Reliable biomarkers for common disorders of gut–brain interaction characterized by abdominal pain, including irritable bowel syndrome (IBS), are critically needed to enhance care and develop individualized therapies. The dynamic and heterogeneous nature of the pathophysiological mechanisms that underlie visceral hypersensitivity have challenged successful biomarker development. Consequently, effective therapies for pain in IBS are lacking. However, recent advances in modern omics technologies offer new opportunities to acquire deep biological insights into mechanisms of pain and nociception. Newer methods for large-scale data integration of complementary omics approaches have further expanded our ability to build a holistic understanding of complex biological networks and their co-contributions to abdominal pain. Here, we review the mechanisms of visceral hypersensitivity, focusing on IBS. We discuss candidate biomarkers for pain in IBS identified through single omics studies and summarize emerging multi-omics approaches for developing novel biomarkers that may transform clinical care for patients with IBS and abdominal pain. Despite the notion that IBS is largely regarded as a benign condition, the cost and impact of IBS on society, the economy, and patients are substantial. It is associated with increased healthcare utilization,3, higher healthcare spending,4–6 decreased productivity,6–8 and impaired quality of life.9–12 For these reasons, developing cost-effective approaches to enhance diagnosis and management of IBS and other disorders characterized by chronic abdominal pain remains a high healthcare priority.Irritable bowel syndrome (IBS) and other disorders of gut–brain interaction (DGBI) are common, chronic conditions that affect tens of millions across the globe. It is estimated that 4–10% 13 While a number of candidate biomarkers targeting both central and peripheral mechanisms of IBS have emerged over the years, achieving major breakthroughs in IBS biomarker development is challenged by the complex interindividual heterogeneity and intraindividual temporal instability that is intrinsic to the pathophysiology of the disorder. Furthermore, identifying and validating biomarkers of visceral hypersensitivity or abdominal pain, a defining feature of IBS, remains particularly challenging. The mechanisms leading to visceral hypersensitivity in IBS are complex, multifactorial, and incompletely characterized. Biological tools that capture quantitative measurements of visceral pain and are valid across the spectrum from experimental animal models to humans are limited. The lack of rigorous surrogate endpoints for visceral pain has further hindered the translation of bench to bedside research in IBS, particularly in drug development, which continues to rely on endpoints defined by subjective patient-reported symptoms.14 However, the ability to confront existing challenges and develop successful biomarkers may be aided through newer ‘omics-based approaches: metataxonomic and metagenomic sequencing of the intestinal microbiome, targeted and untargeted metabolomics, genomics, epigenomics, transcriptomics, proteomics, and connectomics for study of brain networks as well as integrated ‘omics-based studies’. Meanwhile, other evolving platforms such as single-cell RNA sequencing and spatial transcriptomics may present additional opportunities for novel insights the near future.Identifying and validating actionable biomarkers are key strategies for improving the established paradigms of diagnosing, evaluating, and treating IBS.In this review, we will cover key findings related to candidate IBS biomarkers, emphasizing those that are linked to visceral hypersensitivity and chronic abdominal pain. We will review the role and importance of biomarkers in IBS; describe the pathophysiological mechanisms underlying visceral pain in the context of IBS through a critical summary of animal and human studies; evaluate individual ‘omics-based biomarker research for pain in IBS that have been derived from both preclinical and human studies with an emphasis on recent clinical research; and highlight the potential applications of multi-omics based approaches for the future discovery of novel and translationally relevant biomarkers that may help us decipher pain in this common, yet debilitating disorder.15 Through the modern era, progress has been made with the recognition of other peripheral and central features that offer a more nuanced understanding of this complex disorder within a biopsychosocial model. These features include altered motility or transit, impaired intestinal permeability, changes in intestinal secretion, immune cell activation, visceral hypersensitivity, and perturbed neural signaling within the enteric nervous system and bidirectionally along the gut-brain axis  rather than positively identifying pathophysiological targets for IBS therapy. Moreover, available tests do not address the specific feature of abdominal pain in IBS, which is independently correlated with increased symptom severity,21 impaired quality of life,22 and is a primary predictor for important clinical outcomes such as healthcare utilization.23 There is a clear need for usable biological assessments that will support a positive diagnosis of IBS, identify treatable mechanisms that underlie hypersensitivity, allow monitoring of treatment response, and promote cost-effective delivery of precision care. Under ideal circumstances, good biomarkers25 should provide objective, accurate and precise, reproducible, and quantifiable measurements that correlate with patient outcomes and provide clinically meaningful information beyond that which can be ascertained from history alone. Cost, feasibility, as well as patient acceptance are important additional considerations. Useful biomarkers should not require excessively invasive methods that subject patients to a potential risk of harm; acceptability and safety are especially important in the quest for biomarker discovery in IBS, a disorder that is associated with a major burden on patients and the healthcare system but carries an overall benign prognosis.Current guidelines26 While many insights have been gathered through preclinical and clinical studies, the full complexity of visceral hypersensitivity and pain is incompletely understood. Numerous factors including genetic variations; interactions between the gastrointestinal microbiome and host immune system and/or enteric nervous system; and perturbations in molecular events and chemical mediators of visceral nociceptive signaling may all contribute to the peripheral pathogenesis of abdominal pain in IBS.26–28 Centrally mediated mechanisms of pain in IBS that independently influence or augment peripheral pathways may further be affected by psychological factors, emotional distress or mood, chronic stressors, and bidirectional microbiome-gut-brain communication.29 Thus, a number of peripherally- and centrally mediated mechanisms of visceral hypersensitivity or pain have been explored in both preclinical and clinical studies of IBS -IBS mouse model of food-induced visceral hypersensitivity, investigators recently demonstrated local IgE production by B cells and plasma cells with subsequent colonic mast cell activation as a mechanism for food-induced visceral hypersensitivity.32 Other factors that may contribute to mast cell activation and have been examined in animal models of colonic hypersensitivity include increased expression of toll-like receptor 4 in colonic tissue,66, interactions with gut bacteria,68, bile acid-induced stimulation of mast cell-derived nerve growth factor (NGF),69 and stimulation by neuropeptides such as oxytocin via the Ca2+-nitric oxide synthase pathway.70 Activated mast cells may further trigger visceral pain via upregulation and activation of colonic nociceptors.30 In one recent study, Grabauskas et al.33 reported that abnormal synthesis and release of prostaglandin E2 by colonic mast cells activates the EP2 receptor on neighboring sensory neurons to induce visceral hypersensitivity in rats. Other immune-mediated mechanisms of visceral hypersensitivity that have been described through preclinical models include changes in lamina propria macrocyte phenotype or dendritic cells, altered cytokine signaling, and T-cell activation.71–74Although it is well recognized that no perfect animal correlate exists due to the complex clinical heterogeneity of IBS, animal studies serve a critical role in gathering basic insights into the biological mechanisms of visceral pain. Preclinical studies have implicated immune dysregulation involving intestinal immune cells and neural-immune interactions32 inhibition of myosin light chain kinase-mediated intestinal permeability improved visceral hypersensitivity in a Trichinella spiralis-infected PI-IBS mouse model;36 and decreased expression of tight junction protein, occludin, in colonic epithelial cells resulted in visceral hyperalgesia in rats.37 Multiple studies have also demonstrated a role for ion channels including voltage-gated sodium and calcium channels75–77, mechanical-gated Piezo proteins,78 and transient receptor potential (TRP) channels.80 In a trinitrobenzene sulfonic acid-induced chronic visceral hypersensitivity mouse model, administration of selective NaV1.1-activating toxin enhanced mechanosensory responses to suggest that NaV1.1 regulates mechanical hypersensitivity in IBS.42 Others have demonstrated that blockade of voltage-gated calcium channels, CaV2.2 and CaV2.3, inhibits colonic nociceptor excitability41 and that sensitization of TRP vanilloid 1 (TRPV1) mediates visceral hypersensitivity in rodents.40In addition to aberrant immune responses, there is evidence demonstrating contributions to visceral pain from other related and independent mechanisms. Epithelial barrier impairment may modulate colonic hypersensitivity through mucosal immune activation or by stimulating nociceptive drive. The effects of barrier dysfunction on pain have been well described in preclinical research. For example, increased colonic permeability to sodium fluorescein was associated visceral hypersensitivity in PI-IBS;82 Prior studies have shown that antibiotic treatment decreases luminal bacterial counts, enhances bacterial wall adherence, and prevents stress enhanced capsaicin-evoked visceral pain response in mice while sensitivity to colonic distension can be transferred through the fecal microbiota to rodents from patients with IBS.83 Preclinical studies of SCFA effects have shown than intracolonic acetate enhances sensitivity to colorectal distention40 while butyrate-enteric glial cell interactions may regulate NGF and colonic hypersensitivity in rats.48 Bacterial neuroactive mediators such as histamine have also been hypothesized to influence visceral hypersensitivity. In a