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During colon inflammation, Th17 cells and immunosuppressive regulatory T cells (Treg) are thought to play promotive and preventative roles, respectively. Dietary (n-3) PUFA favorably modulate intestinal inflammation in part by downregulating T-cell activation and functionality. We used the Fat-1 mouse, a genetic model that synthesizes long-chain (n-3) PUFA de novo, to test the hypothesis that (n-3) PUFA protect against colonic inflammation by modulating the polarization of Treg and Th17 cells during colitis. Male and female wild-type (WT) and Fat-1 mice were administered dextran sodium sulfate (DSS) in the drinking water (2.5%) to induce acute (5 d DSS) or chronic (3 cycles DSS) colitis and the percentage of Treg and Th17 cells residing locally [colonic lamina propria (cLP)] and systemically (spleen) was determined by flow cytometry. The percentage of Treg in either tissue site was unaffected by genotype (P > 0.05); however, during chronic colitis, the percentage of Th17 cells residing in both the spleen and cLP was lower in Fat-1 mice compared to WT mice (P < 0.05). Colonic mucosal mRNA expression of critical Th17 cell cytokines and chemokine receptors (IL-17F, IL-21, and CCR6) were lower, whereas expression of the Th17 cell suppressive cytokine, IL-27, was greater in Fat-1 mice compared to WT mice during chronic colitis (P < 0.05). Moreover, colon histological scores were improved in Fat-1 mice (P < 0.05). Collectively, these results demonstrate for the first time, to our knowledge, that (n-3) PUFA can modulate the colonic mucosal microenvironment to suppress Th17 cell accumulation and inflammatory damage following the induction of chronic colitis. [ABSTRACT FROM AUTHOR]

Background: The combination of fish oil-derived docosahexaenoic acid (DHA) (22:6; omega 3 [n-3]) and butyrate (4:0), a fiber fermentation product, synergized to enhance colonocyte apoptosis by inducing a p53-independent, oxidation sensitive, mitochondrial Ca(2+) -dependent (intrinsic) pathway.Methods: In this study, the authors probed the specificity of n-6 and n-3 polyunsaturated fatty acid induction of Ca(2+) -dependent proapoptotic events in immortalized young adult mouse colonocytes and determined whether combinations of polyunsaturated fatty acid and butyrate could trigger endoplasmic reticulum (ER) stress conditions, thereby promoting mitochondrial Ca(2+) overload. Cultures were treated with 0 μM to 50 μM of DHA (22:6; n-3), EPA (20:5; n-3), arachidoinic acid (AA) (20:4; n-6), linoleic acid (18:2; n-6), or oleic acid (OA) (18:1; n-9) for a total of 72 hours with or without RU-360 (to inhibit the mitochondrial Ca(2+) uniporter) for 30 minutes before cotreatment with butyrate (0 mM or 5 mM).Results: Combined DHA and butyrate maximally induced apoptosis and mitochondrial-to-cytosolic Ca(2+) levels. By comparison, EPA, a precursor to DHA, was minimally effective. Similarly, AA and OA in combination with butyrate had no effect on mitochondrial Ca(2+) or apoptosis compared with butyrate alone. DHA with or without butyrate cotreatment minimally altered the ER stress-regulated genes DNA damage-inducible transcript 3, the CCAAT enhancer binding protein (C/EBP) homologous protein (CHOP), and eukaryotic initiation factor 2α.Conclusions: The current data indicated that butyrate and DHA, but not EPA, worked in a coordinated fashion to trigger an ER-independent, Ca(2+) -dependent, intrinsic mitochondrial-mediated apoptotic pathway in colonocytes. [ABSTRACT FROM AUTHOR]

Phytochemicals may reduce chronic inflammation and cancer risk in part by modulating T-cell nuclear factor-kappaB (NF-kappaB) activation. Therefore, we examined the effects of curcumin (Cur) and limonin (Lim) feeding on NF-kappaB-dependent CD4(+) T-cell proliferation. DO11.10 transgenic mice (n = 5-7) were fed diets containing 1% Cur or 0.02% Lim combined with either (n-6) PUFA [5% corn oil (CO)] or (n-3) PUFA [4% fish oil+1% corn oil (FO)] for 2 wk, followed by splenic CD4(+) T-cell isolation and stimulation with ovalbumin peptide 323-339 (OVA) and antigen-presenting cells from mice fed a conventional nonpurified rodent diet. Both Cur and Lim diets suppressed (P < 0.05) NF-kappaB p65 nuclear translocation in activated CD4(+) T-cells. In contrast, activator protein-1 (c-Jun) and nuclear factor of activated T-cells c1 were not affected compared with the CO control diet (no Cur or Lim). CD4(+) T-cell proliferation in response to either mitogenic anti-CD3/28 monoclonal antibodies (mAb) or antigenic stimulation by OVA was also suppressed (P < 0.05) by Cur as assessed by carboxyfluorescein succinimidyl ester staining. In contrast, interleukin-2 production was not directly associated with NF-kappaB status. Interestingly, dietary combination with FO enhanced the suppressive effects (P < 0.05) of Cur or Lim with respect to CD4(+) T-cell proliferation in response to anti-CD3/28 mAb. These results suggest that combination chemotherapy (FO+Cur or Lim) may favorably modulate CD4(+) T-cell-mediated inflammation. [ABSTRACT FROM AUTHOR]

To determine if dietary fish oil protects against colon cancer by decreasing oxidative DNA damage at the initiation stage of colon tumorigenesis, oxidative DNA damage, proliferation, and apoptosis were assessed by colonic crypt cell position using quantitative immunohistochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG), Ki-67, and TUNEL assay, respectively. Sixty rats were provided one of two diets (corn oil or fish oil) and dextran sodium sulfate (DSS, an inducer of oxidative DNA damage) treatments (no DSS, 3% DSS, or DSS withdrawal). Fish oil feeding resulted in lower 8-OHdG levels (P = 0.038), higher levels of apoptosis (P = 0.035), and a lower cell proliferative index (P = 0.05) compared with corn oil feeding. In the top third of the crypt, fish oil caused an incremental stimulation of apoptosis with increased DNA damage (P = 0.043), whereas there was no such relationship with corn oil. Because polyps and tumors develop from DNA damage that leads to loss of growth and death control, the significant difference in fish oil vs. corn oil on these variables may account, in part, for the observed protective effect of fish oil against oxidatively induced colon cancer. [ABSTRACT FROM AUTHOR]

