Columnar Phenotype Research Articles

Abstract 555: Role of TRIM31 in the pathogenesis of esophageal adenocarcinoma

Abstract Background: Esophageal adenocarcinoma (EAC) is one the deadliest cancers in United States (US) and the only major cancer in the US with increasing incidence. Gastroesophageal reflux disease (GERD) can cause Barrett’s esophagus (BE), a columnar metaplasia of the esophagus strongly associated with the development of EAC. GERD leading to BE is a common pre-occurrence in EAC patients, but the mechanism remains obscure. To explore the mechanism and its components, we compared gene expression in BE and EAC cells with normal esophageal cells and found high expression of tripartite motif-containing protein 31 (TRIM31) in BE and EAC compared to normal esophagus (NE) by transcriptomic profiling of RNA extracted from human esophageal tissues. E3 ubiquitin ligase TRIM 31 is a new member of TRIM family and it has been reported to have oncogenic potentials in some cancers. However, its role in EAC pathogenesis is yet to be understood. Methods: RNA sequencing and transcriptomic profiling were performed on human NE, BE, and EAC epithelial tissue samples. TRIM31 expression in NE cell line (Het-1A) and EAC cell lines (OE19, Flo-1, OE33, SK-GT-2, and OACM5.1C) were identified by Western blot analysis. The Het-1A cell line, after exposure to acidic pH and bile acids, was assessed for variable TRIM31 expression. Cell morphology and viability analyses of NE and EAC cell lines after exposure to acidic pH and bile acids either alone or in combinations were observed by microscopy and WST-1 assay respectively. Results: Transcriptomic analysis showed that BE and EAC are similar in their differential gene expression with upregulation of the TRIM31 compared to that in NE. Concordant with their columnar phenotype, BE and EAC groups displayed a multitude of differentially expressed genes relative to NE samples; however, less differentially expressed genes were found comparing BE and EAC groups. Western blot demonstrated frequent overexpression of TRIM31 in EAC cell lines with very low expression of TRIM31 in NE epithelial cell line. When NE epithelial cell line Het-1A was exposed to acidic pH as well as bile acids in acidic pH, we saw a change in cell morphology that may correlate with the metaplasia of these cells to more resilient forms, as seen in human BE and EAC. Interestingly, bile acids in acidic pH increased TRIM31 expression in NE cell line Het-1A. Bile acids had a greater effect on decreasing cell viability at high concentrations and that effect is enhanced when bile acids were added in acidic pH. WST-1 cell viability assay revealed that EAC cells were more resistant to bile acids as well as bile acids in acidic pH induced decreased in cell viability compared to that in NE cell line Het-1A. The sustained cell viability of EAC cell lines under these harsh conditions correlates with the upregulation of TRIM31. Conclusions: Thus, TRIM31 may be an important oncogenic factor in GERD-induced EAC development and may be an innovative therapeutic target and marker for EAC. Citation Format: Md Sazzad Hassan, Jesse Mast, Annie Ritter, Saisantosh Ponna, Niranjan Awasthi, Urs von Holzen. Role of TRIM31 in the pathogenesis of esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 555.

Screening Candidate Genes at the Co Locus Conferring to the Columnar Growth Habit in Apple (Malus × Domestica Borkh.).

The columnar growth trait of apple (Malus × domestica Borkh.) is genetically controlled by the Columnar (Co) locus on 10 chromosomes, including several candidate genes. Except for MdCo31, other candidate genes at the Co locus are less elucidated. In this study, a strategy of step-by-step screening was adopted to select 11 candidate genes by experimental cloning, transient expression, and genetic transformation. There existed several SNPs in four genes by sequence alignment in columnar and non-columnar apples. Two genes were detected in the nucleus and three genes in the cell membrane, other genes were located in multiple cellular structures by subcellular location. Ectopic expression demonstrated that more branching occurred in MdCo38-OE by upregulating NtPIN1 and NtGA2ox and enlarged leaves in MdCo41-OE tobaccos by upregulating NtCCDs. Transcripts of MdCo38 and MdCo41 were associated with the Co genotypes in apples. The results indicate that MdCo38 and MdCo41 are involved in the columnar growth phenotype in apple, probably through altering polar auxin transport, active gibberellin levels, and strigolactone biosynthesis.

