Genome-wide gene expression tuning reveals diverse vulnerabilities of M. tuberculosis.

Antibacterial agents target the products of essential genes but rarely achieve complete target inhibition. Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene "vulnerability" is a continuous, quantifiable trait that relates the magnitude of gene inhibition to the effect on bacterial fitness. We developed a CRISPR interference-based functional genomics method to systematically titrate gene expression in Mycobacterium tuberculosis (Mtb) and monitor fitness outcomes. We identified highly vulnerable genes in various processes, including novel targets unexplored for drug discovery. Equally important, we identified invulnerable essential genes, potentially explaining failed drug discovery efforts. Comparison of vulnerability between the reference and a hypervirulent Mtb isolate revealed incomplete conservation of vulnerability and that differential vulnerability can predict differential antibacterial susceptibility. Our results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.

Bayes factor functions for reporting outcomes of hypothesis tests.

Bayes factors represent a useful alternative to P-values for reporting outcomes of hypothesis tests by providing direct measures of the relative support that data provide to competing hypotheses. Unfortunately, the competing hypotheses have to be specified, and the calculation of Bayes factors in high-dimensional settings can be difficult. To address these problems, we define Bayes factor functions (BFFs) directly from common test statistics. BFFs depend on a single noncentrality parameter that can be expressed as a function of standardized effects, and plots of BFFs versus effect size provide informative summaries of hypothesis tests that can be easily aggregated across studies. Such summaries eliminate the need for arbitrary P-value thresholds to define "statistical significance." Because BFFs are defined using nonlocal alternative prior densities, they provide more rapid accumulation of evidence in favor of true null hypotheses without sacrificing efficiency in supporting true alternative hypotheses. BFFs can be expressed in closed form and can be computed easily from z, t, χ2, and F statistics.

Quantum advantage in variational Bayes inference.

Variational Bayes (VB) inference algorithm is used widely to estimate both the parameters and the unobserved hidden variables in generative statistical models. The algorithm-inspired by variational methods used in computational physics-is iterative and can get easily stuck in local minima, even when classical techniques, such as deterministic annealing (DA), are used. We study a VB inference algorithm based on a nontraditional quantum annealing approach-referred to as quantum annealing variational Bayes (QAVB) inference-and show that there is indeed a quantum advantage to QAVB over its classical counterparts. In particular, we show that such better performance is rooted in key quantum mechanics concepts: i) The ground state of the Hamiltonian of a quantum system-defined from the given data-corresponds to an optimal solution for the minimization problem of the variational free energy at very low temperatures; ii) such a ground state can be achieved by a technique paralleling the quantum annealing process; and iii) starting from this ground state, the optimal solution to the VB problem can be achieved by increasing the heat bath temperature to unity, and thereby avoiding local minima introduced by spontaneous symmetry breaking observed in classical physics based VB algorithms. We also show that the update equations of QAVB can be potentially implemented using ⌈logK⌉ qubits and 𝒪(K) operations per step, where K is the number of values hidden categorical variables can take. Thus, QAVB can match the time complexity of existing VB algorithms, while delivering higher performance.

Tree-informed Bayesian multi-source domain adaptation: cross-population probabilistic cause-of-death assignment using verbal autopsy.

Determining causes of deaths (CODs) occurred outside of civil registration and vital statistics systems is challenging. A technique called verbal autopsy (VA) is widely adopted to gather information on deaths in practice. A VA consists of interviewing relatives of a deceased person about symptoms of the deceased in the period leading to the death, often resulting in multivariate binary responses. While statistical methods have been devised for estimating the cause-specific mortality fractions (CSMFs) for a study population, continued expansion of VA to new populations (or "domains") necessitates approaches that recognize between-domain differences while capitalizing on potential similarities. In this article, we propose such a domain-adaptive method that integrates external between-domain similarity information encoded by a prespecified rooted weighted tree. Given a cause, we use latent class models to characterize the conditional distributions of the responses that may vary by domain. We specify a logistic stick-breaking Gaussian diffusion process prior along the tree for class mixing weights with node-specific spike-and-slab priors to pool information between the domains in a data-driven way. The posterior inference is conducted via a scalable variational Bayes algorithm. Simulation studies show that the domain adaptation enabled by the proposed method improves CSMF estimation and individual COD assignment. We also illustrate and evaluate the method using a validation dataset. The article concludes with a discussion of limitations and future directions.

