Genome-wide transcription factor-binding maps reveal cell-specific changes in the regulatory architecture of human HSPCs.

Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.

Role of Sex, Anxiety, and Resilience in the Association between Adverse Childhood Experiences and Irritable Bowel Syndrome.

BACKGROUND & AIMS: Adverse childhood experiences (ACE) are associated with increased risk of irritable bowel syndrome (IBS), a female-predominant chronic abdominal disorder. Factors contributing to this association have not been well-studied. We compared sex differences in ACE for adults with and without IBS and evaluated the impact of anxiety and resilience on the relationship between ACE and IBS. METHODS: Sex and disease differences in total score and ACE subtypes from the ACE Questionnaire in subjects with IBS and control subjects were assessed. Cross-sectional mediation analysis determined if anxiety (Hospital Anxiety and Depression Scale) and resilience (Connor-Davidson Resilience Scale or Brief Resilience Scale) mediated the relationship between ACE and IBS. RESULTS: Of 798 participants studied, 368 met IBS diagnostic criteria (265 women, 103 men) and 430 were healthy control subjects (277 women, 153 men). Prevalence and number of ACE were higher in IBS versus control subjects (P < .001) but similar between IBS women and men. Household mental illness increased odds of having IBS in women (odds ratio [OR], 1.95; 95% confidence interval [CI], 1.35-2.85; false discovery rate [FDR], 0.002) and men (OR, 2.32; 95% CI, 1.26-4.33; FDR, 0.014). Emotional abuse increased odds of having IBS in women (OR, 1.94; 95% CI, 1.23-3.09; FDR, 0.019) and sexual abuse increased odds of IBS in men (OR, 3.54; 95% CI, 1.35-10.38; FDR, 0.027). Anxiety mediated 54% (P < .001) of ACE's effect on IBS risk and resilience mediated 12%-14% (Connor-Davidson Resilience Scale, P = .008; Brief Resilience Scale, P = .018). CONCLUSIONS: Both men and women with a history of ACE are twice as likely to have IBS than those without an ACE. Anxiety mediated the relationship between ACE and IBS in men and women and resilience mediated this relationship only in women.

The Association Between a Mediterranean Diet and Symptoms of Irritable Bowel Syndrome.

BACKGROUND & AIMS: Low adherence to Mediterranean diet (MD) has been shown to be associated with a higher prevalence of irritable bowel syndrome (IBS), but its association with IBS symptoms is not established. We aim to assess the association between MD and IBS symptoms, identify components of MD associated with IBS symptoms, and determine if a symptom-modified MD is associated with changes in the gut microbiome. METHODS: One hundred and six Rome +IBS and 108 health control participants completed diet history and gastrointestinal symptom questionnaires. Adherence to MD was measured using Alternate Mediterranean Diet and Mediterranean Diet Adherence Screener. Sparse partial least squares analysis identified MD food items associated with IBS symptoms. Stool samples were collected for 16S ribosomal RNA gene sequencing and microbial composition analysis in IBS subjects. RESULTS: Alternate Mediterranean Diet and Mediterranean Diet Adherence Screener scores were similar between IBS and health control subjects and did not correlate with Irritable Bowel Syndrome Severity Scoring System, abdominal pain, or bloating. Among IBS participants, a higher consumption of fruits, vegetables, sugar, and butter was associated with a greater severity of IBS symptoms. Multivariate analysis identified several MD foods to be associated with increased IBS symptoms. A higher adherence to symptom-modified MD was associated with a lower abundance of potentially harmful Faecalitalea, Streptococcus, and Intestinibacter, and higher abundance of potentially beneficial Holdemanella from the Firmicutes phylum. CONCLUSIONS: A standard MD was not associated with IBS symptom severity, although certain MD foods were associated with increased IBS symptoms. Our study suggests that standard MD may not be suitable for all patients with IBS and likely needs to be personalized in those with increased symptoms.

Disruption of a GATA2-TAL1-ERG regulatory circuit promotes erythroid transition in healthy and leukemic stem cells.

