Introduction

The number of women choosing surgical and interventional specialties is rising in South Asia. Women in medicine, including gastroenterology, face unique challenges, including gender bias, work-life imbalance, and occupational radiation exposure affecting their training and practice (1,2). There are few studies on the impact of gender-related factors on training and career advancement in gastroenterology in the South Asian subcontinent.

Aims & Methods

The aim was to study the impact of gender on training, career advancement and to understand perceptions of work-life balance and beliefs regarding gender dynamics among gastroenterologists of South Asia. A web-based survey was conducted among trainees and attending physicians of South Asia from 15th November 2021 to 30th March 2022. The survey instrument had four components: demographic features, training in gastroenterology, career advancement in gastroenterology, and work-life balance.

Results

A total of 622 gastroenterologists completed the survey (mean age: 40.65 years; females: 13.18%). Most respondents believed hands-on training (83.2%) and mentorship (56.1%) were important for endoscopy training. Mentorship from a same-gender person was deemed important more often by males as compared to females (18.7% vs. 4.9%, p=0.006).A higher proportion of female respondents believed that gender bias in recruiting and training had negatively impacted their career (37.8% vs 1.9%, p<0.001). Lack of ergonomically designed equipment (9.8% vs 4.3%, p=0.03), radiation hazard (13.4% vs 2.2 %, p<0.001), and as a general health concern (13.4% vs 5.2%, p=0.004) were listed as a significant career deterrent by a higher proportion of females. A higher proportion of males had been in leadership positions in the past five years (36.7% vs 23.2%, p=0.017). A higher proportion of female participants faced an interruption in their career (55% vs 32.7%, p= <0.0001), with the most common reasons being pregnancy (25%) and childcare provision (22%). Male respondents reported a perception that women were promoted at a faster rate than men (32.4% vs 12.2%, p<0.001) and were appreciated more for performing the same tasks (58.9% vs 24.4%, p<0.001). They also believed that men were inherently better at administrative and leadership tasks (51.4 vs 34.1%, p=0.001).More females believed that women were more productive than men (48.8% vs 20.4%, p<0.001) and that a salary gap existed (42.7% vs 28%, p=0.002). Females more often experienced burnout as compared to males (67.1% vs 51.3%, p=0.008). A higher proportion of female participants believed that their workplace commitments had negatively impacted their family planning and parental responsibilities (45.1% vs 38.3%, p=0.024). Similarly, a higher proportion of females believed that their family and childcare commitments had affected career advancement (22% vs 13.1%, p<0.001)

Conclusion

Hands on training and mentorship are considered as important factors for endoscopy training. Gender related factors negatively impact training and career growth of female gastroenterologists. Domestic responsibilities weigh down female gastroenterologists significantly. Views on gender disparity significantly differ between the two genders. A high proportion of gastroenterologists have experienced burnout in the past year. Purposeful steps from leaders and mentors may help in creating a more gender equitable workplace for young gastroenterologists

References

1. Rabinowitz LG. Recognizing Blind Spots - A Remedy for Gender Bias in Medicine? N Engl J Med. 2018;378(24):2253-5.
2. David YN, Dixon RE, Kakked G, et al. Gender-Specific Factors Influencing Gastroenterologists to Pursue Careers in Advanced Endoscopy: Perceptions vs Reality. Am J Gastroenterol. 2021;116(3):539-50.

Introduction

Intraductal Papillary Mucinous Neoplasias (IPMN) are cystic neoplasias of the ducts of the pancreas. They usually occur at advanced age and can progress to invasive adenocarcinomas. While there are morphological and spatial features to assess the progression risk, a mutational footprint defining the progression risk remains to be identified. IPMNs often harbor an activating mutation of either GNAS or KRAS or both, but neither interaction nor signaling crosstalk is well understood.

Aims & Methods

Here, we use a patient-derived induced pluripotent stem cell (iPSC) model system, harboring either a wildtype or mutant GNAS with an inducible oncogenic KRAS^G12D cassette. Various iPSC genotypes were differentiated towards the pancreatic lineage including pancreatic duct-like organoids, followed by subsequent transplantation onto the porcine urinary bladder (PUB) for long-term culture. Dysplasia, mucin expression and cyst size were subsequently analysed.

Results

Already 2 weeks post transplantation, a substantial amount of CK7/CK19 positive pancreatic ductal structures were found in all grafts with a strong cystic phenotype driven by a GNAS^R201C background. The phenotype was still present 8 weeks after transplantation. The induction of KRAS^G12D for 6 weeks resulted in an increased ductal lumen, which was further boosted by GNAS^R201C. In addition, a strong GNAS^R201C dependent expression of Mucin 2, a marker for the intestinal IPMN subtype was documented. Quantification of dysplastic ductal structures revealed an increase in low grade dysplasia driven either by KRAS^G12D or GNAS^R201C. Combination of both mutations was associated with a strong increase in the amount of high grade dysplastic ductal structures. In ductal structures a pro-proliferative phenotype driven by GNAS^R201C background was observed. The amount of p21 positive nuclei was highly increased by KRAS^G12D, while it was significantly attenuated in ductal cells with a double mutated KRAS^G12D GNAS^R201C background, showing a decreased amount of oncogene induced senescence. This was recapitulated in vitro in pancreatic ductal organoids.

Conclusion

The first results show an effect of GNAS^R201C on early dysplasia formation and a further boost of KRAS^G12D driven dysplasia and high-grade dysplasia formation. Additionally, the increased proliferative and decreased senescent phenotype hints at a GNAS^R201C driven bypass of oncogene induced senescence. Our genuine patient-derived iPSC model featuring an endogenous GNAS^R201C locus will allow us to closely investigate the early formation of IPMNs and timewise resolve their transition towards an invasive phenotype to further unravel the mechanisms of IPMN formation and the collaborative pro-oncogenic signaling of KRAS^G12D with GNAS^R201C.