Evaluation of potential alterations related to ADHD in the effective connectivity between the default mode network and cerebellum, hippocampus, thalamus, and primary visual cortex.

Hyperactivity in children with attention-deficit/hyperactivity disorder (ADHD) leads to restlessness and impulse-control impairments. Nevertheless, the relation between ADHD symptoms and brain regions interactions remains unclear. We focused on dynamic causal modeling to study the effective connectivity in a fully connected network comprised of four regions of the default mode network (DMN) (linked to response control behaviors) and four other regions with previously-reported structural alterations due to ADHD. Then, via the parametric empirical Bayes analysis, the most significant connections, with the highest correlation to the covariates ADHD/control, age, and sex were extracted. Our results demonstrated a positive correlation between ADHD and effective connectivity between the right cerebellum and three DMN nodes (intrinsically inhibitory connections). Therefore, an increase in the effective connectivity leads to more inhibition imposition from the right cerebellum to DMN that reduces this network activation. The lower DMN activity makes leaving the resting-state easier, which may be involved in the restlessness symptom. Furthermore, our results indicated a negative correlation between age and these connections. We showed that the difference between the average of effective connectivities of ADHD and control groups in the age-range of 7-11 years disappeared after 14 years-old. Therefore, aging tends to alleviate ADHD-specific symptoms.

Baldur: Bayesian Hierarchical Modeling for Label-Free Proteomics with Gamma Regressing Mean-Variance Trends.

Label-free proteomics is a fast-growing methodology to infer abundances in mass spectrometry proteomics. Extensive research has focused on spectral quantification and peptide identification. However, research toward modeling and understanding quantitative proteomics data is scarce. Here we propose a Bayesian hierarchical decision model (Baldur) to test for differences in means between conditions for proteins, peptides, and post-translational modifications. We developed a Bayesian regression model to characterize local mean-variance trends in data, to estimate measurement uncertainty and hyperparameters for the decision model. A key contribution is the development of a new gamma regression model that describes the mean-variance dependency as a mixture of a common and a latent trend-allowing for localized trend estimates. We then evaluate the performance of Baldur, limma-trend, and t test on six benchmark datasets: five total proteomics and one post-translational modification dataset. We find that Baldur drastically improves the decision in noisier post-translational modification data over limma-trend and t test. In addition, we see significant improvements using Baldur over the other methods in the total proteomics datasets. Finally, we analyzed Baldur's performance when increasing the number of replicates and found that the method always increases precision with sample size, while showing robust control of the false positive rate. We conclude that our model vastly improves over popular data analysis methods (limma-trend and t test) in several spike-in datasets by achieving a high true positive detection rate, while greatly reducing the false-positive rate.

Control of false discoveries in grouped hypothesis testing for eQTL data.

BACKGROUND: Expression quantitative trait locus (eQTL) analysis aims to detect the genetic variants that influence the expression of one or more genes. Gene-level eQTL testing forms a natural grouped-hypothesis testing strategy with clear biological importance. Methods to control family-wise error rate or false discovery rate for group testing have been proposed earlier, but may not be powerful or easily apply to eQTL data, for which certain structured alternatives may be defensible and may enable the researcher to avoid overly conservative approaches. RESULTS: In an empirical Bayesian setting, we propose a new method to control the false discovery rate (FDR) for grouped hypotheses. Here, each gene forms a group, with SNPs annotated to the gene corresponding to individual hypotheses. The heterogeneity of effect sizes in different groups is considered by the introduction of a random effects component. Our method, entitled Random Effects model and testing procedure for Group-level FDR control (REG-FDR), assumes a model for alternative hypotheses for the eQTL data and controls the FDR by adaptive thresholding. As a convenient alternate approach, we also propose Z-REG-FDR, an approximate version of REG-FDR, that uses only Z-statistics of association between genotype and expression for each gene-SNP pair. The performance of Z-REG-FDR is evaluated using both simulated and real data. Simulations demonstrate that Z-REG-FDR performs similarly to REG-FDR, but with much improved computational speed. CONCLUSION: Our results demonstrate that the Z-REG-FDR method performs favorably compared to other methods in terms of statistical power and control of FDR. It can be of great practical use for grouped hypothesis testing for eQTL analysis or similar problems in statistical genomics due to its fast computation and ability to be fit using only summary data.

An empirical Bayes approach to improving population-specific genetic association estimation by leveraging cross-population data.

