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Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is correlated with poor prognosis. HER2-targeting drugs have been successful to treat HER2-positive breast cancer. However, the acquisition of the drug resistance with long-term use is still recognized. Here we report the novel molecular targets to treat HER2-positive breast cancer. HER2 is a key driving forces to proliferate HER2-positive breast cancer cells. Unlike other HER family members (EGFR, HER3, HER4), it recognizes no ligands and forms heterodimers with each other member, leading to the transduction of various cellular signals. Especially, the activated EGFR transactivates HER2, and the phosphorylation of both EGFR and HER2 strongly drives cell proliferation. In this study, we found that the degradation of an endosomal small GTPase, RhoB, by the ubiquitin ligase complex cullin-3 (CUL3)/KCTD10 is essential for both EGFR and HER2 phosphorylation in HER2-positive breast cancer cells. Depletion of CUL3 or KCTD10 drastically reduced the phosphorylation of EGFR and HER2, as well as HER2-positive breast cancer cell proliferation. As mechanisms, we identified a RhoB-interacting scaffold protein and an EGFR phosphatase from human protein arrays prepared by the wheat cell-free protein synthesis system, a unique technology of Ehime University. In HER2-positive breast cancer cells, we showed that the scaffold protein interacts with the EGFR phosphatase at the plasma membrane, which leads to inactivation of the EGFR phosphatase and inhibits EGFR dephosphorylation. Upon CUL3- or KCTD10-knockdwon, accumulated RhoB interacts with the scaffold protein, resulting in the release of active EGFR phosphatase. Using the METABRIC database, we next found that low expression of RhoB mRNA and high expression of scaffold protein correlate with poor prognosis for HER2-positive breast cancer patients. Additionally, high expression of scaffold protein was associated with poor prognosis in immunohistochemistry of HER2-positive breast cancer tissues. These data suggest that high expression of scaffold protein enhances cell proliferative signaling in HER2-positive breast cancers. The inhibitors of the scaffold protein/EGFR phosphatase interaction would be new therapeutic agents for HER2-positive breast cancer cells through EGFR dephosphorylation mediated by activation of the EGFR phosphatase. Citation Format: Kanako Nishiyama, Masashi Maekawa, Akari Murakami, Kaho Utsunomiya, Kana Takemoto, Erina Kusakabe, Haruna Noda, Reina Aoki, Kana Taguchi, Michiko Yamashita, Tomoya Nakagita, Jun Nakayama, Mami Chosei, Takeshi Kiyoi, Yoshiaki Kamei, Hiroyuki Takeda, Yasutsugu Takada, Shigeki Higashiyama. A novel mechanism of phosphatase activation for EGFR by Cullin-3/KCTD10 ubiquitin E3 complex in HER2-positive breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-02.

Background Fibroadenomas (FAs) and phyllodes tumors (PTs) are major benign breast tumors. They are pathologically classified as fibroepithelial tumors, composed of a proliferation of both epithelial and stroma. Although the clinical management of PTs differs from FAs, distinction by core needle biopsy diagnoses is still challenging. Computer-aided diagnosis is playing a pivotal role in accurate and objective evaluation of medical images. This technology opens up a new route to a solution for diagnostic problems. Methods A combined technique of label-free imaging with multi-photon microscopy and artificial intelligence was applied to detect quantitative signatures that differentiate fibroepithelial lesions. Multi-photon excited autofluorescence and second harmonic generation (SHG) signals were detected in tissue sections. A pixel-wise semantic segmentation method using a deep learning framework was used to separate epithelial and stromal regions automatically. Quantitative signatures, the epithelial to stromal area ratio, and the collagen SHG signal strength were investigated for their ability to distinguish between FA and PT lesions. Results Multi-photon microscopy recordings of tissue sections revealed distinct morphology between the epithelia and stroma, and further indicated that stromal regions emit a strong SHG signal which derives from collagen fibrils. However, this signal strength differs between the lesions, suggesting differences of collagenous molecular composition between the two lesions. In order to investigate hypertrophy of the stroma and compare this to the epithelial areas, an image segmentation analysis with a pixel-wise semantic segmentation framework using a deep convolutional neural network was performed. The deep learning-based analysis showed accurate separation of epithelial and stromal regions. Further investigation was conducted to determine if scoring the epithelial to stromal area ratio could be a marker for differentiating fibroadenoma and phyllodes tissues; we determined that most samples can be clearly separated but some are difficult to separate by the signature. Further investigations on the collagen SHG signal strength within the stromal area revealed accurate classification of the breast tissue lesions. Conclusions Molecular and morphological changes detected through the assistance of computational and label-free multi-photon imaging techniques enabled us to propose quantitative signatures for epithelial and stromal alterations in breast tissues.

