Similar Localization Pattern Research Articles

Arabidopsis EXO70A1 recruits Patellin3 to the cell membrane independent of its role as an exocyst subunit.

The exocyst is a well-known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast-two-hybrid (Y2H) analysis, we found that EXO70A1 interacted with the GOLD domain of Patellin3 (PATL3). The direct EXO70A1-PATL3 interaction was supported by in vitro and in vivo experiments. In Arabidopsis, PATL3-GFP colocalized with EXO70A1 predominantly at the cell membrane, and PATL3 localization was insensitive to BFA and TryA23. Remarkably, in the exo70a1 mutant, PATL3 proteins accumulated as punctate structures within the cytosol, which did not colocalize with several endomembrane compartment markers, and was insensitive to BFA. Furthermore, PATL3 localization was not changed in the exo70e2, PRsec6 or exo84b mutants. These data suggested that EXO70A1, but not other exocyst subunits, was responsible for PATL3 localization, which is independent of its role in secretory/recycling vesicle-tethering/fusion. Both EXO70A1 and PATL3 were shown to bind PI4P and PI(4,5)P2 in vitro. Evidence was obtained that the other four members of the PATL family bound to EXO70A1 as well, and shared a similar localization pattern as PATL3. These findings offered new insights into exocyst subunit-specific function, and provided data and tools for further characterization of PATL family proteins.

Distinguishing Cloud and Snow in Satellite Images via Deep Convolutional Network

Cloud and snow detection has significant remote sensing applications, while they share similar low-level features due to their consistent color distributions and similar local texture patterns. Thus, accurately distinguishing cloud from snow in pixel level from satellite images is always a challenging task with traditional approaches. To solve this shortcoming, in this letter, we proposed a deep learning system to classify cloud and snow with fully convolutional neural networks in pixel level. Specifically, a specially designed fully convolutional network was introduced to learn deep patterns for cloud and snow detection from the multispectrum satellite images. Then, a multiscale prediction strategy was introduced to integrate the low-level spatial information and high-level semantic information simultaneously. Finally, a new and challenging cloud and snow data set was labeled manually to train and further evaluate the proposed method. Extensive experiments demonstrate that the proposed deep model outperforms the state-of-the-art methods greatly both in quantitative and qualitative performances.

A novel framework based on biclustering for automatic epileptic seizure detection

Automatic epileptic seizure detection based on electroencephalogram is crucial to epilepsy diagnosis and treatment. However, the large numbers of time series make it quite challenging to establish a high performance automatic detection method. Considering different physiological states of the brain could be characterized by distinct combinations or interactions of similar discontinuous local temporal patterns, a novel framework based on biclustering for automatic epileptic seizure detection is proposed in this paper. First, the CC algorithm is used to identify similar discontinuous local temporal patterns. Then, the bicluster membership matrix using a new similarity measurement is constructed to reduce the dimensionality. At last, the ELM classifier is adopted to discriminate between epileptic seizure and seizure-free EEG signals. With extensive comparative studies and evaluations on the publicly available Bonn epileptic EEG dataset, it indicates that the proposed framework could not only automatically detect or predict an epilepsy seizure with high performances with respect to accuracy, robustness and efficiency, but also implicitly provide valuable knowledge for studying the mechanisms of epilepsy.

NOTCH1 activates the Wnt/β-catenin signaling pathway in colon cancer.

Purpose and MethodsThe translocation of β-catenin/CTNNB1 to the nucleus activates Wnt signaling and cell proliferation; however, the precise mechanism underlying this phenomenon remains unknown. Previous reports have provided evidence that NOTCH1 is involved in the Wnt signaling pathway. Therefore, we sought to determine the mechanism by which NOTCH1 influences the Wnt/β-catenin pathway. We constructed a vector expressing the NOTCH1 intracellular domain (NICD1) and transfected the vector into HCT116 which has low expression of NICD1. Furthermore, inhibition of NOTCH signal pathway in SW480 which has abundant NICD1 expression, was performed by transfection of siNICD1 or DAPT, gamma secretase inhibitor, treatment. In addition, we evaluated NICD1 and β-catenin localization in colon cancer cell lines and in 189 colon cancer tissue samples and analyzed the correlation between the nuclear localization of NICD1 and the clinicopathological features of colon cancer patients.ResultsImmunohistochemical assays demonstrated that NICD1 and β-catenin exhibited a similar localization pattern in colon cancer tissues. In addition, we found that NICD1 induced the translocation of β-catenin to the nucleus and that NICD1 and β-catenin co-localized in the nucleus. Overexpression of NICD1 increased luciferase activity of Wnt signal pathway. On the other hand, reduction of NICD1 reduced luciferase activity of Wnt signaling pathway. In the 189 analyzed colon cancer cases, multivariate COX regression analysis demonstrated the independent prognostic impact of nuclear localization of NICD1(p=0.0376).ConclusionNOTCH1 plays a key role in the Wnt pathway and activation of NOTCH1 is associated with the translocation of β-catenin to the nucleus.

