bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic Plasticity in Morphological and Physiological Responses in Gills of Seawater‐ and Fresh water‐acclimated Asian Sea Bass, Lates calcarifer
Authors: Lee Tsung‐Han , Yang Wen‐Kai , Chao Tse‐Lih , Lee Sih‐Shien , Syu Jyun‐Jie , Chen Wei‐Zhu
Published: June 2021

Osmoregulation is necessary for euryhaline teleosts in maintaining internal ion and water balance in response to environmental salinity changes. In the epithelia of teleostean gills, the ionocytes (i.e. mitochondrion‐rich cells) are thought to be the primary site of osmo/ionoregulation. In the ionocytes, the Na+, K+‐ATPase (NKA) plays a crucial role in triggering ion‐transporting systems. Asian sea bass (Lates calcarifer) is a marine euryhaline teleost and an important economic species in Taiwan, Southeast Asia, and Australia that can be cultured in environments from seawater (SW) to fresh water (FW). However, the modulatory mechanisms of osmoregulation in gills of euryhaline Asian sea bass were not clear to date. Hence the purposes of the present study were to investigate changes in physiological parameters, morphology of apical openings of gill ionoctyes, as well as expression and localization of branchial NKA in the SW‐ and FW‐acclimated Asian sea bass. The results showed that the sea bass was able to maintain their internal physiological parameters including plasma osmolality and muscle water content in a tolerated range. Observed by the scanning electron microscope, different types of apical surfaces of ionocytes with distinct features were found in the afferent epithelia of gills in FW and SW milkfish. On the other hand, the specific activity of branchial NKA was lower in the FW milkfish compared to the SW individuals. The immunohistological observation further revealed that most NKA‐immunoreactive cells in gills of the FW‐ and SW‐acclimated Asian sea bass were distributed in the afferent and interlamellar regions of the filaments. Taken together, these results illustrated that the Asian sea bass was an efficient osmoregulator with these salinity‐dependent alterations to maintain homeostasis of ions and water in environments of different salinities. These findings contributed to the knowledge of phenotypic plasticity of ionocytes and changes in activity of NKA, the crucial enzyme for ionoregulation, in gills of the Asian sea bass upon salinity challenge.

Support or Funding Information

This study was supported by a grant to THL from the Ministry of Science the Technology (MOST), Taiwan (105‐2313‐B‐005‐027‐MY3).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Walk the Walk: Parallels between Gait Parameters and Muscle Force Production in Turkeys
Authors: Vega Karina , Eng Carolyn , Roberts Thomas J.
Published: June 2021

Running and walking involve regular patterns of limb kinematics, which can be characterized by simple parameters such as stride frequency and foot contact time. It is not clear whether these regular patterns of timing are mirrored at the level of muscle function, or whether variable sharing of force among different muscles might result in timing within individual muscles that does not reflect the timing of limb movements. We used direct measurements of force production in the lateral gastrocnemius (LG) muscle of wild turkeys during walking and running to test the hypothesis that stride parameters measured from limb movements would match those measured from muscle force production at all speeds of walking and running. Muscle force was measured via strain gauges attached to the bony tendon of the muscle, and stride parameters were measured with high‐speed video as animals ran on a treadmill at a range of walking and running speeds (1 to 3.5 m/s). Stride frequency and stride time values measured from video matched those determined from the onset of muscle force for all speeds. Time of foot contact measured from video decreased continuously with speed. Time of force development for the LG also decreased with speed, but the time spent in force development in the LG was significantly lower than the time the foot was on the ground for all speeds. The fraction of time the muscle was generating force was relatively constant across speed, with the muscle producing force for approximately 50% of the period of foot ground contact. This observation indicates that there is a temporal sharing of the task of force production between different muscles during the step, and that this sharing may remain relatively fixed across walking and running speeds. Such temporal sharing of force development should be considered in models of locomotor mechanics and energetics, and may be relevant to rehabilitative strategies for gait disorders.

Support or Funding Information

The Integrative Organismal Systems Physiology (IOSP) Undergraduate Summer Research Fellowship that made this research possible was supported by the American Physiological Society and a grant from the National Science Foundation (NSF) Integrative Organismal Systems (Award No. IOS‐1238831). Support from NIH grant AR‐055295 is also gratefully acknowledged.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Strategies to Assess Pharyngeal Pumping Variability in the Nematode C. elegans
Authors: Harris Michael B. , Berlemont Renaud , Ortiz Brianna , Taylor Barbara E.
Published: June 2021

In C. elegans, the feeding behavior of pharyngeal pumping occurs spontaneously in proportion to metabolism and in response to stimuli. Age‐ and health‐related changes in tissue morphology and function correlate with declines in the frequency of pumping. As such, pumping frequency is an established index of C. elegans “health”, and pumping changes illustrate and quantify functional decline. Timing of pharyngeal pumping is controlled by two pairs of pharyngeal motor neurons (MC and M3). Each pump cycle corresponds to the propagation of a single pharyngeal muscle action potential, initiated by MC and terminated by M3. We currently model this system with MC acting as an oscillator and synaptically linked to the pharyngeal muscle. Pumping can change by influences on the pace and regularity of the oscillator and/or of the fidelity of pharyngeal muscle response to neuronal inputs.