Background Metaplastic carcinoma of the breast consists of both invasive ductal carcinoma and metaplastic carcinoma. This rare subtype of cancer has a poor prognosis. The development of metaplastic breast cancer and relationship with BRCA1 are not well known. Here, we report a rare case of germline BRCA1 mutation-positive breast cancer with chondroid metaplasia. Case presentation A 39-year-old Japanese woman with a family history of breast cancer in her mother and ovarian cancer in her maternal grandmother consulted at our hospital with a left breast mass. Needle biopsy for the mass was performed, leading to a diagnosis of invasive breast cancer with chondroid metaplasia. We performed left mastectomy + sentinel lymph node biopsy + tissue expander insertion and replaced with a silicone implant later. Pathological examination revealed that the patient had triple-negative breast cancer. Four courses of doxorubicin+ cyclophosphamide therapy were performed as adjuvant therapy after surgery. We performed genetic counseling and genetic testing, and the results suggested the germline BRCA1 mutation 307 T> A (L63*). She has currently lived without a relapse for 2 years post-surgery. Conclusions There have been only 6 cases of metaplastic breast carcinoma with germline BRCA1 mutations including our case. Patients with BRCA1 mutations may develop basal-like subtypes or M type of triple-negative breast cancer besides metaplastic breast cancers.

Significance There are no biomarkers for schizophrenia (SCZ), a disorder of dysfunctional neural networks. We demonstrate that a master regulator of cytoskeleton (“CRMP2”) and, hence, neural circuitry, may form the basis for such a biomarker because its activity is uniquely imbalanced in SCZ patients. We show that SCZ patients are characterized by an excess of active CRMP2 not only in their brains (where it is correlated with dendritic abnormalities) but also in their peripheral blood lymphocytes. The abundance of active CRMP2 and insufficiency of opposing inactive p-CRMP2 likely disrupts neuronal function. Because peripheral blood CRMP2 appears to reflect intracerebral processes, it could form the basis of a rapid, minimally invasive, sensitive, and specific clinical diagnostic aid for SCZ in young patients., There are no validated biomarkers for schizophrenia (SCZ), a disorder linked to neural network dysfunction. We demonstrate that collapsin response mediator protein-2 (CRMP2), a master regulator of cytoskeleton and, hence, neural circuitry, may form the basis for a biomarker because its activity is uniquely imbalanced in SCZ patients. CRMP2’s activity depends upon its phosphorylation state. While an equilibrium between inactive (phosphorylated) and active (nonphosphorylated) CRMP2 is present in unaffected individuals, we show that SCZ patients are characterized by excess active CRMP2. We examined CRMP2 levels first in postmortem brains (correlated with neuronal morphometrics) and then, because CRMP2 is expressed in lymphocytes as well, in the peripheral blood of SCZ patients versus age-matched unaffected controls. In the brains and, more starkly, in the lymphocytes of SCZ patients

