CAR expression in invasive breast carcinoma and its effect on adenovirus transduction efficiency.

BACKGROUND: Breast cancer is the second leading cause of death in women, with invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) as the two most common forms of invasive breast cancer. While estrogen receptor positive (ER+) IDC and ILC are treated similarly, the multifocality of ILC presents challenges in detection and treatment, worsening long-term clinical outcomes in patients. With increasing documentation of chemoresistance in ILC, additional treatment options are needed. Oncolytic adenoviral therapy may be a promising option, but cancer cells must express the coxsackievirus & adenovirus receptor (CAR) for adenoviral therapy to be effective. The present study aims to evaluate the extent to which CAR expression is observed in ILC in comparison to IDC, and how the levels of CAR expression correlate with adenovirus transduction efficiency. The effect of liposome encapsulation on transduction efficiency is also assessed. METHODS: To characterize CAR expression in invasive breast carcinoma, 36 formalin-fixed paraffin-embedded (FFPE) human breast tumor samples were assayed by CAR immunohistochemistry (IHC). Localization of CAR in comparison to other junctional proteins was performed using a multiplex immunofluorescence panel consisting of CAR, p120-catenin, and E-cadherin. ILC and IDC primary tumors and cell lines were transduced with E1- and E3-deleted adenovirus type 5 inserted with a GFP transgene (Ad-GFP) and DOTAP liposome encapsulated Ad-GFP (DfAd-GFP) at various multiplicities of infection (MOIs). Transduction efficiency was measured using a fluorescence plate reader. CAR expression in the human primary breast carcinomas and cell lines was also evaluated by IHC. RESULTS: We observed membranous CAR, p120-catenin and E-cadherin expression in IDC. In ILC, we observed cytoplasmic expression of CAR and p120-catenin, with absent E-cadherin. Adenovirus effectively transduced high-CAR IDC cell lines, at MOIs as low as 12.5. Ad-GFP showed similar transduction as DfAd-GFP in high-CAR IDC cell lines. Conversely, Ad-GFP transduction of ILC cell lines was observed only at MOIs of 50 and 100. Furthermore, Ad-GFP did not transduce CAR-negative IDC cell lines even at MOIs greater than 100. Liposome encapsulation (DfAd-GFP) improved transduction efficiency 4-fold in ILC and 17-fold in CAR-negative IDC cell lines. CONCLUSION: The present study demonstrates that oncolytic adenoviral therapy is less effective in ILC than IDC due to differences in spatial CAR expression. Liposome-enhanced delivery may be beneficial for patients with ILC and tumors with low or negative CAR expression to improve adenoviral therapeutic effectiveness.

Coxsackievirus and Adenovirus Receptor (CXADR): Recent Findings and Its Role and Regulation in Spermatogenesis.

Coxsackievirus and adenovirus receptor (CXADR) belongs to immunoglobulin superfamily of cell adhesion molecules. It expresses in most tissues, but displays unique and indispensable functions in some tissues such as heart and testis. CXADR is a multifunctional protein that can serve as a viral receptor, a junction structural protein and a signalling molecule. Thus, it exerts a wide range of functions such as facilitating leukocyte transmigration, regulating barrier function and cell adhesion, promoting EMT transition, and mediating spermatogenesis. This review aims to provide an overview and highlights some recent findings on CXADR in the field with emphasis on studies in the testis, upon which future studies can be designed to delineate the roles and regulation of CXADR in spermatogenesis.

Coxsackievirus B3 Infection of Human iPSC Lines and Derived Primary Germ-Layer Cells Regarding Receptor Expression.

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.

CXADR-like membrane protein protects against heart injury by preventing excessive pyroptosis after myocardial infarction.

