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: Metastasis and drug resistance contribute substantially to the poor prognosis of colorectal cancer (CRC) patients. However, the epigenetic regulatory mechanisms by which CRC develops metastatic and drug-resistant characteristics remain unclear. This study aimed to investigate the role of miR-302a in the metastasis and molecular-targeted drug resistance of CRC and elucidate the underlying molecular mechanisms. : miR-302a expression in CRC cell lines and patient tissue microarrays was analyzed by qPCR and fluorescence hybridization. The roles of miR-302a in metastasis and cetuximab (CTX) resistance were evaluated both and . Bioinformatic prediction algorithms and luciferase reporter assays were performed to identify the miR-302a binding regions in the NFIB and CD44 3'-UTRs. A chromatin immunoprecipitation assay was performed to examine NFIB occupancy in the ITGA6 promoter region. Immunoblotting was performed to identify the EGFR-mediated pathways altered by miR-302a. : miR-302a expression was frequently reduced in CRC cells and tissues, especially in CTX-resistant cells and patient-derived xenografts. The decreased miR-302a levels correlated with poor overall CRC patient survival. miR-302a overexpression inhibited metastasis and restored CTX responsiveness in CRC cells, whereas miR-302a silencing exerted the opposite effects. NFIB and CD44 were identified as novel targets of miR-302a. miR-302a inhibited the metastasis-promoting effect of NFIB that physiologically activates ITGA6 transcription. miR-302a restored CTX responsiveness by suppressing CD44-induced cancer stem cell-like properties and EGFR-mediated MAPK and AKT signaling. These results are consistent with clinical observations indicating that miR-302a expression is inversely correlated with the expression of its targets in CRC specimens. : Our findings show that miR-302a acts as a multifaceted regulator of CRC metastasis and CTX resistance by targeting NFIB and CD44, respectively. Our study implicates miR-302a as a candidate prognostic predictor and a therapeutic agent in CRC.
© The author(s).
The sustained expression of the MAFB transcription factor in human islet β-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet β-cell-enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet β-cell population and that expression is postnatally restricted in mouse β-cells by de novo DNA methylation. To gain insight into how MAFB affects human β-cells, we developed a mouse model to ectopically express in adult mouse β-cells using transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse β-cells, suggesting that the human adult β-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet β-cell defects in a mouse mutant lacking MafA in β-cells. Of note, transgenic production of MafB in β-cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives the serotonin biosynthesis critical for adaptive maternal β-cell responses. Together, these studies provide novel insight into the role of MAFB in human islet β-cells.
© 2018 by the American Diabetes Association.
RATIONALE - Lung natural killer cells (NKs) kill a greater percentage of autologous lung parenchymal cells in chronic obstructive pulmonary disease (COPD) than in nonobstructed smokers. To become cytotoxic, NKs require priming, typically by dendritic cells (DCs), but whether priming occurs in the lungs in COPD is unknown.
METHODS - We used lung tissue and in some cases peripheral blood from patients undergoing clinically indicated resections to determine in vitro killing of CD326 lung epithelial cells by isolated lung CD56 NKs. We also measured the cytotoxicity of unprimed blood NKs after preincubation with lung DCs. To investigate mechanisms of DC-mediated priming, we used murine models of COPD induced by cigarette smoke (CS) exposure or by polymeric immunoglobulin receptor (pIgR) deficiency, and blocked IL-15Rα (IL-15 receptor α subunit) trans-presentation by genetic and antibody approaches.
RESULTS - Human lung NKs killed isolated autologous lung epithelial cells; cytotoxicity was increased (P = 0.0001) in COPD, relative to smokers without obstruction. Similarly, increased lung NK cytotoxicity compared with control subjects was observed in CS-exposed mice and pIgR mice. Blood NKs both from smokers without obstruction and subjects with COPD showed minimal epithelial cell killing, but in COPD, preincubation with lung DCs increased cytotoxicity. NKs were primed by CS-exposed murine DCs in vitro and in vivo. Inhibiting IL-15Rα trans-presentation eliminated NK priming both by murine CS-exposed DCs and by lung DCs from subjects with COPD.
CONCLUSIONS - Heightened NK cytotoxicity against lung epithelial cells in COPD results primarily from lung DC-mediated priming via IL-15 trans-presentation on IL-15Rα. Future studies are required to test whether increased NK cytotoxicity contributes to COPD pathogenesis.
