspects of image quality.,Finally, while our methodology systematically characterized key factors influencing CTA performance, we note that our study did not account for many clinical factors that influence the accuracy of CTA in patient care and clinical outcomes. Those include additional patient variability and cardiac conditions, testing, catheterization, and hospitalization.5A computational framework was successfully implemented to objectively estimate task performance in quantifying coronary stenosis using an ideal MLE in cardiac CT. The framework was validated against clinical results in the context of a subset of a large clinical trial (PROMISE) with diverse sites, readers, scanners, acquisition protocols, and patient characteristics. The developed task-based metric of image quality (


The hair follicle (HF) is a highly conserved sensory organ associated with the immune response against pathogens, thermoregulation, sebum production, angiogenesis, neurogenesis and wound healing. Although recent advances in lineage-tracing techniques and the ability to profile gene expression in small populations of cells have increased the understanding of how stem cells operate during hair growth and regeneration, the construction of functional follicles with cycling activity is still a great challenge for the hair research field and for translational and clinical applications. Given that hair formation and cycling rely on tightly coordinated epithelial–mesenchymal interactions, we thus review potential cell sources with HF-inducive capacities and summarize current bioengineering strategies for HF regeneration with functional restoration. In addition, hair in human society greatly affects the quality of life, attractiveness and self-esteem. However, destructive inflammation with various aetiologies and the subsequent replacement of fibres can involve the permanent loss of HFs, which impairs inherent skin function and, especially, psychological well-being. Thus, HF regeneration is in ever-increasing demand and has promising market prospects. HF morphogenesis and regeneration were shown to be dependent on the intensive cooperation of epithelial  and hair-inducive mesenchymal  components, also called epithelial–mesenchymal interaction (EMI). EMI is a prerequisite for functional HF formation, regeneration and cycling, mainly through paracrine mechanisms,2 and has become the theoretical basis of tissue engineering for HF regeneration. Current strategies to regenerate HF in vivo are aimed at simulating EMI, mostly adopting the principle of combining epithelial (Epi-SC and keratinocyte) and mesenchymal (DP cell [DPC] and skin-derived precursors [SKPs]) components. HFs are also a dynamic mini-organ, and their most notable feature is hair cycling  and DPCs plays a significant role in the regulation of hair cycling.4 The activation, stability and sustainability of hair cycling are considered to be a key factor in achieving the longevity of HF function, but HF loss is often accompanied by the termination and elimination of hair cycling. Thus, the achievement of hair cycling regeneration is important for functional HF regeneration. Although hair transplantation has been widely applied, transplanted hair is not maintained in the long term. Moreover, clinical drugs still fail to meet the patents’ needs and even have drastic side effects.5 Therefore, there is a need to explore alternative therapeutic solutions capable of generating functional HFs. Current techniques could make it possible to obtain potential cells in vitro  are a major skin appendage originating from the ectoderm. As a stem cell repository and a hair shaft factory, the HF contributes to remodelling its cutaneous microenvironment, including skin innervation and vasculature.ing Fig. , from petro Fig. , such astro Fig. , SKPs, ktro Fig. . Here, w6 originate from blimp1+ fibroblasts  during embryonic development.8 DPCs have the ability to stimulate epithelial HFSCs and are considered to be a master regulator of HF cycling. Past studies reported that DPCs isolated from rat and guinea pig vibrissae, as well as humans, could also induce HF formation when implanted into recipient non-hairy skin,10 which indicates that DPCs could reprogramme non-hairy epidermis to a follicular fate. Subsequently, DPCs, either fresh or after tissue culture expansion, could also reproduce new HFs if placed in proximity to the epithelium.12 Based on their strong HF-inducive ability, many attempts to coculture DPCs with other cell types to regenerate HFs have been studied, such as the two-dimensional juxtaposition of other epithelia,13 cultured epithelial cells,15 keratinocytes,17 corneal epithelium18 and amnion epithelium.19 DPCs can secrete various factors to initiate HF formation by activating skin epithelial stem cells (Epi-SCs), so the mixture of DPCs and Epi-SCs also promotes functional HF regeneration in vivo.20DPCs, a kind of differentiated dermal cell at the base of HFs,+ DPCs and Versican+ DPCs. CD133+ DPCs have been shown to be a specific subpopulation of cells in DPCs, and they can produce Wnt ligands and mediate signalling crosstalk between the mesenchyme and the epithelial compartment, further promoting adult HF growth and regeneration.21 In addition, Versican+ DPCs exhibit the typical characteristics of aggregation growth,22 on which HF formation is highly dependent.23 In addition, many functional molecules are involved in the positive regulation of DPC HF-inducive capacity 25 and histidine decarboxylase,26 but matricellular protein connective tissue growth factor (CCN2) negatively regulates HF regeneration, physiologically curbing HF formation by the destabilization of β-catenin.27 In wound healing, hedgehog gene activation could shift the dermal fibroblast fate towards DPCs and result in extensive HF neogenesis.28Hoxc genes are able to reprogramme DPCs, and a single Hoxc gene is sufficient to activate dormant DP niches and promote regional HF regeneration through canonical Wnt signalling.29 Monoterpenoid loliolide regulates the HF inductivity of human DPCs by activating the AKT/β-catenin signalling pathway.30 Although DPCs possess the potential to regenerate HFs, freshly isolated human DPCs are not efficient in regenerating new HFs when they are directly transplanted.31 To restore their intrinsic properties, three-dimensional (3D) spheres offer a more physiologically relevant system where cell–cell communication as well as microenvironments have been studied. It has been reported that sphere formation increases the ability of cultured human DPCs to induce HF from mouse epidermal cells,32 in which glucose metabolism33 and epigenetics34 may be important regulators. The natural vitamin E form tocotrienol acts upstream of DP formation to induce HF anagen, dependent on the loss of E-cadherin and activation of β-catenin.35 Pretreatment with 1α,25-dihydroxyvitamin D3 (VitD3) could significantly improve DPC functionality and hair folliculogenesis, which is mediated by the activation of Wnt10b, alkaline phosphatase (ALP) and transforming growth factor β2 (TGF-β2).36 Consistent with VitD3, platelet-rich plasma has been shown to function in HF regeneration by enhancing DPC proliferation,37 which may result from the downregulation of MYC, CCAAT/enhancer-binding protein beta and E2F transcription factor-1 gene.38 Icariin promotes mouse HF growth by increasing IGF-1 secreted by DPCs.39 Utilization keratinocyte-conditioned medium,40 coculture with keratinocytes41 or the addition of BMP642 and basic fibroblast growth factor (FGF)43 to DPC expansion cultures could preserve their HF-inducive capacity. JAK inhibitor regulates the activation of key HF populations, such as the hair germ, and improves the inductive potential of DPCs by controlling a molecular signature enriched in intact, fully inducive DPs.44DPCs with specific marker molecules possess HF-inducive capacity, including CD133ity Fig. , such as45 For instance, DPCs are differentiated dermal cells originating from blimp1+ dermal fibroblasts,7 while SKPs are derived from Sox2+ follicle-associated dermal precursors.46 SKPs are defined as DSCs that reside in the adult HF mesenchyme and can be isolated and expanded in vitro as self-renewing colonies. SKPs have the capacity to differentiate in vitro and in vivo into multiple lineages of different progeny.47 Of note is that SKPs could regenerate the dermal sheath and repopulate DPCs in each growth cycle48 to serially reconstitute HF when subcutaneously engrafted.46 There are also various molecules affecting the inductive potential of SKPs. Trichostatin A, a potent and specific inhibitor of a histone deacetylase, could restore the HF-inducive capacity of SKPs by markedly alleviating culture expansion-induced SKP senescence, increasing the expression and activity of ALP and elevating the acetylation level of histone H3.49 Platelet-derived growth factor (PDGF) could enhance SKP proliferation, increase SKP progeny and improve their HF-inducive capacity, but PDGF deficiency results in a progressive depletion of the stem cell pool and their progeny.50 Many tentative methods to isolate and cultivate SKPs have also been explored,52 particularly in computer-controlled stirred suspension bioreactors, which lead to a greater expansion of viable SKPs.53 This technique allows a large number of SKPs that share a similar expression profile to that in static culture (fivefold greater than in static culture), and both static and bioreactor condition-derived SKPs are able to induce de novo HFs and repopulate their cellular niches.53DSCs are stem cells located in the dermis of the skin. Based on distinct phenotypic properties and different cultural environments, DSCs can be divided into dermal fibroblasts and SKPs.54 Primed HFSCs have faster activation dynamics in the secondary hair germ of telogen HF.55 The activation of primed HFSCs could trigger the subsequent activation of quiescent HFSCs, and the coordination of these two populations could fuel HF regeneration from telogen to anagen.57 HFSCs express a panel of transcription factors, including Sox9, Tcf3, Lhx2 and Nfatc1.59 HFSCs derived from a single rat vibrissa via organ culture could reconstitute HFs in vivo,60 and even aged HFSCs still have the potential to regenerate HFs.61 HFSCs could also differentiate into epidermal and sebaceous gland lineages, participating in the process of skin wound healing, and thus were considered ideal candidates for cutaneous repair and regeneration. Recent studies have reported that basal keratinocytes also have the capacity to facilitate HF regeneration under induction. Transgenic expression of the hairless gene (HR) in progenitor keratinocytes could rescue HF regeneration in Hr(−/−) mice, in which the new HFs resemble wild-type follicles and express markers of early anagen.62 This process may be linked to the regulation of the precise timing of WNT signalling to HR.63 Tuberous sclerosis complex 2 (TSC2)-null fibroblast-like cells grown from human TSC skin hamartomas could also induce neonatal foreskin keratinocytes to form HFs, complete with sebaceous glands, hair shafts, inner and outer root sheath (ORS) cells and the anagen-specific expression of versican, in which the TSC1/TSC2/mTORC1 pathway may play a significant role.64HFSCs are heterogeneous Epi-SCs compartmentalized along the longitudinal axis of HFs. They are divided into quiescent and primed HFSCs. For instance, relatively quiescent HFSCs in the follicular bulge region can serve as a reservoir for transient amplifying cells that are able to produce various cell types during HF regeneration.