Pseudomonas aeruginosa and Klebsiella pneumoniae expressing over 55 diverse serine and metallo-beta-lactamases.QPX7728 is an ultrabroad-spectrum boronic acid beta-lactamase inhibitor, with potent inhibition of key serine and metallo-beta-lactamases being observed in biochemical assays. Microbiological studies using characterized strains were used to provide a comprehensive characterization of the spectrum of beta-lactamase inhibition by QPX7728. The MICs of multiple antibiotics administered intravenously only  and orally bioavailable antibiotics  alone and in combination with QPX7728 (4 μg/ml), as well as comparator agents, were determined against panels of laboratory strains of  Pseudomonas aeruginosa and Klebsiella pneumoniae expressing over 55 diverse serine and metallo-beta-lactamases. QPX7728 significantly enhanced the potency of antibiotics against strains expressing class A extended-spectrum beta-lactamases  and carbapenemases , consistent with the beta-lactamase inhibition demonstrated in biochemical assays. It also inhibited both plasmidic  and chromosomally encoded  class C beta-lactamases and class D enzymes, including carbapenemases, such as OXA-48 from Enterobacteriaceae and OXA enzymes from Acinetobacter baumannii (OXA-23/24/72/58). QPX7728 is also a potent inhibitor of many class B metallo-beta-lactamases . Addition of QPX7728 (4 μg/ml) reduced the MICs for a majority of the strains to the level observed for the control with the vector alone, indicative of complete beta-lactamase inhibition. The ultrabroad-spectrum beta-lactamase inhibition profile makes QPX7728 a viable candidate for further development.QPX7728 is an ultrabroad-spectrum boronic acid beta-lactamase inhibitor, with potent inhibition of key serine and metallo-beta-lactamases being observed in biochemical assays. Microbiological studies using characterized strains were used to provide a comprehensive characterization of the spectrum of beta-lactamase inhibition by QPX7728. The MICs of multiple antibiotics administered intravenously only  and orally bioavailable antibiotics  alone and in combination with QPX7728 (4 μg/ml), as well as comparator agents, were determined against panels of laboratory strains of  Acinetobacter baumannii or metallo-beta-lactamases (MBLs). While these combination products represent advances for the treatment of infections caused by pathogens that are recognized to be urgent or serious threats by the CDC, many pathogens and resistance mechanisms remain unaddressed (The development of beta-lactamase inhibitors (BLIs) in combination with beta-lactams is a powerful strategy to protect beta-lactams from beta-lactamase-mediated hydrolysis and preserve the clinical utility of this widely used group of antimicrobial agents. Four beta-lactam–BLI combination agents that were recently approved by the FDA are ceftolozane-tazobactam (approved in 2014), ceftazidime-avibactam (approved in 2015), meropenem-vaborbactam (approved in 2017), and imipenem-relebactam (approved in 2019); all of them represent significant progress in addressing serious drug-resistant Gram-negative bacterial infections (1–5–ddressed .50) values generally being in the low-nanomolar range. These biochemical results  and Klebsiella pneumoniae  expressing over 55 diverse beta-lactamases. The use of P. aeruginosa as an isogenic background facilitates the detection of the beta-lactamase activity (as an MIC increase) of low-catalytic-efficiency enzymes that rely heavily on the low permeability of the outer membrane  combinations , 18. Thimembrane . The useP. aeruginosa strains were determined  alone and in combination with QPX7728 against the panel of termined  and 2. Stermined , the MICtermined . For theQPX7728 enhanced the potency of multiple beta-lactams against all strains producing class A extended-spectrum beta-lactamases  and carbapenemases . In the absence of QPX7728, the antibiotic MIC values ranged widely, depending on the beta-lactamase, and often were >64 μg/ml. When MICs were determined in combination with QPX7728 at 4 μg/ml, the MIC values against the majority of strains were generally the same as or less than those seen for the control strains containing the vector alone (≤0.06 to 0.25 μg/ml), consistent with the complete inhibition of beta-lactamase activity by QPX7728. In the case in which QPX7728 was combined with cefpodoxime, there were several strains with class A enzymes  that showed a significant (32- to >256-fold effect) but incomplete reversion of resistance . Future studies with purified enzymes will assess whether this can be explained by the decreased activity of QPX7728 against these beta-lactamases when it is tested with the labile agent cefpodoxime  to 3. to P. aeruginosa, A. baumannii, and Enterobacter cloacae) and that had a wide range of MIC values for the antibiotics alone (often >64 μg/ml); inhibition often reduced the MICs to the level observed with the vector-only control strain, indicating the complete inhibition of various class C enzymes  and chromosomally encoded   . The higher MICs of ceftazidime and ceftolozane with QPX7728 (4 μg/ml) for the NDM-producing clones (32 μg/ml) suggest that NDM has very potent activity in the hydrolysis of ceftazidime and ceftolozane, with consequent high MIC values and a ca. 4-fold lower inhibitory potency of QPX7728 against NDM than against VIM   . As expelass B3)  to 3, ino 55 nM) . QPX7728ctively) . When teBacteroides fragilis. QPX7728 also demonstrated inhibitory activity against some but not all MBLs from the IMP subclass; it was more active in antibiotic potentiation activity against the strains producing IMP-1/4/13/15/19 than against the strain producing IMP-26, in good agreement with the findings of previous biochemical experiments   . No actictively) . In the Analysis of the crystal structure of the QPX7728/NDM-1 complex  suggestsIn order to elucidate the structural basis for the lack of QPX7728 activity against beta-lactamase L1, we modeled the putative QPX7728/L1 complex by docking. While we found a bound ligand position consistent with coordination of the two metal ions in a manner closely resembling that seen in the available X-ray structures for other MBLs , QPX7728P. aeruginosa and K. pneumoniae producing various beta-lactamases. QPX7728 inhibits serine class A ESBLs , carbapenemases , and class C beta-lactamases, both plasmidic  and chromosomally encoded . QPX7728 also inhibits class D beta-lactamases, such as oxacillinase OXA-10 and carbapenemases (OXA-48 and OXA-23/40/72/58) from Enterobacteriaceae and A. baumannii, respectively. Of note, metallo-beta-lactamase class B, subclass B1, enzymes  are inhibited by QPX7728.QPX7728 is a new boronate BLI with an unprecedented spectrum of inhibition of beta-lactamases, including the major representatives of serine and metallo-beta-lactamases from all molecular classes found in infections defined to be urgent and serious threats to public health. Microbiological experiments with an extensive collection of engineered strains with beta-lactamases significantly expanded the information available on the beta-lactamase inhibition profile of QPX7728. They also demonstrated that the broad-spectrum inhibitory activity of QPX7728 observed in cell-free biochemical experiments using purified enzymes translates into enhancement of the potency of multiple i.v. administered  and orally bioavailable  beta-lactams against engineered strains of Enterobacteriaceae  was described previously (P. aeruginosa PAM1154 and K. pneumoniae KPM1001 (ATCC 43816) by transformation. PAM1154 lacks MexAB-OprM due to disruption of the oprM gene. The use of P. aeruginosa as an isogenic background facilitates the detection of beta-lactamase activity (as an MIC increase) of the low-catalytic-efficiency enzymes that rely heavily on the low permeability of the outer membrane. The use of a strain that lacks efflux pumps ensures no interference from efflux in interpreting the results. KPM1001 is a wild-type strain of K. pneumoniae containing functional genes encoding efflux pumps, such as AcrAB-TolC, and major porins OmpK35 and OmpK36.The panels of engineered isogenic strains of eviously ; the comThe bacterial isolates were subjected to broth microdilution susceptibility testing, performed according to Clinical and Laboratory Standards Institute (CLSI) methods , using p


Methamphetamine (MA) is a highly addictive psychomotor stimulant drug. In recent years, MA use has increased exponentially on a global scale, with the number of MA-involved deaths reaching epidemic proportions. There is no approved pharmacotherapy for treating MA use disorder, and we know relatively little regarding the neurobiological determinants of vulnerability to this disease. Extracellular signal-regulated kinase (ERK) is an important signaling molecule implicated in the long-lasting neuroadaptations purported to underlie the development of substance use disorders, but the role for this kinase in the propensity to develop addiction, particularly MA use disorder, is uncharacterized. In a previous MA-induced place-conditioning study of C57BL/6J mice, we characterized mice as MA-preferring, -neutral, or -avoiding and collected tissue from the medial prefrontal cortex (mPFC). Using immunoblotting, we determined that elevated phosphorylated ERK expression within the medial prefrontal cortex (mPFC) is a biochemical correlate of the affective valence of MA in a population of C57BL/6J mice. We confirmed the functional relevance for mPFC ERK activation for MA-induced place-preference via site-directed infusion of the MEK inhibitor U0126. By contrast, ERK inhibition did not have any effect upon MA-induced locomotion or its sensitization upon repeated MA treatment. Through studies of transgenic mice with alanine point mutations on T1123/S1126 of mGlu5 that disrupt ERK-dependent phosphorylation of the receptor, we discovered that ERK-dependent mGlu5 phosphorylation normally suppresses MA-induced conditioned place-preference (MA-CPP), but is necessary for this drug’s reinforcing properties. If relevant to humans, the present results implicate individual differences in the capacity of MA-associated cues/contexts to hyper-activate ERK signaling within mPFC in MA Use Disorder vulnerability and pose mGlu5 as one ERK-directed target contributing to the propensity to seek out and take MA. In the wake of the opioid epidemic, the world is currently facing a psychostimulant crisis. The Drug Enforcement Agency of the United States reports a recent, major surge in the amount of methamphetamine (MA) seized by law enforcement, with a 15-fold higher supply and demand for psychostimulants than opioids world-wide . AccordiMA is an indirect dopamine agonist ,4 and prhttps://grantome.com/grant/NIH/R33-DA031441-06), MA Use Disorder is associated with lower levels of glutamatergic neurometabolites in frontal cortical regions . TThe results of the present study provide novel evidence that ERK activation within mPFC is a biomolecular response to a drug-conditioned context that drives the positive affective effects of MA. While the downstream targets of ERK phosphorylation within the mPFC driving MA-conditioned reward are unknown, mGlu5 appears to be a critical target of ERK-directed signaling regulating MA reinforcement and intake.


