bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitophagy regulation after diet and exercise in non‐alcoholic fatty liver disease
Authors: Rosa‐Caldwell Megan E. , Harris Matthew , Brown Jacob L , Lee David E. , Poole Kaylee , Seija Andrew , Brown Lemuel A. , Perry Richard A. , Washington Tyrone A. , Wooten Joshua S. , Greene Nicholas P.
Published: November 2023

Along with the increase in obesity, there has been a concurrent increase in the diagnosis of non‐alcoholic fatty liver disease (NAFLD). NAFLD may begin with fat accumulation in the liver, but can progress to non‐alcoholic steatohepatitis (NASH), fibrosis, and eventual cirrhosis. Current recommendations include weight loss strategies such as caloric restriction and exercise. Aberrant mitochondrial content/quality and mitochondrial autophagy (mitophagy) are common in NAFLD. Exercise is known to improve hepatic mitochondrial health and mitophagy thus mitophagy may be a mediator in the treatment of obesity induced‐NAFLD.

PURPOSE

The purpose of the study was to examine how weight loss from diet or diet combined with physical activity impacts hepatic mitochondrial content, autophagy and mitophagy in NAFLD.

METHODS

48 Male C57BL/6J mice were divided into 1 of 4 groups: low fat diet (LFD, 10% fat, for 18 wks), high fat diet (HFD, 60% fat diet, for 8 wks.), weight loss by diet (D, 60% fat diet for 10 wks then 10% fat diet for 8 wks) or weight loss by diet and physical activity (D/PA, 60% fat diet for 10 wks, then 10% fat diet plus a running wheel for 8 wks). After interventions, livers were collected and analyzed via Western blot for markers of mitophagy. Results were analyzed by one‐way ANOVA with α set at 0.05.

RESULTS

Mitochondrial content and biogenesis markers COX‐IV and PGC‐1α protein contents were approximately 50% less in HFD compared to LFD. COX‐IV content was restored and PGC‐1α content increased 50% with D/PA. Mitophagy marker BNIP3 content was 45% lower in HFD compared to LFD; D/PA had 50% more BNIP3 compared to LFD controls. PINK1 content was 40% higher in D and D/PA animals compared to LFD. P‐PARKIN/PARKIN levels were 40% lower in HFD, D, and D/PA compared to LFD. Whereas, p‐UbSer65 was 3‐fold higher in HFD animals. Autophagome marker LC3II/I ratio was 2‐fold greater in HFD and D/PA animals, yet autophagy flux marker p62 content was 2.5 fold higher in HFD animals compared to LFD, D, and D/PA, with no further differences observed.

CONCLUSION

Obesity‐induced NAFLD causes disruptions in hepatic mitochondrial content, mitophagy, and macroautophagy. While diet appears to restore some of these derangements, diet combined with physical activity ameliorates most of these derangements.
Production of Bioactive Peptides by Neutrophil Elastase Cleavage of E‐cadherin
Authors: Gordon Marilyn , MacNaughton Wallace K
Published: November 2023

BACKGROUND

The inflammatory microenvironment in the gut contains a variety of proteases from numerous sources including inflammatory cells. We have been studying the ability of proteases to induce a switch in the colonic epithelium from a barrier to a repair phenotype (epithelial to mesenchymal transition [EMT]) characterized by increased migration and disrupted homeostasis. EMT involves the degradation of junctional proteins such as E‐cadherin [Ecad], but the functional consequences of Ecad loss remain incompletely understood.

AIM

To test the hypothesis that inflammatory proteases cleave Ecad to produce peptides that alter epithelial homeostasis by altering cell migration, proliferation, or cell death.

METHODS

Recombinant Ecad was incubated with neutrophil elastase [NE] in a minimized cell‐free system and 24 peptides identified by mass spectrometry were chosen for high‐throughput screening based on frequency, p‐value scoring, and accessibility of peptide cleavage site. Synthesized peptides were tested for biological activities including proliferation, cell death, and cell migration. Murine CMT‐93 intestinal epithelial cells were cultured in 96 well plates and wounded 5 days post‐confluency using the EssenBio WoundMaker™ tool. Plates were imaged with the IncuCyte™ live‐cell imaging system for 24–48 hours following exposure to 1, 10, and 100 mg/mL concentrations of peptides. Cells were transfected with a GFP marker for automated cell counting to measure proliferation. Cytotoxicity was determined using Cytotox dye entering cells undergoing membrane degradation. All analysis was done using the IncuCyte™ ZOOM platform in conjunction with ImageJ software.

RESULTS

Basolateral stimulation of human Caco2 and murine CMT93 cells with NE confirmed the ability of NE to produce C‐ and N‐terminal Ecad fragments in vitro. From our synthesized peptides, we identified 10 Ecad peptides that significantly influenced wound closure rates, both positively and negatively. Two peptides (100 mg/mL) significantly inhibited wound healing rate by 13–26% compared to vehicle controls. Six peptides (100 μg/mL) significantly increased wound healing rates by 7–11% and three peptides (10 μg/mL) significantly increased wound healing rates by 6–13% compared to vehicle controls. Preliminary results suggest that several of these peptides also have cytostatic or pro‐proliferative activity.

CONCLUSIONS

Our results suggest that degradation of cell junction proteins by inflammatory proteases can create bioactive peptides that alter epithelial homeostasis and alter cell migration. Our data reveal a novel and complex pathway whereby epithelia respond to inflammatory tissue damage at the cellular level to alter a barrier phenotype to a more dynamic epithelium.