Overexpression of HER2 in breast cancer is correlated with poor prognosis. HER2-targeted drugs, such as trastuzumab and lapatinib, have been successful to treat HER2-positive breast cancers, however, the acquisition of the drug resistance of the cells is recognized. Here we suggest the novel molecular targets to cure HER2-positive breast cancers. The oncogenic roles of Rac1, a Rho family small GTPase, in a variety of cancers have been demonstrated. For example, the elevated expression or hyperactivation of Rac1 is frequently observed in human cancers, correlating with their aggressiveness and poor prognosis. In breast cancers, upregulation of Rac1 GEF (GTP exchange factor) and downregulation of Rac1 GAP (GTPase activating protein) have been reported. Moreover, activation of Rac1 contributed to trastuzumab resistance, which poses a serious problem during chemotherapy. In the present study, we first investigated in detail in which subtypes of breast cancers mRNA expression of Rac1 is correlated with their poor prognosis. Using the METABRIC database, we found that high mRNA expression of Rac1 significantly correlated with the poor prognosis of HER2-positive breast cancer (p=0.0012, High: n=49, Low: n=171). On the other hands, other three types (basal, claudin-low, or luminal-B type) did not show significant correlation between the expression levels of Rac1 mRNA and their prognosis (p=0.15, High: n=97, Low: n=102; p=0.052, High: n=110, Low: n=89; p=0.17, High: n=70, Low: n=391; respectively). In luminal-A type breast cancer, low mRNA expression of Rac1 significantly correlated with poor prognosis (p=0.0046, High: n=492, Low: n=187). We next investigated the molecular mechanism underlying Rac1 activation in HER2-positive breast cancer cells, SKBR-3 cells. We found that Cullin-3 (CUL3, a subunit of a RING ubiquitin E3 ligase complex) and its adaptor protein KCTD10 are essential for Rac1 activation. Mechanistically, CUL3/KCTD10 ubiquitinate RhoB, a Rho family small GTPase that suppresses the activation of Rac1, leading to the degradation RhoB. We also found that HER2 signaling is essential for the activation of Rac1. Conclusions: This study reveals that the novel molecular axis CUL3/KCTD10/RhoB regulates the Rac1 activation in HER2-positive breast cancer cells. The interference of CUL3/KCTD10 complex formation may be a new strategy to the treatment of HER2- and Rac1-positive breast cancers. Citation Format: Murakami A, Maekawa M, Kusakabe E, Yamasawa H, Aoki R, Komatsu S, Taguchi K, Nishiyama K, Yamashita M, Sugimori W, Kawai K, Nakayama J, Araki N, Semba K, Taguchi T, Kamei Y, Takada Y, Higashiyama S. Novel mechanisms of Rac1 activation by the Cullin-3/KCTD10 ubiquitin E3 complex in HER2-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-02.

Breast cancer subtypes have been classified based on the gene expression profiles, and the specifically effective treatments for each subtype have been developed and applied in clinical situations. However, there is no effective treatment for metaplastic breast carcinomas, which are pathological tissue types with poor prognosis. Metaplastic breast carcinomas are almost resistant to existing chemotherapy because it has not been identified the specific therapeutic target molecules. Recently, Lehmann et al. reported that breast cancers with chondroid metaplasia are categorized in Mesenchymal (M) type, which is one subtype out of six triple negative breast cancer (TNBC) subtypes based on drug susceptibility. From the knowledge on this report, we hypothesized that integrative analyses using the gene expression profile data sets obtained from various types of breast cancers with or without cartilage formation may enable us to identify the therapeutic target genes for metaplastic breast carcinomas, especially for breast cancers with chondroid metaplasia. Bioinformatics analyses were performed using the gene expression profile data sets of TNBC, which are registered in Gene Expression Omnibus (GEO). When compared with metaplastic breast carcinomas (28 data sets), 200 genes were specifically fluctuated over 2-fold in breast cancer with chondroid metaplasia (8 data sets). On the other hand, M type (11 data sets) specific 578 genes were identified over 56 TNBC data sets. In addition, 57 genes were overlapped between chondoroid metaplastic 200 genes and M type 578 genes. Therefore, these 57 overlapped genes might be considered as possible poor prognostic factors in breast cancer with chondroid metaplasia. As a result of Gene Ontology analysis on these 57 genes, skeletal system development and extracellular matrix structural constituent were significantly enriched in Biological process and Molecular function, respectively. Also, SCRG1, SOX8 and SOX10, which are genes related to cartilage differentiation, were included. Next, we focused 14 genes because these genes could serve as therapeutic targets. Then, we validated the expression levels of these 14 genes using tissues obtained from surgically resected human breast cancer with chondroid metaplasia dividing into three parts, the invasive ductal carcinoma, the chondroid metaplasia and normal mammary gland. As a result, SCRG1, CD86, HCLS1, EPSTI1, LYZ, and SLA were significantly increased in chondroid metaplasia part, suggesting that these validated genes were identified as possible therapeutic targets for breast cancer with chondroid metaplasia. Further study will help us to investigate a novel and effective therapeutic strategy for metaplastic breast carcinomas. Citation Format: Yamashita M, Kamei Y, Kusakabe E, Yamasawa H, Aoki R, Taguchi K, Nishiyama K, Murakami A, Sawada Y, Takada Y, Imai Y. Exploring novel therapeutic target molecules for metaplastic breast carcinoma using comprehensive genome-wide gene expression analyses [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-07.