recent study of diet-microbial interactions, germ-free mice colonized with high histamine-producing fecal microbiota exhibited visceral hyperalgesia and mast cell activation.31 The gastrointestinal microbiome has generated considerable interest due its potential for shaping visceral sensation through direct effects, interactions with the intestinal immune system and host genetics or diet, and its role in regulating bidirectional communication between the gut and brain.Animal models have further suggested that visceral hypersensitivity may be modulated by the gut microbiome and its mediators  that may act independently or in concert with other aforementioned mechanisms.84–87 altered neuroreceptor signaling ,88–91 guanylate cyclase C (GC-C) signaling,92 stress-induced activation or remodeling of the hypothalamic-pituitary-adrenal (HPA) axis, alteration of descending pain pathways, and sensitization of spinal afferents.93Other mechanisms underlying visceral hypersensitivity that have been identified through preclinical research include neurotransmitter/peptide-mediated hyperalgesia ,95 however, clinical trials of mast cell stabilizers such as ketotifen in patients with IBS have suggested that mast cell-induced visceral hypersensitivity is largely dependent on the actions of mast cell mediators including histamine on submucosal neurons35 rather than overall mast cell density. These findings are consistent with observational studies showing no clear relationship between colonic mucosal immune cells and visceral hypersensitivity in patients with IBS.96 In a recent study, detailed inspection of mast cell mediated pathways revealed that injection of food antigens into rectosigmoid mucosa was associated with increased mast cell activation and higher numbers of IgE+ mast cells in closer proximity to nerve fibers in patients with IBS  compared to controls; distance between IgE+ mast cells with nerve fibers was inversely correlated with abdominal pain severity.32 Higher levels of systemic proinflammatory cytokines have also been described in IBS patients with pain compared to those without;98 however, these data are varied and relationships of plasma or colonic cytokine profiles with changes in circulating immune cells including lymphocytes in the context of visceral pain are not fully delineated in patients with IBS.Evidence gathered from preclinical models of visceral pain and colonic hypersensitivity have guided clinical studies investigating the mechanisms of sensation and pain in patients with IBS, providing opportunities to develop new approaches for diagnosis and treatment. In patients, pain is commonly measured through quantitative traits including sensation thresholds during colonic and/or rectal barostat testing, symptom, or sensation ratings, and neuroimaging-based assessments of brain structure and function. Of the various mechanisms that have been demonstrated in animal studies, many have been examined in humans, with some showing promise as pathways for novel biomarker development. The role of immune dysregulation has been assessed in multiple clinical studies. Two systematic reviews have reported increased mast cell infiltration in the colon and small intestine in patients with IBS compared to healthy controls;38 Clinical studies have also provided data to support preclinical observations identifying ion channels as important factors in visceral pain including studies reporting positive associations between colonic transient receptor potential melastatin 8, rectosigmoid TRPV1, or small intestinal TRPV1 or TRPV3 expression levels and abdominal pain in studies of patients with IBS including multiple IBS subytpes,43–45 as well as associations of TRPV4 activation and pain scores in IBS-D.46 Genetic studies previously demonstrated associations of SCN5A-encoded Nav1.5 sodium channel mutations with increased symptoms including pain.47 Moreover, clinical trial data have suggested calcium channels as promising therapeutic targets for pain in IBS. Treatment with otilonium bromide, which blocks L-type calcium channel, reduced abdominal pain frequency in a randomized placebo-controlled trial 355 patients with IBS ;99 and treatment with pregabalin, which binds the α2δ subunit on voltage-dependent calcium channels, has been shown to reduce pain and increase sensory thresholds in IBS and especially in patients with IBS-D.101Epithelial barrier integrity and the “leaky gut” hypothesis has been examined and linked to visceral hypersensitivity and patients with IBS. In one systematic review and meta-analysis, estimated prevalence of barrier dysfunction in IBS ranged from 4% to 62%, was more common in those with IBS-D or PI-IBS, and positively associated with symptoms including pain.102 Admittedly, quantifying effects of 5-HT on visceral hypersensitivity in IBS has proven challenging. Early studies suggested a correlation between mucosal 5-HT and abdominal pain severity.103 However, others have been unable to confirm these observations through analysis of tryptophan hydroxylase 1 mRNA levels, SERT transcript levels, or 5-HT in rectosigmoid biopsies of IBS patients with and without rectal hypersensitivity.104 In one study, researchers attempted to quantify 5-HT by evaluating both 5-HT and its metabolite, 5-HIAA, in platelet-poor plasma to report lower 5-HIAA levels across all IBS subtypes compared to controls, but no significant differences in 5-HT levels between patients with IBS and controls Although promising, reasons for similarities across subtypes could not be fully explained and laboratory measurement of 5-HIAA levels for IBS remains investigational while relationships with nociception require further study.105 Despite promising preclinical data, measuring the effect of 5-HT on abdominal pain in patients with IBS is exquisitely difficult due to multiple factors including variations in sample processing, continued in vitro 5-HT metabolism post-sampling, the effects of fasting versus post-prandial states, expression and activity of SERT, transport of 5-HT in the liver/lungs/platelets, 5-HT half-life, and stability, as well as the effects of psychological comorbidities, diet, and consumption of tryptophan-rich foods, and medications.106In addition to investigating immune cells, epithelial barrier function, and ion channels, many clinical studies have attempted to characterize the role of 5-HT metabolism in IBS pathophysiology. 5-HT is a key gastrointestinal signaling molecule that that acts on both the enteric and central nervous systems  to modulate gastrointestinal motility, secretion, and nociception. Intestinal enterochromaffin cells produce the vast majority the body’s 5-HT, which is synthesized from tryptophan by tryptophan hydroxylase 1 (the rate-limiting enzyme for 5-HT biosynthesis). 5-HT signaling is ultimately terminated through uptake by serotonin reuptake transporter (SERT) across the plasma membrane where 5-HT is broken down into 5-hydroxy indoleacetic acid (5-HIAA) by monoamine carboxylase.Prevotella-9 genus with abdominal pain in patients with IBS ,58 correlations of fecal bacterial taxa with sensation and pain,59 reduction in pain and discomfort following fecal microbiota transplantation in patients with IBS-D or IBS-M and predominant bloating,60 and associations of Coprococcus and Clostridium XIVa and subcortical regions of the brain involved in pain processing.107 However, a clear relationship between microbial structure and pain in IBS has not been fully established and clinical data are often inconsistent or even contradictory.108–110 Microbial functions and mediators have also been investigated. While some preclinical studies suggested a role for bile acids in inducing visceral hypersensitivity,69 studies in patients have produced conflicting results.111–113 Examination of other microbial metabolites and functions have generated data implicating SCFA and microbial metabolism as contributors to visceral hypersensitivity. Vanhoutvin et al. previously demonstrated direct effects of luminal butyrate administration50 on sensation thresholds in healthy controls while others have reported associations of fecal microbial enzymes involved in animal carbohydrate metabolism with IBS severity.114 One recent study in 29 patients with IBS . and 23 healthy controls further described fecal microbial genes involved in neurotransmitter and SCFA metabolism to be associated with morphometry of the posterior insula, a primary visceral cortex.115Direct effects of the intestinal microbiome on pain sensation have also been suggested through clinical studies conducted in patients with IBS. In recent years, researchers have reported associations of relative abundance of colonic mucosal 52 negative associations of NGF expression in rectosigmoid biopsies and visceral sensitivity thresholds in IBS-D,116 and relief of abdominal pain with linaclotide treatment via GC-C signaling in IBS-C.92 Endocannabinoid-like dietary compounds resulted in improved abdominal pain severity in a randomized controlled trial of 54 patients with IBS  and 12 healthy controls.54 Although convincing clinical trial data supporting the use of cannabinoids for the treatment of visceral pain in IBS are lacking, peripherally restricted agents are recognized as a promising avenue for treatment (reviewed in reference 69). Researchers have also implicated alterations in brain structure, function, and functional connectivity of brain regions related emotional, autonomic, and descending modulatory responses to visceral afferent pain signals117–121 as well as stress-mediated HPA-axis dysregulation in IBS pathophysiology and visceral pain,63 although the specific role of HPA-axis activation in the modulation of abdominal pain has been questioned.122 Integrative models have proposed a role for reciprocal interactions between brain networks referred to as the “brain connectome” that co-exist alongside interacting networks with the gut such as the enteric nervous system and the gut microbiota (i.e. the “gut connectome’) in the development, maintenance, and subjective experience of chronic visceral pain  between the relative abundance of Prevotella genus and Prevotella_9 copri from sigmoid biopsy specimens and abdominal pain based on 16S rRNA gene sequencing in a large cohort of patients with IBS, which included patients of multiple IBS subtypes. Others108 have examined the fecal microbiome in population-based cohorts to demonstrate a relationship between stool microbiome β-diversity by 16S rRNA gene sequencing, differences in the distribution of Ruminococcaceae, Prevotella, and Bacteroides clusters between those with and without abdominal pain, decreased representation of the Prevotella-predominant enterotype in those with pain, and significant differences in abundances of individual taxa between those without and without pain. Notably, Prevotella