The swift rise of omics-approaches allows for investigating microbial diversity and plant-microbe interactions across diverse ecological communities and spatio-temporal scales. The environment, however, is rapidly changing. The introduction of invasive species and the effects of climate change have particular impact on emerging plant diseases and managing current epidemics. It is critical, therefore, to take a holistic approach to understand how and why pathogenesis occurs in order to effectively manage for diseases given the synergies of changing environmental conditions. A multi-omics approach allows for a detailed picture of plant-microbial interactions and can ultimately allow us to build predictive models for how microbes and plants will respond to stress under environmental change. This article is designed as a primer for those interested in integrating -omic approaches into their plant disease research. We review -omics technologies salient to pathology including metabolomics, genomics, metagenomics, volatilomics, and spectranomics, and present cases where multi-omics have been successfully used for plant disease ecology. We then discuss additional limitations and pitfalls to be wary of prior to conducting an integrated research project as well as provide information about promising future directions. Plant disease ecology is inherently interdisciplinary and relies on the disparate fields of microbial ecology, epidemiology, plant physiology, and genetics to inform a critical observation: plants get sick. For more than a century, plant pathologists have observed that virulent microbes  drive disease dynamics in susceptible plants given the right environmental conditions . In natuTechnological advances over the last few decades have enabled a new, more comprehensive approach to plant disease ecology allowing a holistic approach to identifying pathogenesis and its underlying mechanisms. Among these technologies are -omics tools that allow for the comprehensive examination of plant and microbial characteristics along the genotype-phenotype spectrum . UnderstThese developments in omics-approaches to plant disease ecology have been particularly important in light of the current, rapidly changing environment. As increasing biotic and abiotic stressors impact plant health, adapting to and mitigating the effects of these stressors is critical for maintaining both healthy terrestrial ecosystems and productive agricultural systems. For example, globalization and the movement of people, plants, and microbes around the world can drastically change the composition and ecology of habitats. Invasive microbial pathogens and/or soilborne pathogens, especially those with broad host ranges, can cause the death of, in some cases, thousands of trees across a landscape , 6. BothWhile some benefits from more holistic approaches have been realized, further opportunities exist through increasing integration of -omics technologies and platforms. Insights from genetics approaches can be tied to transcriptomics, metabolomics, and spectranomics to link multiple steps in the -omics cascade from genotype to phenotype. This integration, while still in its early stages, holds vast potential for unlocking fundamental and applied insights in microbial plant disease ecology. Understanding the genetic basis for plant pathogen susceptibility, how the plant microbial community influences expression, the cascading metabolomic and volatilomic pathways resulting from infection, and the spectranomic characteristics of foliage present numerous opportunities for intervention that can be tuned and optimized for plant disease management situations.In this review, we discuss exactly these opportunities by beginning with an overview of -omics technologies including the unique challenges that each field faces. Next, we present recent case studies that highlight integration of multi-omics insights. We then offer additional opportunities and considerations for integrating these technologies and touch briefly on expected benefits resulting from this integration. These opportunities and considerations will be discussed in light of our principal goal: driving the development of healthy plants and their microbial communities in a constantly changing environment.Haemophilus influenzae, the first free-living organisms to have its whole genome sequenced via shotgun sequencing [Saccharomyces cerevisiae, first eukaryote to have its whole genome sequenced [Caenorhabditis elegans, the first multicellular eukaryote [de novo sequence assemblies and resequencing of multiple strains of a single species.The field of genomics, the study of the complete genetic makeup of organisms, has grown exponentially in the past 20 years since the milestone publication of the first draft of the human genome in 2001 , 10. Altquencing ; the funequenced ; and theukaryote . FurtherMicrobial genomics is an interdisciplinary field that focuses on the structure, function, evolution, mapping, and editing of genomes for bacteria, fungi, archaea, viruses, and other microscopic organisms. Integration of genomics questions and tools can aid ecological questions, especially those that involve environmental change. For example, it is critical to understand the evolutionary history of the genome to understand if certain elements may change quickly under changing temperatures (rapid evolution). Sometimes, it may be necessary to draw comparisons across genomes to understand microbial functions in related species, or delve into transcriptomics and other –omics. Coupled with transcriptomics and proteomics, functional genomics uses genomic data to study gene and protein expression and function on a genome-wide or system-wide scale. The integration of genomics and transcriptomics has resulted in an increased understanding of various aspects related to plant pathogen biology, plant-pathogen interactions, and plant health , 16.In investigations of plant disease ecology and plant health, it can be helpful to consider two main groups of microbial genomics. Structural genomics, focused on assigning and mapping genes and markers to individual chromosomes, results in a physical map of the whole genome. Functional genomics integrates genome sequences with an organism’s transcriptome (transcripts produced by a given organism) and proteome (encoded proteins) to describe gene functions and interactions , 18. TheFusarium species clear genomic compartmentalization has been identified at both structural and functional levels: a core component of the genome that encodes functions necessary for growth and survival and is shared among Fusarium species, and an accessory component [Verticillium genomes differ in four regions of approximately 300–350 kb each, which are present in V. dahliae Ls.17 and absent in V. alfalfae Ms.102. These four regions contain genes that seem to vary considerably between strains and have functions putatively involved in pathogenicity and virulence of the pathogens [Comparative genomics aims to identify structural and functional genomic elements conserved within species or across different species . Using tomponent , 23. Theathogens , 25. As athogens –28.In addition to elucidating genomic architecture, phylogenomics and population genomics address similar questions, but at different biological scales: relationships among species, and individuals and populations within species, respectively. The types of questions addressed include genome-wide evolutionary and demographic processes affecting population structure, evolutionary processes affecting speciation and the divergence of closely related taxa, and locus-specific effects, acting on specific genes or chromosomes that affect adaptation or defined phenotypes , 29, 30.Metagenomics builds on the tools and theories from molecular genomics to investigate DNA that is sourced directly from the environment. This field involves the extraction and amplification of microbial DNA targets from soil, water, air, plants and animals . AlthougToday the field has evolved to answer questions related to the ecological dynamics of microbial communities, multi-species regulation, mechanisms for microbe-microbe and host-microbe interactions, community coevolution . The terHomo sapiens) where reproductive boundaries and barriers are strongly delineated. Bacteria, on the other hand, are currently assigned to a common species if their reciprocal, pairwise DNA re-association values are ≥ 70% during DNA hybridization experiments [A major challenge in metagenomics is defining the microbial species concept itself. The biological species concept works well with more derived taxa ..Fig 1). Metabolomics began in the 1970s with the expansion of medical analyses of compounds of interest using gas chromatography-mass spectrometry . As techThese advances are not without limitations, however. A key challenge in metabolomics is the sheer diversity of potential metabolites present in any given sample. In contrast to micro-organisms or animals producing on the order of 1,500 and 2,500 unique metabolites respectively, a single plant species can produce upwards of 5,000 unique compounds . All tolTargeted metabolomics approaches address these challenges by constraining profiling to a known set of annotated compounds . PreciseUntargeted metabolomics approaches, in contrast, provide insight into unknowns . ApproacStrictly speaking, volatilomics could be considered a subset of metabolomics . HoweverAs with metabolomics, plants use an astonishing array of compounds for communication ranging from plant hormones, terpenoids, green leaf volatiles, and volatiles produced and released upon attack from pests (herbivore-induced plant volatiles) . These vThe field of volatilomics grew out of the field of metabolomics as advances in gas chromatography coupled with mass spectrometry allowed for more in-depth profiling of gaseous volatile compounds . CriticaFoliar functional trait characterization has emerged in terrestrial ecology as a unifying concept to better understand both natural variability in vegetation function and variability in response to environmental change and stress. The idea of foliar functional traits has been enthusiastically embraced by the remote sensing community as many traits shown to strongly correlate with natural and stress-induced variation in plant function  can be dOnly recently have plant pathologists begun to take advantage of the spectranomics trail blazed by terrestrial ecologists. Recent studies have helped establish in-situ (or foliar) and imaging spectroscopy  as effective tools for early, non-destructive, and scalable biotic stress detection in natural and agroecosystems –86. BothBroadband and multispectral methods relying primarily on visible (VIS) and near-infrared (NIR) reflectance indices, such as normalized difference vegetation index (NDVI), have been used to sense late stage plant disease since the 1980s –103. ChaPlant defence pathways mediate plant microbial interactions in changing environments regulating responses to abiotic and biotic stressors above and belowground –119. A cUnderstanding SA signaling began with early molecular studies as realization dawned that this phenolic secondary metabolite was nearly ubiquitous in the plant kingdom and involved in everything from plant reproduction and photosynthesis to mediating responses to abiotic and biotic stresses , 122. ThFollowing pathway elucidation, modern targeted and untargeted metabolomics approaches have characterized SA signaling cascades from plant stressors –128. TheWhile understanding of the SA pathway may have begun with