Background: The 2019 version 1 of the National Comprehensive Cancer Network guidelines states that magnetic resonance imaging (MRI)-based diagnosis of breast cancer “May be used for staging evaluation to define extent of cancer or presence of multifocal or multicentric cancer in the ipsilateral breast, or as screening of the contralateral breast cancer at time of initial diagnosis.” Therefore, we performed breast MRI to evaluate the extent of spread of breast cancer as part of the preoperative diagnosis. Dual-energy computed tomography (DECT) is a new imaging technology that uses X-ray tube and detectors with two different tube voltages. High-contrast and low-noise images, termed “virtual monochromatic image,” can be obtained using DECT. Recently, clinical cases using DECT have been reported in urology; however, only few cases have been reported on the diagnostic use of DECT for breast cancer. Purpose: Evaluation of the usefulness of DECT in the preoperative diagnosis of extent of breast cancer spread. Patients: Thirty-three patients with breast cancer, who underwent preoperative DECT and MRI, and were operated at our hospital from November 2016 to July 2018. The surgical specimens from the patients were excised horizontally. Patients who underwent preoperative chemotherapy were excluded from the study. Methods: The maximum diameter, including that of the mass and non-mass enhancements in the horizontal sections of each lesion, was retrospectively measured by DECT and MRI. Moreover, the maximum diameter of the invading and intraductal lesions in the horizontal sections of the postoperative pathological sample were retrospectively measured. In addition, the information on the patients’ background, clinical characteristics of the tumor, etc. were retrospectively collected and reviewed from the patients’ electronic medical records. Results: The median age of the patients was 62 years. The median lesion size was 14.3 mm (0-75.8 mm) when measured with DECT, 13.1 mm (0-99.1 mm) with MRI, and 13 mm (3-95 mm) with postoperative pathology. Spearman's rank correlation coefficient was 0.752 (p Subgroup analysis of factors, such as age and menopausal status, showed significant positive correlation in all subgroups. Conclusion: As compared to MRI, DECT is advantageous in that it can simultaneously perform a whole-body metastasis search and scan in the supine position, more closer to the surgical position. Furthermore, in this study, DECT was able to evaluate the extent of spread of lesions, including the intraductal extension, almost similar to that of MRI, suggesting that the diagnostic performance may not differ across subgroups. Additional analysis, such as evaluation of the size of infiltrated area alone, will be conducted and reported in future. Citation Format: Haruna Yamasawa, Akari Murakami, Megumi Takaoka, Erina Kusakabe, Reina Aoki, Kana Taguchi, Kanako Nishiyama, Michiko Yamashita, Wakana Sugimori, Mie Kurata, Mana Fukushima, Riko Kitazawa, Kanako Okada, Megumi Matsuda, Yoshiaki Kamei, Yasutsugu Takada. Evaluating the usefulness of dual-energy computed tomography for the preoperative diagnosis of the extent of spread of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-02-12.