Populations of non-European ancestry are substantially underrepresented in genome-wide association studies (GWAS). As genetic effects can differ between ancestries due to possibly different causal variants or linkage disequilibrium patterns, a meta-analysis that includes GWAS of all populations yields biased estimation in each of the populations and the bias disproportionately impacts non-European ancestry populations. This is because meta-analysis combines study-specific estimates with inverse variance as the weights, which causes biases towards studies with the largest sample size, typical of the European ancestry population. In this paper, we propose two empirical Bayes (EB) estimators to borrow the strength of information across populations although accounting for between-population heterogeneity. Extensive simulation studies show that the proposed EB estimators are largely unbiased and improve efficiency compared to the population-specific estimator. In contrast, even though the meta-analysis estimator has a much smaller variance, it yields significant bias when the genetic effect is heterogeneous across populations. We apply the proposed EB estimators to a large-scale trans-ancestry GWAS of stroke and demonstrate that the EB estimators reduce the variance of the population-specific estimator substantially, with the effect estimates close to the population-specific estimates.

An Introduction to Bayesian Approaches to Trial Design and Statistics for Stroke Researchers.

While the majority of stroke researchers use frequentist statistics to analyze and present their data, Bayesian statistics are becoming more and more prevalent in stroke research. As opposed to frequentist approaches, which are based on the probability that data equal specific values given underlying unknown parameters, Bayesian approaches are based on the probability that parameters equal specific values given observed data and prior beliefs. The Bayesian paradigm allows researchers to update their beliefs with observed data to provide probabilistic interpretations of key parameters, for example, the probability that a treatment is effective. In this review, we outline the basic concepts of Bayesian statistics as they apply to stroke trials, compare them to the frequentist approach using exemplary data from a randomized trial, and explain how a Bayesian analysis is conducted and interpreted.

Local functional connectivity abnormalities in mild cognitive impairment and Alzheimer's disease: A meta-analytic investigation using minimum Bayes factor activation likelihood estimation.

Functional magnetic resonance imaging research employing regional homogeneity (ReHo) analysis has uncovered aberrant local brain connectivity in individuals with mild cognitive impairment (MCI) and Alzheimer's disease (AD) in comparison with healthy controls. However, the precise localization, extent, and possible overlap of these aberrations are still not fully understood. To bridge this gap, we applied a novel meta-analytic and Bayesian method (minimum Bayes Factor Activation Likelihood Estimation, mBF-ALE) for a systematic exploration of local functional connectivity alterations in MCI and AD brains. We extracted ReHo data via a standardized MEDLINE database search, which included 35 peer-reviewed experiments, 1,256 individuals with AD or MCI, 1,118 healthy controls, and 205 x-y-z coordinates of ReHo variation. We then separated the data into two distinct datasets: one for MCI and the other for AD. Two mBF-ALE analyses were conducted, thresholded at "very strong evidence" (mBF ≥ 150), with a minimum cluster size of 200 mm³. We also assessed the spatial consistency and sensitivity of our Bayesian results using the canonical version of the ALE algorithm. For MCI, we observed two clusters of ReHo decrease and one of ReHo increase. Decreased local connectivity was notable in the left precuneus (Brodmann area - BA 7) and left inferior temporal gyrus (BA 20), while increased connectivity was evident in the right parahippocampal gyrus (BA 36). The canonical ALE confirmed these locations, except for the inferior temporal gyrus. In AD, one cluster each of ReHo decrease and increase were found, with decreased connectivity in the right posterior cingulate cortex (BA 30 extending to BA 23) and increased connectivity in the left posterior cingulate cortex (BA 31). These locations were confirmed by the canonical ALE. The identification of these distinct functional connectivity patterns sheds new light on the complex pathophysiology of MCI and AD, offering promising directions for future neuroimaging-based interventions. Additionally, the use of a Bayesian framework for statistical thresholding enhances the robustness of neuroimaging meta-analyses, broadening its applicability to small datasets.

Deep latent variable joint cognitive modeling of neural signals and human behavior.