was decreased in those with pain and could predict the absence of abdominal pain. Unfortunately, these findings again, could not be replicated in an independent general population-based cohort110 despite the use of similar specimen types and the marker-gene (16S) based approach. In general, comparisons of high throughput sequencing data across studies have been hampered by inconsistencies in sample processing, preparation, sequencing techniques and analytical approaches. While the International Human Microbiome Standards Consortium has recognized that stool sampling requires minimal processing to isolate and maintain microbial DNA and RNA and studies have increasingly targeted the V4 to V3-V4 hypervariable regions for 16S sequencing, substantial phenotypic heterogeneity persists across studies due to factors such as sample size, study design, consideration of patient-specific factors , and sequencing approach (16S rRNA vs. random shotgun metagenomic sequencing) which has precluded the identification of a consistent structural bacterial biomarker for pain in IBS.134 While acknowledging current limitations, metagenomic sequencing continues to improve and will be strengthened by initiatives to standardize approaches to study design, sample collection and processing, analysis methods, and data sharing. It has further offered hope by providing researchers the ability to expand on initial observations through investigation of bacterial gene representation and the assessment of non-bacterial members of the gastrointestinal microbiome. Recent studies have brought to light the potential role of the gastrointestinal virome in IBS pathogenesis which may involve interactions with the gastrointestinal bacteriome;136 however, direct links between virome composition and visceral hypersensitivity in IBS have not yet been reported. Fungal dysbiosis, on the other hand, has been directly correlated with pain and visceral hypersensitivity in preclinical and clinical research. Botschuijver et al. previously reported differences in fecal mycobiome composition including α-diversity and fungal biomarker species among healthy controls and patients with normo- and hyper-sensitive IBS based on sequencing of fungal internal transcribed spacer regions amplicons. Corresponding animal experiments demonstrated reduction in visceral hypersensitivity with fungicide treatment, restoration of hypersensitivity with transfer of cecal mycobiomes, mast cell histamine release with fungi stimulation, and evidence for fungal recognition via the Dectin-1/Syk pathway with activation of the host immune system as a driver of visceral hypersensitivity.133 A subsequent study described reversal of post-stress hypersensitivity with fungicidal miltefosine and reduced TRPV1-induced hypersensitivity in an IBS-like rat model well.137 Studies applying high throughput sequencing technologies for evaluation of the gastrointestinal mycobiome, virome, and bacterial genes in modulating visceral sensation in IBS are eagerly awaited. Presently, the available data highlight the importance of considering the full complexity of microbiology ecology through integrated analysis of co-existing bacterial and non-bacterial microbial communities to develop usable microbiome biomarkers for visceral pain. Other important limitations of human microbiome research in IBS include the difficulty of identifying potentially important, but low-abundant taxa, and the incomplete assessment of microbial functions. Moving forward, the clinical relevance of taxonomic profiling and microbial metagenomics may be further advanced through complementary transcriptomics, proteomics, and metabolomics that are designed to measure microbial functions and microbe–host interactions important for mediating nociception in IBS.Preclinical data and human studies show direct links between the gastrointestinal microbiome or microbial byproducts and critical aspects of gastrointestinal physiology including visceral sensation and nociceptive signaling  to report differences between patients with IBS compared to controls and between IBS subtypes as well as a correlation between gastrointestinal metabolites and physiological functions  or symptoms . Measurement of fecal bile acids has been validated a biomarker for bowel functions and clinical phenotype in IBS; however, their role as biomarkers for visceral hypersensitivity appear more limited.146 Targeted assessment of SCFA effects on visceral sensation in preclinical studies have provided evidence for increased colorectal hypersensitivity with luminal administration of SCFA including butyrate.49 However, these data have not been verified in clinical studies, including studies that have reported decreased pain scores according to the barostat protocol with daily administration of butyrate enemas in healthy volunteers.50 Overall, validating luminal SCFA as individual biomarkers for pain or treatment responsiveness in IBS has proven challenging31 due to the dynamic nature  and role of SCFA within the gut. SCFA may also be linked to sensation through unmeasured mechanisms including the metabolic pathways for SCFA production and downstream SCFA effects on host physiology. For these reasons, future investigations of SCFA profiles will benefit from integrated analysis incorporating multi-omics tools that will offer a fuller picture of the role for SCFA as candidate IBS biomarkers and mediators of pain.Metabolomics data serve as a rich source of information on the small molecule mediators, including microbial metabolites, that influence visceral hypersensitivity and could be used as novel biomarkers for pain in IBS . Microbi31 as well as metabolites affecting the serotonin biosynthetic pathway. In a prior study conducted in patients of any IBS subtype, metabolomics assessments demonstrated decreased urinary histamine following a low fermentable oligosaccharide, disaccharide, monosaccharide, and polyol diet, which was associated with reduced symptom severity as well as increased bacterial richness.138 Concentrations of histamine and its metabolites have also been associated with TRP channel sensitization, and therefore recognized as important candidate biomarkers for visceral pain perception in studies of patients with IBS .139 Another recent study analyzed fecal metabolomics data in patients with IBS-D to observe increased tryptophan concentrations in patients with symptom exacerbation.140 Others have studied nontargeted metabolite profiling including volatile organic compounds in breath and fecal metabolite profiles as potential tools, and therefore biomarkers, for identifying clinical symptoms and outcomes.147 Despite promising signals, rigorous assessment with standardization of optimal specimen acquisition and technical methods148 as well as validation in independent cohorts will be required before such metabolomic panels can be adapted as biomarkers for evaluating pain or hypersensitivity in clinical practice.Data derived from metabolomics studies have also identified other important mediators including histamine, which may be produced through mast cell degranulation and as a bacterial metabolite149 The increasing availability of large-scale population-based biobanks have facilitated advancements in genomic research in IBS150 that may accelerate our understanding of genetic risk determinants in IBS. In one genome-wide association study of 53,400 people with IBS, six genes  were found to be associated with IBS susceptibility including four genes linked to mood, anxiety, or expressed in the nervous system. Although genetic mechanisms of visceral hypersensitivity were not specifically examined, these findings provide evidence that genes involving neuronal pathways may play a major part in shaping abnormal brain-gut interactions in IBS.151 Recently, investigators identified new candidate genes involved in degradation of epidermal growth factor (SNX13) and histone methyl-lysine binding protein (L3MBTL4) that were associated pain severity and frequency in IBS.152 The reader is referred to a prior paper that provides an in-depth review on the genetics of visceral sensation in IBS.27 Focused genomic studies have also implicated SERT gene (SLC6A4) polymorphisms in visceral hypersensitivity in IBS. However, a systematic review and meta-analysis examining the relationships between SLC6A4 polymorphisms and IBS revealed no significant overall association. Instead, associations of mutations in the polymorphic region (5-HTTLPR) of SLC6A4 and IBS-C were observed only in the East Asian population to suggest that assessment of genetic variations may be more useful for pharmacogenomics and in predicting responses to drug therapies153 than as biomarkers for IBS. Although large-scale studies have hinted at genetic heritability of IBS and visceral hypersensitivity,151 functional contributions of genes to pain in IBS may be modest, determined by epigenetic modification and gene expression, and influenced or even overshadowed by environmental factors.Preclinical and clinical studies investigating genetic determinants of pain and visceral sensation have focused largely on identifying genetic variations associated with immune dysregulation, barrier function, neurotransmitter biosynthesis and metabolism, cannabinoid receptors, ion channel dysfunction, and G-protein coupled receptor expression alongside other mechanisms.154 Studies of epigenetic contributions to visceral pain in IBS have examined epigenetic modulation of stress-associated activation of the HPA-axis, SERT, and TRPV1. Epigenetic marks defined by decreases in histone acetylation at the corticotrophin-releasing hormone (CRH)- and glucocorticoid-receptors were recently shown to contribute to stress-mediated visceral hypersensitivity in rats.61 Others have published evidence to suggest a role for miRNA in regulating gene expression of SERT and TRPV1. Liao et al.86 previously demonstrated upregulation of miR-24 in patients with IBS and SERT as a potential gene target; treatment with miR-24 inhibitor increased colonic pain and nociceptive thresholds in mice. In another study, colonic miR-199a/b correlated with pain and TRPV1 expression in patients with IBS-D.155 In the future, it is possible that integrated studies examining genetic variants, epigenetic markers, gene expression and gene-environment interactions through complementary analyses of the microbiome and metabolome will offer refined genetic biomarkers targeting diagnosis and drug development for visceral hypersensitivity in IBS.Epigenetic modulation of gene expression has been proposed as another important factor in shaping gene-environment interactions that influence mechanisms of visceral pain in IBS and thus, hold promise as novel diagnostic and prognostic biomarkers. Epigenetic changes that mediate visceral hypersensitivity can include