Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.

Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.

An atlas of protein-protein interactions across mouse tissues.

Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the interactome has therefore been a central objective of high-throughput biology. However, the dynamics of protein interactions across physiological contexts remain poorly understood. Here, we develop a quantitative proteomic approach combining protein correlation profiling with stable isotope labeling of mammals (PCP-SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide a proteome-scale survey of interactome rewiring across mammalian tissues, revealing more than 125,000 unique interactions at a quality comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewired proteins are tightly regulated by multiple cellular mechanisms and are implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.

Next-Generation Machine Learning for Biological Networks.

Machine learning, a collection of data-analytical techniques aimed at building predictive models from multi-dimensional datasets, is becoming integral to modern biological research. By enabling one to generate models that learn from large datasets and make predictions on likely outcomes, machine learning can be used to study complex cellular systems such as biological networks. Here, we provide a primer on machine learning for life scientists, including an introduction to deep learning. We discuss opportunities and challenges at the intersection of machine learning and network biology, which could impact disease biology, drug discovery, microbiome research, and synthetic biology.

A Quantitative Genetic Interaction Map of HIV Infection.

We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary T cells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of how different pathogens hijack and rewire the host during infection.

From data to discovery: AI-guided analysis of disease-relevant molecules in spinal muscular atrophy (SMA).

Spinal Muscular Atrophy is caused by partial loss of survival of motoneuron (SMN) protein expression. The numerous interaction partners and mechanisms influenced by SMN loss result in a complex disease. Current treatments restore SMN protein levels to a certain extent, but do not cure all symptoms. The prolonged survival of patients creates an increasing need for a better understanding of SMA. Although many SMN-protein interactions, dysregulated pathways, and organ phenotypes are known, the connections among them remain largely unexplored. Monogenic diseases are ideal examples for the exploration of cause-and-effect relationships to create a network describing the disease-context. Machine learning tools can utilize such knowledge to analyze similarities between disease-relevant molecules and molecules not described in the disease so far. We used an artificial intelligence-based algorithm to predict new genes of interest. The transcriptional regulation of 8 out of 13 molecules selected from the predicted set were successfully validated in an SMA mouse model. This bioinformatic approach, using the given experimental knowledge for relevance predictions, enhances efficient targeted research in SMA and potentially in other disease settings.

Comprehensive Proteomics Analysis of Polyhydroxyalkanoate (PHA) Biology in Pseudomonas putida KT2440: The Outer Membrane Lipoprotein OprL is a Newly Identified Phasin.

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress.

Every gene can (and possibly will) be associated with cancer.

A PubMed analysis shows that the vast majority of human genes have been studied in the context of cancer. As such, the study of nearly any human gene can be justified based on existing literature by its potential relevance to cancer. Moreover, these results have implications for analyzing and interpreting large-scale analyses.

Network analysis of transcriptomic data uncovers molecular signatures and the interplay of mRNAs, lncRNAs, and miRNAs in human embryonic stem cells.

Growing evidence has shown that besides the protein coding genes, the non-coding elements of the genome are indispensable for maintaining the property of self-renewal in human embryonic stem cells and in cell fate determination. However, the regulatory mechanisms and the landscape of interactions between the coding and non-coding elements is poorly understood. In this work, we used weighted gene co-expression network analysis (WGCNA) on transcriptomic data retrieved from RNA-seq and small RNA-seq experiments and reconstructed the core human pluripotency network (called PluriMLMiNet) consisting of 375 mRNA, 57 lncRNA and 207 miRNAs. Furthermore, we derived networks specific to the naïve and primed states of human pluripotency (called NaiveMLMiNet and PrimedMLMiNet respectively) that revealed a set of molecular markers (RPS6KA1, ZYG11A, ZNF695, ZNF273, and NLRP2 for naive state, and RAB34, TMEM178B, PTPRZ1, USP44, KIF1A and LRRN1 for primed state) which can be used to distinguish the pluripotent state from the non-pluripotent state and also to identify the intra-pluripotency states (i.e., naïve and primed state). The lncRNA DANT1 was found to be a crucial as it formed a bridge between the naive and primed state-specific networks. Analysis of the genes neighbouring DANT1 suggested its possible role as a competing endogenous RNA (ceRNA) for the induction and maintenance of human pluripotency. This was computationally validated by predicting the missing DANT1-miRNA interactions to complete the ceRNA circuit. Here we first report that DANT1 might harbour binding sites for miRNAs hsa-miR-30c-2-3p, hsa-miR-210-3p and hsa-let-7b-5p which may influence pluripotency.

