Monoclonal antibody-based time-resolved fluorescence immunoassays for Daidzein, Genistein, and Equol in blood and urine: Application to the Isoheart Intervention Study

Background: Time-resolved fluorescence immunoassays (TR-FIAs) for phytoestrogens in biological samples are an alternative to mass spectrometric methods. These immunoassays were used to test urine and plasma samples from individuals in a dietary intervention trial aimed at determining the efficacy of dietary isoflavones in reducing the risk of coronary heart disease in postmenopausal women. Methods: We established murine monoclonal TR-FIA methods for daidzein, genistein, and equol. These assays could be performed manually or adapted to an automated analyzer for high throughput and increased accuracy. Analysis of urine was conducted on nonextracted samples. Blood analysis was performed on nonextracted samples for daidzein, whereas genistein and equol required diethyl-ether extraction. Results: Comparison of monoclonal TR-FIA, commercial polyclonal antibody–based TR-FIA, and gas chromatography–mass spectrometry showed correlations (r, 0.911–0.994) across the concentration range observed in the Isoheart study (50 mg/day isoflavones). The concentrations of urinary daidzein and genistein observed during intervention demonstrated good compliance, and a corresponding increase in serum daidzein and genistein confirmed bioavailability of the isoflavone-rich foods; 33 of the 117 volunteers (28.2%) were classified as equol producers on the basis of their urinary equol concentration (>936 nmol/L), and significant differences in the numbers of equol producers were observed between Berlin and the 3 other European cohorts studied. Conclusions: The validated monoclonal TR-FIA methods are applicable for use in large-scale human phytoestrogen intervention studies and can be used to monitor compliance, demonstrate bioavailability, and assess equol producer status

The Immune System GTPase GIMAP6 Interacts with the Atg8 Homologue GABARAPL2 and Is Recruited to Autophagosomes

<div><p>The GIMAPs (GTPases of the <b><u>i</u><u>m</u></b>munity-<b><u>a</u></b>ssociated <b><u>p</u></b>roteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux. </p> </div

Identification of the domains of GIMAP6 and GABARAPL2 required for their interaction.

<p>Panels A-C) HEK293T cells were transfected with 10µg wild-type GIMAP6 or the indicated mutated derivatives in plasmid pcDNA3Biot1His6iresBirA. Biotinylated and associated proteins were recovered by streptavidin-agarose affinity chromatography 48 h after transfection. Western blots of the recovered proteins were probed with HRP-conjugated streptavidin (to show the GIMAP6 proteins) or rat monoclonal antibody MAC446 to GABARAPL2 followed by an HRP-conjugated goat F(ab’)₂ fragment anti-rat IgG. Panel A) GIMAP6 compared with mutations of the putative AIM motif in GIMAP6. Panel B) GIMAP6 compared with N- and C-terminal mutants of the protein as indicated. In panel B, 1-292 corresponds to full-length GIMAP6. Panel C) Mutations within the GTPase domain of GIMAP6 as indicated. Panel D) HEK293T cells were transfected with a plasmid encoding myc-GIMAP6 together with plasmids encoding biotinylated forms of GABARAPL2 as indicated. Cell lysates were prepared and biotinylated and associated proteins recovered by streptavidin-agarose affinity chromatography. Western blots were probed with HRP-conjugated streptavidin (to show the GABARAPL2 proteins) or an anti-myc monoclonal antibody 9E10 followed by an HRP-conjugated goat anti-mouse IgG to detect myc-tagged GIMAP6. The wild-type protein is represented by 1-117. Results in all four panels are representative of data obtained from three independent experiments.</p

GIMAP6 over-expression leads to the induction of GABARAPL2.

