BACKGROUND:Ethanolamine (Etn), a precursor of phosphatidylethanolamine (PE), may alter hepatic lipid homeostasis and gut health; its dietary effects remain undefined. OBJECTIVE:The objective of this study was to determine the effects of dietary Etn on lipid and glucose metabolism and liver/gut health in high-fat diet (HFD)-fed mice, complemented by in vitro hepatocyte assays. METHODS:Ten-wk-old C57BL/6 mice (20 male, 18 female) were fed ad libitum HFD (45% energy from fat) with [Ethanolamine supplementation (ES-group)] or without (CON-group) Etn (8 g/kg diet) for 10 wk. Outcomes included body/liver weight, glucose tolerance test (GTT) results, plasma phosphatidylcholine (PC)/cholesteryl ester (CE)/triacylglycerol (TG) concentrations, hepatic TG/PC/PE concentrations, hepatic endoplasmic reticulum (ER)-stress, and inflammation markers, jejunal morphology/barrier/inflammation genes, and fecal microbiota (α/β diversity). HuH7 cells received 20 μM or 5 mM Etn to assess TG/PC/PE synthesis. STATISTICS:repeated-measures analysis of variance (ANOVA) (GTT), t-test or Wilcoxon (other endpoints), permutational multivariate analysis of variance (PERMANOVA) (β diversity); α=0.05. RESULTS:ES increased hepatic TG in females by 230% compared with CON (P = 0.001), and trended higher in males (P = 0.054); hepatic PC and PE masses were unchanged. In ES males, GTT area under the curve decreased by 22.6% (P = 0.037), and plasma PC, CE, and TG were reduced by: PC -16.6%, CE -24.5%, TG -25.9%, respectively (all P < 0.05). ES males showed higher hepatic Tnf and Cd68 and increased C/EBP homologous protein (CHOP) (all P < 0.05). In vitro, Etn did not alter hepatocellular TG, PC, or PE synthesis (all P > 0.05). Female ES mice exhibited altered fecal β-diversity (PERMANOVA P = 0.006) with early jejunal inflammatory signals (Tnf ↑; P = 0.055). CONCLUSIONS:Dietary Etn modifies hepatic lipid storage and gut microbiota in a sex-dependent manner and improves glucose tolerance in males, whereas in vitro data indicate no direct effect on hepatocyte lipid synthesis.

Abstract Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is characterized and initiated by the excessive accumulation of triacylglycerols (TG) and cholesteryl esters (CE) in the liver. Hepatic TG and CE synthesis, lipolysis and transport are tightly regulated by nutritional status, and disruption of this homeostasis contributes to MASLD pathogenesis. We have found that an endoplasmic reticulum-localized arylacetamide deacetylase (AADAC) catalyzes hepatic TG/CE turnover, and suppresses SREBP- and LXR-regulated lipogenesis and fatty acid esterification. Consequently, AADAC deficiency in mice leads to increased hepatic lipid synthesis, exacerbated steatosis, and impaired whole-body metabolism during Western-type diet feeding. These findings implicate AADAC as an important regulator of hepatic neutral lipid metabolism, linking endoplasmic reticulum cholesteryl ester hydrolysis as a modulator of lipid synthesis, and suggest its potential role in limiting MASLD pathogenesis under conditions of chronic overnutrition.

A 30-year-old man with a history of liver transplantation (LT) for primary sclerosing cholangitis presented with a clinical picture compatible with ascending cholangitis. The patient was started on antibiotic therapy, and a magnetic resonance imaging with cholangiopancreatography (MRCP) revealed biliary dilation. The patient then underwent double-balloon endoscopic retrograde cholangiopancreatography (DBE-ERCP). Endoscopic findings revealed an ulcerated mass obstructing the hepatico-jejunostomy (HJ), and biopsies were taken. Pathology was compatible with nondestructive post-transplant lymphoproliferative disorder, and the patient received treatment with rituximab. Eight weeks after treatment, repeat DBE-ERCP showed resolution of the mass and a patent HJ. To our knowledge, this is the first report of a diagnosis of post-transplant lymphoproliferative disorder at the HJ after LT identified with DBE-ERCP.