Co-expression of podoplanin and fibroblast growth factor 1 predicts poor prognosis in patients with lung squamous cell carcinoma.

Podoplanin and fibroblast growth factor (FGF) 1 have been detected more frequently in lung squamous cell carcinoma (SQCC) compared with lung adenocarcinoma. Furthermore, it has been previous demonstrated that FGF1 is located on the edge of tumor nests in certain lung SQCC sections, which resembles the characteristic expression pattern of podoplanin. Podoplanin and FGF1 have roles in lymphangiogenesis and angiogenesis. Based on their consistently specific expression in lung SQCC and similar localization patterns, the present study aimed to investigate whether the expression of podoplanin in tumor cells is correlated with FGF1 expression in lung SQCC and whether their co-expression has clinicopathological significance, particularly for lymphangiogenesis/angiogenesis. The correlation between podoplanin and FGF1 expression in tumor cells of 82 lung SQCC cases was investigated by immunohistochemical staining and the association between the co-expression of podoplanin and FGF1, and clinicopathological factors such as microvessel density (MVD), was examined in these samples. In addition, the prognostic value of co-expression of podoplanin and FGF1 in tumor cells was determined, and the regulation of FGF1 expression and angiogenesis by podoplanin was examined in vitro in a human lung SQCC cell line. Immunohistochemical analysis demonstrated that there was a significant correlation between podoplanin and FGF1 expression in lung SQCC tumor cells (R=0.591; P<0.0001). Co-expression of podoplanin and FGF1 was significantly associated with larger primary tumor size, advanced TNM stage and higher intratumoral MVD. Survival analysis demonstrated that cases with podoplanin and FGF1 double-positive staining had a significantly lower survival rate compared with cases with double-negative staining. In vitro experiments revealed that podoplanin regulated FGF1 expression and affected tube formation of human umbilical vein endothelial cells. Combined, the results demonstrated that podoplanin was co-expressed with FGF1 in lung SQCC and this co-expression was correlated with poor prognosis.

Intestinal Na+, K+, 2Cl- cotransporter 2 plays a crucial role in hyperosmotic transitions of a euryhaline teleost.

Euryhaline fishes, such as the red drum (Sciaenops ocellatus), must quickly transition between hyperosmotic and hypoosmotic physiological strategies. When freshwater individuals transition to seawater they are exposed to increased diffusive water loss and ion gain. To maintain osmoregulatory balance these animals must drink and absorb seawater through the intestine, followed by ion excretion at the gills. The ability of fishes to transition between strategies can limit the magnitude of osmotic shock that can be tolerated. Here, we demonstrate that red drum can tolerate direct transfer from freshwater to full‐strength seawater with marginal impacts on osmotic balance, as indicated by plasma and muscle ion concentration, as well as muscle water. Seawater transition is concurrent with a significant increase in intestinal fluid volume. Typical patterns of osmoregulatory plasticity were observed in the gill with increased expression of nkcc1 and cftr. Expression changes in the anterior intestine were observed after 24 h for nkcc2 with smaller and later responses observed for slc26a3, slc26a6, and nbc. Immunofluorescence staining demonstrated similar patterns of NKCC localization in freshwater and seawater intestines; however, reduced basolateral staining of V‐type ATPase was observed in seawater. Electrophysiological preparations demonstrated that seawater fish had increased absorptive current in the anterior intestine, which was significantly reduced in the presence of 10 μmol/L bumetanide. Overall, these results suggest that nkcc2 plays a crucial role during hyperosmotic transitions, and may be a more important complement to the well‐known bicarbonate secretion pathway than generally considered.