<p>A) Cells lysates were prepared from three myc-GIMAP6 HEK293 cell lines (lanes 3-5) or two cell lines carrying the corresponding vector (lanes 1-2) and expression of GIMAP6, GABARAPL2, and β-actin analysed by Western blotting as indicated. B) Three T-Rex^TM-HeLa cell lines carrying plasmid pcDNA4.TO (lanes 1-3) or three myc-GIMAP6 T-Rex HeLa cell lines (lanes 4-6) were grown in the presence or absence of 2 µg/ml tetracycline for four days. Cells lysates were prepared and analysed for myc-GIMAP6, GABARAPL2 or β-actin expression, as indicated, by Western blotting. C) A myc-GIMAP6 T-Rex Hela cell line was grown for various times in the presence or absence of tetracycline. After 4 days, some dishes of cells that had been grown in the presence of tetracycline were extensively washed and maintained in the absence of tetracycline for further time intervals. At each time-point, cell lysates were prepared and analysed for GIMAP6, GABARAPL2 or β-actin expression (with primary antibodies: rat anti-GIMAP6 monoclonal antibody MAC 445, rat anti-GABARAPL2 MAC446, and mouse anti-β-actin monoclonal antibody AC-15 respectively, followed by the corresponding HRP-conjugated second antibodies) by Western blotting. The experiment was performed on both clones 5 and 6 from panel B with similar results – that from clone 6 is shown. D) A myc-GIMAP6 T-Rex HeLa cell line was incubated in the presence (plus) or absence (minus) of tetracycline for four days. Lysates were prepared, separated by SDS-PAGE, and Western blotted for the expression of GIMAP6 (using rat monoclonal antibody, MAC445), GABARAPL2 (rat monoclonal antibody, MAC446), or other Atg8 members and SQSTM1, using antibodies as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077782#s2" target="_blank">Materials and Methods</a> section. The results shown are representative of two independent experiments. E) Total RNA was isolated from a myc-GIMAP6 T-Rex HeLa cell line which had been grown in the presence or absence of tetracycline for four days. qPCR was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077782#s2" target="_blank">Materials and Methods</a> section. Expression levels were normalised between samples to GAPDH and then the levels of individual RNA species represented as a fold-stimulation of the plus-tetracycline samples relative to those from cells maintained in the absence of tetracycline. Data are presented as mean ± range of two independent experiments. F) myc-GIMAP6 HEK293 cells (right-hand panels) or the corresponding vector cells (left-hand panels) were treated with 10 µM emetine for the indicated times. Cell lysates were prepared and analysed by SDS-PAGE and Western blotting using either anti-GABARAPL2 rat monoclonal MAC446 or anti-β-ACTIN followed by the appropriate HRP-conjugated secondary antibodies. The result shown is representative of two independent experiments. G) Individual clones of TRex-Jurkat cells carrying GIMAP6 shRNA sequences were either treated for 4 days with 1 µg/ml tetracycline or were similarly maintained but in the absence of tetracycline. Cell lysates were prepared and assayed for GIMAP6 (using mAb MAC445) GABARAPL2 (using mAb MAC446) or β-ACTIN expression. Clones 1-3 carried shRNA1 and clones 4-6, shRNA2 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077782#s2" target="_blank">Materials and Methods</a>). The gap between clones 2 and 3 reflects the removal of intermediate lanes because of inconsistent cell recovery in those samples. </p

Over-expressed GIMAP6 is recruited to MAP1LC3B positive autophagosomes on induction of autophagy.

<p>A) myc-GIMAP6 HEK293 cells were grown on coverslips and were either left untreated (control) or maintained in starvation medium (starved) or treated with mTOR inhibitors AZD8055 (1 µM final concentration) or PP242 (0.4 µM) for 90 min. Cells were then processed for immunocytochemistry using rat anti-human GIMAP6 monoclonal antibody MAC445 or rabbit anti-MAP1LC3B (Sigma product number L7543) followed by an Alexafluor 568-conjugated anti-rat IgG or an Alexafluor 488-conjugated anti-rabbit IgG, respectively. Images were captured using an Olympus FV1000 imaging system. B) Graphical representation of the co-localisation of GIMAP6 puncta with MAP1LC3B puncta, and are shown as a mean percentage ± SD for each autophagic stimulus. Analysis was performed using Imaris software. 7-8 fields of approximately 10 cells/field were viewed for each analysis. C) Cells were grown and treated as in A). They were then stained for GIMAP6 using a rabbit anti-human GIMAP6 polyclonal antiserum and for GABARAPL2 using rat anti-GABARAPL2 mAb MAC446 followed by an Alexafluor 488 conjugated goat anti-rabbit IgG and an Alexafluor 568-conjugated goat anti-rat IgG, respectively. Images were captured using an Olympus FV1000 imaging system. In panels A and C the scale bar represents 13 µm. The results shown in panels A and C are representative of three independent experiments.</p

The C-terminal 10 amino acids of GIMAP6 are required for its recruitment to autophagosomes.