Cyclooxygenase (COX)-derived prostaglandins stimulate uterine contractions and prepare the cervix for parturition. Prior reports suggest Cox-1 knockout (KO) mice exhibit delayed parturition due to impaired luteolysis, yet the mechanism for late-onset delivery remains unclear. Here, we examined key factors for normal onset of parturition to determine whether any could account for the delayed parturition phenotype. Pregnant Cox-1KO mice did not display altered timing of embryo implantation or postimplantation growth. Although messenger RNAs of contraction-associated proteins (CAPs) were differentially expressed between Cox-1KO and wild-type (WT) myometrium, there were no differences in CAP agonist-induced intracellular calcium release, spontaneous or oxytocin (OT)-induced ex vivo uterine contractility, or in vivo uterine contractile pressure. Delayed parturition in Cox-1KO mice persisted despite exogenous OT treatment. Progesterone (P4) withdrawal, by ovariectomy or administration of the P4-antagonist RU486, diminished the delayed parturition phenotype of Cox-1KO mice. Because antepartum P4 levels do not decline in Cox-1KO females, P4-treated WT mice were examined for the effect of this hormone on in vivo uterine contractility and ex vivo cervical dilation. P4-treated WT mice had delayed parturition but normal uterine contractility. Cervical distensibility was decreased in Cox-1KO mice on the day of expected delivery and reduced in WT mice with long-term P4 treatment. Collectively, these findings show that delayed parturition in Cox-1KO mice is the result of impaired luteolysis and cervical dilation, despite the presence of strong uterine contractions.
Copyright © 2018 Endocrine Society.
OBJECTIVE - is the strongest risk factor for gastric cancer; however, the majority of infected individuals do not develop disease. Pathological outcomes are mediated by complex interactions among bacterial, host and environmental constituents, and two dietary factors linked with gastric cancer risk are iron deficiency and high salt. We hypothesised that prolonged adaptation of to in vivo carcinogenic microenvironments results in genetic modification important for disease.
DESIGN - Whole genome sequencing of genetically related strains that differ in virulence and targeted sequencing following prolonged exposure of bacteria to in vitro carcinogenic conditions were performed.
RESULTS - A total of 180 unique single nucleotide polymorphisms (SNPs) were identified among the collective genomes when compared with a reference genome. Importantly, common SNPs were identified in isolates harvested from iron-depleted and high salt carcinogenic microenvironments, including an SNP within (FurR88H). To investigate the direct role of low iron and/or high salt, was continuously cultured under low iron or high salt conditions to assess genetic variation. Exposure to low iron or high salt selected for the FurR88H variant after only 5 days. To extend these results, was sequenced in 339 clinical strains. Among the isolates examined, 17% (40/232) of strains isolated from patients with premalignant lesions harboured the FurR88H variant, compared with only 6% (6/107) of strains from patients with non-atrophic gastritis alone (p=0.0034).
CONCLUSION - These results indicate that specific genetic variation arises within strains during in vivo adaptation to conditions conducive for gastric carcinogenesis.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
Inhibition of proliferation in estrogen receptor-positive (ER) breast cancers after short-term antiestrogen therapy correlates with long-term patient outcome. We profiled 155 ER/human epidermal growth factor receptor 2-negative (HER2) early breast cancers from 143 patients treated with the aromatase inhibitor letrozole for 10 to 21 days before surgery. Twenty-one percent of tumors remained highly proliferative, suggesting that these tumors harbor alterations associated with intrinsic endocrine therapy resistance. Whole-exome sequencing revealed a correlation between 8p11-12 and 11q13 gene amplifications, including and , respectively, and high Ki67. We corroborated these findings in a separate cohort of serial pretreatment, postneoadjuvant chemotherapy, and recurrent ER tumors. Combined inhibition of FGFR1 and CDK4/6 reversed antiestrogen resistance in ER/ coamplified CAMA1 breast cancer cells. RNA sequencing of letrozole-treated tumors revealed the existence of intrachromosomal fusion transcripts and increased expression of gene signatures indicative of enhanced E2F-mediated transcription and cell cycle processes in cancers with high Ki67. These data suggest that short-term preoperative estrogen deprivation followed by genomic profiling can be used to identify druggable alterations that may cause intrinsic endocrine therapy resistance.