Background: The biliary and pancreatic tract is increasingly recognized as a microbial ecosystem rather than a sterile environment. Dysbiosis contributes to inflammation, bile acid alterations, and carcinogenesis, with distinct microbiota profiles linked to progression from benign to malignant conditions. Clinical factors, including gut-liver axis disruption and biliary stenting, may further exacerbate microbial imbalance. Objective: The objective of this study is to synthesize current evidence and identify knowledge gaps on the role of biliary microbiota in pancreaticobiliary carcinogenesis and its implications for diagnosis, prognosis, and therapy. Methods: This scoping review was conducted following PRISMA-ScR guidelines. A systematic search of PubMed, Web of Science, and Scopus was performed for studies published between January 2015 and December 2025, guided by the PICo framework. Results: Included studies primarily characterized changes in microbiota composition to identify microbial biomarkers associated with pancreaticobiliary diseases. Predictive bioinformatics analyses suggest that dysbiosis may promote carcinogenesis through metabolic and inflammatory pathways. Machine learning approaches identified microbiota-based signatures with potential diagnostic value for precancerous lesions, although discrimination remains limited. Biliary dysbiosis was also associated with outcomes related to biliary stenting, chemoprophylaxis, postoperative complications, and responses to chemotherapy or surgery. Conclusions: Integration of microbiota profiling with predictive bioinformatics and machine learning may improve understanding of pancreaticobiliary carcinogenesis. Identifying microbial and functional biomarkers could enable personalized diagnostic and therapeutic strategies, ultimately improving patient outcomes.

Background:A chart audit (pre-intervention, PRE) of endoscopic ultrasound (EUS)-fine needle biopsy (FNB) of solid masses identified a diagnostic yield of 75%. To improve diagnostic yield, a quality improvement intervention targeting tissue procurement and processing was developed and trialed (post-intervention, POST). Intervention:Increase needle passes to ≥3/mass, limit the personnel preparing cytology slides, and replace saline with formalin for cell block preparation. Methods:A POST chart audit was undertaken for solid mass EUS-FNB from January 2024-December 2024, with quarterly reviews. Only a definite diagnosis was used to calculate diagnostic yield. ResultsAbout:241 patients underwent 262 EUS-FNBs. Diagnostic yield was 81% in POST vs. 75% in PRE (p = ns). Single passes decreased to 14% in POST vs. 20% in PRE (p = ns), with similar diagnostic yield (57% vs. 56%, respectively). Number of ≥3 passes increased significantly (47% POST vs. 12% PRE, P < .0001). In POST, 86% of single passes were performed in the first half vs. 14% in the second (P = .012). Only 25% of ≥3 passes were performed in the first half vs. 75% in the second (P = .06). Diagnostic yield improved significantly between the first and second halves (72% vs. 90%, P < .0001). Replacing saline with formalin for cell block improved diagnostic yield (57% vs. 75%, P = .062). Limiting the personnel making slides from 25 nurses (PRE) to 3 endoscopists (POST) had no impact on diagnostic yield. Conclusions:Critical practice assessment, regular audit, and continued reinforcement led to a significant improvement in EUS-FNB diagnostic yield to 90% by increasing the number of needle passes to ≥3/mass.

