ResearchPad - protein-expression https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Clinicopathological and prognostic significance of caveolin-1 and ATG4C expression in the epithelial ovarian cancer]]> https://www.researchpad.co/article/elastic_article_14473 Altered expression of caveolin-1 (CAV1) and autophagy marker ATG4C is observed in various types of human cancers. However, the clinical significance of CAV1 and ATG4C expression in epithelial ovarian cancer (EOC) remains largely unknown. The present study aims to explore the clinicopathological value and prognostic significance of CAV1 and ATG4C expression in EOC.MethodsThe expression pattern and prognostic value of CAV1 and ATG4C mRNA in EOC were analyzed using data from the Cancer Genome Atlas (TCGA) database (N = 373). In addition, immunohistochemistry analysis was performed to detect and assay the expression of CAV1 and ATG4C proteins in tissue microarray of EOC.ResultsBased on TCGA data, Kaplan-Meier analysis indicated that patients with low CAV1 mRNA (p = 0.021) and high ATG4C mRNA (p = 0.018) expression had a significantly shorter overall survival (OS). Cox regression analysis demonstrated that the expression levels of CAV1 (p = 0.023) and ATG4C mRNA (p = 0.040) were independent prognostic factors for OS in EOC. In addition, the Concordance Index of the nomogram for OS prediction was 0.660. Immunohistochemical analysis showed the expression levels of stromal CAV1 and cancerous ATG4C proteins, and high expression of both CAV1 and ATG4C protein in the stroma were found to significantly correlate with the histologic subtypes of EOC, especially with serous subtype.ConclusionsDecreased expression of CAV1 mRNA and increased expression of ATG4C mRNA in EOC can predict poor overall survival. The expression levels of CAV1 protein in stromal cells and ATG4C protein in cancer cells are significantly associated with histologic subtypes of EOC. These findings suggest that CAV1 and ATG4C serve as useful prognostic biomarkers and candidate therapeutic targets in EOC. ]]> <![CDATA[Increased 14-3-3β and γ protein isoform expressions in parasitic eosinophilic meningitis caused by Angiostrongylus cantonensis infection in mice]]> https://www.researchpad.co/article/5c8acccdd5eed0c484990014

The 14-3-3 proteins are cerebrospinal fluid (CSF) markers of neuronal damage during infectious meningitis and Creutzfeldt-Jakob disease. Little is known about dynamic changes in the individual isoforms in response to parasitic eosinophilic meningitis. The purposes of this study were to determine the 14-3-3 protein isoform patterns, examine the kinetics and correlate the severity of blood brain barrier (BBB) damage with the expressions of these markers in mice with eosinophilic meningitis.

Mice were orally infected with 50 A. cantonensis L3 via an oro-gastric tube and sacrificed every week for 3 consecutive weeks after infection. The Evans blue method and BBB junctional protein expressions were used to measure changes in the BBB. Hematoxylin and eosin staining was used to analyze pathological changes in the mice brains following 1–3 weeks of infection with A. cantonensis. The levels of 14-3-3 protein isoforms in serum/CSF and brain homogenates were analyzed by Western blot, and immunohistochemistry (IHC) was used to explore the different isoform distributions of 14-3-3 proteins and changes in BBB junctional proteins in the mice brain meninges. Dexamethasone was injected intraperitoneally from the seventh day post infection (dpi) until the end of the study (21 dpi) to study the changes in BBB junctional proteins. The amounts of Evans blue, tight junction and 14-3-3 protein isoforms in the different groups of mice were compared using the nonparametric Kruskal-Wallis test.

There were significant increases in 14-3-3 protein isoforms β and γ in the CSF in the second and third weeks after infection compared to the controls and first week of infection, which were correlated with the severity of BBB damage in brain histology, and Evans blue extravasation. Using IHC to assess the distribution of 14-3-3 protein isoforms and changes in BBB junctional proteins in the mice brain meninges, the expressions of isoforms β, γ, ε, and θ and junctional proteins occludin and claudin-5 in the brain meninges increased over a 3-week period after infection compared to the controls and 1 week after infection. The administration of dexamethasone decreased the expressions of BBB junctional proteins occludin and claudin-5 in the mice brain meninges.