Introduction

Necrotizing enterocolitis and sepsis are two of the most prevalent life-threatening disorders for preterm neonates. Both of these disorders occur significantly less frequently in breastfed neonates (1). A beneficial role has been identified of tryptophan related factors in mother’s milk but the involvement of its bacterial-derived metabolites, which also act as Aryl hydrocarbon receptor (AhR) activator ligands, remains poorly understood.

Aims & Methods

In this study, we aim to investigate the presence of AhR-ligands in mother milk and in neonatal feces and assess their potential effects on neonatal epithelial cells.

Results

First, we assessed the ability of human milk and formula to activate AhR using an AhR-reporter cell system. Mother milk (n=10) (unlike formula; (n=6)) triggered AhR and increased significantly AhR-activity. In order to identify the composition and levels of AhR-ligands in milk and feces from neonates, targeted liquid chromatography coupled with high resolution mass spectrometry was performed on mother milk (n=40), formula milk (n=3) and feces from preterm (n=50) and full-term (n=10) Dutch neonates at birth. Indole-acetic-acid (IAA) and indole-3-sulfate (I3S) were significantly more abundant in mother milk (IAA [pmol/mL] = 43, IQR 32-57, I3S = 221, IQR 181-291) compared to formula (IAA [pmol/mL] = 5.40, I3S = 33, IQR 31-78). At birth, preterm neonates showed significantly lower fecal levels of indole-lactic-acid (ILA) compared to full-term neonates (preterm [pmol/mL] = 7, IQR 0-18, term = 10, IQR 0-252). Using our AhR-reporter system we confirmed the ability of milk IAA and I3S, and fecal ILA to activate AhR, suggesting that the levels of milk and fecal AhR-agonists are relevant for activating neonatal intestinal AhR.
Finally, to assess the role of milk AhR-ligands on epithelial barrier function, intestinal crypts were isolated from fetuses (16-20 weeks of gestation), grown into organoids and stimulated for 24h with AhR-agonists (physiological concentrations; 0.175-1 nmol/mL) and challenged with TNFα for 4h. The activation of Nf-kB and consequent production of IL-8 was significantly reduced by both IAA and ILA (One-way ANOVA, p<0.01 and p<0.0001) but not by I3S in human fetal derived organoids (p<0.58). In addition, the barrier function was assessed through TEER measurement. All three of the indoles restored barrier integrity damaged by TNFα after 72h (One-way ANOVA, IAA = p<0.001, I3S = p<0.01, ILA = p<0.001).

Conclusion

To conclude, our results suggest that 1) Feces of preterm neonates generate lower levels of ILA compared to full term neonates; 2) Mother milk contains AhR-ligands able to activate AhR in neonatal intestine; 3) Mother milk and neonatal feces-derived AhR-ligands reduce epithelial Nf-kB activation in fetal intestinal organoids; 4) IAA, I3S and ILA could restore intestinal barrier integrity.
All of these results suggest a possible beneficial mechanism of mother milk through intestinal AhR-activation.

References

¹ Herrmann K., et al. Breastfeed. Med. 2014;9:184–190. doi: 10.1089/bfm.2013.0121.

Introduction

Mutations of Adenomatous polyposis coli (APC) are among the most frequent molecular aberrations in colorectal cancer (CRC) and result in oncogenic activation of the Wnt signaling pathway. Previous work showed that despite harboring these mutations, CRC cells require the secretion of Wnt ligands (WNTs) for survival. So far, no systematic analysis to assess the expression and function of all WNTs in CRC has been conducted.

Aims & Methods

We sought to systematically assess the expression of all 19 WNTs in different CRC models. This was performed by bioinformatical analysis of publicly available datasets as well as qRT-PCR and Western blot analysis of CRC organoid lines. Among the highly expressed WNTs, we aimed to investigate their molecular regulation as well as specific oncogenic functions. Employing CRISPR/Cas9 gene editing, we modeled frequent oncogenic mutations in CRC cells and human colon organoids to examine their effect on the expression of WNTs. In order to examine the specific oncogenic effects of selected WNTs, we performed shRNA-based gene knockdown in CRC cell lines and organoids. Using colony formation and CellTiter-Glo (CTG) assays, we assessed the viability of CRC cells and organoids, respectively.

Results

Among all WNTs, especially WNT11 is highly expressed in publicly available datasets and a panel of CRC organoids. Furthermore, the expression of WNT11 is correlated to a signature of high Wnt activation in publicly available datasets of CRC tissues. Introduction of APC mutations through CRISPR/Cas9 in CRC cell lines and human colon organoids resulted in upregulation of WNT11. Inhibition of the downstream transcriptional coactivator of Wnt signaling β-Catenin by small molecule inhibitors and siRNA abrogates the expression of WNT11 in CRC cells and organoids. Knockdown of WNT11 impairs the ​colony formation of CRC cells as well as the viability of CRC organoids in CTG assay.

Conclusion

Our results show that WNT11 is highly expressed in CRC and directly secreted by tumor cells. We show that WNT11 is a target gene of the Wnt pathway, which is upregulated in the context of APC mutations, and that WNT11 supports the proliferation of CRC cells. These results unravel new aspects of the oncogenic effects of APC mutations and identify WNT11 as a potential therapeutical target in APC-mutant CRC. To characterize the oncogenic function of WNT11 in CRC in more detail, further research is needed. The in vivo effects of WNT11 silencing will be addressed in a murine xenograft model.