Support or Funding Information

Funding: Supported by the Canadian Institutes of Health Research (CIHR)
ER stress antagonizes metabolic stress‐induced loss of epithelial barrier function to commensal bacteria via xenophagy
Authors: Lopes Fernando , Keita Åsa , Saxena Alpana , Reys Jose Luiz , Mancini Nicole , Rajabi Ala , Arthur Wang , Baggio Cristiane , Dicay Michael , van Dalen Robert , Ahn Younghee , Rho Jong , MacNaughton Wallace , Jijon Humberto , Söderholm Johan , Philpott Dana , McKay Derek M.
Published: November 2023

Background

Patients with IBD commonly present deregulated barrier function of the intestinal epithelium. We have shown that treatment with dinitrophenol (DNP: uncouples oxidative phosphorylation) dramatically impairs the barrier function of cultured epithelial monolayers, particularly internalization of non‐invasive commensal E. coli. Mitochondria do not function in isolation and are linked to the endoplasmic reticulum (ER). We hypothesized that interaction of mitochondrial dysfunction and ER stress is a key determinant of epithelial‐bacterial interaction, specifically the fate of commensal bacteria that gain access to the intracellular compartment as a consequence of metabolic stress.

Methods

Human colonic biopsies were mounted in Ussing chambers and human colon‐derived epithelial cell lines were cultured on transwell filters or on plasticware. Bacteria were added to the luminal buffer and the cells treated with DNP ± the ER stressor, tunicamycin (TM), and bacterial internalization and translocation assessed. Mechanistic studies involved measuring ATP production, assessment of autophagy (i.e. LC3 activation) and gene knock‐down (KD) by siRNA and CRISPR/cas9.

Results

DNP promoted the translocation and internalization of E. coli in colon tissue and epithelial monolayers: TM reduced this barrier defect, but did not prevent the DNP‐evoked drop in ATP or the rate of epithelial update of inert beads. E. coli, DNP + TM‐treated cells had increased autophagy. The TM antagonist of the DNP effect was lost in cells lacking the autophagy protein, ATG16L1, suggesting that the ER‐stress promoted killing of the internalized bacteria. Of the 3 major arms of the ER stress response, only KD of ATF6 ablated the TM antagonism of the DNP‐evoked barrier effect; the increase in autophagy evoked by TM was absent in ATF6 KD epithelia.

Conclusion

An effective ER stress response, via mobilization of xenophagic response, can ameliorate epithelial barrier function in terms of enhanced killing of bacteria that gain access to the cell as a consequence of mitochondrial dysfunction. We suggest that target induction of ATF6 activity could be beneficial in treating disease characterized by bacterial ‘invasion’ of epithelial cells.

Support or Funding Information

CIHR AIHS HPI NSERC
GLP‐2 acutely diminishes LPS‐related intestinal permeability in rats
Authors: Akiba Yasutada , Kaji Izumi , Maruta Koji , Kaunitz Jonathan D
Published: November 2023

Circulating lipopolysaccharide (LPS; endotoxin) increases the permeability of the gut mucosal barrier, increasing LPS translocation into the circulation, augmenting endotoxemia with consequent systemic inflammation. Although daily treatment for 1 week with glucagon‐like peptide‐2 (GLP‐2) prevents the observed LPS‐related intestinal permeability increase, presumably due to its intestinotrophic effects, we hypothesized that acute administration of GLP‐2 reduces LPS‐related increased intestinal permeability by a mechanism unlikely related to hypertrophy. We thus examined the effect of GLP‐2 on intestinal permeability dynamics in vivo and in Ussing‐chambered intestinal mucosa in vitro.

We assessed intestinal permeability in vivo by measuring the appearance of intraduodenally perfused FITC‐dextran 4000 (FD4) into the portal vein (PV) in rats every 15 min for 90 min at 6 or 24 hr after LPS treatment (5 mg/kg, ip). We also examined FD4 movement from mucosal to serosal solution (m‐to‐s) in Ussing‐chambered muscle‐stripped intestinal tissues of vehicle‐ or LPS‐treated animals.

6 hr after LPS treatment, PV FD4 concentrations gradually increased following bolus intraduodenal infusion of FD4 (0.1 mM). Co‐treatment with the GLP‐2 receptor antagonist GLP‐2(3–33) (1 mg/kg, ip) with LPS enhanced PV FD4 concentrations. In Ussing‐chambered ileum obtained from rats 6 hr after LPS treatment, m‐to‐s movement of FD4 was unchanged compared with controls, whereas FD4 movement was increased in the ileum of GLP‐2(3–33) co‐treated animals. Furthermore, PV GLP‐2 concentrations and proglucagon mRNA expression in ileal mucosa were increased 6 hr after LPS treatment. 24 hr after LPS treatment, PV FD4 concentrations were further increased compared to FD4 concentrations at 6 hr. Exogenous GLP‐2 treatment (100 nmol/kg, ip) 6 hr after LPS treatment reduced FD4 permeability measured 24 hr after LPS treatment.

Increased intestinal permeability 6 hr after LPS administration was aggravated by GLP‐2 receptor antagonism. Systemic LPS releases endogenous GLP‐2, acutely protecting against LPS‐related loss of barrier function due to endotoxemia, followed by increased mRNA expression for the GLP‐2 precursor proglucagon. GLP‐2 treatment 6 hr after LPS administration prevented further increased intestinal permeability measured 24 hr after LPS, suggesting that the anti‐permeabilizing effects of GLP‐2 last longer than the t1/2 of the hormone. Exogenous GLP‐2 treatment may be of value in the prevention of systemic inflammation associated with endotoxemia due to a “leaky gut”.