Repeated movie viewings produce similar local activity patterns but different network configurations

People seek novelty in everyday life, but they also enjoy viewing the same movies or reading the same novels a second time. What changes and what stays the same when re-experiencing a narrative? In examining this question with functional neuroimaging, we found that brain activity reorganizes in a hybrid, scale-dependent manner when individuals processed the same audiovisual narrative a second time. At the most local level, sensory systems (occipital and temporal cortices) maintained a similar temporal activation profile during the two viewings. Nonetheless, functional connectivity between these same lateral temporal regions and other brain regions was stronger during the second viewing. Furthermore, at the level of whole-brain connectivity, we found a significant rearrangement of network partition structure: lateral temporal and inferior frontal regions clustered together during the first viewing but merged within a fronto-parietal cluster in the second. Our findings show that repetition maintains local activity profiles. However, at the same time, it is associated with multiple network-level connectivity changes on larger scales, with these changes strongly involving regions considered core to language processing.

Nucleosome eviction in mitosis assists condensin loading and chromosome condensation.

Condensins associate with DNA and shape mitotic chromosomes. Condensins are enriched nearby highly expressed genes during mitosis, but how this binding is achieved and what features associated with transcription attract condensins remain unclear. Here, we report that condensin accumulates at or in the immediate vicinity of nucleosome-depleted regions during fission yeast mitosis. Two transcriptional coactivators, the Gcn5 histone acetyltransferase and the RSC chromatin-remodelling complex, bind to promoters adjoining condensin-binding sites and locally evict nucleosomes to facilitate condensin binding and allow efficient mitotic chromosome condensation. The function of Gcn5 is closely linked to condensin positioning, since neither the localization of topoisomerase II nor that of the cohesin loader Mis4 is altered in gcn5 mutant cells. We propose that nucleosomes act as a barrier for the initial binding of condensin and that nucleosome-depleted regions formed at highly expressed genes by transcriptional coactivators constitute access points into chromosomes where condensin binds free genomic DNA.

Characterization, expression analysis and localization pattern of toll-like receptor 1 (tlr1) and toll-like receptor 2 (tlr2) genes in grass carp Ctenopharyngodon idella.

In this study, the toll-like receptor 1 (tlr1) and toll-like receptor 2 (tlr2) genes of grass carp Ctenopharyngodon idella were cloned and characterized. tlr1 and tlr2 were found to be highly expressed in immune system organs such as spleen, middle kidney and heart kidney. The expression level of tlr1 and tlr2 was found to be up-regulated at the later stage of viral challenge process. Moreover, subcellular localization indicated that Tlr1 and Tlr2 shared similar localization pattern and both of them may locate in the plasma membrane of transfected cells.

Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell’s nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

Ion Channel Function and Cross-Species Determinants in Viral Assembly of Nonprimate Hepacivirus p7.

Nonprimate hepacivirus (NPHV), the closest homolog of hepatitis C virus (HCV) described to date, has recently been discovered in horses. Even though the two viruses share a similar genomic organization, conservation of the encoded hepaciviral proteins remains undetermined. The HCV p7 protein is localized within endoplasmic reticulum (ER) membranes and is important for the production of infectious particles. In this study, we analyzed the structural and functional features of NPHV p7 in addition to its role during virus assembly. Three-dimensional homology models for NPHV p7 using various nuclear magnetic resonance spectroscopy (NMR) structures were generated, highlighting the conserved residues important for ion channel function. By applying a liposome permeability assay, we observed that NPHV p7 exhibited liposome permeability features similar to those of HCV p7, indicative of similar ion channel activity. Next, we characterized the viral protein using a p7-based trans-complementation approach. A similar subcellular localization pattern at the ER membrane was observed, although production of infectious particles was likely hindered by genetic incompatibilities with HCV proteins. To further characterize these cross-species constraints, chimeric viruses were constructed by substituting different regions of HCV p7 with NPHV p7. The N terminus and transmembrane domains were nonexchangeable and therefore constitute a cross-species barrier in hepaciviral assembly. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectious trans-complemented viral particles. In conclusion, comparison of NPHV and HCV p7 revealed structural and functional homology of these proteins, including liposome permeability, and broadly acting determinants that modulate hepaciviral virion assembly and contribute to the host-species barrier were identified. The recent discovery of new relatives of hepatitis C virus (HCV) enables for the first time the study of cross-species determinants shaping hepaciviral pathogenesis. Nonprimate hepacivirus (NPHV) was described to infect horses and represents so far the closest homolog of HCV. Both viruses encode the same viral proteins; however, NPHV protein functions remain poorly understood. In this study, we aimed to dissect NPHV p7 on a structural and functional level. By using various NMR structures of HCV p7 as templates, three-dimensional homology models for NPHV p7 were generated, highlighting conserved residues that are important for ion channel function. A p7-based trans-complementation approach and the construction of NPHV/HCV p7 chimeric viruses showed that the N terminus and transmembrane domains were nonexchangeable. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectious viral particles. These results identify species-specific constraints as well as exchangeable determinants in hepaciviral assembly.