+Thy1+ subpopulation with high proliferation could be induced into DPCs in the DP medium.65 The mixture of iPSC ectodermal precursor cells in keratinocyte culture medium with RA and bone morphogenetic protein (BMP) could also transform into new DPCs.66 Intriguingly, iPSCs could also be generated from human DPCs upon lentiviral transfection with Oct4, Sox2, Klf4 and c-Myc.67 In addition, iPSCs, post-culture on Matrigel, could differentiate into keratinocytes when treated with keratinocyte serum-free media supplemented with all-trans RA and BMP4.68 Human hair follicular keratinocytes could also be reprogrammed into iPSCs, and keratinocyte-derived iPSCs are further capable of differentiating into keratinocytes,69 which suggests a novel way to provide a source of keratinocytes. Lineage reprogramming is direct cellular reprogramming, which means that targeted cells could bypass the stem cell stage and convert directly to potential cells. Unlike traditional concepts regarding the epigenetic stability of differentiated cells, direct lineage reprogramming can transform one specialized cell type into another using defined factors, which is a more efficient and promising approach for producing functional cells. Treatment with the combination of FGF2, PDGF and 6-bromoindirubin-3′-oxime (BIO) could chemically transform human dermal fibroblasts into DPCs.70 SKPs can be produced from healthy adult fibroblasts via lineage reprogramming, and their cryopreservation can largely preserve their properties and produce more significant yields.71 Another way to rapidly expand SKPs via lineage reprogramming is to expose pre-established dermal fibroblasts to 30-min acid stress prior to isolating SKPs.72 Acute acidic stress treatment of dermal fibroblast cultures greatly improves SKP isolation, growth, yield and multipotency.The differentiation of stem cells into adult cells in response to defined factors is an important application of cell reprogramming. Induced pluripotent stem cells (iPSCs) have similar characteristics to embryonic stem cells in terms of morphology, self-renewal and differentiation capacity. They are not only free from ethical issues but also able to be propagated as autologous cells, which can avoid the complication of immune rejection. Thus, reprogramming of iPSCs into potential cells could be an approach to providing cell sources for HF regeneration. Under retinoic acid (RA), an iPSC-derived LNGFR73 In addition, the efficiency of reprogramming by gene editing is still low. The optimization of the culture system in vitro and the improvement of reprogramming efficiency are challenges for HF regeneration. Therefore, we believe that the construction of 3D culture systems that simulate the in vivo environment may provide an alternative approach, similar to hydrogel scaffold-based cell culture, which contributes to maintaining cell proliferation and growth as well as the potential to regenerate HFs. In addition, chemical reprogramming, a new reprogramming technology, is characterized by high security and efficiency.74 To improve the reprogramming efficiency, the replacement of chemical reprogramming with gene editing has broad prospects in HF regeneration.With progress in HF developmental biology and cellular reprogramming techniques, several cells with the potential for HF regeneration have been identified. These results have greatly expanded the seed cell bank for HF regeneration and solved the problem of the lack of a cell source. However, the problems these cell sources have in common are that their potential to regenerate HF fails to be maintained during long-term culture in vitro. In addition, HFSCs are few in number and extremely difficult to obtain, and SKPs in vitro senesce soon when isolated from their physiological environments.75 At the onset of anagen following stimulation by growth-inducing signals from the DP, primed HFSCs also contribute to new hair shaft production.76 Under induction by such signalling, primed HFSCs undergo rapid proliferation to fuel the initial stage of hair growth, and then, the quiescent HFSCs undergo a second round of activation that replenishes cells lost at the onset of anagen, finally supporting prolonged growth of the hair shaft.56 At the beginning of anagen, activated HFSCs migrate from the bulge to the matrix area and become transit-amplifying cells that proliferate and differentiate to form the new hair shaft.77 Therefore, the coordinated activation of primed HFSCs and quiescent HFSCs is instrumental for the maintenance of hair cycling. The mechanism underlying HFSC homeostasis and hair cycling regulation is a complex molecular controlling process , apoptosis-driven regression (catagen) and relative quiescence (telogen). Therefore, the HF is regarded as a dynamic mini-organ. The activation and maintenance of hair cycling is a prerequisite for the functional regeneration of HFs. HFSCs play an indispensable role in maintaining hair cycling. The HFSC population remains largely quiescent during hair growth, but a subpopulation actively proliferates and promotes the production of the new hair shaft under the control of Axin2 expression.ss Table  that is The dynamic characteristics of HFs enable their sustainable and periodic regeneration. We think that the activation and maintenance of hair cycling are indispensable for achieving functional HF regeneration. Past studies have not only led to a better understanding of the molecular mechanisms of hair cycling, which makes it more possible to find the key molecules therein, but also have revealed the complexity of HF dynamic characteristics. Therefore, it is still difficult to discover the key molecular event in hair cycling.20 Although the new HFs are relatively small, this result proves again that the rearrangement of the EMI and the niches of the potential cells are essential and necessary for HF construction. A mixture of Epi-SCs and SKPs was grafted into excisional wounds in nude mice, and a bilayer structure resembling the epidermis and the dermis formed on the fifth day, followed by de novo HF.80 In the regeneration process, the SKPs formed DPCs in neogenic HFs and abundant dermal cells in the dermis, and the Epi-SCs formed the epidermis and trunk of the HF. More importantly, this experiment also demonstrates that the PI3K-Akt signalling pathway plays a crucial role in the interactions between Epi-SCs and SKPs and de novo HF regeneration, which may suggest potential therapeutic applications in enhancing hair regeneration.80 During the hair growth cycle, HFSCs periodically switch between the active and inactive stages to maintain stem cell populations and generate new HFs, while this potential is impaired in aged HFSCs.81 However, in transplantation assays in vivo, aged HFSCs could still regenerate HFs when supported with the young dermis, while young HFSCs failed to regenerate HFs when combined with the aged dermis, which shows that the ageing skin microenvironment dictates stem cell behaviour and illustrates the dominant role of the niche microenvironment.61 This research discovered that the HF regeneration potential of aged HFSCs can be rejuvenated by neonatal dermis during in vivo transplantation, providing promising new avenues for regenerative and geriatric medicine. In recent years, for the fully functional regeneration of ectodermal organs, a bioengineered organ germ has been developed by reproducing the embryonic processes of organogenesis, including bioengineered HFs.82 Bioengineered HF germ could be reconstituted with embryonic skin-derived epithelial and mesenchymal cells and was able to develop histologically correct HFs when ectopically transplanted. The bioengineered HFs not only properly connected to the host skin epithelium by intracutaneous transplantation and reproduced the stem cell niche and hair cycling but also autonomously connected with nerves and the arrector pili muscle at the permanent region and exhibited piloerection ability.83 Another way to construct bioengineered HF is with pelage and vibrissae reconstituted with embryonic skin-derived cells and adult vibrissa stem cells. After intracutaneous transplantation, this bioengineered HF germ not only develops the correct structures and forms proper connections with surrounding host tissues such as the epidermis, arrector pili muscle and nerve fibres but also shows restored hair cycling and piloerection through the rearrangement of follicular stem cells and their niches, with fully functional hair organ regeneration.84 It is worth mentioning that the in vivo transplantation of bioengineered HFs also achieves the partial restoration of hair cycling, which is a great step forward for functional HF regeneration.Cell-based transplantation without biomaterials is a minimally invasive approach to in vivo HF regeneration. Current cell transplantation mainly involves the transplantation of stem cells or a mixture of epidermal and dermal components. The