Our findings support that 14-3-3 proteins β and γ can potentially be used as a CSF marker of neuronal damage in parasitic eosinophilic meningitis caused by A. cantonensis.

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<![CDATA[Sexual dimorphism of acute doxorubicin-induced nephrotoxicity in C57Bl/6 mice]]> https://www.researchpad.co/article/5c76fe23d5eed0c484e5b5ad

Doxorubicin (DOX) is a chemotherapeutic agent that has been reported to cause nephrotoxicity in rodent models and to a lesser degree in cancer patients. Female rodents have been shown to be protected against several features of DOX-induced nephrotoxicity. Nevertheless, the underlying mechanisms of this sexual dimorphism are not fully elucidated. Therefore, in the current study, we investigated the sex and time-dependent changes in pathological lesions as well as apoptotic and fibrotic markers in response to acute DOX-induced nephrotoxicity. We also determined the effect of acute DOX treatment on the renal expression of the sexually dimorphic enzyme, soluble epoxide hydrolase (sEH), since inhibition of sEH has been shown to protect against DOX-induced nephrotoxicity. Acute DOX-induced nephrotoxicity was induced by a single intra-peritoneal injection of 20 mg/kg DOX to male and female adult C57Bl/6 mice. The kidneys were isolated 1, 3 and 6 days after DOX administration. Histopathology assessment, gene expression of the apoptotic marker, BAX, protein expression of the fibrotic marker, transforming growth factor-β (TGF-β), and gene and protein expression of sEH were assessed. DOX administration caused more severe pathological lesions as well as higher induction of the apoptotic and fibrotic markers in kidneys of male than in female mice. Intriguingly, DOX inhibited sEH protein expression in kidneys of male mice sacrificed at 3 and 6 days following administration, suggesting that induction of sEH is not necessary for acute DOX-induced nephrotoxicity. However, DOX-induced inhibition of renal sEH in male mice may protect the kidney from further DOX-induced injury in a negative feedback mechanism. We also observed lower constitutive expressions of TGF-β and sEH in the kidney of female mice which may contribute, at least in part, to sexual dimorphism of DOX-induced nephrotoxicity.

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<![CDATA[Expression optimization, purification, and functional characterization of cholesterol oxidase from Chromobacterium sp. DS1]]> https://www.researchpad.co/article/5c6dc9e1d5eed0c48452a3da

Cholesterol oxidase is a bifunctional bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This valuable enzyme has attracted a great deal of attention because of its wide application in the clinical laboratory, synthesis of steroid derived drugs, food industries, and its potentially insecticidal activity. Therefore, development of an efficient protocol for overproduction of cholesterol oxidase could be valuable and beneficial in this regard. The present study examined the role of various parameters (host strain, culture media, induction time, isopropyl ß-D-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature) on over-expression of cholesterol oxidase from Chromobacterium sp. DS1. Applying the optimized protocol, the yield of recombinant cholesterol oxidase significantly increased from 92 U/L to 2115 U/L. Under the optimized conditions, the enzyme was produced on a large-scale, and overexpressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver-Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were determined. This study reports a straightforward protocol for cholesterol oxidase production which can be performed in any laboratory.

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<![CDATA[Expression, purification and characterization of the dimeric protruding domain of Macrobrachium rosenbergii nodavirus capsid protein expressed in Escherichia coli]]> https://www.researchpad.co/article/5c5df342d5eed0c484581038

Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1–252 and 253–371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity.