Epidemiological and experimental data suggest that dietary fiber and fat are major determinants of colorectal cancer. However, the mechanisms by which these dietary constituents alter the incidence of colon cancer have not been elucidated. Evidence indicates that dominant gain-of-function mutations short-circuit protooncogenes and contribute to the pathogenesis of cancer. Therefore, we began to dissect the mechanisms whereby dietary fat and fiber, fed during the initiation, promotion and progression stages of colon tumorigenesis, regulate ras p21 localization, expression and mutation frequency. Male Sprague-Dawley rats (140) were provided with corn oil or fish oil and pectin or cellulose plus or minus the carcinogen azoxymethane (AOM) in a 2x2x2 factorial design and killed after 34 weeks. We have previously shown adenocarcinoma incidence in these animals to be 70.3% (52/74) for corn oil + AOM and 56.1% (37/66) for fish oil + AOM (P < 0.05). Total ras expression as well as ras membrane:cytosol ratio was 4- to 6-fold higher in colon tumors than in mucosa from AOM- or saline-injected rats. Expression of ras in the mucosal membrane fraction was 13% higher for animals fed corn oil compared with fish oil feeding (P < 0.05), which is noteworthy since ras must be localized at the plasma membrane to function. The elevated ras membrane:cytosol ratio in tumors was not due to increased farnesyl protein transferase activity or prenylation state, as nearly all detectable ras was in the prenylated form. Phosphorylated p42 and p44 mitogen activated protein kinase (ERK) expression was two-fold higher in tumor extracts compared with uninvolved mucosa from AOM- and saline-injected rats (P < 0.05). The frequency of K-ras mutations was not significantly different between the various groups, but there was a trend toward a greater incidence of mutations in tumors from corn oil fed rats (85%) compared with fish oil fed rats (58%). Our results indicate that the carcinogen-induced changes in ras expression and membrane localization are associated with the in vivo activation of the ERK pathway. In addition, suppression of tumor development by dietary n-3 polyunsaturated fatty acids may be partly due to a combined effect on colonic ras expression, membrane localization, and mutation frequency. [ABSTRACT FROM PUBLISHER]

We have developed a non-invasive method utilizing feces, containing sloughed colonocytes, as a sensitive technique for detecting diagnostic colonic biomarkers. In this study, we used the rat colon carcinogenesis model to determine if changes in fecal protein kinase C (PKC) expression have predictive value in monitoring the neoplastic process. Weanling rats were injected with saline or azoxymethane (AOM) and 36 weeks later fecal samples and mucosa were collected, poly A+RNA isolated, and quantitative RT-PCR performed using primers to PKC βII and ζ Fecal PKC βII and ζ mRNA levels were altered by the presence of a tumor, with tumor-bearing animals having a 3-fold higher (P <0.05) PKC βII expression as compared with animals without tumors. In addition, AOM-injection increased mucosal PKC βII mRNA expression compared with saline controls. No effect of tumor incidence on mucosal PKC βII expression was observed. In contrast, fecal PKC ζ expression was 2.5-fold lower (P <0.05) in animals injected with azoxymethane versus saline. Since tumor incidence exerts a reciprocal effect on fecal PKC βII and ζ mRNA expression, data were also expressed as the ratio between PKC βII and ζ. The isozyme ratio was strongly related to tumor incidence, i.e. ratio for animals with tumors was 2.18 ± 1.25, animals without tumors was 0.50 ± 0.16, P = 0.025. We demonstrate that the expression of fecal PKC βII and ζ may serve as a non-invasive marker for development of colon tumors. A sensitive technique for the detection of colon cancer is of importance since early diagnosis can substantially reduce mortality. [ABSTRACT FROM PUBLISHER]

Tertiary lymphoid tissues (TLTs) are adaptive immune structures that develop during chronic inflammation and may worsen or lessen disease outcomes in a context-specific manner. Immune cell activity governing TLT formation in the intestines is dependent on immune cell aryl hydrocarbon receptor (AhR) activation. Homeostatic immune cell activity in the intestines is further dependent on ligand activation of AhR in intestinal epithelial cells (IECs), yet whether AhR activation and signaling in IECs influences the formation of TLTs in the presence of dietary AhR ligands is not known. To this end, we used IEC-specific AhR deletion coupled with a mouse model of dextran sodium sulfate (DSS)-induced colitis to understand how dietary AhR ligand 3, 3'-diindolylmethane (DIM) influenced TLT formation. DIM consumption increased the size of TLTs and decreased T-cell aggregation to TLT sites in an IEC-specific manner. In DSS-exposed female mice, DIM consumption increased the expression of genes implicated in TLT formation (Interleukin-22, Il-22 ; CXC motif chemokine ligand 13, CXCL13 ) in an IEC AhR-specific manner. Conversely, in female mice without DSS exposure, DIM significantly reduced the expression of Il-22 or CXCL13 in iAhRKO mice, but this effect was not observed in WT animals. Our findings suggest that DIM affects the immunological landscape of TLT formation during DSS-induced colitis in a manner contingent on AhR expression in IECs and biological sex. Further investigations into specific immune cell activity, IEC-specific AhR signaling pathways, and dietary AhR ligand-mediated effects on TLT formation are warranted.

After birth, the development of secondary lymphoid tissues (SLTs) in the colon is dependent on the expression of the aryl hydrocarbon receptor (AhR) in immune cells as a response to the availability of AhR ligands. However, little is known about how AhR activity from intestinal epithelial cells (IECs) may influence the development of tertiary lymphoid tissues (TLTs). As organized structures that develop at sites of inflammation or infection during adulthood, TLTs serve as localized centers of adaptive immune responses, and their presence has been associated with the resolution of inflammation and tumorigenesis in the colon. Here, we investigated the effect of the conditional loss of AhR activity in IECs in the formation and immune cell composition of TLTs in a model of acute inflammation. In females, loss of AhR activity in IECs reduced the formation of TLTs without significantly changing disease outcomes or immune cell composition within TLTs. In males lacking AhR expression in IECs, increased disease activity index, lower expression of functional-IEC genes, increased number of TLTs, increased T-cell density, and lower B- to T-cell ratio were observed. These findings may represent an unfavorable prognosis when exposed to dextran sodium sulfate (DSS)-induced epithelial damage compared with females. Sex and loss of IEC AhR also resulted in changes in microbial populations in the gut. Collectively, these data suggest that the formation of TLTs in the colon is influenced by sex and AhR expression in IECs. NEW & NOTEWORTHY This is the first research of its kind to demonstrate a clear connection between biological sex and the development of tertiary lymphoid tissues (TLT) in the colon. In addition, the research finds that in a preclinical model of inflammatory bowel disease, the expression of the aryl hydrocarbon receptor (AhR) influences the development of these structures in a sex-specific manner.