injection of a mixture containing Epi-SCs and DPCs into nude mice could induce new HFs with the correct histological structures and form a multilayered stratified epidermis containing HF-like structures.85 Because DPCs and dermal fibroblasts originate from common fibroblast progenitors in the developing embryonic mouse skin and have highly correlated gene expression profiles (96%),86 adult dermal fibroblasts can be reprogrammed into a neonatal state, with the capacity of inducing ectopic HF formation similar to DPCs through the epidermal activation of β-catenin.87 Furthermore, treatment with the combination of FGF2, PDGF and BIO in adherent culture followed by suspension culture could induce the generation of DP-like cells from foetus- or adult foreskin-derived fibroblasts. The integration of foetal/adult DP-like cells can be recruited to replenish DP of de novo generated HFs, and the regenerated HF structures were reconstructed in 65% nude mice implanted with foetal DP-like cells and in 70% nude mice with adult DP-like cells.70 Finally, the combination of MITF, SOX10 and PAX3 could directly convert mouse and human fibroblasts into induced melanocytes, which have the ability to generate pigmented epidermis and HF in vivo when properly integrated into the dermal–epidermal junction.88 Healed wounds with the loss of HF are usually filled with a large number of fibroblasts, so the exploration of fibroblast-based reprogramming holds great promise for HF regeneration in situ.Cellular reprogramming is not only a tool for tissue engineering to enrich potential cell sources for the regeneration of HF but also a participant in physiological de novo HF induction. Secreted proteins  from embryonic skin conferred upon non-hair fibroblasts the competency to regenerate HF via the activation of IGF and WNT signalling, thereby endowing non-HF skin with the ability to reproduce HFs, which suggests the involvement of cellular reprogramming.89 The combination of potential cells with biomaterials, such as hydrogels, scaffolds and other self-assembled materials, could contribute to HF regeneration  but also exhibited a more advanced hair cycling stage;90 an average of 71% of the HFs reached anagen stage III, and 19% reached anagen stage IV in reconstituted skin containing β-catenin-expressing CD133+ DPCs, while 67.5% of the HFs in the control remained in anagen I and II, and only 27.5% reached anagen III.90 The stable expression of β-catenin could promote the clonal growth of CD133+ DPCs in vitro in 3D hydrogel culture. An alternative strategy to reproduce EMI is to use a collagen-chitosan scaffold (CCS)-based 3D system containing dissociated epithelial cells and DPCs followed by treatment of the CCS with Wnt-CM from Wnt1a+ bone marrow mesenchymal stem cells. The results suggested that the cell mixture was able to induce hair regeneration in nude mice, and Wnt-CM can maintain the hair induction ability of DPCs in expansion cultures, which may be associated with the activation of the Wnt/β-catenin signalling pathway.91 Therefore, how to enhance the HF induction efficiency of cultured human DPCs is a priority in bioengineering for clinical HF regeneration. It has been reported that DPCs retain HF inductivity best when cultured and transplanted as multicellular aggregates, and DP spheroids could form a structure similar to the natural intercellular organization in vivo.92 Therefore, another strategy is to achieve the scalable fabrication of controllable DP spheroids to regenerate HFs. Scalable production of controllable DP spheroids on polyvinyl alcohol surfaces has high viability and preserves DP characteristics, and at DPC numbers of 5 × 103 to 30 × 103 cells each, both human and rat DP spheroids are able to induce HF neogenesis. Larger DP spheroids exhibit higher HF inductivity. The researchers developed a method that can be automated for mass production of DP spheroids with controllable size and cell number in a wide range,93 although the average diameter of regenerated hair fibre did not significantly change with increasing size of the transplanted DP spheroids. Likewise, the hanging-drop approach could also lead to a controllable 3D spheroid model for the scalable fabrication of inductive DP microtissues. That technique is based on surface tension and the interaction between surface tension and a gravity field that causes the convergence of liquid drops. With the converged drops, DP spheroids could endow high-passaged DP microtissues with many similarities to primary DP. Subcutaneous implantation of these microtissues mixed with new-born mouse epidermal cells has achieved reproducible HF induction in the hypodermis of nude mice, and a large amount of extracellular matrix (ECM) components is found in the intercellular space within the DP microtissue, similar to an anagen DP.94 These models provide the potential to elucidate the native biology of human DP and show promise for the controllable and scalable production of inducive DPCs for future follicle regeneration. Exosomes derived from DP spheroids (3D DP-Exos) are also able to promote the proliferation of DPCs and ORS cells and accelerate anagen onset to influence hair cycling. Local injections of 3D DP-Exos (exosomes) could induce anagen from telogen and prolong anagen in mice. Moreover, DPC spheres treated with Exos could augment HF neogenesis when implanted with mouse epidermal cells,95 which may be associated with high levels of miR-218-5p in Exos.96 Cell surface engineering technology also advances HF regeneration by accurate micro/nanoscale control in cell-biomaterial ensembles and DP spheroid formation. Owing to the security and tuneable thickness at the nanoscale, the nanogel could encapsulate a single cell by layer-by-layer (LbL) self-assembly and further form DPC spheroids by physical cross-linking on nanogel-coated cells. LbL-DPC aggregation is akin to that of primary DPCs and has the capacity to restore HF induction potential in vitro and regenerate HFs in vivo.97 Other biomaterials that contribute to the restoration of HF-inducive ability include human placenta ECM hydrogel,98 synthesized ECM99 and a chitosan/polyvinyl alcohol nanofibre sponge with an open-cell cellular structure,100 which expands the biomaterial libraries for the optimization of the culture environment in vitro to restore the ability of DPCs to regenerate HFs. 3D printing technology for HF regeneration by recapitulating the physiological 3D organization of cells in the HF microenvironment is an innovative biomimetic approach.101 It permits the controllable self-aggregating spheroid formation of DPCs in a physiologically relevant ECM, the initiation of EMI and further HF formation in human skin constructs. Remarkably, the vascularization of HF-bearing human skin constructs increases graft survival and enables efficient human hair growth in mice.101 This method represents a novel bioengineering strategy for the feasible generation of hair-bearing human skin constructs entirely ex vivo from cultured human cells, and adaptation of this new technology by hair researchers, hair restoration surgeons and the pharmaceutical industries will have overwhelming implications in the maintenance and regeneration of HFs.Biomaterials are implantable, inactive materials that can replace or repair damaged tissue with high biocompatibility. Biomaterials can create a 3D environment for cell-to-cell interactions, simulating the function of cell niches to a certain extent, and they have been widely used in wound repair and tissue regeneration.on Figs.  and 2. F103 Self-assembling peptide nanofibres are made of natural amino acids and form hydrogels that surround cells in a manner similar to the ECM. RADA16 (Ac-(RADA)4-CONH2) is a representative example of such peptides that has been shown to promote nerve regeneration and wound healing.105 A PRG (PRGDSGYRGDS) solution functionalized by mixing with RADA16 promoted the proliferation and migration of periodontal ligament fibroblasts.106 The self-assembling peptide hydrogels formed by RADA16 and PRG (RADA-PRG) could facilitate the attachment, proliferation and survival of SKPs, ultimately supporting HF neogenesis in vivo. The transplantation of a combination of culture-expanded SKPs and neonatal epidermal cells into RADA-PRG hydrogel resulted in a significantly increased number of neogenic hairs compared to Matrigel and other peptide hydrogels. This may be attributed to the similarity of the properties of these designer peptide nanofibres to those of ECM molecules.102 In addition, a mixture of culture-expanded Epi-SC and SKPs derived from the adult human scalp in a hydrogel was capable of reconstituting functional HFs, and the mechanisms of the expression of ALP in SKPs in vitro and the maintenance of HF-inducive properties in vivo may be associated with BMP4.103 More importantly, Epi-SC implanted in wounds in combination with SKPs could also form functional sebaceous glands in association with HFs. Normal human neonatal foreskin keratinocytes were induced to differentiate into several cellular components that compose normal HFs, with the expression of anagen-specific versican when grown on a collagen matrix embedded with TSC2-null fibroblast-like cells or with fibroblasts.64 Regenerated HFs were complete with sebaceous glands, hair shafts and inner and ORSs.64Similar to DPCs, long-term culture could also impair the HF-inducive capacity of SKPs. It is likely that SKPs rely on special environments for their self-renewal and stable gene expression. In tissue engineering, scaffolds are created to mimic environments for stem cells to survive, differentiate and form functional tissue structures. In this regard, scaffolds such as hydrogels and matrix could be candidates to support stem cells for organogenesis and regenerate HFs.107 Multifunctional Zn-doped hollow mesoporous silica/polycaprolactone electrospun membranes exhibit excellent antibacterial activity for wound healing and are ~20 times as likely to regenerate HFs as the control.108 Most excitingly, the treatment of deep second-degree scalding injuries with human erythropoietin (EPO) to, whether by the local subcutaneous injection of nanosized rhEPO (recombinant human EPO)/infusion pumping or the topical application of rhEPO