Abstract Background A retrospective chart audit (01/2022-12/2022) of endoscopic ultrasound (EUS)-guided fine needle aspiration biopsy (FNAB) of solid mass lesions revealed a disappointing diagnostic yield of 56% with a single needle pass. To improve this, a quality improvement (QI) intervention where endoscopists (three) were encouraged to perform three needle passes per patient was developed and trialed. Aims To assess intervention impact on improving the diagnostic yield of EUS-FNAB of solid mass lesions over a 9-month study period. Methods A chart audit was completed quarterly for all patients undergoing EUS-FNAB of solid mass lesions from 01/2024-09/2024. Descriptive statistics were completed. Only a definite diagnosis, as confirmed on histological examination, was considered when calculating the diagnostic yield. Results A total of 183 patients (112 M, 71 F), mean age 63±13 years (range 12-88 years), underwent 198 EUS-FNABs by 3 endoscopists over 9 months. A single pass with an FNAB needle was undertaken in 36/198 cases (18%). The diagnostic yield was 20/36 (56%), similar to the pre-intervention year. The solid mass lesions targeted were pancreas (16), lymph nodes (12), subepithelial (3), rectal and retro-peritoneal (2 each), and ampulla (1). One endoscopist performed 25/36 cases (69%) whereas the other two performed 11/36 (31%) and 0/36 cases. Proximity to vasculature and technical difficulty were reasons provided in 11 and 1 case(s), respectively, whereas no reason was documented in 24 cases. There was no difference in these variables between the groups with a definite diagnosis (20/36) vs. those without (16/36). After the first quarter audit, the need to avoid a single needle-pass was reinforced. Figure 1 shows the trend of single vs. 3 or more needle passes for the pre-intervention vs. the post-intervention year. There is a trend towards performing fewer single needle passes (39 pre vs. 36 post) and increasing 3 or more needle passes (25 pre vs. 68 post). This was associated with an improvement in diagnostic yield in the pre (22/39 [56%] vs. 21/25 [84%]) vs. post groups (20/36 [56%] vs. 61/68 [90%]). Conclusions Education, regular audit, and continued reinforcement has demonstrated an improvement in the diagnostic yield of EUS-FNAB by increasing the number of needle passes to 3 or more for solid mass lesions. Funding Agencies None

Ethanolamine phosphate phospholyase (ETNPPL) is an enzyme that irreversibly degrades phosphoethanolamine (p-ETN), an intermediate in the Kennedy pathway of phosphatidylethanolamine (PE) synthesis. Whole body knockout Etnppl mice were fed a high-fat diet (HFD) containing 45% kcal fat for 10 wk. Etnppl-/- female mice were resistant to HFD-induced obesity and had decreased liver weight compared with Etnppl+/+ mice. Furthermore, Etnppl-/- female mice had improved glucose sensitivity and increased energy expenditure compared with Etnppl+/+ mice. Plasma triglyceride (TG) levels were elevated in Etnppl-/- female mice, although the rate of very low-density lipoprotein (VLDL) secretion was not increased. The hepatic expression of PCSK9 was elevated, indicating a possible decrease in VLDL uptake. Interestingly, both plasma and hepatic cholesterol levels were reduced in Etnppl-/- relative to Etnppl+/+ mice. No difference in hepatic phosphatidylcholine, PE, or TG was detected between groups. Histopathological examination of hepatic tissues revealed decreased lipid deposition in Etnppl-/- mice that may be explained by the lower hepatic cholesterol level. Additionally, RNA sequencing analysis showed upregulation in genes related to cholesterol metabolism in Etnppl-/- female mice. In male mice, a slight decrease in weight gain was observed in Etnppl-/- mice compared with Etnppl+/+ mice. No change in plasma and hepatic lipid levels was detected in Etnppl-/- male mice. To conclude, ETNPPL impacts whole body energy expenditure, weight gain, cholesterol metabolism, and hepatic lipoprotein metabolism without altering hepatic phospholipid levels.NEW & NOTEWORTHY Etnppl-/- female mice resisted diet-induced obesity with enhanced energy expenditure and less adipose tissue. In addition, Etnppl-/- female mice fed an HFD showed decreased liver cholesterol deposition. RNA sequencing revealed changes in genes related to cholesterol and lipid metabolism in Etnppl-/- female mice. Etnppl-/- female mice fed an HFD supplemented with cholesterol had no difference in plasma and hepatic cholesterol levels compared with Etnppl+/+ mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the MECP2 gene, potentially disrupting lipid metabolism and leading to dyslipidemia (DLD) and steatotic liver disease (SLD). Although SLD has been described in RTT mouse models, it remains undocumented in humans. We herein describe a 24-year-old woman with RTT who was evaluated for abnormal liver enzymes. Imaging revealed hepatic steatosis, and transient elastography showed a controlled attenuation parameter of 342 dB/m and stiffness of 7.1 kPa. Laboratory investigations excluded secondary causes, including insulin resistance, metabolic syndrome, alcohol use, and new medications. Her Homeostatic Model Assessment for Insulin Resistance score was 1.8, her hemoglobin A1c concentration was 4.8%, and her lipid profile showed elevated triglycerides and low-density lipoprotein, consistent with DLD. Liver biopsy confirmed SLD. This case supports the hypothesis that MECP2 mutations in RTT disrupt lipid metabolism through a unique pathophysiologic mechanism, increasing the risk of DLD and SLD independently of traditional metabolic syndrome factors. It highlights the importance of early screening for liver disease in patients with RTT, despite their young age, to prevent complications. Additionally, it validates MECP2-null mouse models as reliable tools for investigating future therapeutic strategies in RTT.