<p>A stable HEK293 cell line expressing a myc-tagged GIMAP6 lacking the C-terminal 10 amino acids and the myc-GIMAP6 HEK293 cell line were either left untreated or were treated with starvation medium for 90 min. Cells were then processed for immunocytochemistry using rat anti-human GIMAP6 monoclonal antibody MAC445 or rabbit anti-MAP1LC3B (Sigma product number L7543) followed by an Alexafluor 488-conjugated anti-rat IgG or an Alexafluor 568-conjugated anti-rabbit IgG, respectively. Images were captured using an Olympus FV1000 imaging system. GIMAP6 (1-292) indicates the full-length protein and GIMAP6 (1-282) the truncated form. Scale bar represents 16 µm. The results shown are representative of three independent experiments.</p

Endogenously expressed GIMAP6 is relocated to punctate structures in response to starvation A) Jurkat-T cells were grown on poly-L-lysine-treated coverslips and were either left untreated or maintained in starvation medium for 90 min.

<p>Cells were then processed for immunocytochemistry using rat anti-human GIMAP6 monoclonal antibody MAC445 or rabbit anti-MAP1LC3B (Sigma product number L7543) followed by an Alexafluor 488-conjugated anti-rat IgG or an Alexafluor 568-conjugated anti-rabbit IgG, respectively. Note that on starvation cells flatten slightly on to the coverslips and thus have a slightly different appearance with respect to the nucleus. Images were captured using a Nikon N-SIM super-resolution system with a CFI Apo TIRF 100x oil objective lens (N.A. 1.49). Scale bar represents 2.4 µm. B) (i) Cell lysates prepared from control or HUVEC cells starved as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077782#pone-0077782-g006" target="_blank">Figure 6A</a> were analysed by Western blotting using rat mAb MAC445 to GIMAP6 or a rabbit polyclonal antibody to MAP1LC3B as indicated. MAP1LC3B-II is indicated by an arrow. (ii) HUVEC cells were grown and then were either starved or left untreated and subsequently processed for immunocytochemistry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077782#pone-0077782-g006" target="_blank">Figure 6A</a>. Fluorescence microscopy was performed using an Axio Imager.D2 microscope (Carl Zeiss Microscopy) with a 100 x oil emersion objective. The results shown in each panel are representative of three independent experiments.</p

GIMAP6 interacts with GABARAPL2 in mammalian cells and <i>in vitro</i>.

<p>A) Jurkat T-cells engineered to over-express myc-tagged BirA and GIMAP6 carrying a biotinylation target sequence were incubated with or without 1% (v/v) formaldehyde at room temperature for the indicated times. Cell lysates were analysed by Western blotting with a streptavidin-HRP conjugate to reveal biotinylated proteins. The mobilities of biotinylated GIMAP6 and the cross-linked species (X-linked species) are shown. The electrophoretic mobility of molecular weight protein standards run in parallel is indicated. Note that the gap between the 15 and 60 min samples indicates that intermediate tracks have been removed. B) Silver-stained SDS-PAGE gel of purified biotinylated GIMAP6 and co-purifying proteins from myc-birA Jurkat cells stably transfected with either plasmid biot-GIMAP6-His-pCAG-iPuro (Lane 2) or the corresponding vector (Lane 1). The electrophoretic mobilities of biotinylated human GIMAP6 and GABARAPL2 are shown, as are those of molecular weight protein standards run in parallel. The asterisks (*) indicate the location of streptavidin released from the streptavidin-agarose beads. C) The sequence of GABARAPL2. Sequence coverage detected in tryptic peptides by the mass spectrometry analysis are shown in bold and underlined. D) HEK293T cells (3 x 10⁶) were transfected with a plasmid (10 µg) encoding myc-tagged human GIMAP6 together with a plasmid (10 µg) encoding either an HA-tagged GABARAPL2 or the corresponding vector. Post-nuclear supernatants were then either directly separated by SDS-PAGE (lysate) or immunoprecipitated with anti-HA mouse mAb 12CA5 and protein-A Sepharose prior to SDS-PAGE (anti-HA IP). Separated proteins were analysed by Western blotting using either anti-HA mouse mAb 12CA5 or anti-myc mAb 9E10 followed by HRP-conjugated goat anti-mouse IgG. E) Jurkat T cells (approximately 9 x 10⁷) were incubated in PBS with or without 1% (w/v) formaldehyde for 1 h at room temperature. The reaction was terminated by adding 1/10^th volume of 1.25 M glycine, and cells solubilised into 200 µl TX100 lysis buffer containing mammalian protease inhibitors (Sigma). After centrifugation (20000 g, 5 min, 4°C) an equal volume of 2 x CSB was added and the samples either heated at 100°C (boiled) for 30 min to reverse the formaldehyde-induced cross-links or left untreated. Aliquots of the boiled and unboiled (untreated or cross-linked) lysates were then separated on SDS-PAGE gels and then Western blotted, using rat mAb MAC445 to human GIMAP6 - (left panel) or rat mAb MAC446 to GABARAPL2 (right panel) followed by horse-radish peroxidase (HRP) conjugated goat F(ab’)₂ fragment anti-rat IgG. Blots were then developed using Immobilon ECL western blotting substrate. Cross-linked samples are indicated by “X-linked”. The mobilities of GIMAP6 and GABARAPL2 are indicated. F) Glutathione Sepharose 4B-immobilised GST or GST-GIMAP6 was incubated with bacterially expressed purified GABARAPL2. Proteins were then eluted with glutathione and the eluates (two eluted fractions from each column), resolved by SDS-PAGE. Proteins were then visualised either by Coomassie Blue staining or by Western blotting with anti-GABARAPL2 monoclonal antibody MAC446. Results shown in panels A, D, E and F are representative of data obtained in at least two independent experiments. Panel B shows a typical example of one of the three pooled purifications performed to allow identification of GIMAP6-interacting proteins.</p

