Introduction

Arsenic, a potent environmental toxin, has been widely reported to cause significant health problems, including liver damage and disturbances in the metabolic processes. One of the lesser-understood facets of arsenic toxicity is its impact on gut microbiota, the community of microorganisms living in the intestinal tract, which plays a crucial role in host metabolism and overall health. Previous studies have indicated that arsenic exposure can alter the composition of gut microbiota, potentially affecting the host’s metabolism of arsenic and exacerbating its toxic effects. Despite ongoing research, the mechanisms underlying the interaction between arsenic exposure, gut microbiota changes, and arsenic metabolism remain inadequately defined.

Aims & Methods

The primary objective of this study was to elucidate the dynamics of gut microbiota changes under varying levels of arsenic exposure and to understand how these changes affect arsenic metabolism and subsequent liver toxicity. We used C57BL/6 mice as a model system, exposing them to 0, 5, or 10 ppm sodium arsenite (NaAsO2) in their drinking water over a period of six months. We conducted a comprehensive analysis of the gut microbiota, focusing on folic acid (FA)/vitamin B12 (VB12)- and butyrate-synthesizing communities. Additionally, molecular interactions between arsenic and methionine synthase (MTR), an enzyme crucial for FA/VB12 utilization, were assessed using 3D molecular simulations and biochemical assays. The intervention model involved supplementation of FA and VB12 to evaluate their protective effects against arsenic-induced disturbances.

Results

Exposure to NaAsO2 significantly altered gut microbiota, with a notable increase in the abundance of FA/VB12- and butyrate-synthesizing microbiota. This shift was hypothesized to compensate for the disrupted utilization of these nutrients due to arsenic exposure. In higher arsenic exposure levels, however, the microbiota changes were inconsistent, suggesting a potential threshold beyond which the compensatory mechanism may be overwhelmed. Biochemical assays confirmed that arsenic binds to MTR, disrupting its function and further impacting FA/VB12 metabolism. The interaction was substantiated by SEC-UV-DAD and fluorescence-labeled arsenic studies, which demonstrated that arsenic directly interacts with MTR in both non-cellular and cellular systems. Our intervention model using FA and VB12 supplementation showed that it was possible to mitigate the disturbances in gut microbiota, restore MTR levels, enhance arsenic metabolism, and reduce liver injury.

Conclusion

This study provides significant insights into the interaction between arsenic exposure and gut microbiota. We demonstrated that changes in gut microbiota are not merely a consequence of arsenic exposure but are intricately linked to the metabolic processes affected by arsenic, particularly through the disruption of MTR function. The supplementation of FA and VB12 proved effective in blocking these disturbances, suggesting potential therapeutic strategies to combat arsenic toxicity. The findings emphasize the importance of considering gut microbiota in the management and treatment of arseniasis and underscore the complex interplay between environmental toxins, gut microbiota, and host metabolism. Future studies should focus on further delineating the specific pathways and mechanisms by which microbiota influence arsenic metabolism and exploring the potential for microbiota-targeted therapies in arsenic-related diseases.

Clinical Case Summary

A 13-year-old boy presented with abdominal distention, recurrent hypoglycemia, and self-resolving jaundice. Clinical examination revealed growth retardation and hepatosplenomegaly. Blood work revealed anemia, thrombocytopenia, elevated aspartate and alanine transaminase levels, and raised plasma cholesterol and triglycerides. Ultrasound showed an enlarged liver with surface irregularity, splenomegaly, and a dilated portal vein. Etiological work-up for cirrhosis was positive for hepatitis B (HBV) surface antigen (HbsAg) and antibodies for HBV core and envelope antigens (anti Hbc, antiHbe) but an undetectable HBV DNA level. HBV was considered to be an unlikely cause of cirrhosis due to the young age of the patient, hence further metabolic and genetic work-up was done. Tests for Wilson’s and autoimmune liver disease were negative. Liver biopsy revealed a distorted lobular architecture with nodules, thin septae, and portal fibrosis. Hepatocytes showed marked swelling and pyknotic nuclei, and negative HbsAg immunostaining. Molecular analysis revealed a novel mutation in the exon 3 of the PHKG2 gene (c.98G>T, p.Gly33Val). The patient was diagnosed with cirrhosis due to GSD IXc with inactive chronic hepatitis B. His symptoms resolved with an optimized diet of corn starch, high-protein and vitamin supplementation. The importance of liver transplantation was also discussed with the family.