Growing axons rely on local signaling at the growth cone for guidance cues. Semaphorin3A (Sema3A), a secreted repulsive axon guidance molecule, regulates synapse maturation and dendritic branching. We previously showed that local Sema3A signaling in the growth cones elicits retrograde retrograde signaling via PlexinA4 (PlexA4), one component of the Sema3A receptor, thereby regulating dendritic localization of AMPA receptor GluA2 and proper dendritic development. In present study, we found that nimodipine (voltage-gated L-type Ca(2+) channel blocker) and tetrodotoxin (TTX; voltage-gated Na(+) channel blocker) suppress Sema3A-induced dendritic localization of GluA2 and dendritic branch formation in cultured hippocampal neurons. The local application of nimodipine or TTX to distal axons suppresses retrograde transport of Venus-Sema3A that has been exogenously applied to the distal axons. Sema3A facilitates axonal transport of PlexA4, which is also suppressed in neurons treated with either TTX or nimodipine. These data suggest that voltage-gated calcium and sodium channels mediate Sema3A retrograde signaling that regulates dendritic GluA2 localization and branch formation.

Case 1 involved a 75-year-old woman with breast cancer and diffuse large B-cell lymphoma(DLBCL).Although we initially administered the R-CHOP regimen, the breast tumor increased in size and surgery had to be performed.After surgery, the R-CHOP regimen was re-initiated and DLBCL achieved clinical complete response.Case 2 involved a 74-year-old woman with breast cancer and gastric MALT lymphoma.After administration of rituximab and H. pylori eradication, a therapeutic effect was achieved in the lymphoma.A docetaxel and FEC regimen was continuously administered and surgery was performed. Case 3 involved a 62-year-old woman with breast cancer and follicular lymphoma.She presented with a history of DLBCL treatment.We performed mastectomy and sentinel lymph node biopsy, which revealed metastasis of breast cancer, and axillary lymph node dissection was subsequently performed.Considering the pathological stage, adjuvant chemotherapy was needed.We selected the TCH regimen based on her past treatment.In conclusion, it is necessary to treat patients with double presentation of breast cancer and malignant lymphoma through cooperation with different departments.

We report a case of microangiopathic hemolytic anemia(MHA)caused by metastatic breast cancer treated with weekly paclitaxel. A 58-year-old woman was diagnosed with metastatic breast cancer 2 years earlier. She was treated with various chemotherapy regimens and hormonal therapy, before being switched to fulvestrant 3 months earlier. She presented with severe anemia, and was diagnosed with MHA with bone marrow carcinomatosis following bone marrow biopsy. She was treated with weekly paclitaxel and recovered successfully. A subsequent biopsy showed that the bone marrow carcinomatosis had decreased. MHA due to breast cancer is rare and is associated with poor prognosis; however, rapid initiation of chemotherapy may be effective.

The mammalian intestinal epithelium has a unique organization in which crypts harboring stem cells produce progenitors and finally clonal populations of differentiated cells. Remarkably, the epithelium is replaced every 3–5 d throughout adult life. Disrupted maintenance of the intricate balance of proliferation and differentiation leads to loss of epithelial integrity or barrier function or to cancer. There is a tight correlation between the epigenetic status of genes and expression changes during differentiation; however, the mechanism of how changes in DNA methylation direct gene expression and the progression from stem cells to their differentiated descendants is unclear. Using conditional gene ablation of the maintenance methyltransferase Dnmt1, we demonstrate that reducing DNA methylation causes intestinal crypt expansion in vivo. Determination of the base-resolution DNA methylome in intestinal stem cells and their differentiated descendants shows that DNA methylation is dynamic at enhancers, which are often associated with genes important for both stem cell maintenance and differentiation. We establish that the loss of DNA methylation at intestinal stem cell gene enhancers causes inappropriate gene expression and delayed differentiation.

Collapsin response mediator protein 1 (CRMP1) and CRMP2 have been known as mediators of extracellular guidance cues such as semaphorin 3A and contribute to cytoskeletal reorganization in the axonal pathfinding process. To date, how CRMP1 and CRMP2 focally regulate axonal pathfinding in the growth cone has not been elucidated. To delineate the local functions of these CRMPs, we carried out microscale-chromophore-assisted light inactivation (micro-CALI), which enables investigation of localized molecular functions with highly spatial and temporal resolutions. Inactivation of either CRMP1 or CRMP2 in the neurite shaft led to arrested neurite outgrowth. Micro-CALI of CRMP2 in the central domain of the growth cones consistently arrested neurite outgrowth, whereas micro-CALI of CRMP1 in the same region caused significant lamellipodial retraction, followed by retardation of neurite outgrowth. Focal inactivation of CRMP1 in its half region of the growth cone resulted in the growth cone turning away from the irradiated site. Conversely, focal inactivation of CRMP2 resulted in the growth cone turning toward the irradiated site. These findings suggest different functions for CRMP1 and CRMP2 in growth cone behavior and neurite outgrowth. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012