The etiology of major depressive disorder (MDD) remains poorly understood. Our previous studies suggest a role for the aryl hydrocarbon receptor (AhR) in depression. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic environmental contaminant, with a high AhR binding affinity, and an established benchmark for assessing AhR activity. Therefore, this study examined the effect of TCDD on depression-like behaviors. Female mice were fed standard chow or a high-fat diet (HFD) for 11 weeks, and their weight was recorded. Subsequently, they were tested for baseline sucrose preference and splash test grooming. Then, TCDD (0.1 µg/kg/day) or vehicle was administered orally for 28 days, and mice were examined for their sucrose preference and performances in the splash test, forced swim test (FST), and Morris water maze (MWM) task. TCDD significantly decreased sucrose preference, increased FST immobility time, and decreased groom time in chow-fed mice. HFD itself significantly reduced sucrose preference. However, TCDD significantly increased FST immobility time and decreased groom time in HFD-fed mice. A small decrease in bodyweight was observed only at the fourth week of daily TCDD administration in chow-fed mice, and no significant effects of TCDD on bodyweights were observed in HFD-fed mice. TCDD did not have a significant effect on spatial learning in the MWM. Thus, this study demonstrated that TCDD induces a depression-like state, and the effects were not due to gross lethal toxicity. This study further suggests that more studies should examine a possible role for AhR and AhR-active environmental pollutants in precipitating or worsening MDD., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The equine gastrointestinal (GI) microbiome has been described in the context of various diseases. The observed changes, however, have not been linked to host function and therefore it remains unclear how specific changes in the microbiome alter cellular and molecular pathways within the GI tract. Further, non-invasive techniques to examine the host gene expression profile of the GI mucosa have been described in horses but not evaluated in response to interventions. Therefore, the objectives of our study were to (1) profile gene expression and metabolomic changes in an equine model of non-steroidal anti-inflammatory drug (NSAID)-induced intestinal inflammation and (2) apply computational data integration methods to examine host-microbiota interactions., Methods: Twenty horses were randomly assigned to 1 of 2 groups (n = 10): control (placebo paste) or NSAID (phenylbutazone 4.4 mg/kg orally once daily for 9 days). Fecal samples were collected on days 0 and 10 and analyzed with respect to microbiota (16S rDNA gene sequencing), metabolomic (untargeted metabolites), and host exfoliated cell transcriptomic (exfoliome) changes. Data were analyzed and integrated using a variety of computational techniques, and underlying regulatory mechanisms were inferred from features that were commonly identified by all computational approaches., Results: Phenylbutazone induced alterations in the microbiota, metabolome, and host transcriptome. Data integration identified correlation of specific bacterial genera with expression of several genes and metabolites that were linked to oxidative stress. Concomitant microbiota and metabolite changes resulted in the initiation of endoplasmic reticulum stress and unfolded protein response within the intestinal mucosa., Conclusions: Results of integrative analysis identified an important role for oxidative stress, and subsequent cell signaling responses, in a large animal model of GI inflammation. The computational approaches for combining non-invasive platforms for unbiased assessment of host GI responses (e.g., exfoliomics) with metabolomic and microbiota changes have broad application for the field of gastroenterology. Video Abstract., (© 2024. The Author(s).)

Cellular membranes serve as an epicentre combining extracellular and cytosolic components with membranous effectors, which together support numerous fundamental cellular signalling pathways that mediate biological responses. To execute their functions, membrane proteins, lipids and carbohydrates arrange, in a highly coordinated manner, into well-defined assemblies displaying diverse biological and biophysical characteristics that modulate several signalling events. The loss of membrane homeostasis can trigger oncogenic signalling. More recently, it has been documented that select membrane active dietaries (MADs) can reshape biological membranes and subsequently decrease cancer risk. In this review, we emphasize the significance of membrane domain structure, organization and their signalling functionalities as well as how loss of membrane homeostasis can steer aberrant signalling. Moreover, we describe in detail the complexities associated with the examination of these membrane domains and their association with cancer. Finally, we summarize the current literature on MADs and their effects on cellular membranes, including various mechanisms of dietary chemoprevention/interception and the functional links between nutritional bioactives, membrane homeostasis and cancer biology., (© 2022 Federation of European Biochemical Societies.)

Lipids are important biomarkers within the field of disease diagnostics and can serve as indicators of disease progression and predictors of treatment effectiveness. Although lipids can provide important insight into how diseases initiate and progress, mass spectrometric methods for lipid characterization and profiling are limited due to lipid structural diversity, particularly the presence of various lipid isomers. Moreover, the difficulty of handling small-volume samples exacerbates the intricacies of biological analyses. In this work, we have developed a strategy that electromigrates a thin film of a small-volume biological sample directly to the air-liquid interface formed at the tip of a theta capillary. Importantly, we seamlessly integrated in situ biological lipid extraction with accelerated chemical derivatization, enabled by the air-liquid interface, and conducted isomeric structural characterization within a unified platform utilizing theta capillary nanoelectrospray ionization mass spectrometry, all tailored for small-volume sample analysis. We applied this unified platform to the analysis of lipids from small-volume human plasma and Alzheimer's disease mouse serum samples. Accelerated electro-epoxidation of unsaturated lipids at the interface allowed us to characterize lipid double-bond positional isomers. The unique application of electromigration of a thin film to the air-liquid interface in combination with accelerated interfacial reactions holds great potential in small-volume sample analysis for disease diagnosis and prevention.

The advances of modern sequencing techniques have generated an unprecedented amount of multi-omics data which provide great opportunities to quantitatively explore functional genomes from different but complementary perspectives. However, distinct modalities/sequencing technologies generate diverse types of data which greatly complicate statistical modeling because uniquely optimized methods are required for handling each type of data. In this paper, we propose a unified framework for Bayesian nonparametric matrix factorization that infers overlapping bi-clusters for multi-omics data. The proposed method adaptively discretizes different types of observations into common latent states on which cluster structures are built hierarchically. The proposed Bayesian nonparametric method is able to automatically determine the number of clusters. We demonstrate the utility of the proposed method using simulation studies and applications to a single-cell RNA-sequencing dataset, a combination of single-cell RNA-sequencing and single-cell ATAC-sequencing dataset, a bulk RNA-sequencing dataset, and a DNA methylation dataset which reveal several interesting findings that are consistent with biological literature.

