bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fragment-Based Drug Discovery forTrypanosoma bruceiGlycosylphosphatidylinositol-Specific Phospholipase C through Biochemical and WaterLOGSY-NMR Methods
Authors: Ibrahim Mohammed Auwal , Yamasaki Tomoko , Furukawa Koji , Yamasaki Kazuhiko 
Published: February 2022

AbstractGlycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) of Trypanosoma brucei, the causative protozoan parasite of African trypanosomiasis, is a membrane-bound enzyme essential for antigenic variation, because it catalyses the release of the membrane-bound form of variable surface glycoproteins. Here, we performed a fragment-based drug discovery of TbGPI-PLC inhibitors using a combination of enzymatic inhibition assay and water ligand observed via gradient spectroscopy (WaterLOGSY) NMR experiment. The TbGPI-PLC was cloned and overexpressed using an Escherichia coli expression system followed by purification using three-phase partitioning and gel filtration. Subsequently, the inhibitory activity of 873 fragment compounds against the recombinant TbGPI-PLC led to the identification of 66 primary hits. These primary hits were subjected to the WaterLOGSY NMR experiment where 10 fragment hits were confirmed to directly bind to the TbGPI-PLC. These included benzothiazole, chlorobenzene, imidazole, indole, pyrazol and quinolinone derivatives. Molecular docking simulation indicated that six of them share a common binding site, which corresponds to the catalytic pocket. The present study identified chemically diverse fragment hits that could directly bind and inhibit the TbGPI-PLC activity, which constructed a framework for fragment optimization or linking towards the design of novel drugs for African trypanosomiasis.
A1 USING HUMAN NEONATAL ORGANOIDS TO EXPLORE GUT-IMMUNE SYSTEM INTERACTIONS OF THE NEONATAL INTESTINE
Authors: Allaire J M , Sharafianardakani Z , Poon B , Piper H , Jacobson K , Lavoie P , Vallance B 
Published: February 2022

Abstract

Background
Necrotizing Enterocolitis (NEC) affects around 10% of preterm babies and is one of the leading causes of death for newborns. NEC is characterized by exaggerated inflammation of the intestinal mucosa, possibly triggered by aberrant exposure to gut microbes, leading to hypoxic conditions and the death of intestinal tissues. It has been hypothesized that NEC develops when the immature intestine (epithelium and immune system) is unable to properly balance these new microbial interactions. To date, NEC is poorly understood and due to the difficulty of modeling the human neonatal intestine, few therapeutic options are available.
Intestinal epithelial cells (IEC) are important players in promoting beneficial host-microbe interactions in the gut, being the primary barrier that separates the host’s mucosal immune system from luminal microbiota, as well as key players in mediating signaling between microbes and the host. Based on their location, IEC are also subject to injury associated with maladaptive immune responses against gut microbes. Many studies have shown that immune cells (such as T helper 17 cells) interact with IEC to promote gut health and function. These interactions include educating IEC on how to respond to, and fight pathogenic microbes, yet also remain tolerant to commensal microbes.

Aims
This project seeks to develop an in vitro human neonatal intestinal organoid model to study developmental changes in IEC and their functional interactions with neonatal Th17 cells.

Methods
3D organoids were established from human neonatal intestinal biopsies and then co-cultured with the supernatant of differentiated Th17 cells or with recombinant cytokines, IL-17 and IL-22. Changes in barrier function, cell proliferation, production of mucins and anti-microbial peptides (AMP) were analyzed by qPCR and immunostaining.

Results
Using 3D neonatal organoids we observed that the supernatants from neonatal Th17 cells (containing IL-17, IL-22 etc.) promoted the proliferation, differentiation and barrier function of the neonatal epithelium. By using specific recombinant cytokine (IL22, IL17) and neutralizing IL-22 antibodies in parallel, we demonstrated that the high levels of IL-22 produced by neonatal Th17 cells specifically induced proliferation of IEC, AMP and mucus production as compared to control media treated organoids, as shown by increases in Ki67, Reg3γ and Muc2 markers.

Conclusions
This experimental model mimicking the neonatal intestinal environment can be used to study interactions between neonatal IEC and immune cells. Our findings can provide clinically relevant information and clues to how developmental changes in the newborn intestine can influence susceptibility to NEC while demonstrating our development of a simple, yet accurate and clinically applicable model of the neonatal gut.