BACKGROUND:Induced pluripotent stem cells (iPSCs) offer the potential to generate autologous iPSC-derived islets (iPSC islets), however, remain limited by scalability and product safety. METHODS:Herein, we report stagewise characterization of cells generated following a bioreactor-based differentiation protocol. Cell characteristics were assessed using flow cytometry, quantitative reverse transcription polymerase chain reaction, patch clamping, functional assessment, and in vivo functional and immunohistochemistry evaluation. Protocol yield and costs are assessed to determine scalability. RESULTS:Differentiation was capable of generating 90.4% PDX1 + /NKX6.1 + pancreatic progenitors and 100% C-peptide + /NKX6.1 + iPSC islet cells. However, 82.1%, 49.6%, and 0.9% of the cells expressed SOX9 (duct), SLC18A1 (enterochromaffin cells), and CDX2 (gut cells), respectively. Explanted grafts contained mature monohormonal islet-like cells, however, CK19 + ductal tissues persist. Using this protocol, semi-planar differentiation using 150 mm plates achieved 5.72 × 10 4 cells/cm 2 (total 8.3 × 10 6 cells), whereas complete suspension differentiation within 100 mL Vertical-Wheel bioreactors significantly increased cell yield to 1.1 × 10 6 cells/mL (total 105.0 × 10 6 cells), reducing costs by 88.8%. CONCLUSIONS:This study offers a scalable suspension-based approach for iPSC islet differentiation within Vertical-Wheel bioreactors with thorough characterization of the ensuing product to enable future protocol comparison and evaluation of approaches for off-target cell elimination. Results suggest that bioreactor-based suspension differentiation protocols may facilitate scalability and clinical implementation of iPSC islet therapies.

Type IV secretion systems (T4SSs) are central to bacterial pathogenesis. Traditionally known for facilitating DNA transfer via conjugation, T4SSs also mediate biofilm formation. These biofilms are critical for the fitness of adherent-invasive Escherichia coli (AIEC), which are commonly isolated from Crohn's disease patients and are known for propelling gut inflammation. Many AIEC strains carry F-like plasmids encoding the IncF subgroup of T4SSs. Unlike minimized systems with 12 core components, the IncF family is an expanded T4SS with additional genes that enhance conjugation. Here, we show that a biofilm-forming AIEC strain harbors an unusual IncF plasmid that lacks two conserved components essential for T4SS functionality. This strain forms a natural hybrid T4SS where the missing components are supplied by a co-residing chromosomal T4SS on an integrative and conjugative element (ICE). Biochemical assays reveal that this hybrid T4SS drives pilin polymerization and biofilm formation on epithelial cells. Furthermore, we show that a bacterial subpopulation expresses the IncF and ICE-encoded genes in response to host cells, leading to the assembly of biofilms and enhanced fitness in the gut. These findings uncover crosstalk between two evolutionary distant mobile genetic elements to form a hybrid T4SS that mediates biofilm biogenesis by a Crohn's disease-associated pathogen.