Support or Funding Information

Supported by VA Merit Review, NIH R01 DK54221, Shire Pharmaceuticals
Importance of actin dynamics in gastric epithelial repair of damage
Authors: Aihara Eitaro , Medina‐Candelaria Neisha M , Engevik Kristen A , Matthis Andrea L , Montrose Marshall H
Published: November 2023

Background

Superficial injury to the gastrointestinal (GI) epithelium requires a rapid response to prevent further damage. In the stomach, initial gastric epithelial repair involves restitution, during which neighboring cells migrate to cover the damaged area within minutes. This process is regulated by several factors, including calcium and trefoil factor 2 (TFF2). In many cell types, changes in the actin cytoskeleton are necessary for cell migration, however its role within gastric epithelial repair of damage has yet to be elucidated. In this present study, we explore the role of actin in gastric repair of damage in vivo and in vitro gastric organoids (gastroids) using human actin‐GFP (HuGE) transgenic mouse stomach.

Methods

With an anesthetized mouse on the microscope stage, the surgically exposed stomach mucosa was continuously imaged by intravital confocal/two‐photon microscopy. Selective high intensity illumination of 3–5 gastric epithelial surface cells (730 nm light) caused a microscopic lesion in the epithelium via photodamage. Gastroids were created from gastric corpus tissue of HuGE mice. Hoechst33342 (10 μg/ml) was added to image the nucleus. Photodamage was induced in a single epithelial cell nucleus in the gastroid. Dynamic changes in actin‐GFP and damage size were quantified over time.

Results

The unperturbed actin‐GFP intracellular distribution was similar in gastric epithelium between in vivo and in vitro gastroids generated from the HuGE mice. In response to photodamage of either in vivo gastric epithelium or in vitro gastroid epithelium, actin‐GFP fluorescence decreased in the damaged cell. Subsequently, actin‐GFP expression increased in the lateral membranes neighboring the damaged cell, followed by migration inward to close the gap at the basal pole of the dead cell, in parallel with dead cell exfoliation into the lumen. This repair of damage and focal increase of actin‐GFP was significantly blocked by the calcium chelator EDTA (10 mM in the lumen in vivo, 1 mM in the medium in vitro) or the inhibition of actin polymerization (1 μM Cytochalasin D in the medium in vitro). Phalloidin (actin) staining in the gastroids, at 5 and 10 min after photodamage, showed that endogenous actin selectively increased at the leading edge of cell migration/restitution that restored epithelial continuity after cell exfoliation. Conversely, immunofluorescence of E‐cadherin increased in the trailing edge of the same restituting cells. AMD3100 (CXCR4/TFF2 inhibitor, 1 μM) and FAK inhibitor (1 μM) significantly slowed repair of damage but did not impact actin‐GFP dynamics.

Conclusion

Actin dynamics regulated by extracellular calcium but not CXCR4 are a component of gastric restitution.

Support or Funding Information

NIH RO1 DK102551 (M.H. Montrose).
Probiotic Metabolites Promote Anti‐inflammatory Functions of Immune Cells
Authors: Fu Richard H , Wu Danielle C , Yang Wei , Scoffield Jessica , Benveniste Etty N
Published: November 2023

The microbiome is an important factor in human health and disease. Recent reports demonstrate that short‐chain fatty acids (SCFA), type of bacterial metabolites, ameliorate autoimmune diseases, indicating a link between the gut microbiome, immune cells and inflammation. In this study, we investigated the potential health benefits of probiotic bacterial metabolites by evaluating their functions in regulating the differentiation of anti‐inflammatory and pro‐inflammatory immune cells, and determining the underlying mechanisms.

Using conditioned media from the probiotic bacteria, Lactococcus lactis and Lactobacillus reuteri, we demonstrated that bacterial metabolites induced differentiation of immunosuppressive anti‐inflammatory T‐regulatory (Treg) cells. Consistently, butyrate, a bacterial‐derived SCFA, enhanced the differentiation of Treg cells from naïve CD4+ T cells. Furthermore, butyrate inhibited the maturation of pro‐inflammatory antigen‐presenting dendritic cells (DCs) from hematopoietic progenitor cells. Mechanistically, we found that butyrate promoted Treg cell differentiation via increasing expression of transcription factor Foxp3 and the anti‐inflammatory cytokine IL‐10. In addition, it suppressed the maturation of DCs by inhibiting the expression of CD11c and MHC Class II, critical molecules for pathogenic CD4+ T cell activation. Taken together, these studies reveal novel functions of probiotic metabolites, SCFA, in regulating the differentiation/activation of anti‐inflammatory and pro‐inflammatory immune cells, thus inhibiting inflammation and promoting human health.

Support or Funding Information

This work was supported by the National Institute of Health grants NS057563 (E.N.B.)
Effects of Probiotic VSL#3 on Recovery from Dextran Sodium Sulfate‐Induced Colitis in Mice
Authors: Maag Kasey , Cornelius Holly , Edelman Danielle , Walton Kristen LW
Published: November 2023

Inflammatory bowel disease (IBD) affects as many as 1.6 million Americans every year. This condition is a chronic intestinal disease that also includes Crohn's disease and ulcerative colitis. Probiotic bacteria have previously been shown to have beneficial effects in some intestinal conditions such as pouchitis, irritable bowel syndrome, and some models of IBD. We examined the effects of the probiotic cocktail VSL#3 administration for 3 days of recovery after a 5‐day treatment with 3% dextran sodium sulfate (DSS). Clinical signs of colitis, including weight loss, fecal occult blood, and fecal consistency, were observed daily during the treatment and recovery period. At the end of the experiment, samples were collected for histological scoring, fecal bacterial analysis by quantitative PCR, and analysis of colonic pro‐inflammatory cytokine expression. As expected, clinical signs of colitis were observed more frequently in DSS treated mice than in non‐DSS treated mice. However, probiotic‐treated colitic mice had clinical signs of disease similar to those of non‐probiotic treated colitic mice. Histological scoring of colitis showed a trend towards decreased histological colitis with VSL#3 treatment compared to colitic mice. Quantitative PCR for Lactobacillus 16s rRNA showed a decrease in total lactobacilli with DSS treatment that was not affected by administration of VSL#3. In conclusion, 3 days of treatment with VSL#3 following acute DSS‐induced colitis did not significantly improve recovery from colitis.