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<![CDATA[Equilin in conjugated equine estrogen increases monocyte-endothelial adhesion via NF-κB signaling]]> https://www.researchpad.co/article/5c5b5254d5eed0c4842bc66d

The adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules, plays a crucial role in the onset of atherosclerosis. Conjugated equine estrogen, which is widely used for estrogen-replacement therapy, contains both estrone sulfate and various nonhuman estrogens, including equilin. To investigate the association between various estrogen types and atherosclerosis risk, we examined their effect on adhesion-molecule expression in human umbilical vein endothelial cells (HUVECs). In estrogen-treated HUVECs, the mRNA and protein expression levels of adhesion molecules were quantified by real-time polymerase chain reaction and enzyme immunoassay. Additionally, a flow-chamber system was used to assess the effects of estrogens on the adherence of U937 monocytoid cells to HUVECs. Equilin, but not 17β-estradiol (E2) or other types of estrogen, significantly increased the mRNA (P < 0.01) and protein (P < 0.05) expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 as compared with levels in controls. Equilin treatment increased the adherence of U937 monocytoid cells to HUVECs relative to the that in the control (P < 0.05), decreased estrogen receptor (ER)β expression, and increased the expression of proteins involved in nuclear factor kappa-B (NF-κB) activation relative to levels in controls. Furthermore, the accumulation of NF-κB subunit p65 in HUVEC nuclei was promoted by equilin treatment. By contrast, E2 treatment neither increased the number of adhered monocytoid cells to HUVECs nor altered the expression of ERβ or NF-κB-activating proteins. Our findings suggest that in terms of the adhesion of monocytes at the onset of atherosclerosis, E2 may be preferable for estrogen-replacement therapy. Further studies comparing equilin treatment with that of E2 are needed to investigate their differential impacts on atherosclerosis.

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<![CDATA[Individual response to mTOR inhibition in delaying replicative senescence of mesenchymal stromal cells]]> https://www.researchpad.co/article/5c5ca2eed5eed0c48441edbc

Background aims

Delaying replicative senescence and extending lifespan of human mesenchymal stromal cells (MSCs) may enhance their potential for tissue engineering and cell based therapies. Accumulating evidence suggests that inhibitors of the mTOR signaling pathway, such as rapamycin, constitute promising pharmacological agents to retard senescence and extend stemness properties of various progenitor cell types. Here, we investigated whether the ability of rapamycin to postpone replicative senescence varies among bone marrow MSC samples (BM-MSCs) derived from different healthy donors, and explored the molecular mechanisms that drive rapamycin-mediated lifespan increment.

Methods

BM-MSCs at early passages were serially passaged either in absence or continuous presence of rapamycin and the number of cell population doublings until growth arrest was measured. The inhibition of mTOR signaling was assessed by the phosphorylation status of the downstream target RPS6. The expression levels of several senescence and pluripotency markers at early and late/senescent passages were analyzed by RT-qPCR, flow cytometry and western blot.

Results

We found that the lifespan extension in response to the continuous rapamycin treatment was highly variable among samples, but effective in most BM-MSCs. Despite all rapamycin-treated cells secreted significantly reduced levels of IL6, a major SASP cytokine, and expressed significantly higher levels of the pluripotency marker NANOG, the expression patterns of these markers were not correlated with the rapamycin-mediated increase in lifespan. Interestingly, rapamycin-mediated life-span extension was significantly associated only with repression of p16INK4A protein accumulation.

Conclusions

Taken together, our results suggest that some, but not all, BM-MSC samples would benefit from using rapamycin to postpone replicative arrest and reinforce a critical role of p16INK4A protein downregulation in this process.

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<![CDATA[Connective tissue growth factor mediates transforming growth factor β-induced collagen expression in human endometrial stromal cells]]> https://www.researchpad.co/article/5c59feb0d5eed0c4841352f2

Background

Adenomyosis is a medical condition defined by the abnormal presence of endometrial tissue within the myometrium, in which fibrosis occurs with new collagen deposition and myofibroblast differentiation. In this study, the effect of several mediators and growth factors on collagen expression was investigated on human endometrial stromal cells (fibroblasts) derived from adenomyotic endometrium.

Experimental approach

RT-PCR, Western blot analysis, pharmacological interventions and siRNA interference were applied to primary cultured human endometrial stromal cells (fibroblasts). Immunohistochemistry was used to analyze protein expression in adenomyotic endometrium tissue specimens.