Funding Agencies
CAG, CIHRBCCHRI, CCC, C.H.I.L.D. Fdn
A2 MUCUS DEFICIENCY IMPACTS INTESTINAL EPITHELIAL CELL PROLIFERATION INDEPENDENT OF THE MICROBIOTA
Authors: Han X , Tsai K , Allaire J M , Crowley S M , Lorentzian A , Maxwell C , Vallance B 
Published: February 2022

Abstract

Background
Intestinal homeostasis is highly dependent on the proliferation and differentiation of intestinal epithelial cells (IEC). IEC arise from intestinal stem cells (ISCs) that reside at the bottom of intestinal crypts. Following proliferation, the IEC migrate up as transient amplifying (TA) cells, and differentiate into mature IEC subtypes. When this process is disrupted, it can lead to aberrant IEC proliferation and differentiation. Mucus production by secretory goblet cells is also crucial for intestinal homeostasis, as mucus separates the IEC from luminal microbiota. Surprisingly, mice lacking Muc2, the main protein component of mucus, display increased distal colonic IEC proliferation and crypt hyperplasia at baseline, suggesting a relationship between Muc2 production and IEC proliferation.

Aims
We investigated how mucus production impacts IEC proliferation and differentiation in the intestinal crypt.

Methods
We used wildtype (Muc2+/+) and Muc2 deficient (Muc2-/-) littermates to measure distal colon crypt length and IEC proliferation pattern via microscopy. Organoids were also derived from distal colons of Muc2+/+ and Muc2-/- mice, and quantified for size, density and proliferation for 7 days to test whether the hyper-proliferation phenotype was also seen in vitro, thus epithelial-intrinsic. Crypts and organoids were collected for RNA sequencing to examine changes in IEC proliferation pathways. Proliferation assessments were repeated in germ-free (GF) Muc2+/+ and Muc2-/- mice. Muc2-/- mice were also cross-bred with Lgr5-EGFP-IRES-CreERT2 mice to investigate the contribution of ISCs to IEC hyperproliferation resulting from Muc2 deficiency.

Results
Significant crypt hyperplasia was observed in the distal colons of Muc2-/- mice in concert with a &gt; twofold increase in Ki67+ TA cells as compared to Muc2+/+ mice. Similarly, Muc2-/- organoids also displayed significantly greater size, density and an increased number of Ki67+ cells than Muc2+/+ organoids. Hyperproliferation was also seen in GF mice and organoids, suggesting that mucus impacts IEC proliferation independent of the microbiome. Muc2-/- Lgr5-EGFP+ mice showed no significant increase in numbers of Lgr5+ cells, indicating that Muc2 deficiency does not directly impact ISC number, but rather their proliferation and differentiation reflected in increased numbers of TA cells. RNA-sequencing results suggested that changes in lipid metabolism may underlie the increased IEC proliferation seen in Muc2-/- mice.

Conclusions
Mucus not only promotes IEC homeostasis by separating luminal bacteria from the intestinal epithelium, but also intrinsically modifies IEC proliferation independent of the microbiota. Taken together, our results emphasize the importance of mucus in controlling gut health through mechanisms independent of its role in barrier function.

Funding Agencies
CCC, CIHRC.H.I.L.D Foundation
A8 REPEATED SUBMERGENCE OF AIR-LIQUID INTERFACE COLONOID CULTURES IMPAIRS INFLAMMATORY AND REGENERATIVE RESPONSES
Authors: Rees W D , Telkar N , Lin D , Wong M , Poloni C , Fathi A , Kobor M , Zachos N , Ted S 
Published: February 2022

Abstract

Background
Damage in the intestinal epithelium is repaired via de-differentiation of mature intestinal epithelial cells to a stem-like state. Indeed, literature has primarily focused on acute forms of intestinal damage, but there is a lack of models to study how intestinal stem cells function after chronic injury, such as in inflammatory bowel disease (IBD). A previous report found that growth of mouse intestinal organoids in air-liquid interface (ALI) follows by submergence caused differentiation and reversible injury, but this has not been demonstrated in human cells or with repeated cycles of injury. Understanding how chronic damage alters human intestinal stem cell fate and function is imperative to developing novel therapies that repair the epithelium in people with IBD

Aims
To develop a robust in vitro model to differentiate and damage human intestinal epithelial cells, with or without the addition of bacterial flagellin to mimic pathogen exposure.