Support or Funding Information

Missouri Western State University PORTAL Program
Loss of Sprouty2 in the colonic epithelium limits inflammatory cytokine production and enhances Muc2 and Lgr5 expression during colitis
Authors: Schumacher Michael A , Hsieh Jonathan J , Almohazey Dana , Warburton David , Washington M. Kay , Frey Mark R
Published: November 2023

OBJECTIVE

Sprouty2 (Spry2) is an intracellular modulator of MAPK signaling. Its expression is suppressed by tumor necrosis factor (TNF) in airway epithelium, but its regulation and activity in the intestinal tract are unknown. Aberrant MAPK signaling is associated with increased inflammatory cytokine production in diseases including inflammatory bowel disease, raising the possibility that Spry2 may play a role in regulating cytokine expression in the colon. MAPK is also an important regulator of secretory cell differentiation in the intestine. Here we tested the hypothesis that Spry2 regulation of MAPK signaling during colitis controls inflammatory cytokine production and goblet cell function, and thus impacts intestinal response to injury.

METHODS

We generated VillinCre;Spry2FLOXXED mice that lack Spry2 in the intestinal epithelium (Spry2KOIE) and LysMCre;Spry2FLOXXED mice that lack Spry2 in cells of myeloid lineage. Mice were given 3% dextran sodium sulfate (DSS) in drinking water for 4 days followed by 3 days without DSS and colitis severity was assessed. Cytokine levels were determined by qPCR. Epithelial crypt cultures (colonoids) were generated to assess regulation of ISC and cell lineages. The role of MAPK signaling was tested with MEK inhibitor U0126.

RESULTS

DSS colitis caused a 59% reduction of colonic Spry2 (p<0.05). Spry2KOIE mice subjected to DSS had decreased levels of TNF, CXCL2, IL1β, and IFNγ versus wildtype littermates, and were protected from histological damage (injury score reduced by 30%, p=0.037), weight loss, and colon shortening. In contrast, mice with Spry2 deletion in myeloid cells developed acute colitis similar to controls, suggesting an epithelial specific effect of Spry2 in colitis. Spry2KOIE mice showed higher colonic Lgr5 (ISC marker) and Muc2 (goblet cell marker) levels. In vitro, Spry2KOIE‐derived colonoids also showed a 3‐fold increase (p=0.005) in Muc2 expression and elevated Lgr5 expression. Because goblet cell differentiation is thought to be dependent upon MAPK signaling, we analyzed colonoids treated with the MAPK inhibitor, U0126, but surprisingly did not see reversal of the elevated Muc2 in Spry2KOIE colonoids.

CONCLUSION

Our work demonstrates that Spry2 is robustly expressed in the colonic epithelium, and expression is regulated by injury and inflammation. This may represent a protective mechanism against tissue injury by limiting inflammatory cytokine production, thus supporting Lgr5+ ISCs and inducing expression of the protective mucin, Muc2. Elevated Muc2 in Spry2KOIE colonoids was independent of MAPK activation, suggesting loss of Spry2 may elicit colonic protection through an alternative route of inhibiting inflammatory cytokine production.

Support or Funding Information

NIH R01DK095004 (MRF)
G‐protein Coupled Receptor Kinase‐2 Deficiency Protects Mice from Experimentally Induced Colitis
Authors: Steury Michael , Rios‐Arce Naiomy , McCabe Laura , Parameswaran Narayanan
Published: November 2023

G‐protein coupled receptor kinase 2 (GRK2), a member of the serine/threonine family of GPCR kinases are known for their role in GPCR phosphorylation and desensitization. In addition to this canonical role, GRK2 also modulates inflammation via non‐canonical pathways and thus has been shown to be involved in some inflammatory diseases. However, role of GRK2 in inflammatory bowel disease is not known. We hypothesized that the ablation of GRK2 would significantly modulate the pathogenesis of dextran sodium sulfate (DSS)‐induced colitis in mice. To test this, we administered DSS (in drinking water) to GRK2 heterozygous (HET) and wild type (WT) mice for 6 days followed by drinking water for 1 day. We found that GRK2 HET mice were significantly protected from DSS‐induced colitis compared to WT mice. This was demonstrated by decreased weight loss (WT 20±2.8% vs HET 11±3.4% n=6–7), lower disease activity index scores (WT 9.1 vs HET 4.1; n=6–7), and increased colon lengths (WT 4.7 cm vs HET 5.3 cm; n=6–7) in the GRK2 HET compared to WT mice. Additionally, we observed significant decrease in inflammatory cytokines including IL‐17, GM‐CSF, as well as the IL‐6/IL‐10 ratio in the colon without significant differences in cellular infiltration at 7‐day post‐DSS treatment. To further identify the cellular mechanisms by which GRK2 modulates colitis, we investigated the role for GRK2 in a myeloid‐specific GRK2 knockout. Our results demonstrate that GRK2 deficiency in the myeloid cells is sufficient for the protection observed in the heterozygous mice. Together our results indicate that GRK2 is an important mediator of colitis pathogenesis and this is conferred by the myeloid‐specific GRK2. Our results further suggest that GRK2 could be an important therapeutic target in colitis.