gel, achieved excellent skin repair with conical and HF structures, which was related to the combined expression of EPO receptor and β-subunit receptor.110 A novel fibrous membrane  containing quercetin-copper (Qu-Cu) chelates was fabricated by using quercetin and highly bioactive bioceramic (CaCuSi4O10) incorporated in PCL/gelatine electrospun fibres. The fibrous membrane can effectively release Qu and Cu ions to induce the proliferation, migration and differentiation of skin- and HF-related cells, and the Qu, Cu ions and Si ions released from the composite membrane revealed synergistic activity to stimulate HF regeneration and wound healing in burned skin.111 However, drug delivery systems mainly contribute to wound repair, accompanied by the acceleration of wound-induced HF neogenesis (WIHN). WIHN is a regenerative phenomenon separate from physiological regeneration, as its cellular origin is not from the HFSCs in the bulge at the wound edge. In WIHN, a fully functional follicle can regenerate in the centre of a full-thickness wound with a large enough size, and the cellular origin of this process is similar to an embryonic process. The neogenic follicles have similar functions to embryonic HFs, which also have a growth cycle.63 Currently, there are few studies about drug delivery systems for HF regeneration alone outside the wound environment. However, because of their controllability, targeted delivery, sustained release and even intellectuality, we think drug delivery systems hold great promise for HF regeneration in the future.Drug delivery systems consist of molecules with pharmacological activity modified into advanced materials, which have been widely used in skin wound treatment. Currently, it has been reported that many drug delivery systems could promote wound healing as well as HF regeneration. Newly developed multidomain peptide hydrogels have exhibited regenerative potential in a diabetic wound healing model, resulting in wound closure, accelerated HF regeneration and a greater average number of HFs at both the edge and the centre.112 HF organoids can be established from skin stem cells or a mixture of dermal and epidermal components. DP spheroids encapsulated by silk-gelatine hydrogel and HF keratinocytes as well as stem cells could be used to construct in vitro HF organoids. These organoids show enhanced DPC-specific gene expression and ECM production, and their structural features and cell–cell interactions are similar to those of in vivo HFs.113 This simple in vitro DP organoid model system has the potential to provide significant insights into the underlying mechanisms of HF morphogenesis and distinct molecular signals relevant to different stages of the hair cycle and hence can be used for the controlled evaluation of the efficacy of new drug molecules to induce HF regeneration. To date, in vitro skin derivation strategies have focused on first generating keratinocytes and fibroblasts from iPSCs in separate cultures and then combining the two types of cells to form a skin-like bilayer. A major challenge is how to realize the synchronous construction of its appendages. Under initial treatment with the TGF-β inhibitor SB431542 (SB) and recombinant BMP4 and subsequent treatment with FGF2 (FGF) and a BMP inhibitor LDN-193189 (LDN), a homogeneous population of mouse pluripotent stem cells and constituting epidermal and dermal layers in a 3D culture formed skin organoids with a cyst conformation, in which HFs were spontaneously produced de novo. However, the new HFs entered catagen and degenerated during long-term culture.114 These results suggest how skin organoid structures can be generated de novo without the use of embryonic tissue or undefined media, which will be useful for studying the minimal cellular and microenvironment requirements for HF induction. Scalp-derived dermal progenitor cells mixed with foreskin-derived Epi-SCs at a 2:1 ratio could aggregate in suspension to form a large number of HF organoids, and the dermal and epidermal cells self-assembled into distinct epidermal and dermal compartments. The addition of recombinant WNT3a protein to the medium enhanced the formation of these aggregates, and the transplantation of these organoids in vivo achieved HF formation.115 Finally, a 3D integumentary organ system (IOS) obtained by the self-assembly of mesenchymal and Epi-SCs from iPSCs using the clustering-dependent embryoid body transplantation method also regenerated fully functional HF organoids.116 After transplantation into nude mice, the HF organoid in that system could form proper connections to the surrounding host tissues, such as the epidermis, arrector pili muscles and nerve fibres, without tumorigenesis, and show appropriate hair eruption and hair cycles, including the rearrangement of follicular stem cells and their niches.116 These findings reveal the generation of a bioengineered 3D IOS from iPSCs, including appendage organs such as HFs and sebaceous glands, with appropriate connections to surrounding tissues, which significantly advances the technological development of the bioengineered 3D IOS and its potential applications, including an in vitro assay system, an animal model alternative and bioengineered organ replacement therapy.An organoid is defined as a 3D structure grown from organ-specific stem cell types. It can recapitulate key aspects of in vivo organs and avoid many of the disadvantages associated with cell lines.The transition from traditional culture to 3D culture constitutes excellent progress in HF regeneration. 3D culture enhances the proliferation and HF regeneration ability of potential cells, and a mixture of epidermal (Epi-SC) and dermal (SKP and DPCs) components in a 3D system simulates the characteristics of EMI, especially organoid technology and 3D printing technology. In 3D culture, regenerated HFs in vivo not only connect appropriately to surrounding host tissues but also undergo hair cycling activation. However, some problems are less thoroughly addressed. How long can the regenerated HFs last? Can the regenerated HFs go through full hair cycling? Will HF regeneration-induced hair be superior to transplanted hair? We think these are key questions and important challenges in functional HF regeneration.Much progress has been made in the developmental biology and regeneration of HFs. (1) A variety of cells with the potential to regenerate HFs have been identified, including DPCs, SKPs, HFSCs, keratinocytes and reprogrammed cells (iPSCs and fibroblasts), which provide a wide range of cell sources for HF regeneration. (2) We have gained a more comprehensive and in-depth understanding of the hair cycling-related mechanism, which provides the biological basis for finding key molecules to initiate and sustain the hair cycle. (3) Optimization of the in vitro culture system and the construction of a 3D culture environment could overcome the loss of the ability of potential cells to proliferate, self-renew and regenerate HFs caused by 2D culture. (4) The transplantation of a mixture of epidermal and dermal components, such as cell-based transplantation with or without biomaterials and HF organoids, could simulate EMI to a certain extent and successfully induce new HFs with the correct structure in vivo. (5) The HF organoid is a model for the exploration of mechanisms of HF morphogenesis and drug research to reproduce hair. (6) Drug delivery systems are characterized by strong controllability and high security, and they can promote wound healing accompanied by HF regeneration.Since it is an architecturally and functionally complex organ, the HF is much more difficult to regenerate or reconstruct than many other organs. Due to this limitation, HF regeneration is still far from clinical transformation. (1) The sources of potential cells are still poor, largely because of cell ageing in vitro culture and inefficient reprogramming, so there is still a need to optimize the in vitro culture system. (2) The mechanism of hair cycling is very complex, and it is extremely difficult to identify the key molecules. (3) Current strategies simulating EMI are still insufficient. Both cell transplantation and organoid architecture lack the microenvironment of connective tissue, blood vessels and immune cells, which is still quite different from the physiological environment of normal tissues and organs. (4) It is unknown how many new HFs can be regenerated from biomaterials and tissue engineering. Do they allow other essential cells to be recruited to the new follicle? If so, do the attracted cells have the ability to affect organogenesis overall? 5) The cellular reprogramming techniques that contribute to HF regeneration still have low efficiency in vitro. (6) The 3D regeneration of HFs depends on biomaterials that need better external security, controllability and internal stability. Ideal biomaterials need to be safe and nontoxic. Under normal metabolism in the body, they can be kept in a stable state without biological degeneration, and the metabolism or degradation products are harmless and easily metabolized. (7) Whether the regenerated HFs function normally and how long they can last in vivo are less mentioned in past studies.In summary, at the current stage, various attempts are only imitating a partial structure and/or function regeneration of HFs. The combination of different technologies and methodologies will hopefully lead to new progress. For example, the creation of transplantable HFs that closely mimic the structures and functions of native tissue may be accomplished by combining organoid technology with a drug delivery system. Depending on the controllable release of the relevant factors in hair cycling, such as WNT or BMP, hair cycling activation and maintenance of HF organs may be achieved. We also need to continue to optimize the in vitro culture systems of potential cells and look for more efficient reprogramming techniques, such as chemical reprogramming induced by small molecules or genetic reprogramming of genes delivered by biomaterials. Finally, we want to reiterate that, based on existing work, it is worth considering whether the achievement of the activation and maintenance of hair cycling in regenerated HFs could be the heart of the next phase.