histone tail acetylation or modification, differential microRNA (miRNA) expression, and DNA methylation.156 Microarray analysis of rectal mucosal specimens and bioinformatics approaches have recently implicated genes including GRPR, neuropeptide FF (NPFF), and TRPA1 to be associated with abdominal pain in IBS-D.149 Gene expression profiling of colonic biopsy specimens with microarray analysis has also demonstrated pathways involved in 5-HT metabolism to be differentially expressed between IBS and controls, suggesting transcriptional patterns of serotonergic gene expression including TPH1 as potential biomarkers for IBS.157High throughput transcriptomics technologies including microarray and RNA-sequencing (RNA-seq) have emerged as valuable tools for unraveling the complexities of the functional genome and pathobiology of visceral hypersensitivity in IBS. The role of transcriptomics biomarkers for IBS and more specifically, for abdominal pain, has been explored in multiple studies. In a pilot cohort of 9 patients with IBS-D and 9 controls, RNA-seq of rectosigmoid biopsy specimens demonstrated transcriptome changes involving neurotransmitters, ion channels, immune function and cytokines, barrier function, as well as cell adhesion in patients with IBS.158 Transcriptomics has also enabled researchers to investigate mechanisms of stress-induced visceral hyperalgesia related to CGRP, mast cells, and CRH to identify novel gene targets for the diagnosis and treatment of IBS. In a preclinical WAS model, gene expression of colonic mast cells was analyzed by RNA-seq to demonstrate increased expression of genes related to response to CRH, transcription regulation, mast cell activation, inflammation, and proliferation. Findings suggested that CGRP may regulate mast cell activation and mediate stress-induced hypersensitivity through CRH in the brain-gut axis.87 In patients with both IBS-D and IBS-C, transcriptomics analysis of small intestinal biopsies have also demonstrated correlations of abdominal pain and rectal sensation thresholds with changes in duodenal TRPV1 and TRPV3 expression. Results indicated that visceral hypersensitivity in IBS may be mediated by altered intestinal chemosensitivity and that intestinal transcriptomics may be useful for evaluating visceral pain45 Others have applied RNA-seq to study bacterial transcriptomes and gene expression in peripheral blood. These studies have detected differences in bacterial transcriptomes related to butyrate production and neuroendocrine hormones between IBS-D and health140 as well as relationships between expression of inflammatory genes in peripheral blood mononuclear cells and brain regions within the salience network, which is involved in modulating cognitive processing including attention to pain;159 however, direct associations with abdominal pain and colonic sensation were not reported. Although encouraging, findings from recent transcriptomics studies should be interpreted cautiously considering factors such as the relative inaccessibility of the intestine with limited sample collection via traditional biopsy methods and cell-to-cell heterogeneity that may be masked by bulk transcriptomic profiling. While no single transcriptomics-biomarkers have been fully validated for clinical use, transcriptomics including next-generation single-cell RNA-seq represent an important tool for disentangling the genetic mechanisms of abdominal pain in IBS and represents a crucial layer of multi-omics discovery research  rodent model, Wiley et al. demonstrated changes in gene expression within colonic epithelial cells of genes involved in inflammatory and immune responses including cytokine- and chemokine-receptor, adherens, and tight junction genes to suggest alterations in the expression of these genes as prospective biomarkers for impaired permeability and visceral hypersensitivity in IBS.research .Table 424 In a recent study of patients with PI-IBS, fecal metaproteomic specimens assays were used to identify host proteases associated with high proteolytic activity in feces, which has been linked to barrier dysfunction and visceral hypersensitivity. Complementary analyses of the fecal microbiome further revealed that specific members of the intestinal microbiome could suppress proteolytic activity through microbial β-glucoronidase.160 Metaproteomics has also been used to study colonic mucus samples from patients with IBS-D to demonstrate high rates of Brachyspira colonization through identification of bacterial peptides/protein, as well as associated changes in the host mucus proteome involving inflammatory mediators and membrane remodeling proteins. Researchers demonstrated that spirochetosis was not associated with symptom severity or rectal sensitivity, but was correlated with other clinical and quantitative traits including transit, bowel symptoms, mucosal inflammation, and mast cell activation that have been linked pain in IBS.161 While still in the early stages of application, proteomics-based tools may represent a strategy to overcome technical and conceptual challenges in biomarker discover. Similar to other omics strategies, metaproteomics may benefit from complementary single-cell approaches to account for cell-specific heterogeneity.Modern proteomics technologies have shown great promise for addressing key bottlenecks in biomarker development for abdominal pain and IBS, offering a unique advantage of more closely representing endophenotype and clinical phenotype than genomics or transcriptomics-based analyses. Untargeted proteomics may be used as a strategy for high throughput biomarker screening that can be further validated by targeted proteomics approaches.117 compared resting state functional connectivity between normosensitive and hypersensitive patients with IBS to report that visceral sensitivity correlated with changes in functional connectivity of networks involved in salience, sensory processing and interoception. In the future, big neuroimaging data may offer promising brain-based biomarkers for identifying IBS endophenotypes or predicting responses in symptoms such pain to pharmacological treatments and other therapies.120Other omics approaches that have considered in the effort to develop reliable biomarkers capable of measuring pain and treatment response in IBS include “connectomics” which utilizes noninvasive neuroimaging to study brain neural elements and networks. A prospective study of 84 IBS patients from a randomized controlled trial of cognitive behavioral therapy (CBT) examined brain resting state functional connectivity and microstructure to observe reduced functional connectivity between brain regions associated with sensorimotor, default mode, salience, and emotion regulation networks as well as changes in white matter in patients exhibiting treatment responses, measured by IBS symptom severity including features such as pain. However, prediction to CBT response was more accurately predicted by baseline microbiota than by neuroimaging data to suggest microbial composition as a more promising biomarker. In another study, Icenhour et al.143 Another recent study used multi-omics approaches to examine fecal proteolytic activity, which has been associated with increased symptom severity and higher intestinal permeability, in patients with PI-IBS to observe increased abundance of human serine proteases using metaproteomics.160 Transcriptomic analysis of the colonic mucosa revealed that differences in host proteases were not explained by mucosal expression of proteases of protease inhibitors. Instead, complementary metagenomics and metabolomics analyses determined intestinal proteolytic activity to be regulated by the commensal microbiome including Alistipes putredinis and gut microbial β-glucuronidase-mediated production of unconjugated bilirubin, suggesting a novel host-microbial interaction that could represent a future biomarker or therapeutic target for PI-IBS and more specifically, impaired permeability or visceral hypersensitivity. Others have employed 16S rRNA sequencing, bacterial metatranscriptomics, and metabolomics to examine contributions of microbial structure and function to clinical symptoms and phenotype among healthy controls and IBS-D patients with and without symptom exacerbation to identify changes in fecal omics profiles associated with symptom exacerbation including decreased Bifidobacterium longum as well as differences SCFA and neurometabolites and bacterial gene transcripts related to butyrate production and neuroendocrine hormones, highlighting the utility of multi-omics profiling of the colonic microenvironment as an indicator of IBS symptom phenotype.140 Multi-omics studies have also been performed to generate novel insights into the interactions between the gastrointestinal virome, bacteriome, host transcriptome, and metabolome in healthy controls and patients with IBS. In a subset of individuals from a larger multi-omics study, investigators recently reported temporal stability of the intestinal virome, associations between the virome and host colonic genes linked to immune responses and epithelial barrier function, associations of bacteriophage composition with clinical phenotype, as well as correlations between viral contigs and bacterial species, functions, and the primary bile acid chenodeoxycholate.136 These findings, while preliminary, provide a comprehensive assessment of the broader microbial ecosystem and the interactive contributions of bacterial and non-bacterial microbes to IBS pathophysiology through combined omics analyses that could guide the future selection of important diagnostic and/or predictive microbial biomarkers. New machine-learning approaches for integration of paired 16S rRNA sequencing and host transcriptome data from colonic mucosal specimens have further been developed to identify host gene–microbe interactions that are specific to IBS and common to various gut disorders including IBS, colorectal cancer, and inflammatory bowel disease.162 Priya et al. observed overlapping host gene pathways to exhibit disease-specific correlations with microbial taxa. For example, the RAC1 pathway, which is involved in immune response and intestinal mucosal repair, was correlated with Bacteroides massiliensis, Bifidobacterium, and Odoribacter in IBS. Other IBS-specific associations included associations of Prevotella with pathways for sumoylation (DNA damage response and repair proteins), B. massiliensis with arachidonic acid metabolism , Peptostreptococcaceae with HAS2 (colonic epithelium protection), and Streptococcus with DPEP2 (macrophage inflammatory response). Systems biology approaches have also been described through combined analyses of brain connectivity data with other omics datasets in patients with IBS. In a cross-sectional analysis of 102 women  Sarnoff et al. utilized