bluuebunny/crossref_metadata_2025 huggingface dataset:
Emerging regulatory roles of Wnt1 in host‐pathogen interactions in inflammation and colon cancer
Authors: Liu Xingyin , Chen Hongshan , Zhang Yongguo , Lu Rong , Liu Qisha , Sun Jun
Published: November 2023

Salmonella infection is a major public health concern and colonization in humans can be chronic as carrier state. The Wnt signaling plays an essential role in gastrointestinal epithelial proliferation. Our previous studies have demonstrated that both Wnt2 and Wnt1 protein secretion in epithelial cells were elevated after bacterial infection (Liu et al., 2011 and 2012). Bacteria modulate the host by injecting bacterial effectors to the host cells. AvrA is a Salmonella protein that is transported into intestinal epithelial cells by a type 3 secretion system. Our own studies, as well as those of other investigators, have demonstrated that AvrA influences eukaryotic cell pathways by altering ubiquitination and acetylation of target proteins to modulate inflammation, epithelial apoptosis and proliferation. Deubiquitination properties of AvrA leads to upregulation of the Wnt/beta‐catenin signaling pathway, which is critical in initiation of colon cancer. In the current study, we report that Wnt1 plays a role in infection and colon cancer. Different from the bacteria‐induced increase of Wnt2 and Wnt11, pathogenic Salmonella colonization significantly reduces the level of Wnt1 in intestinal epithelial cells in vivo and in vitro. We observed that Wnt1 expression level were down‐regulated by Salmonella infection, but stabilized under AvrA‐deficient Salmonella iin the intestine of Salmonella‐colitis mice. In a chronic Salmonella infected cancer model, the wnt1 protein level is decreased in AvrA+ infected group, consistent with our observation in the Salmonella‐colitis animal model. Furthermore, we assess the functional role of down‐regulation of Wnt1 in the inflammatory response and colon cancer progression. We found that Wnt1 is involved in the regulation of inflammatory pathway in Salmonella‐epithelia cell interaction. Moreover, down‐regulation Wnt1 promotes cancer cell invasion and migration. Interestingly, we found the Wnt1 is down‐regulated in colon cancer patients, which is correlated with colon cancer progression. The results suggest Salmonella effector AvrA and Wnt1 may functionally interrelate in the development of colon cancer. Growing interest and accumulating data on human microbiota implicate that host‐microbe interplay has an important role in the development of colon cancer. A recent study reported that antibody against Salmonella flagellin was higher in colorectal cancer and pre‐cancer cases than controls in two distinct populations in US and the Netherlands and that dietary intake is the one of the mediating factors, suggesting a potential link of Salmonella to colorectal cancer. Our study provides novel insights into mechanisms by which the bacteria effector regulates Wnt1 expression and potential contributes to infectious‐associated colon cancer.

Support or Funding Information

Starting package of Nanjing Medical University to Xingyin Liu and NIH R01 to Jun Sun
How EGF and Insulin Activate Their Receptors
Authors: Leahy Dan
Published: November 2023

Receptors for Epidermal Growth Factor (EGF) and Insulin are Receptor Tyrosine Kinases (RTKs), which consist of an extracellular ligand‐binding region, a single transmembrane (TM) region, and a cytoplasmic kinase. RTKs play essential roles in the development and maintenance of normal organisms, but abnormal RTK activity is frequently associated with cancer and many successful anti‐cancer agents target RTKs. Ligand‐dependent dimerization is an important feature of RTK activation, and structural and biophysical studies of EGFR and other RTKs provide elegant visualization of many ligand‐dependent RTK dimers. The Insulin Receptor and its homologs are constitutive disulfide‐linked dimers, however, that appear to require a ligand‐dependent conformational change to activate. Recent results show that the extracellular regions of both the EGF and Insulin receptors prevent subunit TM regions from interacting in the absence of ligand but that the TMs associate in the presence of ligand. These results update the ligand‐dependent dimerization paradigm for RTK activation by demonstrating (i) the importance of autoinhibition RTK activity in the absence of ligand and (ii) the role of TM separation in governing RTK activity.

Support or Funding Information

NIH GM099321 and NIH GM099092
FFA3 activation stimulates duodenal bicarbonate secretion and prevents NSAID‐induced enteropathy via the GLP‐2 pathway in rats
Authors: Said Hyder , Akiba Yasutada , Kaji Izumi , Kaunitz Jonathan D
Published: November 2023

The duodenal mucosa expresses the short‐chain fatty acid (SCFA) receptors FFA2 and FFA3, implicating SCFAs generated by foregut luminal flora in the pathological condition termed small intestinal bacterial overgrowth (SIBO) in the genesis of functional dyspepsia symptoms. FFA2 activation stimulates duodenal bicarbonate secretion (DBS) via 5‐HT4 receptor activation, but, when cyclooxygenase (COX) is inhibited, injures the mucosa via 5‐HT3 receptor activation. To further explore the effects of SCFAs on the foregut mucosa, we investigated the effects of FFA3 activation on DBS and nonsteroidal anti‐inflammatory drug (NSAID)‐induced enteropathy.

We measured DBS in vivo in a duodenal loop perfused with the selective FFA3 agonists N‐[2‐methylphenyl]‐[4‐furan‐3‐yl]‐2‐methyl‐5‐oxo‐1,4,5,6,7,8‐hexahydro‐quinoline‐3‐carboxamide (MQC) or AR420626 (AR) while measuring released GLP‐2 into the portal vein (PV). Intestinal injury was induced by indomethacin (IND) with or without MQC (1 – 10 mg/kg) or AR (0.01 – 0.1 mg/kg) treatment.