BMP-enriched vesicles serve in endosomal-lysosomal trafficking and function as docking structures for the activation of lysosomal hydrolytic enzymes, notably those involved in the catabolic breakdown of sphingolipids. BMP levels are dysregulated in lysosomal storage disorders, phospholipidosis, metabolic diseases, liver and kidney diseases and neurodegenerative disorders. However, whether BMP alteration is a mediator or simply a marker of pathological states is unclear. Likewise, although BMP acyl chain composition may be altered with disease states, the functional significance of specific BMP species remains to be resolved. Newly developed tools for untargeted lipidomic analysis, together with a deeper understanding of enzymes mediating BMP synthesis and degradation, will help shed further light on the functional significance of BMPs in cellular physiology and pathology.Although understudied relative to many phospholipids, accumulating evidence suggests that bis(monoacylglycero)phosphate (BMP) is an important class of regulatory lipid that plays key roles in lysosomal integrity and function. BMPs are rare in most mammalian tissues, comprising only a few percent of total cellular lipid content, but are elevated in cell types such as macrophages that rely heavily on lysosomal function. BMPs are markedly enriched in endosomal and lysosomal vesicles compared to other organelles and membranous structures, and their unique  Recent analytical, cheminformatic, and biological breakthroughs are revealing the diverse roles of lipids in physiology and disease. The function of lipid mediators and other signaling lipids in biological processes have paved the way for the discovery of new lipid classes and characterization of their mechanistic contributions. The phospholipid class bis(monoacylglycero)phosphate (BMP), first identified in 1967, is reportedly key to lysosome stability and vesicle trafficking, and is dysregulated in diseases such as lysosomal storage disorders . Yet, thBMPs are negatively charged glycerophospholipids localized almost exclusively within the membranes of late endosomal and lysosomal vesicles . Bisacyb. In mosThe measurement of BMPs can be performed by either antibody-based or mass spectrometry (MS)-based approaches, as illustrated in Depending on the methodologies employed, mass spectrometry (MS) can semi-quantify or quantify BMPs and resolve individual BMP species of varying fatty acyl chain length and saturation. Quantification of BMP species can be achieved by including an isotopically labeled internal standard or by comparison to an external BMP calibration curve. Fatty acyl chain compositions of BMPs are annotated using fragmentation patterns in MS/MS spectra. Collision induces dissociation in tandem mass spectrometry fragments precursor molecules, and the subsequent fragment ions are recorded as the MS/MS (or MS2) spectra. The MS/MS spectra of a compound can be considered a fingerprint pointing to the identity of the compound , especiaRecent advances in chromatography and mass spectrometry investigated the separation of BMPs from their phosphatidylglycerol (PG) isomers by other techniques, including ion mobility , as highsn-3 glycerophosphate stereo configuration, while BMPs can reportedly possess sn-1:sn-1′ as well as sn-1:sn-3’ and sn-2:sn-2’ stereoconfiguration [The metabolism of BMPs has not been fully elucidated, and there are competing theories regarding the biosynthesis and catabolism of BMP . BMP is guration ,33. Manyguration  As suggeguration .Manipulating cellular BMP concentrations has been shown to regulate endo-lysosomal lipid metabolism and membrane dynamics . ABHD6 iInhibition of ABHD6 increases total BMP levels as well as long chain and fully saturated BMP species in mouse livers. When animals were fed high fat chow, livers exhibited elevated BMP levels and enrichment of long chain BMP species relative to control animals . KnockdoThree other enzymes have reported hydrolase activity toward BMP species. PA-PLA2/MGL, a member of the carboxylesterase family, has been shown to hydrolyze BMP, assessed by the production of free fatty acids in BMP-rich lipid fractions prepared from liver lysosomes . PancreaThe composition of BMP fatty acyl chains is thought to contribute to the biochemical functions of the various BMP species ,8. In nuImproved analytical methods have prompted the more widespread investigation of BMP acyl chain composition, and several studies have reported varying compositions of BMP in response to cellular stress or pathological conditions. Bouvier et al. demonstrated that fatty acyl chain composition dictates the stability of BMP species under oxidative stress. Supplementation with either PG-18:1/18:1 or PG-22:6/22:6 remodeled BMP acyl chain composition accordingly, and altered BMP sensitivity to oxidative stress-induced degradation in macrophages . While tBMP abundance and side chain composition also vary with the onset of lysosomal storage disease in patient plasma and cultured fibroblasts . NumerouIn an in vivo model of Gaucher disease, fatty acid supplementation increased total BMP species . The autBMP plays a critical but historically underappreciated role in lysosome biology, as recent reports implicate BMP in the regulation of lysosomal stability, function, enzyme activation, and endosomal trafficking. BMP’s negative charge is retained within the acidic environment of the lysosome, allowing it to act as a docking site and essential cofactor  for someASM is one of three types of sphingomyelinase hydrolases that cleave sphingomyelins into ceramides, and is primarily localized in the lysosome , though The cholesterol transfer protein NPC-2 shuttles cholesterol from intralysosomal membranes to the NPC-1 protein in the perimeter/limiting lysosomal membrane ,48. NPC-Lysosomal Hsp70 binds BMP with high affinity and specificity and is known to stabilize lysosomal membranes, protecting against lysosomal membrane permeabilization (LMP) . The extThe phospholipase A2 family represents a diverse group of over 15 enzymes that can be classified into subgroups based on cellular location . Group XWhile Alix reportedly binds BMP, the functional significance of this interaction is debated. Alix is essential in the formation intra-lysosomal vesicles, and it has been proposed that its interaction with BMP at the limiting lysosomal membrane facilitates this process ,7,48,49.BMPs are tied to a broad range of diseases, with direct links to mechanisms of disease initiation or progression, or as markers of disease status. As noted above, BMPs can provide scaffolding to facilitate endosomal trafficking and lysosomal membrane maintenance through association with target proteins. Increased BMP levels have been linked to several well-studied lysosomal lipid storage diseases including NPC, GM1 Gangliosidosis , and GauBiomarkers for lysosomal storage diseases  and a vaLysosomal storage diseases (LSDs) are a group of inherited metabolic disorders caused by genetic defects in lysosome resident proteins ,68, charGangliosides are the main glycolipids of neuronal plasma membranes , and botGaucher disease is caused by an inherited deficiency in acid β-glucosidase, resulting in impaired degradation of glucosylceramide (GC) ,45. UndeIn a macrophage cell line model of Gaucher THP-1 disease in which acid beta-glucosidase was inhibited, BMP levels increased in disease cells compared to control. The increase in BMPs was suggested by the authors to occur mainly as a consequence of lysosomal expansion-associated GC accumulation . Total BElevated BMP levels have been identified in brain tissue from patients with infantile neuronal ceroid lipofuscinoses (INCL) ,74 as weMutations in either the NPC-1 or NPC-2 genes lead to Niemann-Pick C (NPC), also characterized by abnormal lipid storage and accumulation . As mentStargardt disease is an inherited form of macular degeneration characterized by juvenile-onset progressive vision loss, and is most commonly associated with defects in the ATP-binding cassette gene sub family A-4 transporter protein  . ABCA4 pIt has been suggested that BMP may play a role in the progression of Stargardt disease , though Unlike most of the previously described diseases, antiphospholipid syndrome is an autoimmune disease arising primarily from antiphospholipid antibody binding to beta2-glycoprotein1 (B2GP1) ,76, whicCationic amphiphilic drugs (CADs) are compounds that carry a neutral charge in the cytoplasm but are protonated and positively charged upon entering the lysosome, consequently becoming trapped and concentrated within the organelle ,79. PrevComparable to the acquired lysosomal storage disease state triggered by CAD activity, multilamellar and myeloid body formation has been observed in kidney tissue in response to high fat diet, linking this metabolic syndrome with increased risk of kidney disease . Mice thWhile alterations in BMP metabolism clearly correlate with the induction of metabolic disease states including CAD-induced phospholipidosis/acquired LSD and other non-LSD diseases, BMP function in the pathophysiology of these conditions is poorly understood. It was recently reported that BMP fatty acyl side chain composition differed between diet- and drug-induced phospholipidosis and in various human disease states, and that deletion of ABHD6 increased circulating BMP levels, effectively reverting the pathological phenotype . SignatuIn mice, deletion of ABHD6 increased circulating BMP levels, but a corresponding accumulation of hepatic BMP and multilamellar body formation indicative of acquired LSDs was not observed . This imConsidering the critical role BMP plays in endosomal trafficking, including the fusion of late endosomal membranes, it is unsurprising that BMPs have been studied in the context of infection and immune cell function. The BMP-Alix interaction has been reported to contribute to viral invasion. BMP localized to the cytosolic leaflet of late endosomes recruits Alix and its associated proteins . Once boThe antiviral mechanism of these CADs may be explained by the characteristic lysosomal lipid accumulation induced by drug administration, which disrupts the endocytic pathway . As BMP Alterations in endosomal/lysosomal function are key etiologies of aging-associated neurological diseases including Alzheimer’s and Parkinson’s diseases. The involvement of autophagy and lysosomal function in the progression of Alzheimer’s disease and Parkinson’s disease, amongst others, has been reviewed previously . For exaAlthough our understanding of BMPs has unfolded slowly since their discovery over 50 years ago, emerging cellular and clinical observations are beginning to shed light on the contributions of this unique lipid class to tissue homeostasis and pathology. At the cellular level, BMPs are highly localized to late endosomes and lysosomes and appear to play key roles in maintaining lysosomal structural integrity by facilitating cholesterol redistribution and glycolipid trafficking and degradation. Their enriched presence within intraluminal vesicles serves as activating docking sites for several lysosomal enzymes and accessory proteins involved in glycolipid breakdown. Clinically, elevated BMP levels, or alterations in BMP-linked acyl chains, are associated with a variety of pathological states. For example, elevated BMP is a feature of the lipidosis class of lysosomal storage diseases, where the function of key glycolipid degradative enzymes is disrupted but is also observed as a response to treatment with cationic amphiphilic drugs. Considering the variety of states that exhibit BMP dysregulation, an interesting question concerns whether alterations in BMPs might be associated with other diseases such as cancer, where biomarkers for aggressive disease are sorely needed. Moreover, the extent to which BMPs play causative, compensatory or passive roles in each of the pathological states where their levels or acyl chain content are altered defines a key set of questions for future studies.A second series of questions concern the mechanisms underlying BMP accumulation and acyl chain modification in the pathological state. For example, why is the lipid activator of a compromised lysosomal enzyme elevated in LSD when enzyme activation is biochemically upstream of the genetic lesion? Moreover, why do BMP levels respond to CADs when BMP is upstream of the drugs’ presumed targets? While it has been suggested that elevated BMP is simply the outcome of elevated lysosomal content in cells storing glycolipids, the possibility remains to be explored that the lysosomal accumulation of some glycolipid substrates triggers an adaptive mechanism to bolster BMP levels in an effort to promote the degradation of these species. Exploration of the notion that BMP levels may be tunable and regulated in response to environmental conditions or lysosomal substrate load will require a deeper understanding of the enzymes and metabolic pathways contributing BMP synthesis and degradation. Likewise, the unexplored possibility that alterations in the acyl chain incorporation into BMP contribute to its efficacy as a lysosomal enzyme activator or to its own metabolic stability will require a deeper understanding of the metabolic pathways governing BMP esterification.


Aberrant epigenetic modifications in oncogenic pathways can lead to the onset of different cancers. This study aims to explore the role of differential DNA methylation in the regulation of oncogenic signaling pathways by integrating data from multiple sources including methylome, transcriptome and clinical presentation to uncover the effect of methylation changes acting on the four most common cancers. We utilized a differential methylation-parsing pipeline, which extracted differentially methylated biomarkers based on feature selection. Extracted biomarkers were integrated with the matching RNA-Seq and clinical data to determine if these differentially methylated CpGs could serve as potential diagnostic candidates for the four most common cancers. Our results suggested differential methylation of the genes within the NRF2-PI3K pathway may lead to the presentation of various cancer and serve as potential epigenetic biomodifiers.Disruption of signaling pathways that plays a role in the normal development and cellular homeostasis may lead to the dysregulation of cellular signaling and bring about the onset of different diseases, including cancer. In addition to genetic aberrations, DNA methylation also acts as an epigenetic modifier to drive the onset and progression of cancer by mediating the reversible transcription of related genes. Although the role of DNA methylation as an alternative driver of carcinogenesis has been well-established, the global effects of DNA methylation on oncogenic signaling pathways and the presentation of cancer is only emerging. In this article, we introduced a differential methylation parsing pipeline (MethylMine) which mined for epigenetic biomarkers based on feature selection. This pipeline was used to mine for biomarkers, which presented a substantial difference in methylation between the tumor and the matching normal tissue samples. Combined with the Data Integration Analysis for Biomarker discovery (DIABLO) framework for machine learning and multi-omic analysis, we revisited the TCGA DNA methylation and RNA-Seq datasets for breast, colorectal, lung, and prostate cancer, and identified differentially methylated genes within the NRF2-KEAP1/PI3K oncogenic pathway, which regulates the expression of cytoprotective genes, that serve as potential therapeutic targets to treat different cancers. Over the past few decades, the field of cancer research has continuously evolved . DiffereGenomic data has been associated with rich clinical annotations, and DNA sequencing has now been incorporated into standard clinical practice . An emerEpigenetics is the study of heritable phenotypic changes that do not involve alteration in the genomic sequence, and includes mechanisms such as histone modification, altered micro (mi) RNA, or long non-coding (lnc) RNA . MediateAlthough thousands of studies have highlighted the value of using changes in DNA methylation as candidate biomarkers in the detection and treatment of various cancers, only a handful of these targets have been approved for clinical use . Furthermore, a pipeline that identified epigenetic biomarkers with distinct methylation profiles (between specific cancers or tissues) through a supervised feature selection method was used to screen for differentially methylated biomarkers that are involved in the disruption of different pathways. Written in the Python language, the MethylMine pipeline classified biomarkers that are either hypo- or hypermethylated based on the 1st and 3rd quartiles, which are computed from the combined DNA methylation profile of all patient samples corresponding to a specific cancer or tissue type .Extracted biomarkers were integrated with the matching RNA-Seq and clinical data, and then examined using a ML-based application to reflect on potential signaling pathways that might emerge as potential ‘epigenetic switches’ which could then be used as potential therapeutic targets for diagnosing and/or treating tumorigenesis. Using this methodology, we identified a number of CpG sites which are differentially methylated and discuss the potential of using these biomarkers as screening tools for disruptions in the NRF2-PI3K oncogenic signaling pathway, which acts as the major regulator of cytoprotective responses to both endogenous and exogenous stresses from reactive oxygen species (ROS), and serve as potential epigenetic biomodifiers in the diagnosis and treatment of the screened cancers.https://portal.gdc.cancer.gov/, accessed 2018). We downloaded two types of publicly-available data—methylation , and gene expression data (fpkm) using the gdc-client tool [https://github.com/uqjlu8/MethylMine).Datasets of the four most common cancers  were dowent tool  (summaryhttps://github.com/uqjlu8/MethylMine).The MethylMine pipeline was written in the Python (3.8+) language, with the aid of other scientific libraries including: matplotlib , numpy , and panhttps://www.R-progject.org). The tuning of the component and variable number was performed according to the tutorial at http://mixomics.org. The R script used to perform this analysis is available via github (https://github.com/uqjlu8/MethylMine).Transcriptome and methylation data integration (including PLS-DA and correlation studies) were conducted using the Data Integration Analysis for Biomarker discovery (DIABLO) feature within the Bioconductor mixOmics  package Semi-unsupervised clustering: the methylation values of each sample from the TCGA were obtained as beta values, which represent the level of methylation