Research in the HIV-1 field is no exception: the challenges encountered in the development of preventive and curative anti-HIV-1 strategies may well originate in part from inadequate assumptions built on bulk technologies, highlighting the need for new perspectives. The emergence of diverse single-cell technologies set the stage for potential breakthrough discoveries, as heterogeneous processes can now be investigated with an unprecedented depth in topics as diverse as HIV-1 tropism, dynamics of the replication cycle, latency, viral reservoirs and immune control. In this review, we summarize recent advances in the HIV-1 field made possible by single-cell technologies, and contextualize their importance.The need for definitive answers probably explains our natural tendency to seek simplicity. The reductionist “bulk” approach, in which a mean behavior is attributed to a heterogeneous cell population, fulfills this need by considerably helping the conceptualization of complex biological processes. However, the limits of this methodology are becoming increasingly clear as models seek to explain biological events occurring  HIV-1 remains a major public health problem around the world. Although ART succeeds in suppressing viral replication and has had a tremendously positive impact for people living with HIV-1, it fails to eradicate the virus and restore effective anti-HIV-1 immunity: the virus persists in long-lived reservoirs, and viral rebound occurs almost invariably after cessation of therapy. HIV-1 pathogenesis is complex and diverse at multiple levels. In the absence of therapy, steady-state viremia and disease progression rate are highly variable, depending on both host and pathogen factors; the enormous diversity of circulating viral strains is a major hurdle for the development of effective vaccination and cure strategies . This in+ CD4+ T cells, in particular in ART-suppressed individuals  The extremely low frequency of HIV-1ividuals ; the larividuals ; currentividuals . (4) BioDespite these hurdles, great strides were nonetheless made using more standard methods that could be considered conceptual predecessors of newer single-cell technologies, including limiting dilutions, subpopulation partitioning by population cell sorting, digital droplet PCR (ddPCR), immunohistochemistry, conventional confocal microscopy and flow cytometry etc. While these technologies remain major research tools, their low dimensionality, poor resolution, laboriousness or low-throughput are all good reasons to complement them with newer single-cell techniques. Single-cell “multiomic” technologies play a dominant role in the “single-cell revolution,” but other cutting-edge approaches must not be overlooked. In this review, we broadly define “single-cell technologies” as any approach providing quantitative analyses reaching single-cell resolution. For convenience, we grouped these technologies in four global categories based on their key contribution to the field .+ cells is typically achieved by the detection of viral RNA (vRNA), viral DNA (vDNA) or expression of the structural protein p24. Several direct single-cell virus detection imaging methods with signal amplification were developed in the past years, including in situ PCR (in situ hybridization (FISH) techniques, including RNAflow-FISH (flow cytometry) and DNA/RNAscope (microscopy) assays  assays . This te) assays  to achieBy partitioning single cells, capturing their transcripts, and generating sequencing libraries in which the transcripts are mapped to individual cells, single-cell RNA sequencing (scRNA-Seq) and its DNA equivalent (scDNA-Seq) represent unequaled “omic” opportunities. All single-cell sequencing technologies follow the same basic principles. Cells must first be individualized by fluidic technologies, limiting dilutions or single-cell sorting flow cytometry . Single High-throughput cell phenotyping for protein markers is most frequently performed by polychromatic flow cytometry or mass cytometry . In addition to antibodies, polychromatic flow cytometry allows detection of fluorescent dyes and benefits from a large pool of commercially available reagents. However, overlapping fluorescence spectra are a recurrent problem that requires complex compensation. Conversely, mass spectrometry by time-of-flight relies on metal-conjugated antibodies requiring essentially no compensation. Limitations of this technology includes fewer available reagents and a lower acquisition throughput than fluorescent cytometry. Current high-end platforms are designed to achieve high dimensionality . While technical considerations usually slightly reduce the number of channels useable simultaneously compared to the limit of parameters available, the depth of single-cell profiling achieved is still remarkable . For botMicroscopy is often overlooked as a single-cell technology probably because of its traditionally low throughput and semi-quantitative nature. High-resolution time-lapse imaging technologies now provide unprecedented spatial information of unique HIV-1 infected cells in near real-time . AutomatThese recent developments helped the HIV-1 field take the leap toward single-cell technologies. Here, we discuss the contribution of these various new single-cell technologies in the context of HIV-1 research and review concrete examples of their applications.+ T cells were quickly found to be primary targets during productive infection. The evolution of molecular biology and flow cytometry tools came with more precise characterization of cells targeted by HIV-1. The ever-improving capacity to divide cell populations into more and more refined subsets by cell sorting or column enrichment enabled the in vitro interrogation of various immune cells for either their susceptibility to HIV-1 infection or the presence of vDNA by ultrasensitive PCR methods. These approaches provided a wealth of data, with at times conflicting results that may be mostly due to technical considerations. Numerous studies identified several cell populations with high susceptibility to infection, including central memory CD4+ T cells (TCM) (+ T memory stem (TSCM) cells (REG) cells (H17 cells (FH) , CD63, and CD317 (BST-2) that all correlated with T cell activation (Better defining the nature of HIV-1 infected cells has been an active topic of research since the discovery of the virus. CD4ls (TCM) , CD4+ T M) cells , regulatG) cells ; TH17 ce17 cells , 2017, Tlls (FH) . All thetivation . Activatin vitro must be interpreted with caution. Cultured CD4+ T cells drift from their original transcriptional program, especially when exogenous biologically active molecules are applied to maintain survival (ex: IL-2) or promote infection (ex: CD4+ T cell activation by PHA or CD3/CD28 crosslinking). Such models are often a reasonable and necessary compromise because of the rarity of infected cells in people living with HIV-1. This bias can now be avoided to some extent by direct ex vivo detection of infected cells using dual HIV-1 detection by flow cytometry, as described above. Both HIVflow and RNAflow-FISH showed remarkable consistency in providing ex vivo validation of previous bulk observations such as HIV-1 enrichment in cells (1) expressing the activation-associated proteins CD25, HLA-DR, Ki67 and the inhibitory receptors TIGIT, PD-1 or CTLA-4 (+ T (TTM) cells rather than in central and effector memory ; (3) in cells of TH17, TFH and TREG polarizations  traizations . Unfortuizations , cells u+ T cells. Myeloid cells could also represent targets and/or facilitate viral dissemination although definitive in vivo confirmation of productive infection is still lacking. The best current technologies to detect HIV-1+ cells by flow cytometry call for a large number of cells difficult to obtain from blood, typically a poor source of mature myeloid cells. Microscopy studies using DNA/RNA FISH techniques are therefore better suited to address this detection challenge (+ CD21+ follicular dendritic cells (FDCs) and CD68+/CD163+ macrophages in lymph nodes of a viremic donor  and integrated HIV-1 proviruses close to nuclear pores  enabled the visualization in adherent cell lines of clusters of mixed native and fluorescently tagged Gag proteins corresponding to single virions. Combined with photoconvertible fluorescence technology, it showed the nucleation of recently membrane-associated Gag proteins rather than the existence of long-lived stable platforms . Assemblin vivo, given the heterogeneity of immune cells. One strategy involves taking transcriptional snapshots of thousands of unsynchronized single cells and reconstitute the dynamic pattern of gene expression. This approach implies a compromise as the data produced only infer temporality to allow assessment of primary cells infected with native viruses. Using RT-qPCR on single-sorted cells, investigators were able to subdivide SIV replication cycle in various stages defined by the relative presence of multiply spliced versus unspliced vRNAs  method brought molecular evidence that Tat can act as a molecular switch. By following the relative expression of dual reporters of spliced and unspliced vRNA, a novel post-transcriptional mechanism of noise suppression stabilizing the commitment of HIV-1 to its active gene expression state was discovered  (reviewed in + T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA upon LRA stimulation of primary CD4+ T cells from ART participants  agonist Bryostatin-1 preferentially reactivated TEM reservoirs whereas the PEP005 showed broader activity, including on TCM cells , in which single infected-cell are seeded in limiting dilutions among reporter cells to allow the amplification of p24, gives a minimal estimation of the size of replication-competent reservoirs. These methods offer the highest and lowest estimates of “total” and “replication-competent” viral reservoirs. New highly sensitive and specific flow cytometry-based methods based on detection of viral products  provide additional information on the competence of the reservoirs at the single-cell resolution required to associate viral or cellular features to the quantified reservoirs, with estimated VR sizes that are intermediate between standard DNA and Q-VOA quantification  can connect residual HIV-1 transcriptional activity to proviruses in ART-treated donors   . The anaesponses , reveali+ vs CD8+ T cells, + T cell clusters with considerable heterogeneity in inhibitory co-receptor and transcription factor co-expression. Some clusters of severely exhausted CD8+ T cells were found similarly enriched in people afflicted with lung cancer whereas others were differentially represented, suggesting that a common core biology of T cell exhaustion across diseases exists along with more disease-specific defects  studies exemplify the challenges of human studies as well as the potential to make optimal use of the rare cell populations isolated from precious clinical samples. CNS involvement remains a significant issue, as neurocognitive disorders occur in spite of highly effective ART and as the CNS can serve as immune sanctuary. A recent study used scRNA-Seq to define the immune cell landscape in the cerebrospinal fluid (CSF) of virologically suppressed individuals (in vivo and identifying factors with direct contribution to residual immune impairment. Clearly, further investigations will be needed to establish the extent of myeloid dysfunction during HIV-1 infection.Single-cell technologies can also facilitate in-depth studies of anatomic compartments for which sampling is quite limiting in humans. Until recently, the paradigms of protection against HIV-1 largely relied on peripheral blood studies, although virus replication occurs mainly in lymphoid tissues. An approach combining high dimensional mass cytometry, scRNA-Seq, and ATAC-Seq enabled transcriptional and epigenetic-profiling of a novel extrafollicular LN-resident CD69l subset , with noividuals . They foin vitro (The best hope to control the HIV-1 pandemic probably resides in prophylactic vaccines. Although none of the attempted vaccine trials led to a definitive breakthrough, correlates of protection could be identified in human studies (reviewed in in vitro , 2014. Tin vitro , and to in vitro . Furtherin vitro . Deploym+ or CD8+ T cell studies currently appear to lag behind in the HIV field compared to B cell studies and to investigations of cellular immunity in other diseases, the conceptual and technical frameworks now appear to be mature for such cutting-edge investigations in advanced HIV-1 vaccine immunomonitoring.Single-cell transcriptomics and epigenomics strategies, in some cases combined with TCR sequencing, have also been successfully applied to study the T cell response in human diseases . Althoug+ T cell polyfunctionality as a new correlate of vaccine efficacy in the RV144 HIV vaccine trial and delineated qualitative differences in CD4+ T cell responses between different HIV-1 vaccine regimens (Standard biostatistical approaches often failed to appreciate differences in the quality of the immune vaccine response because they rely on expected biological outcome and consequently fail to grasp the inherent complexity of the multi-component nature of immunity. Single-cell technologies have been used to provide high throughput multi-dimensional data, but the lack of computational tools has in many instances led to suboptimal exploitation of the wealth of data generated. To overcome these limitations, analytical frameworks harnessing the full extent of single-cell technologies were tested on multidimensional datasets . For exaregimens . These fregimens . Such stSingle-cell analysis is not a novel concept. However, recently developed technologies are now bringing the resolution and depth of single-cell investigations to the next level in every field of biology. Immunology and cancer are fields that pioneered the use of these new tools. HIV-1 research contributed to the development of many technologies in the past, for example single-cell microscopy applied to investigation of the viral replication cycle. Yet, compared to other areas of biomedical research, the field appears to currently lags behind in fully adopting newer high throughput single-cell technologies, this despite the fact that the very nature of HIV-1 biology would extensively benefit from these tools. The ball is now in the HIV-1 researchers’ court.GS and MD performed the literature review and wrote the manuscript. DK edited the manuscript and provided supervision. All authors read and approved the final version of 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.