A computational approach for detecting physiological homogeneity in the midst of genetic heterogeneity.

The human genetic dissection of clinical phenotypes is complicated by genetic heterogeneity. Gene burden approaches that detect genetic signals in case-control studies are underpowered in genetically heterogeneous cohorts. We therefore developed a genome-wide computational method, network-based heterogeneity clustering (NHC), to detect physiological homogeneity in the midst of genetic heterogeneity. Simulation studies showed our method to be capable of systematically converging genes in biological proximity on the background biological interaction network, and capturing gene clusters harboring presumably deleterious variants, in an efficient and unbiased manner. We applied NHC to whole-exome sequencing data from a cohort of 122 individuals with herpes simplex encephalitis (HSE), including 13 individuals with previously published monogenic inborn errors of TLR3-dependent IFN-α/ß immunity. The top gene cluster identified by our approach successfully detected and prioritized all causal variants of five TLR3 pathway genes in the 13 previously reported individuals. This approach also suggested candidate variants of three reported genes and four candidate genes from the same pathway in another ten previously unstudied individuals. TLR3 responsiveness was impaired in dermal fibroblasts from four of the five individuals tested, suggesting that the variants detected were causal for HSE. NHC is, therefore, an effective and unbiased approach for unraveling genetic heterogeneity by detecting physiological homogeneity.

Intersection of network medicine and machine learning towards investigating the key biomarkers and pathways underlying amyotrophic lateral sclerosis: a systematic review.

BACKGROUND: Network medicine is an emerging area of research that focuses on delving into the molecular complexity of the disease, leading to the discovery of network biomarkers and therapeutic target discovery. Amyotrophic lateral sclerosis (ALS) is a complicated rare disease with unknown pathogenesis and no available treatment. In ALS, network properties appear to be potential biomarkers that can be beneficial in disease-related applications when explored independently or in tandem with machine learning (ML) techniques. OBJECTIVE: This systematic literature review explores recent trends in network medicine and implementations of network-based ML algorithms in ALS. We aim to provide an overview of the identified primary studies and gather details on identifying the potential biomarkers and delineated pathways. METHODS: The current study consists of searching for and investigating primary studies from PubMed and Dimensions.ai, published between 2018 and 2022 that reported network medicine perspectives and the coupling of ML techniques. Each abstract and full-text study was individually evaluated, and the relevant studies were finally included in the review for discussion once they met the inclusion and exclusion criteria. RESULTS: We identified 109 eligible publications from primary studies representing this systematic review. The data coalesced into two themes: application of network science to identify disease modules and promising biomarkers in ALS, along with network-based ML approaches. Conclusion This systematic review gives an overview of the network medicine approaches and implementations of network-based ML algorithms in ALS to determine new disease genes, and identify critical pathways and therapeutic target discovery for personalized treatment.

Melanoma 2.0. Skin cancer as a paradigm for emerging diagnostic technologies, computational modelling and artificial intelligence.

We live in an unprecedented time in oncology. We have accumulated samples and cases in cohorts larger and more complex than ever before. New technologies are available for quantifying solid or liquid samples at the molecular level. At the same time, we are now equipped with the computational power necessary to handle this enormous amount of quantitative data. Computational models are widely used helping us to substantiate and interpret data. Under the label of systems and precision medicine, we are putting all these developments together to improve and personalize the therapy of cancer. In this review, we use melanoma as a paradigm to present the successful application of these technologies but also to discuss possible future developments in patient care linked to them. Melanoma is a paradigmatic case for disruptive improvements in therapies, with a considerable number of metastatic melanoma patients benefiting from novel therapies. Nevertheless, a large proportion of patients does not respond to therapy or suffers from adverse events. Melanoma is an ideal case study to deploy advanced technologies not only due to the medical need but also to some intrinsic features of melanoma as a disease and the skin as an organ. From the perspective of data acquisition, the skin is the ideal organ due to its accessibility and suitability for many kinds of advanced imaging techniques. We put special emphasis on the necessity of computational strategies to integrate multiple sources of quantitative data describing the tumour at different scales and levels.

Network analytics for drug repurposing in COVID-19.