Quantitative morphometric analysis of intrinsic and extrinsic skin ageing in individuals with Fitzpatrick skin types II-III.

Skin ageing is an intricate physiological process affected by intrinsic and extrinsic factors. There is a demand to understand how the skin changes with age and photoexposure in individuals with Fitzpatrick skin types I-III due to accelerated photoageing and the risk of cutaneous malignancies. To assess the structural impact of intrinsic and extrinsic ageing, we analysed 14 skin parameters from the photoprotected buttock and photoexposed dorsal forearm of young and ageing females with Fitzpatrick skin types II-III (n=20) using histomorphic techniques. Whilst the minimum viable epidermis (Emin ) remained constant (Q > 0.05), the maximum viable epidermis (Emax ) was decreased by both age and photoexposure (Q ≤ 0.05), which suggests that differences in epidermal thickness are attributed to changes in the dermal-epidermal junction (DEJ). Changes in Emax were not affected by epidermal cell proliferation. For the first time, we investigated the basal keratinocyte morphology with age and photoexposure. Basal keratinocytes had an increased cell size, cellular height and a more columnar phenotype in photoexposed sites of young and ageing individuals (Q ≤ 0.05), however no significant differences were observed with age. Some of the most striking changes were observed in the DEJ, and a decrease in the interdigitation index was observed with both age and photoexposure (Q ≤ 0.001), accompanied by a decreased height of rête ridges and dermal papilla. Interestingly, young photoexposed skin was comparable to ageing skin across many parameters, and we hypothesise that this is due to accelerated photoageing. This study highlights the importance of skin care education and photoprotection from an early age.

MdCo31 interacts with an RNA polymerase II transcription subunit 32 to regulate dwarf growth with short internodes in columnar apple

The dominant Co locus controls the columnar growth phenotype of apple (Malus × domestica) trees. Candidate gene MdCo31, encoding 2-oxoglutarate-dependent dioxygenase, causes dwarf growth with short internodes in transgenic plants by reducing the abundance of biologically active gibberellin. However, the pathway regulating MdCo31 in the dwarfism of apple trees remains unclear. In this study, expression of MdCo31 was proved to be negatively correlated with internode length in F1 populations created by crossing columnar parents, and with dwarfism in transgenic apple plantlets. Yeast (Saccharomyces cerevisiae) two-hybrid screening identified the RNA polymerase II transcription subunit MdMED32 as putative interactor of MdCo31. Bimolecular fluorescence complementation, co-immunoprecipitation, and dual-luciferase reporter assays confirmed this interaction both in vivo and in vitro. Ectopic expression of MdMED32 in Nicotiana tabacum led to a dwarf phenotype, similar to that of MdCo31 transgenic apple plants. Expression of GA2ox1 and GA20ox1, encoding key enzymes of gibberellin metabolism, was upregulated in transgenic plants. Transient transcriptional activity demonstrated that MdMED32 functioned as an activator, promoting expression of MdGA2ox1 and MdGA20ox1. These findings indicate that the interaction between MdCo31 and MdMED32 functions in the regulation of internode length in columnar apple.

The Characterization of Columnar Apple Gene MdCoL Promoter and Its Response to Abscisic Acid, Brassinosteroid and Gibberellic Acid.