Mycobacterium tuberculosis (Mtb) infections in high-prevalence settings is unfeasible due to unclear treatment durability, drug toxicity, logistical constraints related to directly observed treatment strategy (DOTS) and the lengthy treatment protocols. Together, these factors promote non-adherence, contributing to relapse and establishment of drug-resistant Mtb strains. Although antibiotic treatment of drug-susceptible Mtb is generally effective, drug-resistant TB has a treatment efficacy below 50% and can, in a proportion, develop into progressive, untreatable disease. Other immune compromising co-infections and/or co-morbidities require more complex prevention/treatment approaches, posing huge financial burdens to national health services. Novel TB treatment strategies, such as host-directed therapeutics, are required to complement pathogen-targeted approaches. Pre-clinical studies have highlighted promising candidates that enhance endogenous pathways and/or limit destructive host responses. This review discusses promising pre-clinical candidates and forerunning compounds at advanced stages of clinical investigation in TB host-directed therapeutic (HDT) efficacy trials. Such approaches are rationalized to improve outcome in TB and shorten treatment strategies.Bacille Calmette-Guérin (BCG) is the only licenced tuberculosis (TB) vaccine, but has limited efficacy against pulmonary TB disease development and modest protection against extrapulmonary TB. Preventative antibiotic treatment for  We present a comprehensive, up-to-date review of host-directed therapeutics (HDTs) to improve clinical outcome in tuberculosis (TB) disease, summarizing all compounds, primarily repurposed drugs, tested in human clinical trials. Where available, results from each phase of these investigations are discussed. The crisis is exacerbated by co-infections and co-morbidities, drug-resistant (DR) Mycobacterium tuberculosis (Mtb) strains and a rise in the reservoir of latent infection. Host immune status plays a determining role in TB disease outcome. It is also well-known that Mtb itself imposes several evasion strategies and prompts the host to elicit an immune response that favours its persistence. Adjunctive treatments aimed at “re-educating” the immune system are realistic alternative approaches to tailor host anti-TB responses. The use of host-directed therapeutics (HDTs) is intended to increase the success of TB treatment by immunomodulation and/or immune augmentation. Here, immunomodulation alludes to down-regulating non-productive inflammation and modifying the immune response. In contrast, immune augmentation is considered in the framework of synergizing with anti-TB treatment regimens of drug susceptible (DS)- and DR-TB to improve long-term outcome and promote cure.Tuberculosis (TB) remains the leading cause of death by infection worldwide.2 Repurposed compounds are more likely to be investigated in human clinical trials. In this regard, prior safety and regulatory approval increases the likelihood of fast-tracked implementation of drugs as appropriate immune response modifiers. Here we introduce HDT agents at advanced testing stages and highlight promising candidates for future HDT evaluations. These candidates may reveal favourable clinical outcomes and translate into useful adjunctive treatment strategies in our fight against TB.HDTs are, therefore, considered crucial to achieving the 2035 World Health Organization (WHO) End TB goals.Mtb infection. There is, however, no clear consensus of what constitutes clinically protective immunity. First-line innate immune defences play a central role in TB pathogenesis, albeit insufficient to clear infection. For this reason, T-helper (TH)-1 and CD8 T-cell adaptive responses are considered crucial for effective anti-TB immunity.4 Conversely, type-I interferon (IFN) and typical TH2 responses are associated with disease progression, contributing to disease susceptibility. Additionally, regulatory T cells (Tregs) may inhibit protective immunity.6 Theoretically, each of these pathways constitutes potential and ‘druggable’ targets. However, this notion is complicated by the complex course of progressive TB disease, including stages such as initial infection, protracted latency and overt disease8 Furthermore, other factors such as genetic diversity and co-morbidities (e.g. type-2 diabetes and HIV infection) also have a role to play.TB disease is perceived as a paradigm of host immune failure. In contrast, latency is considered a proxy of immunological control of 7 highlighting the importance of immunotherapeutics treating the full TB spectrum. Realistically, a single immunotherapeutic agent is unlikely to be effective in the full TB spectrum. This has led to the concept of precision medicine approaches, since patient groups are likely to vary in their need for HDTs directed at immunomodulation and/or immune augmentation. For example, the treatment requirements from HDTs for individuals with advanced TB disease or even post-TB lung disorders are likely to differ considerably from those required for latently infected community members or healthy contacts of TB patients.A more recent concept is that TB represents a dynamic spectrum of mycobacteria at varying states of replication,Mtb and realignment of the response to support immune protection. Within the context of repurposed drugs, we summarize the four main mechanisms by which these adjunctive therapies are thought to improve outcome in TB  disrupting and penetrating the granuloma to expose Mtb bacilli to anti-TB treatment.The National Institutes of Health (NIH) clinicaltrials.gov resource database of privately and publicly funded human clinical trials lists investigations on adjunct therapies for various forms of TB. A literary search of human clinical trials, animal model studies and preliminary in vitro cohort studies was performed to identify current research highlighting repurposed drugs, HDTs and adjunctive candidates for TB treatment. Many of these have confirmed effective therapeutic manipulation of host immunity against  TB Fig. ; namely,9 These eicosanoid products serve as signalling molecules, modulating inflammation and cell death. A delicate balance in eicosanoid levels is crucial for Mtb control and regulating the production of pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-α . Several modulators of the arachidonic acid pathway have been evaluated in humans as TB HDT.Catabolism of arachidonic acid by cyclooxygenase (COX) enzymes produces prostaglandins, whereas lipoxygenase (LOX) metabolism yields leukotrienes.Mtb infection (>45 days post-infection in mice) promote TB disease progression by down-regulating cell-mediated immunity.10 NSAID are commonly prescribed analgesic and anti-inflammatory medications worldwide and have shown promise as HDT in several pre-clinical studies.13 NSAID exert their effects by inhibiting COX activity, thereby interrupting formation of pro-inflammatory and immunosuppressive mediators such as prostaglandins and leukotrienes.14 Thus, the rationale for use of NSAID as HDT encompasses the inhibition of pro-inflammatory COX enzymes to attenuate excessive inflammation-induced tissue pathology and to improve host bactericidal mechanisms in individuals with active TB disease.Emerging evidence shows that heightened levels of prostaglandins at late stages of 15 Earlier trials also demonstrated reduced TB meningitis-associated strokes and mortality . For example, aspirin was investigated as an HDT candidate in a randomized trial during early TB treatment with dexamethasone in adult TB meningitis. Findings suggest that aspirin reduces new brain infarcts and related deaths through a mechanism involving inhibition of thromboxane-A2 and increased levels of protectins.Mtb replication and greater survival rates in a TB-susceptible murine model18; thus rationalizing the prospective use as adjunctive therapy to improve TB outcome. Accordingly, individuals with latent TB, who fail to develop active TB disease, display balanced levels of PGE-2 and lipoxins.18 Lipoxins also negatively regulate protective TH1 responses. This was demonstrated by increased IFN-γ, interleukin (IL)-12 and nitric oxide synthase (NOS)-2 mRNA levels and reduced mycobacterial burden in 5-LOX-deficient mice.19 At present, no trials evaluating 5-LOX inhibitors as HDT complementing standard TB treatment are registered on the clinicaltrials.gov resource database. The 5-LOX inhibitor, zileuton, is however, approved for treating asthma which could be repurposed as TB HDT and tested to elucidate whether modulation of this pathway improves TB treatment outcomes.Eicosanoids were previously suggested as targets for therapeutic exclusion in TB. However, data demonstrate the protective role of prostaglandin E (PGE)-2 during early infection, either by direct supplementation or via inhibition of 5-Lipoxygenase (5-LOX). Inhibiting 5-LOX has been linked to restricted lung pathology, lower type-I IFN production, reduced 21 In TB, hyper-activation of the inflammatory response often results in tissue pathology and oedema, leading to tissue dysfunction and chronic inflammation. The rationale for using anti-inflammatory corticosteroids as adjunctive treatment of active TB disease mechanistically involves modulation of inflammatory and apoptotic gene transcription pathways.22 This occurs by binding to intracellular receptors and modulating gene transcription in target tissues, thus modulating inflammatory mediator function, suppressing the humoral immune response and inhibiting leucocyte infiltration to the site of disease.24 These effects are thought to reduce chronic, non-productive inflammation and favour the host antimicrobial response.Corticosteroids have been employed as adjunctive therapy for a range of inflammatory conditions and disease states, including bacterial and viral meningitis, pneumonia and sepsis.28 In particular, trials testing the efficacy of adjunctive dexamethasone treatment on the risk of death or disability in TB meningitis demonstrated improved patient survival rate  disease.32 Taken together, it is evident that more investigation is needed to establish conclusive outcomes for the risks and benefits of corticosteroids as adjunctive therapy for advanced TB disease.Importantly, since data suggest that the effects and benefits of corticosteroid adjunctive therapy are organ specific, its use in extrapulmonary TB requires careful consideration on a case-specific basis. Meta-analyses refute long-term treatment efficacy of corticosteroids. In fact, studies involving high-dose corticosteroid treatment observed an increased risk of side effects.33 Altogether, the anti-inflammatory effects of PDE-i serve to modulate chronic inflammation and cytokine storms associated with infectious disease, while improving antibacterial responses and reducing bacillary load. This suggests relevance, not only during active TB disease, but also in clearing non-productive inflammation for conditions such as TB-IRIS and extrapulmonary TB.Phosphodiesterase inhibitors (PDE-i) are small-molecule inhibitors that reduce inflammation by increasing intracellular cyclic adenosine monophosphate (cAMP) and cGMP.34 The PDE-4-i, roflumilast has also shown promise as an effective HDT in a TB mouse model when used with isoniazid. Supporting evidence illustrated reduced TNF-α production, thwarted neutrophil recruitment and reduced lung bacillary burden.35 Similar findings were reported for another selective PDE-4-i, CC-11050, in a TB rabbit model.36 Analogues of thalidomide, such as CC-3052, have also shown to possess similar PDE-4-i properties and demonstrated potential as TB HDT by reducing lung pathology and inflammation.37 These promising pre-clinical screenings of PDE-i have led to safety and efficacy testing of adjunctive