bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microbiologique Microfilm™ EBEc – a Rapid Method for the Simultaneous Enumeration of Enterobacteriaceae and Escherichia coli
Authors: Mai Tam L , Ming Ziwen , Nguyen Van , Shapovalova Anna , Samadpour Mansour 
Published: February 2020

Abstract

Background
The Enterobacteriaceae and generic Escherichia coli are routinely enumerated in foods as part of product release criteria, or in the case of swabs, for environmental monitoring.

Objective
Microbiologique Microfilm™ EBEc is intended to provide a rapid and easy-to-use method for simultaneous enumeration of Enterobacteriaceae and E. coli on foods and environmental surfaces. Methods: This study evaluated the performance of Microfilm™ EBEc against ISO methods (ISO 21528-2:2017 for Enterobacteriaceae and ISO 16649-2: 2001 for E. coli) in 20 food matrixes and two environmental surfaces. Inclusivity, exclusivity, lot-to-lot reproducibility, ruggedness and stability studies were also performed on Microfilm™ EBEc.

Results
No significant differences and high correlation coefficients (R2) were observed between the Microfilm™ EBEc and the corresponding ISO methods in spiked food matrixes and environmental samples. Inclusivity studies showed expected results for all the E. coli and Enterobacteriaceae strains tested. In terms of exclusivity testing, all the strains tested failed to grow on Microfilm™ EBEc. A lot-to-lot study showed no significant differences in mean difference (log10) counts among the three lots of the Microfilm™ EBEc. Ruggedness studies showed no significant differences in mean difference (log10) counts at varying incubation temperatures and times. Stability studies on the Microfilm™ EBEc showed that it is stable at 2–25°C for 12 months, and at 45°C for 6 weeks.

Conclusions
The results of this study indicate that the Microfilm™ EBEc is equivalent to the corresponding ISO methods for enumeration of Enterobacteriaceae and E. coli. Highlights: Microfilm™ EBEc offers a convenient and relatively fast test method for simultaneous enumeration of Enterobacteriaceae and E. coli in 24 h and has an advantage over the corresponding ISO methods that require two assays on the same sample for enumeration of Enterobacteriaceae and E. coli Gram-negative indicator groups.
Testing for Stanozolol, Using UPLC–MS-MS and Confirmation by UPLC–q-TOF-MS, in Hair Specimens Collected from Five Different Anatomical Regions
Authors: Gheddar Laurie , Raul Jean-Sébastien , Kintz Pascal 
Published: February 2020

Abstract
An athlete challenged the result from an in-competition doping test which returned with an adverse analytical finding for stanozolol, claiming it was due to supplement contamination. Her lawyer asked the laboratory to analyze several hair specimens simultaneously collected from five different anatomical regions, head, arm, leg, pubis and armpit, to document the pattern of drug exposure. A specific UPLC–MS-MS method was developed. After decontamination with dichloromethane, stanozolol was extracted from hair in the presence of stanozolol-d3 used as internal standard, under alkaline conditions, with diethyl ether. Linearity was observed for concentrations ranging from 5 pg/mg to 10 ng/mg. The method has been validated according to linearity, precision and matrix effect. Concentrations of stanozolol in head hair, pubic hair, arm hair, leg hair and axillary hair were 73, 454, 238, 244 and 7,100 pg/mg, respectively. The concentration of stanozolol in head hair is in accordance with data published in the literature. When comparing the concentrations, body hair concentrations were higher than the concentration found in head hair. These results are consistent with a better incorporation rate of stanozolol in body hair when compared to head hair. The simultaneous positive concentrations in different hair types confirm the adverse analytical finding in urine of the top athlete, as the measured concentrations do not support the theory of contamination. For the first time, an anabolic agent was simultaneously tested in hair collected from five different anatomical regions from the same subject, with a large distribution of concentrations, due to anatomical variations, and these findings will help interpretation in further doping cases when documented with hair.
A58 REGULATION OF INTESTINAL EPITHELIAL THYMIC STROMAL LYMPHOPOIETIN GENE EXPRESSION BY RETINOIC ACID RECEPTOR ALPHA
Authors: Mahmood R , Chan R , Beck P , Jijon H 
Published: February 2020