Traditionally, this phenomenon has been reported as an occurrence frequency, describing the number of pumping events observed per unit time over a period of observation. We have found variation in pumping frequency between subjects in an experimental group, and the relative coarseness of measures of occurrence frequency may interfere with the resolution of subtle, yet significant, treatment effects. Recently developed technologies facilitate precise recording of the electrical signatures of pharyngeal pumping in the form of an electropharyngeogram (EPG), allowing specific identification of timing of individual pumping events. This precision allows not only characterization of the occurrence frequency, but also the variability of pumping. We are considering that changes in variability may be a more sensitive measure of treatment effects.

We have developed a strategy to normalize pumping occurrence in EPG recordings of individual subjects, allowing relevant comparisons between subjects. We have applied a series of variability assessments to quantify pumping patterns and identify variation stemming from changes in fidelity of drive from MC to the pharyngeal muscle. Finally, we have applied assessments that we speculate can distinguish influences at or above the MC oscillator. This presentation describes the theory and practice of normalization and variability assessments. The use of this analysis strategy on an experimental dataset is described in a companion presentation.

Support or Funding Information

Work reported in this publication was supported by the National Heart Lung and Blood Institute and National Institute of General Medical Sciences of the National Institutes of Health under awards 1R15HL126105 and 1SC2GM112570. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health or any other funding body.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscle DRP‐1 and FIS‐1 Regulation by IL‐6 Signaling
Authors: Fix Dennis , VanderVeen Brandon N. , Hardee Justin , Carson James A.
Published: June 2021

Introduction

The IL‐6 cytokine family signals through the ubiquitously expressed glycoprotein 130 (gp130) receptor to induce signal transducer and activator of transcription 3 (STAT3) and extracellular signal‐regulated kinase 1/2 (ERK1/2). Evidence suggests that IL‐6 can negatively regulate muscle mitochondrial quality control and disrupt muscle oxidative metabolism. Mitochondrial fission is a critical component of skeletal muscle mitochondria maintenance and oxidative metabolism, which is regulated impart by the expression of FIS‐1 and DRP‐1 proteins. The purpose of this study is to examine muscle DRP‐1 and FIS‐1 expression by IL‐6 signaling.

Methods

Fully differentiated C2C12 myotubes were treated with 100ng of IL‐6 for 24 hours in the presence of gp130siRNA, C188‐9 (STAT3 inhibitor), or PD98059 (ERK1/2 inhibitor). Additionally, male C57BL/6 (B6) and muscle specific gp130 knockout mice (KO) were electroporated in the quadriceps muscles with 50ng of IL‐6 and monitored for two weeks.

Results

IL‐6 induced DRP‐1 and FIS‐1 expression in myotubes and skeletal muscle. Knock down of gp130 in myotubes and KO in skeletal muscle inhibited the IL‐6 induction of DRP‐1 and FIS‐1. STAT3 inhibition in myotubes did not alter IL‐6 induced DRP‐1 and FIS‐1 expression, while ERK 1/2 inhibited the IL‐6 induction. Collectively, these results provide evidence for skeletal muscle mitochondrial fission regualtion through an IL‐6/gp130/ERK 1/2 signaling axis.

Support or Funding Information

This work was supported by National Institutes of Health grant # NCI‐R01CA121249 (JAC).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ADGRG6/GPR126 as a Novel Therapeutic Target in Pulmonary Arterial Hypertension
Authors: Muthusamy Abinaya , Gorr Matthew W. , Chinn Amy M. , Insel Paul A.
Published: June 2021

Pulmonary arterial hypertension (PAH) is a chronic and fatal disease of the lungs. The 3‐year survival rate is 50–60%, as current therapies of PAH have limited effect on mortality. An important feature of PAH is the increased proliferation and contractile activity of pulmonary arterial smooth muscle cells (PASMCs). We cultured patient‐derived PASMCs and performed RNA‐seq on PASMCs from control and PAH lungs. We found that the expression of a G protein‐coupled receptor (GPCR) ADGRG6 (adhesion GPCR G6, or GPR126) is ~10‐fold higher in PAH patient PASMCs compared to control PASMCs. To test its functional activity, we overexpressed ADRGR6 in HEK293 cells, and found that proliferation of the cells decreased by 60% at 48 hours after transfection. Using qPCR, we observed that expression of cell cycle/proliferation markers CDK1, CDK2, and MKI67 was decreased after 48 hours, while expression of apoptosis markers VIM, ITGAM, and PCDH7 was increased. Additional preliminary data using flow cytometry indicate that cells overexpressing ADGRG6 are stalled at the S/G2 cell cycle checkpoint. These data suggest that ADRGRG6 activation could cause reduced proliferation in smooth muscle cells and be a viable target for PAH therapy.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multidimensional Clustering of Regenerative Alveolar Duct Cells after Murine Pneumonectomy
Authors: Ysasi Alexandra , Bennett Robert , Valenzuela Cristian , Servais Andrew , Wagner Willi , Tsuda Akira , Ackermann Maximilian , Mentzer Steven
Published: June 2021