There is evidence that the orphan nuclear receptor 4A1 (NR4A1, Nur77) is overexpressed in exhausted CD8 + T cells and regulates PD-L1 in tumors. This study investigated the effects of potent bis-indole-derived NR4A1 antagonists on reversing T-cell exhaustion and downregulating PD-L1 in colon tumors/cells. NR4A1 antagonists inhibited colon tumor growth and downregulated expression of PD-L1 in mouse colon MC-38-derived tumors and cells. TILs from MC-38 cell-derived colon tumors and splenic lymphocytes exhibited high levels of the T-cell exhaustion markers including PD-1, 2B4, TIM3+ and TIGIT and similar results were observed in the spleen, and these were inhibited by NR4A1 antagonists. In addition, treatment with NR4A1 antagonists induced cytokine activation markers interferon γ, granzyme B and perforin mRNAs and decreased TOX, TOX2 and NFAT in TIL-derived CD8 + T cells. Thus, NR4A1 antagonists decrease NR4A1-dependent pro-oncogenic activity and PD-L1 expression in colon tumors and inhibit NR4A1-dependent T-cell exhaustion in TILs and spleen and represent a novel class of mechanism-based drugs that enhance immune surveillance in tumors., (© 2023. The Author(s).)

We study a discrete-time multi-type Wright-Fisher population process. The mean-field dynamics of the stochastic process is induced by a general replicator difference equation. We prove several results regarding the asymptotic behavior of the model, focusing on the impact of the mean-field dynamics on it. One of the results is a limit theorem that describes sufficient conditions for an almost certain path to extinction, first eliminating the type which is the least fit at the mean-field equilibrium. The effect is explained by the metastability of the stochastic system, which under the conditions of the theorem spends almost all time before the extinction event in a neighborhood of the equilibrium. In addition to the limit theorems, we propose a maximization principle for a general deterministic replicator dynamics and study its implications for the stochastic model., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In this paper, we introduce Gene Knockout Inference (GenKI), a virtual knockout (KO) tool for gene function prediction using single-cell RNA sequencing (scRNA-seq) data in the absence of KO samples when only wild-type (WT) samples are available. Without using any information from real KO samples, GenKI is designed to capture shifting patterns in gene regulation caused by the KO perturbation in an unsupervised manner and provide a robust and scalable framework for gene function studies. To achieve this goal, GenKI adapts a variational graph autoencoder (VGAE) model to learn latent representations of genes and interactions between genes from the input WT scRNA-seq data and a derived single-cell gene regulatory network (scGRN). The virtual KO data is then generated by computationally removing all edges of the KO gene-the gene to be knocked out for functional study-from the scGRN. The differences between WT and virtual KO data are discerned by using their corresponding latent parameters derived from the trained VGAE model. Our simulations show that GenKI accurately approximates the perturbation profiles upon gene KO and outperforms the state-of-the-art under a series of evaluation conditions. Using publicly available scRNA-seq data sets, we demonstrate that GenKI recapitulates discoveries of real-animal KO experiments and accurately predicts cell type-specific functions of KO genes. Thus, GenKI provides an in-silico alternative to KO experiments that may partially replace the need for genetically modified animals or other genetically perturbed systems., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Although the role of the Wnt pathway in colon carcinogenesis has been described previously, it has been recently demonstrated that Wnt signaling originates from highly dynamic nano-assemblies at the plasma membrane. However, little is known regarding the role of oncogenic APC in reshaping Wnt nanodomains. This is noteworthy, because oncogenic APC does not act autonomously and requires activation of Wnt effectors upstream of APC to drive aberrant Wnt signaling. Here, we demonstrate the role of oncogenic APC in increasing plasma membrane free cholesterol and rigidity, thereby modulating Wnt signaling hubs. This results in an overactivation of Wnt signaling in the colon. Finally, using the Drosophila sterol auxotroph model, we demonstrate the unique ability of exogenous free cholesterol to disrupt plasma membrane homeostasis and drive Wnt signaling in a wildtype APC background. Collectively, these findings provide a link between oncogenic APC, loss of plasma membrane homeostasis and CRC development., (© 2023. The Author(s).)

Background: Obese females are more likely to suffer from depression and are also more likely to be resistant to current medications. This study examined the potential antidepressant-like effects of 1,4-dihydroxy-2-napthoic acid (DHNA), a selective aryl hydrocarbon receptor modulator (SAhRM), in obese female mice., Methods: Obesity was established by feeding C57BL/6N female mice a high fat diet (HFD) for 9-10 weeks. Subsequently, mice were subjected to unpredictable chronic mild stress (UCMS) or remained unstressed. Daily administration of vehicle or 20 mg/kg DHNA began three weeks prior or on the third week of UCMS. Mice were examined for depression-like behaviors (sucrose preference, forced swim test (FST), splash and tape groom tests), anxiety (open-field test, light/dark test, novelty-induced hypophagia), and cognition (object location recognition, novel object recognition, Morris water maze)., Results: UCMS did not alter, and DHNA slightly increased, weight gain in HFD-fed females. HFD decreased sucrose preference, increased FST immobility time, but did not alter splash and tape tests' grooming time. UCMS did not have additional effects on sucrose preference. UCMS further increased FST immobility time and decreased splash and tape tests' grooming time; these effects were prevented and reversed by DHNA treatment. HFD did not affect behaviors in the cognitive tests. UCMS impaired spatial learning; this effect was not prevented nor reversed by DHNA., Conclusions: DHNA protected against UCMS-induced depression-like behaviors in HFD-fed female mice. DHNA neither improved nor worsened UCMS-induced impairment of spatial learning. Our findings indicate that DHNA has high potential to act as an antidepressant in obese females., Competing Interests: Conflict of interest The authors have no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The use of nanoparticles in agrichemical formula and food products as additives has increased their chances of accumulation in humans via oral intake. Due to their potential toxicity, it is critical to understand their fate and distribution following oral intake. Cerium oxide nanoparticle (CeO 2 NP) is commonly used in agriculture and is highly stable in the environment. As such, it has been used as a model chemical to investigate nanoparticle's distribution and clearance. Based on their estimated human exposure levels, 0.15-0.75 mg/kg body weight/day of CeO 2 NPs with different sizes and surface charges (30-50 nm with negative charge and <25 nm with positive charge) were gavaged into C57BL/6 female mice daily. After 10-d, 50% of mice in each treatment were terminated, with the remaining being gavaged with 0.2 mL of deionized water daily for 7-d. Mouse organ tissues, blood, feces, and urine were collected at termination. At the tested levels, CeO 2 NPs displayed minimal overt toxicity to the mice, with their accumulation in various organs being negligible. Fecal discharge as the predominant clearance pathway took less than 7-d regardless of charges. Single particle inductively coupled plasma mass spectrometry analysis demonstrated minimal aggregation of CeO 2 NPs in the gastrointestinal tract. These findings suggest that nanoparticle additives >25 nm are unlikely to accumulate in mouse organ after oral intake, indicating limited impacts on human health., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