Myocardial infarction (MI) results in cardiomyocyte death and ultimately leads to heart failure. Pyroptosis is a type of the inflammatory programmed cell death that has been found in various diseased tissues. However, the role of pyroptosis in MI heart remains unknown. Here, we showed that CXADR-like membrane protein (CLMP) was involved in pyroptosis in the mouse MI heart. Our data showed that CLMP was strongly expressed in fibroblasts of the infarcted mouse hearts. The Clmp+/- mice showed more serious myocardial fibrosis and ventricular dysfunction post-MI than wild-type (Clmp+/+ ) mice, indicating a protective effect of the fibroblast-expressed CLMP against MI-induced heart damage. Transcriptome analyses by RNA sequencing indicated that Il-1ß mRNA was significantly increased in the MI heart of Clmp+/- mouse, which indicated a more serious inflammatory response. Meanwhile, cleaved caspase-1 and Gasdermin D were significantly increased in the Clmp+/- MI heart, which demonstrated enhanced pyroptosis in the Clmp knockdown heart. Further analysis revealed that the pyroptosis mainly occurred in cardiac fibroblasts (CFs). Compared to wild-type fibroblasts, Clmp+/- CFs showed more serious pyroptosis and inflammatory after LPS plus nigericin treatment. Collectively, our results indicate that CLMP participates in the pyroptotic and inflammatory response of CFs in MI heart. We have provided a novel pyroptotic insight into the ischaemic heart, which might hold substantial potential for the treatment of MI.

Naturally occurring variants in the transmembrane and cytoplasmic domains of the human Coxsackie- and adenovirus receptor have no impact on virus internalisation.

The Coxsackie- and adenovirus receptor (CAR) mediates homophilic cell-cell contacts and susceptibility to both human pathogenic viruses through its membrane-distal immunoglobulin domain. In the present study, we screened five missense variants of the human CAR gene for their influence on adenovector or Coxsackievirus entry into Chinese hamster ovary cells. The CAR variants facilitated virus internalisation to a similar extent as wild type CAR. This underlines CAR's presumed invariance and essential physiological role in embryogenesis.

The Coxsackievirus and Adenovirus Receptor, a Required Host Factor for Recovirus Infection, Is a Putative Enteric Calicivirus Receptor.

Human norovirus (HuNoV) is a leading cause of acute gastroenteritis in both developed and developing countries. Studies of HuNoV host cell interactions are limited by the lack of a simple, robust cell culture system. Due to their diverse HuNoV-like biological features, including histo-blood group antigen (HBGA) binding, rhesus enteric caliciviruses (ReCVs) are viable surrogate models for HuNoVs. In addition, several ReCV strains can be propagated to high titers in standard nonhuman primate cell lines while causing lytic infection and cell death. To identify the ReCV entry receptor, we performed CRISPR/Cas9 library screening in Vero cells, which identified the coxsackievirus and adenovirus receptor (CAR) as a candidate ReCV entry receptor. We showed that short interfering RNA, anti-human CAR (hCAR) monoclonal antibody RmcB treatment, and recombinant hCAR ectodomain blocked ReCV replication in LLC-MK2 cells. CRISPR/Cas9-targeted knockout of CAR in LLC-MK2 and Vero cells made these cell lines resistant to ReCV infection, and susceptibility to infection could be restored by transient expression of CAR. CHO cells do not express CAR or HBGAs and are resistant to ReCV infection. Recombinant CHO cells stably expressing hCAR or the type B HBGA alone did not support ReCV infection. However, CHO cells expressing both hCAR and the type B HBGA were susceptible to ReCV infection. In summary, we have demonstrated that CAR is required for ReCV infection and most likely is a functional ReCV receptor, but HBGAs are also necessary for infection.IMPORTANCE Because of the lack of a simple and robust human norovirus (HuNoV) cell culture system surrogate, caliciviruses still represent valuable research tools for norovirus research. Due to their remarkable biological similarities to HuNoVs, including the utilization of HBGAs as putative attachment receptors, we used rhesus enteric caliciviruses (ReCVs) to study enteric calicivirus host cell interactions. Using CRISPR/Cas9 library screening and functional assays, we identified and validated the coxsackievirus and adenovirus receptor (CAR) as a functional proteinaceous receptor for ReCVs. Our work demonstrated that CAR and HBGAs both are necessary to convert a nonsusceptible cell line to being susceptible to ReCV infection. Follow-up studies to evaluate the involvement of CAR in HuNoV infections are ongoing.

Identification of novel human adenovirus candidates using the coxsackievirus and adenovirus receptor for cell entry.