a multi-omics data integration analysis method for biomarker discovery (DIABLO) to integrate fecal microbiome, metabolome, resting-state connectome, and clinical data to identify brain-gut-microbiota signatures that differentiated IBS subtypes from healthy controls.163 Bloating and visceral sensitivity were negatively associated beneficial taxa such as Blautia obeum and with brain connectivity involving the orbitofrontal cortex. Although visceral perception was not directly assessed, these findings hold significant potential as they may pave the way for future research in biomarker development through the study of multidimensional omics data.Advancements in metataxonomics, metagenomics, metabolomics, genomics, epigenomics, transcriptomics, proteomics, and even connectomics have made it apparent that integrated analyses of complementary omics data will be crucial for understanding the complex biological networks that underlie the interactions between the gastrointestinal microbiome and human physiology in a clinically meaningful manner. Through multi-omics discovery science, researchers have begun to delineate novel biological pathways and dynamic microbe–host interactions that may drive IBS pathophysiology; these insights may further facilitate the identification of candidate biomarkers with diagnostic, prognostic, or therapeutic value . In a lo164Studies such as these offer invaluable information on the host–microbe interactions that mark and shape IBS pathophysiology, while outlining powerful strategies for data integration. Increasingly, researchers have begun embracing integrative multi-omics as a powerful and systematic approach to studying the many layers of molecular and biological events that link genotype to clinical symptoms, with immense potential for identifying reliable candidate biomarkers for visceral pain in IBS. Indeed, some have begun combining multi-omics with digital therapeutics to demonstrate that changes in IBS symptom severity can be modeled through a mixture of genomic and microbiome predictors, suggesting that prognostic multi-omics biomarkers for IBS symptomology are already on the horizon.Prevotella or microbial β-glucoronidase as well as non-bacterial microbes; metabolomics have identified critical mediators of host physiology; genetic or epigenetic and transcriptomic research have shed light on important genetic differences involving ion channels, serotonin metabolism, immune activation, and HPA-axis dysregulation; proteomics has provided opportunities to infer functional microbe–host interactions within the gut; while connectomics has enabled researchers to employ new technical and conceptual approaches to studying brain signatures as biomarkers for visceral pain perception. In parallel, advanced computational tools and statistical methods designed to combine large-scale data from different omics approaches are continuously emerging, allowing researchers to screen for biomarkers that could be used for IBS and visceral hypersensitivity through simultaneous investigation of multiple functional layers within overlapping biological systems that include contributions from both the host and the resident microbiome. As major advancements are underway, there remain important limitations of omics-based tools that will need to be addressed before these approaches can be used in the clinical setting. Analysis of compositional microbiome-based biomarkers for pain in IBS is challenged by significant heterogeneity across studies and an incomplete understanding of microbiota–host interactions. Metabolomics research holds significant promise, but inherent technical limitations of metabolite quantification remain and clinical validation is required to fully interpret the pathophysiological consequences of targeted and untargeted metabolomics data. Genetic contributions to pain in IBS may be largely shaped by epigenetics, gene expression, and environmental variables. Standardization and refinement of neuroimaging techniques including the incorporation of multimodal imaging modalities will be needed to more fully understand the impact of the central nervous system on bidirectional brain–gut interactions. Furthermore, bulk omics-based approaches aimed at conducting high throughput analyses of biological molecules and samples may mask or fail to capture microbe- or cell-specific variations in qualitative and quantitative functions.165 For these reasons, further research including longitudinal work, large-scale studies, integration of novel single-cell technologies, and incorporation of clinical meta-data is necessary to validate the findings and deep biological insights that are being gathered through innovative multi-omics science. Yet, with the arrival of the omics era, the biomedical research community is now positioned to investigate dynamic microbe-host interactions and that can be leveraged to accelerate precision medicine. Together, multi-omics approaches hold immense potential for driving novel biomarker prediction, development, and implementation to enhance the diagnosis and treatment of visceral pain in IBS.High throughput single-omics technologies have fueled significant progress in our understanding of the biological mechanisms of visceral pain and provided new opportunities for biomarker discovery research in IBS as well as other disorders of gut–brain interaction and diseases of the digestive tract for which mechanisms of pain are not yet fully defined. Individually, omics technologies have revealed key mechanisms within microbial communities and host molecular pathways that may underlie the development of visceral hypersensitivity in IBS. Microbiome studies have detected structural and functional features including individual taxa such as


The most consistent finding was hypo-frontality in the clozapine-resistant group compared with the clozapine-responding group with possible differences in frontal-striatal-basal ganglia circuitry as well as the GABA level between the two treatment-resistant groups. Additional statistically significant findings were reported when comparing clozapine-resistant patients with the FLR group, including lower cortical thickness and brain volume of multiple brain regions as well as lower Glx/Cr level in the dorsolateral prefrontal cortex. Both treatment-resistant groups were found to have extensive differences in neurobiological features in comparison with the FLR group. Overall results suggested treatment-resistant schizophrenia is likely to be a neurobiological distinct type of the illness. Clozapine-resistant and clozapine-responding schizophrenia are likely to have both shared and distinct neurobiological features. However, conclusions from existing studies are limited, and future multi-center collaborative studies are required with a consensus clinical definition of patient samples, multimodal imaging tools, and longitudinal study designs.This systematic review aimed to review neuroimaging studies comparing clozapine-resistant schizophrenia patients with clozapine-responding patients, and with first-line antipsychotic responding (FLR) patients. A total of 19 studies including 6 longitudinal studies were identified. Imaging techniques comprised computerized tomography (CT,  Despite adequate trials of at least 2 antipsychotic medications with sufficient dose and duration, ~15–30% of patients experience persistent symptoms5, and are defined as treatment-resistant schizophrenia (TRS)6. At present, clozapine is the only antipsychotic medication that is effective in ameliorating symptoms of TRS7 with evidence showing clinical efficacy in both short and long-term studies11. However, 30–70% of patients with TRS show inadequate response to clozapine12, and are categorized as ultra-treatment-resistant schizophrenia (UTRS)6. UTRS patients are associated with poorer clinical and functional outcomes compared to non-treatment-resistant schizophrenia (NTRS) and TRS patients who responded to clozapine5. Though delay in clozapine prescription is consistently identified as a factor associated with poor clozapine response13, multiple barriers to clozapine prescription are suggested including concerns of side effects14. Understanding factors associated with clozapine non-response, including neurobiological mechanisms may contribute to the development of targeted interventions to improve outcomes. Of the many studies examining predictors or associated factors contributing to clozapine responses, relatively few consistent effects were reported including younger age, fewer negative symptoms at onset and paranoid schizophrenia subtypes15; consistent biological predictors were lacking16.The clinical course of schizophrenia shows huge interpatient variability, ranging from complete remission to persistent severe symptoms with significant functional impairment17, with a growing focus on presynaptic dopaminergic dysfunction18. A meta-analysis of 44 studies using positron emission tomography (PET) or single-photon emission computed tomography (SPECT) reported a significant increase in synthesis and release of striatal dopamine in patients with schizophrenia19. Furthermore, the magnitude of dopamine synthesis increase predicted the clinical efficacy of dopamine receptor antagonist antipsychotics, and thus the treatment response21. In contrast, a cross-sectional study found a lower dopamine synthesis capacity in TRS than NTRS patients22. Coupled with the lower affinity of clozapine for dopamine receptors23, a different underlying of pathophysiology for TRS was proposed, as well as the possibility of using response to antipsychotic medications to characterize biologically distinct subtypes of illness24.Earlier studies suggested that a disturbance in dopaminergic transmission is central to schizophrenia1H magnetic resonance spectroscopy (1H-MRS) studies, glutamate hypothesis is a complementary theory of schizophrenia, highlighting the role of upstream N-methyl-D-aspartate (NMDA) receptor hypofunction in causing a downstream cascade of neurotoxicity26. In more recent cross-sectional studies, TRS patients showed significantly higher glutamate levels in the anterior cingulate cortex (ACC) than the NTRS patients27 and healthy controls28. Moreover, higher ACC glutamate levels were associated with poorer treatment response to non-clozapine antipsychotics29. Meta-analyses of structural magnetic resonance imaging (MRI) studies reported subcortical volumetric reduction in schizophrenia patients compared to healthy subjects31. Specifically, cortical thinning was observed in the dorsolateral prefrontal cortex (DLPFC) of TRS patients compared to NTRS patients32. These neuroimaging studies highlighted the possibility of different biological mechanisms in TRS and NTRS and suggest reducing ACC glutamate levels may be a strategy for intervention in TRS.Evidenced by the