Columnar apple was an important germplasm resource to develop compact cultivars for labor-saving cultivation and to study fruit tree architecture. MdCoL is a strong candidate gene for controlling the columnar phenotype in apple. In this study, a 2000 bp upstream region of MdCoL was cloned as a full-length promoter, named MdCoLp1. To gain a better understanding of the characterization of the MdCoL promoter, cis-acting elements and the binding sites of transcription factors were predicted and analyzed, and four binary expression vectors consisting of the GUS reporter gene under the control of the MdCoL promoter was transformed into Arabidopsis thaliana to analyze the response to abscisic acid (ABA), brassinosteroid (BR) and gibberellic acid (GA3) of MdCoL promoters. Multiple transcription factors involving TCP, BEL1 and BES1/BZR1 and other transcription factor (TF) binding sites were predicted on the promoter of MdCoL. Histochemical staining showed that both full-length and 5′ truncated promoters could initiate GUS expression. The GUS activity was the most in leaf and stem, and mainly concentrated in the fibrovascular tissue, followed by root, and the least activity was observed in silique and flower. In addition, MdCoL expression was mainly localized in the quiescent center (QC) and lateral root growing point of root tip and the vascular tissue of stem and leaf by in situ hybridization. The results of exogenous hormones treatment showed that ABA and BR could activate the activity of the MdCoL promoter, while GA3 had opposite effects. In columnar apple seedlings, ABA treatment could upregulate the expression of MdCoL, but GA3 and BR restrained the transcription level of MdCoL. These results provide the foundation for deciphering the regulatory network of hormones affecting MdCoL transcription.

Molecular Mechanisms Regulating the Columnar Tree Architecture in Apple

The columnar apple cultivar ‘McIntosh Wijcik’ was discovered as a spontaneous mutant from the top of a ‘McIntosh’ tree in the early 1960s. ‘McIntosh Wijcik’ exhibits the columnar growth phenotype: compact and sturdy growth, short internodes, and very few lateral shoots. Classical genetic analysis revealed that the columnar growth phenotype of ‘McIntosh Wijcik’ is controlled by a single dominant gene, Co. This review focuses on the advances made toward understanding the molecular mechanisms of columnar growth in the last decade. Molecular studies have shown that an 8.2 kb insertion in the intergenic region of the Co locus is responsible for the columnar growth phenotype of ‘McIntosh Wijcik’, implying that the insertion affects the expression patterns of adjacent genes. Among the candidate genes in the Co region, the expression pattern of MdDOX-Co, putatively encoding 2-oxoglutarate-dependent dioxygenase (DOX), was found to vary between columnar and non-columnar apples. Recent studies have found three functions of MdDOX-Co: facilitating bioactive gibberellin deficiency, increasing strigolactone levels, and positively regulating abscisic acid levels. Consequently, changes in these plant hormone levels caused by the ectopic expression of MdDOX-Co in the aerial organs of ‘McIntosh Wijcik’ can lead to dwarf trees with fewer lateral branches. These findings will contribute to the breeding and cultivation of new columnar apple cultivars with improved fruit quality.

Forkhead box F1 induces columnar phenotype and epithelial-to-mesenchymal transition in esophageal squamous cells to initiate Barrett's like metaplasia

Multiple genome-wide association studies (GWAS) have linked Forkhead Box F1 (FOXF1) to Barrett's esophagus (BE). Understanding whether FOXF1 is involved in initiation of Barrett's metaplasia could allow FOXF1 to be used for risk stratification and for therapy. Two-dimensional cell cultures and three-dimensional organoid cultures and well-annotated human biopsies were used to determine the role of FOXF1 in BE pathogenesis. Multiple established esophageal squamous and BE cell lines were tested in gain- and loss-of-function studies. Initiation of a BE-like metaplastic change was evaluated by measuring characteristic cytokeratins and global gene expression profiling and by culturing organoids. Epithelial–mesenchymal transition (EMT) was evaluated by immunostaining for E-cadherin, vimentin and Snail, and by cell motility assay. Columnar esophageal epithelium of BE patients exhibited higher expression of FOXF1 compared to normal squamous esophageal epithelium of GERD patients (P < 0.001). Acidic bile salts induced nuclear FOXF1 in esophageal squamous cells. FOXF1 overexpression in normal esophageal squamous cells: (a) increased columnar cytokeratins and decreased squamous cytokeratins, (b) converted squamous organoids to glandular organoids, and (c) switched global gene profiles to resemble that of human BE epithelium (P = 2.1685e − 06 for upregulated genes and P = 8.3378e − 09 for downregulated genes). FOXF1 inhibition in BE cell lines led to loss of BE differentiation markers, CK7, and mucin 2. Also, FOXF1 induced EMT and promoted cell motility in normal esophageal squamous epithelial cells. FOXF1-induced genes mapped to pathways such as Cancer, Cellular Assembly and Organization, DNA Replication, Recombination, and Repair. In conclusion, FOXF1 promotes a BE-like columnar phenotype and cell motility in esophageal squamous epithelial cells, which may have a critical role in BE development. FOXF1 should be studied further as a biomarker for BE and as a target for BE treatment.