at a single CpG site . Semi-unsupervised hierarchical clustering was performed on the probe sites across the four cancer types  that were found to be differentially methylated by the MethylMine pipeline using the “Euclidean” method via a custom Python (3.8) script using the numpy and seaborn modules.https://string-db.org/.Interaction network: protein-protein interaction network of proteins correspond to CpG sites that were found to be differentially methylated between the four cancer types—breast, colon, lung and prostate were constructed using the online STRING program  using thp value) and −log(fold change) between the tumor and normal tissue of each cancer type was visualized on a volcano plot to examine the difference in methylation of individual CpG sites using the volcano function within the Python bioinfokit module within Python where the cutoffs are: p < 0.05 and the −log2 (fold change) > 1 (hypermethylation) or <−1 (hypomethylation).Methylation comparison: differentially methylated CpG-sites (β) between normal tissue and the primary tumor of four cancer types were identified using a custom Python script, which firstly calculates the significance between the primary tumor and normal tissue of each cancer type using the non-parametric Mann–Whitney’s test. The log10 was created, and visualized in the Cytoscape software [Enrichment analysis was performed for transcriptomic targets using the online tool g: Profiler (g:GOSt)—ler/gost , with thsoftware  using thhttps://github.com/uqjlu8/MethylMine. Instructions on implementation of pipeline is available through the github page. Input matrix files (beta) need to be presented in the following format. Although we only presented the filtering of four different cancer types in this manuscript, the current version of the MethylMine pipeline can filter up to 12 cancer types simultaneously.The MethylMine pipeline was implemented in the Python language and is available to the public through our github page—http://mixomics.org. Instructions on implementation of DIABLO is available through the mixomics webpage or through CRAN—https://cran.r-project.org/.The DIABLO framework was implemented in the mixOmics R Bioconductor package and is downloadable through their website—https://cytoscape.org/. Tutorials for implementation of Cytoscape is available through their webpage.The Cytoscape software is available for download through the Cytoscape website—https://portal.gdc.cancer.gov/.The TCGA datasets analyzed in this article is publically available through the GDC Data Portal—An understanding of the genes and pathways altered in different cancers is essential to identify potential therapeutic options for diagnosing and treatment of tumorigenesis. In this article, we identified several oncogenic signaling pathways that have been recognized to be genetically altered in most cancers. In this section, we investigated the methylation profile of genes associated with four oncogenic signaling pathways ) that were found to be disrupted in most cancers, and identified methylated genes which could serve as potential targets for cancer therapeutics.To identify differential methylation in ARF-MDM2-p53 pathway-associated genes, DNA methylation data  from primary tumor and corresponding normal tissue samples were extracted for comparative analysis. This analysis focused on the CpGs within the promoter regions  of the four analyzed cancers , and ideA survey of literature suggests the hypermethylation of the associated proteins, including PIK3R5, TBX2 and TWIST1 individually, have multiple biological functions in various cancers ,71, and To identify differential methylation in the NRF2-KEAP1 and PIK3K pathway associated genes, DNA methylation data  from primary tumor and corresponding normal tissue samples were extracted for comparative analysis. This analysis focused on the CpGs within the promoter regions  of the four analyzed cancers , and idehippo = 318, nwnt = 343), TCGA-COAD , TCGA-LUSC , and TCGA-PRAD .The Hippo and Wnt signaling pathways play a crucial role in maintaining tissue homeostasis and organ size by orchestrating cell proliferation, differentiation, and apoptosis. These pathways are highly complex and are frequently dysregulated in human cancers. To identify differential methylation in the Hippo and Wnt pathway associated genes, DNA methylation data  from primary tumor and corresponding normal tissue samples were extracted for comparative analysis. This analysis focused on the CpGs within the promoter regions  of the four analyzed cancers , and ideGenes that are commonly hypermethylated across the four cancers in the Hippo and Wnt pathways include APC, CCND2 FZD, TCFs and WNTS , suggesting a link between the two pathways during oncogenesis. Furthermore, the hypomethylation of RASSF6 protein in lung cancer samples was observed C, which hippo = 318, nwnt = 343), TCGA-COAD , TCGA-LUSC , and TCGA-PRAD .The retinoblastoma (RB) pathway plays an important role in the cell cycle progression by acting as a regulator of cell proliferation and growth suppression. To identify differential methylation of genes associated with the RB suppressor pathway, DNA methylation data  from primary tumor and corresponding normal tissue samples were extracted for comparative analysis. This analysis focused on the CpGs within the promoter regions  of the four analyzed cancers , and ideGiven that comparative analysis identified hypermethylated genes that were commonly found in breast, colorectal, lung and prostate cancer samples from the TCGA initiative, we sought to propose a method that could identify pathway-associated genes with a distinct methylation profile between the tumor and matching normal tissue. The MethylMine pipeline was written in the Python (3.8+) language to identify distinct methylation biomarkers based on a feature selection method. This pipeline filters for differentially methylated CpG biomarkers by comparing the Q1 (25th percentile) and Q3 (75th percentile) of sample methylation of tumor and normal tissue samples, respectively, across each CpG site  are above 0.6 (60% methylated), while Q3 of the normal tissue (of target cancer) and the Q3 of the primary tumor and normal tissue of other cancers are less than 0.3 (30% methylated). For cancer-specific hypomethylated CpG sites, the Q3 of the primary tumor (of target cancer) are below 0.3 (30% methylated), while the Q1 of the normal tissue (of target cancer) and the Q1 of the primary tumor and normal tissue of other cancers are more than 0.6 (60% methylated). For tissue-specific hypermethylation, the Q1 of the primary tumor and normal tissue of the target cancer is more than 0.6 (60% methylated), and the Q3 of the primary tumor and normal tissue of the target cancer is less than 0.3 (30% methylated). For tissue-specific hypomethylation, the Q3 of primary tumor and normal tissue of the target cancer is less than 0.3 (30% methylated), and the Q1 of the primary tumor and normal tissue of other cancers are more than 0.6 (60% methylated). These criteria were selected based on previously analyzed data (not shown), and has been found to be effective in selecting for CpG locis with distinct methylation profiles between the tumor and matching tissues and can be adjusted to suit the user’s needs. Using this algorithm, a list of 1053 CpG sites were found to be differentially methylated between the four cancers. A list of the MethylMine extracted probes is summarized in To determine if the MethylMine pipeline can be used to filter for differentially methylated CpG sites that were either cancer-specific or tissue-specific, the beta values of all filtered CpG sites of the primary tumor and normal tissue samples across the four cancers  were extracted using a custom Python script (available through our github page). A semi-unsupervised clustering  was performed separately on samples that displayed cancer-specific hypomethylation, cancer-specific hypermethylation, and tissue-specific hypomethylation , and relClustering of MethylMine-filtered CpG sites based on the feature selection  were defined by pronounced differences in the methylation of specific cancers and/or tissues, suggesting the pipeline used is effective in parsing for biomarkers with distinct methylation patterns between the different tissues and/or cancers. By further separating the filtered CpG samples into the MethylMine features , we were able to get a clearer picture of the CpG loci that were either hypo- or hypermethylated across the different tissues, when a semi-supervised clustering analysis was performed. This analysis presented panels of biomarkers that were specific for each cancer and/or tissue. To determine the association of the differentially methylated genes and its effects on the regulation of associated signaling pathways, a multi-omic data integration approach was used .To identify highly correlated multi-omic (methylation and mRNA) cancer-discriminating signatures between breast, colorectal, lung and prostate cancers, the DIABLO  framework from the mixOmics package in R was used to integrate and analyze the methylation signatures of MethylMine-filtered CpG sites . To deteWhen both the RNA-Seq and CpG (meth) are plotted individually , with thHere, we can see the MethylMine-filtered CpG sites are highly correlated with the integrated RNA-Seq mRNA transcripts , with a Following the performance analysis, the correlation between