Methods
Human colonoid monolayers were seeded on Transwell inserts for 10 days until fully confluent and then differentiated by removing the apical media to create ALI growth conditions for 7 days. To induce damage, media was added to the apical side of the Transwell, with or without the addition of flagellin in the basolateral compartment. Following submergence induced damage, the apical media was removed and collected for chemokine analysis, and the cells were grown back in ALI for 3 days to recover them from injury. This cycle was repeated 5 times to induce chronic damage. Cells were collected for qPCR analysis, immunofluorescence imaging, RNA sequencing and DNA methylation analysis

Results
Repeated rounds of damage impaired the ability of intestinal epithelial cells (IECs) to respond to TLR stimulation (a decrease in basolateral IL-8 with each round), likely due to a decrease in TLR signaling pathways, as demonstrated by GSEA and qPCR. Chronic submergence damage led to an increase in differentiation of cells expressing MUC2, SLC26a3 and CHGA, and a decrease in stemness as shown by qPCR for BMI1, HOPX, and LGR5. After several rounds of damage, colonoid monolayers were unable to regrow as monolayers after passaging, likely due to a decrease in YAP signaling. We also identified mRNA expression and DNA methylation changes in genes associated with IBD and colon cancer.

Conclusions
We have developed a novel chronic damage model of recurrent IEC injury, which possibly mimics pathologies seen in people with inflammatory bowel disease. This model can be used to understand how chronic damage alters the ability of IECs to respond to pathogens and regenerate to repair and protect the epithelium from further damage.

Funding Agencies
CCC
A12 STEPWISE COORDINATION OF COLONIC NEUTROPHILS AND INNATE LYMPHOID CELLS IN THE ONSET AND RESOLUTION OF CLOSTRIDIOIDES DIFFICILE TOXIN-INDUCED INJURY
Authors: Flannigan K L , Serra A , Hirota S A 
Published: February 2022

Abstract

Background
While our understanding and use of treatments for Clostridioides difficile infection (CDI) has improved, initial CDI still carries significant morbidity and mortality owing to heterogeneity in host immune responses. Further, host immunity is a critical modulator of fecal microbiota transplantation (FMT) success in CDI. Thus, understanding the host immune response during CDI is essential.

Aims
To assess the cellular immune responses that trigger the onset and resolution of injury and inflammation in CDI.

Methods
Colonic injury and inflammation triggered by CDI was modelled in mice using intrarectal installation of C. difficile toxins A and B (TcdA/B). Colonic tissue was collected at various timepoints following TcdA/B exposure to assess gene expression (qPCR), cytokine production (ELISA) and immune cell responses (flow cytometry). Knockout mice and neutralizing antibodies were used to deplete cytokines or cells.

Results
Examinion of colonic gene expression at different times following TcdA/B exposure found a dominant transcriptional signature related to neutrophil adhesion and diapedesis. In addition to the typical neutrophil chemokines Cxcl1 and Cxcl2, TcdA/B exposure also increased expression of neutrophil effector genes including Elane (neutrophil elastase). Neutrophil influx in response to TcdA/B was a critical driver of intestinal injury as antibody-mediated depletion of neutrophils lead to significantly less damage in the colon following TcdA/B exposure. Along with neutrophil influx, there were high levels of antimicrobial gene expression in the colon after TcdA/B exposure including RegIIIγ, S100a8, and Socs3, all genes regulated by IL-22. Upon further investigation, IL-22 was a significant mediator in the host response to TcdA/B exposure as it was upregulated &gt;150-fold in the colon and originated from type 3 innate lymphoid cells (ILC3). Further, TcdA/B exposure in IL-22-/- mice lead to significantly more colonic damage compared to wildtype (WT) mice. Subsequent screening of previously published RNAseq data from IL-22-treated mouse colonic organoids identified various upregulated proteins involved in immune regulation, including the gene Slpi that encodes a protein (secretory leukocyte peptidase inhibitor) that inhibits leukocyte proteases, including neutrophil elastase. While TcdA/B challenge robustly induced the expression of Slpi in the colon of WT mice, IL-22-/- mice failed to express increased levels of Slpi and had greater levels of neutrophil elastase activity in the colon.

Conclusions
Together these data suggest a stepwise immune response to TcdA/B where ILC3 produce IL-22 to induce epithelial release of SLPI that attenuates the damaging effects of early neutrophil responses. Strategies to upregulate IL-22 may help control damage triggered by CDI and promote resolution of injury.