It was recently reported that the hydroxyflavones quercetin and kaempferol bind the orphan nuclear receptor 4A1 (NR4A1, Nur77) and act as antagonists in cancer cells and tumors, and they inhibit pro-oncogenic NR4A1-regulated genes and pathways. In this study, we investigated the interactions of flavone, six hydroxyflavones, seven dihydroxyflavones, three trihydroxyflavones, two tetrahydroxyflavones, and one pentahydroxyflavone with the ligand-binding domain (LBD) of NR4A1 using direct-binding fluorescence and an isothermal titration calorimetry (ITC) assays. Flavone and the hydroxyflavones bound NR4A1, and their K D values ranged from 0.36 µM for 3,5,7-trihydroxyflavone (galangin) to 45.8 µM for 3'-hydroxyflavone. K D values determined using ITC and K D values for most (15/20) of the hydroxyflavones were decreased compared to those obtained using the fluorescence assay. The results of binding, transactivation and receptor-ligand modeling assays showed that K D values, transactivation data and docking scores for these compounds are highly variable with respect to the number and position of the hydroxyl groups on the flavone backbone structure, suggesting that hydroxyflavones are selective NR4A1 modulators. Nevertheless, the data show that hydroxyflavone-based neutraceuticals are NR4A1 ligands and that some of these compounds can now be repurposed and used to target sub-populations of patients that overexpress NR4A1.

We present scTenifoldXct, a semi-supervised computational tool for detecting ligand-receptor (LR)-mediated cell-cell interactions and mapping cellular communication graphs. Our method is based on manifold alignment, using LR pairs as inter-data correspondences to embed ligand and receptor genes expressed in interacting cells into a unified latent space. Neural networks are employed to minimize the distance between corresponding genes while preserving the structure of gene regression networks. We apply scTenifoldXct to real datasets for testing and demonstrate that our method detects interactions with high consistency compared with other methods. More importantly, scTenifoldXct uncovers weak but biologically relevant interactions overlooked by other methods. We also demonstrate how scTenifoldXct can be used to compare different samples, such as healthy vs. diseased and wild type vs. knockout, to identify differential interactions, thereby revealing functional implications associated with changes in cellular communication status., Competing Interests: Declaration of interests Y.Y., G.L., Y.Z., and J.J.C. are listed as inventors on a patent application related to this work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The aryl hydrocarbon receptor (AhR) is overexpressed in many tumor types and exhibits tumor-specific tumor promoter and tumor suppressor-like activity. In colon cancer, most but not all studies suggest that the AhR exhibits tumor suppressor activity which is enhanced by AhR ligands acting as agonists. Our studies investigated the role of the AhR in colon tumorigenesis using wild-type and AhR-knockout mice, the inflammation model of colon tumorigenesis using mice treated with azoxymethane (AOM)/dextran sodium sulfate (DSS) and APC S580/+ ; Kras G12D/+ mice all of which form intestinal tumors. The effects of tissue-specific AhR loss in the intestine of the tumor-forming mice on colonic stem cells, organoid-initiating capacity, colon tumor formation and mechanisms of AhR-mediated effects were investigated. Loss of AhR enhanced stem cell and tumor growth and in the AOM/DSS model AhR-dependent suppression of FOXM1 and downstream genes was important for AhR-dependent anticancer activity. Furthermore, the effectiveness of interleukin-22 (IL22) in colonic epithelial cells was also dependent on AhR expression. IL22 induced phosphorylation of STAT3, inhibited colonic organoid growth, promoted colonic cell proliferation in vivo and enhanced DNA repair in AOM/DSS-induced tumors. In this mouse model, the AhR suppressed SOCS3 expression and enhanced IL22-mediated activation of STAT3, whereas the loss of the AhR increased levels of SOCS3 which in turn inhibited IL22-induced STAT3 activation. In the APC S580/+ ; Kras G12D/+ mouse model, the loss of the AhR enhanced Wnt signaling and colon carcinogenesis. Results in both mouse models of colon carcinogenesis were complemented by single cell transcriptomics on colonic intestinal crypts which also showed that AhR deletion promoted expression of FOXM1-regulated genes in multiple colonic cell subtypes. These results support the role of the AhR as a tumor suppressor-like gene in the colon., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