The apple gene responsible for columnar tree shape reduces the abundance of biologically active gibberellin.

Ectopic expression of the apple 2-oxoglutarate-dependent dioxygenase (DOX, 2ODD) gene, designated MdDOX-Co, is thought to cause the columnar shape of apple trees. However, the mechanism underlying the formation of such a unique tree shape remains unclear. To solve this problem, we demonstrated that Arabidopsis thaliana overexpressing MdDOX-Co contained reduced levels of biologically active gibberellin (GA) compared with wild type. In summary: (i) with biochemical approaches, the gene product MdDOX-Co was shown to metabolize active GA A4 (GA4 ) to GA58 (12-OH-GA4 ) in vitro. MdDOX-Co also metabolized its precursors GA12 and GA9 to GA111 (12-OH-GA12 ) and GA70 (12-OH-GA9 ), respectively; (ii) Of the three 12-OH-GAs, GA58 was still active physiologically, but not GA70 or GA111 ; (iii) Arabidopsis MdDOX-Co OE transformants converted exogenously applied deuterium-labeled (d2 )-GA12 to d2 -GA111 but not to d2 -GA58 , whereas transformants converted applied d2 -GA9 to d2 -GA58 ; (iv) GA111 is converted poorly to GA70 by GA 20-oxidases in vitro when GA12 is efficiently metabolized to GA9 ; (v) no GA58 was detected endogenously in MdDOX-Co OE transformants. Overall, we conclude that 12-hydroxylation of GA12 by MdDOX-Co prevents the biosynthesis of biologically active GAs in planta, resulting in columnar phenotypes.

The apple columnar gene candidate MdCoL and the AP2/ERF factor MdDREB2 positively regulate ABA biosynthesis by activating the expression of MdNCED6/9.

MdCoL, which encodes a putative 2OG-Fe(II) oxygenase, is a strong candidate gene for control of the columnar growth phenotype in apple. However, the mechanism by which MdCoL produces the columnar trait is unclear. Here, we show that MdCoL influences abscisic acid (ABA) biosynthesis through its interactions with the MdDREB2 transcription factor. Expression analyses and transgenic tobacco studies have confirmed that MdCoL is likely a candidate for control of the columnar phenotype. Furthermore, the ABA level in columnar apple trees is significantly higher than that in standard apple trees. A protein interaction experiment has showed that MdCoL interacts with MdDREB2. Transient expression and electrophoretic mobility shift assays have demonstrated that MdDREB2 binds directly to the DRE motif in the MdNCED6 and MdNCED9 (MdNCED6/9) gene promoters, thereby activating the transcription of these ABA biosynthesis genes. In addition, a higher ABA content has been detected following co-overexpression of MdCoL-MdDREB2 when compared with the overexpression of MdCoL or MdDREB2 alone. Taken together, our results indicate that an interaction between MdCoL and MdDREB2 promotes the expression of MdNCED6/9 and increases ABA levels, a phenomenon that may underlie the columnar growth phenotype in apple.

Exploring DNA Variant Segregation Types Enables Mapping Loci for Recessive Phenotypic Suppression of Columnar Growth in Apple.