Support or Funding Information

Funding provided through NIH Grants 4R01AT007695‐04, 5R01DK101050‐03, 5R21AI099404‐02
Targeting Palmitoyl Acyltransferase ZDHHC21 Improves Epithelial Barrier Dysfunction Resulting from Burn Induced Systemic Inflammation
Authors: Haines Ricci J , Wang Chunyan , Zhang Shimin , Eitnier Rebecca A , Wang Fang , Yang Clement GY , Wu Mack H
Published: November 2023

Clinical studies in burn patients demonstrate a close association between leaky guts and increased incidence or severity of sepsis and other complications. Severe thermal injury triggers intestinal inflammation contributing to intestinal epithelial hyperpermeability, which exacerbates systemic response leading to multiple organ failure and sepsis. In this study, we identified a significant function of a particular palmitoyl acyltransferase (PAT), ZDHHC21, in mediating signaling events required for gut hyperpermeability induced by inflammation. Using qPCR, we showed that ZDHHC21 mRNA production was enhanced two‐fold when intestinal epithelial cells were treated with TNFα/IFNγ in vitro. Additionally, using electric cell‐substrate impedance sensing (ECIS) assays and FITC‐dextran permeability assays, and pharmacologically targeting PATs with 2‐bromopalmitate (2BP) or specific targeting of ZDHHC21 with siRNA, showed significant epithelial barrier function improvement when treated with TNFα/IFNγ. Using the ABE assay and click chemistry, we showed that TNFα/IFNγ treatment of intestinal epithelial cells results in enhanced detection of total palmitoylated proteins, and this response is inhibited by 2BP. We further explored the impact of targeting palmitoylation using ZDHHC21 deficient mice or wild‐type mice treated with 2BP and determined intestinal barrier function using mucosal permeability assays. These experiments showed that mice with impaired ZDHHC21 expression or pharmacological inhibition resulted in attenuated intestinal barrier dysfunction caused by thermal injury. Furthermore, H&E staining of small intestine and transmission electron microscopy (TEM) revealed that mice with a genetic interruption of ZDHHC21 had attenuated villus structure disorganization resulting from thermal injury induced inflammation. Taken together, these results suggest an important role of ZDHHC21 in mediating gut hyperpermeability resulting from thermal injury.

Support or Funding Information

VA Merit IBX000799
Flufenamic Acid Prevents Vibrio cholerae El Tor Variant‐Induced Diarrhea via AMPK Activation and Suppression of NF‐κB‐Mediated Inflammation
Authors: Pongkorpsakol Pawin , Satitsri Saravut , Wongkrasant Preedajit , Srimanote Potjanee , Chatsudthipong Varanuj , Muanprasat Chatchai
Published: November 2023

Pathogenesis of cholera diarrhea caused by infection with Vibrio cholerae El Tor variant (EL), a current epidemic strain of Vibrio cholerae that causes severe diarrhea in several regions, involves the nuclear factor kappa B (NF‐κB)‐mediated inflammatory responses including cyclooxygenase‐2 (COX‐2) upregulation and prostaglandin E2 (PGE2)‐induced intestinal fluid secretion and barrier disruption. The aim of this study was to evaluate the anti‐diarrheal efficacy of flufenamic acid (FFA), an non‐steroidal anti‐inflammatory drug (NSAID) that has previously been reported to stimulate AMP‐activated protein kinase (AMPK), a negative regulator of NF‐κB. We found that intraperitoneal injection of FFA (20 mg/kg) effectively reduced EL‐induced intestinal fluid secretion and intestinal barrier leakage in a mouse closed‐loop model of EL infection. In addition, FFA activated AMPK and suppressed p65 NF‐κB nuclear translocation, mRNA expression of proinflammatory cytokines and protein expressions of COX‐2 and inducible nitric oxide synthase (iNOS) in the mouse intestine infected with EL. In human intestinal epithelial cells (T84 cells), FFA induced tight junction assembly via AMPK activation. Mechanistic studies using immunoblot analysis and molecular docking prediction demonstrated that FFA activated AMPK in T84 cells by directly stimulating Ca(2+)/calmodulin dependent kinase kinase beta (CaMKKβ). Taken together, our results indicate that FFA and related compounds may be repurposed for the treatment of cholera caused by EL infection.

Support or Funding Information

This work was supported by grants BRG5980008 from the Thailand Research Fund and Mahidol University and by the Faculty of Science, Mahidol University and Faculty of graduate studies, Mahidol University.
Socio‐Cognitive Considerations to Broaden Participation and Support Student Success in STEM
Authors: Trujillo Gloriana
Published: November 2023

What can educators do to enhance the success and retention of all students with an interest in pursuing STEM pathways? In recent years, calls from multiple federal entities to improve undergraduate STEM education have mainly emphasized cognitive teaching techniques that are applicable in many learning environments. Taking into consideration the many contexts in which educators engage with students, as well as the social nature of learning, certain socio‐cognitive elements such as self‐efficacy, sense of belonging, and science identity may also be important to monitor in learning environments. What educators can do to assess these elements in their classrooms, how to translate and implement lessons from social psychology research, and further, what educators might adapt in their learning environments based on these findings will be discussed.
Engaging students in the large‐lecture biology classroom
Authors: Elfring Lisa K.
Published: November 2023

In the current era of higher‐education belt‐tightening, many institutions are dealing with resource shortages by increasing class sizes. This is particularly true for introductory‐level courses (and, at some institutions, upper‐division courses as well) in biology and other STEM disciplines. There is ample evidence from the learning sciences that students' active engagement in classes helps their learning: self‐testing, peer collaboration, explaining course concepts to others, and spaced retrieval practice are all techniques that demonstrably improve learning regardless of discipline (for examples, see Brown et al., Make It Stick, 2014). Creating classroom environments that promote these student practices is important to help students to build strong learning habits, but can seem overwhelming to those who teach large science classes.