The aryl hydrocarbon receptor (AhR) is a key regulator in the microbiome-gut-brain axis, and AhR-active microbial metabolites modulate multiple neuronal responses. We recently demonstrated that 3,3'-diindolylmethane (DIM) and 1,4-dihydroxy-2-naphthoic acid (DHNA), two selective AhR modulators (SAhRMs), act as antidepressants in female mice. Thus, to examine the role of intestinal AhR in depression, anxiety, and spatial learning, this study employed transgenic mice in which the AhR was knockout only in the intestinal epithelium (AhRΔIEC). Additionally, this study examined whether the antidepressant effects of dietary DIM and DHNA is mediated by intestinal AhR. AhRΔIEC and WT female mice were fed daily with vehicle, 20 mg/kg DIM or DHNA for three weeks prior to four weeks of unpredictable chronic mild stress (UCMS). Mice were examined for weight gain, anhedonia-like behavior (sucrose preference test), anxiety levels (open field, light/dark, elevated plus maze, novelty-induced hypophagia, and marble burying tests), and spatial learning (Morris water maze). UCMS reduced weight gain in AhRΔIECs, but not WTs. Moreover, UCMS initially reduced sucrose preference in both AhRΔIECs and WTs, but over 4 weeks of UCMS, AhRΔIECs develop resilience to UCMS-induced anhedonia. Additionally, AhRΔIECs exhibit slightly reduced anxiety in certain tests and faster spatial learning. DIM and DHNA acted as antidepressants in both AhRΔIECs and WTs. Thus, this study suggests that intestinal AhR plays differential roles, mitigating stress effects on weight gain, and increasing stress effects on mood. However, the site of antidepressant action of SAhRMs, such as DIM and DHNA, is not dependent on the expression of intestinal AhR., Competing Interests: Conflict of interest The authors have no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Among all racial/ethnic groups, people who identify as African American/Blacks have the second highest colorectal cancer (CRC) incidence in the USA. This disparity may exist because African American/Blacks, compared to other racial/ethnic groups, have a higher prevalence of risk factors for CRC, including obesity, low fiber consumption, and higher intakes of fat and animal protein. One unexplored, underlying mechanism of this relationship is the bile acid-gut microbiome axis. High saturated fat, low fiber diets, and obesity lead to increases in tumor promoting secondary bile acids. Diets high in fiber, such as a Mediterranean diet, and intentional weight loss may reduce CRC risk by modulating the bile acid-gut microbiome axis. The purpose of this study is to test the impact of a Mediterranean diet alone, weight loss alone, or both, compared to typical diet controls on the bile acid-gut microbiome axis and CRC risk factors among African American/Blacks with obesity. Because weight loss or a Mediterranean diet alone can reduce CRC risk, we hypothesize that weight loss plus a Mediterranean diet will reduce CRC risk the most., Methods: This randomized controlled lifestyle intervention will randomize 192 African American/Blacks with obesity, aged 45-75 years to one of four arms: Mediterranean diet, weight loss, weight loss plus Mediterranean diet, or typical diet controls, for 6 months (48 per arm). Data will be collected at baseline, mid-study, and study end. Primary outcomes include total circulating and fecal bile acids, taurine-conjugated bile acids, and deoxycholic acid. Secondary outcomes include body weight, body composition, dietary change, physical activity, metabolic risk, circulating cytokines, gut microbial community structure and composition, fecal short-chain fatty acids, and expression levels of genes from exfoliated intestinal cells linked to carcinogenesis., Discussion: This study will be the first randomized controlled trial to examine the effects of a Mediterranean diet, weight loss, or both on bile acid metabolism, the gut microbiome, and intestinal epithelial genes associated with carcinogenesis. This approach to CRC risk reduction may be especially important among African American/Blacks given their higher risk factor profile and increased CRC incidence., Trial Registration: ClinicalTrials.gov NCT04753359 . Registered on 15 February 2021., (© 2023. The Author(s).)

Coffee is one of the most widely consumed beverages worldwide, and epidemiology studies associate higher coffee consumption with decreased rates of mortality and decreased rates of neurological and metabolic diseases, including Parkinson's disease and type 2 diabetes. In addition, there is also evidence that higher coffee consumption is associated with lower rates of colon and rectal cancer, as well as breast, endometrial, and other cancers, although for some of these cancers, the results are conflicting. These studies reflect the chemopreventive effects of coffee; there is also evidence that coffee consumption may be therapeutic for some forms of breast and colon cancer, and this needs to be further investigated. The mechanisms associated with the chemopreventive or chemotherapeutic effects of over 1000 individual compounds in roasted coffee are complex and may vary with different diseases. Some of these mechanisms may be related to nuclear factor erythroid 2 (Nrf2)-regulated pathways that target oxidative stress or pathways that induce reactive oxygen species to kill diseased cells (primarily therapeutic). There is evidence for the involvement of receptors which include the aryl hydrocarbon receptor (AhR) and orphan nuclear receptor 4A1 (NR4A1), as well as contributions from epigenetic pathways and the gut microbiome. Further elucidation of the mechanisms will facilitate the potential future clinical applications of coffee extracts for treating cancer and other inflammatory diseases.

The intersection of protein and lipid biology is of growing importance for understanding how cells address structural challenges during adhesion and migration. While protein complexes engaged with the cytoskeleton play a vital role, support from the phospholipid membrane is crucial for directing localization and assembly of key protein complexes. During angiogenesis, dramatic cellular remodeling is necessary for endothelial cells to shift from a stable monolayer to invasive structures. However, the molecular dynamics between lipids and proteins during endothelial invasion are not defined. Here, we utilized cell culture, immunofluorescence, and lipidomic analyses to identify a novel role for the membrane binding protein Annexin A2 (ANXA2) in modulating the composition of specific membrane lipids necessary for cortical F-actin organization and adherens junction stabilization. In the absence of ANXA2, there is disorganized cortical F-actin, reduced junctional Arp2, excess sprout initiation, and ultimately failed sprout maturation. Furthermore, we observed reduced filipin III labeling of membrane cholesterol in cells with reduced ANXA2, suggesting there is an alteration in phospholipid membrane dynamics. Lipidomic analyses revealed that 42 lipid species were altered with loss of ANXA2, including an accumulation of phosphatidylcholine (16:0&#95;16:0). We found that supplementation of phosphatidylcholine (16:0&#95;16:0) in wild-type endothelial cells mimicked the ANXA2 knock-down phenotype, indicating that ANXA2 regulated the phospholipid membrane upstream of Arp2 recruitment and organization of cortical F-actin. Altogether, these data indicate a novel role for ANXA2 in coordinating events at endothelial junctions needed to initiate sprouting and show that proper lipid modulation is a critical component of these events., (© 2022 Federation of American Societies for Experimental Biology.)

There is a growing prevalence of inflammatory bowel disease (IBD), a chronic inflammatory condition of the gastrointestinal tract, among the aging population. Ghrelin is a gut hormone that, in addition to controlling feeding and energy metabolism, has been shown to exert anti-inflammatory effects; however, the effect of ghrelin in protecting against colitis in old mice has not been assessed. Here, we subjected old female C57BL/6J mice to dextran sulfate sodium (DSS) in drinking water for six days, then switched back to normal drinking water, administered acyl-ghrelin or vehicle control from day 3 to 13, and monitored disease activities throughout the disease course. Our results showed that treatment of old mice with acyl-ghrelin attenuated DSS-induced colitis. Compared to the DSS group, ghrelin treatment decreased levels of the inflammation marker S100A9 in the colons collected on day 14 but not on day 8, suggesting that the anti-inflammatory effect was more prominent in the recovery phase. Ghrelin treatment also significantly reduced F4/80 and interleukin-17A on day 14. Moreover, acyl-ghrelin increased mitochondrial respiration and activated transcriptional activity of the peroxisome proliferator-activated receptor gamma (PPARγ) in Caco-2 cells. Together, our data show that ghrelin alleviated DSS-induced colitis, suggesting that ghrelin may promote tissue repair in part through regulating epithelial metabolism via PPARγ mediated signaling.