Luminal perfusion with MQC or AR (0.1 – 10 μM) dose‐dependently increased DBS accompanied by GLP‐2 release into PV. The effect of MQC was inhibited by co‐perfusion of an FFA3 antagonist N‐(2‐methylphenyl)‐4‐[5‐(2‐trifluoromethoxy‐phenyl)‐furan‐2‐yl]‐2‐methyl‐5‐oxo‐1,4,5,6,7,8‐hexahydro‐quinoline‐3‐carboxamide (CF3‐MQC, 30 μM). AR‐induced increased DBS was reduced by iv injection of the GLP‐2 receptor antagonist GLP‐2(3–33) (3 nmol/kg). IND‐induced small intestinal ulcer formation was dose‐dependently reduced by intragastric administration of MQC or AR. The GLP‐2 receptor antagonist GLP‐2(3–33) (1 mg/kg, ip) or CF3‐MQC (1 mg/kg, ig) reversed AR‐induced protection of intestinal mucosa from NSAID‐induced enteropathy.

These results suggest that in contrast to FFA2 activation, which injures the mucosa via the 5‐HT pathway, FFA3 activation enhances mucosal defenses and prevents NSAID‐induced enteropathy via the GLP‐2 pathway. Dual effects of SCFA receptor activation may help explain the diversity of functional symptoms related to foregut bacterial overgrowth (SIBO).

Support or Funding Information

VA Merit Review, NIH R01 DK54221
Butyrate Enhances MCT1 Association with CD147 via GPR109A Activation‐Dependent Mechanisms
Authors: Chatterjee Ishita , Kumar Anoop , Anbazhagan Arivarasu N , Alrefai Waddah A , Borthakur Alip , Dudeja Pradeep K
Published: November 2023

Butyrate, a key short‐chain fatty acid (SCFA) produced by colonic luminal bacteria, serves as the primary fuel for the colonocytes and exerts multiple beneficial effects on gut epithelial health. Efficient intracellular absorption of butyrate is important for these effects. Monocarboxylate transporter 1 (MCT1) plays a major role in intestinal butyrate absorption. Localization of MCT1 on the luminal membrane of colonocytes is critical for its optimal absorptive function. We have earlier shown substrate (butyrate)‐induced enhancement of butyrate absorption in human intestinal epithelial C2BBE cells. This enhancement was secondary to increased abundance of MCT1 protein on the apical plasma membrane following butyrate‐induced activation of GPR109A, a Gαi‐coupled receptor (GPCR), involving nutrient sensing mechanisms. Since membrane targeting of MCT1 has been shown to be governed by its interaction with CD147, an ancillary glycoprotein, current studies examined whether butyrate increases MCT1/CD147 interactions via GPR109A‐dependent mechanisms.

Methods

MCT1 function was measured as p‐chloromercuri‐benzene sulfonate [pCMBS (MCT1 inhibitor)]‐sensitive 14C‐butyrate uptake, MCT1/CD147 interactions by co‐immunoprecipitation, MCT1 phosphorylation by immunoblotting of immunoprecipitated MCT1, GPR109A silencing by siRNA.

Results

Butyrate (10 mM, 1 hr) significantly increased MCT1‐CD147 interaction in human intestinal C2BBE cells. GPR109A silencing or inhibition by pertussis toxin (PTX) abrogated these effects. GPR109A activation is known to decrease intracellular cyclic AMP that could alter MCT1 phosphorylation, and in turn could modulate its interaction with CD147. Therefore, we examined MCT1 phosphorylation in response to butyrate and role of GPR109A. Butyrate (10 mM, 1 hr) increased serine‐phosphorylated MCT1 that was attenuated in response to PTX inhibition of GPR109A. Further, okadeic acid, a protein phosphatase inhibitor, augmented butyrate enhancement of MCT1 function, presumably via maintaining MCT1 phosphorylation status critical for its membrane targeting.

Conclusion

Our results highlight distinct role of GPR109A as a SCFA sensor in mediating substrate‐induced enrichment of MCT1 apical membrane abundance via modulating MCT1 phosphorylation and increasing MCT1 interaction with CD147.

Support or Funding Information

Supported by NIH(NIDDK) and Department of Veteran Affairs
Modulation of CD4+ T Cell Differentiation by the Probiotic Lactobacillus reuteri
Authors: Collins Fraser , Britton Robert A , McCabe Laura R , Parameswaran Narayanan
Published: November 2023

Probiotic bacteria are micro‐organisms that when taken in adequate amounts have beneficial effects on the host. Previous studies have demonstrated that probiotic bacteria and specifically Lactobacillus reuteri have important immunoregulatory effects; regulating differentiation of T cells through interactions with dendritic cells or in cultures of peripheral blood mononuclear cells (PBMC). However, it is not known whether or how L. reuteri or its secreted factors can directly influence T cell differentiation. To test this, we isolated Naïve CD4+CD62L+ T cells from the spleen of male C57BL/6 mice by magnetic isolation and measured their cytokine response to treatment with live, dead or whole and fractionated conditioned medium (components separated based on size) from L. reuteri (ATCC 6475). For live bacterial studies, cells were treated with multiplicity of infection (MOI) of 1, 10 or 100. For conditioned media studies cells were treated with whole, >100kD, 50–100kD, 30–50kD, 10–30kD, 3–10kD and <3kD fractions. After 4 days, cells were stained and analyzed by flow cytometry for expression of the cytokines IL‐10, IL‐17A, IFNγ and TGFβ (Latency Associated Peptide). Live L. reuteri significantly increased expression of IL‐10, IL‐17A and IFNγ but not TGFb in a dose dependent manner. Heat killed L. reuteri did not affect expression of these cytokines except for IL‐10 and IL‐17A at the highest dose. Compared to these studies, L. reuteri conditioned media significantly increased expression of IL‐10, IL‐17A, IFNγ and TGFb. Examination of fractionated conditioned media (by size, 3Kd to >100Kd) indicated that all fractions elicited increased cytokine expression compared to control with the greatest effect observed in the >100kD fraction. Taken together, our data demonstrate that live probiotic L. reuteri and its secreted factors are able to directly influence naïve T cell differentiation. Dead bacteria only affected cytokine expression at high doses suggesting an important role for the secretory products of live L. reuteri in the regulation of T‐cell differentiation and cytokine secretion.