Lung regeneration occurs in a variety of adult mammals after surgical removal of one lung (pneumonectomy). Previous studies of murine post‐pneumonectomy compensatory lung growth have identified areas of increased growth in subpleural alveolar ducts. To identify transcriptional patterns in these regenerative “hotspots”, we isolated single cells from alveolar ducts at post‐pneumonectomy days 1, 3 and 7. We used laser microdissection, enzymatic digestion and microfluidic isolation to capture individual cells from alveolar ducts, then performed single‐cell transcriptional analysis using the C1 integrated microfluidic circuit (Fluidigm) and a custom PCR panel designed for lung growth and repair genes. The multi‐dimensional data set was analyzed using visualization software based on the tSNE similarity analysis algorithm (viSNE, Cytobank). The similarity analysis identified six clusters of cells. To obtain a provisional cell type identity for these clusters, previously obtained post‐pneumonectomy cell sorted bulk transcriptional data was projected on the viSNE map using a similarity matrix analysis. The viSNE cluster with the highest overall level of gene expression was genotypically similar to myofibroblasts (64.04% of Acta2+ cells), endothelial progenitor cells (66.67% of sorted CD31+, CD34+cell samples), and type II pneumocytes (83.33% of sorted CD45−, MHC Class II+, phosphine+ cell samples). Another cluster genotypically overlapped primarily with sorted endothelial cells (94.74% of sorted CD31+ cell samples), but also some monocytes (50% of sorted CD11b+ cell samples) and alveolar macrophages (61.54% of sorted CD45+, CD11b−, CD11c+, F4/80+, Gr1− cell samples). One cluster was present only after pneumonectomy (99.23% post‐pneumonectomy, 0.77% un‐operated control), suggesting time point dependent gene activation. The transcriptional pattern of the six clusters was analyzed for genes associated with lung repair, matrix production and angiogenesis. The data demonstrated that multiple cell types (clusters) transcribed genes linked to these basic functions. We conclude that the coordinated gene expression across multiple clusters is likely a response to a shared regenerative microenvironment within the subpleural alveolar ducts.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
N‐Glycan and Heparan Sulphate Components of the Extracellular Matrix are Essential for Laminar Shear Stress Response of the Epithelial Sodium Channel (ENaC)
Authors: Ashley Zoe , Barth Daniel , Baldin Jan Peter , Fronius Martin
Published: June 2021

The epithelial sodium channel (ENaC), a mechanosensitive ion channel, is known to be activated by laminar shear stress (LSS) [1]. We hypothesise that the large extracellular loops of ENaC interact with the complex extracellular matrix (ECM) and that the application of LSS deforms both the extracellular matrix (ECM) and the interacting ENaC components resulting in a change in ENaC activity. This study investigated whether or not the N‐glycan and heparan sulphate components of the ECM are specifically involved in the LSS activation of ENaC.

Human α, β and γ ENaC subunits were co‐expressed in oocytes surgically harvested from female Xenopus laevis. Two‐electrode voltage‐clamp was used to determine ENaC activity in response to LSS when different components of the ECM were degraded by neuraminidase and heparinase III enzymes. A significant LSS response was observed in ENaC expressing oocytes, compared to native (no ENaC) oocytes; normalized LSS current of 5.5 ± 1.1 compared to 0.06 ± 0.03 (p = 0.003, n =8–14). In ENaC expressing oocytes cleavage of N‐glycan from the ECM with neuraminidase (0.2U/mL for 2h at 21°C) significantly reduced the normalised LSS response from 4.5 ± 0.4 to 1.7 ± 0.4 (p=0.003, n=8). Destruction of heparan sulphate components of the ECM with heparinase III (0.5 U/mL for 30min at 21°C) significantly reduced the LSS response from 5.1 ± 1.2 to 1.2 ± 0.5 (p=0.009, n=12). Data indicates that both removal of N‐glycan and cleavage of heparan sulphate from the ECM reduce the activation of ENaC by LSS, indicating that they are crucial components of the ECM for mechanosensation by ENaC.

Support or Funding Information

This study was supported by the Royal Society Marsden fund.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exposure of proximal tubule cells to fluid shear stress alters transcription of metabolic pathway enzymes
Authors: Ren Qidong , Eshbach Megan L. , Rittenhouse Natalie L. , Long Kimberly R. , Rbaibi Youssef , Locker Joseph , Poholek Amanda C. , Weisz Ora A.
Published: June 2021

Cells lining the proximal tubule (PT) of the kidney are specialized for efficient recovery of ions, glucose, and proteins from the glomerular filtrate. Despite their high glucose levels, PT cells in vivo utilize gluconeogenic rather than glycolytic pathways to maintain the robust metabolic activity needed to carry out their functions. In contrast, the reliance of PT cell cultures on glycolytic metabolism has been a significant limitation in studying the regulation of PT function in vitro. We discovered recently that opossum kidney (OK) cells cultured under continuous fluid shear stress (FSS) develop morphological and functional characteristics that more closely resemble PT cells in vivo compared with cells cultured under static conditions. We performed RNA Seq on OK cells maintained under static conditions or exposed to FSS for up to 96 h. Principal component analysis confirmed significant time‐ and FSS‐dependent changes in gene expression. Consistent with enzyme activity profiles described in the PT, we observed an increase in transcripts encoding enzymes in the gluconeogenic pathway in OK cells cultured under FSS, and a concomitant reduction of transcripts encoding glycolytic enzymes. Current studies are underway to extend and validate our observations using complementary approaches. Our data provide further support that OK cell culture under FSS provides an improved model of the PT.