BACKGROUND: There are over 100 known human adenovirus (HAdV) types, which are able to cause a broad variety of different self-limiting but also lethal diseases especially in immunocompromised patients. Only limited information about the pathogenesis and biology of the majority of these virus types is available. In the present study, we performed a systematic screen for coxsackievirus and adenovirus receptor (CAR)-usage of a large spectrum of HAdV types. METHODS: To study receptor usage we utilized a recombinant HAdV library containing HAdV genomes tagged with a luciferase and GFP encoding transgene. We infected CHO-CAR cells stably expressing the CAR receptor and to much information with tagged viruses (HAdV3, 14, 16, 50, 10, 24, 27, 37 and 69) and measured luciferase expression levels 26 and for some viruses (AdV10, - 24 and - 27) 52 h post-infection. As positive control, we applied human adenovirus type 5 (HAdV5) known to use the CAR receptor for cell entry. For viruses replication studies on genome level we applied digital PCR. RESULTS: Infection of CHO-CAR and CHO-K1 cells at various virus particle numbers per cell (vpc) revealed that HAdV10, 24, and 27 showed similar or decreased luciferase expression levels in the presence of CAR. In contrast, HAdV3, 14, 16, 50, 37 and 69 resulted in increased luciferase expression levels in our initial screening experiments. CAR usage of HAdV3, 14, 50, and 69 was not studied before, and therefore we experimentally confirmed CAR usage for these HAdV as novel viruses utilizing CAR as a receptor. To rule out that replication of HAdV in transduced CHO cells is responsible for increased transduction rates we performed replication assays on virus genome level, which revealed that there is no HAdV replication. CONCLUSION: In the present study, we screened a HAdV library and identified novel human HAdV using the CAR receptor. To our knowledge, this is the first description of CAR usage for HAdV 3, 14, 50, and 69.

Adenovirus Receptor Expression in Cancer and Its Multifaceted Role in Oncolytic Adenovirus Therapy.

Oncolytic adenovirus therapy is believed to be a promising way to treat cancer patients. To be able to target tumor cells with an oncolytic adenovirus, expression of the adenovirus receptor on the tumor cell is essential. Different adenovirus types bind to different receptors on the cell, of which the expression can vary between tumor types. Pre-existing neutralizing immunity to human adenovirus species C type 5 (HAdV-C5) has hampered its therapeutic efficacy in clinical trials, hence several adenoviral vectors from different species are currently being developed as a means to evade pre-existing immunity. Therefore, knowledge on the expression of appropriate adenovirus receptors on tumor cells is important. This could aid in determining which tumor types would benefit most from treatment with a certain oncolytic adenovirus type. This review provides an overview of the known receptors for human adenoviruses and how their expression on tumor cells might be differentially regulated compared to healthy tissue, before and after standardized anticancer treatments. Mechanisms behind the up- or downregulation of adenovirus receptor expression are discussed, which could be used to find new targets for combination therapy to enhance the efficacy of oncolytic adenovirus therapy. Additionally, the utility of the adenovirus receptors in oncolytic virotherapy is examined, including their role in viral spread, which might even surpass their function as primary entry receptors. Finally, future directions are offered regarding the selection of adenovirus types to be used in oncolytic adenovirus therapy in the fight against cancer.

The Cxadr-Adam10 complex plays pivotal roles in tight junction integrity and early trophoblast development in mice.

Understanding preimplantation embryo development has important implications for assisted reproductive technologies (ARTs) after the introduction of in vitro fertilisation and embryo transfer because most embryonic losses occur during pre/peri-implantation. Recent studies have shown that tight junctions (TJs) are important components for embryos to develop to the blastocyst stage. However, their biological function after cavitation has not been extensively studied. We examined TJ assembly focusing on coxsackievirus and adenovirus receptor (Cxadr) and A disintegrin and metalloproteinase 10 (Adam10) using siRNA and/or an Adam10-specific inhibitor (GI254023X). TJ-associated genes, including occludin and tight junction protein 1 (Tjp1), were downregulated in the Cxadr knockdown (KD) embryos but were unaltered in Adam10 KD embryos. However, Adam10 KD or chemical inhibition affected subcellular localisation of Adam10, Cxadr, and Tjp1, leading to disrupted TJ assembly. Furthermore, Cxadr KD or GI254023X-treated blastocysts showed a relatively smaller outgrowth area and aberrant expression of transcription factor AP-2γ, a trophoblast-specific marker in the in vitro embryo outgrowth assay. In summary, we demonstrated that the Cxadr-Adam10 complex might moderate TJ integrity/stability and play pivotal roles during early embryonic development. Collectively, understanding the establishment of the TJ complex and its integrity will provide insight into translational research for predicting and selecting developmental competency for ART.