Recombination: The birds and the yeast

Nonhuman Genetics Apes and mice have a specific gene, PRDM9 , that is associated with genomic regions with high rates of recombination, called hotspots. In species with PRDM9 , hotspots move rapidly within the genome, varying among populations and closely related species (see the Perspective by Lichten). To investigate recombination hotspots in species lacking PRDM9 , Singhal et al. examined bird genomes, which lack a PRDM9 gene. They looked closely at the genomes of finch species and found that recombination was localized to the promoter regions of genes and highly conserved over millions of years. Similarly, Lam and Keeney examined recombination localization within yeast, which also lacks PRDM9 . They found a similar more-or-less fixed pattern of hotspots. Thus, recombination in species lacking a PRDM9 gene shows similar patterns of hotspot localization and evolution. Science , this issue p. [913][1], p. [928][2]; see also p. [932][3] [1]: /lookup/doi/10.1126/science.aad5404 [2]: /lookup/doi/10.1126/science.aad0843 [3]: /lookup/doi/10.1126/science.aad0814

Nonparadoxical evolutionary stability of the recombination initiation landscape in yeast.

The nonrandom distribution of meiotic recombination shapes heredity and genetic diversification. Theoretically, hotspots--favored sites of recombination initiation--either evolve rapidly toward extinction or are conserved, especially if they are chromosomal features under selective constraint, such as promoters. We tested these theories by comparing genome-wide recombination initiation maps from widely divergent Saccharomyces species. We find that hotspots frequently overlap with promoters in the species tested, and consequently, hotspot positions are well conserved. Remarkably, the relative strength of individual hotspots is also highly conserved, as are larger-scale features of the distribution of recombination initiation. This stability, not predicted by prior models, suggests that the particular shape of the yeast recombination landscape is adaptive and helps in understanding evolutionary dynamics of recombination in other species.

A mutual titer-enhancing relationship and similar localization patterns between Citrus exocortis viroid and Hop stunt viroid co-infecting two citrus cultivars.

BackgroundCitrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are commonly found simultaneously infecting different citrus cultivars in Taiwan. A crucial question to be addressed is how accumulations of these two viroids affect each other in an infected plant. In this study, we investigated the relationship between the two viroids at macroscopic and microscopic levels.MethodsCEVd and HSVd titers were examined by real-time RT-PCR in 17 plants of two citrus cultivars (blood orange and Murcott mandarin) every 3 months (spring, summer, fall and winter) from 2011 to 2013. Three nonparametric tests (Spearman’s rank correlation coefficient, Kendall’s tau rank correlation coefficient and Hoeffding’s inequality) were performed to test the correlation between CEVd and HSVd. Cellular and subcellular localizations of the two viroids were detected by digoxigenin- and colloidal gold-labeled in situ hybridization using light and transmission electron microscopy.ResultsThe two viroids were unevenly distributed in four different types of citrus tissues (rootstock bark, roots, twig bark and leaves). Compared with blood orange, Murcott mandarin was generally more susceptible to CEVd and HSVd infection. Both viroids replicated and preferentially accumulated in the underground tissues of the two citrus cultivars. Except for blood orange at high temperatures, significant positive correlations were observed between the two viroids in specific tissues of both cultivars. Relative to concentrations under single-infection conditions, the CEVd population significantly increased under double infection during half of the 12 monitored seasons; in contrast, the population of HSVd significantly increased under double infection during only one season. At cellular/subcellular levels, the two viroids showed similar localization patterns in four tissues and the cells of these tissues in the two citrus cultivars.ConclusionsOur findings of titer enhancement, localization similarity, and lack of symptom aggravation under CEVd and HSVd double infection suggest that the two viroids have a positive relationship in citrus. The combination of molecular and cellular techniques used in this study provided evidence of titer correlation and localization of co-infecting viroids in the host. These methods may thus be useful tools for exploring viroid–viroid and viroid–host interactions.