Columnar apples trees, originated from a bud mutation ‘Wijcik McIntosh,’ develop a simple canopy and set fruit on spurs. These characteristics make them an important genetic resource for improvement of tree architecture. Genetic studies have uncovered that columnar growth habit is a dominant trait and is caused by a retroposon insertion that induces the expression of the neighboring gene Co encoding a 2OG-Fe(II) oxygenase. Here we report the genetic mapping of two loci of recessive suppressors (genes) c2 (on Chr10) and c3 (on Chr9) that are linked to repression of the retroposon-induced Co gene expression and associated columnar phenotype in 275 F1 seedlings, which were developed from a reciprocal cross between two columnar selections heterozygous at the Co locus. The mapping was accomplished by sequencing a genomic pool comprising 18 columnar seedlings and another pool of 16 standard seedlings that also carry the retroposon insertion, and by exploring DNA variants of segregation types that are informative for mapping recessive traits in apple. The informative segregation types include <hk × hk>, <lm × ll>, <nn × np>, <lm × mm>, and <pp × np>, where each letter denotes one of the four DNA bases and the letters in bold represent variants in relation to the reference genome. The alleles in each first and third positions are assumed in linkage with the recessive suppressors’ allele in the two parents, respectively. Using RNA-seq analysis, we further revealed that the Co gene together with the differentially expressed genes under loci c2 and c3 formed a co-expression gene-network module associated with growth habit, in which 12 MapMan Bins were enriched.

Columnar growth phenotype in apple results from gibberellin deficiency by ectopic expression of a dioxygenase gene.

The apple cultivar McIntosh Wijcik, which is a mutant of 'McIntosh', exhibits a columnar growth phenotype (short internodes, few lateral branches, many spurs, etc.) that is controlled by a dominant Co gene. The candidate gene (MdDOX-Co), encoding a 2-oxoglutarate-dependent dioxygenase, is located adjacent to an insertion mutation. Non-columnar apples express MdDOX-Co in the roots, whereas columnar apples express MdDOX-Co in the aerial parts as well as in the roots. However, the function of MdDOX-Co remains unknown. Here, we characterized tobacco plants overexpressing MdDOX-Co. The tobacco plants showed the typical dwarf phenotype, which was restored by application of gibberellin A3 (GA3). Moreover, the dwarf tobacco plants had low concentrations of endogenous bioactive gibberellin A1 (GA1) and gibberellin A4 (GA4). Similarly, 'McIntosh Wijcik' contained low endogenous GA4 concentration and its dwarf traits (short main shoot and internodes) were partially reversed by GA3 application. These results indicate that MdDOX-Co is associated with bioactive GA deficiency. Interestingly, GA3 application to apple trees also resulted in an increased number of lateral branches and a decrease in flower bud number, indicating that gibberellin (GA) plays important roles in regulating apple tree architecture by affecting both lateral branch formation (vegetative growth) and flower bud formation (reproductive growth). We propose that a deficiency of bioactive GA by ectopic expression of MdDOX-Co in the aerial parts of columnar apples not only induces dwarf phenotypes but also inhibits lateral branch development and promotes flower bud formation, and assembly of these multiple phenotypes constructs the columnar tree form.

Barrett's esophagus and esophageal cancer: Links to microbes and the microbiome.