External validation of molecular subtype classifications of colorectal cancer based on microsatellite instability, CIMP, BRAF and KRAS.

BACKGROUND: Competing molecular classification systems have been proposed to complement the TNM staging system for a better prediction of survival in colorectal cancer (CRC). However, validation studies are so far lacking. The aim of this study was to validate and extend previously published molecular classifications of CRC in a large independent cohort of CRC patients. METHODS: CRC patients were recruited into a population-based cohort study (DACHS). Molecular subtypes were categorized based on three previously published classifications. Cox-proportional hazard models, based on the same set of patients and using the same confounders as reported by the original studies, were used to determine overall, cancer-specific, or relapse-free survival for each subtype. Hazard ratios and confidence intervals, as well as Kaplan-Meier plots were compared to those reported by the original studies. RESULTS: We observed similar patterns of worse survival for the microsatellite stable (MSS)/BRAF-mutated and MSS/KRAS-mutated subtypes in our validation analyses, which were included in two of the validated classifications. Of the two MSI subtypes, one defined by additional presence of CIMP-high and BRAF-mutation and the other by tumors negative for CIMP, BRAF and KRAS-mutations, we could not confirm associations with better prognosis as suggested by one of the classifications. For two of the published classifications, we were able to provide results for additional subgroups not included in the original studies (men, other disease stages, other locations). CONCLUSIONS: External validation of three previously proposed classifications confirmed findings of worse survival for CRC patients with MSS subtypes and BRAF or KRAS mutations. Regarding MSI subtypes, other patient characteristics such as stage of the tumor, may influence the potential survival benefit. Further integration of methylation, genetic, and immunological information is needed to develop and validate a comprehensive classification that will have relevance for use in clinical practice.

Relationship between Cell Receptors and Tumor Cell Sensitivity to Oncolytic Enteroviruses.

Replicative ability of 5 oncolytic enterovirus strains was evaluated on a panel of 18 human normal and tumor cells. The capacity of each cell line to support replication of enterovirus strains varied. Cell lines weakly replicating one virus could be highly sensitive to another viral strain. Differences in the expression of CXADR cell receptor did not correlate with susceptibility to infection and replication of Coxsackie B virus, but complete inactivation of CXADR gene and poliovirus receptor gene (PVR) led to loss of the sensitivity to Coxsackie B5 and poliovirus, respectively. Detection of additional expression markers will contribute to understanding the causes of different sensitivity of tumor cells to viruses.

Coxsackievirus Adenovirus Receptor Loss Impairs Adult Neurogenesis, Synapse Content, and Hippocampus Plasticity.

UNLABELLED: Although we are beginning to understand the late stage of neurodegenerative diseases, the molecular defects associated with the initiation of impaired cognition are poorly characterized. Here, we demonstrate that in the adult brain, the coxsackievirus and adenovirus receptor (CAR) is located on neuron projections, at the presynapse in mature neurons, and on the soma of immature neurons in the hippocampus. In a proinflammatory or diseased environment, CAR is lost from immature neurons in the hippocampus. Strikingly, in hippocampi of patients at early stages of late-onset Alzheimer's disease (AD), CAR levels are significantly reduced. Similarly, in triple-transgenic AD mice, CAR levels in hippocampi are low and further reduced after systemic inflammation. Genetic deletion of CAR from the mouse brain triggers deficits in adult neurogenesis and synapse homeostasis that lead to impaired hippocampal plasticity and cognitive deficits. We propose that post-translational CAR loss of function contributes to cognitive defects in healthy and diseased-primed brains. SIGNIFICANCE STATEMENT: This study addressed the role of the coxsackievirus and adenovirus receptor (CAR), a single-pass cell adhesion molecule, in the adult brain. Our results demonstrate that CAR is expressed by mature neurons throughout the brain. In addition, we propose divergent roles for CAR in immature neurons, during neurogenesis, and at the mature synapse. Notably, CAR loss of function also affects hippocampal plasticity.