Evaluation of LBP-Based Facial Emotions Recognition Techniques to Make Consistent Decisions

Decision making is one of the smouldering problems in day to day works. Human emotions play crucial role in decision-making systems. While person is in high emotion he cannot make proper decision. Robust local binary pattern (RLBP) operator is more powerful to recognize the emotions and extends the features of local binary pattern (LBP). However, there are some precincts like discriminating bright faces against dark features and vice versa and intra-class variances increase. The RLBP solves this problem by finding minimum of LBP codes and their complements. However, it miss the mark for different local structures a similar feature is obtained, weak contrast local patterns and similar strong contrast local patterns. Hence, the discriminative robust local binary pattern (DRLBP) method is proposed to retain the contrast information of image patterns next to considering both edge and texture information. Nevertheless, LBP family methods are highly sensitive to noise. To trounce these drawbacks this paper extends fuzzy rule-based DRLBP which is more robust to noise, low contrasted, uneven lighting conditions, variations in expressions and rotation variant images.

Abstract 4997: Phosphorylation of SLAP by the KIT-D816V oncogenic mutant suppresses SLAP-mediated negative regulation of KIT signaling

Abstract The SRC-like-adaptor protein (SLAP) is a negative regulator of mitogenic signaling. SLAP shares considerable structural homology with SRC, apart from lacking a kinase domain. SLAP is known to be expressed in a variety of cells and regulates cell surface receptor signaling by direct association. Here we show that SLAP interacts with the type III receptor tyrosine kinase KIT and also with its oncogenic mutant KIT-D816V. This mutant has been described as a driving mutant in several cancers including mastocytosis and certain types of acute myeloid leukemia. The association is mediated through phosphotyrosine residues in KIT and the SLAP-SH2 domain. Despite high structural similarity between SLAP and SRC, these tyrosine residues are different from the ones mediating SRC binding to KIT. This association results in increased ubiquitination and degradation of KIT. siRNA-mediated depletion of SLAP selectively potentiates wild-type KIT downstream signaling but not KIT-D816V signaling. Although SLAP displays similar localization pattern with both ligand-stimulated KIT and KIT-D816V, only KIT-D816V can phosphorylate SLAP on three tyrosine residues (Y120, Y258 and Y273). This phosphorylation takes place when SLAP interacts with KIT-D816V. Mutations on these tyrosine residues in SLAP restores normal SLAP regulation on oncogenic KIT-D816V. Taken together, our data suggest that the oncogenic mutant of KIT can bypass normal cellular regulation of signaling by phosphorylating regulatory proteins such as SLAP. Citation Format: Julhash U. Kazi, Jianmin Sun, Lars Rönnstrand. Phosphorylation of SLAP by the KIT-D816V oncogenic mutant suppresses SLAP-mediated negative regulation of KIT signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4997. doi:10.1158/1538-7445.AM2015-4997

Network-Based Analysis on Orthogonal Separation of Human Plasma Uncovers Distinct High Density Lipoprotein Complexes.

High density lipoprotein (HDL) particles are blood-borne complexes whose plasma levels have been associated with protection from cardiovascular disease (CVD). Recent studies have demonstrated the existence of distinct HDL subspecies; however, these have been difficult to isolate and characterize biochemically. Here, we present the first report that employs a network-based approach to systematically infer HDL subspecies. Healthy human plasma was separated into 58 fractions using our previously published three orthogonal chromatography techniques. Similar local migration patterns among HDL proteins were captured with a novel similarity score, and individual comigration networks were constructed for each fraction. By employing a graph mining algorithm, we identified 183 overlapped cliques, among which 38 were further selected as candidate HDL subparticles. Each of these 38 subparticles had at least two literature supports. In addition, GO function enrichment analysis showed that they were enriched with fundamental biological and CVD protective functions. Furthermore, gene knockout experiments in mouse model supported the validity of these subparticles related to three apolipoproteins. Finally, analysis of an apoA-I deficient human patient's plasma provided additional support for apoA-I related complexes. Further biochemical characterization of these putative subspecies may facilitate the mechanistic research of CVD and guide targeted therapeutics aimed at its mitigation.

Nuclear β-catenin localization supports homology of feathers, avian scutate scales, and alligator scales in early development.