two meta-analyses of 33. Despite growing evidence supporting structural brain changes and differential neural mechanisms between NTRS and TRS, the neuroimaging evidence comparing TRS groups, including those who respond to clozapine (CRS) with UTRS is sparse and inconsistent16. An improved, systematically derived understanding of the neurobiological substrates of TRS as well as differences between CRS and UTRS would contribute to understanding the mechanisms of clinical efficacy of clozapine in TRS, and could facilitate greater application of existing clozapine treatment as well as facilitate development of novel interventions.Over decades, understanding the mechanisms of clozapine in TRS has been challenging due to the heterogeneity of TRS and the complex interaction between receptors and neurotransmitter systemsThe aim of this review was to address the current research gap by systematically reviewing neuroimaging studies comparing patients with UTRS with other stages of the illness, particularly TRS responding to clozapine to provide insight on potential distinct neural mechanisms among UTRS patients. The results may provide further information on the possible presence of unique neurobiological characteristics of different subtypes of schizophrenia corresponding to characteristics of treatment response.Systematic searches of relevant articles were conducted from the electronic database, including Ovid Medline, Embase, Pubmed, PsychINFO, and Web of Science, from inception to 20 August 2022. An additional search was conducted from 21–30 August 2022 to confirm the inclusion of all relevant studies. Studies that were published in English in a peer-reviewed journal with study samples including schizophrenia, schizoaffective disorder, and schizophreniform disorders according to Diagnostic and Statistical Manual of Mental Health (DSM) or International Classification of Diseases (ICD) criteria, having an operationalized definition of UTRS status with comparison of CRS and patients with other treatment response characteristics using any form of neuroimaging approach were included. All cross-sectional and longitudinal studies were included. Conference abstracts, theses, and editorials were excluded. References from other review articles were examined for relevant studies. The review protocol was registered in the public domain .An electronic database search was conducted using the following syntax as search terms: (“ultra-treatment resistant”) OR (“ultra-resistant schizophrenia”) OR (“non clozapine responder schizophrenia”) OR (“clozapine resistant schizophrenia”) OR (“treatment refractory schizophrenia”) OR (“clozapine nonresponder”) AND . The results of the systematic review are reported based on the PRISMA guidelines Fig. .Fig. 1PRAfter removal of duplicates, one of the reviewers (TP) conducted a first-level screening of the titles and abstracts. References of included studies were screened for eligibility. Eligible studies were selected for full review and data extraction. A second reviewer (SKWC) independently conducted the review process. Inconsistencies were resolved through consensus meetings.Basic information from the eligible studies was extracted independently by two reviewers, including study characteristics , participant details (number of subjects per group), clinical characteristics, statistical analysis used, and main study outcomes. Due to different definitions of UTRS, details of the inclusion criteria and definition of each patient group were documented. Inconsistencies were resolved through consensus meetings.To align the different names and definitions of the illness status used in various studies, the following definitions are used in the current review: first-line responders (FLR) are those who show adequate response to non-clozapine antipsychotic medications; treatment-resistant schizophrenia (TRS) are those who failed to show an adequate response to at least 2 previous non-clozapine antipsychotics with at least 6 to 8-week trials; TRS that respond to clozapine (CRS) are TRS patients with symptomatic improvement after receiving adequate clozapine trials; TRS that do not respond to clozapine (UTRS) are TRS who failed to show an optimal response after adequate clozapine trials. Studies were grouped based according to brain region of interest and imaging modalities used.Joanna Briggs Institute (JBI) appraisal tools were used to assess the methodological quality of all included studies. This tool allows consistent and transparent judgment of the quality of parameters including recruitment procedures, sample size, and the degree of appropriateness of statistical analysis. JBI critical appraisal tools for cohort and longitudinal studies were used as appropriate. These tools are comprised of 10 questions that address study design, the methodology, and the statistical analysis used and for each question, the risk of bias assessed using ‘Yes’, ‘No’, ‘Unclear’, and ‘Not applicable’. Two raters (T.P and D.M) completed the critical appraisal tool independently and any discrepancies were resolved through discussion.A total of 308 studies were identified based on search keywords; 19 studies fulfilled the inclusion criteria and were finally included in the current review  and FLR, patients with both treatment-resistant groups had significantly smaller overall gray matter volume (GM) and all patient groups had smaller whole brain volume and white matter (WM) volume than the healthy controls. However, no significant differences were seen between the UTRS and the CRS37. In a longitudinal study of response to clozapine, marginally more cortical thinning of the left medial frontal cortex and the right middle temporal cortex was seen for the UTRS in comparison with clozapine responders, but overall differences in brain volumes and cortical thickness between patients and healthy controls were reported at either time point38.Five studies reported the global structural differences between UTRS patients and patients with other treatment response characteristics Table . An earl39. However, the significance disappeared after adding head motion as a regressor. Furthermore, CRS patients had overall lower FA than health controls, the FLR, and the UTRS patients, though none of the post-hoc analyses survived corrections for multiple testing39. Only one functional MRI study investigated whole brain functional connectivity and reported that UTRS had the weaker connectivity in 3 networks including the cerebella-frontal, cingulo-frontal-temporal, and fronto-parietal when compared with healthy controls but no differences when compared to other patient groups40. The connectivity between controls, FLR, and CRS patients was found to be similar.Only one study reported the white matter microstructure using track-based spatial analysis of diffusion tensor imaging data, and a significantly higher fractional anisotropy (FA) of the right superior longitudinal fasciculus in the UTRS patients compared with the CRS patients was found36. These findings suggest that enlarged frontal sulcal widening could be a unique structural characteristic of UTRS. UTRS showed significantly greater cortical thinning in the left medial frontal cortex than CRS longitudinally38, while no differences were seen when investigated cross-sectionally41. Another MRI study reported no differences in cortical thickness between UTRS and CRS but a significantly greater mean diffusivity was found in UTRS compared to CRS after controlling for age and gender42. However, a greater cortical thinning in the frontal regions was observed in UTRS compared with healthy controls and FLR regardless of age, sex, chlorpromazine equivalent (CPZ) daily dose, and PANSS total scores41. In addition, UTRS demonstrated a significantly greater mean diffusivity in the ACC compared to healthy controls after controlling for age and gender42.Thirteen studies reported results of analyses of frontal lobes; three used CT, three used MRI, three used SPECT, and four used MRS Table . A large99Tc-labeled hexamethyl-propylene-aminoxine (HMPAO) SPECT studies were conducted  in UTRS compared to CRS at baseline. Furthermore, no changes in cortical frontal perfusion were observed in the UTRS group, whereas the responder group had a significant reduction in cortical frontal perfusion44. The later SPECT study found a significant increase in frontal/caudate perfusion ratio in the clozapine responder group but not in the non-responder group45. When compared with HC, a significantly lower perfusion value at the left DLPFC was noted in UTRS at baseline44. Furthermore, improvement of digit span-forward was significantly correlated with increase in percentage change in the right frontal/caudate perfusion ratio, whereas a significant relationship between improvement of word fluency and increase in percentage change in both right and left frontal /caudate perfusion ratio was seen.Three ed Table . In the 46, nor Glx/creatinine (Glx/Cr) level47, likewise for the glutathione level in the dorsal anterior cingulate cortex (dACC)48 (Table 47. A recent study found a higher gamma-aminobutyric acid (GABA) level in mid-cingulate cortex (MCC) in UTRS compared with CRS after controlling for smoking status, sex, education, GM/(GM + WM), and age but no differences in Glx levels were found49. Furthermore, all four spectroscopy studies reported no significant relationship between the levels of these neuro-metabolites and clinical or cognitive function scores.Three spectroscopy studies found no group differences (clozapine responders or non-responders) in glutamate or Glx (glutamate and glutamine) levels in the dorsolateral prefrontal cortex (DLPFC)48 Table . One stu45. When compared with FLR, an extensive reduction of GM volume in the right parietal operculum was observed among UTRS37.Two studies (one MRI and one SPECT) examined the parietal lobes Table  without 41, and no significant difference in perfusion ratio between the groups43. However, UTRS showed a significant GM reduction in the right lateral occipital cortex when compared with healthy controls, whereas the CRS patients showed a significant GM reduction in the lateral occipital cortex when compared with the FLR group37. Although no significant differences in cortical thinning in occipital lobes were found between UTRS and CRS, a significantly greater cortical thinning in UTRS was found in occipital gyri when compared with healthy controls, in addition to a more extensive thinning in the left occipital gyri than FLR41.Three studies (two MRI and one SPECT) investigated the occipital lobes region Table  reportin38. Other studies did not find any difference in cortical thickness41, no volumetric differences in lateral ventricle, hippocampus, and amygdala50, no GM volume differences of temporal cortex37, nor cerebral perfusion differences at the anterior