Scholarly Work - Microbiology & Cell Biology
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/3494
Browse
9 results
Search Results
Item Profiling Luminal pH in Three-Dimensional Gastrointestinal Organoids Using Microelectrodes(MyJove Corporation, 2024-07) Lyon, Katrina; Bansil, Rama; Bimczok, DianeThe optimization and detailed characterization of gastrointestinal organoid models require advanced methods for analyzing their luminal environments. This paper presents a highly reproducible method for the precise measurement of pH within the lumina of 3D human gastric organoids via micromanipulator-controlled microelectrodes. The pH microelectrodes are commercially available and consist of beveled glass tips of 25 µm in diameter. For measurements, the pH microelectrode is advanced into the lumen of an organoid (>200 µm) that is suspended in Matrigel, while a reference electrode rests submerged in the surrounding medium in the culture plate. Using such microelectrodes to profile organoids derived from the human gastric body, we demonstrate that luminal pH is relatively consistent within each culture well at ~7.7 ± 0.037 and that continuous measurements can be obtained for a minimum of 15 min. In some larger organoids, the measurements revealed a pH gradient between the epithelial surface and the lumen, suggesting that pH measurements in organoids can be achieved with high spatial resolution. In a previous study, microelectrodes were successfully used to measure luminal oxygen concentrations in organoids, demonstrating the versatility of this method for organoid analyses. In summary, this protocol describes an important tool for the functional characterization of the complex luminal space within 3D organoids.Item Antiviral responses in a Jamaican fruit bat intestinal organoid model of SARS-CoV-2 infection(Springer Science and Business Media LLC, 2023-10) Hashimi, Marziah; Sebrell, T. Andrew; Hedges, Jodi F.; Snyder, Deann; Lyon, Katrina N.; Byrum, Stephanie D.; Mackintosh, Samuel G.; Crowley, Dan; Cherne, Michelle D.; Skwarchuk, David; Robison, Amanda; Sidar, Barkan; Kunze, Anja; Loveday, Emma K.; Taylor, Matthew P.; Chang, Connie B.; Wilking, James N.; Walk, Seth T.; Schountz, Tony; Jutila, Mark A.; Bimczok, DianeBats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB, Artibeus jamaicensis) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Upon infection with SARS-CoV-2, increased viral RNA and subgenomic RNA was detected, but no infectious virus was released, indicating that JFB organoids support only limited viral replication but not viral reproduction. SARS-CoV-2 replication was associated with significantly increased gene expression of type I interferons and inflammatory cytokines. Interestingly, SARS-CoV-2 also caused enhanced formation and growth of JFB organoids. Proteomics revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.Item Severe Acute Respiratory Syndrome Coronavirus 2 Is Detected in the Gastrointestinal Tract of Asymptomatic Endoscopy Patients but Is Unlikely to Pose a Significant Risk to Healthcare Personnel(Elsevier, 2022-06) Cherne, Michelle D.; Gentry, Andrew B.; Nemudraia, Anna; Nemudryi, Artem; Hedges, Jodi F.; Walk, Heather; Blackwell, Karlin; Snyder, Deann T.; Jerome, Maria; Madden, Wyatt; Hashimi, Marziah; Sebrell, T. Andrew; King, David B.; Plowright, Raina K.; Jutila, Mark A.; Wiedenheft, Blake; Bimczok, DianeBackground and aims. Recent evidence suggests that the gut is an additional target for severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection. However, whether SARS-CoV-2 spreads via gastrointestinal secretions remains unclear. To determine the prevalence of gastrointestinal SARS-CoV-2 infection in asymptomatic subjects, we analyzed gastrointestinal biopsy and liquid samples from endoscopy patients for the presence of SARS-CoV-2. Methods. We enrolled 100 endoscopic patients without known SARS-CoV-2 infection (cohort A) and 12 patients with a previous COVID-19 diagnosis (cohort B) in a cohort study performed at a regional hospital. Gastrointestinal biopsies and fluids were screened for SARS-CoV-2 by polymerase chain reaction (PCR), immunohistochemistry, and virus isolation assay, and the stability of SARS CoV-2 in gastrointestinal liquids in vitro was analyzed. Results. SARS-CoV-2 ribonucleic acid was detected by PCR in the colonic tissue of 1/100 patients in cohort A. In cohort B, 3 colonic liquid samples tested positive for SARS-CoV-2 by PCR and viral nucleocapsid protein was detected in the epithelium of the respective biopsy samples. However, no infectious virions were recovered from any samples. In vitro exposure of SARS-CoV-2 to colonic liquid led to a 4-log-fold reduction of infectious SARS-CoV-2 within 1 hour (P ≤ .05). Conclusion. Overall, the persistent detection of SARS-CoV-2 in endoscopy samples after resolution of COVID-19 