Support or Funding Information

This project was supported by funding from NIH NCCIH 1RO1AT007695‐01
Protective Actions of 5‐HT4 Receptors in the Colonic Epithelium
Authors: Spohn Stephanie N. , Scott Rachel , O'Neill Conor , MacNaughton Cathy , Dicay Michael , Bianco Francesca , Bonora Elena , Lavoie Brigitte , Wilcox Rebecca L , De Giorgio Roberto , MacNaughton Wallace , Sharkey Keith A , Mawe Gary M
Published: November 2023

5‐HT4 receptors are expressed in colonic epithelium, and activation with 5‐HT4 receptor agonists causes mucus secretion from goblet cells, chloride secretion from enterocytes, and 5‐HT release from enterochromaffin cells. We tested whether this receptor could serve a protective role in models of colitis and under basal conditions. CD‐1 mice were given DSS (4% w/v for 5 days in drinking water) or TNBS (7.5 mg/mL) on day 0. 5‐HT4 receptor agonist (Tegaserod, 1mg/kg), vehicle, or antagonist (GR 113808) plus agonist was delivered by enema daily day 1–7 (prevention) or day 6–15 (recovery) to determine the effects of epithelial 5‐HT4 receptor activation in colitis. To test the actions of 5‐HT4 receptors under basal conditions, GR 113808 was administered to normal mice by enema for 10 days. Colitis was evaluated using disease activity index (DAI) and histological damage scores (HDS). Possible protective actions such as cell proliferation in colonic tissue and migration and resistance to oxidative stress in CaCo‐2 cell culture were also explored. To investigate function, we evaluated propulsive motility in the guinea pig distal colon. Intraluminal tegaserod treatment in both DSS and TNBS‐inflamed animals significantly attenuated the development of, and accelerated the recovery from, colitis in an antagonist‐sensitive manner. However, administration of Tegaserod (1 mg/kg) by intraperitoneal injection had no effect. TNBS‐induced dysmotility in guinea pigs was reversed by 5‐HT4 receptor agonist treatment, and this action was blocked by the atagonist. Increased cell proliferation was detected in the form of increased Ki‐67 immunoreactivity in crypts of inflamed animals treated with Tegaserod. We also observed areas of accelerated healing of scratches and increased resistance to H2O2 induced oxidative stress in CaCo‐2 cells. Interestingly, antagonist treatment alone in normal mice resulted in significant increases in DAI, HDS, and bacterial translocation in mice, as well as disrupted motility patterns in guinea pig colon. In summary, activation of 5‐HT4 receptors on the colonic epithelium reduces the development of, and accelerates recovery from, colitis. It is likely acting through increased wound healing by increased cell migration and cell proliferation, as well as resistance to apoptosis. Additionally, 5‐HT4 receptor activation protects colonic motility. Taken together, these data suggest that epithelial 5‐HT4 receptors have a protective effect in the normal and inflamed colon.

Support or Funding Information

Funded by DK62267
Elevated D‐2‐Hydroxyglutarate during colitis drives progression to colorectal cancer
Authors: Theiss Arianne , Han Jie , Genta Robert , Sweetman Lawrence
Published: November 2023

Although the association between inflammatory bowel diseases (IBD) and an increased risk of developing colorectal cancer is well‐established, the pathogenesis of colitis‐associated cancer is poorly understood. Our previous study of quantitative metabolic profiling of urine serially collected from mice during azoxymethane (AOM)‐dextran sodium sulfate (DSS)‐induced colitis‐associated cancer identified that urine D‐2‐hydroxyglutarate (D2HG) levels during colitis directly correlated with severity of polyp formation. Levels of D2HG are normally low and accumulation of D2HG has been linked to oncogenesis in glioma and acute myeloid leukemia. Here, we determined whether elevated D2HG during colitis drives progression to colorectal cancer. Wild‐type C57BL/6 male mice were i.p. injected with 7.6 mg/kg AOM followed by 2 cycles of 3% DSS (1 week DSS and 3 weeks recovery). During DSS, mice were i.p. injected with 25 mg/kg D2HG or vehicle once daily. Body weight, severity of diarrhea, and presence of blood in the stool were monitored daily during DSS administration and twice per week during recovery phases. Excised colons were observed for polyp number/size and embedded for dysplasia scoring. Mice injected with D2HG exhibited similar body weight loss, severity of diarrhea, and presence of blood in the stool during DSS as vehicle‐injected mice. However, mice injected with D2HG during colitis exhibited delayed recovery of body weight and significantly more and larger (>6mm in diameter) colonic polyps with the majority of polyps being high‐grade dysplasia or intramucosal adenocarcinoma. These results suggest that elevated D2HG during colitis enhances progression to colon cancer.