As the field of computational cognitive neuroscience continues to expand and generate new theories, there is a growing need for more advanced methods to test the hypothesis of brain-behavior relationships. Recent progress in Bayesian cognitive modeling has enabled the combination of neural and behavioral models into a single unifying framework. However, these approaches require manual feature extraction, and lack the capability to discover previously unknown neural features in more complex data. Consequently, this would hinder the expressiveness of the models. To address these challenges, we propose a Neurocognitive Variational Autoencoder (NCVA) to conjoin high-dimensional EEG with a cognitive model in both generative and predictive modeling analyses. Importantly, our NCVA enables both the prediction of EEG signals given behavioral data and the estimation of cognitive model parameters from EEG signals. This novel approach can allow for a more comprehensive understanding of the triplet relationship between behavior, brain activity, and cognitive processes.

Untangling the Evolution of the Receptor-Binding Motif of SARS-CoV-2.

The spike protein determines the host-range specificity of coronaviruses. In particular, the Receptor-Binding Motif in the spike protein from SARS-CoV-2 contains the amino acids involved in molecular recognition of the host Angiotensin Converting Enzyme 2. Therefore, to understand how SARS-CoV-2 acquired its capacity to infect humans it is necessary to reconstruct the evolution of this important motif. Early during the pandemic, it was proposed that the SARS-CoV-2 Receptor-Binding Domain was acquired via recombination with a pangolin infecting coronavirus. This proposal was challenged by an alternative explanation that suggested that the Receptor-Binding Domain from SARS-CoV-2 did not originated via recombination with a coronavirus from a pangolin. Instead, this alternative hypothesis proposed that the Receptor-Binding Motif from the bat coronavirus RaTG13, was acquired via recombination with an unidentified coronavirus. And as a consequence of this event, the Receptor-Binding Domain from the pangolin coronavirus appeared as phylogenetically closer to SARS-CoV-2. Recently, the genomes from coronaviruses from Cambodia (bat_RShST182/200) and Laos (BANAL-20-52/103/247) which are closely related to SARS-CoV-2 were reported. However, no detailed analysis of the evolution of the Receptor-Binding Motif from these coronaviruses was reported. Here we revisit the evolution of the Receptor-Binding Domain and Motif in the light of the novel coronavirus genome sequences. Specifically, we wanted to test whether the above coronaviruses from Cambodia and Laos were the source of the Receptor-Binding Domain from RaTG13. We found that the Receptor-Binding Motif from these coronaviruses is phylogenetically closer to SARS-CoV-2 than to RaTG13. Therefore, the source of the Receptor-Binding Domain from RaTG13 is still unidentified. In accordance with previous studies, our results are consistent with the hypothesis that the Receptor-Binding Motif from SARS-CoV-2 evolved by vertical inheritance from a bat-infecting population of coronaviruses.

Reliability of dynamic causal modelling of resting-state magnetoencephalography.

This study assesses the reliability of resting-state dynamic causal modelling (DCM) of magnetoencephalography (MEG) under conductance-based canonical microcircuit models, in terms of both posterior parameter estimates and model evidence. We use resting-state MEG data from two sessions, acquired 2 weeks apart, from a cohort with high between-subject variance arising from Alzheimer's disease. Our focus is not on the effect of disease, but on the reliability of the methods (as within-subject between-session agreement), which is crucial for future studies of disease progression and drug intervention. To assess the reliability of first-level DCMs, we compare model evidence associated with the covariance among subject-specific free energies (i.e., the 'quality' of the models) with versus without interclass correlations. We then used parametric empirical Bayes (PEB) to investigate the differences between the inferred DCM parameter probability distributions at the between subject level. Specifically, we examined the evidence for or against parameter differences (i) within-subject, within-session, and between-epochs; (ii) within-subject between-session; and (iii) within-site between-subjects, accommodating the conditional dependency among parameter estimates. We show that for data acquired close in time, and under similar circumstances, more than 95% of inferred DCM parameters are unlikely to differ, speaking to mutual predictability over sessions. Using PEB, we show a reciprocal relationship between a conventional definition of 'reliability' and the conditional dependency among inferred model parameters. Our analyses confirm the reliability and reproducibility of the conductance-based DCMs for resting-state neurophysiological data. In this respect, the implicit generative modelling is suitable for interventional and longitudinal studies of neurological and psychiatric disorders.

ACTOR: a latent Dirichlet model to compare expressed isoform proportions to a reference panel.

