Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase

Background

Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis.

Methods

In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling.

Results

Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk.

Conclusion

These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes.

     

The potential hazards of Aspergillus sp. in foods and feeds,and the role of biological treatment: A review

The contamination of food and feed by Aspergillus has become a global issue with a significant worldwide economic impact. The growth of Aspergillus is unfavourable to the development of food and feed industries, where the problems happen mostly due to the presence of mycotoxins, which is a toxic metabolite secreted by most Aspergillus groups. Moreover, fungi can produce spores that cause diseases, such as allergies and asthma, especially to human beings. High temperature, high moisture, retarded crops, and poor food storage conditions encourage the growth of mold, as well as the development of mycotoxins. A variety of chemical, biological, and physical strategies have been developed to control the production of mycotoxins. A biological approach, using a mixed culture comprised of Saccharomyces cerevisiae and Lactobacillus rhamnosus resulted in the inhibition of the growth of fungi when inoculated into fermented food. The results reveal that the mixed culture has a higher potential (37.08%) to inhibit the growth of Aspergillus flavus (producer of Aflatoxin) compared to either single culture, L. rhamnosus NRRL B-442 and S. cerevisiae, which inhibit the growth by 63.07% and 64.24%, respectively.     

Proteomic analysis of human osteoarthritis synovial fluid

Background

Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography.

Results

We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples.

Conclusions

We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis.     

Involvement of NHERF1 in apical membrane localization of MRP4 in polarized kidney cells

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette protein superfamily, confers resistance to nucleoside and nucleotide analogs as well as camptothecin derivatives. MRP4 also mediates the efflux of certain cyclic nucleotides, eicosanoids, conjugated steroids, and uric acid. Depending on the cell type, MRP4 may localize to either apical or basolateral membranes in polarized cells. The adaptor protein NHERF1 has previously been implicated in MRP4 internalization in non-polarized cells. We have now found that NHERF1 levels are very low in polarized MDCKI cells which express MRP4 on basolateral membranes relative to polarized LLC-PK1 cells which express MRP4 on apical membranes. Furthermore, ectopic expression of FLAG-tagged NHERF1 in MDCKI cells and in MDCKI cells stably expressing eGFP-tagged MRP4 causes endogenous MRP4 and eGFP-MRP4, respectively, to traffic to the apical membranes. These data establish NHERF1 as a major determinant of MRP4 trafficking to apical membranes of mammalian kidney cells.     

Hypermethylation of Genes Detected in Urine from Ghanaian Adults with Bladder Pathology Associated with Schistosoma haematobium Infection

Purpose

Schistosoma haematobium is associated with chronic bladder damage and may subsequently induce bladder cancer in humans, thus posing a serious threat where the parasite is endemic. Here we evaluated aberrant promoter DNA methylation as a potential biomarker to detect severe bladder damage that is associated with schistosomiasis by analyzing urine specimens.

Materials and Methods

A quantitative methylation-specific PCR (QMSP) assay was used to examine the methylation status of seven genes (RASSF1A, RARβ2, RUNX3, TIMP3, MGMT, P16, ARF) in 57 urine samples obtained from volunteers that include infected and uninfected by S. haematobium from an endemic region. The Fishers Exact Test and Logistic Regression analysis were used to evaluate the methylation status with bladder damage (as assessed by ultrasound examination) in subjects with S. haematobium infection.

Results

RASSF1A and TIMP3 were significant to predict severe bladder damage both in univariate (p = 0.015 and 0.023 respectively) and in multivariate (p = 0.022 and 0.032 respectively) logistic regression analysis. Area under the receiver operator characteristic curves (AUC-ROC) for RASSF1A and TIMP3 to predict severe bladder damage were 67.84% and 63.73% respectively. The combined model, which used both RASSF1A and TIMP3 promoter methylation, resulted in significant increase in AUC-ROC compared to that of TIMP3 (77.55% vs. 63.73%.29; p = 0.023).

Conclusions

In this pilot study, we showed that aberrant promoter methylation of RASSF1A and TIMP3 are present in urine sediments of patients with severe bladder damage associated with S. haematobium infection and that may be used to develop non-invasive biomarker of S. haematobium exposure and early molecular risk assessmentof neoplastic transformation.     