Results

Of the tested mediators, transforming growth factor β1 (TGFβ1) and its isoforms were effective to induce collagen and connective tissue growth factor (CTGF) expression. Collagen and CTGF induction by TGFβ1 could be reduced by the inhibitors targeting DNA transcription, protein translation, and Smad2/3 signaling. Interestingly, TGFβ1 induced Smad2/3 phosphorylation and CTGF mRNA expression, but not collagen mRNA expression, suggesting that TGFβ1 mediates collagen expression through CTGF induction and Smad2/3 activation. In parallel, TGFβ1 and CTGF also induced expression of heat shock protein (HSP) 47, a protein required for the synthesis of several types of collagens. However, only CTGF siRNA knockdown, could compromise TGFβ1-induced collagen expression. Finally, the immunohistochemistry revealed vimentin- and α-SMA-positive staining for (myo)fibroblasts, TGFβ1, collagen, and CTGF in the subepithelial stroma region of human adenomyotic endometria.

Conclusion and implications

We reveal here that TGFβ1, collagen, and CTGF are expressed in the stroma of adenomyotic endometria and demonstrate that TGFβ1 can induce collagen production in endometrium-derived fibroblasts through cellular Smad2/3-dependent signaling pathway and CTGF expression, suggesting that endometrial TGFβ may take part in the pathogenesis of adenomyosis and ectopic endometrium may participate in uterine adenomyosis.

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<![CDATA[In silico identification of critical proteins associated with learning process and immune system for Down syndrome]]> https://www.researchpad.co/article/5c58d652d5eed0c484031b96

Understanding expression levels of proteins and their interactions is a key factor to diagnose and explain the Down syndrome which can be considered as the most prevalent reason of intellectual disability in human beings. In the previous studies, the expression levels of 77 proteins obtained from normal genotype control mice and from trisomic Ts65Dn mice have been analyzed after training in contextual fear conditioning with and without injection of the memantine drug using statistical methods and machine learning techniques. Recent studies have also pointed out that there may be a linkage between the Down syndrome and the immune system. Thus, the research presented in this paper aim at in silico identification of proteins which are significant to the learning process and the immune system and to derive the most accurate model for classification of mice. In this paper, the features are selected by implementing forward feature selection method after preprocessing step of the dataset. Later, deep neural network, gradient boosting tree, support vector machine and random forest classification methods are implemented to identify the accuracy. It is observed that the selected feature subsets not only yield higher accuracy classification results but also are composed of protein responses which are important for the learning and memory process and the immune system.

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<![CDATA[The value of diffusion kurtosis imaging in assessing mismatch repair gene expression of rectal carcinoma: Preliminary findings]]> https://www.researchpad.co/article/5c61e8d4d5eed0c48496f200

Purpose

To determine the correlation between the parameters of MR diffusion kurtosis imaging (MR-DKI) and mismatch repair gene expression (MMR) for rectal carcinomas.

Materials and methods

Data from 80 patients with rectal carcinoma were analyzed in this prospective study. High-resolution T2WI and DKI (b = 0, 800 and 1600 s/mm2, respectively) were performed. Mean kurtosis (MK) and mean diffusivity (MD) from DKI were measured. MMR-positive expression and HER-2 expression were classified into two groups. For comparison between different grades, the Mann-Whitney U test, receiver operating characteristic curve, and Spearman’s correlation analysis were used for statistical analyses.

Results

The MK values in identifying positive MMR expressions (MLH1, MSH2, and MSH6) were more reliable than the MD values (rs value: 0.772 vs. 0.448, 0.733 vs. 0.499, and 0.828 vs. 0.633 respectively, P<0.01). Receiver operating curve analysis showed that the performances of the MK values were better than those of the MD values (z = 2.835, 2.000, and 2.827, respectively, P<0.05), while the performances of the MK and MD-MK values were not statistically significant (z = 0.808, 1.557, and 0.596, respectively, P>0.05). Similarly, MK values were better than MD values in identifying HER2 expression (z = 2.795, P<0.05).

Conclusions

MK derived from DKI demonstrated a greater correlation than MD with MMR expression. It also showed better performance in differentiating between high- and low-grade positive MMR expression and HER2 expression. Thus, DKI may be valuable for the prognoses and evaluation of non-invasive therapies.