Abstract Background A chart audit to review endoscopic ultrasound (EUS)-fine needle aspiration biopsy (FNAB) of solid mass lesions from 01/2022-12/2022 identified a diagnostic yield of 75%. To improve this, a quality improvement intervention including increasing the number of needle passes to 3/case, improving needle pass documentation in endoscopy reports, reducing the number of individuals making cytology slides, and using formalin as transport medium for cell block preparation instead of saline was developed and trialed for 9 months. Aims To assess intervention impact on improving the diagnostic yield of EUS-FNAB of solid mass lesions. Methods Three endoscopists were provided targeted education and a chart audit was completed for all patients undergoing EUS-FNAB of solid mass lesions from 01/2024-09/2024. Descriptive statistics were completed. Only a definite diagnosis, as confirmed on histological examination, was considered when calculating the diagnostic yield. Results A total of 183 patients (112 M, 71 F), mean age 63±13 years (range 12-88 years), underwent 198 EUS-FNABs by 3 endoscopists (who made cytology slides, ensured transport medium and completed documentation). Pancreatic masses were the most common (122/198, 62%). A 22-gauge FNAB needle was used in 194/198 (98%) cases. A total of 420 needle passes were performed for 191 patient cases (mean 2.2/case) compared with mean of 1.9/case pre-intervention. Documentation improved, 7 cases (3.5%) did not have the number of needle passes specified compared with 51 cases (26%) pre-intervention. Tissue samples were transported in formalin for histology in 197 cases, on cytology slides in 165 cases, and in formalin for cell block preparation in 42 cases, similar to pre-intervention. Overall, a definite diagnosis was achieved in 156/198 cases (79%) compared with 149/200 (75%) in the pre-intervention year. Stratifying for needle passes, a definite diagnosis was achieved in 20/36 (56%), 69/87 (79%), 56/63 (89%), and 5/5 (100%) cases that had 1, 2, 3, and >3 needle passes, respectively. The number of passes was seen to independently impact diagnostic yield regardless of type of solid mass, endoscopist, type/size of needle used, and whether or not tissue was provided for cell block preparation. Conclusions Although we did not reach our goal of 3 needle passes per case, documentation and the number of tissue samples transported in formalin improved. The results suggest 3 or more needle passes per case may improve diagnostic yield and attempts should be made to avoid performing single needle pass FNAB. Funding Agencies None

Ethanolamine phosphate phospholyase (ETNPPL) is an enzyme that irreversibly degrades phospho-ethanolamine (p-ETN), an intermediate in the Kennedy pathway of phosphatidylethanolamine (PE) biosynthesis. PE is the second most abundant phospholipid in mammalian membranes. Disturbance of hepatic phospholipid homeostasis has been linked to the development of metabolic dysfunction-associated steatotic liver disease (MASLD). We generated whole-body Etnppl knockout mice to investigate the impact of genetic deletion of Etnppl on hepatic lipid metabolism. Primary hepatocytes isolated from Etnppl-/- mice showed increased conversion of [3H]ethanolamine to [3H]p-ETN and [3H]PE compared to Etnppl+/+ mice. Male and female Etnppl+/+ and Etnppl-/- mice were fed either a chow or a western-type diet (WTD). Irrespective of diet, Etnppl-/- mice had elevated fasting levels of total plasma cholesterol, triglyceride (TG) and apolipoprotein B100 (VLDL particles). Interestingly, hepatic TG secretion was unchanged between groups. Although hepatic lipids (phosphatidylcholine (PC), PE, TG, and cholesterol) were not different between mice, RNA sequencing analysis showed downregulation in genes related to cholesterol biosynthesis in Etnppl-/- mice. Furthermore, hepatic low-density lipoprotein receptor-related protein1 (LRP1) protein level was lower in female Etnppl-/- mice, which may indicate reduced uptake of remnant VLDL particles from circulation. Hepatic PE levels were only increased in WTD-fed female Etnppl-/- mice, not chow diet-fed mice. However, hepatic lipid accumulation and metabolic dysfunction-associated steatohepatitis (MASH) development were unchanged between Etnppl+/+ and Etnppl-/- mice. To conclude, ETNPPL has a role in regulating plasma lipoprotein metabolism independent of hepatic TG levels.