Abstract

Background
We have targeted the retinoic acid receptor alpha (RARα) isoform in murine intestinal epithelial cells (IECs) to explore the role of epithelial intrinsic retinoid signaling in intestinal immune homeostasis. We noted a significant decrease in the CD11c+ and CD103+ dendritic cell (DC) subpopulations in the intestines of intestinal-epithelial specific RARα-deficient (RARα villin) mice. In the present study, we identified Thymic Stromal Lymphopoietin (TSLP) as a contributing factor that influences intrinsic RARα signaling within IECs. This cytokine preferentially stimulates CD103+ DCs to a more tolerogenic phenotype in the GI tract, induces Tregs and drives TH2 polarization. TSLP also influences immune homeostasis by modulating the activation of myeloid cells, inhibition of proinflamamtory DCs, and production of IL-12. Notably, TSLP levels are decreased in Crohn’s disease (CD) where inflammation is driven by IL-12.

Aims
We hypothesize that RA intrinsic signaling within IECs modulates TSLP expression via activation of RARα. We sought to generate a RARα -/- murine intestinal epithelial cell line utilizing the CRISPR/CAS9 system to examine the effects of RARa ablation. Second, we aim to establish RARα’s role in regulating intestinal epithelial TSLP expression in both the RARα villin mice and the CRISPR-generated RARα -/- cell line.

Methods
Exon 5 in the open reading frame of the RARα gene in Mode-K murine IECs was targeted using CRISPR/CAS9. CRISPR cleavage and knockdown of RARα activity was confirmed via surveyor/luciferase assays. The cell lines were phenotyped with regards to their morphology, proliferative ability, and viability. Next, we examined TSLP mRNA expression in the colons of RARα villin mice and the CRISPR RARα -/- knockout cell line via qRT-PCR under baseline and stimulated conditions. Wild-type (WT) and RARα-/- cells were stimulated with RARα-selective agonist, BMS753, and muramyl dipeptide (MDP), a NOD2-selective agonist.

Results
TSLP expression in surface colonic epithelium was elevated 2.3-fold in RARα villin mice compared to WT litter mates. TSLP expression was elevated 10-fold in the Mode-K RARα-/- cells versus parent cells at baseline. BMS753 stimulation resulted in a 15-fold increase in TSLP expression in the RARα-/- cells compared to baseline. When stimulated with MDP, a NOD2-selective agonist known to stimulate TSLP expression, TSLP expression was elevated 60-fold in the RARα-/- cells, but significantly impaired compared to the WT cells (400-fold).

Conclusions
TSLP expression is controlled by RARα in colonic IECs where it may act as a repressor of TSLP promoter transactivation under baseline conditions. This suggests an important role for RA on myeloid and T cell function via effects on intestinal epithelial TSLP expression.

Funding Agencies
CCCUniversity of Calgary, Department of Medicine
A83 INTESTINAL EPITHELIAL CELL STEMNESS AND DIFFERENTIATION ARE REGULATED BY THE HIPPO PATHWAY EFFECTOR YAP1
Authors: Fallah S , Beaulieu J 
Published: February 2020

Abstract

Background
The high rate of cell turnover in the intestinal epithelium is supported by the LGR5+ crypt base columnar (CBC) stem cells, which are located at the lower part of the gland. Among of the various factors and signals like Wnt and Notch, YAP1 (yes associated protein) also plays an important role in stemness of CBC stem cells. YAP1 is the effector of the Hippo pathway. Hippo Pathway restricts the cells proliferation, tissues overgrowth and cancer formation through the phosphorylation and inactivation of the YAP1 protein. When active, YAP1 transfers into nucleus, forms the complex with TEADs transcription factors and promotes the transcription of genes involved in cell growth and proliferation.

Aims
In the present study, we investigated the role of the YAP1 in the colorectal cancer multipotent HT29 cell line, which contain cancer stem cells (CSC).

Methods
For approaching to this goal, YAP1 expression was knocked down using shRNAs in HT29 cells. Then stem cells and intestinal cell lineages (secretory goblet, Paneth and enteroendocrine and absorptive) markers expression was analyzed using qPCR and Western blot.