Support or Funding Information

Tsinghua MD Scholars Program

NIH R01 DK101484

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Persistent disruption of lateral junctional complexes and actin cytoskeleton in parotid salivary glands following radiation treatment
Authors: Wong Wen Yu , Pier Maricela , Limesand Kirsten H.
Published: June 2021

Xerostomia and hyposalivation are debilitating side effects for patients treated with ionizing radiation for head and neck cancer. Despite technological advances, collateral damage to the salivary glands remains a significant problem for patients and severely diminishes their quality of life. During the wound healing process, restoration of junctional contacts is necessary to maintain polarity, structural integrity, and orientation cues for secretion. However, little is known about whether these structural molecules are impacted following radiation damage and more importantly following tissue restoration. Here, we evaluated changes in adherens junctions and cytoskeletal regulators in an injury model where mice were irradiated with 5 Grey (5Gy) and a restoration model where mice injected post‐radiation with insulin like growth factor 1 (IGF1) are capable of restoring salivary function. Using co‐immunoprecipitation, we determined that there is a decrease in β‐catenin bound to E‐cadherin following damage that is restored to untreated levels with IGF1. Via its adaptor proteins, β‐catenin links the cadherins to the cytoskeleton and part of this regulation is mediated through Rho‐associated coiled‐coil kinase (ROCK) signaling. In our model, F‐actin organization is also disrupted and there is an induction of ROCK activity. These findings illustrate that radiation induces a disruption of the cadherin‐catenin complex, alters F‐actin organization, and induces ROCK signaling at early and chronic stages where salivary hyposalivation is observed. Understanding the role of these components and its regulation may be important in tissue restoration where a coordinated and polarized epithelium are necessary for functionality.

Support or Funding Information

This work was supported in part by NIH R01 DE023534 received by Kirsten Limesand and stipend support for Wen Yu Wong was provided by the Cancer Biology Training Grant, T32CA009213‐38

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Early life stress induces vascular expression of pro‐oxidant, proinflammatory genes in adulthood in an HDAC9‐dependent manner
Authors: Pettway Yasminye D. , Ho Dao H. , McPherson Kasi , Seifert Michael E. , Pollock Jennifer S.
Published: June 2021

Early life stress (ELS) has been correlated with an increased risk of developing cardiovascular disease (CVD) in adulthood; however, mechanisms linking ELS and CVD risk are unknown. Toll‐like receptor (TLR) activation, specifically TLR4, has been implicated as a key link between inflammation and CVD. TLR4 signaling can activated NADPH oxidases (NOXs) that produce superoxide to increase CVD risk. Further, multiple studies have found that NF‐kB activation, an important downstream step in the TLR4 signaling pathway, requires the activation of certain NOX isoforms, including NOX2 and NOX4. We utilized a mouse model of maternal separation with early weaning (MSEW) to investigate the effect of ELS on the expression and activation of genes encoding these pro‐oxidant, proinflammatory proteins. MSEW mice were separated 4hr/day during postnatal day (PD)2–5, 8 hr/day during PD6‐16, and weaned at PD17. Normally‐reared control mice were left undisturbed and weaned at PD21. Experiments were conducted on 3–6 month old male mice. We have previously shown that MSEW induces NOX‐dependent superoxide production and vascular endothelial dysfunction. We also found increased gene expression of NOX2, NOX4, and TLR4 in the aorta of MSEW mice. Recent immunohistochemical studies have also revealed increased aortic TLR4 protein expression in MSEW mice (n=5/group). Due to similar patterns of expression of genes involved in the TLR4 signaling pathway, we further hypothesized that a common upstream transcription factor could be a driver of these ELS‐induced gene changes. We focused this study on histone deacetylases (HDACs), as numerous studies have implicated HDACs in the modulation of proinflammatory responses and the progression of a variety of CVDs. Interestingly, we found that MSEW induced aortic HDAC9 protein expression, exclusive of other HDAC isoforms. A 3‐hr ex vivo pretreatment with trichostatin A (TSA), a pan‐inhibitor of HDACs, improved vascular endothelial function in MSEW mice (n=5/group, p=0.047). Further, ex vivo TSA treatment led to a trend of decreased expression of NOX2, NOX4, and TLR4 genes (n=4–8/group). These results suggest that ELS leads to HDAC9‐dependent pro‐oxidative and proinflammatory gene expression that increase CVD risk in adulthood. They also further emphasize the role of TLR4 signaling on superoxide production and vascular endothelial function. Future studies aim to clarify the role of TLR4 signaling in these phenotypes, as well as the effect of increased HDAC9 expression on the transcriptional regulation of TLR4 and other genes that could confer CVD risk in adulthood.