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<![CDATA[Development of a novel fusion protein with Anaplasma marginale and A. centrale MSP5 improved performance of Anaplasma antibody detection by cELISA in infected and vaccinated cattle]]> https://www.researchpad.co/article/5c5217b1d5eed0c4847943a4

Detection of antibodies to Anaplasma spp. using commercial competitive enzyme-linked immunosorbent assay (ccELISA) is based on the recombinant major surface protein 5 fused to maltose binding protein (MBP-MSP5) or glutathione S-transferase (GST-MSP5). To avoid false positive reactions due to the presence of antibodies against E. coli MBP in cattle, previous sera absorption is required. This study evaluated the replacement of MBP-MSP5 or GST-MSP5 antigens by the truncate MSP5 (residues 28–210) of A. marginale (tMSP5m), A. centrale (tMSP5c) and fusion protein MSP5 (tMSP5cm), expressed without N-terminus transmembrane helix in the ccELISA test. Immunoreactivity was evaluated by western blot using monoclonal antibodies against the tMSP5 and by in-house cELISA (hcELISA) with purified tMSP5m, tMSP5c or tMSP5cm using sera from cattle infected with A. marginale (n = 226) or vaccinated with A. centrale (n = 173) and uninfected cattle (n = 216). Results of hcELISA were compared with those of ccELISA. Recombinant protein was expressed highly soluble (> 95%) in E. coli without a molecular chaperone. Specificity of the hcELISA-tMSP5m, -MSP5c or -tMSP5cm was identical to (99.5%) and greater than that in ccELISA (96.3%). Sensitivity of hcELISA-tMSP5m and ccELISA was identical (95.5%), but lower than that of hcELISA-tMSP5cm (96.2%) and -tMSP5c (97.2%). The analysis of vaccinated cattle by hcELISA-tMSP5c showed sensitivity of 99.4%. In summary, the generation of fusion MSP5 A. marginale-A. centrale protein without transmembrane helix was a very effective method to express the recombinant protein highly soluble in the bacterial cytoplasm and contributed to an increased test performance for detecting antibodies in cattle naturally infected with A. marginale or vaccinated with A. centrale.

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<![CDATA[Protein refolding based on high hydrostatic pressure and alkaline pH: Application on a recombinant dengue virus NS1 protein]]> https://www.researchpad.co/article/5c6448dbd5eed0c484c2f04f

In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion bodies (IBs) generated by recombinant proteins expressed by Escherichia coli strains. The method was successfully applied to a recombinant form of the dengue virus (DENV) non-structural protein 1 (NS1). The minimal pH for IBs solubilization at 1 bar was 12 while a pH of 10 was sufficient for solubilization at HHP: 2.4 kbar for 90 min and 0.4 kbar for 14 h 30 min. An optimal refolding condition was achieved by compression of IBs at HHP and pH 10.5 in the presence of arginine, oxidized and reduced glutathiones, providing much higher yields (up to 8-fold) than association of HHP and GdnHCl via an established protocol. The refolded NS1, 109 ± 9.5 mg/L bacterial culture was recovered mainly as monomer and dimer, corresponding up to 90% of the total protein and remaining immunologically active. The proposed conditions represent an alternative for the refolding of immunologically active recombinant proteins expressed as IBs.

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<![CDATA[Blue light irradiation and its beneficial effect on Dupuytren’s fibroblasts]]> https://www.researchpad.co/article/5c42438ed5eed0c4845e05ab

Dupuytren’s contracture is a fibroproliferative disorder affecting the palmar fascia of the hand. Most affected are the ring fingers, and little fingers of middle-aged men. Symptomatic for this disease is the increased proliferation and differentiation of fibroblasts to myofibroblasts, which is accompanied by an elevated α-SMA expression. The present study evaluated the therapeutic benefit of blue light (λ = 453 nm, 38 mW/cm2, continuous radiance, spot size 10–12 cm2) as well as the molecular mechanism mediating this effect. It could be determined that blue light significantly diminished the induced α-SMA protein expression in both normal palmar fibroblasts and Duypuytren’s fibroblasts. The beneficial effect mediated by this irradiance, radiant exposure and wavelength was associated with an elevated reactive oxygen species generation. Furthermore, the data underlines the potential usefulness of blue light irradiation as a promising therapy option for Dupuytren’s disease, especially for relapse prevention, and may represent a useful strategy to treat further fibrotic diseases, such as keloids, hypertrophic scarring, and scleroderma.