CC-11050 with the standard 6-month multi-drug therapy. This phase-II open-label human clinical trial is currently recruiting South African TB patients . Outcomes from ongoing studies should provide greater insight and justification for further trials investigating concomitant NAC for treatment of multi-drug-resistant (MDR)-TB patients  tyrosine kinase, has shown promise in both acute and chronic 54 In this regard, metformin (class: biguanide) treatment improves glucose control in diabetic patients and restores dysfunctional immunity associated with hyperglycaemia.57 In the context of TB, the immunomodulatory effects of metformin have been shown to promote macrophage autophagy by activating the expression of AMP-activated protein kinase (AMPK) and reactive oxygen species (ROS) production. Altogether these effects inhibit Mtb growth, reduce inflammation and prevent lung damage.58 These findings have promoted metformin as a candidate for therapeutic prevention and adjunctive treatment approaches in TB.55Poor glycaemic control is a risk factor for TB disease onset, mortality, treatment failure and relapse.62 A number of pre-clinical studies further demonstrated that metformin synergizes with the antimicrobial properties of rifampicin and reduces intracellular Mtb growth. This occurs in an AMPK-dependant manner, through inhibition of pro-inflammatory cell proliferation, thereby reducing disease severity.64 Other in vitro and in vivo findings show that metformin promotes ROS production, required for fusion of the phagosome-lysosome complex to aid in phagocytosis- and autophagy-induced killing of Mtb.65 Metformin also has a direct effect on the bacterial respiratory chain complex, which plays an important role in bacterial persistence and tolerance.66 Retrospective evaluation of clinical trial data demonstrated that metformin treatment of type-II diabetics with TB is associated with fewer lung cavities, lower proportion of individuals with advanced disease and improved sputum culture conversion rate 2 months post-treatment initiation.67 Retrospective analyses also showed that type-II diabetics receiving metformin treatment have a lower TB risk profile compared to those using sulfonylureas.68 Interestingly, diabetics on metformin treatment have a lower chance of having latent Mtb infection (LTBI), as measured by a positive T-Spot TB test.64In vitro and in vivo murine studies have further shown that metformin treatment reduces inflammation in TB by promoting expansion of anti-inflammatory cell types, particularly alternatively activated macrophages.Mtb in heathy subjects.69 Moreover, metformin has illustrated beneficial effects for non-diabetic indications such as obesity, polycystic ovary syndrome and Alzheimer’s disease.70 The first prospective trial testing the addition of metformin to DOTS, the ‘Metformin for TB/HIV Host-directed Therapy’ (METHOD) trial, is currently in planning and will evaluate the proportion of smear negative TB patients at month two post-treatment. This trial will also test the efficacy of metformin on lung function, severity of lung involvement and HIV viral load (R34-AI124826-01).Most clinical testing has been limited to evaluating the effects of metformin on diabetic TB patients. This begs the question of whether these same properties translate to non-diabetic TB patients in a clinical setting. Pre-clinical evidence of a recent in vitro study showed metformin-mediated modulation of cellular metabolism, immune function and gene transcription involved in innate immune responses to Mtb proliferation.While the TB community is anxiously awaiting results from clinical trials testing these and other drugs with anti-inflammatory or antihyperglycemic properties, it remains important to carefully consider and assess ideal dosages to prevent excessive anti-inflammatory responses, which too may favour Vitamins are furthest along the pipeline of TB HDT testing in human clinical trials Table . This is72 At a pre-clinical level, the protective effects of vitD has been linked to enhanced innate immune production of ROS, IL-1β, IFN-γ and cathelicidin,74 while positive trials have shown a reduction in inflammatory mediators including matrix metalloproteinases (MMPs) (Table Vitamin D (vitD), acting via its vitD receptor (VDR), regulates gene expression of cytokines and immune mediators in activated cells. In the antimicrobial immune response, VDR is upregulated following ligation of TLRs, which induces antimicrobial peptides such as cathelicidins and defensins. Thus, vitD as an adjunctive therapy may enhance the immune response and favourable disease outcome in TB. At least 22 current and completed trials of vitD as TB HDT are listed on the clinicaltrials.gov database. Inconsistencies in trial outcomes have, however, impeded interpretation of HDT efficacy. While some studies on vitD supplementation during TB treatment demonstrate clinical and radiological involvement in patients with vitD deficiency Table , others 75 Therefore, evidence of the direct benefit of vitA has been weak, at best demonstrating modest improvements in the weight of TB patients.76 VitA supplementation with zinc yielded similar results75 (Table 77Vitamin A (vitA) deficiency has also been associated with incident TB and correlated with increased mortality in HIV/TB co-infected individuals. VitA supplementation is thought to strengthen the immune system and reduce mortality; however, information on vitA supplementation in conjunction with TB treatment has been inconsistent. The handful of completed case-control studies investigating vitA supplementation during TB treatment mainly report findings as part of a dietary multivitamin supplement.75 Table . A more Although the overall findings in the area of vitamins as TB HDT is promising, significant challenges exist that impede objective interpretation of data. This mainly stems from heterogeneous study design with discrepancies in nutritional/dietary intake and route of administration, amongst others. We propose that meticulous study design may overcome these challenges and may provide more conclusive data for the use of vitamin supplementation to improve TB outcome.79 Pre-clinical studies and clinical trial data have shown that PBA synergises with vitD to upregulate expression of the anti-mycobacterial peptide cathelicidin, restrict Mtb uptake, reduce Mtb intracellular growth in macrophages, upregulate chemokine secretion and induce autophagy.81 These benefits of PBA has been verified in a randomized controlled trial in Bangladesh, demonstrating its potential as TB HDT (Table Sodium phenylbutyrate (PBA), a biological aromatic fatty acid, has been approved for treating various diseases, including urea cycle disorders, cancer, muscular dystrophy and Parkinson’s disease. Its functions include inhibition of histone deacetylase and endoplasmic reticulum stress.86 Despite promising outcomes in animal and in vitro models, clinical use of ICIs may favour progression to active TB disease, potentially attributed to excessive inflammation and focal necrosis.87 Such therapies thus require careful consideration regarding method, dose and timing of administration to minimise potential negative effects.Other immunomodulatory biologics include immune checkpoint inhibitors (ICIs). The ICIs currently attracting the most attention in TB include nivolumab and ipilimumab. Nivolumab is a monoclonal antibody targeted against programmed death (PD) 1 protein, while ipilimumab targets cytotoxic T-lymphocyte-associated antigen 4 (CTLA4). Typically, signalling via immune checkpoints inhibits T- and B-cell function. However, in the context of TB, immune regulatory checkpoints are dysregulated and associated with T-cell exhaustion.89 In contrast, boosting TH cytokine responses could serve as a feasible strategy for those with acute/recent Mtb infection.91Therapeutic modulation of immunity via cytokines is another method to support host defences. Cytokines play crucial roles in immune cell function and can theoretically serve as promising candidates for inclusion in adjunctive immunotherapies. This, however, is strictly dependent on role of a given cytokine in host immunity. Reducing excessive cytokine responses appears as a promising HDT strategy for individuals with active DR- and DS-TB.Mtb bacilli to anti-TB treatment  reductase enzymes, which are essential in lipid metabolism and inflammatory pathways. Since the lipid-rich macrophage is a favourable environment for gy Table .106–107 Another drug, auranofin, is an organogold compound that induces transcription of heme-oxygenase-1. This inducible heme-degrading enzyme exerts anti-inflammatory properties and decreases free radical production, while enhancing oxygen-mediated killing and bactericidal activity in TB disease. A trial in South Africa is currently recruiting TB patients to test the safety and efficacy of auranofin as an adjunctive TB HDT . These TCRs, in turn, may be transferred into recipient effectors (such as NK or T cells), theoretically giving rise to genetically modified therapeutic cellular products.109 Indeed, there has also been promising outcomes in MDR/extensively drug-resistant (XDR)-TB patients receiving mesenchymal stromal cells (MSC) as a single infusion of bone marrow-derived autologous MSC.111 These MSC are well-known for their safety, anti-inflammatory and immunomodulatory properties and may thus also be applicable to various forms of TB disease.112 Additionally, microRNAs (miRs) have been implicated as potential adjunctive HDTs to regulate immune responses in TB and improve outcome. This approach holds promise by using miR for repairing and replenishing miR stores or administering anti-miRs to inhibit rogue miR that may otherwise induce pathology.114 Although having solid theoretical foundation and promising outlooks for the future, cell-based therapies and miRs in the context of TB remains in its infancy, with much still to be uncovered.Within the scope of cancer management, cell-based therapies are receiving growing interest. Indeed, such strategies may form a good template for HDTs in the TB field. As reviewed by Rao et al., in the context of MDR-TB, adoptive cell therapies and screening techniques could identify useful non-cross-reactive 115 Autophagy-targeting small molecules have shown promise in the context of Mtb infection. In pre-clinical testing, gefitinib (targeting EGFR), fluoxetine (a serotonin reuptake inhibitor), baicalin  induce autophagy and enhance intracellular Mtb clearance.117 Indeed, antimicrobial drugs such as loperamide, verapamil and standard anti-TB drugs (such as INH and pyrazinamide) themselves promote autophagy and may work synergistically with autophagy-inducing small molecules as adjunctive therapy to standard treatment for TB patients.120Autophagy-activating compounds may represent promising adjunctive therapies against TB disease. A review by Paik et al. discusses autophagy mediators targeting VDR signalling, the AMPK pathway, sirtuin 1 activation and nuclear receptors.122) and vaccine strategies may modulate immunity, and have been proposed as potential adjunctives to the TB treatment regimen. There is evidence supporting beneficial effects of Bacille Calmette-Guérin (BCG) re-vaccination in adolescents and adults. Results indicate that BCG re-vaccination reduces the rate of sustained QuantiFERON (QFT) conversion and displays improved long-term innate or trained immunity and adaptive responses, thus leading to effective control of mycobacterial infection.129 A comprehensive review by Schaible et al. in 2017 evaluates strategies to improve vaccine efficacy against TB by targeting innate immunity.130 Here, they propose that short-term modulation of the local immune response to BCG vaccination may result in long-term protective immunity against Mtb infection. Examples