Esophageal cancer represents a major problem globally, with an estimated 398,000 esophageal squamous cell cancers (ESCC) and 52,000 esophageal adenocarcinomas (EAC) diagnosed worldwide in 2012 [1]. In the United States, EAC is more common than ESCC, and EAC in the US increased dramatically between 1997 and 2006 before stabilizing [2]. Furthermore, esophageal cancer remains particularly deadly, with a five-year survival rate of less than 20% [3]. EAC is thought to arise from Barrett’s esophagus (BE), a metaplastic differentiation from normal squamous epithelium to a columnar phenotype. Accepted risk factors for BE and progression to EAC include age, male sex, obesity, gastroesophageal reflux disease (GERD), and smoking [4, 5]. Unfortunately, less is known about the association between esophageal dysbiosis, inflammation, and the pathogenesis of BE and EAC. Most of the existing data on esophageal microbiota in BE and esophageal cancer are derived from small cross-sectional studies with limited information on causality. As such, this represents an important area for future research because changes in the esophageal microbiota may relate to modifiable factors such as antibiotic use and proton pump inhibitors (PPIs). The esophageal microbiota was initially thought to be primarily related to transient esophageal exposure to the oral microbiota [6], but more recently, culture-independent studies have shown the esophageal microbiota to represent its own niche [7]. In addition to swallowed oral bacteria, the esophagus is also exposed to refluxed gastric microbes, and while the esophageal microbiota resembles both oral and gastric microbiota, it is equal to neither [7, 8]. This review provides an overview of differences that have been described between the normal esophageal microbiota, BE, and EAC. We also discuss novel approaches to esophageal microbial sampling and consider clinical exposures that may alter the esophageal microbiota.

A root-localized gene in normal apples is ectopically expressed in aerial parts of columnar apples

The columnar apple ‘McIntosh Wijcik’, which is a mutation of ‘McIntosh’ shoots, has short internodes, thick stems, upright growth, poor lateral branches, and increased spur density. These columnar traits are controlled by a single dominant gene known as Co. We previously identified a putative dioxygenase gene (designated as 91071) as a promising Co candidate (expressed in the shoot apices of ‘McIntosh Wijcik’). However, tissue expression and function of the 91071 gene in noncolumnar apples is still not clear. In this study, we used reverse transcription polymerase chain reaction to demonstrate that the 91071 gene is mainly expressed in the roots of noncolumnar apples, whereas it is also expressed in shoot apices and leaves of columnar apples. In situ hybridization revealed that the 91071 gene is expressed at the primordium of lateral roots and root tips of both noncolumnar and columnar apple trees and in the shoot meristem and leaf primordium in the columnar apple ‘McIntosh Wijcik’. Grafting experiments of noncolumnar scion onto columnar rootstocks revealed that the columnar growth phenotype is not transmissible from rootstock to scion. These results indicated that ectopic expression of the 91071 gene in aerial parts causes columnar growth, whereas the expression of the 91071 gene in roots does not produce columnar growth. Furthermore, transgenic apples overexpressing the 91071 gene showed larger median adventitious root length and higher median number of lateral roots than control apples. Our result suggests that the 91071 gene may be related to root development.

Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (Malus × domestica).

Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant 'McIntosh Wijcik', which was discovered as a bud mutation from 'McIntosh', exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in 'McIntosh Wijcik' but not in 'McIntosh'. The 91071-gene was located approximately 16kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202bp retroposon insertion in 'McIntosh Wijcik' is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.

Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett's metaplasia.

Metaplasia can result when injury reactivates latent developmental signaling pathways that determine cell phenotype. Barrett's esophagus is a squamous-to-columnar epithelial metaplasia caused by reflux esophagitis. Hedgehog (Hh) signaling is active in columnar-lined, embryonic esophagus and inactive in squamous-lined, adult esophagus. We showed previously that Hh signaling is reactivated in Barrett's metaplasia and overexpression of Sonic hedgehog (SHH) in mouse esophageal squamous epithelium leads to a columnar phenotype. Here, our objective was to identify Hh target genes involved in Barrett's pathogenesis. By microarray analysis, we found that the transcription factor Foxa2 is more highly expressed in murine embryonic esophagus compared with postnatal esophagus. Conditional activation of Shh in mouse esophageal epithelium induced FOXA2, while FOXA2 expression was reduced in Shh knockout embryos, establishing Foxa2 as an esophageal Hh target gene. Evaluation of patient samples revealed FOXA2 expression in Barrett's metaplasia, dysplasia, and adenocarcinoma but not in esophageal squamous epithelium or squamous cell carcinoma. In esophageal squamous cell lines, Hh signaling upregulated FOXA2, which induced expression of MUC2, an intestinal mucin found in Barrett's esophagus, and the MUC2-processing protein AGR2. Together, these data indicate that Hh signaling induces expression of genes that determine an intestinal phenotype in esophageal squamous epithelial cells and may contribute to the development of Barrett's metaplasia.