Changes in gene regulation and expression govern orderly transitions from hematopoietic stem cells to terminally differentiated blood cell types. These transitions are disrupted during leukemic transformation, but knowledge of the gene regulatory changes underpinning this process is elusive. We hypothesized that identifying core gene regulatory networks in healthy hematopoietic and leukemic cells could provide insights into network alterations that perturb cell state transitions. A heptad of transcription factors (LYL1, TAL1, LMO2, FLI1, ERG, GATA2, and RUNX1) bind key hematopoietic genes in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and have prognostic significance in acute myeloid leukemia (AML). These factors also form a densely interconnected circuit by binding combinatorially at their own, and each other's, regulatory elements. However, their mutual regulation during normal hematopoiesis and in AML cells, and how perturbation of their expression levels influences cell fate decisions remains unclear. In this study, we integrated bulk and single-cell data and found that the fully connected heptad circuit identified in healthy HSPCs persists, with only minor alterations in AML, and that chromatin accessibility at key heptad regulatory elements was predictive of cell identity in both healthy progenitors and leukemic cells. The heptad factors GATA2, TAL1, and ERG formed an integrated subcircuit that regulates stem cell-to-erythroid transition in both healthy and leukemic cells. Components of this triad could be manipulated to facilitate erythroid transition providing a proof of concept that such regulatory circuits can be harnessed to promote specific cell-type transitions and overcome dysregulated hematopoiesis.

Colonic mucosal microbiota is associated with bowel habit subtype and abdominal pain in patients with irritable bowel syndrome.

Mucosal microbiota differ significantly from fecal microbiota and may play a different role in the pathophysiology of irritable bowel syndrome (IBS). The aims of this study were to determine if the composition of mucosal microbiota differed between IBS, or IBS bowel habit (BH) subtypes, and healthy controls (HCs). Sigmoid colon mucosal biopsies were obtained from 97 Rome-positive patients with IBS (28% IBS-constipation, 38% IBS-diarrhea, 24% IBS-mixed, and 10% IBS-unsubtyped) and 54 HCs, from which DNA was extracted. 16S rRNA gene sequencing and microbial composition analysis were performed. Group differences in α and ß diversity and taxonomic level differences were determined using linear regression while controlling for confounding variables. IBS BH subtype was associated with microbial α diversity (P = 0.0003) with significant differences seen in the mucosal microbiota of IBS-constipation versus IBS-diarrhea (P = 0.046). There were no significant differences in α or ß diversity in the mucosal microbiota of IBS versus HCs (P = 0.29 and 0.93, respectively), but metagenomic profiling suggested functional differences. The relative abundance of Prevotella_9 copri within IBS was significantly correlated with increased abdominal pain (r = 0.36, P = 0.0003), which has not been previously reported in IBS. Significant differences in the mucosal microbiota were present within IBS BH subtypes but not between IBS and HCs, supporting the possibility of IBS BH subtype-specific pathogenesis. Increased Prevotella copri may contribute to symptoms in patients with IBS.NEW & NOTEWORTHY Gut mucosal microbiota differs significantly from fecal microbiota in irritable bowel syndrome (IBS) and may play a different role in its pathophysiology. Investigation of colonic mucosal microbiota in the largest cohort of patients with IBS and healthy controls accounting for confounding variables, including diet demonstrated significant differences in mucosal microbiota between IBS bowel habit subtypes but not between IBS and healthy controls. In addition, the study reported gut microbiota is associated with abdominal pain in patients with IBS.

Effect of Exclusion Diets on Symptom Severity and the Gut Microbiota in Patients With Irritable Bowel Syndrome.