Introduction

Crohn’s disease (CD), a variant of inflammatory bowel disease, is a complex, debilitating, chronic and relapsing disorder of the gastrointestinal tract. If poorly controlled, patients are at risk of intestinal obstruction and infection due to the formation of strictures, fistulae and narrowing of the intestinal lumen, often requiring surgical intervention. As the precise aetiology remains unclear, current treatments for CD are limited. An altered composition of native gut microbiota may play a role in CD pathogenesis, with recent faecal microbiota transplantation (FMT) studies revealing variable therapeutic efficacy in CD management. Orally administered Emu Oil, derived from Emu adipose tissue, has demonstrated anti-inflammatory and reparative properties in models of intestinal disease.

Aims & Methods

The current study aimed to determine whether Emu Oil-modulated FMT could reduce disease severity and modulate the gut microbiota in a mouse model of trinitrobenzene sulfonic acid (TNBS)-induced Crohn’s-like colitis. Female ARC(s) mice were assigned to FMT donor (n=5/group) and recipient (n=10/group) groups. Donor mice were orally administered either Water (80μl), Olive Oil (OO) or Emu Oil (160μl) daily (days -7 to 0); fresh faeces were collected (days -1 to 3) and pooled per group. Recipient mice were administered TNBS (3mg; day 0) and donor faecal supernatant (120μl; day 3) intrarectally and euthanised on day 6. Daily bodyweight and disease activity index (DAI), colonoscopically-assessed disease severity, organ weights, intestinal permeability (fluorescein isothiocyanate [FITC]-dextran uptake), histologically-assessed colonic damage (disease severity, crypt depth), intestinal barrier function (mucin-secreting cells), acute inflammation (myeloperoxidase [MPO]) and faecal microbiome (16S rRNA amplicon sequencing) were assessed. p<0.05 was considered statistically significant.

Results

TNBS induced bodyweight loss in all groups, which normalised to day 0 values on the final day of the experimental period. Daily DAI remained elevated throughout the trial in all groups; only Emu Oil FMT normalised DAI on day 6 compared with day 0 (p<0.05). TNBS increased colonoscopically-assessed disease severity scores (p<0.05), with no significant Emu Oil nor OO FMT effect. Emu Oil FMT decreased liver weights and increased small intestinal weights relative to bodyweight, reduced histologically assessed disease severity and increased colonic mucin counts compared with Water and OO FMT (p<0.05). The most abundant genera across all groups were Muribaculaceae, Eisenbergiella and Bacteroides; while alpha and beta diversity, taxonomic profiles, FITC-dextran, crypt depth and MPO activity remained unchanged in all treatment groups (p>0.05).

Conclusion

Emu Oil-modulated FMT improved histologically-assessed indicators of intestinal disease and barrier function, compared with Water FMT and OO FMT. Interestingly, Emu Oil-modulated FMT affected liver and small intestinal organs weights, suggesting a systemic effect in this setting of disease. Importantly, key genera across taxonomic profiles were identified, however, the microbiome and remaining disease indicators were largely unchanged between treatments. Overall, Emu Oil-modulated FMT demonstrates therapeutic potential in CD management. Future studies would benefit from a deeper assessment of the gastrointestinal microbiome through long-read whole genome sequencing to further determine the role of the microbiome in EO FMT efficacy compared with conventional FMT.

Introduction

The depth of early gastric cancer is an important factor in determining the treatment plan such as endoscopic resection or surgery. We developed an artificial intelligence (AI) program using deep neural networks to diagnose mucosal and submucosal cancers using endoscopic images. Performance evaluation tests were conducted for clinical applications.

Aims & Methods

A validation dataset was established comprising 204 patients with early gastric cancer who underwent endoscopic resection or surgery at our hospital during 2018–2021. The lesion sites were the upper part in 16 (8%), middle part in 106 (52%), and lower part in 82 (40%) patients. The median lesion size (range) was 14 (2–90) mm; the lesion depth was mucosal cancer in 136 (67%) and submucosal cancer in 68 (33%) patients. Five white-light images per case at a minimum were used (median: 8, range: 5–22). The primary endpoint was sensitivity of mucosal cancer, defined as the lower limit of the 95% confidence interval (CI) > 75%, based on previous reports on the depth diagnosis of early gastric cancer. The secondary endpoints were specificity and accuracy.