I will describe and model several course‐structure and teaching strategies I have used to create a large‐class learning environment that promotes meaningful engagement with important biology concepts and supports students as they practice the skills of active engagement. Each can be adopted on its own or in combination with any other; this incremental approach allows for customization to each instructor's personal style and the content matter taught, as well as a lower “activation energy” than a wholesale course redesign.
The t‐STEM Initiative: Faculty Inquiry in the Context of Institutional Commitments to Student Success
Authors: Jewett Sarah , LaCourse William
Published: November 2023

In the midst of busy semester schedules, it is often difficult to create time and space for faculty to meet with colleagues in their own departments, much less the colleagues at other institutions. Within the context of the STEM Transfer Student Success Initiative (t‐STEM), a broad inter‐institutional commitment to student success, chemistry faculty across institutions committed to a collaborative inquiry in which they exchanged professional knowledge to determine how best to support student learning. The chemistry faculty members who are engaged in this generative process represent the partner institutions of Anne Arundel Community College (AACC), Community College of Baltimore County (CCBC), Howard Community College (HCC), Montgomery College (MC) and the University of Maryland, Baltimore County (UMBC).*

This shared faculty experience led to a deep awareness of the curriculum and pedagogy of general chemistry coursework across institutions. Beginning from the analytic framework of the Significant Discussions publication (League for Innovation in the Community College, 2010), the team identified key differences and similarities in course experiences. Because the comparative analysis yielded many more similarities than differences, the team focused on shared curricular challenges, and created content for new instructional resources. Rather than supplementing resources for transfer students at four‐year institutions, as is a more typical outcome of this kind of work, these resources were designed to promote the success for all STEM students across institutions at any point in their academic journey.

This work was undergirded by three critical and collaborative aspects of the process, including awareness, analysis, and action. In this way, the work reaches beyond the traditional boundaries of articulation and curricular alignment, and creates a new analytic space for curricular inquiry and collaboration around student learning. This focused attention contributes to a compelling national model for STEM student success, especially within the broader context of a significant inter‐institutional initiative that also addresses pre‐transfer advisement, peer mentorship, professional engagement and campus involvement. This presentation will highlight key aspects of the model, and share practices that can help other two‐year and four‐year teams to establish or enhance their own collaborative work.
miR‐125a‐5p a Novel Regulator of SLC26A6 in Intestinal Epithelial Cells
Authors: Anbazhagan Arivarasu Natarajan , Priyamvada Shubha , Alrefai Waddah A. , Saksena Seema , Gill Ravinder K. , Dudeja Pradeep K.
Published: November 2023

Putative Anion Transporter‐1 (PAT‐1 or SLC26A6) is expressed on apical membrane of the intestinal epithelial cells primarily in small intestine and functions as a Cl−/HCO3−, Cl−/formate or Cl−/oxalate exchanger. PAT‐1 has high affinity for oxalate and plays a crucial role in oxalate homeostasis. Mice deficient in Slc26a6 exhibit enhanced oxalate absorption resulting in nephrolithiasis. SLC26A6 has emerged as a potential target for reducing the risk of kidney stone formation. In this regard microRNAs (miRs) are small RNAs that play a key role in modulating gene expression via blocking the translation and/or promoting mRNA degradation of the target gene. In silico analysis of PAT‐1‐3¢UTR using Targetscan 7.1 revealed potential binding sites for several miRNAs. We hypothesized that PAT‐1 expression in intestinal epithelial cells is modulated by microRNAs. 3¢‐untranslated region (UTR) of PAT‐1 (263 bp) was cloned into pmirGLO vector upstream of luciferase. 3¢UTR PAT‐1 was transiently transfected in Caco‐2, T‐84 and HT‐29 cells and dual luciferase reporter activity was measured. Levels of microRNA, mRNA and protein expression were determined in transiently transfected Caco‐2 cells by RT‐PCR and western blotting, respectively. Significant decrease in the relative luciferase activity was observed in PAT‐1‐3¢UTR transfected Caco‐2 (53.87±3.02 p<0.0005) HT‐29 (50.51±3.05 p<0.0001) and T‐84 (44.93±4.47 p<0.0005) cells compared to empty vector. These data indicate the presence of intracellular factors in intestinal epithelial cells that target 3¢UTR of PAT‐1. In silico anaylsis showed highest context score for miR‐125a‐5p among the microRNAs targeting PAT‐1. Co‐transfection with microRNA mimic‐125a‐5p resulted in a further decrease in the relative luciferase activity of PAT‐1‐3¢UTR (~26% p<0.0001). In parallel, the decrease in luciferase activity by miR‐125a‐5p mimic concomitantly decreased PAT‐1 mRNA (~49% p<0.004) and protein expression (~44% p<0.006) in Caco‐2 cells. These data demonstrate a novel regulatory mechanism of intestinal PAT‐1 expression via miR‐125a‐5p and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for counteracting defective SLC26A6‐mediated intestinal oxalate secretion leading to nephrolithiasis.

Support or Funding Information

NIDDK and Dept of Veteran Affairs
Pharmacological but not genetically alteration of neural Epo modifies the CO2/H+central chemosensitivity in postnatal mice at birth
Authors: Laouafa Sofien , Perrin‐Terrin Anne‐Sophie , Jeton Florine , Elliot‐Portal Elizabeth , Tam Rose , Bodineau Laurence , Voituron Nicolas , Soliz Jorge
Published: November 2023