The relative proportion of RNA isoforms expressed for a given gene has been associated with disease states in cancer, retinal diseases, and neurological disorders. Examination of relative isoform proportions can help determine biological mechanisms, but such analyses often require a per-gene investigation of splicing patterns. Leveraging large public data sets produced by genomic consortia as a reference, one can compare splicing patterns in a data set of interest with those of a reference panel in which samples are divided into distinct groups, such as tissue of origin, or disease status. We propose A latent Dirichlet model to Compare expressed isoform proportions TO a Reference panel (ACTOR), a latent Dirichlet model with Dirichlet Multinomial observations to compare expressed isoform proportions in a data set to an independent reference panel. We use a variational Bayes procedure to estimate posterior distributions for the group membership of one or more samples. Using the Genotype-Tissue Expression project as a reference data set, we evaluate ACTOR on simulated and real RNA-seq data sets to determine tissue-type classifications of genes. ACTOR is publicly available as an R package at https://github.com/mccabes292/actor.

A meta-inference framework to integrate multiple external models into a current study.

It is becoming increasingly common for researchers to consider incorporating external information from large studies to improve the accuracy of statistical inference instead of relying on a modestly sized data set collected internally. With some new predictors only available internally, we aim to build improved regression models based on individual-level data from an "internal" study while incorporating summary-level information from "external" models. We propose a meta-analysis framework along with two weighted estimators as the composite of empirical Bayes estimators, which combines the estimates from different external models. The proposed framework is flexible and robust in the ways that (i) it is capable of incorporating external models that use a slightly different set of covariates; (ii) it is able to identify the most relevant external information and diminish the influence of information that is less compatible with the internal data; and (iii) it nicely balances the bias-variance trade-off while preserving the most efficiency gain. The proposed estimators are more efficient than the naïve analysis of the internal data and other naïve combinations of external estimators.

Tip dating and Bayes factors provide insight into the divergences of crown bird clades across the end-Cretaceous mass extinction.

The origin of crown birds (Neornithes) remains contentious owing to conflicting divergence time hypotheses obtained from alternative sources of data. The fossil record suggests limited diversification of Neornithes in the Late Mesozoic and a substantial radiation in the aftermath of the Cretaceous-Palaeogene (K-Pg) mass extinction, approximately 66 Ma. Molecular clock studies, however, have yielded estimates for neornithine origins ranging from the Early Cretaceous (130 Ma) to less than 10 Myr before the K-Pg. We use Bayes factors to compare the fit of node ages from different molecular clock studies to an independent morphological dataset. Our results allow us to reject scenarios of crown bird origins deep in the Early Cretaceous, as well as an origin of crown birds within the last 10 Myr of the Cretaceous. The scenario best supported by our analyses is one where Neornithes originated between the Early and Late Cretaceous (ca 100 Ma), while numerous divergences within major neoavian clades either span or postdate the K-Pg. This study affirms the importance of the K-Pg on the diversification of modern birds, and the potential of combined-evidence tip-dating analyses to illuminate recalcitrant 'rocks versus clocks' debates.

SC-MEB: spatial clustering with hidden Markov random field using empirical Bayes.

Spatial transcriptomics has been emerging as a powerful technique for resolving gene expression profiles while retaining tissue spatial information. These spatially resolved transcriptomics make it feasible to examine the complex multicellular systems of different microenvironments. To answer scientific questions with spatial transcriptomics and expand our understanding of how cell types and states are regulated by microenvironment, the first step is to identify cell clusters by integrating the available spatial information. Here, we introduce SC-MEB, an empirical Bayes approach for spatial clustering analysis using a hidden Markov random field. We have also derived an efficient expectation-maximization algorithm based on an iterative conditional mode for SC-MEB. In contrast to BayesSpace, a recently developed method, SC-MEB is not only computationally efficient and scalable to large sample sizes but is also capable of choosing the smoothness parameter and the number of clusters. We performed comprehensive simulation studies to demonstrate the superiority of SC-MEB over some existing methods. We applied SC-MEB to analyze the spatial transcriptome of human dorsolateral prefrontal cortex tissues and mouse hypothalamic preoptic region. Our analysis results showed that SC-MEB can achieve a similar or better clustering performance to BayesSpace, which uses the true number of clusters and a fixed smoothness parameter. Moreover, SC-MEB is scalable to large 'sample sizes'. We then employed SC-MEB to analyze a colon dataset from a patient with colorectal cancer (CRC) and COVID-19, and further performed differential expression analysis to identify signature genes related to the clustering results. The heatmap of identified signature genes showed that the clusters identified using SC-MEB were more separable than those obtained with BayesSpace. Using pathway analysis, we identified three immune-related clusters, and in a further comparison, found the mean expression of COVID-19 signature genes was greater in immune than non-immune regions of colon tissue. SC-MEB provides a valuable computational tool for investigating the structural organizations of tissues from spatial transcriptomic data.