Funding Agencies
Lloyd Sutherland Chair in GI Research, Canadian Research Chair
A15 GIARDIA DUODENALIS INDUCES REGIOSPECIFIC ALTERATIONS TO INTESTINAL MUCOSAL GLYCOSYLATION PATTERNS
Authors: Fekete E , Allain T , Buret A 
Published: February 2022

Abstract

Background
Disruption of intestinal barrier function is important in the pathogenesis of numerous intestinal diseases and can lead to increased intestinal permeability. During infection with the protozoan parasite Giardia duodenalis, intestinal permeability is increased in part due to disruption of the intestinal mucus barrier. Intestinal mucus is composed primarily of the heavily glycosylated MUC2 mucin. Glycosylation of mucins is important for maintaining the structure of the mucus gel, and glycans are key mediators of host-microbe interactions, as they provide binding sites to microbes and can be degraded as a nutrient source. Changes to mucin glycosylation patterns have been noted during intestinal inflammation and bacterial infection, and may contribute to altered intestinal permeability and dysbiosis. We hypothesized that intestinal mucosal glycosylation patterns may be disrupted during Giardia infection, and that this may contribute to Giardia-induced barrier dysfunction.

Aims
Characterize changes to mucosal glycosylation patterns in the small and large intestines during in vivo Giardia infection.

Methods
3–4 week old C57BL/6 mice were infected with Giardia duodenalis strain GS/M for 7 days. Tissue sections from the jejunum and colon were collected and stained with various fluorescein-coupled lectins (CONA, DBA, PNA, WGA, SNA, UEA-1) and fluorescence was quantified and normalized to tissue area. Quantitative PCR (qPCR) was performed for glycosyltransferase genes in the jejunum and colon.

Results
In the jejunum, abundance of N-acetylglucosamine increased upon infection, while sialic acid and fucose abundance were significantly decreased in infected mice compared to controls. Conversely, in the distal colon, mannose and sialic acid abundance increased significantly upon infection.
Expression of mucin-associated glycosyltransferase genes was also altered in the small and large intestines of Giardia infected mice. In the jejunums of infected mice, expression of the sulfotransferase Chst4 decreased, while the fucosyltransferase Fut2 and the sialyltransferase St6GalNAc1 increased in comparison to controls. In both the jejunum and distal colon, expression of the core 2 synthase C2GnT1 increased, while expression of the core 1 synthase C1GalT1 was similar between control and infected mice.

Conclusions
Glycosylation patterns and the expression of glycosyltransferase genes are altered in both the small and large intestines of Giardia-infected mice. Disruptions to mucin glycans appear to be regiospecific, suggesting that unique mechanisms are involved in the regulation of mucosal glycosylation during parasitic infection throughout different regions of the gut. These findings uncover a novel mechanism in the pathogenesis of Giardia infection, and may be important in understanding intestinal barrier dysfunction in a variety of different gastrointestinal diseases.

Funding Agencies
CCC
A16 ESOPHAGEAL ORGANOID PROLIFERATION AND DIFFERENTIATION ARE ALTERED BY LOSS OF MSH2
Authors: Rolland M , Gonneaud A , Jean D , Giroux V 
Published: February 2022

Abstract

Background
The stratified epithelium of the esophagus includes Krt15+ basal stem cells that display self-renewing and regenerative capacity, and multipotency. However, the mechanisms that specifically control their functions remain unknown. Interestingly, RNA sequencing and GSEA revealed an enrichment of a gene set associated with DNA repair in Krt15+ cells vs Krt15- cells. We also observed that Msh2 (DNA mismatch repair pathway) is the most significantly upregulated gene in Krt15+ stem cells.

Aims
To determine the effect of Msh2 loss on self-renewal and differentiation of esophageal organoids.

Methods
Esophageal epithelial cells were isolated from a wild-type mouse. Using flow cytometry, esophageal Krt15+ (GFP+) and Krt15- (GFP-) cells were sorted from Krt15-CrePR1 (R26mT/mG) mice. All cell populations were grown as organoids and Msh2 was depleted using a CRISPR/Cas9 approach. Impact of Msh2 loss on self-renewal and differentiation in esophageal epithelial organoids was evaluated through organoid formation assays, WST-1 proliferation assays and histological analysis.