Results
The results showed the reduction of the expression of stem cells markers including LGR5 in YAP1 knockdown HT29 cells compare with control. Expression of the goblet cells markers (MUC2 and trefoil factor 3) and absorptive cells markers (sucrase-isomaltase and dipeptidylpeptidase IV) were significantly increased in YAP1 knockdown cells but Paneth (DEFA5 and lysozyme) and enteroendocrine (CHGA) were not detected. Finally, examination of the main transcription factors for intestinal differentiation revealed an increase in CDX2 expression.

Conclusions
These results suggest that YAP1 is involved in the maintenance of colorectal cancer stem cells while preventing intestinal differentiation in both secretory and absorptive lineages through the repression of CDX2.

Funding Agencies
CIHR
A91 SARCOMATOID CARCINOMA OF THE COLON PRESENTING AS IRON DEFICIENCY ANEMIA
Authors: Masckauchan M , Soulellis C A 
Published: February 2020

Abstract

Background
Gastrointestinal malignancy is a common cause of iron deficiency and occult gastrointestinal bleeding among adults in developed countries. Given the high prevalence of colorectal cancer and adenomatous polyps in our population, it is imperative that these patients undergo early colonoscopy. Sarcomatoid carcinoma (SCA) of the colon is a very rare condition, with less than 30 cases reported in the literature. Given the highly aggressive nature of this neoplasm, it is important to familiarize oneself with its presentation, histopathology and treatment options.

Aims
Describe a case of SCA of the colon in a cirrhotic patient, presenting initially as iron deficiency anemia of unknown etiology.

Methods
Case report and literature review.

Results
A 69-year-old female presented to the emergency department with complaints of progressive weakness and failure to thrive. Past medical history was significant for chronic kidney disease, NASH cirrhosis Child-Pugh C and recurrent hepatocellular carcinoma despite previous radiofrequency ablation. Investigations demonstrated de novo iron deficiency anemia with the following blood results: hemoglobin of 96, iron of 8.6, iron saturation of 20% and ferritin of 56. Subsequently, gastroenterology was consulted in order to assess for an occult source of bleeding. A gastroscopy was first performed in which no abnormalities were found. She then underwent a colonoscopy, which demonstrated a very large indurated nodular ulcer in the ascending colon, suggestive of invasive neoplasm (Figure A). Indeed, the biopsies demonstrated a malignant spindle cell neoplasm consistent with SCA. On immunohistochemistry, the tumor cells stained positive for keratin AE1/AE3, weakly positive for SMA and S-100, but negative for SMM, HMB45, DOG1, C-KIT and CD31. Given her severe comorbidities, no therapeutic options were available for this patient, thus further staging investigations were not pursued. Unfortunately, the patient rapidly deteriorated and died less than 8 weeks following her diagnosis.

Conclusions
Sarcomatoid carcinoma of the colon is an extremely rare neoplasm with very poor prognosis. Histologically, this tumor is characterized by a typical biphasic pattern, composed of both mesenchymal and epithelial cells. However, as demonstrated in this case, some SCA display a rather predominantly mesenchymal pattern, rendering the diagnosis difficult using morphology only. Immunostaining is therefore used to identify carcinoma features and to rule out similar tumors such as sarcoma, GIST, mesothelioma and melanoma. SCA of the colon is exceedingly aggressive, with remarkably rapid tumor growth and early distant metastasis. Indeed, median survival is reported to be less than 6 months. Given its rarity, no well-defined treatment guidelines exist, however most patients are managed with early radical surgery, adjuvant chemotherapy and close follow-up.

Funding Agencies
None
A110 DGAT1 MUTATION OF LIPID TRAFFICKING ASSOCIATED WITH A RARE CASE OF CONGENITAL DIARRHEA
Authors: Little R , Gonska T 
Published: February 2020

Abstract

Background
Congenital diarrheas and enteropathies (CODEs) are rare and cause severe chronic diarrhea in children under 2 years of age.1 Current genomic research has identified several monogenic defects resulting in epithelial disorders of nutrient and electrolyte transport, enzymes function, metabolism and trafficking.