Support or Funding Information

Cancer Prevention Research Institute of Texas (CPRIT) RP150638
Estrogen receptor beta (ERβ) activation plays a therapeutic role in murine models of inflammatory bowel disease (IBD) via inhibiting P2X3 and P2X7 receptors
Authors: Ma Bei , Jiang Qian , Li Wenxin , Li Zhaoshen
Published: November 2023

IBD is an inflammatory condition of the bowel system, represented by cumulating and recurring abdominal pain, diarrhea and bloody stools. ERβ has been shown to play a therapeutic role in IBD, while P2X3 receptor (P2X3R) and P2X7 receptor (P2X7R) have been known to promote the initiation and exacerbation of IBD. However, the mechanism how ERβ exerts therapeutic effects and its relationship with P2X3R and P2X7R in IBD are still unknown. In our study, animal behavior tests, histological staining, western blotting, ELISA, and visceromotor reflex recording were used to determine whether the therapeutic effect of ERβ in IBD was through down‐regulation of the expression and function of P2X3R and P2X7R. Based on the successful model of IBD characterized by more weight loss, higher DAI scores and significant inflammatory responses, the expressions of P2X3, P2X7 and ERβ in different tissues including rectocolon, dorsal root ganglia (DRG) and dorsal horn of spinal cord (SCDH) were examined. We found that in colitis group, the expressions of ERβ in rectocolon, DRG and SCDH were all significantly reduced, while that of P2X3R were significantly increased in rectocolon and DRG, but not SCDH, and P2X7R was significantly increased only in rectocolon, but not DRG and SCDH, compared with control group. After administration of A317491 or BBG, antagonists of P2X3R and P2X7R, the weight loss, DAI scores and inflammatory responses of colitis rats in antagonists‐treated groups were all significantly reversed compared with those of control group. Likewise, when agonists of ERβ, DPN and ERB‐041 were given, there was also such a therapeutic role, and at the same time, so did ERβ transfection with recombinant lentivirus. As to the expressions of P2X3R and P2X7R, DPN could significantly decreased P2X3R in all the three fields, namely rectocolon, DRG and SCDH, while P2X7R was decreased only in rectocolon. However, ERB‐041 could decrease P2X3R and P2X7R both in rectocolon only, but not DRG. Furthermore, DPN inhibited visceromotor reflex by colonic extension and secretion of proinflammatory factors, IL‐1b, TNF‐a and IL‐6, in colitis rats. In brief, these results suggest that ERβ may play an important therapeutic role in IBD through down‐regulation of P2X3R and P2X7R. Therefore, it will provide certain evidences and new therapeutic strategies for clinical treatment towards IBD.

Support or Funding Information

This study is supported by National Natural Science Foundation of China (No, 31471103).
Transfection of Rat Intestinal Epithelial Cells with a Stable Mitogen‐Activated Protein Kinase Phosphatase (MKP)‐1 Mutant
Authors: Talavera Maria Margarita , Crowell Sara , Nelin Leif Daniel , Liu Yusen
Published: November 2023

MAPK phosphatases (MKPs) are critical modulators of the innate immune response in both immune cells and intestinal epithelial cells (IECs). MKP's regulate the innate immune response via MAPK inactivation by dephosphorylation of specific Tyr‐Xaa‐Thr motif, where Xaa represents amino acid residues specific to a MAPK subfamilies. We have shown that peak MKP‐1 expression in LPS stimulated rIEC‐6 cells was 60 minutes, limiting the regulation of the inflammatory response to a short interval. The objective of this study was to create a unique transfected rIEC‐6 cell line that expresses a more stable MKP‐1 mutant protein. rIEC‐6 cells were transfected with one of three plasmids using either Fugene or Viofectin transfection reagent and a fluorescent tag (EGFP‐Cl). The three plasmids were designated either empty vector control or WT‐Flag‐tagged MKP‐1 plasmid or Flag‐tagged MKP‐1 S359/364D plasmid. The Flag MKP‐1S/D mutant carries a specific serine to aspartate mutation in the C‐terminus created using site‐directed mutagenesis. Successful clone selection was achieved with increasing concentration of G418 over a period of 14 days. Clones were grown in DMEM growth media for 24 hours. Cell lysate harvested for protein and vector expression quantified using Western blot analysis. Results demonstrated an 18× fold increase in Flag MKP‐1S/D expression compared to WT‐Flag MKP‐1 protein in rIEC‐6 cells. In conclusion, expression of a more stable MKP‐1 protein in intestinal epithelial cells will enable further study of the critical role of MKP‐1 in the intestinal inflammatory response.

Support or Funding Information

Liu (PI) R21 AI113930 NIH/NIAID
Zinc Deficiency Contributes to Alcohol Consumption‐Induced Defect of Mitochondrial Electron Transport Chain and Overproduction of Reactive Oxygen Species in the Live of Rats
Authors: Sun Qian , Zhong Wei , Zhang Wenliang , Sun Xinguo , Zhou Zhanxiang
Published: November 2023

Clinical studies demonstrated that patients with alcoholic liver disease (ALD) have decreased hepatic zinc levels; however the effect of zinc deficiency on the function of hepatic mitochondria has not been well studied. The present study was undertaken to determine if zinc deficiency causes mitochondrial electron transport chain (ETC) defect and if defected ETC function could increase hepatic oxidative stress. Adult male Wistar rats were pair‐fed with the Lieber‐DeCarli control or ethanol liquid diet for 5 months. Chronic alcohol exposure significantly increased hepatic levels of triacylglycerol and free fatty acid and induced apoptotic cell death. Zinc levels in both the whole liver and the purified hepatic mitochondria were significantly reduced by alcohol exposure in association with perturbed expression of zinc transporters. Chronic alcohol exposure also increased the levels of 4‐hydroxynonenal (4HNE) in the whole liver as well as the purified mitochondria. The expression of hepatic mitochondrial respiratory complex I, III, IV, and V were repressed by chronic alcohol exposure, which was in consistence with a significant decrease of the hepatic ATP production. Examination of the mitochondrial biogenesis markers demonstrated that chronic alcohol exposure significantly decreased AMPK, PGC1α, NRF1 and TFAM levels in association with downregulation of mitochondrial DNA expression. In order to define the link between zinc deficiency and the defect of mitochondrial respiratory complexes, HepG2 cells were treated with zinc chelator, TPEN, for 6h. Experimental zinc deficiency significantly decreased the protein levels of mitochondrial respiratory complex I, III, and IV. In addition, p‐AMPK, PGC1α, NRF1 and TFAM levels as well as mitochondrial DNA expression were significantly decreased by TPEN treatment. Consequently, mitochondrial respiratory complex I, III, or IV was silenced by corresponding shRNA, respectively. HepG2 cells with decreased expression of complex I, III, or IV by gene manipulation exhibited a decrease in mitochondrial membrane potential and an increase in free radical production compared to the cells transfected with scrambled shRNA. These results suggest that hepatic zinc deficiency critically mediates alcohol‐diminished mitochondrial biogenesis and mitochondrial DNA levels which, at least partially, accounts for alcohol‐induced defect of mitochondrial ETC and overproduction of reactive oxygen species.