CCR6 Functions as a New Coreceptor for Limited Primary Human and Simian Immunodeficiency Viruses

More than 12 chemokine receptors (CKRs) have been identified as coreceptors for the entry of human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2), and simian immunodeficiency viruses (SIVs) into target cells. The expression of CC chemokine receptor 6 (CCR6) on Th17 cells and regulatory T cells make the host cells vulnerable to HIV/SIV infection preferentially. However, only limited information is available concerning the specific role of CCR6 in HIV/SIV infection. We examined CCR6 as a coreceptor candidate in this study using NP-2 cell line-based in-vitro studies. Normally, CD4-transduced cell line, NP-2/CD4, is strictly resistant to all HIV/SIV infection. When CCR6 was transduced there, the resultant NP-2/CD4/CCR6 cells became susceptible to HIV-1HAN2, HIV-2MIR and SIVsmE660, indicating coreceptor roles of CCR6. Viral antigens in infected cells were detected by IFA and confirmed by detection of proviral DNA. Infection-induced syncytia in NP-2/CD4/CCR6 cells were detected by Giemsa staining. Amount of virus release through CCR6 has been detected by RT assay in spent culture medium. Sequence analysis of proviral DNA showed two common amino acid substitutions in the C2 envelope region of HIV-2MIR clones propagated through NP-2/CD4/CCR6 cells. Conversely, CCR6-origin SIVsmE660 clones resulted two amino acid changes in the V1 region and one change in the C2 region. The substitutions in the C2 region for HIV-2MIR and the V1 region of SIVsmE660 may confer selection advantage for CCR6-use. Together, the results describe CCR6 as an independent coreceptor for HIV and SIV in strain-specific manner. The alteration of CCR6 uses by viruses may influence the susceptibility of CD4+ CCR6+ T-cells and dendritic cell subsets in vivo and therefore, is important for viral pathogenesis in establishing latent infections, trafficking, and transmission. However, clinical relevance of CCR6 as coreceptor in HIV/SIV infections should be investigated further.     

The Epac1 Signaling Pathway Regulates Cl? Secretion via Modulation of Apical KCNN4c Channels in Diarrhea

The apical membrane of intestinal epithelia expresses intermediate conductance K⁺ channel (KCNN4), which provides the driving force for Cl⁻ secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl⁻ secretion via stimulation of Rap2-phospholipase Cϵ-[Ca²⁺]_i signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl⁻ secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl⁻ secretion and apical K⁺ conductance (I_K(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2′-O- methyladenosine 3′,5′-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K⁺ channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed ∼83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate I_K(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated I_K(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases.     

Accessory cholera enterotoxin, Ace, from Vibrio cholerae: structure, unfolding, and virstatin binding

Vibrio cholerae accessory cholera enterotoxin (Ace) is the third toxin, along with cholera toxin (CT) and zonula occludens toxin (Zot), that causes the endemic disease cholera. Structural characterization of Ace has been restricted because of the limited production of this toxic protein by V. cholerae. We have cloned, overexpressed, and purified Ace from V. cholerae strain O395 in Escherichia coli to homogeneity and determined its biological activity. The unfolding of the purified protein was investigated using circular dichroism and intrinsic tryptophan fluorescence. Because Ace is predominantly a hydrophobic protein, the degree of exposure of hydrophobic regions was identified from the spectral changes of the environment-sensitive fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) that quenches the fluorescence of tryptophan residues of Ace in a concentration-dependent manner. Results showed that bis-ANS binds one monomeric unit of Ace with a 1:1 stoichiometry and a K' of 0.72 μM. Ace exists as a dimer, with higher oligomeric forms appearing upon glutaraldehyde cross-linking. This study also reports the binding of virstatin, a small molecule that inhibits virulence regulation in V. cholerae, to Ace. The binding constant (K=9×10(4) M(-1)) and the standard free energy change (ΔG°=-12 kcal mol(-1)) of Ace-virstatin interaction have been evaluated by the fluorescence quenching method. The binding does not affect the oligomeric status of Ace. A cell viability assay of the antibacterial activity of Ace has been performed using various microbial strains. A homology model of Ace, consistent with the experimental results, has been constructed.