metabolomics, it quickly progressed to inquiries into the genetic basis for observed phenoma, particularly related to systemic acquired resistance (SAR)—the ability of plants to acquire long-term resistance to pathogenic microbes , 137. GeThe SA signaling pathway and systemic acquired resistance not only affects the plant pathogen microbial community, but also a whole host of other organisms . To do sThe story of how these aspects of SA-regulated plant disease ecology were discovered is inextricably intertwined with the early history of multi-omics approaches. As integrated multi-omics has emerged, our resolution of the processes underlying SA plant defense signaling has improved and, with it, our ability to use this knowledge for positive intervention in managed natural systems. Early vegetative spectroscopic studies addressing the retrieval of foliar biochemicals primarily targeted pigments, nitrogen, protein, structural elements (cellulose and lignin) and mineral compounds , 153. SoSpectroscopic methods such as fluorescence spectroscopy, Fourier-transform infrared spectroscopy, and chemometrics are used to better assess and understand plant-microbe interactions, but these methods require sampling and processing , 156. ThCombining spectranomics and metabolomics would not be without its challenges: the assessment of plant biochemical traits at the canopy scale still faces considerable limitations from complexities related to canopy structure/density and the subsequent impact on light reflectance. Additionally, historical knowledge of the VIS-NIR-SWIR spectral features  associated with plant metabolites is limited because typically other approaches have been used such as UV, MIR, X-ray, Raman or FTIR spectroscopy . These mThe observation that soils have the capacity to suppress plant disease has been known for decades, some would argue for almost a century or longer in agricultural settings . Yet undArabidopsis as a model, researchers were able to tease apart the relationship between plant nutrient levels and host defense [In order to apply basic knowledge of the soil microbial community identities and functions to address problems in the applied plant sciences such as conservation, restoration, forestry, and agriculture, it is essential to consider a multi-pronged approach. For instance, plant nutrient deficiencies have long been known to affect both crop yield, but also overall defense against pathogens. Using  defense . The aut defense . The autAnother application of a multi-omics approach is identifying more specific microbes that could be involved as biological control agents (BCA) against soilborne plant diseases and the creation of synthetic communities. Biocontrol research has typically taken a targeted approach that focuses on using a single or a few microbes to to suppress pathogenicity. With the development of high throughput sequencing techniques, such as amplicon-based sequencing, entire microbiomes were and are continuing to be identified. There is now need to shift away from community description toward functional understanding of these microbial communities in their capacity to not only suppress disease, but potentially have one microbe or a suite of microbes elicit antagonistic interactions to hinder plant disease , 166.What is still needed for understanding the microbial ecology of soil suppression and the potential for biological applications to suppress plant disease are studies that seek predictive patterns and results. Specifically the spatial-temporal distribution of soil suppression and whether certain pathogens are consistently suppressed by specific taxa or functional groups of soil microbes. For instance certain sustainable land management practices such as the use of cover crops , organicBurkholderia phytofirmans PsJN which should how various extra-cytoplasmatic functional group elements  were critical in facilitating other bacteria to sense changes in their surrounding environment such as temperature or moisture and shift their metabolic activity to survive the change. This paper used a dual -omic approach, by using high throughput sequencing to identify both the key player, Burkholderia, other bacteria, and basic metabolomics [A promising area of -omics research is understanding the role of endophytes in plant disease. Endophytes are microbial organisms that live within plant tissue without causing visible symptoms and are essential to plant health and well being . There ibolomics . More rebolomics , howeverbolomics .An additional promising frontier is the integration of spectranomics with genomics to construct multi-dimensional phylogenies that capture the evolutionary dynamics of leaf chemistry, structure, and more. Recent work by Meireles et al. showed that spectroscopy is capable of robustly capturing phylogenetic signals and that broad plant groups, orders, and families can be identified from reflectance spectra . Using eIntegrating multi-omic approaches can help us understand who are the microbial players in the system, how they disperse and are distributed across time and space, and what are their individual and combined functions. As detailed in the case studies for this review, -omics can provide a richer picture of the mechanisms behind fundamental topics in plant disease ecology such as plant defence, stress response, and the potential for disease suppression. This is especially the case when compared to simply utilizing one -omic alone. These approaches, when integrated correctly at the right scale and for the right research question, promise to reveal a multi-dimensional view of plant disease. However, there are still many pitfalls and limitations toward integrating and using these approaches that researchers must be aware of before designing a research proposal and project. Below, we outline some of the promises as well as the pitfalls of -omics driven studies, as well as future directions for using these approaches for plant disease research.When designing projects that could benefit from multi-omic approaches, there is a need to move away from strictly observational research questions to those that are hypothesis driven. Although the number of published research articles has grown exponentially in the past decade to reflect breakthroughs in high throughput sequencing, few have been applied to questions within the fields of environmental and conservation genomics that ask experimental questions . A shiftAnother major limitation to conducting multi-omics are taxonomic or chemical compound databases that are currently in their nascent phases as more microbes are sequenced and discovered worldwide. Still, a handful of databases do exist that are well curated and are the standard for conducting -omics research. For both amplicon-based studies and metagenomic studies, open-source databases include RDP , Silva , GreengeAdditional limitations to integrating multi-omics approaches in plant disease ecology include cost-per-sample  and difficulties in integrating disparate data-sets. -Omics information inherently comes in different formats with different means of preprocessing, analyzing, and interpreting the final results. Integrating those results across the genotype-phenotype spectrum can be challenging.Addressing these limitations and realizing the potential of multi-omics research necessitates collaboration. Indeed, the motivation for writing this review was to spark such collaborations. No one person can be an expert across multi-omics domains; effectively conducting multi-omics research necessitates a integration of expertise just as it necessitates integration of datasets. This aspect of collaboration is perhaps the most promising feature of multi-omics research. Integrating a diversity of disciplines engenders an integration of perspectives. The challenges apparent in one discipline will likely benefit from fresh perspectives from another. Integrating these approaches will allow research collaborations to answer questions and address intractable problems that were previously not possible.A multi-omics approach to solving problems in plant disease ecology has already led to breakthroughs in understanding plant defense, detecting plant stress, and managing disease with suppressive soils. This approach also seems poised to create breakthroughs in our understanding of endophytes. The promise of multi-omics in plant disease ecology extends beyond these areas, however, and looks to create breakthroughs in our understanding of how microbial communities respond in a changing environment for years to come .


Prostate cancer is the most common malignancy in men, with a high mortality rate when disease progresses to metastasis and therapeutic resistance. Evidence implicates inflammation as a driver of prostate cancer risk and has a significant impact on processes in the tumor microenvironment that facilitate progression to advanced therapeutically resistant disease. In this review, we discuss the sources of inflammation in the prostate, the functional contribution of the critical inflammatory effectors to prostate cancer initiation and metastatic progression, and the therapeutic challenges that they impose on treatment of advanced disease and overcoming therapeutic resistance. Full understanding of the role of inflammation in prostate cancer progression to advanced metastatic disease and tumor relapse will aid in the development of personalized predictive biomarkers and therapy to reduce the burden and mortality in prostate cancer patients.Prostate cancer is the most common malignancy among men, and progression to metastasis and the emergence of therapeutically resistant disease confers a high mortality rate. Growing evidence implicates inflammation as a driver of prostate cancer development and progression, resulting in increased cancer risk for prostate cancer. Population-based studies revealed that the use of antinflammatory drugs led to a 23% risk reduction prostate cancer occurrence, a negative association that was stronger in men who specifically used COX-2 inhibitors. Furthermore, patients that were taking aspirin had a 21% reduction in prostate cancer risk, and further, long-term users of daily low dose aspirin had a 29% prostate cancer risk reduction as compared to the controls. Environmental exposure to bacterial and viral infections, exposure to mutagenic agents, and genetic variations predispose the prostate gland to inflammation, with a coordinated elevated expression of inflammatory cytokines . It is the dynamics within the tumor microenvironment that empower these cytokines to promote survival and growth of the primary tumor and facilitate disease progression by navigating the immunoregulatory network, phenotypic epithelial-mesenchymal transition (EMT), angiogenesis, anoikis resistance, and metastasis. In this review, we discuss the sources of inflammation in the prostate, the functional contribution of the critical inflammatory effectors to prostate cancer initiation and metastatic progression, and the therapeutic challenges that they impose on treatment of advanced disease and overcoming therapeutic resistance. Growing mechanistic evidence supports the significance of inflammation in localized prostate cancer, and the systemic impact of the process within the tumor microenvironment on disease progression to advanced therapeutically-resistant prostate cancer. Rigorous exploitation of the role of inflammation in prostate cancer progression to metastasis and therapeutic resistance will empower the development of precise biomarker signatures and effective targeted therapeutics to reduce the clinical burden and lethal disease in the future.  