BACKGROUND & AIMS: Altered fecal microbiota have been reported in irritable bowel syndrome (IBS), although studies vary, which could be owing to dietary effects. Many IBS patients may eliminate certain foods because of their symptoms, which in turn may alter fecal microbiota diversity and composition. This study aimed to determine if dietary patterns were associated with IBS, symptoms, and fecal microbiota differences reported in IBS. METHODS: A total of 346 IBS participants and 170 healthy controls (HCs) completed a Diet Checklist reflecting the diet(s) consumed most frequently. An exclusion diet was defined as a diet that eliminated food components by choice. Within this group, a gluten-free, dairy-free, or low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols diet was further defined as restrictive because they often are implicated in reducing symptoms. Stool samples were obtained from 171 IBS patients and 98 HCs for 16S ribosomal RNA gene sequencing and microbial composition analysis. RESULTS: Having IBS symptoms was associated with consuming a restrictive diet (27.17% of IBS patients vs 7.65% of HCs; odds ratio, 3.25; 95% CI, 1.66-6.75; P value = .006). IBS participants on an exclusion or restrictive diet reported more severe IBS symptoms (P = .042 and .029, respectively). The composition of the microbiota in IBS patients varied depending on the diet consumed. IBS participants on an exclusion diet had a greater abundance of Lachnospira and a lower abundance of Eubacterium (q value, <.05), and those on a restrictive diet had a lower abundance of Lactobacillus (q value, <.05). CONCLUSIONS: Restrictive diets likely are consumed more by IBS patients than HCs to reduce GI symptom severity. Dietary patterns influence the composition of the fecal microbiota and may explain some of the differences between IBS and HCs.

The Colonic Mucosal MicroRNAs, MicroRNA-219a-5p, and MicroRNA-338-3p Are Downregulated in Irritable Bowel Syndrome and Are Associated With Barrier Function and MAPK Signaling.

BACKGROUND & AIMS: Alterations in microRNA (miRNA) and in the intestinal barrier are putative risk factors for irritable bowel syndrome (IBS). We aimed to identify differentially expressed colonic mucosal miRNAs, their targets in IBS compared to healthy controls (HCs), and putative downstream pathways. METHODS: Twenty-nine IBS patients (15 IBS with constipation [IBS-C], 14 IBS with diarrhea [IBS-D]), and 15 age-matched HCs underwent sigmoidoscopy with biopsies. A nCounter array was used to assess biopsy specimen-associated miRNA levels. A false discovery rate (FDR) < 10% was considered significant. Real-time polymerase chain reaction (PCR) was used to validate differentially expressed genes. To assess barrier function, trans-epithelial electrical resistance (TEER) and dextran flux assays were performed on Caco-2 intestinal epithelial cells that were transfected with miRNA-inhibitors or control inhibitors. Protein expression of barrier function associated genes was confirmed using western blots. RESULTS: Four out of 247 miRNAs tested were differentially expressed in IBS compared to HCs (FDR < 10%). Real-time PCR validation suggested decreased levels of miR-219a-5p and miR-338-3p in IBS (P = .026 and P = .004), and IBS-C (P = .02 and P = .06) vs. HCs as the strongest associations. Inhibition of miR-219a-5p resulted in altered expression of proteasome/barrier function genes. Functionally, miR-219a-5p inhibition enhanced the permeability of intestinal epithelial cells as TEER was reduced (25-50%, P < .05) and dextran flux was increased (P < .01). Additionally, inhibition of miR-338-3p in cells caused alterations in the mitogen-activated protein kinase (MAPK) signaling pathway genes. CONCLUSION: Two microRNAs that potentially affect permeability and visceral nociception were identified to be altered in IBS patients. MiR-219a-5p and miR-338-3p potentially alter barrier function and visceral hypersensitivity via neuronal and MAPK signaling and could be therapeutic targets in IBS.

Lysine methyltransferase 2D regulates pancreatic carcinogenesis through metabolic reprogramming.