Support or Funding Information

This research was funded by PO1HL69999 and the UAB PREP Program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Local Colonic Delivery of Vasoactive Intestinal Peptide (VIP) Nanomedicine Alleviates Colitis in Mice
Authors: Jayawardena Dulari , Anbazhagan Arivarasu Natarajan , Onyuksel Hayat , Dudeja Pradeep K.
Published: June 2021

Inflammatory Bowel Disease (IBD) is an unmet medical need encompassing two entities; Ulcerative Colitis (UC) and Crohn's Disease (CD). We have previously demonstrated the superior therapeutic effects of systemically administered VIP nanomedicine (VIP in sterically stabilized micelles; VIP‐SSM), in alleviating colitis in mice. In addition, we have also characterized a luminally expressed VPAC1 receptor in human and mouse colonic mucosa. Thus, the current studies were undertaken to determine if delivering the nanomedicine directly to the colonic lumen could have similar therapeutic benefits.

Methods

VIP‐SSM was prepared by mixing aqueous solutions of lipid and peptide in a predetermined molar ratio and was characterized for its size by dynamic light scattering. Colitis was induced in C57Bl/6 mice with 3.5% w/v dextran sulfate sodium (DSS) in drinking water for 7 days. Animals were randomly divided into 5 groups; (Control, DSS, VIP‐SSM, DSS+VIP‐SSM and DSS+ VIP). On the 5th day mice were anesthetized with ketamine/xylazine and catheterized to intra rectally instill 100 μls of, VIP‐SSM (0.25 nmol), free VIP (0.25 nmol) or SSM (100 nmol). At day 7, mice were switched to tap water and sacrificed at day 10. Body weights of mice were recorded daily. Colon appearance was observed for diarrheal phenotype, distal colonic mucosa was used for RNA and protein isolation for qPCR and western blot analysis, respectively. Frozen sections of colon were used for H & E and immunostaining.

Results

The prepared nanoparticle hydrodynamic particle sizes were approximately 15 nm. At day 10, VIP‐SSM treated mice showed significant improvement in body weight compared to DSS and free VIP treated DSS mice. The diarrheal phenotype seen with DSS indicated by loose stool in colon and reduced length was alleviated with VIP‐SSM (showing presence of solid fecal pellets) and improved colon length. In parallel, reduced expression of ion transporters such as SLC26a3 or down regulated in adenoma (DRA) seen in colitis was abrogated with VIP‐SSM intra rectal treatment but not with the free VIP peptide. Furthermore, increased pro‐inflammatory cytokine mRNA expression and damaged distal colonic histology and goblet cell loss seen in DSS was also significantly alleviated with VIP‐SSM treatment. The VPAC1 receptor expression was similar across all groups.

Conclusions

These results demonstrate that local administration of VIP nanomedicine is effective in alleviating severe inflammation and associated diarrhea in colitis. Thus, delivering VIP‐SSM nanoparticle orally with site‐specific release to the colon could be a potential option in managing UC in future.

Support or Funding Information

NIH/NIDDK, Department of Veteran Affairs

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of Sprouty2 enhances IL‐33 expression and protects against experimental colitis.
Authors: Schumacher Michael A. , Hsieh Jonathan J. , Liu Cambrian Y. , Almohazey Dana , Warburton David , Washington M. Kay , Frey Mark R.
Published: June 2021

OBJECTIVE

Sprouty2 (Spry2) is an intracellular modulator of MAPK signaling. It is present in the intestinal tract, but its function there is unknown. Aberrant MAPK signaling is associated with altered cytokine production in disorders such as inflammatory bowel disease, raising the possibility that Spry2 may affect the response to colitis. In particular, MAPK regulates IL‐33 expression in response to bacterial infection, and IL‐33 has been shown to be protective in some forms of colitis. Furthermore, both MAPK and IL‐33 regulate secretory cell differentiation. Here we tested the hypothesis that Spry2 control of MAPK signaling alters cytokine production and/or secretory cell development, thus altering the intestinal response to injury.

METHODS

Mice that lack Spry2 in the intestinal epithelium (VillinCre;Spry2flox/flox, hereafter Spry2KOIE) and littermate controls 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 and epithelial marker expression were determined by qPCR in challenged and unchallenged mice. GSK3beta phosphorylation was determined by western blot. In vitro, IEC6 and YAMC intestinal epithelial cells were treated with GSK3beta and MAPK inhibitors and cytokine expression was assessed.