and posterior temporal lobe43 between the two groups. When compared with FLR and HC, significant cortical thinning was found in UTRS41. A bilateral pattern of decreased GM volume was also found in the superior and middle temporal gyri when compared between UTRS and healthy controls37. Compared with FLR, a significant reduction of the GM volume in superior, middle, and inferior temporal gyri was seen in the CRS group37.Five studies examined temporal lobe regions Table  with onl51 and longitudinal studies52. However, UTRS showed a smaller mean striatal volume, globus pallidus, nucleus accumbens, pre- and post-commissural putamen, and pulvinar nucleus volume when compared against FLR51. Furthermore, both patient groups were found to have a reduction in volume of putamen and hippocampus compared with healthy controls50 but no differences were shown between UTRS and HC51.Six studies examined the basal ganglia region; three used MRI, one MRS, and two SPECT techniques Table . All thr47. Lastly, one 99Tc-labeled HMPAO SPECT study44 noted decreased perfusion in the bilateral basal ganglia in UTRS compared to CRS, while the other indicated a decreased perfusion in the left basal ganglia in CRS instead43. When compared with HC, UTRS had significantly lower perfusion in the basal ganglia44. Furthermore, another 99Tc-labeled HMPAO SPECT study found a significant increase in right and left frontal/caudate perfusion ratio in the CRS compared with the UTRS45.The study examining glutamatergic function reported a significantly higher Glx/Cr in CRS than UTRS in putamen after controlling for CPZ but no other significant differences in metabolites were detected in putamen between UTRS and other comparison groups51 or a longitudinal study50. A smaller pulvinar nucleus volume in UTRS compared to FLR and HC and a smaller mean thalamus volume was found between UTRS and HC after controlling for age, sex, total brain volume, education, tobacco use, life history of substance dependence or abuse51. In the two 99Tc-labeled HMPAO SPECT studies, both showed CRS had a significant decrease of perfusion in thalamus44.Four studies examined the thalamus region, two using MRI and two SPECT Table . No sign37.Only one MRI study examined the cerebellum region Table , reporti53. Response to antipsychotic medications is further suggested as a subtyping strategy for patients with schizophrenia54. Despite the plethora of literature on biomarkers in predicting treatment-resistant schizophrenia as well as clozapine response16, there were only 19 neuroimaging studies identified in the current review specifically comparing UTRS patients with patients with other characteristics of treatment responsiveness, in particular clozapine response. Among these studies, comparisons of frontal lobe properties were reported most frequently (13 studies) and generated the greatest number of differences between groups. Four out of five studies reported a significant difference between UTRS and CRS, including lowered cortical thickness and widening of sulci, suggesting a general reduction of the frontal lobe volume in UTRS patients in comparison with patients who responded to clozapine. Furthermore, two SPECT studies both reported a significant reduction in perfusion of the frontal region and the frontal/caudate perfusion ratio in UTRS patients. One MRI study reported UTRS had a significantly greater mean diffusivity in ACC than CRS. Studies of other brain regions are much fewer, and all reported no differences in brain volumes. Only one MRS study reported a significantly lower Glx/Cr level in putamen in UTRS compared with CRS, one MRS study found a significantly higher GABA level in mid-cingulate cortex (MCC) in UTRS compared with CRS, and two SPECT study reported lower perfusion and lack of perfusion changes with clozapine treatment in the thalamus among the UTRS patients compared with the CRS patients. These findings suggested that the most pervasive and significant neurobiological differences between the UTRS patients compared with patients who responded to clozapine are likely to be in the frontal region. In fact, this is also reflected by the largely negative findings of global brain volume and cortical thickness comparison between the two treatment-resistant group patients. Furthermore, compared with healthy controls, the UTRS group was found to have lower brain functional connectivity of three networks, all involving the frontal region. But no difference in the CRS patients was found in comparison with controls. These findings align with previous reports on the presence of low prefrontal cortex activities in clozapine-resistant patients16 and relationship of hypo-frontality and clozapine treatment in an earlier systematic review55. Furthermore, an earlier systematic review identified 5 studies comparing clozapine responders and clozapine non-responders also similarly found involvement of the frontal region53.Certain neurobiological features of treatment-resistant schizophrenia appear categorically different from treatment-responsive patients and could be considered biomarkers53. Though all the positive findings were reported in only a single study, coupled with the findings of the frontal lobe region, it is possible that frontal-striatal-basal ganglia circuitry function may represent a distinct neurobiological marker of UTRS. However, further exploratory studies are required.Studies of other regions comparing UTRS and CRS patients are relatively few, and mostly with negative findings apart from basal ganglia and thalamus. One SPECT study reported both reduction in perfusion of the bilateral basal ganglia and thalamus in UTRS patients compared with the clozapine response group and two studies reported a lack of perfusion changes in the UTRS with clozapine initiation. Another SPECT study also suggested significantly lowered frontal/caudate perfusion ratio in the UTRS patients. Furthermore, one MRS study reported significantly higher Glx/Cr in CRS than UTRS in putamen. Previous review also reported the caudate volume and basal ganglia perfusion were related to clinical response to clozapine without specific comparison of UTRS and CRS56. A counterintuitive result of an early brain imaging study also found brain dysmorphology is related to better symptom improvement with clozapine57. These results place more complexity into the relationship between white matter microstructure and the clozapine responses of TRS patients.Only one DTI study compared the white matter microstructure of the two treatment-resistant groups. Surprisingly the CRS patients were found to have the lowest FA compared with all other groups . In particular, they had a significantly lower FA of the right superior longitudinal fasciculus compared with the UTRS. Though it was no longer significant after correction for multiple testing and adding the head motion as regressor, the preliminary results of this single study may indicate the possible presence of a unique pattern of white matter microstructure of patients who respond to clozapine. Lower FA indicates lower homogeneity of white matter tractography and poorer white matter integrity. However, a study of Williams syndrome found higher FA of the superior longitudinal fasciculus tract associated with poorer visual-spatial functioningB receptor-mediated inhibitory neurotransmission was reported58 as well as the binding properties of clozapine with the GABAB receptor59. Therefore, it is possible that the GABA level may be a biomarker of clozapine-resistant TRS patients. However, replication studies are required. Furthermore, given that the MCC was the only region examined, studies of GABA level in other brain regions particularly in the frontal lobes are needed to further our understanding. Studies of Glx/Cr were largely negative with only one reporting a significantly higher Glx/Cr level in CRS than UTRS in putamen.There were only three MRS studies comparing the two treatment-resistant patient groups and only one study of GABA, that reported significantly higher GABA level in mid-cingulate cortex (MCC) in UTRS compared with CRS. A unique relationship between clozapine and GABAThere were more significant findings when comparing UTRS with the FLR group including lower cortical thickness and brain volume of multiple brain regions as well as lowered Glx/Cr level in the dorsolateral prefrontal cortex. In fact, both treatment-resistant groups were found to have extensive differences in neurobiological features in comparisons with the FLR group. These suggested that the treatment-resistant patient groups have biologically distinct features compared with the FLR group and are likely to be a subtype of schizophrenia. Within the treatment-resistant patient group, the clozapine response group and clozapine-resistance group may share certain neurobiological features. However, a distinct hypo-frontality, abnormalities of frontal-striatal-basal ganglia circuitry as well as the GABA level differences may be neurobiological features differentiating the two treatment-resistant groups.60, thus presence of multiple neurobiological characteristics of TRS and UTRS are possible. Various symptom mixtures of TRS patients might have also contributed to the diversity of results. Therefore, the conclusions with the current literature are far from complete in the pursuit of the understanding of neurobiological nature of different TRS groups and mechanisms of the TRS development. Only five studies adopted a longitudinal design, limiting examination of the effects of clozapine. On the other hand, clinical study of treatment-resistant populations with large sample sizes is challenging. Future multi-center collaborative studies are needed to examine neurobiological markers of clozapine-resistant schizophrenia using the current consensus definition of patient samples6, multimodal imaging tools and a longitudinal study design. Studying neurobiological changes because of the neuromodulation interventions of TRS could also be a viable strategy. Moreover, studies with better symptom characteristics of UTRS and focusing on specific symptom dimensions such as hallucination might better inform the neurobiological mechanisms of TRS and UTRS.One of the strengths of this review study is covering results from multiple imaging modalities whilst focusing on specific regions of the brain to provide a comprehensive coverage of the neurobiological characteristics of UTRS. Secondly, the comparison was focusing specifically on differences between UTRS and CRS as well as FLR and HC and only studies with clearly stated operational definitions of different treatment outcome groups of schizophrenia were included. However, the limited sample size, diverse sample definitions and imaging modalities of existing studies make the conclusions difficult. Furthermore, heterogeneity of the TRS has been reported before, some developed TRS during the first episode and others after multiple relapsesThis systematic review of neuroimaging studies comparing clozapine-resistant schizophrenia patients with patients responding to clozapine and patients responding to other antipsychotic medications found 17 studies with variable definitions of patient samples and study methodologies. The most consistent finding was the hypo-frontality of the clozapine-resistant group compared with the clozapine responsive group with a possible difference of frontal-striatal-basal ganglia circuitry as well as the GABA level between the two treatment-resistant patient groups. Extensive neurobiological differences were seen between the two treatment-resistant patient groups and patients responding to other antipsychotics. These suggest the treatment-resistant schizophrenia is likely to be a neurobiological subtype of schizophrenia. Clozapine-resistant and clozapine-response schizophrenia are likely to have some shared neurobiological features, but possible distinct features in the frontal lobe, frontal-striatal-basal ganglia circuitry as well as the GABA level. However, available studies are limited and define the need for multi-center collaborative studies using a consensus definition of patient samples, multimodal imaging tools, and longitudinal study designs.Supplementary table