Aims
To discuss a rare case of congential diarrhea.

Methods
Case Report.

Results
A 10-month-old Ashkenazi Jewish boy was referred to Pediatric Gastroenterology for loose stools and failure to thrive. Born at 27 weeks, he was a NICU inpatient for his first 3 months of life. Explosive loose stools were first noticed after discharge and treated as cow’s milk protein allergy. At aged 10 months he was admitted to his local hospital with irritability, abdominal distension, foul smelling loose stools and developmental delay. Despite an intake of 130kcal/kg/day he failed to gain weight (weight and length &lt; 3rd centile). His abdomen was distended but there was no organomegaly on examination.
On transfer to Sick Kids he underwent extensive work up with low albumin 24 g/L but normal CBC, amylase, lipase and IgA and
anti-TTG negative. The lipid profile showed hypertriglyceridemia 2.98 mmol/L (normal &lt;0.85), low HDL 0.81 mmol/L but normal total cholesterol. Low apoliprotein A1 0.79 g/L (0.92 - 1.96), low apoliprotein B 0.54 g (0.59 - 1.46) and vitamin E deficiency 9.6 umol/L (14.5 - 33.0). Normal fecal elastase (&gt; 500 ug/g stool). Upper GI endoscopy showed duodenal villi blunting with non-specific chronic inactive duodenitis on histopathology.
Due to ongoing poor weight gain he commenced TPN. A repeat endoscopy four weeks after admission revealed patchy villous blunting, crypt hyperplasia and enterocyte vacuolization. However, ultrastructural electron microscopy examination showed normal enterocyte brush border with well-formed microvilli with no inclusions or vesicular bodies. With a differential diagnosis of chylomicron retention disorder, abetalipoproteinemia or hypolipoproteinemia he was commenced on a low-fat diet with MCT feeds. A 72-hour fecal fat collection on this diet was within normal range 5.4 mmol/day (2–7).
One year later his weight is now on 3rd centile and his albumin levels have normalized. However, triglycerides remain elevated. Whole exome sequencing identified two pathogenic variants in the DGAT1 gene; c.629_631delCCT, pSer210del and c751 + 2T&gt;C, IVSB+2T&gt;C.

Conclusions
Diacylglycerol acyltransferase (DGAT)1 catalyzes triglyceride biosynthesis.2 Loss of function DGAT1 gene mutations are described in several cases studies3,4 characterized by electrolyte transport–related diarrhea, protein-losing enteropathy and growth failure. Our cases adds to the understanding of the phenotypic and histological spectrum of DGAT1 mutations. Management strategies currently focus on growth with dietary lipid restriction, while substituting fat-soluble vitamins and essential fatty acids.

Funding Agencies
CAG
A119 AN EX VIVO MODEL TO STUDY THE GUT SEROTONERGIC SYSTEM RESPONSE TO LIVE AND HEAT-KILLED LACTOBACILLUS RHAMNOSUS STRAIN JB-1
Authors: Javkar T , Paul M , Stanisz A , Forsythe P 
Published: February 2020

Abstract

Background
Enterochromaffin (EC) cells are one of the most abundant enteroendocrine cells in the intestinal epithelium, responsible for producing and storing the largest pool of serotonin or 5-hydroxytryptamine (5-HT) in the body. 5-HT has been shown to be important for modulating a large number of gastrointestinal reflexes in health and disease. 5-HT can stimulate extrinsic (vagal and spinal afferents) or intrinsic primary afferent neurons (IPANs) which are involved in motility, secretion and vasodilation within the intestines. Where EC cell localized enzyme tryptophan hydroxylase (TpH) isoform 1 is responsible for 5-HT synthesis, serotonin reuptake transporter (Sert) and monoamine oxidase A (Mao A) are responsible for termination by uptake and metabolism of 5-HT respectively. Our previous research has demonstrated the effects Lactobacillus rhamnosus (JB-1) on the firing frequency of spinal nerve fibres and motility. Increasing interest is being focused on potential health benefits of heat-inactivated microbes and purified bacterial components. However, the effect of these heat-killed bacteria on the intestinal epithelium cells, particularly on EC cells, is unknown.