Results

Sensitivity of this program for the diagnosis of mucosal cancer was 91.2% (95% CI: 85.2%–94.9%), indicating the usefulness of this program. Specificity was 66.2% (95% CI: 54.3%–76.3%), and accuracy was 82.8% (95% CI: 77.1%–87.4%).

Conclusion

The AI program developed for diagnosing the depth of early gastric cancer is useful and expected to be applied clinically in the near future.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with the lowest 5-year survival rate of all cancers due to late diagnosis and lack of effective treatment. One of the challenges of developing molecular diagnosis and molecular targeted therapy for PDAC is the “mismatch” between research models and patients.

Aims & Methods

This study aimed i) to identify common proteins among different research models and patients and accordingly ii) to identify potential hub proteins and signaling pathways involved in the PDAC pathogenesis by using proteomics-based system modeling of multimodal datasets derived from multiple research models and patients. Mouse PDAC cells (derived from a genetically engineered mouse model with KrasG12D/Pdx1-Cre), PDAC orthotopically tumors in C57B/L6 mice (n=5), mouse PDAC spheroids, mouse pancreatic organoids, mouse PDAC organoids, normal mouse pancreatic tissues (n=5), human PDAC tumor tissue samples from treatment-naїve (n=20), neoadjuvant chemotherapy (n=22), and human PDAC organoids were included. Mass spectrometry-based proteomics, Rstudio, Cytoscape 3.10 and pathway enrichment analysis (using ingenuity pathways analysis, including human PDAC transcriptomic in addition) were used.

Results

In the comparison of PDAC models and patients, shared proteins were identified as follows: 60% between the mouse PDAC cells and the mouse PDAC spheroids, 78% between the mouse PDAC cells and the mouse PDAC tissue, 56% between the mouse PDAC spheroids and the mouse PDAC tissue, 66% between the mouse PDAC cells and the mouse PDAC organoids, 54% between the mouse PDAC spheroids and the mouse PDAC organoids, 38% between the mouse PDAC and the human PDAC tissues, 31% between the human PDAC tumor and the human PDAC organoids, and 60% between the mouse and the human PDAC organoids. A total of 1,367 common proteins were identified within all the PDAC models. Among these proteins ACTB, HSP90AA1, HSPA8, HSP90AB1, HSPA4, EEF2, RPS3, RPS20 and GAPDH were the top 10 hub proteins (with >300 node connections). Upon comparison of all eight models, eight proteins were identified as unique to PDAC, namely FAHD1, ARSA, NEK9, SERPINB9, LAMTOR2, SNRPG, TMED3, and RAB1A. There was a correlation in terms of z-score regarding signaling pathways between the mouse PDAC and human PDAC (p<0.001), particularly in activations of nerve growth factor signaling, integrin signaling, and neuregulin signaling. Central hub proteins commonly found in the mouse models included vinculin (membrane-cytoskeletal protein) as one of the high connectivity nodes and β-catenin and probable ubiquitin carboxyl-terminal hydrolase FAF-X (involved in axonal growth and neuronal cell migration) as high centrality nodes. In patients, central hub proteins that were dysregulated after chemotherapy included 14-3-3 protein zeta/delta (encoded by YWHAZ gene), β-catenin and SRC. Among the 47 proteins that were associated with altered metabolism after the chemotherapy in patients, CD44, CTNND1, APOA 1, PARVA, and SCRBS1 were nodes with low degrees, and desmosomes (DSP, KP3, PPL), PRTN3, thrombospondin-2 (THBS2), and DMBT1 were the most down-regulated proteins without nodes, and APOA1, caveolin-1 (encoded by CAV1), LRP1 were the most up-regulated proteins with nodes.

Conclusion

The system modeling identified the common proteins among the research models and patients and the hub proteins and signaling pathways, suggesting the potential translational targets of PDAC.