Abnormal resting-state effective connectivity in large-scale networks among obsessive-compulsive disorder.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a chronic mental illness characterized by abnormal functional connectivity among distributed brain regions. Previous studies have primarily focused on undirected functional connectivity and rarely reported from network perspective. METHODS: To better understand between or within-network connectivities of OCD, effective connectivity (EC) of a large-scale network is assessed by spectral dynamic causal modeling with eight key regions of interests from default mode (DMN), salience (SN), frontoparietal (FPN) and cerebellum networks, based on large sample size including 100 OCD patients and 120 healthy controls (HCs). Parametric empirical Bayes (PEB) framework was used to identify the difference between the two groups. We further analyzed the relationship between connections and Yale-Brown Obsessive Compulsive Scale (Y-BOCS). RESULTS: OCD and HCs shared some similarities of inter- and intra-network patterns in the resting state. Relative to HCs, patients showed increased ECs from left anterior insula (LAI) to medial prefrontal cortex, right anterior insula (RAI) to left dorsolateral prefrontal cortex (L-DLPFC), right dorsolateral prefrontal cortex (R-DLPFC) to cerebellum anterior lobe (CA), CA to posterior cingulate cortex (PCC) and to anterior cingulate cortex (ACC). Moreover, weaker from LAI to L-DLPFC, RAI to ACC, and the self-connection of R-DLPFC. Connections from ACC to CA and from L-DLPFC to PCC were positively correlated with compulsion and obsession scores (r = 0.209, p = 0.037; r = 0.199, p = 0.047, uncorrected). CONCLUSIONS: Our study revealed dysregulation among DMN, SN, FPN, and cerebellum in OCD, emphasizing the role of these four networks in achieving top-down control for goal-directed behavior. There existed a top-down disruption among these networks, constituting the pathophysiological and clinical basis.

Personalized treatment selection via product partition models with covariates.

Precision medicine is an approach for disease treatment that defines treatment strategies based on the individual characteristics of the patients. Motivated by an open problem in cancer genomics, we develop a novel model that flexibly clusters patients with similar predictive characteristics and similar treatment responses; this approach identifies, via predictive inference, which one among a set of treatments is better suited for a new patient. The proposed method is fully model based, avoiding uncertainty underestimation attained when treatment assignment is performed by adopting heuristic clustering procedures, and belongs to the class of product partition models with covariates, here extended to include the cohesion induced by the normalized generalized gamma process. The method performs particularly well in scenarios characterized by considerable heterogeneity of the predictive covariates in simulation studies. A cancer genomics case study illustrates the potential benefits in terms of treatment response yielded by the proposed approach. Finally, being model based, the approach allows estimating clusters' specific response probabilities and then identifying patients more likely to benefit from personalized treatment.

Integrated biomarker profiling for predicting the response of type 2 diabetes to metformin.

AIM: To explore biomarkers that can predict the response of type 2 diabetes (T2D) patients to metformin at an early stage to provide better treatment for T2D. METHODS: T2D patients with (responders) or without response (non-responders) to metformin were recruited, and their serum samples were used for metabolomic analysis to identify candidate biomarkers. Moreover, the efficacy of metformin was verified by insulin-resistant mice, and the candidate biomarkers were verified to determine the biomarkers. Five different machine learning methods were used to construct the integrated biomarker profiling (IBP) with the biomarkers to predict the response of T2D patients to metformin. RESULTS: A total of 73 responders and 63 non-responders were recruited, and 88 differential metabolites were identified in the serum samples. After being verified in mice, 19 of the 88 were considered as candidate biomarkers. Next, after metformin regulation, nine candidate biomarkers were confirmed as the biomarkers. After comparing five machine learning models, the nine biomarkers were constructed into the IBP for predicting the response of T2D patients to metformin based on the Naïve Bayes classifier, which was verified with an accuracy of 89.70%. CONCLUSIONS: The IBP composed of nine biomarkers can be used to predict the response of T2D patients to metformin, enabling clinicians to start a combined medication strategy as soon as possible if T2D patients do not respond to metformin.