Aims
Small intestinal organoids are shown to recapitulate in vivo characteristics of the small intestine epithelium. The present study aims to assess the suitability of intestinal organoids to study bacterial effects on the serotonergic system in the gut. Here we determined changes in the gene expression of key mediators in the serotonergic system [serotonin reuptake transporter (Sert), tryptophan hydroxylase-1 (Tph-1) and monoamine oxidase A (Mao A)] in response to live and heat-killed JB-1.

Methods
Male C57bl/6 mice aged 6–8 weeks were used for both ex vivo and in vivo experiments. Jejunal organoids were grown from whole crypts isolated using DTT-EDTA solution. Live and heat-killed JB-1 bacteria were used as treatments. Gene expression analysis was performed on jejunal organoids and jejunum tissue using qRT-PCR.

Results
JB-1 induced a significant increase in gene expression of Sert, Mao A and Tph-1. No significant difference was observed between the effects of live and heat-killed bacteria. In contrast the JB-1 increased expression of the peptide hormone CCK. Effects of JB-1 on gene expression in organoid culture were reflective of changes observed in in vivo experiments involving feeding of the bacteria.

Conclusions
Ex vivo organoid culture could be a useful tool in studying mechanisms underlying bacterial effects on serotonergic signalling. The observation that heat-killed bacteria produced comparable effects to the live organism suggests the possibility of isolating active 5-HT modulating components from these strains. Future research will focus on identifying such bacterial components and how their effects on gene expression influence serotonin availability

Funding Agencies
None
A120 INFLUENCE OF THE MICROBIOTA ON THE POSTNATAL DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM IS DEPENDENT ON MOUSE STRAIN
Authors: Huang J , Popov J , Markovic F , Ratcliffe E 
Published: February 2020

Abstract

Background
Gastrointestinal function depends on the normal formation of the enteric nervous system (ENS) during fetal and postnatal development. Prior research in an outbred strain of mice (NIH Swiss) has shown that the absence of the gut microbiome in germ-free (GF) mice results in morphological and functional abnormalities of the ENS compared to specific pathogen free (SPF) mice, including an alteration in proportion of nitrergic neurons. Increasing research has been suggesting that the genetic background of the host can impact the host response to the GF state.

Aims
We tested the hypothesis that the absence of the microbiome in an inbred mouse strain (C57BL/6) could influence the development of the ENS during early postnatal life.

Methods
C57BL/6 GF and SPF mice were sacrificed at postnatal day 3 (P3) and P28 (n=4–5 per group). Ileum and colon were collected at P3 and P28 and processed for whole mount preparations. The neuronal network in the myenteric plexus was visualized by immunohistochemistry using antibodies against the pan-neuronal marker PGP9.5. Neuronal cell bodies and nitrergic neurons were identified by immunolabeling with antibodies to the neuronal marker HuC/D and to neuronal nitric oxide (nNOS). Nerve fibre density was quantified by measuring the percentage of PGP9.5-positive pixels (μm2) compared to the whole field using an image analysis program (Volocity; reported as %). Proportions of nitrergic to myenteric neurons were determined by manually counting (blinded) the number of nNOS-positive neurons and dividing by the total number of HuC/D-positive cells per field (reported as %).

Results
We found a significant increase in nerve density at P3 in the GF compared to SPF mice in both ileum (43% vs. 37%; p=0.03) and colon (45% vs. 39%; p=0.03). No significant differences, however, were identified between GF and SPF mice at P28 in either ileum (27% vs. 25%; n.s.) or colon (31% vs. 32%; n.s.). At P3, no significant differences in proportion of nitrergic neurons were seen in the GF compared to SPF ileum (27% vs. 27%; n.s.).

Conclusions
In contrast to earlier observations in the NIH Swiss mice, in which GF mice had decreased nerve density and an increase in nitrergic neurons at P3, our findings reveal an opposite response in nerve density in the C57BL/6 mice and no change in nitrergic neurons. These results suggest that the genetic strain of the mouse model can influence the host response to changes in the microbiome. Further studies are needed to further elucidate potential underlying mechanisms.

Funding Agencies
NSERC