To better understand the potential of drug repurposing in COVID-19, we analyzed control strategies over essential host factors for SARS-CoV-2 infection. We constructed comprehensive directed protein-protein interaction (PPI) networks integrating the top-ranked host factors, the drug target proteins and directed PPI data. We analyzed the networks to identify drug targets and combinations thereof that offer efficient control over the host factors. We validated our findings against clinical studies data and bioinformatics studies. Our method offers a new insight into the molecular details of the disease and into potentially new therapy targets for it. Our approach for drug repurposing is significant beyond COVID-19 and may be applied also to other diseases.

Drug Repurposing for COVID-19 by Constructing a Comorbidity Network with Central Nervous System Disorders.

In the post-COVID-19 era, treatment options for potential SARS-CoV-2 outbreaks remain limited. An increased incidence of central nervous system (CNS) disorders has been observed in long-term COVID-19 patients. Understanding the shared molecular mechanisms between these conditions may provide new insights for developing effective therapies. This study developed an integrative drug-repurposing framework for COVID-19, leveraging comorbidity data with CNS disorders, network-based modular analysis, and dynamic perturbation analysis to identify potential drug targets and candidates against SARS-CoV-2. We constructed a comorbidity network based on the literature and data collection, including COVID-19-related proteins and genes associated with Alzheimer's disease, Parkinson's disease, multiple sclerosis, and autism spectrum disorder. Functional module detection and annotation identified a module primarily involved in protein synthesis as a key target module, utilizing connectivity map drug perturbation data. Through the construction of a weighted drug-target network and dynamic network-based drug-repurposing analysis, ubiquitin-carboxy-terminal hydrolase L1 emerged as a potential drug target. Molecular dynamics simulations suggested pregnenolone and BRD-K87426499 as two drug candidates for COVID-19. This study introduces a dynamic-perturbation-network-based drug-repurposing approach to identify COVID-19 drug targets and candidates by incorporating the comorbidity conditions of CNS disorders.

Benchmarking causal reasoning algorithms for gene expression-based compound mechanism of action analysis.

BACKGROUND: Elucidating compound mechanism of action (MoA) is beneficial to drug discovery, but in practice often represents a significant challenge. Causal Reasoning approaches aim to address this situation by inferring dysregulated signalling proteins using transcriptomics data and biological networks; however, a comprehensive benchmarking of such approaches has not yet been reported. Here we benchmarked four causal reasoning algorithms (SigNet, CausalR, CausalR ScanR and CARNIVAL) with four networks (the smaller Omnipath network vs. 3 larger MetaBase™ networks), using LINCS L1000 and CMap microarray data, and assessed to what extent each factor dictated the successful recovery of direct targets and compound-associated signalling pathways in a benchmark dataset comprising 269 compounds. We additionally examined impact on performance in terms of the functions and roles of protein targets and their connectivity bias in the prior knowledge networks. RESULTS: According to statistical analysis (negative binomial model), the combination of algorithm and network most significantly dictated the performance of causal reasoning algorithms, with the SigNet recovering the greatest number of direct targets. With respect to the recovery of signalling pathways, CARNIVAL with the Omnipath network was able to recover the most informative pathways containing compound targets, based on the Reactome pathway hierarchy. Additionally, CARNIVAL, SigNet and CausalR ScanR all outperformed baseline gene expression pathway enrichment results. We found no significant difference in performance between L1000 data or microarray data, even when limited to just 978 'landmark' genes. Notably, all causal reasoning algorithms also outperformed pathway recovery based on input DEGs, despite these often being used for pathway enrichment. Causal reasoning methods performance was somewhat correlated with connectivity and biological role of the targets. CONCLUSIONS: Overall, we conclude that causal reasoning performs well at recovering signalling proteins related to compound MoA upstream from gene expression changes by leveraging prior knowledge networks, and that the choice of network and algorithm has a profound impact on the performance of causal reasoning algorithms. Based on the analyses presented here this is true for both microarray-based gene expression data as well as those based on the L1000 platform.

kboolnet: a toolkit for the verification, validation, and visualization of reaction-contingency (rxncon) models.