Prostate cancer is the most common malignancy in men, with one in six American men developing prostate cancer within their lifetime, it is estimated that 191,930 American men will be diagnosed with prostate cancer in 2020 [The link between chronic inflammation and malignancies has been well established, and chronic inflammation has been identified as a major cause of approximately 20% of human cancer cases [In this review, we will discuss the current understanding of the role of inflammation in the prostate tumor microenvironment and progression to therapeutically resistant metastatic prostate cancer. Understanding the biological mechanisms by which inflammation contributes to prostate cancer progression may reveal targets for therapeutics to reduce the burden of prostate cancer in the future. Mechanistic insights into the influence/drivers of inflammation on the tumor microenvironment will enhance our knowledge regarding the contribution of this complex process of prostate cancer progression and therapeutic resistance.Inflammation is a critical immune process that occurs in response to tissue damage caused by injury or infection. Inflammatory processes aim to clear pathogenic material and debris from damaged tissue areas and initiate wound healing. While this process is an essential defense mechanism to fight invading pathogens, persistent chronic inflammation can cause a further tissue damage. This can result in the release of reactive oxygen and nitrogen species from cells, as well as increased genome instability leading to an increased risk of cancer . When thInflammation in early prostate carcinogenesis can be histologically indicated by proliferative inflammatory atrophy (PIA) , which aBacterial Infections: bacterial infections can elicit an inflammatory response in the prostate leading to prostatitis; these infections include sexually transmitted infections (STIs). Previous studies have suggested that an STI diagnosis is more likely to exhibit an elevation in serum prostate specific antigen (PSA) and men with a history of diagnosis with an STI are more likely to develop prostate cancer ,13. HoweViruses-Link to COVID19: viral infections are another potential source of local inflammation within the prostate; however, this area is not well understood, and it is not as extensively studied as bacterial infections. There is conflicting evidence regarding the relationship between prostate cancer and some viruses infecting the prostate. For example, in a case-control study there was no relationship found between human papillomavirus (HPV) infection and symptoms of prostatitis . Howeverhttps://coronavirus.jhu.edu/map.html, 20 September 2020) and more than 205,000 deaths in the United States. The available antibody tests checking for an antibody response to SARS-CoV-2 infection are used in order to determine virulence of infection and case fatality rates in COVID-19 patients. The mechanism of viral SARs infection induces an increase in circulating pro-inflammatory cytokines, including IL-6, IL-8, TGF-β, CCL2, and TNFα as compared to uninfected controls, and this elevation is sustained through viral clearance and recovery in patients [COVID-19 Link: while there is little evidence that viral infections lead to inflammation within the prostate glandular tissue, systemic inflammation that is driven by viral infection easily creates a “favorable” environment for tumor development in various organs, including the prostate. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic that erupted in December 2019, led to the novel coronavirus disease (COVID-19), which has resulted in a global pandemic with over 31,890,248 people infected worldwide  and advanced prostate cancer, biochemical recurrence, and prostate cancer mortality. Obesity leads to chronic systemic inflammation, with greater circulating inflammatory cytokines such as TNF-α, leptin, CRP, MIC1 and IL-6 ,54. In tfed mice . Taken tTrauma to Prostate Epithelium: Chemical irritation to the prostate epithelium can result in the release of pro-inflammatory cytokines and development of chronic inflammation, this can occur as a result of urine reflux . StudiesGenetic Predisposition: a growing body of evidence has identified several genes and their variants involved in inflammation and immune function, which are linked to increased prostate cancer risk. The most studied gene linked to prostate cancer susceptibility is ribonuclease L (RANSEL), which encodes an enzyme that is induced by interferon that degrades viral RNA and modify innate immune responses . SeveralThe macrophage inhibitory cytokine 1 (MIC1) is a member of the TGF-β superfamily that modulates macrophage mediated inflammation and is expressed by prostate cancers, and circulating levels can be linked to disease outcomes . StudiesGlutathione S-transferase protects against cell damage from oxidative stress, which can be a result of inflammation . MethylaAn inflammatory reaction is constantly elicited inside a malignant tissue, which results in an extraordinary dynamic of immune cell infiltration, angiogenesis, and fibroblast proliferation . The tumProstate cancer cells produce several inflammatory factors, which modify the tumor microenvironment in order to contribute to tumor cell growth, survival, invasion and progression. Prostate cancer cells and surrounding prostate stroma cells express high levels of inflammatory cytokines including interleukins IL-1, IL-6, and IL-8 in order to promote prostate tumor proliferation and survival ,83,84. IImmunity: it is becoming increasingly evident that inflammation induces cancer development and resistance to therapy. Recent single cell analyses studies have revealed key insights into the Immune cell regulation and its contribution towards inflammatory microenvironment. In a recent study, Trujillo et al. discussed tumor segregation by T-cell inflamed phenotype and compared the genomic determinants of T-cell inflamed versus T-cell non-inflamed tumors . This stThe production of inflammatory cytokines, such as chemokine ligand 2 (CCL2) and IL-8, lead to an infiltration of immune cells such as macrophages. TAMs are a prominent cell type in the tumor microenvironment and they are associated with a poor prognosis in many types of cancers, including prostate cancer . These cAngiogenesis: angiogenesis is a vital process during tumorigenesis for providing homeostasis to the tumor microenvironment. Inflammation plays a supportive role in facilitating this process, TNF in the microenvironment stimulates the release of pro-angiogenic factors IL-6, IL-8, TGF-β, and vascular endothelial growth factor (VEGF) by cancer cells via the induction of NF-κB . The infEpithelial–Mesenchymal Transition (EMT): inflammatory signals confer more invasive properties of a tumor by promoting the process of epithelial to mesenchymal transition (EMT) in cancer cells. Cancer cells adopt a mesenchymal cell phenotype by losing the cellular polarity and adhesion to the basement membrane to become more invasive and migratory . TGF-β hWhile the prognosis for most men with prostate cancer is good, those who develop advance metastatic castration resistant prostate cancer (mCRPC) have poor survival outcomes. Reliable prognostic markers would be useful as measures to predict prostate cancer risk, patient cancer progression, treatment response, and survival outcome. The use of inflammatory markers has been investigated as prognostic tools due to contribution of inflammation to prostate carcinogenesis. In a meta-analysis of over 16,000 men the neutrophil-lymphocyte ratio (NLR) was associated with poor overall survival, progression-free survival, and recurrence-free survival in men with prostate cancer and mCRPC irrespective of tumor stage and treatment . MoreoveOther circulating inflammatory factors that have been analyzed in various cancers for association with prostate cancer prognosis are CRP and lactate dehydrogenase (LDH) ,107,108.Circulating inflammatory cytokines have been interrogated with precision in order to establish the relationship between inflammation and prostate cancer. Perhaps, the most well studied in cancer is IL-6, as a marker of chronic inflammation. Serum levels of IL-6 are elevated in patients with metastatic and CRPC and, IL-6 significantly correlated with tumor stage, and it is inversely correlated with tumor survival and therapeutic response ,83,113. Prostate cancer primarily metastasizes to the bone, and metastatic prostate cancer has a reduced five-year rate of survival of 30% from 99% in localized prostate cancer cases . InflammAs tumor cells undergo EMT, they adopt more invasive properties, lose their cell-cell adhesions, and detach from the ECM. Following detachment, cells are subject to anoikis, a form of cell death, which occurs in the absence of connections between the cells and ECM. However, tumor cells that develop resistance to anoikis can migrate freely in the circulation, leading to metastasis , contribUpon acquiring resistance to anoikis, tumor cells are able to migrate and invade distant sites, as schematically illustrated on Gene set enrichment analysis (GSEA), revealed that gene signatures that were related to interferon- and inflammatory cytokines signaling as well as apoptosis were the most significantly associated with the gene expression profile of AR-negative cancer epithelial cells, with an inverse association with DNA repair-associated gene signatures . MoreoveAs prostate cancer progresses, there is emergence of androgen-independent state and tumors leading to metastatic castration-resistant prostate cancer, mCRPC. Patients who progress to this advanced disease have poor survival outcomes and they are highly resistant to therapeutic modalities, resulting in treatment failure, tumor recurrence, and patient lethality .Androgen-deprivation therapy (ADT): growing evidence supports the role of inflammatory pathways in promoting progression to androgen-independent mCRPC disease. Recent studies have revealed that IL-1 represses activity of AR in prostate cancer cells and promotes the progression of prostate cancer to androgen-independent disease . IL-23 pTaxane chemotherapy: taxane chemotherapy using microtubule-targeting drugs, such as docetaxel (1st line chemotherapy) and cabazitaxel (2nd line taxane chemotherapy), are standard modalities for the treatment for metastatic CRPC. Studies have investigated the ability of cytokines and macrophages to contribute to therapeutic resistance to taxane chemotherapy in prostate cancer patients impacting progression to lethal disease. In men with CRPC, elevated levels of circulating inflammatory cytokines IL-4, IL-6, and MIC1 were associated with resistance to docetaxel after one cycle of treatment . MacrophRadiotherapy: radiation therapy is commonly used for the treatment of localized prostate cancer. However, radiation resistance emerges and presents a therapeutic challenge among individual tumors . RadioreDetailed analyses of viral- and bacterial-infections, genetic variants, cytokines, and chemokines have paved an avenue for cornerstone studies to the next-gen prostate cancer diagnostics. In this context, future clinical trials should focus on the interventional studies in order to demonstrate the clinical utility of inflammatory markers in cancer.Exposure to numerous infectious and non-infectious agents can promote inflammation; however, the link between this exposure and prostate cancer development and progression requires further analysis. Future studies should investigate the cellular and molecular