Support or Funding Information

R01AA020212 and 2R01AA018844
Liver‐Directed Knockout and Transgene Delivery of Arginase‐1 in Mice
Authors: Funk Colin D , Ballantyne Laurel , Schulze Andreas
Published: November 2023

Arginase‐1 (Arg1) catalyzes the distal step in the liver‐based urea cycle that converts arginine to urea and ornithine. Genetic deficiency of this enzyme in humans, unlike proximal urea cycle disorders that lead to hyperammonemia and neonatal crisis, leads to neurological deficits (e.g. spastic diplegia) that manifest around 2–4 years of age and progress throughout childhood. We previously generated a tamoxifen‐inducible Arg1 deficient mouse model (Arg1‐Cre) that leads to a phenotype much more severe than in humans with lethality occurring ≈2 weeks after loss of enzyme function. The purpose of this study was to evaluate if liver‐selective Arg1 loss is sufficient to recapitulate the phenotype observed in global Arg1 knockout mice, as well as to gauge the effectiveness of a gene therapy protocol to rescue the phenotype. Liver‐selective Arg1 deletion was induced by using an adeno‐associated viral (AAV)‐thyroxine binding globulin (TBG) promoter‐Cre recombinase vector (1.5×1011 genome copies (g.c.), intraperitoneally (i.p.) injected) administered to mice (n=6) with loxP sites flanking exons 7 and 8 of the Arg1 gene (Arg1 floxed mice; Arg1fl/fl). An AAV vector expressing an Arg1‐enhanced green fluorescent protein (Arg1‐eGFP) transgene from a strong promoter (hybrid cytomegalovirus enhancer/chicken β‐actin) was used in a gene therapy approach to “rescue” tamoxifen‐treated Arg1‐Cre mice. The results indicate that liver‐selective loss of Arg1 (>95 % deficient) leads to a phenotype resembling the whole body knockout of Arg1 with lethality around 2 weeks after Cre‐induced gene disruption in all mice. Delivery of Arg1‐eGFP AAV (n=10 male; 1.5×1011g.c. i.p. administered 2 weeks prior to tamoxifen treatment) rescues ≈ half of Arg1 global knockout mice (survival >4 months) but a significant proportion still succumb to the enzyme deficiency even though liver expression and enzyme activity of the fusion protein reach levels approaching that found in wild‐type animals. This raises a conundrum relating to liver‐specific expression of Arg1. On the one hand, loss of expression in this organ appears to be both necessary and sufficient to explain the lethal phenotype of the genetic disorder in mice. On the other hand, the gene therapy rescue studies suggest that loss of extra‐hepatic Arg1 expression factors into disease etiology. Further studies will be necessary to illuminate the detailed mechanisms for pathogenesis of Arg1‐deficiency.

Support or Funding Information

Canada Research Chairs Program
Amoxicillin decreases intestinal microbial diversity and increases stress‐associated behaviors in zebrafish
Authors: Deprey Kirsten L. , Uno Jennifer K
Published: November 2023

Anxiety disorders affect approximately 18% of the population in the U.S. Research indicates that antibiotic exposure may contribute to the onset of anxiety. The gut‐brain axis is a bidirectional communication pathway between the gut and the brain. Evidence supports the role of the microbiota in this relationship. This study employs zebrafish to observe the impact of amoxicillin‐induced changes to the microbiota on stress‐related behaviors. We hypothesize that a decrease in intestinal microbial diversity will increase anxious behavior. Adult zebrafish were administered amoxicillin or vehicle and challenged via death‐induced odors and a net‐stress. Top‐to‐bottom and side‐to‐side movement over time was recorded. Intestinal bacterial‐DNA was extracted and bacterial phyla was examined and quantified by qPCR. Results confirmed amoxicillin significantly decreased the quantity and diversity of intestinal bacterial communities, indicated by a 20% change in universal gut bacteria in the treated fish (n=14, p value <0.05), and a slight increase in Lactobacillus. Antibiotic treatment also correlated to behavioral changes in the fish, illustrated by a significant decrease in top‐to‐bottom and side‐to‐side movement in fish (35%, 54% decrease in top‐to‐bottom and side‐to‐side, respectively, n=12 p value <0.01, p value <0.001). Behavioral studies after reestablishment of the gut microbiota also demonstrated a significant decrease in top‐to‐bottom swimming (n = 12, p value <0.01). Collectively, these results signify that decreased intestinal microbial diversity may elicit anxiety in zebrafish. These data warrant further investigation into the mechanism behind which microbiota‐induced behavioral changes are associated with antibiotics. This study was supported by the APS and Elon University.