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<![CDATA[BRASSINOSTEROID-SIGNALING KINASE 3, a plasma membrane-associated scaffold protein involved in early brassinosteroid signaling]]> https://www.researchpad.co/article/5c3d00fcd5eed0c4840374aa

Brassinosteroids (BRs) are steroid hormones essential for plant growth and development. The BR signaling pathway has been studied in some detail, however, the functions of the BRASSINOSTEROID-SIGNALING KINASE (BSK) family proteins in the pathway have remained elusive. Through forward genetics, we identified five semi-dominant mutations in the BSK3 gene causing BSK3 loss-of-function and decreased BR responses. We therefore investigated the function of BSK3, a receptor-like cytoplasmic kinase, in BR signaling and plant growth and development. We find that BSK3 is anchored to the plasma membrane via N-myristoylation, which is required for its function in BR signaling. The N-terminal kinase domain is crucial for BSK3 function, and the C-terminal three tandem TPR motifs contribute to BSK3/BSK3 homodimer and BSK3/BSK1 heterodimer formation. Interestingly, the effects of BSK3 on BR responses are dose-dependent, depending on its protein levels. Our genetic studies indicate that kinase dead BSK3K86R protein partially rescues the bsk3-1 mutant phenotypes. BSK3 directly interacts with the BSK family proteins (BSK3 and BSK1), BRI1 receptor kinase, BSU1 phosphatase, and BIN2 kinase. BIN2 phosphorylation of BSK3 enhances BSK3/BSK3 homodimer and BSK3/BSK1 heterodimer formation, BSK3/BRI1 interaction, and BSK3/BSU1 interaction. Furthermore, we find that BSK3 upregulates BSU1 transcript and protein levels to activate BR signaling. BSK3 is broadly expressed and plays an important role in BR-mediated root growth, shoot growth, and organ separation. Together, our findings suggest that BSK3 may function as a scaffold protein to regulate BR signaling. The results of our studies provide new insights into early BR signaling mechanisms.

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<![CDATA[Context-explorer: Analysis of spatially organized protein expression in high-throughput screens]]> https://www.researchpad.co/article/5c366800d5eed0c4841a6d01

A growing body of evidence highlights the importance of the cellular microenvironment as a regulator of phenotypic and functional cellular responses to perturbations. We have previously developed cell patterning techniques to control population context parameters, and here we demonstrate context-explorer (CE), a software tool to improve investigation cell fate acquisitions through community level analyses. We demonstrate the capabilities of CE in the analysis of human and mouse pluripotent stem cells (hPSCs, mPSCs) patterned in colonies of defined geometries in multi-well plates. CE employs a density-based clustering algorithm to identify cell colonies. Using this automatic colony classification methodology, we reach accuracies comparable to manual colony counts in a fraction of the time, both in micropatterned and unpatterned wells. Classifying cells according to their relative position within a colony enables statistical analysis of spatial organization in protein expression within colonies. When applied to colonies of hPSCs, our analysis reveals a radial gradient in the expression of the transcription factors SOX2 and OCT4. We extend these analyses to colonies of different sizes and shapes and demonstrate how the metrics derived by CE can be used to asses the patterning fidelity of micropatterned plates. We have incorporated a number of features to enhance the usability and utility of CE. To appeal to a broad scientific community, all of the software’s functionality is accessible from a graphical user interface, and convenience functions for several common data operations are included. CE is compatible with existing image analysis programs such as CellProfiler and extends the analytical capabilities already provided by these tools. Taken together, CE facilitates investigation of spatially heterogeneous cell populations for fundamental research and drug development validation programs.