The Coxsackievirus and Adenovirus Receptor: Glycosylation and the Extracellular D2 Domain Are Not Required for Coxsackievirus B3 Infection.

UNLABELLED: The coxsackievirus and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily (IgSF) and functions as a receptor for coxsackie B viruses (CVBs). The extracellular portion of CAR comprises two glycosylated immunoglobulin-like domains, D1 and D2. CAR-D1 binds to the virus and is essential for virus infection; however, it is not known whether D2 is also important for infection, and the role of glycosylation has not been explored. To understand the function of these structural components in CAR-mediated CVB3 infection, we generated a panel of human (h) CAR deletion and substitution mutants and analyzed their functionality as CVB receptors, examining both virus binding and replication. Lack of glycosylation of the CAR-D1 or -D2 domains did not adversely affect CVB3 binding or infection, indicating that the glycosylation of CAR is not required for its receptor functions. Deletion of the D2 domain reduced CVB3 binding, with a proportionate reduction in the efficiency of virus infection. Replacement of D2 with the homologous D2 domain from chicken CAR, or with the heterologous type C2 immunoglobulin-like domain from IgSF11, another IgSF member, fully restored receptor function; however, replacement of CAR-D2 with domains from CD155 or CD80 restored function only in part. These data indicate that glycosylation of the extracellular domain of hCAR plays no role in CVB3 receptor function and that CAR-D2 is not specifically required. The D2 domain may function largely as a spacer permitting virus access to D1; however, the data may also suggest that D2 affects virus binding by influencing the conformation of D1. IMPORTANCE: An important step in virus infection is the initial interaction of the virus with its cellular receptor. Although the role in infection of the extracellular CAR-D1, cytoplasmic, and transmembrane domains have been analyzed extensively, nothing is known about the function of CAR-D2 and the extracellular glycosylation of CAR. Our data indicate that glycosylation of the extracellular CAR domain has only minor importance for the function of CAR as CVB3 receptor and that the D2 domain is not essential per se but contributes to receptor function by promoting the exposure of the D1 domain on the cell surface. These results contribute to our understanding of the coxsackievirus-receptor interactions.

Marine Lectins DlFBL and HddSBL Fused with Soluble Coxsackie-Adenovirus Receptor Facilitate Adenovirus Infection in Cancer Cells BUT Have Different Effects on Cell Survival.

Cancer development and progression are usually associated with glycosylation change, providing prognostic and diagnostic biomarkers, as well as therapeutic targets, for various cancers. In this work, Dicentrarchus labrax fucose binding lectin (DlFBL) and Haliotis discus discus sialic acid binding lectin (HddSBL) were genetically fused with soluble coxsackie-adenovirus receptor (sCAR), and produced through a bacterial expression system. Results showed that recombinant sCAR-DlFBL not only facilitated adenovirus Ad-EGFP infection in K562/ADR and U87MG cells, but also enhanced the cytotoxicity of adenovirus harboring gene encoding Pinellia pedatisecta agglutinin (PPA) or DlFBL (Ad-PPA or Ad-DlFBL) on U87MG cells through inducing apoptosis. Recombinant sCAR-HddSBL facilitated Ad-EGFP infection, but dramatically counteracted the cytotoxicity of both Ad-PPA and Ad-DlFBL in U87MG cells. Further analysis revealed that sCAR-HddSBL, but not sCAR-DlFBL, significantly upregulated transcription factor E2F1 levels in U87MG cells, which might be responsible for the adverse effect of sCAR-HddSBL on Ad-PPA and Ad-DlFBL. Taken together, our data suggested that sCAR-DlFBL could be further developed to redirect therapeutic adenoviruses to infect cancer cells such as U87MG, and the sCAR-lectin fusion proteins for adenoviral retargeting should be carefully examined for possible survival signaling induced by lectins, such as HddSBL.