BACKGROUND: Computational models of cell signaling networks are extremely useful tools for the exploration of underlying system behavior and prediction of response to various perturbations. By representing signaling cascades as executable Boolean networks, the previously developed rxncon ("reaction-contingency") formalism and associated Python package enable accurate and scalable modeling of signal transduction even in large (thousands of components) biological systems. The models are split into reactions, which generate states, and contingencies, that impinge on reactions; this avoids the so-called "combinatorial explosion" of system size. Boolean description of the biological system compensates for the poor availability of kinetic parameters which are necessary for quantitative models. Unfortunately, few tools are available to support rxncon model development, especially for large, intricate systems. RESULTS: We present the kboolnet toolkit ( https://github.com/Kufalab-UCSD/kboolnet , complete documentation at https://github.com/Kufalab-UCSD/kboolnet/wiki ), an R package and a set of scripts that seamlessly integrate with the python-based rxncon software and collectively provide a complete workflow for the verification, validation, and visualization of rxncon models. The verification script VerifyModel.R checks for responsiveness to repeated stimulations as well as consistency of steady state behavior. The validation scripts TruthTable.R, SensitivityAnalysis.R, and ScoreNet.R provide various readouts for the comparison of model predictions to experimental data. In particular, ScoreNet.R compares model predictions to a cloud-stored MIDAS-format experimental database to provide a numerical score for tracking model accuracy. Finally, the visualization scripts allow for graphical representations of model topology and behavior. The entire kboolnet toolkit is cloud-enabled, allowing for easy collaborative development; most scripts also allow for the extraction and analysis of individual user-defined "modules". CONCLUSION: The kboolnet toolkit provides a modular, cloud-enabled workflow for the development of rxncon models, as well as their verification, validation, and visualization. This will enable the creation of larger, more comprehensive, and more rigorous models of cell signaling using the rxncon formalism in the future.

Analysis of asymptomatic Drosophila models for ALS and SMA reveals convergent impact on functional protein complexes linked to neuro-muscular degeneration.

BACKGROUND: Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by mutations in ubiquitous proteins involved in RNA metabolism, namely SMN, TDP-43 and FUS. Although this suggests the existence of common disease mechanisms, there is currently no model to explain the resulting motor neuron dysfunction. In this work we generated a parallel set of Drosophila models for adult-onset RNAi and tagged neuronal expression of the fly orthologues of the three human proteins, named Smn, TBPH and Caz, respectively. We profiled nuclear and cytoplasmic bound mRNAs using a RIP-seq approach and characterized the transcriptome of the RNAi models by RNA-seq. To unravel the mechanisms underlying the common functional impact of these proteins on neuronal cells, we devised a computational approach based on the construction of a tissue-specific library of protein functional modules, selected by an overall impact score measuring the estimated extent of perturbation caused by each gene knockdown. RESULTS: Transcriptome analysis revealed that the three proteins do not bind to the same RNA molecules and that only a limited set of functionally unrelated transcripts is commonly affected by their knock-down. However, through our integrative approach we were able to identify a concerted effect on protein functional modules, albeit acting through distinct targets. Most strikingly, functional annotation revealed that these modules are involved in critical cellular pathways for motor neurons, including neuromuscular junction function. Furthermore, selected modules were found to be significantly enriched in orthologues of human neuronal disease genes. CONCLUSIONS: The results presented here show that SMA and ALS disease-associated genes linked to RNA metabolism functionally converge on neuronal protein complexes, providing a new hypothesis to explain the common motor neuron phenotype. The functional modules identified represent promising biomarkers and therapeutic targets, namely given their alteration in asymptomatic settings.

Characterization and comparison of gene-centered human interactomes.

The complex web of macromolecular interactions occurring within cells-the interactome-is the backbone of an increasing number of studies, but a clear consensus on the exact structure of this network is still lacking. Different genome-scale maps of human interactome have been obtained through several experimental techniques and functional analyses. Moreover, these maps can be enriched through literature-mining approaches, and different combinations of various 'source' databases have been used in the literature. It is therefore unclear to which extent the various interactomes yield similar results when used in the context of interactome-based approaches in network biology. We compared a comprehensive list of human interactomes on the basis of topology, protein complexes, molecular pathways, pathway cross-talk and disease gene prediction. In a general context of relevant heterogeneity, our study provides a series of qualitative and quantitative parameters that describe the state of the art of human interactomes and guidelines for selecting interactomes in future applications.