Introduction

Dysregulated T-cell immune response-mediated inflammation is implicated in various liver diseases, yet mechanisms governing liver immune homeostasis control and therapies for limiting T-cell overactivation remain unclear. This study aims to elucidate the role of immunoregulatory myeloid cells (MCregs) in liver injury and their potential to modulate CD4⁺ T-cell hyperactivation via extracellular vesicle (EV)-mediated metabolic regulation.

Aims & Methods

Concanavalin A (ConA) mice and autoimmune hepatitis (AIH) patients were examined for alterations in purine metabolism associated with liver injury severity and therapeutic response. The expression of extracellular purine catabolism nucleases, such as CD39 and CD73, on CD11b⁺Gr-1⁺ myeloid cells (MCs) was assessed, with particular focus on liver injury conditions. The study investigated the natural expansion of the CD39⁺CD73⁺Gr-1^highCD11b⁺ regulatory MC (MCreg) subset during early liver injury phases and its effects on CD4⁺ T-cell hyperactivation and liver injury through in vitro and in vivo experiments. Mechanistic insights into MCreg-mediated immunosuppression were explored, particularly regarding the release of CD73^high EVs and their impact on extracellular AMP conversion, cAMP pathway activation, and inhibition of glycolysis and cytokine secretion in activated CD4⁺ T cells.

Results

Alterations in purine metabolism were observed in ConA mice and AIH patients, correlating with liver injury severity and therapeutic response. CD39 and CD73 expression was markedly upregulated on CD11b⁺Gr-1⁺ myeloid cells under liver injury conditions. The CD39⁺CD73⁺Gr-1^highCD11b⁺ MCreg subset expanded naturally during early-stage liver injury and demonstrated efficacy in suppressing CD4⁺ T-cell hyperactivation and liver injury both in vitro and in vivo. The release of CD73^high EVs by MCregs facilitated extracellular AMP conversion to immunosuppressive metabolites (e.g., adenosine and inosine), activating the cAMP pathway and inhibiting glycolysis and cytokine secretion in activated CD4⁺ T cells.

Conclusion

These findings shed light on the mechanisms governing immune homeostasis during early liver injury and suggest that MCreg or MCreg-EV therapy may offer a targeted approach for preventing diverse liver diseases induced by T-cell overactivation.

Clinical Case Summary

Diaphragmatic flutter (DF) is a rare condition characterized by rhythmic, repetitive, and involuntary contractions of the diaphragm and other respiratory muscles, with variable clinical presentation including epigastric pulsations, sleep disturbances, dyspnea, hyperventilation, hiccups, abdominal pain, gastroesophageal reflux and belching, often leading to delays in diagnosis and ineffective treatments. Diagnostic tools were previously limited to thoracic videofluoroscopy and electromyography.
Patient 1: A 22yr male, with abdominal spasms, constant nausea and reflux during adolescence with poor response to omeprazole.Endoscopy and gastric emptying scintigraphy did not reveal the root of the problem. The condition was intermittent, and psychological evaluation was normal. Due to disabling symptoms, high-resolution esophageal manometry (HREM) was performed, revealing absent motility and additionally observing abdominal spasms associated with oscillating increases in intragastric pressure, pharyngeal contraction and repetitive diaphragmatic spasms. Biofeedback therapy (BT) was conducted with symptomatic control.
Patient 2: A 47yr, male with a 10-year history of abdominal spasms, evaluated by neurology, which ruled out structural abnormalities via cranial magnetic resonance imaging and electromyography. He received multiple medications without symptomatic improvement. The condition was intermittent, with periods of remission. Upon symptom recurrence HREM was performed, showing normal motility but a pattern similar to the previous case. BT achieved optimal symptom control.
Discussion: DF is a rare entity. Cases reported in the literature are generally initially managed by neurologists, with minimal involvement from gastroenterologists. However, it is proposed that HREM assessment, which demonstrates a specific pattern, can facilitate diagnosis without the need for invasive studies. Likewise, BT appears to be effective in the short term, improving patients' quality of life.

References

Ramirez JD, Gonzales M, Hoyos JA, Grisales L. Diaphragmatic flutter: a case report and literature review. Neurología. 2015;30(4): 249-251. 2

Iliceto G, Thompson PD, Day BL, et al. Diaphragmatic flutter, the moving umbilicus syndrome, and "belly dancer's" dyskinesia. Mov Disord 1990;5:15–22

Disclosure

Nothing to disclose