RESULTS

Colonic Spry2 levels were reduced in DSS colitis by 59% (p<0.05). Spry2KOIE mice subjected to DSS had lower levels of pro‐inflammatory cytokines versus wildtype littermates, and were protected from histological damage (injury score reduced by 30%, p=0.037), weight loss, and colon shortening. Unchallenged Spry2KOIE mice displayed elevated GSK3beta phosphorylation on the inactivating Ser9 site in epithelial cells (88% increase, p<0.01). These mice also had increased IL‐33 expression (80% increase, p<0.05) and more DCLK1+ tuft cells in the colon (2.2‐fold increase, p<0.01). In vitro, inactivation of GSK3beta in both IEC6 and YAMC cells mimicked the increased IL‐33 observed in vivo; this effect was attenuated by ERK or p38 MAPK inhibitors. Expression of the epithelial‐derived cytokine CXCL2 was unaffected by GSK3beta inhibition, suggesting an IL‐33 specific response.

CONCLUSION

Colonic epithelial Spry2 is downregulated in response to injury and inflammation. This may represent an integration point of environmentally‐driven MAPK signals that leads to altered Wnt signaling, elevated IL‐33 expression, and promotion of intestinal tuft cell development as a protective or regenerative mechanism.

Support or Funding Information

NIH R01DK095004

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Early weaning in pigs induces long‐term alterations in intestinal nutrient transporter function and expression
Authors: Li Yihang , Rajput Mrigendra , Fernandez Karina Matos , Moeser Adam J.
Published: June 2021

Early life adversity (ELA) is associated with increased disease risk throughout the lifespan but the developmental mechanisms underlying the association remain elusive. Altered intestinal nutrient transporter function has been implicated as a mechanism contributing to a number of diseases associated with ELA such as Type 2 diabetes and inflammatory and functional GI disorders in humans and impaired health and growth performance in agricultural animals. Using an early weaning stress model of ELA in pigs, we investigated the link between ELA and long‐term intestinal nutrient transporter function and expression. Yorkshire breed female piglets were weaned from their sows at 15 d (Early Weaning; EW) or 28 d of age (Late Weaning; LW). Early and LW piglets were co‐housed and received the same diets. At 70 d of age, mid‐jejunum and distal ileum were harvested from EW and LW pigs and mounted on Ussing chambers to evaluate Na+‐dependent electrogenic nutrient transporter function, determined as the change in short‐circuit current (ΔIsc) in response to select luminal nutrients. Villus morphology and epithelial gene expression of nutrient transporters were assessed. Compared with age‐matched LW pigs, EW pigs exhibited reduced jejunal and ileal Na+ glucose linked transporter (SGLT1) function (by ~30%; P < 0.05) and amino transporter function for glutamate, glutamine, and alanine by (~30–35%; P < 0.05). In contrast, jejunal and ileal lysine transporter function was upregulated (by 110% and 25%, respectively; P < 0.05) in EW pigs. Gene expression for nutrient transporters (SGLT1, EAAT3, and B0AT1) were reduced (P < 0.05) while the lysine transporter CAT1 was enhanced in the EW jejunum. There were no differences observed in villus morphology between EW and LW pigs. Enteric neuronal inhibitors tetrodotoxin, atropine and propranolol, enhanced ΔIsc responses to glucose, lysine and alanine transporter function in EW but not in LW pig ileum. Together, these data show that EW in pigs induces lasting and divergent alterations in small intestinal nutrient transporter function and expression which is mediated in part by the enteric nervous system. These findings could provide new insight into the mechanisms of increased disease risk linked with ELA and thus lead to novel dietary or therapeutic strategies.

Support or Funding Information

NIH R01 HD072968USDA‐NIFA #2017‐67015‐26673

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Helicobacter pylori Alters the Expression of Circadian Clock Components PER2 and BMAL1 During Infection
Authors: Teal Emma L. , Li Jing , Chakrabarti Jayati L. , Steele Nina , Rosselot Drew , Sundaram Nambirajan , Hawkins Jennifer , Hong Christian , Helmrath Michael A. , Diwan Tayyab L. , Zavros Yana L.
Published: June 2021

Background

Individuals with persistent Helicobacter pylori (H. pylori) infection are known to have an increased risk of gastric adenocarcinoma development. The circadian clock has been shown to be intimately involved in various gastrointestinal functions and clock components BMAL1 and PER2 have been found to be disrupted in colorectal cancer tissues. Aged individuals have a dampened expression of such components which may further increase their susceptibility to gastric cancer development.

Hypothesis

Persistent infection of H. pylori induces an unregulated proliferation of gastric epithelial cells via activation of BMAL1 leading to a predisposition to gastric cancer development.

Methods

Gastric organoids (hFGOs) were generated from either primary gastric tissue derived from aged (> 55 years of age) and young (14–20 years) individuals. Gastric organoids were then infected with H. pylori. BMAL1 and PER2 expression was then quantified over a period of 24 hours. Proliferation and expression of gastric cancer markers were quantified by qRT‐PCR.