Characterisation of two genes encoding cytochrome P450 mono-oxygenases involved in gibberellin biosynthesis in apple (Malus × domestica Borkh.)

SummaryGibberellins are important phytohormones that regulate plant stem elongation. This study reports the isolation of the MdKO and MdKAO genes from apical stem tissue of the apple (Malus × domestica Borkh.) cultivar ‘Fuji’. The genes encode the CytP450 mono-oxygenases, ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO), which play key roles in the central steps of the gibberellin biosynthesis pathway. The genomic DNA sequences of both MdKO and MdKAO contained eight exons and seven introns. The coding sequence of MdKO consisted of 1,545 bp and encoded a predicted polypeptide of 514 amino acids, whereas the coding sequence of MdKAO was 1,512 bp and encoded a putative polypeptide of 503 amino acids. The transcripts MDP0000201930 and MDP0000326359 that spanned chromosome 8 from locations 3,980,774 – 3,983,650 and chromosome 2 from locations 26,788,993 – 26,793,838 in the ‘Golden Delicious’ apple genome, corresponded to the products of MdKO and MdKAO, respectively. Homology studies on the deduced amino acid sequences of MdKO and MdKAO indicated that both shared a high level of similarity (66 – 94%) with KO and KAO proteins from other plant species. Quantitative real-time PCR analyses showed that the level of transcription of MdKAO was significantly lower in the columnar parent, and in F1 progeny with the columnar phenotype, than in the standard parent and F1 progeny with the standard phenotype. This was confirmed in the columnar wild-type and in corresponding standard phenotype mutants. It is conceivable that lower expression of the MdKAO gene was responsible for the reduced concentration of bio-active gibberellins in the apical tissues of columnar apple stems.

Bile Acid at Low pH Reduces Squamous Differentiation and Activates EGFR Signaling in Esophageal Squamous Cells in 3-D Culture

BackgroundBarrett's esophagus is a preneoplastic metaplasia in which the normal squamous epithelium of the esophagus changes to an intestinal, columnar phenotype due to long-term gastro-esophageal reflux. The major components of this reflux are bile and stomach acid. Previous in vitro studies on the effect of bile and acid on esophageal cells have predominantly relied on transformed esophageal squamous cells or cancer cells grown in monolayer culture. DiscussionIn this study, we expanded our previous work using an immortalized primary esophageal squamous cell line (EPC1). We demonstrate that EPC1 cells form a multi-layer, stratified epithelium when grown on polyester transwell filters in media supplemented with calcium. When exposed to short pulses of bile and pH 5, but not either condition alone, EPC1 cells demonstrate a reduction in stratification layers and reduced expression of squamous epithelium-specific genes. Bile at pH 5 also causes activation of epidermal growth factor receptor and down-stream pathways. Blocking epidermal growth factor receptor activation partially attenuates the effects of bile acid and pH 5. These results suggest that bile at low pH, but not bile or low pH alone, promotes loss of differentiation status of stratified squamous esophageal epithelium in vitro, possibly by initiating a mucosal repair response through epidermal growth factor activation.

Mo1937 MicroRNA-192 Promotes Transition of Squamous to Columnar Phenotype: Novel Insights Into the Pathogenesis of Barrett's Esophagus