Support or Funding Information

This study was supported by the APS and Elon University.
MicroRNAs Target Decidual Angiogenic Factors During Early Pregnancy in BPH/5, a Spontaneous Mouse Model of Preeclampsia
Authors: Xia June , Baxter Samuel , Grenier Jen , Davisson Robin L , Sones Jenny L
Published: November 2023

Preeclampsia (PE) is a hypertensive disease that affects up to 10% of pregnant women worldwide. It presents as late gestational hypertension/proteinuria and only resolves upon delivery of the fetus and placenta. Despite being a leading cause of maternal and fetal morbidity/mortality, the underlying mechanism(s) is unknown. Previous research indicates that abnormal placental development plays a key role in PE. Proper placental formation is dependent on successful implantation and decidualization, the essential process of uterine angiogenesis prior to trophoblast invasion and placentation. Using the BPH/5 spontaneous genetic mouse model of PE, we have previously described profound periimplantation defects in early pregnancy with the principle dysregulated factor being cyclooxygenase 2 (Cox2). Preliminary data indicates that uterine Cox2 misexpression plays a causal role in PE. As Cox2 is heavily regulated by micro(mi)RNAs during the periimplantation period in mice, we hypothesized that miRNAs play a key role in the periimplantation defects seen in the preeclamptic BPH/5 mouse. To test this hypothesis, miRNA sequencing was performed on BPH/5 and C57 control implantation sites collected at e7.5 of pregnancy, the day of peak decidualization. Twenty‐one differentially expressed miRNAs were identified (≥1.5‐fold difference, p < 0.05) with the top 9 being downregulated in BPH/5 implantation sites. Bioinformatic analyses identified potential targets related to angiogenesis. Interestingly, one of these was miR‐146a (−2.2‐fold vs C57), which targets vascular endothelial growth factor (VEGF)‐A. Vegf mRNA levels in e7.5 BPH/5 implantation sites are comparable to C57 as measured by qRT‐PCR, however VEGF164 protein is significantly upregulated in BPH/5 e7.5 implantation sites (1.013 ± 0.25 vs C57: 0.1580 ± 0.1, n =7, p<0.05). Further studies are warranted to confirm if miR‐146a is playing a causal role in the VEGF164 expression pattern. PE is characterized by an imbalance in placental angiogenic factors; however, regulation of these factors during decidualization in the context of PE is unknown. This will provide better understanding of the relationship of miRNAs and angiogenic factors in the decidua before placenta formation and the onset of the maternal syndrome in BPH/5 mice. These findings may provide insights into the origins of the angiogenic imbalance observed in PE pregnancies.

Support or Funding Information

Studies described herein were supported in part by a seed grant from the Cornell Center for Reproductive Genomics, using funds obtained as part of the NICHD National Centers for Translational Research in Reproduction and Infertility (NCTRI), award number P50HD076210 (PI: P.E. Cohen).
The Essence of Quiescence: Understanding the roles of histone modification H4K20me3 and the histone modifying enzyme Suv4‐20h2 in cellular quiescence
Authors: Corvalan Adriana Z , Evertts Adam G , Lowry William E , Coller Hilary A
Published: November 2023

The ability to transition between a proliferative state and quiescence, a reversible exit from the cell cycle, is essential for proper development. During the formation of tumors by cancer cells, the ability to exit and re‐enter the cell cycle in response to environmental cues is lost. This shift from the proliferative to quiescent state is associated with changes in gene expression and chromatin landscape, with quiescent cells exhibiting increased chromatin compaction. Using a high throughput mass spectrometry‐based approach to monitor histone modifications in proliferating and quiescent cells, we discovered that histone H4K20 is differentially methylated in proliferating versus quiescent fibroblasts, with an 8‐fold increase of H4K20me3 in quiescent cells. Levels of H4K20me3 are regulated by Suv4‐20h2 and loss of this enzyme results in decreased chromatin compaction, an increase in the fraction of cells in S phase, and a defect in entry to quiescence. Using ChIP‐Seq, we found that H4K20me3 modifications are present in intergenic regions and promoters of genes that are important for proliferation and pluripotency in a cultured cell line. By investigating the hair follicle stem cells isolated directly from mouse skin, we found that H4K20me3 levels are higher in quiescent hair follicle stem cells isolated from skin during the resting phase of the hair follicle cycle than when the same cell lineage is proliferating in the anagen phase of the hair follicle cycle. Further, H4K20me3 levels are higher in the stem cells of the hair follicle compared to other cell types in the skin such as the basal epidermal cells and dermal fibroblasts. These results support a model in which Suv4‐20h2‐mediated H4K20me3 deposition plays a role in entry and exit of the cell cycle and chromatin architecture.

Support or Funding Information

Ruth L. Kirschstein National Research Service Award GM007185.
LncRNA H19 inhibits gut epithelial regeneration via Wnt co‐receptor LRP6 by interacting with RNA‐binding protein HuR
Authors: Chen Gang , Rao Jaladanki N. , Zou Tongtong , Xiao Lan , Chung Hee Kyoung , Gorospe Myriam , Wang Jian‐Ying
Published: November 2023

Long noncoding RNAs (lncRNAs) are involved in a variety of physiological processes and disease states by modulating gene expression through a wide range of distinct mechanisms. H19 is a conserved lncRNA in mammalian tissues and acts as a molecular sponge or primary template for given microRNAs to regulate proliferation and differentiation. This study tested the hypothesis that H19 regulates intestinal epithelial cell (IEC) proliferation by altering Wnt‐signaling pathway.

Methods

Studies were conducted in human colonic epithelial cells (Caco‐2 line) and H19 function was examined by its ectopic overexpression.

Results

H19 overexpression by transient transfection decreased the level of Wnt co‐receptor LRP6 protein but it did not alter cellular abundances of Lrp6 mRNA, suggesting the repression of LRP6 translation by H19. Since H19 exon1 encodes miR‐675, we investigated the possibility that H19 derives its functionality from miR‐675 and demonstrated that ectopic overexpression of either miR‐675‐3p or miR‐675‐5p failed to inhibit LRP6 expression. We further found that H19 directly bound to the RNA‐binding protein HuR (activator of LRP6 expression) and inhibited HuR‐induced stimulation of LRP6 translation. Elevation of H19 also inhibited IEC proliferation.

Conclusions

These data indicate that H19 inactivates Wnt‐signaling by inhibiting LRP6 translation through competitive inhibition of HuR binding to Lrp6 mRNA, thus down‐regulating intestinal epithelial renewal.