The central chemosensitivity to CO2/H+ is considered essential for breathing during the maturation of the mammalian respiratory network. In this study we tested the hypothesis that at birth, the neural Epo is involved in the breathing stimulation induced by the activation of central CO2/H+ chemoreceptors. Measurements were performed on newborn Tg21 (overexpressing cerebral Epo), Epo‐TAgh (cerebral Epo deficient mice) and their control (WT‐Tg21 and WT‐Epo‐TAgh) mice aged of 4 day. Fictive breathing (in‐vitro) response to CO2‐induced acidosis or metabolic acidosis was measured by extracting the brainstem with a part of the spinal cord and placing it in a chamber where it was superfused with an artificial cerebrospinal fluid (aCSF). The output signal from the respiratory network was recorded by a suction electrode placed on the fourth ventral root (C4) representing the inspiratory signal sent to the diaphragm via the phrenic nerve allowing to determine fictive breathing frequency, amplitude and neural ventilation (Frequency × amplitude). Our results did not show differences between preparations from control and transgenic animals Tg21 and Epo‐TAgh for the fictive breathing frequency and amplitude. However, when Tg21 brainstem preparations were incubated for 1h with sEpoR (the antagosnist of Epo), or with inhibitors of ERK/Akt (thus blocking the activation of the Epo molecular pathway), the fictive breathing response to CO2‐induced acidosis was blunted and showed a significant decrease of the neural ventilation (Tg21‐sEpoR: from 111.7±41.1 to 100.0±20.6; & Tg21‐ERK/Akt inhibitors: from 112.3±20.3 to 100.8±23.8; P<0.05) due to a significant decrease of the amplitude (Tg21‐sEpoR: from 100.0±17.1 to 47.4±11.0; and Tg21‐ERK/Akt inhibitors: from 104.6±10.6 to 54.9±8.4; P<0.05). Our data suggest that the variation of the Epo/sEpoR ratio is central in the breathing modulation, and open up clinical perspectives based on the use of Epo drugs at birth in hypoventilation cases.

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Founded by FRQS, Région Rhône‐Alpes and consulat de France à Québec
Cellular localization of spinal A2A receptors giving rise to phrenic motor facilitation in rats
Authors: Seven Yasin Baris , Perim Raphael R , Hobson Orinda R , Simon Alec , Mitchell Gordon S
Published: November 2023

Cervical spinal adenosine 2A (A2A) receptor activation elicits plasticity in the phrenic motor system. For example, spinal A2A receptor‐dependent phrenic motor facilitation (pMF) is observed following severe acute intermittent hypoxia (Nichols et al., 2012), and can be elicited by intrathecal A2A receptor agonist administration (Golder et al., 2008). Since the specific cells expressing the relevant A2A receptors for this form of phrenic motor plasticity are not known, we tested the hypothesis that the relevant A2A receptors are expressed in phrenic motor neurons per se. To test this hypothesis, we combined neurophysiology, neuropharmacology, immunofluorescence and RNA interference. Intrapleural injections of cholera toxin B subunit (CtB) were used to label phrenic motor neurons. Intrapleural injections of siRNAs (3 consecutive days) targeting A2A receptor mRNA (siA2A), or non‐targeting siRNAs (siNT) were used to knock‐down A2A expression within phrenic motor neurons. Successful knock‐down of A2A receptors was confirmed by ~45% decrease in A2A receptor immunolabeling within CtB identified phrenic motor neurons (p<0.05). Minimal A2A receptor expression could be detected in adjacent astrocytes or microglia; further, there was no evidence for knockdown in C4 interneurons or non‐phrenic motor neurons. Selective phrenic motor neuron knock‐down using this method was similar to prior reports using siRNAs targeting either TrkB or PKCθ. In anesthetized, paralyzed and ventilated rats, pMF induced by intrathecal injections of the A2A receptor agonist CGS21680 was significantly attenuated in the siA2A (37%) versus the siNT group (147%; p<0.05). There were no significant effects of siA2A on phrenic burst frequency. Our results support the hypothesis that the A2A receptors giving rise to A2A receptor‐induced PMF are expressed within phrenic motor neurons versus glia, non‐phrenic motor neurons or segmental interneurons.

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Supported by NIH HL69064 and the McKnight Brain Institute.
Phrenic long‐term facilitation (pLTF) following acute intermittent hypoxia (AIH) is TrkB and PI3K/Akt dependent in rats with motor neuron death from intrapleural CTB‐saporin injections
Authors: Nichols Nicole L. , Tanner Miles
Published: November 2023

Intrapleural injections of cholera toxin B fragment conjugated to saporin (CTB‐SAP) selectively kills respiratory motor neurons, and mimics motor neuron death observed in rats with amyotrophic lateral sclerosis (ALS) (Nichols et al., 2015). This CTB‐SAP model allows us to study the impact of respiratory motor neuron death on breathing without many complications attendant to ALS, and is sufficient to enhance phrenic long‐term facilitation (pLTF, a form of phrenic motor facilitation; Nichols and Mitchell, ibid, 2014) after 7 days of CTB‐SAP (CTB‐SAP 145±22%; control 54±5%). However, the mechanism responsible for this enhancement is unknown. pLTF in naïve rats predominately requires 5HT2 receptors, the new synthesis of BDNF, and MEK/ERK signaling; however, there is an alternative mechanism for pLTF that requires A2A receptors, the new synthesis of TrkB, and PI3K/Akt signaling. Preliminary data suggest that A2A receptors are increased in the phrenic motor nucleus of CTB‐SAP treated rats (Craig et al., ibid) suggesting that CTB‐SAP rats utilize the alternative mechanism for pLTF. Here, we tested the hypothesis that pLTF is TrkB and PI3K/Akt, not BDNF and MEK/ERK, dependent in anesthetized, paralyzed and ventilated adult Sprague Dawley rats. Specifically, AIH (3, 5 min bouts of 10.5% O2) induced pLTF was studied 7 days following bilateral, intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control). Intrathecal C4 delivery included either: 1) small interfering RNA that targeted BDNF or TrkB mRNA; 2) U0126 (MEK/ERK inhibitor); or 3) PI828 (PI3K/Akt inhibitor). Our preliminary data suggest that pLTF in CTB‐SAP treated rats is TrkB dependent (siTrkB: 1%, n = 1; siBDNF: 127%, n=1), whereas pLTF in controls is BDNF dependent (siTrkB: 35%, n = 1; siBDNF: 11%, n=1). U0126, not PI828, nearly abolished pLTF in controls (U0126: 13%, n=2; PI828: 52%, n=2); in contrast, pLTF was attenuated in CTB‐SAP treated rats following U0126 and PI828 (U0126: 71%, n=2; PI828: 85%, n=2). This suggests that CTB‐SAP treated rats express pLTF primarily via an alternative mechanism that requires the new synthesis of TrkB and both MEK/ERK and PI3K/Akt signaling. Future studies will be focused on completing this analysis and testing whether pLTF in CTB‐SAP treated rats is abolished following C4 intrathecal, A2A receptor antagonist treatment. This project increases our understanding of respiratory plasticity and its implications for breathing following motor neuron death.