changes that occur in individuals who have been exposed to inflammation inducing agents and how this may drive aggressive prostate cancer. The recent global COVID-19 pandemic provides a unique opportunity to examine how exposure to viral infections, and the resulting systemic inflammation and circulating inflammatory markers, can impact disease progression and outcomes in patients with cancer; this will be particularly in the context of prostate cancer due to the commonality of androgen signaling and TMPRSS2 involvement.Progression to CRPC resistance and therapeutic resistance holds significant challenges to patient mortality; understanding the inflammatory signals that drive these processes can lead to effective therapeutic targeting by existing anti-inflammatory agents (as summarized on While several inflammatory cytokines have been implicated in disease progression and resistance, the mechanisms and pathways by which this occurs are not fully understood. Future studies should utilize both in vitro and in vivo models in order to target these inflammatory cytokines and examine the downstream effects on the expression of key factors that are involved in EMT, anoikis resistance, and metastasis. This may reveal more specific targets to combat the mechanism of resistance as the multifunctional roles of many inflammatory cytokines make them difficult to target safely in clinical situations. As the precise role of inflammation in the emergence of therapeutic resistance still needs to be exploited, identifying inflammatory signatures in different stages of prostate cancer progression and the impact of various therapeutic strategies on such signatures in patients with advanced prostate cancer may reveal critical mediators of therapeutic resistance and attractive new targets for overcoming this resistance .This review exploits the functional contribution of inflammatory cytokines to the development and progression of prostate cancer to metastasis, and the emergence of therapeutic resistance and treatment failure. Inflammatory cytokines have shared functions contributing to diverse processes that landscape the prostate tumor microenvironment and cancer progression to metastasis, as illustrated in


Survival outcomes for resectable metastatic colorectal cancer have improved over the past decade. This is due in part to improvements made in imaging technology, locoregional treatment, and systemic treatment. The focus of this review is to summarize and analyze the existing information available on systemic therapy in the setting of colorectal cancer with liver metastases. It is written with structured viewpoints including patient selection, sequencing of treatment, choice of systemic therapy for upfront resectable disease and conversion chemotherapy for unresectable colorectal cancer. Post-treatment surveillance and future research directions are also discussed. This review will help treating physicians make decisions for the treatment of liver metastases of colorectal cancer.The liver is the dominant site of metastasis for patients with colorectal cancer. For those with isolated liver metastases, surgical resection with systemic therapy has led to long-term remission in as high as 80% of patients in well-selected cohorts. This review will focus on how systemic therapy should be integrated with resection of liver metastases; in particular, the use of clinical risk scores based on clinicopathological features that help with patient selection, various approaches to the treatment of micro-metastatic disease (peri-operative versus post-operative chemotherapy), as well as conversion chemotherapy for those with initially upfront unresectable disease will be discussed. According to the World Health Organization (WHO) Global Cancer Observatory (GLOBOCAN) 2018 data, colorectal cancer (CRC) is the third most common cancer diagnosed (10.2%) and the second-leading cause of mortality amongst all cancers 9.2%) ,4. The f.2% ,4. TAs there is a clear survival impact, surgical resection of R0 resectable liver metastases is the treatment of choice when possible. The criteria to select patients for potentially curative resection is constantly being redefined, and this varies between institutions but is largely dependent on the experience of the multi-disciplinary medical team. Considerations that need to be accounted for include patient factors, disease factors, and anatomic factors.There are several well-established clinical risk scores that are used to stratify patients based on their likelihood of recurrence  7,10,1111,12. ToEven though these scores have been widely used to stratify patients based on their likelihood of recurrence, they were developed based on patients who underwent surgery before 2000, when prognostic information of tumor mutations were not known. Multiple groups have shown that somatic mutations in RAS are associated with inferior progression-free and overall survival in colorectal cancer ,14. RetrMore recently validated risk scores have included factors like KRAS mutation status and presence of extrahepatic disease, as in the Genetic and Morphological Evaluation (GAME) score , and KRAThe optimal timing and sequencing of chemotherapy, chemoradiotherapy , and surgery should be individualized for each patient. This decision is undertaken by a multi-disciplinary tumor board and should account for the following: synchronous versus metachronous liver metastases, presence of a symptomatic primary tumor, resectability of liver metastasis, and response to systemic therapy. There is no evidence that adding a biological agent to a cytotoxic doublet improves the outcome in the presence of resectable metastases compared with a cytotoxic doublet alone in combination with resection of the metastases. In general, patients with a symptomatic primary tumor  should have treatment of the primary tumor prioritized. If liver metastases are upfront resectable, concurrent resection of liver metastases can be undertaken if the peri-operative risks are acceptable. If not, pre-operative chemotherapy may be given, followed by reassessment with close-interval imaging. It is important to proceed to liver metastatectomy once resection is deemed feasible and not to continue with prolonged chemotherapy to avoid veno-occlusive disease or chemotherapy-associated steatohepatitis. Those with upfront resectable metachronous liver metastases  and low clinical risk of recurrence can be considered for upfront resection. Following resection, the benefit of adjuvant chemotherapy or surveillance alone will need to be discussed with the patient. In those with upfront resectable disease, studies have looked at delivering systemic chemotherapy either for six months post-operatively or in a sandwich fashion, i.e., three months before and three months after resection of hepatic metastases. In those with resectable disease, the goal of delivering chemotherapy pre-operatively is to “test tumor biology”, that is, to allow an observational period to determine whether new lesions will appear soon after systemic treatment, rendering surgery futile. Conversely, we know that the pathologic response to pre-operative chemotherapy is predictive of prognosis after resection of colorectal liver metastases; the five-year OS in those attaining pathological complete response is 76%, compared to 45% in those who do not .To date , there aAlthough the addition of anti-epidermal growth factor receptor (EGFR) antibodies such as cetuximab and panitumumab have shown to produce a survival benefit for patients with RAS/RAF wild-type tumors in the palliative setting ,32, thisThe use of oxaliplatin-based adjuvant chemotherapy for the treatment of micro-metastatic disease has long been accepted as the standard of care for patients with stage 3 colon cancer due to the consistent, significant disease-free and overall survival benefits . HoweverThe purported clinical benefits are seen only in R0 resection ; there is no role for partial palliative resection of metastases. As such, the consideration of upfront chemotherapy in the presence of initially unresectable liver metastases with the hope of downstaging to resectable disease seems to be a reasonable approach, but in reality it only occurs in 12% of cases . HoweverMultiple regimens for conversion chemotherapy in the presence of unresectable CRC with liver metastases have been studied, but the optimal chemotherapy regimen has not yet been established . BroadlyOxaliplatin-based (FOLFOX/XELOX) and irinotecan-based doublets (FOLFIRI/XELIRI) result in similar response rates, between 34% and 59% ,52,53,54Bevacizumab is a humanized monoclonal antibody against vascular endothelial growth factor (VEGF) which has shown survival benefit in the palliative setting ,58,59. TAddition of anti-EGFR agents (cetuximab or panitumumab) to doublet chemotherapy in the setting of unresectable liver metastases results in response rates of 41–70%, translating to R0 resection rates of 27–38% ,72,73,74Post-treatment surveillance is indicated for patients who would be considered for a second potentially curative surgical procedure. Approximately 60% of cancers recur a year after the complete resection of colorectal liver metastases, and about one-quarter of patients who undergo surveillance after initial treatment of liver metastases can undergo curative-intent treatment again . The fivThis year, the Federal Drug Agency (FDA) approved anti-PD1 pembrolizumab for first-line treatment of patients with metastatic CRC with microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) CRC based on the findings of the KEYNOTE-177 study . CompareThe lack of OS benefit with modern post-operative chemotherapy after liver resection indicates that patient selection needs improvement. One emerging area is the potential use of circulating tumor DNA (ctDNA), as it defines minimal residual disease, reflecting the existence of micro-metastases after surgical resection . FollowiIn conclusion, the management of CRC liver metastasis requires risk stratification and a multi-disciplinary input. Those patients with upfront resectable disease and low clinical risk should undergo resection followed by a risk–benefit discussion regarding adjuvant chemotherapy post-operatively or surveillance. In patients who fulfill the criteria of the EORTC 40983 study, peri-operative chemotherapy may be considered. The peri-operative chemotherapy approach may also be considered for resectable but high-risk cases to test tumor biology in order to avoid futile surgery. For unresectable liver metastases, a good response to systemic therapy may provide an opportunity for liver metastatectomy. It is important to note that the goal of conversion chemotherapy is surgical resection rather than maximal response; hence, regular imaging at close intervals is required to determine the optimal time for resection in order to reduce the risk of chemotherapy-induced liver injury. The choice of treatment regimen for conversion chemotherapy depends on tumor burden, RAS/RAF mutation status, primary tumor sidedness, exposure to previous adjuvant chemotherapy, and pre-existing toxicities. Regardless of the regimen chosen prior to resection, a total of six months of peri-operative systemic therapy is recommended. Chemotherapy based on 5FU with or without oxaliplatin is recommended in the post-operative setting, as there is no proven benefit for irinotecan, bevacizumab, or EGFR inhibitors when there is no evaluable disease. More reliable biomarkers are required in this setting to better select patients for treatment in order to optimize the current standards of care.