of interventions for such modulation may involve regulating neutrophil, Treg and MDSC recruitment to the vaccination site, preventing disadvantageous cell death pathways, modulating vaccination-induced inflammatory responses, and regulating anti-inflammatory cytokine profiles (e.g. IL-10).130 These strategies are aimed at processes that would otherwise negatively influence T-cell priming, function and proliferation upon vaccination; and many of which are in fact analogous to the rationale of HDT strategies discussed here.Although falling beyond the classification of HDT, antibiotics : eicosanoid modulators (NSAID and lipoxygenase inhibitors), inflammatory mediators (corticosteroids and tyrosine kinase inhibitors), metformin, ICIs, cytokine modulating therapy, statins, auranofin, cell-based therapies, miR and autophagy modulating drugs.Mtb-infected individuals and LTBI, we propose promising outcomes associated with host-strengthening preventative strategies, including vitamin supplementation, PBA (acting in synergy with vitD), NAC and BCG re-vaccination. We further propose HDTs to supplement new vaccines which may include: PDE-i to regulate effects of immunosuppressive subsets such as MDSC, ICIs to modulate cell death pathways, cytokine therapy to regulate anti-inflammatory cytokine profiles and perhaps eicosanoid and inflammatory mediators to modulate vaccine-induced inflammatory responses and potentiate vaccine-specific responsiveness and durability.As TB preventative therapy, HDTs could, for example, alter bacillary cell entry or enhance anti-mycobacterial properties of lung phagocytes. Ideally, this would prevent infection, while also averting disease development in latently infected individuals. For TB contacts, those in high-exposure settings, recent Mtb infection and TB disease. In this context, several HDTs remain highly controversial and require more investigation into their potentially severe off-target and associated effects. Two major examples include, ICIs and cytokine therapies. Although offering preventative HDTs to latently infected individuals remains a promising avenue, distinguishing latent infection from early active TB is however, challenging in high-exposure regions. Applicability of HDTs to MDR-TB, TB treatment shortening, TB/HIV and TB-derived lung diseases, although highlighted in some studies, have not been considered for all HDTs. This leaves much to be answered in the context of the TB spectrum. Furthermore, many theoretically sound approaches remain in their infancy in the TB field and require further investigation, hopefully showing promise and advancing to clinical trial status. Here we propose keeping a watchful eye on autophagy modulators, cell-based and miR therapies.One of the major shortcomings of HDTs includes off-target and associated side effects. These drawbacks require further evaluation against the backdrop of other aspects such as storage stability, delivery method, formulation and timing of administration at different phases of Mtb infection. These goals are hoped to be achieved through clever strategies involving multimodular approaches, including implementation of adjunct HDT as standard of care for TB patients.Considering the spectrum of TB disease formats and complexity of host immunity, adjunct HDT is unlikely to be efficient as a ‘one-size-fits-all’ approach. Even so, personalized medicine is also not feasible in high-burdened TB regions, making a case for a precision medicine approach, tailored to phenotypic disease groups. Therefore, developing biosignatures translating into an efficient, rapid, point-of-care pre-screening test is of great interest. In this way, immune profiling or patient stratification according to the degree of lung involvement or risk of disease relapse, will be a game changer for TB treatment strategies. The field remains inspired in the face of our ambitious goal to eradicate TB disease, and the even greater aspiration of preventing


Gene-based association tests aggregate genotypes across multiple variants for each gene, providing an interpretable gene-level analysis framework for genome-wide association studies (GWAS). Early gene-based test applications often focused on rare coding variants; a more recent wave of gene-based methods, e.g. TWAS, use eQTLs to interrogate regulatory associations. Regulatory variants are expected to be particularly valuable for gene-based analysis, since most GWAS associations to date are non-coding. However, identifying causal genes from regulatory associations remains challenging and contentious. Here, we present a statistical framework and computational tool to integrate heterogeneous annotations with GWAS summary statistics for gene-based analysis, applied with comprehensive coding and tissue-specific regulatory annotations. We compare power and accuracy identifying causal genes across single-annotation, omnibus, and annotation-agnostic gene-based tests in simulation studies and an analysis of 128 traits from the UK Biobank, and find that incorporating heterogeneous annotations in gene-based association analysis increases power and performance identifying causal genes. Gene-based association tests are statistical methods used in genome-wide association studies (GWAS) to identify genes that affect heritable traits. Gene-based tests are formed by aggregating genotypes across multiple genetic variants for each gene, often including only variants that are likely to affect gene function or regulation. In this work, we present a unified framework to integrate heterogeneous classes of functional variants in gene-based association analysis. This approach enables us to simultaneously assess multiple distinct biological mechanisms underlying GWAS association signals, and to construct powerful omnibus tests by aggregating across functional classes for each gene. We evaluated the performance of gene-based association test methods and strategies to identify causal genes by conducting extensive simulation studies, and by analyzing 128 human traits from the UK Biobank and comparing our results against lists of high-confidence putative causal genes. Our analysis suggests that incorporating heterogeneous functional variants in gene-based association tests increases power to detect gene-based association and helps identify causal genes. Genome-wide association studies (GWAS) have identified thousands of genetic loci associated with complex traits ; howeverIn gene-based analysis, variants are often grouped or weighted by putative functional effect, for example, a common strategy for exome analysis is to include only rare non-synonymous or loss-of-function (LoF) variants in gene-based tests such as SKAT and the CMC burden test , 5. A moIncorporating multiple types of annotation in gene-based analysis provides several advantages over analysis methods using annotations of a single type. First, including variants from multiple annotation categories is expected to increase accuracy , since signals that overlap a single annotation type  may be driven by linkage disequilibrium (LD) or pleiotropic regulatory effects , 13. SecHere, we present a statistical framework and computational tool to integrate heterogeneous functional annotations with GWAS association summary statistics for gene-based analysis. We analyze a diverse set of functional annotation data including multiple tissue-specific eQTL annotation data sets, multiple epigenetic annotation sets mapping regulatory elements to putative target genes, coding variant annotations, and TSS annotations. We compare the performance of single-annotation, omnibus, and annotation-agnostic  gene-based analysis methods through simulation studies, and by analyzing GWAS summary statistics from the UK Biobank . Our conWe first outline a statistical framework and open-source tool for gene-based analysis with heterogeneous functional annotations. Next, we describe simulations to evaluate 1) the Type I error rates of gene-based test statistics, 2) statistical power, and 3) specificity to identify causal genes. Finally, we discuss applications to empirical data using GWAS summary statistics from the UK Biobank. We assess 1) the empirical power of gene-based tests by comparing the numbers of significant independent gene-based associations discovered for each UK Biobank trait, and 2) concordance with benchmark gene lists compiled from the ClinVar database  and the GAMBIT  is an open-source tool for calculating and combining annotation-stratified gene-based tests using GWAS summary statistics (single-variant association z-scores). Broadly, GAMBIT’s strategy is to first separately calculate single-annotation gene-based association tests stratified by functional annotation class, and aggregate across classes for each gene to construct omnibus gene-based tests  proximity-based annotations, 2) coding annotations, 3) UTR regions, 4) enhancer and promoter regions, and 5) eQTL predictive weights. Each of these annotation classes comprises multiple subclasses; for example, annotations include non-synonymous, splice-site, and other variant categories; and eQTL variants are stratified by tissue. Briefly, we annotated coding and UTR variants using TabAnno  and EPACn = 50,000; and for each locus-scenario-We simulated GWAS summary statistics at 2,000 loci using haplotype data from the European subset of the 1000 Genomes Project (1KGP) Phase 3 reference panel . Briefly10p-value, which assigns equal weight to all variants within 500kbp of the gene body, 3) the omnibus test (“GAMBIT”) -log10p-value, and 4-8) -log10p-values for gene-based tests using each annotation class individually  ranking each gene by distance between its transcription start site (TSS) and the most significant independent single variant at the locus, 2) the Pascal SOCS test -logWe also compared statistical power for each of the gene-based test methods at both causal and non-causal proximal genes at each simulated locus . For proTo compare the power of gene-based tests in empirical data, we evaluated the numbers of significant independent gene-based associations detected for each method across 128 approximately independent GWAS traits in the UK Biobank  and telomerase reverse transcriptase (TERT) were top-ranked by the omnibus test, but not top-ranked based on TSS-to-top-SNP distance, while all other benchmark genes for skin cancer were top-ranked by both methods or by neither. At the TERT locus, the lead GWAS variant was intronic, whereas the lead variants for TYR, OCA2, and MC1R were nonsynonymous. Unsurprisingly, the latter three benchmark genes were also top-ranked based on coding variant gene-based p-values; however, only TERT was top-ranked based on CT-TWAS.Further inspection revealed a number of loci of biological or clinical interest. In the analysis of skin cancer in the UK Biobank, three melanin or melanogenesis-related genes . By . By 44])Second, we analyzed 128 traits from the UK Biobank to evaluate performance in empirical data across a range of complex traits and genetic architectures, and confirmed that incorporating annotations of many types and across many tissues increases power relative to standard methods. While our analysis of concordance with gold-standard causal genes was limited by the relatively small numbers of benchmark genes identified for UK Biobank traits and the inherent difficulty establishing causal genes underlying regulatory associations, we found suggestive evidence that incorporating diverse annotation types in gene-based analysis can improve performance identifying causal genes relative to standard approaches  and gene based tests using a single annotation type.Finally, we provide a unifying framework and easy-to-use software tool to incorporate heterogeneous functional annotations in gene-based analysis. From its inception, gene-based analysis was built