Mesenchymal stem cells (MSC) are multipotent stem cells that can differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes. Osteoblast differentiation is reduced during osteoporosis development, resulting in reduced bone formation. Further, MSC isolated from different donors possess distinct osteogenic capacity. In this study, we used single-cell multiomic analysis to profile the transcriptome and epigenome of MSC from four healthy donors. Data were obtained from ~1300 to 1600 cells for each donor. These cells were clustered into four groups, indicating that MSC from different donors have distinct chromatin accessible regulatory elements for regulating gene expression. To investigate the mechanism by which MSC undergo osteogenic differentiation, we used the chromatin accessibility data from the single-cell multiome data to identify individual-specific enhancer–promoter pairs and evaluated the expression levels and activities of the transcriptional regulators. The MSC from four donors showed distinct differentiation potential into osteoblasts. MSC of donor 1 showed the largest average motif activities, indicating that MSC from donor 1 was most likely to differentiate into osteoblasts. The results of our validation experiments were consistent with the bioinformatics prediction. We also tested the enrichment of genome-wide association study (GWAS) signals of several musculoskeletal disease traits in the patient-specific chromatin accessible regions identified in the single-cell multiome data, including osteoporosis, osteopenia, and osteoarthritis. We found that osteoarthritis-associated variants were only enriched in the regions identified from donor 4. In contrast, osteoporosis and osteopenia variants were enriched in regions from donor 1 and least enriched in donor 4. Since osteoporosis and osteopenia are related to the density of bone cells, the enrichment of variants from these traits should be correlated with the osteogenic potential of MSC. In summary, this study provides large-scale data to link regulatory elements with their target genes to study the regulatory relationships during the differentiation of mesenchymal stem cells and provide a deeper insight into the gene regulatory mechanism. Single-cell sequencing technologies have been widely used in the detection of multiple omics data at the single-cell level. It provides a deeper characterization of cellular diversity and states  and alloMesenchymal stem cells (MSC) are multipotent cells having the capability to self-renew and differentiate into different cell types under defined conditions . There aIn this study, using the MSC derived from four donors, we profiled the transcriptome and epigenome of the mesenchymal stem cells simultaneously. Single-cell multiome technology provided a more confident way to link the regulatory elements with gene expression to explore the gene regulatory mechanism during differentiation. We used the single-cell multiome data to identify the co-accessible regulatory elements and predict the regulation relationships between genes and the key transcription factors involved in osteogenic differentiation. These analysis results would help to elucidate the regulatory mechanism of MSC differentiation in osteoblast cells.Poietics™ Normal Human Bone Marrow Derived Mesenchymal Stem Cells (hMSC) were obtained from the bone marrow of healthy adult volunteers through bilateral punctures of the posterior iliac crests, as performed by Lonza. Four lots of Poietics™ hMSC, each from a different donor, were purchased from Lonza. Demographic information is listed in g for 5 min to remove culture media. The final single cell suspension was washed 3 times with PBS buffer without calcium and magnesium, plus 0.4% BSA. The single-cell suspension was then assessed for cell viability and quantity by using a hemocytometer under a microscope. Cells were then lysed with 0.1× lysis buffer for 4 min to isolate nuclei, based on the protocol of Demonstrated Protocol Nuclei Isolation Complex Sample ATAC GEX Sequencing, CG000375 RevB . A final nuclei concentration of 3000/µL was used for targeted cell recovery of 10,000 cells. Following the Chromium NextGEM Multiome ATAC GEX User Guide, CG000338_RevB , tagmentation of nuclei preparation was first performed. Thereafter, briefly, along with the single cell multiome gel beads and partition oil, the single nuclei master mixture containing tagmented single nuclei suspension was transferred onto a Next GEM Chip J, and the was chip loaded to the Chromium Controller for GEM generation and barcoding, followed by pre-amplification PCR, ATAC library preparation, cDNA synthesis, and cDNA library preparation. At each step, the quality of cDNA, ATAC library, and cDNA library was examined by Bioanalyzer. The resulting ATAC and cDNA libraries were sequenced separately, cDNA library for 28 bp and 91 bp and ATAC library for 50 bp paired-end sequencing on Illumina NovaSeq 6000.The single cell multiome analysis was conducted using a 10X Chromium single cell system  and a NovaSeq 6000 sequencer . After the cell monolayer reached 70–80% confluency, the MSC from each of the four donors were collected and combined in equal numbers to create a mixed cell preparation. One million of the MSC mixed cells were centrifuged at 300× These RNA+ATAC multiome data were firstly demultiplexed using genotype-free pipeline from souporcell  to separMotif analysis was conducted following the suggested workflow in Signac. We used chromVAR  and JASPp-value using the Wilcox rank-sum test and the area under curve (‘AUC’) statistics. We used the Beniamini–Hochberg method to correct the p-values. Subject-specific key regulators were defined as the transcription factors with significantly differentially expressed genes and significantly different motif activities as compared to the cells of other individuals.For each individual, subject-specific transcriptional regulators were selected by jointly analyzing their gene expression levels and motif activities. The R package Presto was used to evaluate the ability of each gene or motif to separate cell groups by calculating a p-value for the correlation between the accessibility signal and used the Beniamini–Hochberg method to correct the p-values. Only significantly correlated enhancer and promoter pairs with false discoverary rate adjusted p value (FDR) < 0.05 were used in the following analysis.For the single-cell ATAC data, we used Cicero  to identDue to high heterogeneity across individuals and relative homogeneity within individuals, we used a pseudobulk approach to minimize false positive identification. Previous reports  suggest GO analysis was conducted using ‘clusterProfiler’ . We extrFor subject-specific open chromatin regions identified from pseudobulk ATAC-seq data, we examined heritability based on the GWAS data of several traits related to bone diseases, including osteoporosis , osteope® 8-well glass culture slides, and, once the cell monolayer had reached full confluency, the differentiation media were added into the chambers. Following four weeks of cultivation, the osteogenic differentiation status of the four MSC samples was assessed using ScienCell™’s Alizarin Red S (ARS) staining quantification assay (ARed-Q). This approach provided a precise and sensitive means of semi-quantifying ARS in a monolayer of stained cells. In order to compare the staining outcomes of the four MSC samples, we employed a Python program. Initially, the program reads each image and extracts the CIELAB [The Mesenchymal Stem Cell Osteogenesis Kit  was utilized to induce the differentiation of MSC into osteocytes. Osteogenesis differentiation media were prepared according to the instruction of the kit. The MSC were cultured in Falcone CIELAB  color ofe CIELAB  of the dWe performed single-cell multiome sequencing on MSC obtained from four donors. Single-cell suspensions were obtained from each donor and then combined in equal numbers. Taking the advantage of the 10x Chromium Next GEM Single Cell Multiome ATAC + Gene Expression sequencing technology, we profiled gene expression and chromatin accessibility simultaneously. We collected 1635, 1342, 1496, and 1356 cells from each donor, respectively. The MSC were divided into four distinct groups, each consisting of a comparable number of cells and exhibiting clear separation between them a. As theDLX5  and motif activities levels  in donor 1  is believed to function as skeletal stem cells ,36, capaWhile here we focus on heterogeneity among donors, a further study on the gene expression, chromatin status, and relevant gene regulatory network of markers associated with osteoporosis, osteopenia, and osteoarthritis will help us understand the potential driving force for the disease progression and potential targets for therapeutic use.MAPK and COLL1. Further insights into the functional characteristics of these networks can be gained by performing knockdown experiments on key regulator genes and examining the resultant changes in genes, gene ontology, and pathways. It is important to note that our current study is based on data from only four donors. Expanding our dataset to include more individuals will increase our confidence in the validity of our discoveries. Consequently, a future study will focus on collecting data from a larger pool of donors.One of the next steps in our analysis is to validate the presence of other well-known networks associated with osteogenesis, such as