While there has been extensive attention to the ‘demand side’ – or use and adverse consequences, including mortality – of the ‘opioid crisis’ presently unfolding across North America, few considerations have focused on the supply side. This paper examines the supply side dynamics of this unprecedented public health phenomenon. We provide evidence for several interrelated supply-side elements that have contributed to the present public health crisis. We observe that initially, persistently high levels of prescription opioid availability and use exposed large proportions of the North American population to opioids, resulting in correspondingly high levels of medical and non-medical use . While various intervention measures to control prescription opioid availability and use have been implemented in recent years, leading to eventual reductions in opioid dispensing levels, these occurred late in the crisis’s evolution. Moreover, these supply reductions have not been met by corresponding reductions in opioid use or demand levels. These growing discrepancies between opioid demand and prescription-based sources have left major gaps in opioid supplies. In response to such supply gaps, highly potent and toxic illicit opioid products have rapidly proliferated across North America, and become a core driver of the dramatic spikes in opioid overdose fatality levels in recent years. These supply-related interrelations are corroborated by a corresponding increase in illicit opioid-related fatalities, which arose just as medical opioid supplies began to decrease in many jurisdictions. Improved analyses and understanding of the supply-side dynamics of the opioid crisis are urgently needed in order to inform future intervention and policy development. Meanwhile, the high mortality toll related to illicit, highly toxic opioid exposure requires sustained solutions, including supply-oriented measures  towards improved public health protection. North America has been experiencing an unprecedented public health crisis involving the non-medical use of opioid drugs, and consequentially, a massive toll of accidental opioid-related mortality . In 2018, there were 47,590 opioid-related deaths in the United States and a corresponding 4,614 opioid-related deaths in Canada; a threefold increase from a mere decade ago, , 4. NatuThe emergence of highly potent and toxic synthetic opioid products  has been identified as the distinct contributing factor to recent spikes in opioid-related mortality in the United States (U.S.) and Canada , 10. TheAn important challenge therefore remains in adequately understanding the ‘supply side’ dynamics of the present opioid death crisis. Illicit drug markets and related dynamics are complex phenomena involving multitudinal factors, many of which are difficult to dissect and understand yet essential for evidence-based analysis and interventions . One docSubstantial evidence supports the former perspective, namely that the recent 'wave' of toxic illicit opioid supply has emerged as an interrelated consequence within pre-existing opioid demand and supply dynamics. The broader context of the phenomenon of increasing opioid-related mortality in North America originally evolved in the early 2000s, centrally driven by substantial increases in population-level dispensing of prescription  opioids, consequent increases in non-medical use, and related morbidity and mortality . SpecifiThese distinct increases in medically sourced or diverted opioid product supplies in North America were gradually reflected in patterns of drug-related mortality. Both the U.S. and Canada began experiencing substantial increases in opioid-related deaths due to PO-related fatalities between 2002 and 2012 , 28, 29,Between 2010 and 2012, North American intervention systems began to more actively respond to the emerging PO-related crisis, enacting and/or reinforcing a multitude of interventions aimed at constraining the excessive levels of PO-related harms , 35. SucIt is around this period of increasing restraints on POs, from about 2012 onwards, when illicit and distinctly potent toxic opioid products  initially emerged to make their mark on North American drug markets, with correspondingly rising impact on drug mortality outcomes –51. MoreThere are various case studies and – with some presenting counter-factual scenarios - that provide plausibly supporting evidence for the explanatory observations offered. For example, within Canada, the province of Quebec has traditionally featured substantially lower – by up to a three-fold magnitude - levels of PO dispensing compared to all other provinces, and consequently lower population-level exposure to POs , 74, 75.In the above, we have presented evidence and potential explanatory frameworks to examine the supply side dynamics of the unprecedented opioid mortality epidemic as has been unfolding across North America. A substantial body of data suggests that the current crisis, with excessive numbers of deaths related to synthetic opioids, ought to be understood as an evolutionary consequence of prolonged high PO availability and exposure, followed by substantial restrictions in availability in the context of persistent non-medical opioid use demand at the population level. Likely, these restrictive interventions implemented over the past decade have resulted in growing PO supply gaps in spite of persistent demand, and that were compensated for through an influx of highly potent and toxic illicit opioid products. Notably, while opioid overdose fatality levels have risen steeply due to illicit opioid products in recent years, this does not necessarily imply an expansion in the volume of high-risk opioid use or overdose incidents. Rather, the likelihood of overdose incidents and fatality outcomes has likely become elevated through the use of highly potent and toxic opioid products , 48, 52.Given the relative ease of clandestine chemical production and distribution by the creative and adaptive forces behind illicit drug production and supply, it surely required little ingenuity to effectively recognize and respond to these extensive demands for opioid supply, especially in regions of North America where demand for prescription opioid products for non-medical use had been persistently high for long periods , 71, 72.It is becoming increasingly clear that ‘supply side’ considerations are not only essential for our understanding of the present opioid crisis, but also essential for informing effective interventions and solutions delivered on the ground. Concretely, while multiple, extensive ‘demand side’ interventions targeting non-medical opioid use and its adverse health risks  have been implemented and expanded, and there is substantial room for further scale-up and select additional intervention programming (e.g., ED-based pharmacotherapy treatment provision or referral , 85, theHowever, improved empirical understanding of the causal supply dynamics and structures driving the present opioid mortality crisis, and of non-medical drug supply mechanics more generally, are urgently required – if for nothing else than to better prevent such tragedies in the future. An estimated 250,000 lives lost to opioids, and historic reversals in population-level life expectancy for an entire generation of North Americans in barely a decade should provide more than good and sufficient reason for doing so.


It convened a multidisciplinary panel of experts, including research scientists, clinicians, bioinformaticians, laboratory biologists, biostatisticians, and EQA experts. A themed discussion was conducted on EQA strategies towards such assays during the symposium. This article describes the logistical challenges identified and summarizes the opinions and recommendations derived from these discussions, which may inform the development of an inaugural EQA program for NGS HIVDR in the near future.Next-generation sequencing (NGS) is likely to become the new standard method for HIV drug resistance (HIVDR) genotyping. Despite the significant advances in the development of wet-lab protocols and bioinformatic data processing pipelines, one often-missing critical component of an NGS HIVDR assay for clinical use is external quality assessment (EQA). EQA is essential for ensuring assay consistency and laboratory competency in performing routine biomedical assays, and the rollout of NGS HIVDR tests in clinical practice will require an EQA. In September 2019, the 2 External quality assessment (EQA), also referred to as an external quality assessment scheme (EQAS), plays a vital role in assuring that a laboratory performs biomedical assays competently . EQA is Conventional EQA relies on three main approaches: proficiency testing (PT), rechecking/retesting, and on-site evaluation . By leveFor decades, Sanger sequencing (SS) has been applied as the standard HIV drug resistance (HIVDR) genotyping method for research, surveillance, and patient care purposes . SeveralThe VQA HIVDR panels include HIV-positive plasma from donors with HIV or virus stocks derived from the expansion of HIV positive specimens in cell culture, diluted in a plasma matrix. PT specimens are first characterized for viral load (VL) and HIV DR-associated mutations (DRMs) using commercially available assays. The five-specimen panels are then distributed to the designated client laboratories, where they are genotyped for HIVDR using commercial and/or in-house-developed SS assays. All laboratory data derived from such tests are then submitted and compiled. A well-defined scoring algorithm is then used to compare the sequences from different laboratories to a group consensus, and the EQA assessment results are then communicated to the laboratories and relevant requesting agencies directly ,14. SimiWhile SS has been considered the “gold standard” for HIVDR genotyping, its intrinsic limitations, such as inconsistent detection of minority resistance variants (MRVs), necessitate the development of new HIVDR tests with improved sensitivity ,16,17. And International Symposium on NGS HIVDR held in Winnipeg, Canada, in September 2019. This article summarizes the proceedings from the discussions specifically on the logistical challenges and considerations for establishing an EQA program for NGS HIVDR assays.While it is well-appreciated that the generalized adoption of NGS HIVDR assays requires fully validated, standard operating procedures (SOPs) for sample processing and a sophisticated bioinformatics pipeline for effective data analysis, one essential but still missing aspect of ensuring assay consistency and reliability is an EQA program that functions . NGS HIVRecognizably, EQA for NGS HIVDR testing is a new field for which limited knowledge and experience are available currently, and extensive research and development efforts are still required. An operational EQAP that executes such EQA functionalities has yet to be established. Most of the research efforts in this regard have been devoted to the development of effective data assessment and scoring criteria for the evaluation of laboratory competence in performing such assays ,22,24. HLike EQAPs for other biomedical assays, the operation of an EQAP for NGS HIVDR testing may be divided into six main task areas, including (1) organization and administration, (2) laboratory recruitment, (3) reference material preparation and distribution, (4) data collection, (5) data assessment, and (6) EQA reporting . AccordiIt is noteworthy that, while the NIAID VQA program is taken as an exemplar EQAP for SS-based HIVDR assays in As an exemplar “disruptive” technology, NGS can revolutionize the conventional SS-based HIVDR genotyping practice and can enable sensitive and quantitative MRV detection. Many commercial and in-house-developed NGS HIVDR assays have been developed together with sophisticated bioinformatics pipelines. Meanwhile, the gradual cost reductions for both NGS instruments and related consumables have converted NGS from a high-end research tool into an affordable and accessible technology for general HIVDR laboratories. NGS may soon become the new standard for HIVDR testing in research and surveillance, as well as clinical monitoring purposes. Therefore, appropriate EQAPs will become imperative for ensuring the quality of data from the laboratories performing such assays. Due to the uniqueness of NGS HIVDR assays and the complexity of data derived from such tests, the existing EQA strategies and EQAPs targeting SS-based HIVDR genotyping are not optimal for these new assays. Technical and logistical challenges involved in the development and implementation of NGS-specific EQAPs remain to be resolved and require additional research.