OBJECTIVE: Despite advances in the identification of epigenetic alterations in pancreatic cancer, their biological roles in the pathobiology of this dismal neoplasm remain elusive. Here, we aimed to characterise the functional significance of histone lysine methyltransferases (KMTs) and demethylases (KDMs) in pancreatic tumourigenesis. DESIGN: DNA methylation sequencing and gene expression microarrays were employed to investigate CpG methylation and expression patterns of KMTs and KDMs in pancreatic cancer tissues versus normal tissues. Gene expression was assessed in five cohorts of patients by reverse transcription quantitative-PCR. Molecular analysis and functional assays were conducted in genetically modified cell lines. Cellular metabolic rates were measured using an XF24-3 Analyzer, while quantitative evaluation of lipids was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Subcutaneous xenograft mouse models were used to evaluate pancreatic tumour growth in vivo. RESULTS: We define a new antitumorous function of the histone lysine (K)-specific methyltransferase 2D (KMT2D) in pancreatic cancer. KMT2D is transcriptionally repressed in human pancreatic tumours through DNA methylation. Clinically, lower levels of this methyltransferase associate with poor prognosis and significant weight alterations. RNAi-based genetic inactivation of KMT2D promotes tumour growth and results in loss of H3K4me3 mark. In addition, KMT2D inhibition increases aerobic glycolysis and alters the lipidomic profiles of pancreatic cancer cells. Further analysis of this phenomenon identified the glucose transporter SLC2A3 as a mediator of KMT2D-induced changes in cellular, metabolic and proliferative rates. CONCLUSION: Together our findings define a new tumour suppressor function of KMT2D through the regulation of glucose/fatty acid metabolism in pancreatic cancer.

Sigmoid colon mucosal gene expression supports alterations of neuronal signaling in irritable bowel syndrome with constipation.

Peripheral factors likely play a role in at least a subset of irritable bowel syndrome (IBS) patients. Few studies have investigated mucosal gene expression using an unbiased approach. Here, we performed mucosal gene profiling in a sex-balanced sample to identify relevant signaling pathways and gene networks and compare with publicly available profiling data from additional cohorts. Twenty Rome III+ IBS patients [10 IBS with constipation (IBS-C), 10 IBS with diarrhea (IBS-D), 5 men/women each), and 10 age-/sex-matched healthy controls (HCs)] underwent sigmoidoscopy with biopsy for gene microarray analysis, including differential expression, weighted gene coexpression network analysis (WGCNA), gene set enrichment analysis, and comparison with publicly available data. Expression levels of 67 genes were validated in an expanded cohort, including the above samples and 18 additional participants (6 each of IBS-C, IBS-D, HCs) using NanoString nCounter technology. There were 1,270 differentially expressed genes (FDR < 0.05) in IBS-C vs. HCs but none in IBS or IBS-D vs. HCs. WGNCA analysis identified activation of the cAMP/protein kinase A signaling pathway. Nine of 67 genes were validated by the NanoString nCounter technology (FDR < 0.05) in the expanded sample. Comparison with publicly available microarray data from the Mayo Clinic and University of Nottingham supports the reproducibility of 17 genes from the microarray analysis and three of nine genes validated by nCounter in IBS-C vs. HCs. This study supports the involvement of peripheral mechanisms in IBS-C, particularly pathways mediating neuronal signaling. NEW & NOTEWORTHY Peripheral factors play a role in the pathophysiology of irritable bowel syndrome (IBS), which, to date, has been mostly evident in IBS with diarrhea. Here, we show that sigmoid colon mucosal gene expression profiles differentiate IBS with constipation from healthy controls. These profiling data and analysis of additional cohorts also support the concept that peripheral neuronal pathways contribute to IBS pathophysiology.

Veliparib in combination with radiotherapy for the treatment of MGMT unmethylated glioblastoma.

BACKGROUND: The O 6 -methylguanine methyltransferase (MGMT) gene is frequently unmethylated in patients with glioblastoma (GBM), rendering them non-responsive to the standard treatment regime of surgery followed by concurrent radiotherapy (RT) and temozolomide. Here, we investigate the efficacy of adding a PARP inhibitor, veliparib, to radiotherapy to treat MGMT unmethylated GBM. METHODS: The inhibition of PARP with veliparib (ABT-888), a potent and orally bioavailable inhibitor in combination with RT was tested on a panel of patient derived cell lines (PDCLs) and patient-derived xenografts (PDX) models generated from GBM patients with MGMT unmethylated tumors. RESULTS: The combination of veliparib and RT inhibited colony formation in the majority of PDCLs tested. The PDCL, RN1 showed significantly reduced levels of the homologous repair protein, Mre11 and a heightened response to PARP inhibition measured by increased apoptosis and decreased colony formation. The oral administration of veliparib (12.5 mg/kg, twice daily for 5 days in a 28-day treatment cycle) in combination with whole brain RT (4 Gy) induced apoptosis (Tunel staining) and decreased cell proliferation (Ki67 staining) in a PDX of MGMT unmethylated GBM. Significantly longer survival times of the PDX treated with the combination treatment were recorded compared to RT only or veliparib only. CONCLUSIONS: Our results demonstrate preclinical efficacy of targeting PARP at multiple levels and provide a new approach for the treatment of MGMT unmethylated GBM.