A novel Cre recombinase reporter mouse strain facilitates selective and efficient infection of primary immune cells with adenoviral vectors.

Replication-deficient recombinant adenoviruses are potent vectors for the efficient transient expression of exogenous genes in resting immune cells. However, most leukocytes are refractory to efficient adenoviral transduction as they lack expression of the coxsackie/adenovirus receptor (CAR). To circumvent this obstacle, we generated the R26/CAG-CARΔ1(StopF) (where R26 is ROSA26 and CAG is CMV early enhancer/chicken ß actin promoter) knock-in mouse line. This strain allows monitoring of in situ Cre recombinase activity through expression of CARΔ1. Simultaneously, CARΔ1 expression permits selective and highly efficient adenoviral transduction of immune cell populations, such as mast cells or T cells, directly ex vivo in bulk cultures without prior cell purification or activation. Furthermore, we show that CARΔ1 expression dramatically improves adenoviral infection of in vitro differentiated conventional and plasmacytoid dendritic cells (DCs), basophils, mast cells, as well as Hoxb8-immortalized hematopoietic progenitor cells. This novel dual function mouse strain will hence be a valuable tool to rapidly dissect the function of specific genes in leukocyte physiology.

The Intracellular Domain of the Coxsackievirus and Adenovirus Receptor Differentially Influences Adenovirus Entry.

UNLABELLED: The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule used as a docking molecule by some adenoviruses (AdVs) and group B coxsackieviruses. We previously proposed that the preferential transduction of neurons by canine adenovirus type 2 (CAV-2) is due to CAR-mediated internalization. Our proposed pathway of CAV-2 entry is in contrast to that of human AdV type 5 (HAdV-C5) in nonneuronal cells, where internalization is mediated by auxiliary receptors such as integrins. We therefore asked if in fibroblast-like cells the intracellular domain (ICD) of CAR plays a role in the internalization of the CAV-2 fiber knob (FK(CAV)), CAV-2, or HAdV-C5 when the capsid cannot engage integrins. Here, we show that in fibroblast-like cells, the CAR ICD is needed for FK(CAV) entry and efficient CAV-2 transduction but dispensable for HAdV-C5 and an HAdV-C5 capsid lacking the RGD sequence (an integrin-interacting motif) in the penton. Moreover, the deletion of the CAR ICD further impacts CAV-2 intracellular trafficking, highlighting the crucial role of CAR in CAV-2 intracellular dynamics. These data demonstrate that the CAR ICD contains sequences important for the recruitment of the endocytic machinery that differentially influences AdV cell entry. IMPORTANCE: Understanding how viruses interact with the host cell surface and reach the intracellular space is of crucial importance for applied and fundamental virology. Here, we compare the role of a cell adhesion molecule (CAR) in the internalization of adenoviruses that naturally infect humans and Canidae. We show that the intracellular domain of CAR differentially regulates AdV entry and trafficking. Our study highlights the mechanistic differences that a receptor can have for two viruses from the same family.

CXADR is required for AJ and TJ assembly during porcine blastocyst formation.

Coxsackie virus and adenovirus receptor (CXADR) is a member of the immunoglobulin superfamily as well as a member of the junctional adhesion molecule family of adhesion receptor. In human pre-implantation embryos, CXADR was detected and co-localized with tight junction (TJ) proteins on the membrane of the trophectoderm. However, its physiological roles were not elucidated in terms of blastocyst formation. Here, we reported expression patterns and biological functions of CXADR in porcine pre-implantation embryos. The transcripts of CXADR were detected at all stages of pre-implantation. Particularly, its expression dramatically increased and preferentially localized at the edge of cell-cell contacts, rather than in the nucleus from the eight-cell stage onwards. CXADR expression was knocked down (KD) by microinjecting double-stranded RNA into one-cell parthenotes. The vast majority of CXADR KD embryos failed to develop to the blastocyst stage, and a few developed KD blastocysts did not expand fully. Analysis of adherens junction (AJ)- and TJ-associated genes/proteins using qRT-PCR, immunocytochemistry and assessment of TJ permeability using FITC-dextran uptake assay revealed that the developmental failure and relatively small cavities are attributed to the defects of TJ assembly. In summary, CXADR is necessary for the AJ and TJ assembly/biogenesis during pre-implantation development.