Following viral infection, genetically manipulated mice lacking immunoregulatory function may develop colitis and dysbiosis in a strain-specific fashion that serves as a model for inflammatory bowel disease (IBD). We found that one such model of spontaneous colitis, the interleukin (IL)-10 knockout (IL-10−/−) model derived from the SvEv mouse, had evidence of increased Mouse mammary tumor virus (MMTV) viral RNA expression compared to the SvEv wild type. MMTV is endemic in several mouse strains as an endogenously encoded Betaretrovirus that is passaged as an exogenous agent in breast milk. As MMTV requires a viral superantigen to replicate in the gut-associated lymphoid tissue prior to the development of systemic infection, we evaluated whether MMTV may contribute to the development of colitis in the IL-10−/− model. Viral preparations extracted from IL-10−/− weanling stomachs revealed augmented MMTV load compared to the SvEv wild type. Illumina sequencing of the viral genome revealed that the two largest contigs shared 96.4–97.3

Background and aimThe liver is susceptible to ischemia-reperfusion injury (IRI) during hepatic surgery, when the vessels are compressed to control bleeding, or liver transplantation, when there is an obligate period of ischemia. The hallmark of IRI comprises mitochondrial dysfunction, which generates reactive oxygen species, and cell death through necrosis or apoptosis. Cyclosporine (CsA), which is a well-known immunosuppressive agent that inhibits calcineurin, has the additional effect of inhibiting the mitochondrial permeability transition pore (mPTP), thereby, preventing mitochondrial swelling and injury. NIM-811, which is the nonimmunosuppressive analog of CsA, has a similar effect on the mPTP. In this study, we tested the effect of both agents on mitigating warm hepatic IRI in a murine model.Materials and methodsBefore ischemic insult, the mice were administered with intraperitoneal normal saline (control); CsA at 2.5, 10, or 25 mg/kg; or NIM-811 at 10 mg/kg. Thereafter, the mice were subjected to partial warm hepatic ischemia by selective pedicle clamping for 60 min, followed by 6 h of recovery after reperfusion. Serum alanine transaminase (ALT) was measured, and the liver tissue was examined histologically for the presence of apoptosis and the levels of inflammatory cytokines.ResultsCompared with the control mice, the mice treated with 10 and 25 mg/kg of CsA and NIM-811 had significantly lower ALT levels (P <0.001, 0.007, and 0.031, respectively). Moreover, the liver tissue showed reduced histological injury scores after treatment with CsA at 2.5, 10, and 25 mg/kg and NIM-811 (P = 0.041, <0.001, 0.003, and 0.043, respectively) and significant decrease in apoptosis after treatment with CsA at all doses (P = 0.012, 0.007, and <0.001, respectively). Levels of the pro-inflammatory cytokines, particularly interleukin (IL)-1β, IL-2, IL-4, IL-10, and keratinocyte chemoattractant/human growth-regulated oncogene significantly decreased in the mice treated with the highest dose of CsA (25 mg/kg) than those in the control mice.ConclusionsPremedication with CsA or NIM-811 mitigated hepatic IRI in mice, as evidenced by the decreased ALT and reduced injury on histology. These results have potential implications on mitigating IRI during liver transplantation and resection.

BACKGROUND Ischemia/reperfusion injury (IRI) is an inherent problem in organ transplantation, owing to the obligate period of ischemia that organs must endure. Cyclosporine A (CsA), though better know as an immunosuppressant, has been shown to mitigate warm IRI in a variety of organ types, including the liver. However, there is little evidence for CsA in preventing hepatic IRI in the transplant setting. MATERIAL AND METHODS In the present study, we tested the effect of CsA on hepatic IRI in a large-animal ex vivo model of donation after circulatory death (DCD). Porcine donors were pre-treated with either normal saline control or 20 mg/kg of CsA. Animals were subject to either 45 or 60 minutes of warm ischemia before hepatectomy, followed by 2 or 4 hours of cold storage prior to reperfusion on an ex vivo circuit. Over the course of a 12-hour perfusion, perfusion parameters were recorded and perfusate samples and biopsies were taken at regular intervals. RESULTS Peak perfusate lactate dehydrogenase was significantly decreased in the lower-ischemia group treated with CsA compared to the untreated group (4220 U/L [3515-5815] vs 11 305 [10 100-11 674]; P=0.023). However, no difference was seen between controls and CsA-treated groups on other parameters in perfusate alanine or asparagine aminotransferase (P=0.912, 0.455, respectively). Correspondingly, we found no difference on midpoint histological injury score (P=0.271). CONCLUSIONS We found minimal evidence that CsA is protective against hepatic IRI in our DCD model.