Support or Funding Information

NIH (JYW) and Department of Veterans Affairs (JYW, JNR)
Role of lncRNAs in Regulating Endothelial Function
Authors: Huang Tse‐Shun , Wang Kuei‐Chun , Li Julie Yi‐Shuan , Chang Ting‐Yu , Subramaniam Shankar , Shyy John , Wang Hsei‐Wei , Chien Shu
Published: November 2023

Long non‐coding RNAs (lncRNAs) are shown to carry out functions in wide varieties of pathological and physiological processes such as progress of cancer and maintenance of homeostasis in circulation system. The roles of lncRNAs in atherosclerosis (vessel wall thickening) remain unknown. We propose that lncRNAs regulate endothelial functions to control vascular homeostasis. The chemical (such as therapeutic drug Statins) and mechanical (such as blood flow pattern) stimuli modulate lncRNAs to regulate endothelial status.

With the state‐of‐the‐art RNA sequencing technique, we identified several lncRNAs that are enriched in the endothelial cells under various hemodynamic flow patterns. Among these flow‐regulated lncRNAs, we demonstrated that LINC00341 was up‐regulated by atheroprotective flow, which led to the reduction of vascular adhesion molecule‐1 (VCAM1) expression and the consequential monocyte‐endothelial interactions. Overexpression of LINC00341 repressed the VCAM1 expression, as well as monocyte adhesion, caused by TNFα and atheroprone oscillatory shear stress; knocking down LINC00341 abrogated the atheroprotective effect of Statin (i.e. the repression of VCAM1 and monocyte adhesion). Thus, we conclude that LINC00341 plays an important role in vascular homeostasis. Several modes of action for lncRNAs have been purposed, including alteration of protein complexes targeting specificity by direct incorporation. To investigate the molecular mechanisms by which LINC00341 represses VCAM1 expression, we found that LINC00341 is associated with the immunoprecipitates of Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2), the catalytic subunit of polycomb repressive complex 2 that transfers a methyl group to K27 amino acid of histone H3. We further used the systems biology approach that combines computational and molecular biology analyses to investigate the integrated system of lncRNAs and their regulated gene networks to elucidate the mechanisms by which lncRNAs regulate vascular functions. The knowledge gained in this study will help us to understand the roles of lncRNAs in atherosclerosis and benefit the development of therapeutic strategies.

Support or Funding Information

This study is supported by the Ministry of Science and Technology, Taiwan (103‐2917‐I‐564‐030) and the National Institutes of Health, US (HL106579)
Cranial Neural Crest Migration Sculpts Endothelial Patterning in Avians with Anti‐Angiogenic Factors
Authors: McKinney Mary Cathleen , Kulesa Paul
Published: November 2023

The embryonic neural crest and endothelial cells arise in separate locations but migrate to form intimately‐linked distinct structures in the vertebrate head. The lack of in vivo observations of these cell populations simultaneously in the same embryo has led to several unanswered mechanistic questions. Here, we take advantage of fluorescent labeling of cranial neural crest cells within Tie1 transgenic quail embryos Tg(tie1:H2B‐eYFP) to examine the dynamics of cranial neural crest and endothelial cell migration. In vivo time‐lapse analysis reveals that migrating neural crest and endothelial cells 1) move in opposing directions along a common dorsolateral migratory pathway, 2) display physical interactions that result in changes mainly in endothelial cell trajectories, 3) sort onto discrete pathways after interacting. Deletion of premigratory cranial neural crest cells results in changes to the endothelial cell movements and the vascular pattern. To examine the underlying molecular signals, we find that neural crest cells express anti‐angiogenic factors confirmed by fluorescence in‐situ hybridization and protein analysis. We specifically test the role of Angiopoetin‐2 and show that gain‐ or loss‐of‐function in neural crest cells leads to alterations in the migration patterns of the neural crest and endothelial cells. Together, our data support a model hypothesis that migrating neural crest cells express anti‐angiogenic factors in order to prevent occlusion of their migration by premature vessel formation

Support or Funding Information

Stowers Institute for Medical Research
Regulation of liver energy balance by nuclear receptors
Authors: Moore David D
Published: November 2023

The liver is crucial for maintaining whole body energy balance. For example, the liver responds to fasting by secreting glucose and to feeding by storing it as glycogen. In response to overnutrition, the liver stores excess fat, which is correlated with insulin resistance. The nuclear receptors FXR and PPARα are activated in the fed or fasted liver, respectively, and regulate many metabolic processes. Among such processes, autophagy is a catabolic pathway that recycles nutrients upon starvation and maintains cellular energy homeostasis. We have found that both FXR and PPARα regulate hepatic autophagy. Pharmacologic activation of the bile acid receptor FXR strongly suppresses the induction of autophagy in the fasting state, while pharmacologic activation of PPARα reverses the normal suppression of autophagy in the fed state, inducing autophagic lipid degradation, or lipophagy.

Neonatal cholestasis is a potentially life‐threatening condition. We have identified four individuals from two unrelated families with neonatal cholestasis and mutations in NR1H4, which encodes the bile acid receptor FXR. Clinical features of NR1H4‐related cholestasis include neonatal onset with rapid progression to end‐stage liver disease, vitamin‐K independent coagulopathy, low‐to‐normal serum gamma‐glutamyl transferase activity, elevated serum alpha‐fetoprotein, and undetectable liver bile salt export pump (ABCB11) expression. The coagulopathy is due to FXR transcriptional regulation of multiple components of the coagulation pathway. Genome wide binding studies indicate that FXR may also regulate a number of other liver secreted proteins. The process of secretion is very energy intensive, and it is well known that fasting suppresses secretion via post‐transcriptional mechanisms. We suggest that nuclear receptors also provide nutrient sensitive transcriptional input to liver secretion, with FXR licensing this process while PPARα suppresses it. Overall, we conclude that nuclear receptors act via complementary, interlocking mechanisms to maintain proper energy balance in the liver.
Jnk Signaling In Hepatic Disease
Authors: Davis Roger J
Published: November 2023