the CpG sites and RNA-Seq transcripts (mRNA) were visualized using two different methods. Firstly, a circos plot  was usedMoreover, a correlation circle plot (CCP) was used to highlight the correlation between the integrated mRNA and CpG sites. Corresponding to the circos plot, the CCP suggested there was a negative correlation between most CpG sites and the mRNA transcripts. The single CpG site (cg21393713), which overlapped with other mRNA sites, is located within the promoter region of the autism susceptibility gene (AUTS) 2 gene, a component of a polycomb group multiprotein complex required to maintain the transcriptionally repressed state of many genes, including the Hox genes throughout development. Although predominantly associated with neurological disorders , dysregulation of AUTS2 has been implicated in both lung adenocarcinoma and prostate cancer .Furthermore, a clustered image map (cim) was employed to visualize the multi-omic molecular signature expression for each sample , with thPathway enrichment analysis has helped researchers gain a mechanistic insight into compiled gene lists by identifying biological pathways that are enriched in gene lists more than would be expected by chance . It is aThe enrichment map showed pathways that were dysregulated as a result of abnormal methylation between the four cancers screened—breast, colorectal, lung and prostate. The enrichment analysis appeared to highlight processes that are essential in normal cellular processes such as regulators of phosphorylation  and receptor-binding integrin . We found that most of the genes were connected together to form a giant linked network component involved in positive regulatory processes. Interestingly, the enrichment analysis  did not To determine if MethylMine identified differentially methylated CpG sites (in the four cancers) that are dysregulated in a common oncogenic signaling pathway, samples of filtered CpGs were analysed using a semi-supervised analysis. To reduce noise, only CpG sites located within the promoter region of associated genes were analyzed A. ClusteCombined with the pathway analysis, the heatmap revealed the differential methylation of different tumors, such as CYBA in prostate cancer and CSMD2 in lung cancer, which were previously reported to be involved in the progression of the respective cancers by being involved in the production of superoxide and/or hydrogen peroxides , which pAberrant DNA methylation is a widespread feature in cancer that can lead to the transcriptional regression of genes and alterations in different biological and molecular pathways that may play a role in the progression of oncogenesis. Although many studies have described the effects of aberrant DNA methylation on the onset and progression of cancer , a surveThe ARF-MDM2-p53 pathway (commonly referred to as the p53 pathway) has become the focus of many investigations due to its importance in tumor suppression, as shown by its genetic inactivation in more than half of human cancers and its functional impairment occurring in most of the remaining cancers, suggesting the reactivation of p53 as a treatment option that is potentially effective for a wide range of human cancers ,87,88,89The KEAP1-NRF2 and PI3K pathway is the major regulator of cytoprotective responses to endogenous and exogenous stresses caused by reactive oxygen species (ROS) and electrophiles . ROS defThe Hippo and Wnt signaling pathways play a crucial role in maintaining tissue homeostasis and organ size by orchestrating cell proliferation, differentiation, and apoptosis. These pathways are highly complex and have been frequently found to be dysregulated in human cancers. The Hippo pathway consists of a large network of proteins that controls the growth of different tissue during development and growth, as well as in different diseases such as cancer , while tThe RB pathway has been a focus in cancer therapy ,110, as Finally, to identify genes that are differentially methylated between the four cancers screened, we used a combination of our differential methylation parsing pipeline—MethylMine—and unsupervised clustering to identify an initial panel of methylation sites, which are differentially methylated across the four selected cancers—breast, colorectal, lung and prostate. Furthermore, multi-omic integration and pathway enrichment analysis were conducted to identify signaling pathways, which may be dysregulated during the onset of the four cases as a result of these aberrant methylations. In contrast to previous studies that used inferential statistical methods  to mine for differentially methylated genes between different sample groups, the MethylMine pipeline extracted differentially methylated biomarkers by comparing the quartiles of methylation values at each probe across both (primary) tumor and normal tissue samples of selected cancers. The assumption here is that most of the methylation pattern of at least 75% of the samples across a CpG site would be above a certain beta value  if the CpG was hypermethylated. As opposed to inferential statistical methods, we found biomarkers extracted using the MethylMine pipeline returned biomarker panels that presented a marked difference between the different tissues and cancers , and reqInterestingly, given the same dataset, the MethylMine pipeline extracted different a set of biomarkers then the comparative analysis performed above , as thisTo determine if MethylMine-extracted CpG sites influenced the activity of associated genes, the 1053 CpG extracted CpG sites were integrated with the corresponding RNA-Seq transcriptomic data using the N-integration method within the DIABLO module of the mixOmics package. This integration allowed the merge of CpG sites with its corresponding mRNA transcripts so we could determine if the expressed genes (mRNA transcript) were influenced by the methylation pattern of corresponding CpG loci. The integrated data was then analyzed using an enrichment analysis to identify differentially methylated pathways that were disrupted in the four cancers. Interestingly, the enrichment analysis highlighted pathways that were predominantly involved in the regulation of developmental and phosphorylation processes . This suGiven this, we sought to determine if MethylMine identified differentially methylated biomarkers that overlapped with any of the four signaling pathways analyzed. By using a combination of interaction analysis and semi-supervised clustering, we identified a total of 15 differentially methylated genes, which corresponded to the NRF2-KEAP1 and PIK3 pathway B. FurtheDespite the boost in the post-genomic era, there is an urgent need to explore the large volume of methylation data that is curated in publicly available databases in order to explore the commonalities and differences between signaling pathways that may drive the progression of cancer. To explore the use of epigenetic modifiers in various cancers, we revisited the TCGA dataset, and performed a comparative methylation analysis, which suggested the hypermethylation of genes associated with four canonical oncogenic signaling pathways. Although they have individual functions, all four pathways shared common proteins and transcription factors, which could be targeted by different biomodifiers to diagnose or treat cancer. As changes in DNA methylation may result in altered signaling pathways leading to the progression of cancer, here we demonstrated the potential of altering methylation-based targets within these pathways to change their phenotypic expressions, and ultimately switch the pathway back into a non-disease state. Furthermore, we described a differential methylation parsing pipeline MethylMine that filters for differentially methylated biomarkers by comparing the different quartiles between the (primary) tumor and normal tissue of each CpG site of corresponding cancers. By integrating the corresponding RNA-Seq transcript data, we were able to screen for transcripts and by extension, genes that were expressed and enriched in different signaling pathways. As the MethylMine framework filtered for differentially methylated CpGs between different cancer types, we propose this algorithm can be used to screen for cancer-specific CpG markers. Our analysis suggested the pipeline used is effective in filtering for differential methylation of genes associated with the NRF2-KEAP1 and PIK3 pathway, which acts as a major regulator of cytoprotective responses to endogenous and exogenous stresses via ROS. Additionally, genes that encode for ROS-generating NADPH oxidases  were also detected using the current pipeline and have been found to play a role in most cancers. Therefore, the MethylMine pipeline is able to screen for differentially methylated biomarkers with distinct changes in methylation profile between the different cancers and tissues. Additionally, this algorithm will be improved in future versions to screen for pan-cancer biomarkers in order to mine for biomarkers that are shared between different cancers to uncover epigenetic modifiers that can be used to target multiple cancers. As such, the use of DNA methylation as an epigenetic modifier is promising, opening an avenue for identification of novel drug targets, resistance patterns, and the development of combination therapies to treat multiple targets across various signaling pathways to target different cancers.