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<![CDATA[TLR3 deficiency exacerbates the loss of epithelial barrier function during genital tract Chlamydia muridarum infection]]> https://www.researchpad.co/article/5c3fa57fd5eed0c484ca5130

Problem

Chlamydia trachomatis infections are often associated with acute syndromes including cervicitis, urethritis, and endometritis, which can lead to chronic sequelae such as pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, and tubal infertility. As epithelial cells are the primary cell type productively infected during genital tract Chlamydia infections, we investigated whether Chlamydia has any impact on the integrity of the host epithelial barrier as a possible mechanism to facilitate the dissemination of infection, and examined whether TLR3 function modulates its impact.

Method of study

We used wild-type and TLR3-deficient murine oviduct epithelial (OE) cells to ascertain whether C. muridarum infection had any effect on the epithelial barrier integrity of these cells as measured by transepithelial resistance (TER) and cell permeability assays. We next assessed whether infection impacted the transcription and protein function of the cellular tight-junction (TJ) genes for claudins1-4, ZO-1, JAM1 and occludin via quantitative real-time PCR (qPCR) and western blot.

Results

qPCR, immunoblotting, transwell permeability assays, and TER studies show that Chlamydia compromises cellular TJ function throughout infection in murine OE cells and that TLR3 deficiency significantly exacerbates this effect.

Conclusion

Our data show that TLR3 plays a role in modulating epithelial barrier function during Chlamydia infection of epithelial cells lining the genital tract. These findings propose a role for TLR3 signaling in maintaining the integrity of epithelial barrier function during genital tract Chlamydia infection, a function that we hypothesize is important in helping limit the chlamydial spread and subsequent genital tract pathology.

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<![CDATA[Heat stress induces proteomic changes in the liver and mammary tissue of dairy cows independent of feed intake: An iTRAQ study]]> https://www.researchpad.co/article/5c3fa54ed5eed0c484ca2dbc

Heat stress decreases milk yield and deleteriously alters milk composition. Reduced feed intake partially explains some of the consequences of heat stress, but metabolic changes in the mammary tissue and liver associated with milk synthesis have not been thoroughly evaluated. In the current study, changes of protein abundance in the mammary tissue and liver between heat-stressed cows with ad libitum intake and pair-fed thermal neutral cows were investigated using the iTRAQ proteomic approach. Most of the differentially expressed proteins from mammary tissue and liver between heat-stressed and pair-fed cows were involved in Gene Ontology category of protein metabolic process. Pathway analysis indicated that differentially expressed proteins in the mammary tissue were related to pyruvate, glyoxylate and dicarboxylate metabolism pathways, while those in the liver participated in oxidative phosphorylation and antigen processing and presentation pathways. Several heat shock proteins directly interact with each other and were considered as central “hubs” in the protein interaction network. These findings provide new insights to understand the turnover of protein biosynthesis pathways within hepatic and mammary tissue that likely contribute to changes in milk composition from heat-stressed cows.

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<![CDATA[Overexpression of prostate specific membrane antigen by canine hemangiosarcoma cells provides opportunity for the molecular detection of disease burdens within hemorrhagic body cavity effusions]]> https://www.researchpad.co/article/5c366808d5eed0c4841a6ea5

Background

Canine hemangiosarcoma (cHSA) is a highly metastatic mesenchymal cancer that disseminates by hematogenous and direct implantation routes. Therapies for cHSA are generally ineffective, in part due to advanced clinical disease stage at the time of diagnosis. The validation of conventional molecular methods for detecting novel biomarkers preferentially expressed by cHSA could lead to more timely diagnosis, earlier therapeutic interventions, and improved outcomes. In humans, prostate-specific membrane antigen (PSMA) is a transmembrane protein overexpressed by prostate carcinoma and tumor-associated endothelium of various solid cancer histologies. Importantly, the preferential overexpression of PSMA by certain cancers has been leveraged for the development of diagnostic molecular imaging reagents and targeted therapeutics. Recently, PSMA has been qualitatively demonstrated to be expressed in cHSA cell lines, however, quantitative PSMA expressions and the potential utility of PSMA transcript identification in biologic fluids to support the presence of microscopic cHSA burden has not been reported. Therefore, this study sought to characterize the differential quantitative expressions of PSMA between cHSA and non-malignant tissues, and to determine the potential diagnostic utility of PCR-generated PSMA amplicons as a surrogate of rare cHSA cells dwelling within peritoneal and pericardial cavities.