The cJun NH2‐terminal kinase (JNK) signaling pathway is implicated in the pathogenesis of diabetes. High fat diet‐induced obesity causes activation of JNK in insulin target tissues. JNK‐deficient mice are resistant to the effects of feeding a high fat diet, including protection against insulin resistance and failure of obesity development. We have used tissue‐specific JNK‐deficient mice to probe the mechanism of JNK regulation of insulin resistance and obesity. We show that JNK plays different roles in multiple tissues and that the phenotype of whole body JNK‐deficient mice reflects the interactions between these different JNK‐dependent processes. The molecular mechanisms of JNK function will be discussed.
An active microrheological approach to determine material properties of early avian embryos
Authors: Kosa Edina , Akos Zsuzsa , Filla Michael , Isai Dona G , Ghazvini Saba , Dhar Prajna , Czirok Andras
Published: November 2023

To understand how cells facilitate large‐scale tissue rearrangements during morphogenesis, we utilize high resolution time‐lapse microscopy of early embryonic tissues (cells and ECM) of avian embryos. To determine the material properties of embryonic tissues, we microinject magnetic microparticles that can be rotated by an external magnetic field. The ECM microenvironment is visualized by fluorescently labeled antibodies, microinjected into the extracellular space. We calibrate our microrheology method in hydrogels. Our data demonstrate that tissues harden gradually both (i) in time (at the same anatomical location) as tissues mature, and (ii) at more anterior locations within the same embryo. (iii) The apparent elastic modulus strongly depends on the size of the probe: large aggregates of magnetic crystals co‐rotate with the surrounding ECM. In contrast, individual, 5–10 um long ferromagnetic crystals often appear to rotate freely. The posterior embryo is, in particular, a very loose, fluid‐like assembly of cells and ECM.

Support or Funding Information

R01‐GM102801
Co‐Incident Upregulation of Shh and Hoxa 13 by Fgf in The Developing Limb Bud
Authors: Real Daniel , Watson Billy A , Feenstra Jennifer M , Pira Charmaine U , Oberg Kerby C
Published: November 2023

Sonic hedgehog (Shh) is secreted from the zone of polarizing activity (ZPA) within the distal‐posterior limb bud mesoderm and regulates pattern and proliferation along the anterior‐posterior limb axis. Fibroblast growth factor 8 (Fgf8) is expressed in the distal limb bud from a rope‐like thickening called the apical ectodermal ridge (AER) and regulates proximal‐distal outgrowth and patterning. Once established, Shh and Fgf8 maintain each other's expression via a reciprocal loop, tightly regulating progressive limb development. Intermediates between Fgf and Shh are still being characterized. Gene arrays following application of Fgf2 to the posterior margin of developing chick wings showed up‐regulation of not only Shh, but also Hoxa13. Hoxa13 expression is a marker of the developing autopod or handplate. In the absence of Hoxa13, there is increased anterior apoptosis and disruption of the normal anterior‐posterior patterning. We hypothesized that the upregulation of Hoxa13 by Fgf in the posterior margin of the limb bud is important for the up‐regulation of Shh.

To test this hypothesis Fgf2 or PBS soaked beads were implanted in the posterior limb margin of stage 23 chick wing buds. Embryos were harvested 24 hours after bead implantation for Hoxa13 whole mount in‐situ hybridization (WMISH).

The results showed that Fgf induced ectopic Hoxa13 expression 24 hours after bead implantation. This confirmed the gene array data that showed a 13 fold increase in Hoxa13 following Fgf2 application. Limb‐specific Shh expression is regulated by the ZPA regulatory sequence (ZRS) which contains Hox binding sites important for full regulatory activity. Future studies are needed to establish whether Hoxa13 binding to the ZRS can facilitate Shh up‐regulation.
Lmx1b Binds to a Gdf5‐Associated Enhancer Element Active During Limb Dorsalization
Authors: Haro Endika , Pernu Lauren , Pira Charmaine U , Oberg Kerby C
Published: November 2023

Lmx1b is a homeodomain transcription factor expressed in the dorsal limb mesoderm that mediates dorsal patterning. Mice lacking functional Lmx1b develop double ventral limbs, whereas ectopic over‐expression in chicken exhibits double dorsal limbs. Although, Lmx1b‐regulated genes have been identified in developing limb buds by gene arrays, no direct targets have been confirmed.

To elucidate direct target genes we performed an Lmx1b‐specific chromatin immunoprecipitation followed by massive parallel sequencing (Lmx1b‐ChIP‐seq). From the recovered genomic intervals, potential regulatory sequences (PRS) were determined by “in silico” comparison to published enhancer‐associated (H3K27ac, P300, RNAPolII or Med12 ) ChIP‐seq data in stage‐matched limbs. Since dorsal‐ventral patterning is a conserved process across divergent vertebrates, PRS were screened for conservation. Finally, intervals matching these criteria were compared to genes differentially expressed in the presence of Lmx1b. Functional validation of selected PRS was performed using an enhancer‐reporter construct in a chick limb bioassay.

Of the 1287 identified genomic intervals bound by Lmx1b, nearly half were intergenic, suggesting that Lmx1b acts at distant regulatory regions rather than at gene promoters. 1022 Lmx1‐bound intervals correlated with enhancer‐associated markers and conservation was evident in 316 intervals that were associated to 121 differentially expressed genes in the presence of Lmx1b. Within the selected PRSs we identified an active enhancer located 78 Kb from Gdf5 that drives reporter activity in the limb and joints. The activity of this Lmx1b‐bound enhancer overlaps Gdf5 expression suggesting a role for Lmx1b in its regulation. Further studies are needed to characterize this enhancer and the role of Lmx1b in regulating Gdf5 expression during limb dorsalization.