A long noncoding RNA signature for ulcerative colitis identifies IFNG-AS1 as an enhancer of inflammation.

High-throughput technologies revealed new categories of genes, including the long noncoding RNAs (lncRNAs), involved in the pathogenesis of human disease; however, the role of lncRNAs in the ulcerative colitis (UC) has not been evaluated. Gene expression profiling was used to develop lncRNA signatures in UC samples. Jurkat T cells were activated by PMA/ionomycin subsequently interferon-γ (IFNG) and tumor necrosis factor (TNF)-α protein levels were assessed by ELISA. Anti-sense molecules were designed to block IFNG-AS1 expression. A unique set of lncRNAs was differentially expressed between UC and control samples. Of these, IFNG-AS1 was among the highest statistically significant lncRNAs (fold change: 5.27, P value: 7.07E-06). Bioinformatic analysis showed that IFNG-AS1 was associated with the IBD susceptibility loci SNP rs7134599 and its genomic location is adjacent to the inflammatory cytokine IFNG. In mouse models of colitis, active colitis samples had increased colonic expression of this lncRNA. Utilizing the Jurkat T cell model, we found IFNG-AS1 to positively regulate IFNG expression. Novel lncRNA signatures differentiate UC patients with active disease, patients in remission, and control subjects. A subset of these lncRNAs was found to be associated with the clinically validated IBD susceptibility loci. IFNG-AS1 was one of these differentially expressed lncRNAs in UC patients and found to regulate the key inflammatory cytokine, IFNG, in CD4 T cells. Taking these findings together, our study revealed novel lncRNA signatures deregulated in UC and identified IFNG-AS1 as a novel regulator of IFNG inflammatory responses, suggesting the potential importance of noncoding RNA mechanisms on regulation of inflammatory bowel disease-related inflammatory responses.

Transcriptomic and CRISPR/Cas9 technologies reveal FOXA2 as a tumor suppressor gene in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with low survival rates and limited therapeutic options. Thus elucidation of signaling pathways involved in PDAC pathogenesis is essential for identifying novel potential therapeutic gene targets. Here, we used a systems approach to elucidate those pathways by integrating gene and microRNA profiling analyses together with CRISPR/Cas9 technology to identify novel transcription factors involved in PDAC pathogenesis. FOXA2 transcription factor was found to be significantly downregulated in PDAC relative to control pancreatic tissues. Functional experiments revealed that FOXA2 has a tumor suppressor function through inhibition of pancreatic cancer cell growth, migration, invasion, and colony formation. In situ hybridization analysis revealed miR-199a to be significantly upregulated in pancreatic cancer. Bioinformatics and luciferase analyses showed that miR-199a negatively but directly regulates FOXA2 expression through binding in its 3'-untranslated region (UTR). Evaluation of the functional importance of miR-199a on pancreatic cancer revealed that miR-199a acts as an inhibitor of FOXA2 expression, inducing an increase in pancreatic cancer cell proliferation, migration, and invasion. Additionally, gene ontology and network analyses in PANC-1 cells treated with a small interfering RNA (siRNA) against FOXA2 revealed an enrichment for cell invasion mechanisms through PLAUR and ERK activation. FOXA2 deletion (FOXA2Δ) by using two CRISPR/Cas9 vectors in PANC-1 cells induced tumor growth in vivo resulting in upregulation of PLAUR and ERK pathways in FOXA2Δ xenograft tumors. We have identified FOXA2 as a novel tumor suppressor in pancreatic cancer and it is regulated directly by miR-199a, thereby enhancing our understanding of how microRNAs interplay with the transcription factors to affect pancreatic oncogenesis.