Results

H. pylori infection correlated with a significant upregulation of cytoplasmic BMAL1 expression within the gastric fundus of infected individuals. Robust circadian rhythmicity of clock genes Bmal1 and Per2 were observed in human hFGOs. Phosphorylation (inactivation) of GSK‐3b showed circadian changes in hFGOs over 52 hours. H. pylori infection of hFGOs resulted in sustained GSK‐3b phosphorylation, increased BMAL1 expression and epithelial cell hyperproliferation when compared to uninfected controls. Organoids derived from elderly patients exhibited a dampened rhythmicity of of clock genes Bmal1 and Per2. Dampened rhythmicity correlated with increased gastric cancer biomarkers of intestinal metaplasia including Cdx1, Cdx2 and MUC2.

Conclusions

Chronic H. pylori infection induces a hyperproliferative response in gastric epithelial cells by inactivating GSK‐3β leading to the activation of BMAL1:CLOCK. This may be a plausible mechanism that predisposes individuals with persistent H. pylori infection to gastric cancer development and also heightens this risk in aged individuals.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nucleophosmin Is Essential for Stimulation of Rac1 Nuclear Accumulation by RNA‐binding Protein HuR in the Intestinal Epithelium
Authors: Liu Lan , Chung Hee Kyoung , Kalakonda Sudhakar , Yu Tingxi , Xiao Lan , Rao Jaladanki N. , Wang Jian‐Ying
Published: June 2021

The mammalian intestinal epithelium is a rapidly self‐renewing tissue in the body and its homeostasis is tightly regulated by numerous factors including the RNA‐binding protein HuR. HuR is highly expressed in the intestinal mucosa and acts as a key regulator of the epithelial homeostasis by regulating the stability and translation of target mRNAs. We have recently demonstrated that targeted deletion of HuR in intestinal epithelial cells (IECs) inhibits growth of the small intestinal mucosa and delays healing after injury in mice partially by altering subcellular distribution of a small GTP‐binding protein Rac1. HuR knockout in IECs increases the levels of cytoplasmic Rac1 in vivo as well as in vitro, but the exact mechanism underlying this process remains unknown. Nucleophosmin (NPM) is a phosphoprotein that binds to proteins containing nuclear localization signals for their import and functions as a molecular chaperone. In this study, we investigated the role of NPM in HuR‐dependent nuclear accumulation of Rac1 in the intestinal epithelium.

Methods

Studies in vivo were conducted in intestinal epithelial tissue‐specific HuR knockout (IE‐HuR−/−) mice, and experiments in vitro were carried out in cultured IEC‐6 cells that were derived from rat small intestinal crypts. Functions of NPM and HuR in vitro were investigated via their gene silencing by using siRNAs specifically targeting NPM (siNPM) or HuR (siHuR) and their overexpression. Levels of NPM, Rac1, and HuR proteins were measured by Western blotting analysis, and the cellular distributions of Rac1 and NPM were examined by immunofluorescent staining.

Results

The levels of NPM protein in the intestinal mucosa decreased significantly in IE‐HuR−/− mice compared with control littermates. Decreased NPM was associated with elevated cytoplasmic Rac1 in the HuR‐deficient epithelium, along with a decrease in nuclear Rac1 abundance. In cultured IEC‐6 cells, HuR silencing by transient transfection with siHuR also decreased cellular NPM, but it increased the levels of cytoplasmic Rac1. NPM was found to physically interact with Rac1 and formed NPM/Rac1 complex in normal IEC‐6 cells. NPM silencing by transfection with siNPM increased cytoplasmic Rac1 levels and decreased nuclear Rac1 abundance. Furthermore, ectopically expressed NPM abolished the increased levels of cytoplasmic Rac1 in HuR‐silenced cells and restored the nuclear Rac1 levels to near normal.

Conclusions

These results indicate that 1) HuR regulates NPM expression in the intestinal epithelium; and 2) HuR regulates Rac1 subcellular trafficking by altering NPM.

Support or Funding Information

National Institutes of Health (JYW) and US Department of Veterans Affairs (JYW and JNR)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An iron supplement containing L. plantarum increases ferric iron and up‐regulates the ferric reductase Dcytb in human Caco‐2/HT29 MTX co‐cultures
Authors: Scheers Nathalie , Önning Gunilla , Björklund Malin , Sandberg Ann‐Sofie
Published: June 2021

Iron absorption from a bread meal, added an encapsulated iron‐containing supplement, may be increased if the lactic acid bacteria L. plantarum (DSM 9843) is present. The aim of this study was to investigate the mechanism by which L. plantarum increases iron absorption. We have previously shown that lactic fermentation of mixed vegetables increased iron absorption in humans and voltammetric studies revealed that the level of ferric iron [Fe3+ (H2O)6] was increased in the fermented vegetables, compared to unfermented control. In this study, we detected a similar increase in ferric iron in the in vitro digested bread meal containing the supplement with L. plantarum. In a co‐culture of intestinal Caco‐2 cells and mucus‐producing goblet cells (HT29 MTX E12), we observed that the ferric reductase Dcytb increased in the presence of L. plantarum‐containing iron supplements while the production of mucin (muc5a1c) decreased independent of L. plantarum. This suggests that the L. plantarum‐effect on iron absorption is mediated through enhancing the Fe3+/Dcytb system.