on the premise that aggregating functional variants at the gene level can increase statistical power and help identify causal genes in GWAS . Early gThe omnibus testing strategy used here is expected to perform best under sparse alternatives, e.g. when one or few annotation classes harbor causal variants at a given locus. When a larger fraction of annotation classes harbor independent signals at a single gene locus, this omnibus strategy may have less power than one that explicitly accounts for multiple simultaneous signal sources. While we did not explore this possibility in our simulations, it is an interesting question which we defer to future work.Previous studies have evaluated the performance of gene-based tests under misspecification, e.g. by varying the proportion of causal variants and correlation structure of causal effects , 49. In The utility of incorporating annotations in gene-based analysis depends crucially on the accuracy and comprehensiveness of the underlying annotation data sets. While we considered the case that causal variants may be misspecified, our simulations assumed that the confidence weights assigned to regulatory elements are well-calibrated, and that causal eQTL variants are annotated. Violations of these assumptions will reduce both power and accuracy in gene-based analysis, and may in part account for differences between our results with empirical versus simulated data. Current transcriptomic and epigenomic studies are generally limited to a subset of human tissues and cell-types, and are derived from data sets of limited sample size e.g., , 47). Th. Th47]).In summary, our work builds upon and generalizes previous gene-based association methods, providing a flexible framework for gene-based analysis with heterogeneous annotations that can be readily adapted when new annotation resources are developed and released.We describe 1) gene-based association test statistics, 2) procedures to aggregate variants within each class of functional variation for gene-based analysis, 3) functional annotation data sources 4) procedures to simulate GWAS data using real genotype and functional annotation data, and 5) GWAS data from the UK Biobank to which we applied our methods.Here, we review statistical methods to aggregate multiple variants for gene-based, region-based, or pathway association analysis. For convenience, we assume a quantitative trait and ignore the presence of covariates; however, our results can easily be adapted to other settings.TL = (w⊤RZw)−1/2w⊤Z, where w is a vector of single-variant weights, Z is a vector of single-variant association statistics , and RZ is the correlation matrix of z-scores. Under the null hypothesis of no association, TL follows the standard normal distribution. The L-type test statistic TL can be computed from GWAS summary statistics  and covariance estimates, and can be written either as linear combinations of single-variant association statistics or as linear combinations of genotypes a, where a = −1 [For non-eQTL effects, we simulated the genetic effect for each causal variant e a = −1 , 66, 67.We used GWAS summary statistics  for a set of 1,403 traits in the UK Biobank  cohort cBenchmark genes for each of the selected UK Biobank traits were identified using the ClinVar  and HumaS1 TableDescriptive statistics for eQTL variant predictive weights used to calculate TWAS test statistics.(TEX)Click here for additional data file.S2 TableDescriptive statistics for regulatory element annotation data sets used to calculate weights between enhancers and target genes.(TEX)Click here for additional data file.S1 Fig10-p-values) by the maximum value at the corresponding locus within each replicate. This procedure ensures that curves reflect performance ranking genes at each locus individually. We obtained similar results using the quantile rank of gene-based scores within each locus for each method rather than dividing by the maximum value.Receiver Operating Characteristic  and Precision-Recall (bottom) curves for each gene-based testing approach (curve color) when either coding, eQTL, enhancer, or UTR variants are causal (plot columns) given locus heritability (TIF)Click here for additional data file.S2 Fig10-p-values) by the maximum value at the corresponding locus. This procedure ensures that curves reflect performance ranking genes at each locus individually. We obtained similar results using the quantile rank of gene-based scores within each locus for each method rather than dividing by the maximum value.ROC and Precision-Recall curves for each gene-based association or ranking method across benchmark loci present in both HPO and ClinVar . To aggregate results across benchmark loci and UK Biobank traits, we use standardized scores for each method calculated by dividing gene-based scores Click here for additional data file.S3 FigMost significant single-annotation test (x-axis) for genes with one or more gene-based p-value ≤ 5e-6. The proportion of benchmark genes (the union of HPO and ClinVar gene lists) and other genes (not present in either benchmark genes list) for which the indicated annotation class is most significant is shown on the y-axis with 95% confidence intervals. Benchmark genes are strongly enriched for coding associations , which is expected due to the selection criteria used to construct benchmark gene lists (described in (TIF)Click here for additional data file.S4 FigEmpirical cumulative distribution function (ECDF) of minor allele frequency (MAF) in the UK Biobank stratified by stratified by functional annotation. Overall, annotated functional variants tend to have lower MAF than intergenic variants, particularly for nonsense and missense variants, as expected.(TIF)Click here for additional data file.S5 Figα = 0.05 with Bonferroni adjustment for the number of eGenes in each tissue. In this case, the top tissue was “Liver” with 27 significant genes out of 3,314 total eGenes . Top-Tissue p-values are compared with CT-TWAS p-values , which aggregate across all 47 tissues, restricted to Liver eGenes. CT-Q is calculated using the sum of squared single-tissue TWAS z-scores (similar to SKAT); CT-A is calculated by combining single-tissue TWAS p-values using ACAT; and CT-M is calculated from the minimum single-tissue p-value using the multivariate normal joint density of all single-tissue z-scores (described in Comparison of Cross-Tissue TWAS (CT-TWAS) p-values, and p-values using only the top single tissue, for disorders of lipoid metabolism using GWAS summary statistics from the UK Biobank. The top tissue was defined as the tissue with the largest number of significant genes using FWER threshold (TIF)Click here for additional data file.S6 Fig10(p)-values are shown for p ≥ 10−50; 10 genes with outlying p < 10−50 are not displayed. The squared Pearson correlation between z-scores is 0.995; differences in z-scores between GAMBIT and S-PrediXcan are presumably due to differences in the LD reference data. S-PrediXcan uses precomputed LD files which are packaged together with predictive weights, whereas GAMBIT calculates LD interactively from a reference panel .Comparison of TWAS/PrediXcan p-values calculated by GAMBIT versus S-PrediXcan  using GWAS summary statistics for HDL cholesterol from the Global Lipids Genetics Consortium . Results(TIF)Click here for additional data file.S1 Data(CSV)Click here for additional data file.


The development of atherosclerosis is accompanied by the functional deterioration of plaque cells, which leads to the escalation of endothelial inflammation, abnormal vascular smooth muscle cell phenotype switching and the accumulation of lipid-laden macrophages within vascular walls. Autophagy, a highly conserved homeostatic mechanism, is critical for the delivery of cytoplasmic substrates to lysosomes for degradation. Moderate levels of autophagy prevent atherosclerosis by safeguarding plaque cells against apoptosis, preventing inflammation, and limiting the lipid burden, whereas excessive autophagy exacerbates cell damage and inflammation and thereby accelerates the formation of atherosclerotic plaques. Increasing lines of evidence suggest that long noncoding RNAs can be either beneficial or detrimental to atherosclerosis development by regulating the autophagy level. This review summarizes the research progress related to 1) the significant role of autophagy in atherosclerosis and 2) the effects of the lncRNA-mediated modulation of autophagy on the plaque cell fate, inflammation levels, proliferative capacity, and cholesterol metabolism and subsequently on atherogenesis. Cardiovascular disease (CVD) is among the leading causes of morbidity and mortality around the world. The critical pathological foundation of CVD is atherosclerosis, which is characterized by the formation of atheromatous plaques in large- and medium-sized arteries, a damaged endothelium, fatty deposits and the build-up of fibrous caps , 2. The Autophagy, an evolutionarily conserved catabolic process, is the orderly turnover of cellular components that deliver harmful pathogens, damaged protein aggregates and organelles to the lysosome for degradation . Basal a-/-) mice, the SQSTM1/p62 levels are clearly amplified in the atherosclerotic aortae, and increases in the age/plaque burden are associated with further enhancements in these levels [Autophagy occurs in all major atherosclerotic plaque cells present around the necrotic core and in the fibrous cap, e.g., endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and macrophages . In the e levels , which se levels . In geneLong noncoding RNAs (lncRNAs) are defined as non-protein-coding transcripts longer than 200 nucleotides . Accordi-/- mice exhibit significantly lower levels of ATG-6/Beclin-1 protein and a decreased ratio of microtubule-associated protein 1 light chain 3 II to I (LC3-II/I) [Based on the available evidence, the defective autophagy occurs in ECs during atherogenesis. ECs isolated from high-fat diet (HFD)-fed apoEC3-II/I) . In addiC3-II/I) . NeverthC3-II/I) .2O2-induced cytotoxicity and mitochondrial-dependent apoptosis by repressing the generation of reactive oxygen species (ROS), which results in inhibition of the formation of thick plaques and increases in the collagen content in atherosclerotic lesions [EC apoptosis contributes to atherogenesis . Lunasin lesions .in vitro study suggested that lower levels of ATG-5, ATG-12 and LC3-II proteins and enhanced SQSTM1/p62 accumulation are concomitant with an impaired cell viability of young HUVECs [-/- mice substantially promotes autophagy and ameliorates apoptosis in the aortic endothelium. Furthermore, ABO robustly decreases the atherosclerotic plaque size and improves the stability of plaques [-/- mice decreases the ATG-13 protein level in the plaque endothelium, prevents EC death and stabilizes atherosclerotic lesions [The role of endothelial autophagy in cell survival and atherosclerosis progression depends on the autophagy intensity levels. Wang et al. reported that the levels of ATG-5 and ATG-12 are decreased in ECs from the aortae of atherosclerotic mice. An g HUVECs . The inj plaques . Neverth plaques , 23. Pen lesions . Similar lesions .in vivo and in vitro. In LDL receptor-deficient (LDLR-/-) mice, systemic miR-100 overexpression reduces the plaque area by 45% [-/- mice treated with morin hydrate exhibit attenuated atherosclerosis development, decreased serum levels of pro-inflammatory factors  and enhanced autophagy in plaques. In vitro studies have revealed that morin hydrate-treated HUVECs display decreased inflammation, and these effects can be counteracted by the autophagy blocker 3-MA, which suggests that the anti-atherogenic and anti-inflammatory effects of morin hydrate are largely associated with the induction of autophagy [Pankratz et al. observed that miR-100 can suppress m-TOR complex 1 (mTORC1) signaling, enhance endothelial autophagy and decrease the expression of adhesion molecules a by 45% . Additioutophagy .