Alterations in the mitochondrial responses to PENAO as a mechanism of resistance in ovarian cancer cells.

OBJECTIVE: The purpose of this study was to test PENAO, a promising new organoarsenical that is in phase 1 testing in patients with solid tumours, on a range of ovarian cancer cell lines with different histotypes, and to understand the molecular basis of drug resistance exhibited by the endometrioid ovarian cancer cell line, SKOV-3. METHODS: Proliferation arrest and cell death induced by PENAO in serous (OVCAR-3), endometrioid (SKOV-3, TOV112D), clear cell (TOV21G) and mucinous (EFO27) ovarian cancer cells in culture, and anti-tumour efficacy in a murine model of SKOV-3 and OVCAR-3 tumours, were measured. Cells were analysed for cell cycle arrest, cell death mechanisms, reactive oxygen species production, mitochondrial depolarisation, oxygen consumption and acid production. RESULTS: PENAO demonstrated promising anti-proliferative activity on the most common (serous, endometrioid) as well as on rare (clear cell, mucinous) subtypes of ovarian cancer cell lines. No cross-resistance with platinum-based drugs was evident. Endometrioid SKOV-3 cells were, however, shown to be resistant to PENAO in vitro and in a xenograft mouse model. This resistance was due to an ability to cope with PENAO-induced oxidative stress, notably through heme oxygenase-1 induction, and a shift in metabolism towards glycolysis. The adaptive glycolytic shift in SKOV-3 was targeted using a mTORC1 inhibitor in combination with PENAO. This strategy was successful with the two drugs acting synergistically to inhibit cell proliferation and to induce cell death via apoptosis and autophagy. CONCLUSION: Mitochondria/mTOR dual-targeting therapy may constitute a new approach for the treatment of recurrent/resistant forms of epithelial ovarian cancer.

Employing pancreatic tumor γ-glutamyltransferase for therapeutic delivery.

γ-Glutamyltransferase (γGT) is a cell surface enzyme that catalyzes hydrolysis of the bond linking the glutamate and cysteine residues of glutathione and glutathione-S-conjugates. We have observed that human pancreatic tumor cells and tumor-associated stellate cells express high levels of this enzyme when compared to normal pancreatic epithelial and stellate cells. Detection of the protein in tumor sections correlated with γGT activity on the surface of the cultured tumor and stellate cells. We tested whether the tumor γGT could be employed to deliver a therapeutic to the tumor endothelial cells. GSAO is a glutathione-S-conjugate of a trivalent arsenical that is activated to enter endothelial cells by γGT cleavage of the γ-glutamyl residue. The arsenical moiety triggers proliferation arrest and death of the endothelial cells by targeting the mitochondria. Human pancreatic tumor and stellate cell γGT activated GSAO in culture and γGT activity positively correlated with GSAO-mediated proliferation arrest and death of endothelial cells in Transwell and coculture systems. A soluble form of γGT is found in blood, and we measured the rate of activation of GSAO by this enzyme. We calculated that systemically administered GSAO would circulate through the pancreatic blood supply several times before appreciable activation by normal blood levels of γGT. In support of this finding, tumor γGT activity positively correlated with GSAO-mediated inhibition of pancreatic tumor angiogenesis and tumor growth in mice. Our findings indicate that pancreatic tumor γGT can be used to deliver a therapeutic to the tumor.

Relationships between tryptophan-related gut metabolites, brain activity, and autism symptomatology.