Feathers are an evolutionary novelty found in all extant birds. Despite recent progress investigating feather development and a revolution in dinosaur paleontology, the relationship of feathers to other amniote skin appendages, particularly reptile scales, remains unclear. Disagreement arises primarily from the observation that feathers and avian scutate scales exhibit an anatomical placode-defined as an epidermal thickening-in early development, whereas alligator and other avian scales do not. To investigate the homology of feathers and archosaur scales we examined patterns of nuclear β-catenin localization during early development of feathers and different bird and alligator scales. In birds, nuclear β-catenin is first localized to the feather placode, and then exhibits a dynamic pattern of localization in both epidermis and dermis of the feather bud. We found that asymmetric avian scutate scales and alligator scales share similar patterns of nuclear β-catenin localization with feathers. This supports the hypothesis that feathers, scutate scales, and alligator scales are homologous during early developmental stages, and are derived from early developmental stages of an asymmetric scale present in the archosaur ancestor. Furthermore, given that the earliest stage of β-catenin localization in feathers and archosaur scales is also found in placodes of several mammalian skin appendages, including hair and mammary glands, we hypothesize that a common skin appendage placode originated in the common ancestor of all amniotes. We suggest a skin placode should not be defined by anatomical features, but as a local, organized molecular signaling center from which an epidermal appendage develops.

Isolation and expression of grass carp toll-like receptor 5a (CiTLR5a) and 5b (CiTLR5b) gene involved in the response to flagellin stimulation and grass carp reovirus infection

Toll-like receptor 5 (TLR5), a member of Toll-like receptors (TLRs) family and is responsible for the bacterial flagellin recognition in vertebrates, play an important role in innate immunity. In the study, two TLR5 genes of grass carp (Ctenopharyngodon idellus), named CiTLR5a and CiTLR5b, were cloned and analyzed. Both CiTLR5a and CiTLR5b are typical TLR proteins, including LRR motif, transmembrane region and TIR domain. The full-length cDNA of CiTLR5a is 3054 bp long, with a 2646 bp open reading frame (ORF), 78 bp 5′ untranslated regions (UTR), and 330 bp 3′ UTR. The full-length cDNA of CiTLR5b is 3326 bp, with a 2627 bp ORF, 95 bp 5′ UTR, and 594 bp 3′ UTR. Phylogenetic analysis showed that CiTLR5a and CiTLR5b were closed to the TLR5 of cirrhinus mrigala, cyprinus_carpio, and danio rerio. Subcellular localization indicated that CiTLR5a and CiTLR5b shared similar localization pattern and may locate in the plasma membrane of transfected cells. Real-time quantitative PCR revealed CiTLR5a and CiTLR5b were constitutively expressed in all examined tissues, whereas the highest expressed tissue differed. Following exposure to flagellin and GCRV, CiTLR5a and CiTLR5b were up-regulated significantly. Moreover, the downstream genes of TLR5 signal pathway such as MyD88, NF-κB, IRF7, IL-1β, and TNF-α also up-regulated significantly, whereas the IκB gene was down-regulated, suggesting that CiTLR5a and CiTLR5b involved in response to flagellin stimulation and GCRV infection. The results obtained in the study would provide a new insight for further understand the function of TLR5 in teleost fish.

HER2 and β-catenin protein location: importance in the prognosis of breast cancer patients and their correlation when breast cancer cells suffer stressful situations.

In human breast cancer, β-catenin localization has been related with disease prognosis. Since HER2-positive patients are an important subgroup, and that in breast cancer cells a direct interaction of β-catenin/HER2 has been reported, in the present study we have explored whether β-catenin location is related with the disease survival. The study was performed in a tumor bank from patients (n = 140) that did not receive specific anti-HER2 therapy. The proteins were detected by immunohistochemistry in serial sections, 47 (33.5%) patients were HER2-positive with a long follow-up. HER2-positive patients that displayed β-catenin at the plasma membrane (completely surrounding the tumour cells) showed a significant better disease-free survival and overall survival than the patients showing the protein on other locations. Then we explored the dynamics of the co-expression of β-catenin and HER2 in human MCF-7 and SKBR3 cells exposed to different stressful situations. In untreated conditions MCF-7 and SKBR3 cells showed very different β-catenin localization. In MCF-7 cells, cadmium administration caused a striking change in β-catenin localization driving it from plasma membrane to cytoplasmic and perinuclear areas and HER2 showed a similar localization patterns. The changes induced by cadmium were compared with heat shock, H2O2 and tamoxifen treatments. In conclusion, this study shows the dynamical associations of HER2 and β-catenin and their changes in subcellular localizations driven by stressful situations. In addition, we report for the first time the correlation between plasma membrane associated β-catenin in HER2-positive breast cancer and survival outcome, and the importance of the protein localization in breast cancer samples.