points to the gut as a long term reservoir for SARS-CoV-2. Since no infectious virions were recovered and SARS-CoV-2 was rapidly inactivated in the presence of colon liquids, it is unlikely that performing endoscopic procedures is associated with a significant infection risk due to undiagnosed asymptomatic or persistent gastrointestinal SARS-CoV-2 infections.Item Transfer and persistence of bovine immunoglobulins in lambs fed a colostrum replacer(Wiley, 2022-07) Johnson, Thea; Jacobson, Bryan Tegner; Jones, Kerri; Mosdal, Cassie; Jones, Steve; Vitkovic, Maia; Kruppenbacher, Sam; Sebrell, Andy; Bimczok, DianeBackground. Colostrum-derived antibodies are crucial for the protection of newborn lambs from infectious diseases. Several colostrum replacer products that contain bovine antibodies are on the market. We investigated the absorption and persistence of bovine antibodies from a powdered colostrum replacer in newborn lambs. Methods. We tested a lamb colostrum replacer containing bovine serum in lambs that were separated from their dams at birth. Immunoglobulin G (IgG) uptake was analysed by ELISA, and the persistence of antigen-specific antibodies was analysed by parainfluenza 3 virus (PI-3) neutralisation assay. Results. Serum antibody ELISA performed on days 1 and 14 revealed IgG levels of 17.9 ± 2.8 and 27.5 ± 2.5 mg/ml, respectively. PI-3 antibodies derived from the colostrum replacer were present for 86.3 ± 10.6 days. Conclusions. Antibodies derived from bovine serum protein delivered to lambs via a commercial colostrum replacer are readily absorbed and persist for months, suggesting that these products may offer adequate protection.Item A High-Throughput Metabolic Microarray Assay Reveals Antibacterial Effects of Black and Red Raspberries and Blackberries against Helicobacter pylori Infection(MDPI AG, 2021-07) Goodman, Candace; Lyon, Katrina N.; Scotto, Aitana; Smith, Cyra; Sebrell, Thomas A.; Gentry, Andrew B.; Bala, Ganesh; Stoner, Gary D.; Bimczok, DianeHelicobacter pylori infection is commonly treated with a combination of antibiotics and proton pump inhibitors. However, since H. pylori is becoming increasingly resistant to standard antibiotic regimens, novel treatment strategies are needed. Previous studies have demonstrated that black and red berries may have antibacterial properties. Therefore, we analyzed the antibacterial effects of black and red raspberries and blackberries on H. pylori. Freeze-dried powders and organic extracts from black and red raspberries and blackberries were prepared, and high-performance liquid chromatography was used to measure the concentrations of anthocyanins, which are considered the major active ingredients. To monitor antibiotic effects of the berry preparations on H. pylori, a high-throughput metabolic growth assay based on the Biolog system was developed and validated with the antibiotic metronidazole. Biocompatibility was analyzed using human gastric organoids. All berry preparations tested had significant bactericidal effects in vitro, with MIC90 values ranging from 0.49 to 4.17%. Antimicrobial activity was higher for extracts than powders and appeared to be independent of the anthocyanin concentration. Importantly, human gastric epithelial cell viability was not negatively impacted by black raspberry extract applied at the concentration required for complete bacterial growth inhibition. Our data suggest that black and red raspberry and blackberry extracts may have potential applications in the treatment and prevention of H. pylori infection but differ widely in their MICs. Moreover, we demonstrate that the Biolog metabolic assay is suitable for high-throughput antimicrobial susceptibility screening of H. pylori.Item P38 MAPK signaling mediates retinoic acid‐induced CD103 expression in human dendritic(2020-11) Roe, Mandi M.; Hashimi, Marziah; Swain, Steve; Woo, Krista M.; Bimczok, DianeRetinoic acid (RA) is an active derivative of vitamin A and a key regulator of immune cell function. In dendritic cells (DCs), RA drives the expression of CD103 (integrin αE), a functionally relevant DC subset marker. In this study, we analyzed the cell type specificity and the molecular mechanisms involved in RA-induced CD103 expression. We show that RA treatment caused a significant up-regulation of CD103 in differentiated monocyte-derived DCs and blood DCs, but not in differentiated monocyte-derived macrophages or T cells. DC treatment with an RA receptor α (RARα) agonist led to an increase in CD103 expression similar to that in RA treatment, whereas RARA gene silencing with small interfering RNA blocked RA-induced up-regulation of CD103, pointing to a major role of RARα in the regulation of CD103 expression. To elucidate RA-induced signaling downstream of RARα, we used Western blot analysis of RA-treated DCs and showed a significant increase