Gut microbial metabolites have been theorized to play a causative role in the pathophysiology of autism spectrum disorder (ASD). This hypothesis is based on results from mechanistic preclinical studies and several correlational studies showing differences in gut microbial composition between ASD subjects and neurotypical (NT) controls. However, alterations in how the human brain interacts with the gut microbiome in ASD have not been examined. In this cross-sectional, case-control observational study, fecal metabolomics, task-based functional magnetic resonance imaging (fMRI), and behavioral assessments were obtained from 43 ASD and 41 NT children aged 8-17. The fMRI tasks were based on socio-emotional and sensory paradigms that commonly show strong evoked brain differences in ASD participants. General linear models and mediational modeling were applied to examine the links between tryptophan metabolism and evoked brain activity and behavior. Results indicated that fecal levels of specific tryptophan-related metabolites were associated with: 1) brain activity atypicalities in regions previously implicated in ASD (i.e., insula and cingulate); and 2) ASD severity and symptomatology (i.e., ADOS scores, disgust propensity, and sensory sensitivities). Importantly, activity in the mid-insula and mid-cingulate significantly mediated relationships between the microbial tryptophan metabolites, indolelactate and tryptophan betaine, and ASD severity and disgust sensitivity. To our knowledge, this is the first study to elucidate how interactions between gut metabolites and brain activity may impact autism symptomatology, particularly in functional brain pathways associated with vagal and interoceptive/emotion processing.

Sex Differences in Colonic Mucosal Microbiome of Irritable Bowel Syndrome Patients Compared to Healthy Controls.

BACKGROUND: Irritable bowel syndrome (IBS) is a female-predominant disorder of brain-gut interactions. Our previous study on colonic mucosal microbiota demonstrated significant differences between IBS bowel habit subtypes and showed that gut microbiota is associated with abdominal pain in IBS patients. However, there is no consensus on sex-related differences in mucosal microbiota in IBS compared to healthy controls (HC). We aimed to identify sex-related differences in the mucosal microbes associated with IBS. METHODS: Sigmoid mucosal biopsies were obtained from 97 Rome+ IBS patients and 54 healthy controls (HC). Mucosal microbiome was characterized using 16S rRNA sequencing and analyzed and general linear models were used to test group differences between IBS diagnosis and sex. Sex-specific relationships between mucosal microbiome and IBS symptoms were assessed using sparse partial least squares (sPLS) regression. RESULTS: Beta diversity was significantly different between men and women overall (p=.03) but not within IBS or HC. IBS women showed lower abundance of Catenibacterium and Ruminoclstridium_9 and increased abundance of Bacteroides, Escherichia/Shigella, Lachnoclostridium and Ruminococcaceae compared to men with IBS (p<0.05). However, healthy women had a lower abundance of six distinct genera compared to healthy men. In women, higher IBS symptoms were associated with an increased abundance of bacteria including prevotella_9, and paraprevotella, however, in men, IBS symptoms were associated with increased abundances of genera such as Dialister. Interestingly, increased abundance of Desulfovibrio was associated with higher symptoms in women but lower symptoms in men. CONCLUSION: There are distinct sex-related differences in the mucosal microbiome between IBS and healthy participants supporting the importance of studying sex-specific mechanisms in IBS pathophysiology.

TOPORS E3 ligase mediates resistance to hypomethylating agent cytotoxicity in acute myeloid leukemia cells.

Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However, acquired resistance and treatment failure are commonplace. To address this, we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line, MDS-L, and identify TOPORS as a loss-of-function target that synergizes with HMAs, reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable, we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall, our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML.

Differential brainstem connectivity according to sex and menopausal status in healthy men and women.