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Supported by NIH K99/R00 HL119606
Mechanisms of cross‐talk inhibition between phrenic motor facilitation elicited by 5‐HT2B and 5‐HT7 receptors
Authors: Perim Raphael R , Fields Daryl P , Mitchell Gordon S
Published: November 2023

Intermittent spinal serotonin receptor activation elicits long‐lasting phrenic motor facilitation (pMF), a model of respiratory motor plasticity. When activated alone, spinal Gq protein‐coupled serotonin type‐2 receptors (5‐HT2) initiate pMF by a mechanism that requires ERK MAP kinase signaling and new synthesis of BDNF (i.e. the Q pathway to pMF). Activation of spinal Gs protein‐coupled serotonin type‐7 receptors (5‐HT7) also elicits pMF, but by a distinct mechanism that requires Akt (not ERK) signaling and new TrkB (not BDNF) synthesis. When both receptor subtypes are activated simultaneously, pMF is attenuated or even abolished, demonstrating powerful cross‐talk inhibition between these competing mechanisms. However, we know little concerning the mechanisms of cross‐talk inhibition between these competing pathways to pMF. Here, we tested the hypotheses that: 1) concurrent 5‐HT2 subtype‐B (5‐HT2B) and 5‐HT7 receptors undermine pMF; 2) 5‐HT7 receptors inhibit the 5‐HT2B receptor induced Q pathway to pMF via a protein kinase A dependent mechanism; and 3) 5‐HT2B receptors inhibit the 5‐HT7 receptor induced S pathway to pMF via a NADPH oxidase dependent mechanism. Selective agonists for 5‐HT2B (BW 723C86 hydrochloride) and 5HT7 receptors (AS‐19) were administered intrathecally at C4 (3×, 5‐min injections) in anesthetized, vagotomized and ventilated rats. After spinal 5‐HT2B or 5‐HT7 receptor activation alone, integrated phrenic nerve burst amplitude increased progressively (i.e. pMF). In contrast, concurrent 5‐HT2B and 5‐HT7 activation failed to elicit pMF. With concurrent receptor activation, pMF was restored by inhibiting NADPH oxidase activity (apocynin) or protein kinase A activity (Rp‐8‐Br‐cAMPS), consistent with our hypotheses. We suggest an elaborated model accounting for inter‐receptor cross‐talk interactions regulating serotonin‐induced phrenic motor facilitation.

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Supported by NIH HL69064 and the McKnight Brain Institute.
Phrenic Motor Neuron Expression of Receptors Known to Elicit Phrenic Motor Facilitation After Daily Acute Intermittent Hypoxia Preconditioning
Authors: Allen Latoya Lynn , Satriotomo Irawan , Simon Alec K. , Mitchell Gordon S.
Published: November 2023

Spinal serotonin receptor activation elicits a form of respiratory motor plasticity known as phrenic motor facilitation (pMF). One specific form of pMF is phrenic long‐term facilitation (pLTF) following acute intermittent hypoxia (AIH) exposure. pMF exhibits metaplasticity, defined as a future change in the capacity to express plasticity based on experience. For example, repetitive AIH preconditioning enhances AIH‐induced pLTF.

Distinct cellular mechanisms give rise to pMF, including the Q and S pathways, which are induced by 5HT2A/5HT2B and 5HT7 or adenosine 2A signaling, respectively. Although Q and S pathways each elicit pMF when acting alone, they are mutually inhibitory; thus, pLTF following moderate AIH results predominantly via the Q pathway, and is partially restrained by concurrent S pathway activation. Our working hypothesis is that repetitive AIH preconditioning causes pLTF metaplasticity by: 1) reducing inhibitory interactions between the S and Q pathways, and 2) increasing the relative contribution of the S pathway to pLTF. To begin exploring this global hypothesis, we began by investigating the impact of repetitive AIH on the phrenic motor neuron expression of receptors initiating the Q (5HT2A,5HT2B) versus S (5HT7, A2A) pathways to pMF. Four days after intrapleural injections of Cholera toxin B fragment (CTB) to identify phrenic motor neurons, rats were exposed to normoxia or daily AIH (10, 5 min episodes per day, 5 min intervals; 14 days). The rats were perfused and tissue sections were analyzed for 5HT2A, 5HT2B, 5HT7 and A2A receptors, and a downstream mediator of the S pathway (EPAC2) via immunofluorescence. Following dAIH: 1) 5HT2A, 5HT2B and A2A receptor expression were unchanged; but 2) 5HT7 and EPAC2 expression were increased versus controls. Increased 5HT7 and EPAC2 protein expression may support enhanced contributions of the S pathway to pMF, but do not provide information concerning the possibility that cross‐talk inhibition between the Q and S pathways was reduced. Priming a system with dAIH may increase the capacity for respiratory plasticity, and may be a useful therapeutic intervention to treat disorders that cause respiratory motor deficits, including spinal injury, sleep apnea or motor neuron disease.

Support or Funding Information

Supported by: T32 Neuromuscular Plasticity Training Program NICHD: T32‐HD043730, NIH HL69064, and the McKnight Brain Institute. We acknowledge Dr. Mark Brownfield for providing us with the serotonin receptor antibodies.