The ability to detect harmful chemicals rapidly is essential for the survival of all animals. InCaenorhabditis elegans(C. elegans), repellents trigger an avoidance response, causing animals to move away from repellents. Dihydrocaffeic acid (DHCA) is a water-soluble repellent and nonflavonoid catecholic compound that can be found in plant products. Using aXenopus laevis(X. laevis) oocyte expression system, we identified a candidate dihydrocaffeic acid receptor (DCAR), DCAR-1. DCAR-1 is a novel seven-transmembrane protein that is expressed in the ASH avoidance sensory neurons ofC. elegans. dcar-1mutant animals are defective in avoidance response to DHCA, and cell-specific expression ofdcar-1in the ASH neurons ofdcar-1mutant animals rescued the defect in avoidance response to DHCA. Our findings identify DCAR-1 as the first seven-transmembrane receptor required for avoidance of a water-soluble repellent, DHCA, inC. elegans.

Parkinson's disease (PD) is a neurodegenerative disorder that results in a progressively debilitating loss of motor function and hypokinesia and is characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Recent evidence suggests that caspase-3-dependent proteolytic cleavage and nuclear translocation of the δ isoform of protein kinase C (PKCδ) may be required for oxidative stress-induced dopaminergic cell death. Whereas several proteins have been postulated to contribute to dopaminergic neuron loss, the signaling cascades that mediate this selective neuron loss in PD are not well understood. The presynaptic protein α-synuclein (α-syn), mutations in which cause familial PD, has been implicated in pathways that influence both neuronal protection and apoptosis. However, the activities of α-syn in PD have not been elucidated at the molecular level, and whether α-syn is neuroprotective or neurotoxic remains controversial. This Journal Club discusses recent research indicating that α-syn may protect against dopaminergic cell death by down-regulating PKCδ, a key molecule that mediates apoptosis in these cells. These findings are the first steps toward the understanding of critical signaling pathways that might be important in PD pathogenesis and represent potential targets for developing new therapies.

Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1+ cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1+ cells had proliferative potential. Foxl1+ cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1+ cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.

Net sodium balances in humans are maintained through various ion transporters expressed along the entire nephron. Among these ion transporters, epithelial sodium channels (ENaC) located along the aldosterone-sensitive distal nephron (ASDN) play a pivotal role in the homeostasis of sodium balance. This is supported by analyses of inherited hypertensive disorders, showing that genes encoding ENaC and other modulatory proteins cause hereditary hypertension, such as Liddle syndrome. Among various modulating proteins, E3 ubiquitin ligase, Nedd4L, binds the PY motif of ENaC COOH terminals and catalyzes ubiquitination of the NH 2 terminus of the protein for subsequent degradation. Both evolutionarily conserved and evolutionarily new C2 domains of human Nedd4L, a cryptic splice variant resulting in a disrupted isoform product formed by a frame-shift mutation, were reported previously. We focused on one of the isoforms, isoform I, generated by SNP (rs4149601), and studied its expression and interactions with other isoforms by molecular biological, immunohistochemical, and electrophysiological methods. We found that isoform I may interact with other human isoforms in a dominant-negative fashion. Such interactions might abnormally increase sodium reabsorption. Taken together, our analyses suggest that the human Nedd4L gene, especially the evolutionarily new isoform I, is a candidate gene for hypertension.