Background Brainstem nuclei play a critical role in both ascending monoaminergic modulation of cortical function and arousal, and in descending bulbospinal pain modulation. Even though sex-related differences in the function of both systems have been reported in animal models, a complete understanding of sex differences, as well as menopausal effects, in brainstem connectivity in humans is lacking. This study evaluated resting-state connectivity of the dorsal raphe nucleus (DRN), right and left locus coeruleus complex (LCC), and periaqueductal gray (PAG) according to sex and menopausal status in healthy individuals. In addition, relationships between systemic estrogen levels and brainstem-network connectivity were examined in a subset of participants. Methods Resting-state fMRI was performed in 50 healthy men (age, 31.2 ± 8.0 years), 53 healthy premenopausal women (age, 24.7 ± 7.3 years; 22 in the follicular phase, 31 in the luteal phase), and 20 postmenopausal women (age, 54.6 ± 7.2 years). Permutation Analysis of Linear Models (5000 permutations) was used to evaluate differences in brainstem-network connectivity according to sex and menopausal status, controlling for age. In 10 men and 17 women (9 premenopausal; 8 postmenopausal), estrogen and estrogen metabolite levels in plasma and stool were determined by liquid chromatography-mass spectrometry/mass spectrometry. Relationships between estrogen levels and brainstem-network connectivity were evaluated by partial least squares analysis. Results Left LCC-executive control network (ECN) connectivity showed an overall sex difference (p = 0.02), with higher connectivity in women than in men; however, this was mainly due to differences between men and pre-menopausal women (p = 0.008). Additional sex differences were dependent on menopausal status: PAG-default mode network (DMN) connectivity was higher in postmenopausal women than in men (p = 0.04), and PAG-sensorimotor network (SMN) connectivity was higher in premenopausal women than in men (p = 0.03) and postmenopausal women (p = 0.007). Notably, higher free 2-hydroxyestrone levels in stool were associated with higher PAG-SMN and PAG-DMN connectivity in premenopausal women (p < 0.01). Conclusions Healthy women show higher brainstem-network connectivity involved in cognitive control, sensorimotor function, and self-relevant processes than men, dependent on their menopausal status. Further, 2-hydroxyestrone, implicated in pain, may modulate PAG connectivity in premenopausal women. These findings may relate to differential vulnerabilities to chronic stress-sensitive disorders at different life stages.

Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness.

Background and Aims: Decoding pancreatic ductal adenocarcinoma heterogeneity and the consequent therapeutic selection remains a challenge. We aimed to characterize epigenetically regulated pathways involved in pancreatic ductal adenocarcinoma progression. Methods: Global DNA methylation analysis in pancreatic cancer patient tissues and cell lines was performed to identify differentially methylated genes. Targeted bisulfite sequencing and in vitro methylation reporter assays were employed to investigate the direct link between site-specific methylation and transcriptional regulation. A series of in vitro loss-of-function and gain-of function studies and in vivo xenograft and the KPC (LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx1-Cre) mouse models were used to assess pancreatic cancer cell properties. Gene and protein expression analyses were performed in 3 different cohorts of pancreatic cancer patients and correlated to clinicopathological parameters. Results: We identify Hepatocyte Nuclear Factor 4A (HNF4A) as a novel target of hypermethylation in pancreatic cancer and demonstrate that site-specific proximal promoter methylation drives HNF4A transcriptional repression. Expression analyses in patients indicate the methylation-associated suppression of HNF4A expression in pancreatic cancer tissues. In vitro and in vivo studies reveal that HNF4A is a novel tumor suppressor in pancreatic cancer, regulating cancer growth and aggressiveness. As evidenced in both the KPC mouse model and human pancreatic cancer tissues, HNF4A expression declines significantly in the early stages of the disease. Most importantly, HNF4 loss correlates with poor overall patient survival. Conclusion: HNF4A silencing, mediated by promoter DNA methylation, drives pancreatic cancer development and aggressiveness leading to poor patient survival.

Making Meaning Together: Co-designing a Social Robot for Older Adults with Ikigai Experts.

A sense of meaning and purpose in life-known in Japan as one's ikigai-can lead to better health outcomes, an improved sense of well-being, and longer life as people age. The design of socially assistive robots, however, has so far focused largely on the more hedonic aims of supporting positive affect and happiness through interactions with robots. To explore how social robots might be able to support people's ikigai, we performed (1) in-depth interviews with 12 'ikigai experts' who formally support and/or study older adults (OAs)' ikigai and (2) 5 co-design workshop sessions with 10 such experts. Our interview findings show that expert practitioners define ikigai in a holistic way in their everyday experience and practice, incorporating physical, social, and mental activities that relate not only to the individual and their behaviors, but also to their relationships with other people and to their connection with the broader community (3 levels of ikigai). Our co-design workshops showed that ikigai experts were overall positive towards the use of social robots to support OAs' ikigai, particularly in the roles of an information-provider and social enabler that connects OAs to other people and activities in their communities. They also point out areas of potential risk, including the need to maintain OAs' independence, relationships with others, and privacy, which should be considered in design. This research is the first to explore the co-design of social robots that can support people's sense of ikigai-meaning and purpose-as they age.