The processes of recurrence and metastasis, through which cancer relapses locally or spreads to distant sites in the body, accounts for more than 90% of cancer-related deaths. At present there are very few treatment options for patients at this stage of their disease. The main obstacle to successfully treat advanced cancer is the cells' ability to change in ways that make them resistant to treatment. Understanding the cellular mechanisms that mediate this cancer cell plasticity may lead to improved patient survival. Epigenetic reprogramming, together with tumor microenvironment, drives such dynamic mechanisms favoring tumor heterogeneity, and cancer cell plasticity. In addition, the development of new approaches that can report on cancer plasticity in their native environment have profound implications for studying cancer biology and monitoring tumor progression. We herein provide an overview of recent advancements in understanding the mechanisms regulating cell plasticity and current strategies for their monitoring and therapy management. Tumor heterogeneity can be inter-tumoral, if genetic variations are found among different patients with tumors of the same type, or intra-tumoral, involving different cancer cells in the same tumor. In particular, intra-tumor heterogeneity can be caused by genetic variation, modulation in the expression of a gene, transition among cellular states or environmental changes . Thus, iInitially, two models were proposed to justify intra-tumor heterogeneity: the “clonal evolution” model and the “cancer stem-like cell” (CSC) model. The first contemplates differences among cancer cells due to stochastic alterations in genes; according to this theory, clones which gain a growth advantage are selected over time , 4. The Solid tumors are consisted of several sub-clonal cells populations, which compete in a Darwinian manner under the selective pressures of endogenous and exogenous factors, leading to the clonal evolution of a dominant subclone that will characterize the tumor's molecular landscape. Hence, it will be highly heterogeneous and will dynamically change during the disease progression, so longitudinal sampling is essential to define therapeutic strategies.Currently, cancer molecular profile is evaluated through “solid biopsies” from primary tumor or metastatic nodule; however, this approach has several issues: (i) biopsies are not representative of the whole tumor mass; (ii) often tumor site is not accessible; (iii) frequently, biopsies cannot be serially performed; (iv) each metastasis could have a different genomic landscape; (v) finally, therapeutic selective pressure has to be considered too –28. To oThe rapid turnover of cancer cells results in the constant release into the bloodstream of: (i) cell-free circulating tumor DNA (ctDNA); (ii) tumor derived RNA (predominantly micro-RNAs and long-non-coding-RNA) ; (iii) cCTCs are (probably) intravasated or passively spread from the primary and/or secondary tumor sites into the bloodstream, and could be responsible for the beginning of distant metastases.9 blood cells), especially in early cancer stage, and can vary between cancer types  technologies such as density gradient stratification, membrane filtration, photoacoustic detection, dielectric mobility, and microfluidic separation are based on CTCs physical properties  that are different from those of other blood cells , 38. HowFinally, innovative developed approaches to CTCs/CSCs isolation are based on: (v) CTCs functional features such as protein secretion and cell migration  that allow the attachment of these cells to synthetic substrates co-treated with specific matching molecules ; (vi) naDespite different approaches, in our opinion none of them completely satisfy the necessary requirements since low purity, loss of CTCs, and a narrow detection range still need to be tackled.in-vitro and in in-vivo models have been created and novel approaches have been used to study this interaction and its remodeling  and capable to form tumors when injected into immune deficient mice  is the most frequent and deadly glial tumor ; it is min-vitro studies have shown that GSCs preserve their capability for recapitulating their primary heterogeneity also after many cell divisions, and temozolomide (TMZ) does not influence this capacity  and poor prognosis  in the metastatic stage –146. Aggin vitro models demonstrated that melanoma cells are able to form perfusable, vasculogenic-like channels, a biological phenomenon called vasculogenic mimicry (VM) (3D cry (VM) . The trecry (VM) , thus pocry (VM) .On the other hand, a large number of molecular studies jointly revealed a strong stem signature in aggressive melanoma, with still unknown practical significance –152. In Several authors have demonstrated how it is possible to isolate CSCs in most solid malignancies. However, several aspects and molecular features regarding cell stemness still remain uncovered; this means that even if most markers across different cancer are the same , a commoFuture research studies will be needed in order to improve our understanding of the complex phenomenon of cancer cell plasticity. The recent insights on the role of plasticity in cancer progression and relapse highlights the need to develop new and combinatorial therapies, that aim to: (i) inhibit specific cell markers; (ii) interfere with stemness and EMT signaling pathways; (iii) affect also components of the tumor microenvironment.GF and AN wrote the paper. CS conceived the idea, supervised, and edited the manuscript. All authors discussed and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Spouses are commonly involved in their partners’ diabetes management by supporting and regulating  their diet. Little is known, however, about what characteristics are associated with how and how often spouses are involved in this context. This study examined whether sociodemographic and marital characteristics helped explain some of the variability in diet-related spousal involvement in promoting a partner’s adherence to a diabetes diet; specifically, whether gender, race/ethnicity, marital quality, and marital length were related to the frequency of spousal engagement in health-related social support and two types of health-related social control. Gender and race/ethnicity were examined as exploratory moderators of the associations between marital characteristics and spousal involvement. Data from two different data sets of older adults (55+ years) whose partners had type 2 diabetes were examined among four racial/ethnic groups . Regression analyses that controlled for patients’ co-morbid health conditions revealed gender and racial/ethnic differences in the frequency of spousal involvement. In addition, marital quality was related to the frequency of support and positive forms of social control among most participants, particularly African American spouses. No associations between marital length and any type of spousal involvement were found, nor were there any gender differences in any of these associations. These findings provide insight into the importance of sociodemographic characteristics and marital quality in understanding spousal involvement in a partner’s diabetes management.