Support or Funding Information

The work was financially supported by Probi AB

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unlocking gut health with the prebiotic galactooligosaccharide (GOS) and probiotic L. reuteri to keep pathogenic S. typhimurium at bay
Authors: Forshee Micah D. , Sharda Daniel R.
Published: June 2021

The microbiome is a dynamic community that can positively and negatively influence host health. Lactobacillus reuteri is a probiotic that has received much attention for its ability to inhibit pathogens such as Salmonella typhimurium, Escherichia coli, and Clostridium difficile. It does so by its unique ability to metabolize glycerol into the antimicrobial compound 3‐HPA, which is commonly referred to as reuterin. The ability to secrete reuterin is dependent not only on glycerol availability but also the concentration of glucose. In fact, there appears to be a “goldilocks” ratio between glucose and glycerol as either too much or too little glucose significantly diminishes reuterin production. Since L. reuteri primarily resides in distal regions of the intestine and colon where most of the glucose has already been absorbed, it seems unlikely that reuterin production would be promoted at the physiological level via this mechanism.

Prebiotics are carbohydrates that are indigestible by the host and remain for enzymatic digestion by intestinal probiotics. Inulin and galactooligosaccharides (GOS) are two widely studied prebiotics that are known for their ability to promote the growth of a wide range of lactobacilli, and have been shown to promote L. reuteri growth to varying degrees. Here, we asked if prebiotics such as inulin and GOS promote the production of reuterin in the absence of glucose. L. reuteri were cultured in TSB with or without glycerol in the presence of either glucose, inulin, or GOS, and assessed for their ability to produce reuterin. While inulin did not enhance the production of reuterin, GOS induced reuterin production, although 45% less than that of glucose. Moreover, unlike the dose‐dependence observed with glucose, incubation with GOS induced similar reuterin production regardless of concentration. This suggests that an enzymatic equilibrium may exist where glucose/galactose is cleaved from GOS only as needed by L. reuteri. Finally, to confirm the biopotency of reuterin production, we cultured S. typhimurium with supernatants from L. reuteri that were grown with various carbohydrates. Supernatant dilutions as low as 1:15 were able to significantly retard growth of S. typhimurium with ratios of 1:1 completely inhibiting growth. Together, these results suggest that prebiotics such as GOS may be able to elicit physiologically relevant production of reuterin, which may shape the flora of the microbiome and reduce incidence and severity of pathological infections. Further, as GOS are particularly abundant in breast milk, it suggests a possible link for early immunoprotection from intestinal pathogens while the infant is still immunologically naïve.

Support or Funding Information

Olivet Nazarene University Honors Program and Department of Biological Sciences

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepcidin interaction with ferroportin in the Xenopus oocyte expression system
Authors: Ruwe T Alex , Vieth Kyle R. , Aschemeyer Sharraya , Qiao Bo , Ganz Tomas , Nemeth Elizabeta , Mackenzie Bryan
Published: June 2021

The cellular iron‐export protein ferroportin (Fpn), expressed in enterocytes and macrophages, is essential for iron homeostasis. Fpn operates under the control of the iron‐regulatory hormone hepcidin. The canonical mechanism by which hepcidin suppresses Fpn activity comprises hepcidin binding by Fpn and the subsequent internalization and ubiquitin‐dependent degradation of the transport protein [Drakesmith H et al (2015) Cell Metab 22, 777–787]. Here we explored hepcidin inhibition of 55Fe efflux from RNA‐injected Xenopus oocytes expressing human Fpn. Hepcidin treatment induced a rapid inhibition (t½ = 8.2 ± [SEM] 0.5 min; r2 > 0.99, P < 0.001) of Fpn‐mediated 55Fe efflux that was not dependent on endocytosis, a conclusion based on the following four observations: (i) Inhibition of Fpn by hepcidin was only modestly temperature‐dependent (Q10 = 2.5 ± [SEM] 1.4 over the range 17–27 °C) whereas endocytosis is expected to be substantially more temperature‐dependent, Q10 in the order of 5–17 [Weigel PH and Oka JA (1981) J Biol Chem 256, 2615–2617]. (ii) Inhibition of Fpn by hepcidin proceeded even in the presence of blockers of clathrin‐mediated (Dynasore) and clathrin‐independent (filipin, genistein) endocytosis. (iii) Hepcidin treatment inhibited 55Fe efflux in oocytes expressing the K8R mutant, in which 8 Lys residues required for ubiquination and endocytosis of Fpn were mutated to Arg, to the same degree as in wildtype Fpn. (iv) Confocal LSM imaging of wildtype Fpn‐GFP and K8R‐Fpn‐GFP expression in oocytes revealed no detectable endocytosis of either protein after 30‐min treatment with hepcidin. Our data reveal that hepcidin can directly block Fpn activity independent of Fpn internalization. We speculate that the Xenopus oocyte lacks the specific signal by which hepcidin triggers Fpn internalization. The Xenopus oocyte is therefore an ideal model system in which to study the mechanism by which hepcidin directly blocks Fpn activity.

Support or Funding Information

NIH–NIDDK grant R01 DK107309

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.