Construction of a radiogenomic association map of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PDAC) persists as a malignancy with high morbidity and mortality that can benefit from new means to characterize and detect these tumors, such as radiogenomics. In order to address this gap in the literature, constructed a transcriptomic-CT radiogenomic (RG) map for PDAC. METHODS: In this Institutional Review Board approved study, a cohort of subjects (n = 50) with gene expression profile data paired with histopathologically confirmed resectable or borderline resectable PDAC were identified. Studies with pre-operative contrast-enhanced CT images were independently assessed for a set of 88 predefined imaging features. Microarray gene expression profiling was then carried out on the histopathologically confirmed pancreatic adenocarcinomas and gene networks were constructed using Weighted Gene Correlation Network Analysis (WCGNA) (n = 37). Data were analyzed with bioinformatics analyses, multivariate regression-based methods, and Kaplan-Meier survival analyses. RESULTS: Survival analyses identified multiple features of interest that were significantly associated with overall survival, including Tumor Height (P = 0.014), Tumor Contour (P = 0.033), Tumor-stroma Interface (P = 0.014), and the Tumor Enhancement Ratio (P = 0.047). Gene networks for these imaging features were then constructed using WCGNA and further annotated according to the Gene Ontology (GO) annotation framework for a biologically coherent interpretation of the imaging trait-associated gene networks, ultimately resulting in a PDAC RG CT-transcriptome map composed of 3 stage-independent imaging traits enriched in metabolic processes, telomerase activity, and podosome assembly (P < 0.05). CONCLUSIONS: A CT-transcriptomic RG map for PDAC composed of semantic and quantitative traits with associated biology processes predictive of overall survival, was constructed, that serves as a reference for further mechanistic studies for non-invasive phenotyping of pancreatic tumors.

A network biology approach to identify crucial host targets for COVID-19.

The ongoing global pandemic of COVID-19, caused by SARS-CoV-2 has killed more than 5.9 million individuals out of ∼43 million confirmed infections. At present, several parts of the world are encountering the 3rd wave. Mass vaccination has been started in several countries but they are less likely to be broadly available for the current pandemic, repurposing of the existing drugs has drawn highest attention for an immediate solution. A recent publication has mapped the physical interactions of SARS-CoV-2 and human proteins by affinity-purification mass spectrometry (AP-MS) and identified 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Here, we taken a network biology approach and constructed a human protein-protein interaction network (PPIN) with the above SARS-CoV-2 targeted proteins. We utilized a combination of essential network centrality measures and functional properties of the human proteins to identify the critical human targets of SARS-CoV-2. Four human proteins, namely PRKACA, RHOA, CDK5RAP2, and CEP250 have emerged as the best therapeutic targets, of which PRKACA and CEP250 were also found by another group as potential candidates for drug targets in COVID-19. We further found candidate drugs/compounds, such as guanosine triphosphate, remdesivir, adenosine monophosphate, MgATP, and H-89 dihydrochloride that bind the target human proteins. The urgency to prevent the spread of infection and the death of diseased individuals has prompted the search for agents from the pool of approved drugs to repurpose them for COVID-19. Our results indicate that host targeting therapy with the repurposed drugs may be a useful strategy for the treatment of SARS-CoV-2 infection.

Multi-omic molecular profiling and network biology for precision anaesthesiology: a narrative review.

Technological advancement, data democratisation, and decreasing costs have led to a revolution in molecular biology in which the entire set of DNA, RNA, proteins, and various other molecules - the 'multi-omic' profile - can be measured in humans. Sequencing 1 million bases of human DNA now costs US$0.01, and emerging technologies soon promise to reduce the cost of sequencing the whole genome to US$100. These trends have made it feasible to sample the multi-omic profile of millions of people, much of which is publicly available for medical research. Can anaesthesiologists use these data to improve patient care? This narrative review brings together a rapidly growing literature in multi-omic profiling across numerous fields that points to the future of precision anaesthesiology. Here, we discuss how DNA, RNA, proteins, and other molecules interact in molecular networks that can be used for preoperative risk stratification, intraoperative optimisation, and postoperative monitoring. This literature provides evidence for four fundamental insights: (1) Clinically similar patients have different molecular profiles and, as a consequence, different outcomes. (2) Vast, publicly available, and rapidly growing molecular datasets have been generated in chronic disease patients and can be repurposed to estimate perioperative risk. (3) Multi-omic networks are altered in the perioperative period and influence postoperative outcomes. (4) Multi-omic networks can serve as empirical, molecular measurements of a successful postoperative course. With this burgeoning universe of molecular data, the anaesthesiologist-of-the-future will tailor their clinical management to an individual's multi-omic profile to optimise postoperative outcomes and long-term health.