-/- mice can profoundly reduce the plaque size, and the underlying mechanisms are related to inhibition of the VSMC switch to a synthetic phenotype [During atherosclerosis, VSMCs can switch from a differentiated, “contractile” phenotype to a dedifferentiated, “synthetic” phenotype, which is characterized by high proliferative and migratory capacities . Injectihenotype .-/- mice, the medial thickness of the aorta and the size of atherosclerotic plaques are significantly increased [-/- mice increase the LC3-II/I ratio, decrease the SQSTM1/p62 levels in plaque VSMCs and enlarge the lesion area in the aortic root [Autophagy suppresses the synthetic phenotype in VSMCs and cell proliferation . Grootaencreased . Howevertic root . The mec-/- mice exhibit a higher apoptotic death rate, and the mice had larger stenosis areas and intraplaque hemorrhage regions in their carotid arteries [Autophagy in VSMCs prevents cell death and attenuates the instability of atheromatous plaques . VSMCs iarteries , 34. Nevarteries . In addiarteries . Altoget-/- mice enhances cell autophagy and alleviates lipid deposition in plaques [In vivo studies have shown that spermidine significantly increases the LC3-II/I ratio, decreases the levels of SQSTM1/p62, and diminishes lipid deposition and necrotic core formation in aortic atherosclerotic plaques [Autophagy can also impede atherogenesis by preventing excessive lipid loading in VSMCs. Li et al. observed that the downregulation of sterol regulatory element-binding cleavage-activating proteins (SREBPs) rescues dysfunctional autophagy in VSMCs, which leads to decreased accumulation of lipid droplets (LDs), free cholesterol (FC) and cholesteryl ester (CE). Furthermore, the VSMC-specific knockdown of SREBPs in apoE plaques . Similar plaques .Lipophagy, a special type of autophagy, refers to an important acidic cholesterol hydrolysis pathway through which cytoplasmic LD-associated CE is transported to lysosomes . This li-/- apoE-/- mice. In peroxiredoxin-1-deficient murine peritoneal macrophages, the ratio of autolysosomes to autophagosomes is decreased, which leads to impaired lipophagic flux and increased levels of total cholesterol (TC) and CE [Jeong et al. observed dysfunctional lipophagy and an increased number of macrophage foam cells in plaques of peroxiredoxin-1) and CE . In addi) and CE . These e) and CE . These f-/- mice with recombinant CREG significantly decreases the plaque areas by reducing the number of macrophages and inhibiting inflammation in aortae [-/- mice increases the CD206+ macrophage (M2) numbers, and this effect is accompanied by augmented autophagy, weakened inflammatory responses and smaller atherosclerotic lesion areas in the aorta [Liu et al. showed that ATG-5 deficiency suppresses anti-inflammatory M2 macrophage generation , which in aortae . Moreovehe aorta .-/- mice [.The downregulation of GAS5 prevents atherosclerosis progression in apoE-/- mice . In card-/- mice . Moreove-/- mice .In vitro studies have shown that the downregulation of MALAT1 notably enhances the secretion of interleukin-6 (IL-6) and IL-8 from human aortic ECs [MALAT1 mitigates atherosclerotic plaque formation by decreasing myeloid cell adhesion to ECs and reducing proinflammatory cytokine production . In vitrrtic ECs . The overtic ECs . Similarrtic ECs . Other srtic ECs  or the mrtic ECs . MALAT1-Both LPS and ox-LDL can induce excessive autophagy and increase the TGFB2-OT1 levels . TGFB2-OMALAT1 can inhibit atherosclerosis development through several mechanisms –62. In sThe expression of the lncRNA BANCR is significantly increased in human atherosclerotic plaques. The overexpression of BANCR in VSMCs clearly increases the LC3-II/I ratio and promotes cell proliferation, and these effects are counteracted by the JNK inhibitor SP600125 . Moreove-/- mice injected with lentiviral vector-siRNA-FA2H-2 exhibit defective autophagy, increased expression of mixed lineage kinase domain-like protein (MLKL), VCAM-1, MCP-1, and IL-6, and larger lesion areas in the aortic roots and aortic valves. In vitro studies have shown that the downregulation of FA2H-2 in VSMCs and ECs significantly increases the expression of MLKL, suppresses autophagic flux and amplifies the production of proinflammatory cytokines . Furthermore, pretreatment with 3-MA or ATG-7-shRNA reverses the effects induced by FA2H-2 knockdown, which demonstrates that FA2H-2 attenuates atherosclerotic plaque progression by enhancing autophagy in VSMCs and ECs to alleviate inflammation.Guo et al.  found thin vivo can increase VSMC inflammation [-/- mice and human VSMCs. Further study showed that the H19-mediated suppression of the dual-specificity phosphatase 5 (DUSP-5)-ERK1/2 axis mitigates VSMC autophagy [The lncRNA H19 reportedly exacerbates atherosclerosis and induces ischemic stroke . The oveammation . Moreoveutophagy . H19-indThe overexpression of DYNLRB2-2 in THP-1 macrophages markedly increases the ABCA1-mediated efflux of cholesterol and reduces intracellular LDs. The upregulation of DYNLRB2-2 also augments the expression of p-AMPK, LC3-II and ATG-6/Beclin-1 and decreases the levels of phospho-m-TOR and SQSTM1/p62. Furthermore, the effects of DYNLRB2-2 on macrophage lipid accumulation are attenuated by pretreatment with the AMPK signaling pathway inhibitor compound C or the autophagy blocker 3-MA . AnotherThe TLR-4/NF-κB pathway has been well established as a facilitator of atherogenesis –73. The LncRNA-induced autophagy is also involved in other factors that influence the progression of arteriosclerosis, such as hypertension and obesity , 82. Pla-/- mice robustly mitigates atherosclerosis development by inhibiting ADAM10 [LncRNAs are appealing pharmacological targets due to their general cell type-specific functions, but many promising approaches for targeting lncRNAs remain in the preclinical phase . Recombig ADAM10 . The injg ADAM10 . Additiog ADAM10 . Similarg ADAM10 . Thus, tThe entire process of atherogenesis is accompanied by autophagy impairment. Basal autophagy is beneficial in early atherosclerosis but severely dysfunctional in advanced atherosclerotic plaques. A beneficial level of autophagy in plaque cells, but not excessive autophagy, can protect cells against apoptosis, mitigate vascular inflammation and calcification, and alleviate lipid accumulation in atherosclerotic plaques. Additionally, autophagy can usually be overactivated in response to many stresses, including intracellular stresses  , 97, andThe roles of other forms of autophagy in addition to macroautophagy, such as microautophagy, chaperone-mediated autophagy, mitophagy, aggrephagy, and xenophagy, in atherosclerosis have not been adequately addressed. By acting as critical determinants of autophagy, the lncRNA-miRNA and lncRNA-lncRNA axes exert profound influences on atherogenesis. Intriguingly, Liu et al. reported that the lncRNA NBR2 and AMPK kinase form a feed-forward loop under chronic energy stress conditions , which m


Lung cancer is the worldwide leading cause of death from cancer. Epigenetic modifications such as methylation and changes in chromatin accessibility are major gene regulatory mechanisms involved in tumorigenesis and cellular lineage commitment. We aimed to characterize these processes in the context of neuroendocrine (NE) lung cancer. Illumina 450K DNA methylation data were collected for 1407 lung cancers including 27 NE tumors. NE differentially methylated regions (NE-DMRs) were identified and correlated with gene expression data for 151 lung cancers and 31 human tissue entities from the Genotype-Tissue Expression (GTEx) consortium. Assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) were performed on eight lung cancer cell lines, including three NE cell lines, to identify neuroendocrine specific gene regulatory elements. We identified DMRs with methylation patterns associated with differential gene expression and an NE tumor phenotype. DMR-associated genes could further be split into six functional modules, including one highly specific gene module for NE lung cancer showing high expression in both normal and malignant brain tissue. The regulatory potential of NE-DMRs was further validated in vitro using paired ATAC- and RNA-seq and revealed both proximal and distal regulatory elements of canonical NE-marker genes such as CHGA, NCAM1, INSM1, as well as a number of novel candidate markers of NE lung cancer. Using multilevel genomic analyses of both tumor bulk tissue and lung cancer cell lines, we identified a large catalogue of gene regulatory elements related to the NE phenotype of lung cancer. EGFR mutations and ALK gene fusions [Lung cancer is broadly divided into different histological subtypes, including adenocarcinoma (AC), squamous cell carcinoma (SqCC), large cell carcinoma (LCC), large cell neuroendocrine carcinoma (LCNEC), and small cell lung cancer (SCLC). Correct diagnosis of lung cancer histology is clinically important as it can dictate treatment  in both early and late stage disease ,2, and i fusions . On the  fusions ,5,6,7,8.In the most recent WHO guidelines , LCNEC aCHGA), neural cell adhesion molecule (NCAM1/CD56), and the insulinoma-associated 1 (INSM1) gene. Taken together, this study defines a compendium of epigenetic regulators of the NE phenotype of lung cancer which serves as a foundation for future investigations into the gene regulatory networks involved in the development and progression of NE lung cancer. In the current study, we aimed to define the epigenetic landscape of NE lung tumors and to identify putative regulatory elements of the NE phenotype of lung cancer by using global DNA methylation analysis of 1407 lung cancers of all histological subtypes and combined assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) of eight cell lines representative of the major histological subtypes. On the basis of the integrative analyses of DNA methylation patterns and gene expression data in both lung cancer and normal human tissue, we were able to define NE-specific gene regulatory elements. The epigenetic state of these were highly predictive of proximal gene expression, and associated genes could be mapped onto six distinct modules representing both tumor intrinsic and extrinsic processes. Importantly, one of the identified modules constituted a neural 178-gene module highly expressed in brain tissue with the capacity to accurately identify tumors of the NE lineage. Then, we demonstrated by combined ATAC- and RNA-seq that NE specific differentially methylated regions (DMRs) overlap regions of differentially accessible chromatin and that correlations observed in primary tumors between DNA methylation and gene expression could also be captured in an in-vitro context. Predictably, we showed that NE-associated gene regulatory elements were enriched for genes involved in neural differentiation and development, and identified a number of high-confidence regulatory elements controlling the expression of canonical NE-marker genes such as chromogranin A . To form the final set of NE differentially methylated regions (NE-DMRs), we assigned each significant probe a 101 bp genomic window and merged probes with overlapping windows (n = 3214 probes mapping to 1295 regions) for each sample. This yielded a final set of 14,144 NE-DMRs. After filtering, a final dataset interrogating 459,790 genomic positions for the 1407 tumors profiled by DNA methylation arrays remained. A total of 16,063 differentially methylated probes (CpGs) was observed between NE tumors versus normal lung and non-NE tumors , a more stringent choice than that obtained by Bonferroni correction (approximate correlation cut-off = 0.4). This yielded 1306 significant