Background: Our recent study demonstrated that selective aryl hydrocarbon receptor modulators (SAhRMs), such as 1,4-dihydroxy-2-napthoic acid (DHNA) act as antidepressants in female mice. Given that some effects of certain SAhRMs are known to also be mediated via estrogen receptor signaling, this study examined whether the effects of SAhRMs on mood, emotional state, and cognition are sex-dependent., Methods: C57BL/6N mice were fed with vehicle or 20 mg/kg DHNA for three weeks prior to four weeks of unpredictable chronic mild stress (UCMS). Mice were examined for depression-like behaviors (sucrose preference, forced swim test (FST), splash test, tape groom test), emotional state (open-field test, light/dark test, marble burying, novelty-induced hypophagia, elevated-plus maze), and cognition (object location recognition, novel object recognition, Morris water maze)., Results: In females, UCMS decreased sucrose preference and increased FST immobility time; both effects were prevented by DHNA. In males, UCMS increased FST immobility time, and increased the latency to groom in the splash test. These effects were not mitigated by DHNA. However, in males, UCMS induced an increase in novelty-induced locomotion, an increase in the time spent in the light compartment in the L/D test, and an increase in the time spent with an object in a novel location. These effects were prevented by DHNA., Conclusions: Our findings indicate that DHNA has high potential to act as antidepressants in females. However, given classical interpretation, DHNA did not appear to act as an antidepressant in males. Nonetheless, our findings indicate that DHNA can mitigate stress effects and reactivity in males., Competing Interests: Conflict of interest The authors have no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Unsubstituted flavone induced CYP1A1, CYP1B1 and UGT1A1 gene expression in Caco2 cells and was characterized as an aryl hydrocarbon receptor (AhR) agonist. The structure-activity relationships among 15 mono- and dihydroxyflavones showed that addition of one or two hydroxyl groups resulted in active (e.g.: 5- and 6- mono- and 5,6-dihydroxyflavones) and inactive (e.g.: 7-mono, 7,4' and 6,4'-dihydroxyflavones) AhR ligands. Ligand docking studies of flavone, mono- and dihydroxyflavones to the human AhR resulted in similar docking scores that varied from -3.48 to -4.58 kcal/mol and these values did not distinguish between AhR-active and AhR-inactive mono- and dihydroxyflavones. The AhR-inactive flavones were subsequently investigated as AhR antagonists by determining their activities as inhibitors of TCDD-induced expression of CYP1A1, CYP1AA2 and UGT 1A1 gene expression in Caco2 cells. Initial studies with 7,4'-dihydroxyflavone showed that this compound was an AhR antagonist in Caco2 cells and resembled the activity of the classical AhR antagonist CH223191. With few exceptions most of the remaining AhR-inactive compounds in terms of inducing AhR responsive genes were also AhR antagonists. Thus, based on modeling studies, mono- and dihydroxyflavones bind with similar affinities to the AhR and exhibit AhR agonist or antagonist activities, however, the structural requirements (substitution patterns) for predicting these opposing activities were not apparent and could only be determined using bioassays., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stephen H. Safe reports financial support was provided by Texas A&M University. Stephen H. Safe reports a relationship with Texas A&M University that includes: non-financial support., (Copyright © 2022 Elsevier B.V. All rights reserved.)

High-throughput sequencing technology provides unprecedented opportunities to quantitatively explore human gut microbiome and its relation to diseases. Microbiome data are compositional, sparse, noisy, and heterogeneous, which pose serious challenges for statistical modeling. We propose an identifiable Bayesian multinomial matrix factorization model to infer overlapping clusters on both microbes and hosts. The proposed method represents the observed over-dispersed zero-inflated count matrix as Dirichlet-multinomial mixtures on which latent cluster structures are built hierarchically. Under the Bayesian framework, the number of clusters is automatically determined and available information from a taxonomic rank tree of microbes is naturally incorporated, which greatly improves the interpretability of our findings. We demonstrate the utility of the proposed approach by comparing to alternative methods in simulations. An application to a human gut microbiome data set involving patients with inflammatory bowel disease reveals interesting clusters, which contain bacteria families Bacteroidaceae, Bifidobacteriaceae, Enterobacteriaceae, Fusobacteriaceae, Lachnospiraceae, Ruminococcaceae, Pasteurellaceae, and Porphyromonadaceae that are known to be related to the inflammatory bowel disease and its subtypes according to biological literature. Our findings can help generate potential hypotheses for future investigation of the heterogeneity of the human gut microbiome., (© The Author 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Early stage estrogen receptor α (ERα, ESR1)-positive breast cancer patients can develop more aggressive endocrine-resistant tumors that express constitutively active mutant forms of ERα including ERα-Y537S and ERα-D538G. These patients are treated with selective ER down regulators (SERDs) such as the ERα antagonist fulvestrant. Previous studies show that histone deacetylase (HDAC) inhibitors downregulate ERα and since some dietary derived short chain fatty acids (butyrate, propionate and acetate) exhibit HDAC inhibitory activity we investigated their effects as SERDs in MCF-7 and T47D cells expressing wild-type and mutant ERα-D538G and ERα-Y537S. The SCFAs exhibited SERD-like activity in both cell lines expressing wild-type and mutant ERα. The results for propionate and butyrate correlated with parallel induction of histone acetylation and this was also observed for the HDAC inhibitors Panobinostat, Vorinostat and Entinostat which also downregulated wild-type and mutant ERα and induced histone acetylation. Although acetate induced ERα degradation the mechanisms may be independent of the HDAC inhibitory activity of this compound. These results suggest that high fibre diets that induce formation of SCFAs may have some clinical efficacy for treating ER-positive endocrine resistant breast cancer patients and this is currently being investigated., Competing Interests: None., (AJCR Copyright © 2022.)

Background: Current pharmaceutical treatments for depression are sometimes ineffective and may have unwanted side effects that interfere with patient compliance. This study examined the potential antidepressant-like effects of dietary- and microbial-derived aryl hydrocarbon receptor (AhR) ligands, 3,3'-diindolylmethane (DIM) and 1,4-dihydroxy-2-naphthoic acid (1,4-DHNA)., Methods: Female C57BL/6 mice were subjected to unpredictable chronic mild stress (UCMS) or were unstressed. For three weeks prior to UCMS mice were fed daily with vehicle or 20 mg/kg DIM, 1,4-DHNA or AhR-inactive isomer 3,7-DHNA; another group was subjected to two weeks UCMS before ligand administration began. Mice were examined for anhedonia-like behavior as measured by the sucrose preference test. Additionally, anxiety levels of the mice were examined before UCMS and ligand administration began and at the end in the open field, light/dark, elevated plus maze, novelty-induced hypophagia, and marble burying tests. At the end of the experiment they were also examined in the Morris water maze (MWM) task., Results: Both DIM and 1,4-DHNA, but not 3,7-DHNA, successfully prevented and reversed UCMS-induced anhedonia-like behavior. Furthermore, both DIM and DHNA had little to no effect on anxiety levels and did not induce spatial learning deficits., Limitations: Additional studies are required to determine to what degree the antidepressant-like effects of DIM and 1,4-DHNA can be attributed to their activities as AhR ligands., Conclusions: Our findings indicate that dietary and microbial-derived AhR ligands may have clinical applications as potential antidepressants. Future studies are necessary to elucidate the role of AhR in depression-like states and the underlying mechanisms of action., (Copyright © 2022 Elsevier B.V. All rights reserved.)