Circular RNAs (circRNAs) are endogenous, single-stranded, most frequently non-coding RNA (ncRNA) molecules that play a significant role in gene expression regulation. Circular RNAs can affect microRNA functionality, interact with RNA-binding proteins (RBPs), translate proteins by themselves, and directly or indirectly modulate gene expression during different cellular processes. The affected expression of circRNAs, as well as their targets, can trigger a cascade of events in the genetic regulatory network causing pathological conditions. Recent studies have shown that altered circular RNA expression patterns could be used as biomarkers in psychiatric diseases, including schizophrenia (SZ); moreover, circular RNAs together with other cell molecules could provide new insight into mechanisms of this disorder. In this review, we focus on the role of circular RNAs in the pathogenesis of SZ and analyze their biomarker and therapeutic potential in this disorder.  The rapid development of novel DNA and RNA sequencing technologies has shed light on the mechanisms of multifactorial disorders, including their probability, origin, development, and pathogenesis ,2,3.Schizophrenia (SZ) is a serious psychiatric disorder that is caused by both environmental and molecular factors . This diIn 2020, the number of published studies aimed at the molecular portrait of SZ has exceeded tens of thousands. However, a significant number of genes, molecular mechanisms, and pathways that are involved in SZ origin and pathogenesis still remain unknown or unclear . UnderstGenome-wide association studies (GWASs) have described only approximately 200 genome SNPs’ susceptibility loci for SZ . Recent It has long been considered that non-coding RNAs (ncRNAs), initially found in eukaryotes, are nothing more than non-functional “junk” RNAs accumulated during the long evolutionary process. Nowadays, many studies have shown that a significant part of this genomic “dark matter” could affect RNA silencing and post-transcriptional regulation of gene expression. As a result of recent advances in high throughput sequencing technologies, thousands of transcribed non-coding RNAs have been described in the vast majority of eukaryotic organisms . These mCircular RNAs (circRNAs) are most frequently non-coding, endogenous, single-stranded RNA molecules that play an important role in cell life. CircRNAs are covalently closed RNA molecules and often originate from protein-coding genes. Previously, it was thought that circular RNAs result from non-functional splicing errors that sometimes occur during the post-transcriptional processes in cells . CurrentRecent studies have shown a significant impact of circRNAs on the origin and development of SZ, as well as an important role of circRNAs in other non-coding and protein-coding gene expression regulation . RecentlMolecular genetic data for circRNAs’ activity in SZ remain limited, despite significant progress made in functional and prediction analysis for this type of RNA ,24,25. TOne analysis of dorsolateral prefrontal cortex (DLPFC) of postmortem healthy and schizophrenic human brains showed significant changes in circRNA expression. A comparison of schizophrenic individuals and healthy controls resulted in the identification of 574 differentially expressed circRNAs, and more than two-thirds of them  were downregulated in SZ. Most importantly, the vast majority of these differentially expressed circular RNAs had miRNA targets and were considered to be potential sponges for microRNAs regulating their activity in the brain . HoweverIt has been also shown that neuronal-enriched circHomer1a  apparently played an important role in cognition processes, as its expression was downregulated in SZ postmortem prefrontal cortex (PFC) area of the human brain, while an intronic circCUL4A  was significantly upregulated .PLEKHA2) was significantly upregulated in peripheral blood mononuclear cells transcriptome ..126].The importance of research and development advances in the molecular therapy for SZ cannot be overestimated, since SZ is the most common variant of psychiatric disorders with cognitive and sensory symptoms and a prominent hereditary component. We suppose that future studies should focus on a deep search for prognostic biomarkers based on genomic, transcriptomic, and epigenetic data and focus on the development of molecular tools and delivery systems to target tissues and organs.


Even though the thymus is exquisitely sensitive to acute insults like infection, shock, or common cancer therapies such as cytoreductive chemo- or radiation-therapy, it also has a remarkable capacity for repair. This phenomenon of endogenous thymic regeneration has been known for longer even than its primary function to generate T cells, however, the underlying mechanisms controlling the process have been largely unstudied. Although there is likely continual thymic involution and regeneration in response to stress and infection in otherwise healthy people, acute and profound thymic damage such as that caused by common cancer cytoreductive therapies or the conditioning regimes as part of hematopoietic cell transplantation (HCT), leads to prolonged T cell deficiency; precipitating high morbidity and mortality from opportunistic infections and may even facilitate cancer relapse. Furthermore, this capacity for regeneration declines with age as a function of thymic involution; which even at steady state leads to reduced capacity to respond to new pathogens, vaccines, and immunotherapy. Consequently, there is a real clinical need for strategies that can boost thymic function and enhance T cell immunity. One approach to the development of such therapies is to exploit the processes of endogenous thymic regeneration into novel pharmacologic strategies to boost T cell reconstitution in clinical settings of immune depletion such as HCT. In this review, we will highlight recent work that has revealed the mechanisms by which the thymus is capable of repairing itself and how this knowledge is being used to develop novel therapies to boost immune function. Generation of a diverse but tolerant T cell repertoire, which is critical for adaptive immune function, is dependent on the development and maturation of T cell precursors in the thymus. The process of T cell development is reliant on the interactions with the stromal microenvironment, comprised of highly specialized thymic epithelial cells (TECs), endothelial cells (ECs), mesenchymal cells, dendritic cells (DCs) and macrophages. However, despite its importance for generating and maintaining T cells, thymic function is extremely sensitive to acute damage such as that caused by everyday insults like stress and infection, as well as more profound injuries such as that caused by cytoreductive therapies. Nevertheless, the thymus also has a remarkable capacity to regenerate itself from these acute injuries , 2, althGiven the poor outcomes that are associated with deficient T cell immunity, there is a clear clinical need for therapies that can boost thymic function in periods of acute injury or reverse age-related thymic involution. In this review, we will outline what we know about how the thymus is damaged during different modalities of insult and the work that has been done to develop therapeutic strategies to boost thymic function; either ensuing acute insult such as following HCT, or in aged individuals to boost responses to vaccines or immunotherapy .Thymic involution is a routine response to acute insult incurred by multiple triggers including emotional and physical distress, malnutrition, and opportunistic bacterial and viral infections. These can be modeled using approaches such as synthetic corticosteroid treatment, such as dexamethasone ; nutrienContinuous export of naïve cells from the thymus, or recent thymic emigrants (RTEs), is essential for effective immune response to acute and chronic infections , 17. HowStreptococcus suis infection promotes thymic atrophy specifically by inducing increased activation of pro-apoptotic pathways and apoptotic cell death in thymocytes  mice , and redhi TECs , a member of the IL-10 family that typically targets non-hematopoietic cells such as epithelial cells and fibroblasts . In thisSimilar to other tissues , IL-22 iBmpr1a and Bmpr2 were identified on TEC populations, with a higher expression of the non-redundant Bmpr2 on cTECs , is a fibroblast growth factor and acts as a mitogen targeting TECs, inducing epithelial proliferation in several organs –108. In pathways , 108, prpathways , and Fgf in TECs . KGF mod in TECs .KGF−/− mice demonstrated the critical role of KGF on thymus function and immune reconstitution after insult, modeled by both syngeneic and allogeneic bone marrow transplant  equating to an enhanced number of engraftment niches, and increased TEC proliferation . Althougansplant . The samansplant . Howeveransplant , highlig− mTECs to Aire+ mTECs which subsequently present MHC-II peptides that drive the elimination of self-reactive T cells during negative selection (Receptor activator of nuclear factor kappa-B ligand (RANKL), a member of the Tumor necrosis factor (TNF) superfamily, is implicated in multiple physiological roles in the periphery, primarily in bone biology . RANKL helection . RANKL ielection , 118–120election . Importaelection .The prominent role of RANKL in mTEC biology points to the ability of RANKL to modulate thymic regeneration and output. RANKL administration shows an enhancement of thymic function after bone marrow transplantation by boosting TEC subsets, including TEC progenitor niches . MoreoveGiven the fact that Cell development requires the input of hematopoietic progenitors, and the fact that the supply of those progenitors is severely limited after acute injury , 127, on+ progenitors in the BM . IL-7 has a non-redundant role as a survival molecule in lymphoid tissues in mice and humans, most importantly in the thymus where IL-7 is critical for appropriate thymocyte development. An elegant study by Shitara et al.  showed t T cells , an abse T cells , and dis T cells . MoreoveN3 cells , 140, esIL-7 is produced primarily by non-hematopoietic stromal cells such as TECs and signals by binding to the heterodimeric IL-7 receptor (IL-7R), comprised of IL-7R?  and the cytokine receptor γ-chain , and induces an anti-apoptotic pro-survival signaling cascade via the activation of phosphoinositide 3-kinase (PI3K) and the Janus Kinase (JAK)-STAT pathway. The expression of IL-7R? on developing thymocytes occurs in a cyclical pattern, with expression seemingly dependent on the fluctuating need for IL-7 signaling at different stages of thymocyte maturation , demonstThese critical roles of IL-7 in both thymocyte development and in peripheral T cell homeostasis  reveal IGiven the impact of sex steroids on thymic function , 150, suIn addition to sex steroids, several other hormones and metabolic components have been implicated in thymic function and their modulation has been shown to improve thymopoiesis, particularly in the aged. Administration of the appetite stimulating hormone Ghrelin led to improved thymic cellularity and thymic output in aged mice , and simex vivo culture systems that use Notch-1 stimulation of hematopoietic precursor cells has been demonstrated . TEPC have been successfully isolated from fetal thymi and induced to generate a new thymus in athymic recipients –182, andde novo formation of whole organs ex vivo that can be transplanted into patients as required  and others as yet unpublished.