positive and 1399 significant negative correlation coefficients between NE-DMRs and genes. From the way the test was set up, a single NE-DMR was allowed to correlate with the expression of multiple genes, and a given gene could be correlated to multiple NE-DMRs. In total, the 2705 significant correlation coefficients mapped to 2075 unique DMRs and 1110 unique genes , and INSM1, but not SYP. GO-term analysis was also performed on NE-DMR correlated, downregulated genes in NE tumors  consortium. Of the 1110 genes, 1086 genes could be matched, and were analyzed for the presence of transcriptional gene modules using k-means clustering for dimensionality reduced gene expression data (see methods). Six gene modules were identified in the GTEx data (GTEx modules), showing transcriptional differences across tissue types . Gene onE tumors . To assess the gene modules derived from the GTEx normal tissue collection, we clustered the 151 lung tumors from Karlsson et al. , findingp < 0.05, We performed ATAC-seq and complementary RNA-seq on five NSCLC and three NE lung cancer cell lines. After initial processing and filtering, a total of 108,973 regions (peaks) of open chromatin remained, and of these peaks, 13,987 peaks showed differential presence in NE vs. NSCLC cell lines , and INSM1. Hierarchical clustering using 338 (of 540) matching genes in the cohort of 151 lung tumors from Karlsson et al. [Among the 13,987 peaks, 977 were associated with transcription start site (TSS +/− 1000 bp) in 923 unique protein coding genes. The 977 TSS peaks showed high Pearson correlation values with corresponding RNA sequencing data for matched genes (mean of positive correlations = 0.73). Filtering of the 977 TSS peaks versus gene expression correlation (absolute Pearson correlation > 0.7) resulted in 564 peaks in 540 unique protein coding genes that, as expected, could separate NE from NSCLC cell lines using hierarchical clustering A. Gene oNext, we next investigated ATAC-seq peaks statistically different between NE and non-NE cell lines but distal of TSSs (10 kbp). For each peak, we identified, within a ±1 Mbp window, all genes with a Pearson correlation >0.7 between RNA-seq and ATAC-seq peak counts , with the hypothesis that peak gene pairs could represent putative distal regulatory elements. Hierarchical clustering of matching genes (n = 5866) in the 151-sample tumor cohort almost perfectly discerned the histological subtypes D. SimilaFinally, to investigate whether the neural GTEx module (module 6) accounted for the NE component of the NE cell lines, we extracted both ATAC-seq peaks in transcription start site regions (TSS +/− 1000 bp) of module six genes and ATAC-seq peaks distal to the TSS of these genes. Clustering of the TSS ATAC-seq peaks separated NE cell lines from non-NE cell lines, with the exception of one NSCLC cell line (H23) G, which CHGA, NCAM1 (CD56), and INSM1. Gene ontology enrichment analysis of the 585 genes showed overrepresentation of multiple gene ontology terms associated with cell proliferation, biosynthesis, metabolic processes, but also a number of cell development and neural associated terms  also showed significant Pearson correlation between RNA expression and ATAC-seq signal in the eight cell lines. Of the 827 genes, 585 (71%) had ATAC-seq peaks in the TSS region (TSS +/− 1000 bp), including diagnostic NE marker genes such as ed terms . Notablyed terms I. FigureTo validate the regulatory potential of tumor-derived DMRs in ATAC-seq data, first, we adjusted the 14,144 DMRs to a consensus window size of 500 bp. Of the 14,144 DMRs, 33.5% overlapped with an ATAC-seq peak. Of the 2075 DMRs with significant correlation to gene expression data, 45.4% overlapped (n = 942), indicating that a large proportion of NE-DMRs represent bona fide regulatory elements.Conversely, of all 13,987 NE-associated ATAC-seq peaks, 5.8%  overlapped with a DMR region. Similar overlap frequencies were seen for the 10 kbp distal peaks irrespective of window size . In contrast, for the 564 TSS peaks, 13.7% overlapped with a DMR. This larger overlap was likely due to bias in CpG probe positioning on the Illumina platform. Nonetheless, our results indicate that high correlations between DNA methylation and gene expression in bulk tumor material can be used to capture putative regulatory associations.With respect to distal regulatory interactions, integration of bulk tumor methylation sequencing and cell line ATAC-seq data supported the existence of several candidate DMRs involved in the regulation of NE-specific gene expression .DLL3 [DYRK1B gene, approximately 200 kbp distal to the DLL3 TSS, and is significantly correlated with DLL3, but not DYRK1B, gene expression in both primary tumor samples and cell lines. Expression analysis of genes in the region surrounding DLL3 demonstrates the putative demarcation of a regulatory domain by the aforementioned NE-DMR with markedly reduced expression of several genes between DLL3 and the ATAC-supported distal NE-DMR, but with higher expression of genes both upstream and downstream of the silent genomic segment consistent with a putative looping-type interaction  but not the fourth gene, synaptophysin (SYP), since this gene was not correlated to any of the derived NE-DMRs. However, SYP did have two distal regions of open chromatin highly correlated to the expression of the gene and significantly enriched in NE cell lines as compared with NSCLC. In support of the tumor-based analyses, regions of open chromatin in the TSS regions of the neural module genes could differentiate ATAC sequenced NE from NSCLC cell lines, with the exception of H23, an NSCLC cell line showing some expression of neural module genes. To test our hypothesis, we used high-resolution DNA methylation profiles of >1400 lung cancers of all histological types, allowing us to identify 14,144 NE-DMRs. These DMRs, on average, were linked to 9.6 unique genes per DMR, of which 2075 DMRs and 1110 unique genes showed significant correlations between DNA methylation and gene expression in lung cancer. To understand the composition of these genes, i.e., to determine if they grouped in biological subsets, we analyzed the expression of these genes broadly in 29 non-malignant human tissue types, identifying six modules of tightly regulated genes. One of these modules appeared as a neural gene module, based on functional annotation and high expression in normal, as well as malignant, brain tissue. This module was capable of predicting NE lung tumors with high accuracy and included three currently used diagnostic marker genes for NE classification , as having distal ATAC-seq peaks and overlapping a tumor DMR in bulk tissue analysis. ASCL1 is a transcription factor critical for development of pulmonary neuroendocrine cells in the developing lung, with DLL3 as a downstream target [DLL3 TSS and the distal peak region showed no expression of genes in between the TSS and distal region, consistent with a chromatin-loop mediating the regulatory function , and that, for example, 5-Aza-2′-deoxycytidine treatment of cancer cell lines could reactivate expression of the gene [To extend our analyses of NE specific transcription, we performed ATAC- and RNA-seq of the NE and NSCLC cell lines. Despite the heterogeneity of U-1906 and H23 , both showing moderate expression of neural module genes at RNA sequencing, the ATAC-seq data provided robust evidence for NE specific regions of open chromatin. In fact, NE-associated TSS regions of open chromatin were strongly enriched for neural associated genes, and TSS associated genes were able to recapitulate the major lung cancer histological groups including NE tumors in bulk tumor gene expression data. The latter was true also in bulk tumor tissue for genes associated with distal ATAC-seq peaks D–F. Apprm target ,25. Analfunction B. Althouthe gene ,27. CleaFinally, we investigated the potential of our data in addressing the need for more specific and sensitive clinical NE markers . Given tA DNA methylation dataset of 1407 lung tumors and 88 histologically normal lung samples was compiled from previously published research studies and the TCGA data portal (obtained as level 3 data) ,30, or wDNA methylation profiling using Illumina Human Methylation 450K beadchips was performed for 36 cases specifically for this study at the Centre for Translational Genomics, Lund University and Clinical Genomics Lund, SciLifeLab using protocols and methods as previously described . DNA metp < 0.01) comparing NE tumors versus normal lung and non-NE, respectively, was used to identify positions in the genome associated with NE lung cancer. Differentially methylated regions (NE-DMRs) were formed by assigning each significant probe a 101 bp genomic window and merging probes with overlapping windows by their median value. Each NE-DMR was annotated to genomic transcripts using the UCSC hg19 “known gene” database contained in the Bioconductor package “TxDb.Hsapiens.UCSC.hg19.knownGene” (db-version 1.1). Transcripts were further matched to gene symbols using the Bioconductor package “org.Hs.eg.db” (db-version 2.1) and each NE-DMR was annotated with transcript, promoter, and exon overlaps. Illumina CpG ID’s were carried over to the NE-DMRs and CpG island or shore overlaps were assigned using the UCSC genome browser RefCgi-track (hg19 data freeze). To explore putative distal regulatory associations we linked each NE-DMR to TSSs inside of 500 kb up- or downstream from the NE-DMR-midpoint. The choice of 500 kb was motivated by similar analyses performed by the FANTOM [The Mann–Whitney U test . The dimensionality of the variation patterns of the genes in the GTEx data was reduced using multidimensional scaling  of the row-standardized expression matrix and visualized in 3 dimensions in R (R package scatterplot3d). Expression modules were derived using k-means clustering of the MDS output . Agglomerative hierarchical clustering using Euclidean distance and Ward’s method (R function hclust with method “ward.D2”) was used as a complementary approach to evaluate the six expression modules. Statistical overrepresentation GO-term analysis was performed using the Panther web tool (www.pantherdb.org).To analyze the expression of correlated genes from the NE-DMR analysis in normal tissues, average gene expression data for 53 sample types corresponding to 31 human tissues and transformed fibroblasts and lymphocytes were obtained from the GTEx consortium  curves in R (function roc in package pROC). Gene module expression was further assessed in RNA sequencing data from Djureinovic et al.  includinTo assess NE-DMR methylation patterns and gene expression correlations in brain-derived samples, we obtained 450K and RNA-seq data from the Genomic Data Commons (GDC) portal  for 25 gATAC-seq was performed on the following eight cell lines: five NSCLC, NCI-H2228 (ATCC Cat# CRL-5935 RRID:CVCL_1543), LC-2/ad (ECACC Cat# 94072247 RRID:CVCL_1373), U-1752 (RRID:CVCL_0565) , with miRNA-seq data (FPKM) and processed ATAC-seq data for NE and NSCLC cell lines are available through doi:10.17632/wmt6bvrwx8.1. DNA methylation data for additional NE tumors are available through GSE149521 at Gene Expression Omnibus.We used a multilayered genomic approach to identify hundreds of putative causal regulators of the neuroendocrine tumor phenotype. Our approach identified regulatory elements capable of discriminating NE from non-NE lung cancer using two methodologically distinct approaches, bulk tumor analysis and in vitro experiments. Importantly, both layers of analysis identified largely the same set of genes as prominent features of the NE phenotype and even converged on a common set of regulatory elements with high correlations to proximal or distal gene expression. Future studies utilizing ATAC-seq optimized for frozen tissue  in combi