Introduction: The gastroesophageal reflux of acid and bile can cause esophagitis, and inflammation can trigger epithelial-mesenchymal transition (EMT), the process in which epithelial cells acquire phenotypic features of mesenchymal cells. EMT also occurs during carcinogenesis. Chronic exposure to acid and bile salts has been reported to cause non-neoplastic Barrett's epithelial cells in vitro to undergo malignant transformation and acquire EMT characteristics. Noting that these EMT changes occurred in cultures of initially-benign epithelial cells devoid of inflammatory cells, we hypothesized that acid and bile alone (in the absence of inflammation and neoplasia) might trigger EMT in Barrett's metaplasia. To explore this issue, we assessed features of EMT in non-neoplastic and dysplastic Barrett's epithelial cells, and we studied the effects of acid and bile salts on EMT in non-neoplastic Barrett's cells. Methods: We used: a) 2 telomerase-immortalized, non-neoplastic Barrett's epithelial cell lines (BAR-T and BAR-T10); b) BAR-T cells treated for 20 weeks with intermittent exposure to acidic bile salts (BEC-20W), and c) 3 immortalized, dysplastic Barrett's cell lines (CPB, CPC and CPD). BAR-T cells were exposed to acidic media (pH 5.5) containing conjugated bile acids (400 μM) either once for 10-minutes or for 10 minutes TID for 3 days. We evaluated features of EMT using optic morphology, Western blot and immunofluorescence (IF) for E-cadherin (CDH1) and fibronectin1 (FN1) proteins, and cell migration assays. Results: Dysplastic and BEC-20W cells had lower levels of CDH1 (an epithelial cell marker) and higher levels of FN1 (a mesenchymal cell marker) than non-neoplastic BART cells. By IF, dysplastic and BEC-20W cells demonstrated lower levels of membranous CDH1 and higher levels of cytoplasmic FN1 than BAR-T cells. Dysplastic and BEC-20W cells had a spindle-shaped appearance by optic morphology, and exhibited significantly higher migration rates than BAR-T cells (Table). In BAR-T cells, a single 10-minute exposure to acidic bile salts decreased CDH1 and increased FN1 protein levels by 6 hours. Exposure to acidic bile salts for 3 days significantly increased migration rates of BAR-T (21.7±3.3 SEM to 45.1±3.6) and BAR-T10 cells (13.2±1.8 to 22.6±2.5). Conclusion: Non-dysplastic Barrett's epithelial cells exposed to acidic bile salts for 20 weeks show evidence of EMT, as do dysplastic Barrett's epithelial cells. In non-neoplastic Barrett's cells, brief exposures to acidic bile salts induce EMT features including decreased CDH1 and increased FN1 levels, and increased cell migration. These data suggest that the gastroesophageal reflux of acid and bile might induce EMT in Barrett's metaplasia, even in the absence of inflammation and neoplasia. Conceivably, reflux-induced EMTmight contribute to remodeling and carcinogenesis in Barrett's esophagus. Migration Rates (average cells ± SEM per ≤14 fields)

Appearance of remodelled and dendritic cell-rich alveolar-lymphoid interfaces provides a structural basis for increased alveolar antigen uptake in chronic obstructive pulmonary disease

RationaleThe alveolar pathology in chronic obstructive pulmonary disease (COPD) involves antigen-driven immune events. However, the induction sites of alveolar adaptive immune responses have remained poorly investigated.ObjectivesTo explore the hypothesis that...

Evaluation of the hormonal state of columnar apple trees (Malus x domestica) based on high throughput gene expression studies

The columnar phenotype of apple trees (Malus x domestica) is characterized by a compact growth habit with fruit spurs instead of lateral branches. These properties provide significant economic advantages by enabling high density plantings. The columnar growth results from the presence of a dominant allele of the gene Columnar (Co) located on chromosome 10 which can appear in a heterozygous (Co/co) or homozygous (Co/Co) state. Although two deep sequencing approaches could shed some light on the transcriptome of columnar shoot apical meristems (SAMs), the molecular mechanisms of columnar growth are not yet elaborated. Since the influence of phytohormones is believed to have a pivotal role in the establishment of the phenotype, we performed RNA-Seq experiments to study genes associated with hormone homeostasis and clearly affected by the presence of Co. Our results provide a molecular explanation for earlier findings on the hormonal state of columnar apple trees. Additionally, they allow hypotheses on how the columnar phenotype might develop. Furthermore, we show a statistically approved enrichment of differentially regulated genes on chromosome 10 in the course of validating RNA-Seq results using additional gene expression studies.