High-fiber plant foods contain lignans that are converted to bioactive enterolignans, enterolactone (ENL) and enterodiol (END) by gut bacteria. Previously, we conducted an intervention study to gain mechanistic insight into the potential chemoprotective effects of flaxseed lignan supplementation (secoisolariciresinol diglucoside; SDG) compared to a placebo in 42 men and women. Here, we expand on these analyses to further probe the impact of the microbial metabolite phenotype on host gene expression in response to lignan exposure. We defined metabolic phenotypes as high- or low-ENL excretion based on the microbial metabolism of SDG. RNA-seq was used to assess host gene expression in fecal exfoliated cells. Stratified by microbial ENL excretion, differentially expressed (DE) genes in high- and low-ENL excreter groups were compared. Linear discriminant analysis using the ENL phenotypes identified putative biomarker combinations of genes capable of discriminating the lignan treatment from the placebo. Following lignan intervention, a total of 165 DE genes in high-ENL excreters and 1450 DE genes in low-ENL excreters were detected. Functional analysis identified four common upstream regulators (master genes): CD3, IFNG, IGF1 and TNFRSF1A. Our findings suggest that the enhanced conversion of flaxseed lignan to ENL is associated with a suppressed inflammatory status.

Intestinal organoids (IO), known as "mini-guts", derived from intestinal crypts, are self-organizing three-dimensional (3D) multicellular ex vivo models that recapitulate intestine epithelial structure and function and have been widely used for studying intestinal physiology, pathophysiology, molecular mechanisms of host-pathogen interactions, and intestinal disease in mammals. However, studies on avian IO are limited and the development of long-term cultures of IO model for poultry research is lacking. Therefore, the objectives of this study were to generate crypt-derived organoids from chicken intestines and to optimize conditions for cell growth and enrichments, passages, and cryopreservation. Crypts were collected from the small intestines of birds at embryonic d-19 and ceca from layer and broiler chickens with ages ranging from d 1 to 20 wk, embedded in a basement membrane matrix, and cultured with organoid growth media (OGM) prepared in house. The crypt-derived organoids were successfully grown and propagated to form 3D spheres like structures that were cultured for up to 3 wk. Organoids were formed on d one, budding appeared on d 3, and robust budding was observed on d 7 and beyond. For cryopreservation, dissociated organoids were resuspended in a freezing medium. The characteristics of IO upon extended passages and freeze-thaw cycles were analyzed using reverse transcription (RT)-PCR, immunoblotting, and live cell imaging. Immunoblotting and RT-PCR using E-cadherin (the marker for epithelial cells), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5, the marker for stem cells), chromogranin A (the marker for enteroendocrine cells), lysozyme (the marker for Paneth cells), and mucin (the biomarker for goblet cells) confirmed that IO were composed of heterogeneous cell populations, including epithelial cells, stem cells, enteroendocrine cells, Paneth cells, and goblet cells. Furthermore, OGM supplemented with both valproic acid and CHIR99021, a glycogen synthase kinase 3β inhibitor and a histone deacetylase inhibitor, increased the size of the avian IO (P < 0.001). To the best of our knowledge, this is the first comprehensive report for establishing long-term, organoid culture models from small intestines and ceca of layer and broiler chickens. This model will facilitate elucidation of the mechanisms impacting host-pathogen interactions, eventually leading to the discovery of pathogen intervention strategies in poultry., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Gene knockout (KO) experiments are a proven, powerful approach for studying gene function. However, systematic KO experiments targeting a large number of genes are usually prohibitive due to the limit of experimental and animal resources. Here, we present scTenifoldKnk, an efficient virtual KO tool that enables systematic KO investigation of gene function using data from single-cell RNA sequencing (scRNA-seq). In scTenifoldKnk analysis, a gene regulatory network (GRN) is first constructed from scRNA-seq data of wild-type samples, and a target gene is then virtually deleted from the constructed GRN. Manifold alignment is used to align the resulting reduced GRN to the original GRN to identify differentially regulated genes, which are used to infer target gene functions in analyzed cells. We demonstrate that the scTenifoldKnk-based virtual KO analysis recapitulates the main findings of real-animal KO experiments and recovers the expected functions of genes in relevant cell types., Competing Interests: D.O., Y.Z., G.L., Y.Y., J.Z.H., and J.J.C. are listed as inventors on a patent application related to this work., (© 2022 The Author(s).)

Despite recent progress recognizing the importance of aryl hydrocarbon receptor (Ahr)-dependent signaling in suppressing colon tumorigenesis, its role in regulating colonic crypt homeostasis remains unclear. To assess the effects of Ahr on intestinal epithelial cell heterogeneity and functional phenotypes, we utilized single-cell transcriptomics and advanced analytic strategies to generate a high-quality atlas for colonic intestinal crypts from wild-type and intestinal-specific Ahr knockout mice. Here we observed the promotive effects of Ahr deletion on Foxm1 -regulated genes in crypt-associated canonical epithelial cell types and subtypes of goblet cells and deep crypt-secretory cells. We also show that intestinal Ahr deletion elevated single-cell entropy (a measure of differentiation potency or cell stemness) and RNA velocity length (a measure of the rate of cell differentiation) in noncycling and cycling Lgr5 + stem cells. In general, intercellular signaling cross-talk via soluble and membrane-bound factors was perturbed in Ahr-null colonocytes. Taken together, our single-cell RNA sequencing analyses provide new evidence of the molecular function of Ahr in modulating putative stem cell driver genes, cell potency lineage decisions, and cell-cell communication in vivo . PREVENTION RELEVANCE: Our mouse single-cell RNA sequencing analyses provide new evidence of the molecular function of Ahr in modulating colonic stemness and cell-cell communication in vivo . From a cancer prevention perspective, Ahr should be considered a therapeutic target to recalibrate remodeling of the intestinal stem cell niche., (©2021 American Association for Cancer Research.)