BODY: BACKGROUND/AIMS: Three-dimensional endomicroscopy using optical coherence tomography (3D-OCT) provides volumetric images of tissue microstructure without requiring extrinsic contrast agents. Commercially available OCT systems have limited frame rates and typically use proximal rotation and pullback mechanisms to scan the imaging beam across the tissue. These two factors limit the ability to generate high quality en face images of aberrant mucosal surface patterns, which correlate with the presence and grade of dysplasias. In this study, we present a prototype ultrahigh speed 3D-OCT system with an advanced imaging probe using distal micromotor scanning. This system enables en face 3D-OCT images that visualize the mucosal surface patterns in a variety of GI structures in vivo. METHODS: The study was performed at the VA Boston Healthcare System (VABHS) under a study protocol approved by the VABHS, Harvard Medical School and M.I.T. We used a prototype 3D-OCT system that has an imaging speed (axial scan rate) of 600 kHz, which is ~10 times faster than commercially available 3D-OCT imaging systems. Scanning of the imaging beam is performed using a micromotor at the distal end of the OCT probe. The probe was passed through the accessory channel of an Olympus double channel endoscope (GIF-2TH180) to allow coregistration of images with biopsies. RESULTS: The combined advantages of ultrahigh speed and distal scanning of the new 3D-OCT system and probe enables visualization of mucosal surface patterns with unprecedented clarity. Fig. 1(A) demonstrates a volumetric reconstruction of a 3D-OCT dataset acquired in the gastric body of a patient. While cross sectional images give information on the layered architecture of the tissue, gastric folds and pit pattern can be visualized in the en face direction. Fig. 1(C) shows the squamocolumnar junction (SCJ) of a patient with Barrett's esophagus (BE), where the squamous epithelium (SE) appears as a smooth layer, whereas the area with BE has a well-defined pit pattern architecture. Fig. 1(D) shows a region of the esophagus a BE patient immediately after radiofrequency ablation (RFA), where residual BE that is missed by the RFA application can be observed. CONCLUSIONS: This study demonstrates that ultrahigh speed 3D-OCT system with a distal scanning micromotor probe can generate cross-sectional and en face images from the same dataset, yielding a more comprehensive evaluation of tissue architecture. Moreover, imaging can be performed even after biopsies or ablations, and can survey margins of endoscopic resections which is not possible with confocal endomicroscopy using exogenous contrast agents. Therefore, this novel imaging system has the potential to greatly improve the clinical utility of 3D-OCT. ACKNOWLEDGEMENT: NIH 5R01-CA075289-16, R44-CA101067-06, AFOSR FA9550-12-1-0499, and FA9550-10-10551. Figure 1: (A) Volumetric reconstruction of a 3D-OCT dataset acquired in the gastric body of a patient showing cross sectional as well as en face visualization of the gastric folds and pit patterns. (B) Zoomed view of the en face image shown in (A). (C) Squamocolumnar junction (SCJ) of a patient with Barrett's esophagus (BE) showing a region with smooth squamous epithelium and the BE region with well-defined pit patterns. (D) A region of a patient with BE immediately after radiofrequency ablation (RFA), where the encircled area shows BE glands that are likely missed by the RFA. Scale bars: 500um.

Background & Aims: Intestinal epithelial stem cells that express leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) and/or B cell specific Moloney murine leukemia virus integration site 1 (Bmi1) continuously replicate and generate differentiated cells throughout life. Previously, Paneth cells were suggested to constitute an epithelium-intrinsic niche that regulates the behavior of these stem cells. However, ablating Paneth cells has no effect on the maintenance of functional stem cells. Here, we show definitively that a small subset of mesenchymal subepithelial cells expressing the winged-helix transcription factor forkhead box l1 (Foxl1) are a critical component of the intestinal stem cell niche. Methods: We genetically ablated Foxl1+ mesenchymal cells in adult mice using 2 separate models by expressing either the human or simian diphtheria toxin receptor under Foxl1 promoter control. Conclusions: Killing Foxl1+ cells by diphtheria toxin administration led to an abrupt cessation of proliferation of both epithelial stem- and transit-amplifying progenitor cell populations that was associated with a loss of active Wnt signaling to the intestinal epithelium. Therefore, Foxl1-expressing mesenchymal cells constitute the fundamental niche for intestinal stem cells. Keywords: Intestinal Stem Cell Niche, Wnt, Mesenchyme