Results
At baseline, organoids depleted for Msh2 formed more poorly differentiated and less well-differentiated organoids than controls. Lower expression of differentiation gene Krt13 was also observed in Msh2-depleted organoids, confirming an altered differentiation pattern. Furthermore, these organoids showed a higher organoid formation rate and proliferation by WST-1 assay, suggesting that self-renewal capacity and viability are increased when Msh2 is depleted. Interestingly, following radiation, organoids depleted for Msh2 showed higher residual levels of p-H2AX (DNA damage marker), suggesting that their capacity to cope with DNA damages is altered. As mentioned above, we previously reported that Msh2 is the most upregulated gene in Krt15+ vs Krt15- cells. Therefore, to determine if Msh2 role is distinct in both populations, we depleted Msh2 in Krt15+ and Krt15- cells-derived organoids. Interestingly, our preliminary results suggest that Msh2 deletion led to increased p-H2AX and decreased Krt13 levels in Krt15+ organoids but not in Krt15- organoids.

Conclusions
Our results show that Msh2 is potentially a key contributor of esophageal stemness in homeostatic and injured conditions.

Funding Agencies
CIHRCanada Research Chair
A25 CRITICAL APPRAISAL OF GI ENDOSCOPY CLINICAL PRACTICE GUIDELINES DURING THE COVID-19 PANDEMIC
Authors: Tham D , Gimpaya N , Gholami R , Pattni C , Seleq S , Bansal R , Fujiyoshi M A , Ramkissoon A , Lisondra J , Ariaratnam J , Scaffidi M , Khan R , Grover S 
Published: February 2022

Abstract

Background
Clinical Practice Guidelines (CPGs) are integral during a pandemic, offering guidance to clinicians through uncertainty. Existing literature has established that the need for rapid publication of CPGs during previous infectious disease outbreaks resulted in less rigorous guidelines. CPGs were rapidly developed since the onset of the pandemic in December 2019, providing guidance in gastrointestinal (GI) endoscopy, an area where COVID-19 may pose risk of transmission.

Aims
To evaluate the quality of GI endoscopy guidelines developed during the COVID-19 pandemic and to compare these with (a) endoscopy CPGs developed prior to the pandemic; (b) CPGs for other endoscopic topics unrelated to COVID-19; and, (c) non-endoscopic CPGs published during the pandemic.

Methods
We systematically searched Medline, Embase and Scopus for CPGs published by GI societies from January 1, 2018 to December 31, 2020. A grey literature search was conducted. Two authors screened full-texts. In this interim analysis, CPGs were grouped based on publication year: before 2020, or 2020. Endoscopy CPGs published in 2020 were categorized as COVID or non-COVID related. Two authors independently assessed the CPGs using the AGREE II tool, consisting of six domains for evaluating guidelines. A domain score of 60 was set as a threshold to indicate good quality.

Results
There were 70 endoscopy guidelines and 27 CPGs focused on other GI topics. The mean overall scores were 69% (±12%) for endoscopy CPGs published before 2020 (n=28), and 51% (±23%) for CPGs published in 2020 (n=42). For individual AGREE II domains, mean scores for pre-2020 CPGs ranged from 33.11 (±17.39) in Applicability to 81.55 (±10.37) in Clarity of Presentation. For CPGs published during COVID-19, mean domain scores ranged from 34.18 (±10.52) in Applicability to 75.26 (±13.85) in Clarity of Presentation. 21 of 42 CPGs published in 2020 were related to COVID. Mean overall scores were 35% (±20%) for COVID-related CPGs and 67% (±13%) for non-COVID-19 CPGs. For COVID-19 CPGs, scores ranged from 27.88 (±20.31) in Rigour of Development to 69.58 (±10.81) in Scope and Purpose. For non-COVID CPGs, the scores ranged from 37.30 (±8.93) in Applicability to 84.52 (±5.93) in Clarity of Presentation.

Conclusions
The difference in overall scores between COVID-19 endoscopy CPGs and non-COVID endoscopy CPGs may suggest that the urgency to disseminate COVID-19 information decreased CPG quality or completeness of reporting. This interim analysis is limited by the lack of distinction between peer-reviewed CPGs and non-peer reviewed recommendations. Given the importance of CPGs in clinical decision making, it is important to ensure that the rapid development of guidelines does not compromise quality and rigour.

Funding Agencies
None