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
OBJECTIVE - The mutant γ-aminobutyric acid type A (GABA ) receptor γ2(Q390X) subunit (Q351X in the mature peptide) has been associated with the epileptic encephalopathy, Dravet syndrome, and the epilepsy syndrome genetic epilepsy with febrile seizures plus (GEFS+). The mutation generates a premature stop codon that results in translation of a stable truncated and misfolded γ2 subunit that accumulates in neurons, forms intracellular aggregates, disrupts incorporation of γ2 subunits into GABA receptors, and affects trafficking of partnering α and β subunits. Heterozygous Gabrg2 knock-in (KI) mice had reduced cortical inhibition, spike wave discharges on electroencephalography (EEG), a lower seizure threshold to the convulsant drug pentylenetetrazol (PTZ), and spontaneous generalized tonic-clonic seizures. In this proof-of-principal study, we attempted to rescue these deficits in KI mice using a γ2 subunit gene (GABRG2) replacement therapy.
METHODS - We introduced the GABRG2 allele by crossing Gabrg2 KI mice with bacterial artificial chromosome (BAC) transgenic mice overexpressing HA (hemagglutinin)-tagged human γ2 subunits, and compared GABA receptor subunit expression by Western blot and immunohistochemical staining, seizure threshold by monitoring mouse behavior after PTZ-injection, and thalamocortical inhibition and network oscillation by slice recording.
RESULTS - Compared to KI mice, adult mice carrying both mutant allele and transgene had increased wild-type γ2 and partnering α1 and β2/3 subunits, increased miniature inhibitory postsynaptic current (mIPSC) amplitudes recorded from layer VI cortical neurons, reduced thalamocortical network oscillations, and higher PTZ seizure threshold.
SIGNIFICANCE - Based on these results we suggest that seizures in a genetic epilepsy syndrome caused by epilepsy mutant γ2(Q390X) subunits with dominant negative effects could be rescued potentially by overexpression of wild-type γ2 subunits.
Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.
Food intake occurs in bouts or meals, and numerous meal-generated signals have been identified that act to limit the size of ongoing meals. Hormones such as cholecystokinin (CCK) are secreted from the intestine as ingested food is being processed, and in addition to aiding the digestive process, they provide a signal to the brain that contributes to satiation, limiting the size of the meal. The potency of CCK to elicit satiation is enhanced by elevated levels of adiposity signals such as insulin. In the present experiments we asked whether CCK and insulin interact at the level of the blood-brain barrier (BBB). We first isolated rat brain capillary endothelial cells that comprise the BBB and found that they express the mRNA for both the CCK1R and the insulin receptor, providing a basis for a possible interaction. We then administered insulin intraperitoneally to another group of rats and 15min later administered CCK-8 intraperitoneally to half of those rats. After another 15min, CSF and blood samples were obtained and assayed for immunoreactive insulin. Plasma insulin was comparably elevated above baseline in both the CCK-8 and control groups, indicating that the CCK had no effect on circulating insulin levels given these parameters. In contrast, rats administered CCK had CSF-insulin levels that were more than twice as high as those of control rats. We conclude that circulating CCK greatly facilitates the transport of insulin into the brain, likely by acting directly at the BBB. These findings imply that in circumstances in which the plasma levels of both CCK and insulin are elevated, such as during and soon after meals, satiation is likely to be due, in part, to this newly-discovered synergy between CCK and insulin.
Copyright © 2016 Elsevier Inc. All rights reserved.
The endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide (AEA), are endogenous ligands for the cannabinoid receptors (CB1 and CB2) and are implicated in a wide array of physiological processes. These neutral arachidonic acid (AA) derivatives have been identified as efficient substrates for the second isoform of the cyclooxygenase enzyme (COX-2). A diverse family of prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) is generated by the action of COX-2 (and downstream prostaglandin synthases) on 2-AG and AEA. As the biological importance of the endocannabinoid system becomes more apparent, there is a tremendous need for robust, sensitive, and efficient analytical methodology for the endocannabinoids and their metabolites. In this chapter, we describe methodology suitable for carrying out oxygenation of endocannabinoids by COX-2, and analysis of products of endocannabinoid oxygenation by COX-2 and of endocannabinoids themselves from in vitro and cell assays.