Abnormal DNA methylation has been described in human inflammatory conditions of the gastrointestinal tract, such as inflammatory bowel disease (IBD). As other complex diseases, IBD results from the balance between genetic predisposition and environmental exposures. As such, DNA methylation may be the consequence  of both, genetic susceptibility variants and/or environmental signals such as cytokine exposure. We attempted to discern between these two non-excluding possibilities by performing a combined analysis of published DNA methylation data in intestinal mucosal cells of IBD and control samples. We identified abnormal DNA methylation at different levels: deviation from mean methylation signals at site and region levels, and differential variability. A fraction of such changes is associated with genetic polymorphisms linked to IBD susceptibility. In addition, by comparing with another intestinal inflammatory condition  we propose that aberrant DNA methylation can also be the result of unspecific processes such as chronic inflammation. Our characterization suggests that IBD methylomes combine intrinsic and extrinsic responses in intestinal mucosal cells, and could point to knowledge-based biomarkers of IBD detection and progression.  Inflammatory bowel disease (IBD) comprises Crohn’s disease (CD) and Ulcerative Colitis (UC), two chronic and progressive inflammatory conditions of the gastrointestinal (GI) tract that affect 2.2 million people in Europe and 1.4 million in United States . In CD, While genetics explains a fraction of inheritance of IBD  , environExplaining the origin of DNA methylation changes in IBD may be of interest when exploiting their potential as biomarkers. Currently, the most used biomarkers for IBD are C-Reactive Protein and Calprotectin, although they are not specific for inflammation of intestinal origin, limiting their clinical use . InsteadTo identify DNA methylation changes in cells of the intestinal mucosa associated with IBD, we reanalysed bead-array methylation data from different datasets  & 2. To After filtering (see Methods), we tested for the association between IBD and DNA methylation at 392810 CpG sites (81 control and 204 IBD patients) using a linear model. In such a model, we adjusted for sex, age, dataset, and surrogate variables identified during data preprocessing . To account for statistical inflation, we used criteria of effect size (change in mean methylation of at least 10% between controls and IBD) and FDR-adjusted p value <0.05. Using these criteria, we identified 4205 differentially methylated positions (DMPs), out of which 436 were hypo- and 3769 were hypermethylated in IBD  and S1. A subset of DMPs mapped close to each other, suggesting a non-random association with particular genomic loci. To explore this observation, we performed a region-level analysis in the same combined dataset. This led to the identification of 55 differentially methylated regions (DMRs), 31 hypo and 24 hyper methylated in IBD  and S2. In addition, to mean methylation differences at the probe and region levels , methylation variation has been associated with disease and cancer susceptibility . To explIn summary, the intestinal mucosa of IBD displays large non-random methylome abnormalities characterized by high variability, but also by absolute changes in mean DNA methylation at particular loci.DMPs distinguishing IBD from control tissues were assessed for genomic distribution, in terms of gene-centric and CpG island (CGI)-centric context. DMPs were relatively absent from CGIs, gene promoters, or the vicinity of transcription start sites (TSS) -c). InstOverall, abnormal DNA methylation in IBD is relatively absent from CGIs. At the biological level, DNA methylation changes are enriched in inflammation-related pathways. Such changes may occur downstream of cytokine signalling. Alternatively, they may represent early changes linked to genetic susceptibility.JAK3, KRT8, and HLA genes, confirming the findings of previous studies [DNA methylation may represent an intermediary between genotype and disease susceptibility, and such genetic influences on DNA methylation within a defined genomic context are known as methylation quantitative trait loci (mQTLs). Among differentially methylated genes with a significant genetic association, we found  studies ,29–31. M studies ,32. Of nADAM15, SMAD3, RNF186, and IRF5, respectively. Briefly, we found that SNP-CpG pairs overlap regulatory loci, discernible by H3K27ac histone marks and the presence of a CpG island (in the case of ADAM15).We also tested the overlap between IBD-DMPs and CpGs participating in blood mQTLs as defined by McRae et al. . AlthougThese findings suggest that at least a fraction of IBD abnormal methylome is in direct relationship with upstream genetic susceptibility variants.As the IBD methylome is both, related to inflammation and genetic susceptibility, it may also be largely unspecific. We therefore chose coeliac disease (CeD), a chronic inflammatory condition of the GI tract with a well-characterized genetic component, to get further insight into methylome specificity. In addition, DNA methylation data for epithelial and immune components of CeD were analysed separately . When weIn summary, there is a significant overlap in DNA methylation changes associated with IBD and CeD, including the HLA region.IBD is a complex pathology with a wide range of clinical trajectories. Despite such heterogeneity, we show here that non-random changes in DNA methylation associated with IBD are robust to main clinical parameters and consistent across several studies.There are intrinsic limitations of DNA methylation analyses relative to standard genetic profiling, such as confounding, reverse causation, and cellular heterogeneity ,21. InteDifferent characteristics of DNA methylation, such as its relative stability, make this mark an ideal sensor of disease risk and progression. Indeed, several studies have been able to use DNA methylation as a marker of IBD in blood samples ,35,36. BIn agreement with the largest study selected for our meta-analysis , genes nIn our study, we identified 4532 CpG sites that simultaneously display differential variation and differential methylation (DVMCs) associated with IBD. In most cases, IBD mucosal cells displayed higher variation at those DVMCs relative to control cells. Although this hypervariability may represent cellular variation , it has been suggested that a stochastic component of methylation variation at certain genomic locations may characterize pathological conditions ,38. Of nJAK3, KRT8, HLA genes, all of them associated with a role in IBD pathogenesis [In terms of genomic distribution, we found that DMPs are relatively absent from CGIs. Instead, they could be associated with other regulatory regions such as enhancers, for example, in association with SNPs. Indeed, GWAS performed in multiple complex diseases have shown that SNPs of susceptibility are enriched in enhancer regions, and DNA methylation could be an intermediary in this process ,44. Illuogenesis –49.SMAD3 has been reported to be allele-specific and to regulate the expression of the gene [ADAM15, RNF186, and IRF5.The presence of CpGs participating in both IBD-DMPs as well as mQTLs suggests that a considerable number of the DMPs identified in our metanalysis are regulated by SNP-genotypes in cis. However, very few of these are associated with IBD. This observation points to the possibility that, although fine-mapping aims to identify the SNPs responsible for the disease-association, other nearby SNPs in strong linkage disequilibrium could be the ones implicated in the mQTLs, drawing the methylation patterns reported. Additionally, we describe a picture in which most of the IBD-DMPs seem to be genotype-independent, since they do not participate in any mQTL, at least in blood. Regarding the SNPs associated to IBD as well as to the methylation levels of IBD-DMPs, it is interesting that the methylation of a CpG island 4 kb upstream of the cg24032190-DMP identified in the first intron of the gene . TherefoRegarding coeliac epithelial DMPs also found altered in IBD, it is important to note that most of them were located in the HLA region. This locus presents strong linkage disequilibrium and encodes a number of genes related to immune response and immune regulation through self-recognition ,51, and Our findings illustrate an aberrant DNA methylation landscape in IBD, independent of IBD subtype and other clinical and pathological features. The enrichment of abnormal DNA methylation in inflammatory pathways and genes suggests a direct role for this mark downstream of cytokine signalling and/or a risk genotype. Such a landscape may be a more general indicator of intestinal chronic inflammation, although evidence from purified epithelial cells suggests that those changes are not primarily explained by an inflammatory status . Such efDataset selection criteria included: methylome data obtained from intestinal mucosa , availability of healthy controls and IBD samples , in data obtained using Human Infinium Bead Arrays (Illumina’s HM450 or EPIC arrays), an established technology to detect DNA methylation . Tables All methylation data and sample information were downloaded from Gene Expression Omnibus (GEO) and Array Express public repositories, and analysed using R/Bioconductor packages . NormaliAfter filtering, 392810 CpG sites common to all datasets were used to identify principal components (PC) of variation and plotted using PC regression and multidimensional scaling (MDS) plots. Strong associations were observed between PCs and known variables , with age and anatomical location partially confounded by the dataset of origin. As additional quality control, DNA methylation values were used to predict age and sex and contrast with downloaded phenotype information . Sex was inferred from the median total intensity signal on XY chromosomes and permitted the identification of eight sex mismatches that were removed from the analysis. Age prediction was performed using Horvath’s coefficients , as implAs validation of our aggregated analysis, we performed independent region-level analyses to test for the association between IBD and DNA methylation in three datasets, where enough power made it possible . There was a significant overlap among those three analyses, with 905 common gene symbols . We also performed a leave-one-out cross-validation approach. To this end, we successively removed each of the six datasets of the study and performed differential methylation analysis at the probe and region levels . Two different diagrams are shown due to limitations of this visualization, but they illustrate that there is a common set of CpG sites differentially methylated across all or most datasets, and an important overlap with our final list of differentially methylated probes. Similar results were obtained when differential methylation was studied at the region level (DMRs).In addition to the obvious batch effect of the dataset of origin, DNA methylation is known to be influenced by genotype, sex, age, and cell composition. As all of these factors are potential confounders, we tried to minimize or account for their effect using different strategies. Those factors where data was available  were modelled in a linear regression. In the particular case of sex where the effect on DNA methylation is strong, we removed an important part of such effect by filtering out all probes mapping to chromosomes X and Y, as described above. The effect of genotype was addressed a posteriori, in our mQTL analyses. For all other factors , we were able to assess their association with the main components of variation before and after adjustment for latent variables identified using surrogate variable analysis (SVA) . In partAssociations were tested for 392810 CpG sites, across 285 samples (81 control and 204 IBD samples). Methylation data was modelled at the probe and region levels using a linear model with Bayesian adjustment . Sex andTo identify methylation quantitative trait loci (mQTL), single nucleotide polymorphisms (SNPs) associated with IBD risk were obtained from a fine-mapping study of IBD with single-variant resolution . Two indClick here for additional data file.