Persistent inflammation can trigger altered epigenetic, inflammatory, and bioenergetic states. Inflammatory bowel disease (IBD) is an idiopathic disease characterized by chronic inflammation of the gastrointestinal tract, with evidence of subsequent metabolic syndrome disorder. Studies have demonstrated that as many as 42% of patients with ulcerative colitis (UC) who are found to have high-grade dysplasia, either already had colorectal cancer (CRC) or develop it within a short time. The presence of low-grade dysplasia is also predictive of CRC. Many signaling pathways are shared among IBD and CRC, including cell survival, cell proliferation, angiogenesis, and inflammatory signaling pathways. Current IBD therapeutics target a small subset of molecular drivers of IBD, with many focused on the inflammatory aspect of the pathways. Thus, there is a great need to identify biomarkers of both IBD and CRC, that can be predictive of therapeutic efficacy, disease severity, and predisposition to CRC. In this study, we explored the changes in biomarkers specific for inflammatory, metabolic, and proliferative pathways, to help determine the relevance to both IBD and CRC. Our analysis demonstrated, for the first time in IBD, the loss of the tumor suppressor protein Ras associated family protein 1A (RASSF1A), via epigenetic changes, the hyperactivation of the obligate kinase of the NOD2 pathogen recognition receptor (receptor interacting protein kinase 2 [RIPK2]), the loss of activation of the metabolic kinase, AMP activated protein kinase (AMPKα1), and, lastly, the activation of the transcription factor and kinase Yes associated protein (YAP) kinase, that is involved in proliferation of cells. The expression and activation status of these four elements are mirrored in IBD, CRC, and IBD-CRC patients and, importantly, in matched blood and biopsy samples. The latter would suggest that biomarker analysis can be performed non-invasively, to understand IBD and CRC, without the need for invasive and costly endoscopic analysis. This study, for the first time, illustrates the need to understand IBD or CRC beyond an inflammatory perspective and the value of therapeutics directed to reset altered proliferative and metabolic states within the colon. The use of such therapeutics may truly drive patients into remission.

Background Induced pluripotent stem cells (iPSCs) offer potential to revolutionize regenerative medicine as a renewable source for islets, dopaminergic neurons, retinal cells, and cardiomyocytes. However, translation of these regenerative cell therapies requires cost-efficient mass manufacturing of high-quality human iPSCs. This study presents an improved three-dimensional Vertical-Wheel® bioreactor (3D suspension) cell expansion protocol with comparison to a two-dimensional (2D planar) protocol. Methods Sendai virus transfection of human peripheral blood mononuclear cells was used to establish mycoplasma and virus free iPSC lines without common genetic duplications or deletions. iPSCs were then expanded under 2D planar and 3D suspension culture conditions. We comparatively evaluated cell expansion capacity, genetic integrity, pluripotency phenotype, and in vitro and in vivo pluripotency potential of iPSCs. Results Expansion of iPSCs using Vertical-Wheel® bioreactors achieved 93.8-fold (IQR 30.2) growth compared to 19.1 (IQR 4.0) in 2D ( p < 0.0022), the largest expansion potential reported to date over 5 days. 0.5 L Vertical-Wheel® bioreactors achieved similar expansion and further reduced iPSC production cost. 3D suspension expanded cells had increased proliferation, measured as Ki67 + expression using flow cytometry (3D: 69.4% [IQR 5.5%] vs. 2D: 57.4% [IQR 10.9%], p = 0.0022), and had a higher frequency of pluripotency marker (Oct4 + Nanog + Sox2 + ) expression (3D: 94.3 [IQR 1.4] vs. 2D: 52.5% [IQR 5.6], p = 0.0079). q-PCR genetic analysis demonstrated a lack of duplications or deletions at the 8 most commonly mutated regions within iPSC lines after long-term passaging (> 25). 2D-cultured cells displayed a primed pluripotency phenotype, which transitioned to naïve after 3D-culture. Both 2D and 3D cells were capable of trilineage differentiation and following teratoma, 2D-expanded cells generated predominantly solid teratomas, while 3D-expanded cells produced more mature and predominantly cystic teratomas with lower Ki67 + expression within teratomas (3D: 16.7% [IQR 3.2%] vs.. 2D: 45.3% [IQR 3.0%], p = 0.002) in keeping with a naïve phenotype. Conclusion This study demonstrates nearly 100-fold iPSC expansion over 5-days using our 3D suspension culture protocol in Vertical-Wheel® bioreactors, the largest cell growth reported to date. 3D expanded cells showed enhanced in vitro and in vivo pluripotency phenotype that may support more efficient scale-up strategies and safer clinical implementation.