Specificity of GIMAP6-GABARAPL2 interactions.

<p>A) HEK293T cells were transfected with 10 µg GABARAPL2 in pcDNA3Biot1His6iresBirA and 10 µg myc-tagged human GIMAP-encoding plasmids as indicated. 48 h later, cell lysates were prepared and biotinylated GABARAPL2 and associated proteins recovered on streptavidin-agarose beads. Aliquots of the lysates and recovered proteins were separated by SDS-PAGE and blotted for biotinylated proteins using HRP-conjugated streptavidin or myc-tagged proteins using mouse anti-myc mAb 9E10 followed by HRP-conjugated goat anti-mouse IgG. B) HEK293T cells were transfected with 10 µg human GIMAP6 in pcDNA3Biot1His6iresBirA and 10 µg HA-tagged Atg8-encoding plasmids as indicated. 48 h later, cell lysates were prepared and biotinylated GIMAP6 and associated proteins recovered on streptavidin-agarose beads. Aliquots of the lysates and recovered proteins were separated by SDS-PAGE and blotted for biotinylated proteins using HRP-conjugated streptavidin or HA-tagged proteins using mouse anti-HA mAb 12CA5 followed by HRP-conjugated goat anti-mouse IgG. Blots were developed using Immobilon ECL western blotting substrate. C) Biotinylated proteins were purified from the Biot-GIMAP6-His myc-BirA-Jurkat cell line or the corresponding vector-only cell line using streptavidin-agarose. Western blots of lysates prepared directly from the cells or of the purified proteins were then probed with HRP-conjugated streptavidin to visualise biotinylated GIMAP6 or with MAb MAC446 to GABARAPL2 or antibodies (as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077782#s2" target="_blank">Materials and Methods</a> section) to other Atg8 family members or β-ACTIN as described by the suppliers. Results in all three panels are representative of data obtained from at least two independent experiments.</p

Digital neocolonialism and massive open online courses (MOOCs): colonial pasts and neoliberal futures

Through evaluating dominant MOOC platforms created by Western universities, I argue that MOOCs on such platforms tend to embed Western-centric epistemologies and propagate this without questioning their global relevance. Consequently, such MOOCs can be detrimental when educating diverse and complex participants as they erode local and indigenous knowledge systems. Arguing that the digital divide is an exacerbation of historical inequalities, I draw parallels between colonial education, specifically across Sub-Saharan Africa, and ‘digital neocolonialism’ through Western MOOC platforms. I analyse similarities in ideology, assumptions, and methods of control. Highlighting evolving forms of coloniality, I include contemporary problems created by neoliberal techno-capitalist agendas, such as the commodification of education. Balance is needed between the opportunities offered through MOOCs and the harms they cause through overshadowing marginalised knowledges and framing disruptive technologies as the saviour. While recommending solutions for inclusion of marginalised voices, further problems such as adverse incorporation are raised.Cambridge Trust is my sponsor for my Ph