of p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, DCs cultured with RA and a p38 MAPK inhibitor had a significantly reduced expression of CD103 compared with DCs cultured with RA only, indicating that p38 MAPK is involved in CD103 regulation. In summary, these findings suggest that the RA-induced expression of CD103 is specific to DCs, is mediated primarily through RARα and involves p38 MAPK signaling.Item SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits immune suppression(2021-06) Nemudryi, Artem; Nemudraia, Anna; Wiegand, Tanner; Nichols, Joseph; Snyder, Deann T.; Hedges, Jodi F.; Cicha, Calvin; Lee, Helen; Vanderwood, Karl K.; Bimczok, Diane; Jutila, Mark A.; Wiedenheft, BlakeOver 950,000 whole-genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been determined for viruses isolated from around the world. These sequences are critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We isolate one of these mutant viruses from a patient sample and use viral challenge experiments to link this isolate (ORF7aΔ115) to a growth defect. ORF7a is implicated in immune modulation, and we show that the C-terminal truncation negates anti-immune activities of the protein, which results in elevated type I interferon response to the viral infection. Collectively, this work indicates that ORF7a mutations occur frequently, and that these changes affect viral mechanisms responsible for suppressing the immune response.Item Functional Properties of Helicobacter pylori VacA Toxin m1 and m2 Variants(2020-04) Caston, Rhonda R.; Sierra, Johanna C.; Foegeding, Nora J.; Truelock, Mandy D.; Campbell, Anne M.; Frick-Cheng, Arwen E.; Bimczok, Diane; Wilson, Keith T.; McClain, Mark S.; Cover, Timothy L.Helicobacter pylori colonizes the gastric mucosa and secretes a pore-forming toxin (VacA). Two main types of VacA, m1 and m2, can be distinguished by phylogenetic analysis. Type m1 forms of VacA have been extensively studied, but there has been relatively little study of m2 forms. In this study, we generated H. pylori strains producing chimeric proteins in which VacA m1 segments of a parental strain were replaced by corresponding m2 sequences. In comparison to the parental m1 VacA protein, a chimeric protein (designated m2/m1) containing m2 sequences in the N-terminal portion of the m region was less potent in causing vacuolation of HeLa cells, AGS gastric cells, and AZ-521 duodenal cells and had reduced capacity to cause membrane depolarization or death of AZ-521 cells. Consistent with the observed differences in activity, the chimeric m2/m1 VacA protein bound to cells at reduced levels compared to the binding levels of the parental m1 protein. The presence of two strain-specific insertions or deletions within or adjacent to the m region did not influence toxin activity. Experiments with human gastric organoids grown as monolayers indicated that m1 and m2/m1 forms of VacA had similar cell-vacuolating activities. Interestingly, both forms of VacA bound preferentially to the basolateral surface of organoid monolayers and caused increased cell vacuolation when interacting with the basolateral surface compared to the apical surface. These data provide insights into functional correlates of sequence variation in the VacA midregion (m region).Item A new twist on the graduate student journal club: Using a topic‐centered approach to promote student engagement(2020-02) Bimczok, Diane; Graves, JohnJournal clubs are widely used as an educational tool in graduate life science programs. In journal clubs, students are assigned to read specific journal articles to achieve a broad knowledge in their field of study and to gain competence in reading and assessing scientific publications. However, students often show low motivation to read assigned articles, and under‐prepared students contribute little to in‐class discussions. In order to promote student engagement in graduate‐level journal clubs, we used an inverted, student‐centered format that focuses on a scientific question or topic rather than specific publications. Both the weekly topics and the scientific publications were selected by the students and focused on aspects of the students' thesis research. For each weekly topic, students were asked to find papers, read the papers and summarize the findings during class, which led to the presentation of a variety of approaches and viewpoints. This approach trained students in literature search, focused reading and oral presentation skills and provided a broad overview of the research in the selected topic areas. Student feedback showed a high level of acceptance of the new format. We propose this inverted journal club format as a useful alternative to traditional formats, because it focuses on a different scientific skill set and leads to increased student engagement through its student‐centered approach.