Background:Updated 2016 Helicobacter pylori consensus guidelines recommend treatment for 14 days with concomitant therapy (proton-pump inhibitor (PPI)-amoxicillin-metronidazole-clarithromycin (PAMC) or bismuth-based quadruple therapy (PPI-bismuth-metronidazole-tetracycline, PBMT)) as first line, PBMT or PPI-amoxicillin-levofloxacin (PAL) as second or third line, and PPI-amoxicillin-rifabutin (PAR) as fourth line for 10 days.Objectives:This was a retrospective cohort study to describe and compare the efficacy of anti-Helicobacter treatment regimens over the periods 2007-2015 and 2016-2021 as well as antibiotic resistance.Methods:A modified intention-to-treat (mITT) analysis was used to analyze the success rate of therapies. mITT includes all patients who were prescribed H. pylori treatment and had at least one follow-up test-of-cure. This included patients who could not complete treatment or were non-adherent with treatment. Risk factors for treatment failures were analyzed by univariate and multivariate logistic regression. Resistance testing was done in a small subset of patients.Results:H. pylori-positive patients who received treatment in Edmonton, Alberta were included in a mITT analysis: 334/387(86%) from 2007 to 2015 and 193/199 (97%) from 2016 to 2021. During 2016-2021, 78% (150/193) of patients underwent cumulative guideline-based treatment with a successful cure in 80% (120/150) of patients. In those who were newly diagnosed, the cure rate was 88% (52/59) versus those with previous treatment failure 75% (68/91) (P < 0.05, risk difference [RD] 14%, 95% confidence interval [CI] 1.7-26.3%). The most effective first-line regimens were PAMC for 14 days (87% [45/52]) in 2016-2021 and sequential therapy in 2007-2015 (83% [66/80]) (P = 0.535, RD 4%, 95% CI -8.5-16.5%). When other treatments failed, success with PAR was 50% (2/4) from 2007 to 2015 and 57% (21/37) from 2016 to 2021. Recent (2016-2021) resistance rates to clarithromycin and metronidazole are high at 78% (50/64) and 56% (29/52), respectively. From 2007 to 2015, clarithromycin and metronidazole resistance rates were 80% (36/45) and 83% (38/46), respectively. Levofloxacin resistance increased significantly from 2007-2015 to 2016-2021 (28% [13/46] to 61% [35/57], P < 0.05, RD 33%, 95% CI 11.6-54.4%).Conclusions:Algorithmic treatment with PAMC first line followed by PBMT, PAL, and PAR cures H. pylori in 88% of newly diagnosed patients. PAR therapy shows suboptimal cure rates (50-57% success) but can be considered as third instead of fourth line given increasing levofloxacin resistance rates. Antibiotic resistance in H. pylori is common to clarithromycin, metronidazole, and levofloxacin and frequently accounts for treatment failures.