Methods

Quantitative gene and protein expressions for PSMA were compared between one normal endothelial and six cHSA cell lines by RT-PCR, western blot analysis, and fluorescent microscopy. Additionally, gene and protein expressions of PSMA in normal canine tissues were characterized. Graded expressions of PSMA were determined in spontaneously-arising cHSA tumor samples and the feasibility of qualitative PCR as a molecular diagnostic to detect PSMA transcripts in whole blood from healthy dogs and hemorrhagic effusions from cHSA-bearing dogs were evaluated.

Results

PSMA gene and protein expressions were elevated (up to 6-fold) in cHSA cells compared with non-malignant endothelium. By immunohistochemistry, protein expressions of PSMA were detectable in all cHSA tissue samples evaluated. As predicted by human protein atlas data, PSMA’s expression was comparably identified at substantial levels in select normal canine tissues including kidney, liver, and intestine. In young healthy pet dogs, PSMA amplicons could not be identified in circulating whole blood yet were detectable in hemorrhagic effusions collected from pet dogs with confirmed cHSA or PSMA-expressing cancer.

Conclusions

PSMA is quantitatively overexpressed in cHSA compared to normal endothelium, but its protein expression is not restricted to only cHSA tumor tissues, as specific visceral organs also substantively express PSMA. Optimized qualitative PCR methods failed to amplify PSMA amplicons sufficiently for visible detection in circulating whole blood derived from healthy young dogs, yet PSMA transcripts were readily identifiable in hemorrhagic effusions collected from pet dogs with histologically confirmed cHSA or PSMA-expressing cancer. While preliminary, findings derived from a limited cohort of normal and diseased pet dogs provocatively raise the potential value of PSMA amplicon detection as an ancillary molecular diagnostic test for supporting the presence of microscopic cHSA disease burden within hemorrhagic body cavity effusions.

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<![CDATA[Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo]]> https://www.researchpad.co/article/5c141f0dd5eed0c484d296ca

Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.

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<![CDATA[Mismatch repair deficiency and aberrations in the Notch and Hedgehog pathways are of prognostic value in patients with endometrial cancer]]> https://www.researchpad.co/article/5c12cf7cd5eed0c484914705

The aim of this study was to investigate the prognostic value of the Hedgehog (Gli, Patched-1, Shh, Smo) and Notch (Jag1, Notch2, Notch3) pathway members, in comparison to a panel of proteins (ER, PgR, HER2/neu, Ki67, p53, p16, PTEN and MMR) previously suggested to be involved in the pathogenesis of endometrial cancer, in association with clinical outcome and standard clinicopathological characteristics. A total of 204 patients with histological diagnosis of endometrial cancer treated from 2004 to 2013 were included. The evaluation of protein expression was assessed by immunohistochemistry. Univariate analysis showed that higher Ki67 labeling, expression of PTEN, p16, Notch2 and Notch3 proteins, as well as MMR proficiency were associated with increased relapse and mortality rate. Additionally, Patched-1 protein expression was associated with worse DFS, while p53 overexpression was associated with worse OS. In multivariate analyses, patients with MMR proficient tumors had more than double risk for death than patients with MMR deficient (MMRd) tumors (adjusted HR = 2.19, 95% CI 1.05–4.58, p = 0.036). Jag1 positivity conferred reduced mortality risk (HR = 0.48, 95% CI 0.23–0.97, p = 0.042). However, as shown by hierarchical clustering, patients fared better when their tumors expressed high Jag1 protein in the absence of Notch2 and Notch3, while they fared worse when all three proteins were highly expressed. Patched-1 positivity conferred higher risk for relapse (HR = 2.04, 95% CI 1.05–3.96, p = 0.036).

Aberrant expression of key components of the Notch and Hedgehog signaling pathways, as well as MMRd may serve as independent prognostic factors for recurrence and survival in patients with endometrial cancer.

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