Single-step concentration and purification of adenoviruses by coxsackievirus-adenovirus receptor-binding capture and elastin-like polypeptide-mediated precipitation.

A single-step method for quick concentration and purification of adenoviruses (Ads) was established by combining coxsackievirus and adenovirus receptor (CAR)-binding capture with elastin-like polypeptide (ELP)-mediated precipitation. The soluble ELP-CAR fusion protein was expressed in vector-transformed E. coli and purified to high purity by two rounds of inverse transition cycling (ITC). After demonstration of the specific binding of fusion protein, a recombinant Ad (rAd), namely rAd/GFP, was pulled down from the culture medium and extract of rAd-transduced cells using ELP-CAR protein, with recovery of 76.2 % and 73.3 %, respectively. The rAd was eluted from the ELP-CAR protein and harvested by one round of ITC, with recoveries ranging from 30.6 % to 34.5 % (virus titration assay). Both ELP-CAR-bound and eluted rAds were able to transduce CAR-positive cells, but not CAR-negative cells (fluorescent microscopy). A further viral titration assay showed that the ELP-CAR-bound rAd/GFP had significantly lower transduction efficiency than the eluted rAd, and there was less of a decrease when tested in the presence of fetal bovine serum. In addition, rAd/GFP was efficiently recovered from the "spiked" PBS and tap water with recovery of ~74 % or ~60 %. This work demonstrates the usefulness of the ELP-CAR-binding capture method for concentration and/or purification of Ads in cellular and environmental samples.

CXADR-like membrane protein (CLMP) in the rat ovary: stimulation by human chorionic gonadotrophin during the periovulatory period.

CXADR-like membrane protein (CLMP) is a novel cell-cell adhesion molecule. The present study investigated the spatiotemporal expression pattern of CLMP and its regulation in the rat ovary during the periovulatory period. Real-time polymerase chain reaction analysis revealed that Clmp mRNA was rapidly stimulated in intact ovaries by 4h after human chorionic gonadotrophin (hCG) treatment. In situ hybridisation analysis demonstrated that Clmp mRNA expression was stimulated in theca cells at 4h after hCG and remained elevated until 12h. Clmp mRNA was also upregulated in granulosa cells and was present in forming corpora lutea. Our data indicate that the protein kinase A but not the protein kinase C pathway regulates the expression of Clmp mRNA in granulosa cells. Phosphatidylinositol 3 kinase and p38 kinase are also involved in regulating Clmp mRNA expression. The stimulation of Clmp mRNA by hCG requires new protein synthesis. Furthermore, inhibition of epidermal growth factor receptor activation significantly inhibited Clmp mRNA expression, whereas inhibition of prostaglandin synthesis or progesterone action had no effect. The stimulation of CLMP in the rat ovary may be important in cell adhesion events during ovulation and luteal formation such as maintaining the structure and communication of ovarian follicular and luteal cells.

Disruption of the coxsackievirus and adenovirus receptor-homodimeric interaction triggers lipid microdomain- and dynamin-dependent endocytosis and lysosomal targeting.

The coxsackievirus and adenovirus receptor (CAR) serves as a docking factor for some adenovirus (AdV) types and group B coxsackieviruses. Its role in AdV internalization is unclear as studies suggest that its intracellular domain is dispensable for some AdV infection. We previously showed that in motor neurons, AdV induced CAR internalization and co-transport in axons, suggesting that CAR was linked to endocytic and long-range transport machineries. Here, we characterized the mechanisms of CAR endocytosis in neurons and neuronal cells. We found that CAR internalization was lipid microdomain-, actin-, and dynamin-dependent, and subsequently followed by CAR degradation in lysosomes. Moreover, ligands that disrupted the homodimeric CAR interactions in its D1 domains triggered an internalization cascade involving sequences in its intracellular tail.