Oncogenic K-Ras promotes proliferation in quiescent intestinal stem cells

K-Ras is a monomeric GTPase that controls cellular and tissue homeostasis. Prior studies demonstrated that mutationally activated K-Ras (K-RasG12D) signals through MEK to promote expansion and hyperproliferation of the highly mitotically active transit-amplifying cells (TACs) in the intestinal crypt. Its effect on normally quiescent stem cells was unknown, however. Here, we have used an H2B-Egfp transgenic system to demonstrate that K-RasG12D accelerates the proliferative kinetics of quiescent intestinal stem cells. As in the TAC compartment, the effect of mutant K-Ras on the quiescent stem cell is dependent upon activation of MEK. Mutant K-Ras is also able to increase self-renewal potential of intestinal stem cells following damage. These results demonstrate that mutant K-Ras can influence intestinal homeostasis on multiple levels

Development of Escherichia coli Asparaginase II for Immunosensing: A Trade-Off between Receptor Density and Sensing Efficiency

The clinical success of Escherichia coli l-asparaginase II (EcAII) as a front line chemotherapeutic agent for acute lymphoblastic leukemia (ALL) is often compromised because of its silent inactivation by neutralizing antibodies. Timely detection of silent immune response can rely on immobilizing EcAII, to capture and detect anti-EcAII antibodies. Having recently reported the use of a portable surface plasmon resonance (SPR) sensing device to detect anti-EcAII antibodies in undiluted serum from children undergoing therapy for ALL (Aubé et al., <i>ACS Sensors</i> <b>2016</b>, <i>1</i> (11), 1358–1365), here we investigate the impact of the quaternary structure and the mode of immobilization of EcAII onto low-fouling SPR sensor chips on the sensitivity and reproducibility of immunosensing. We show that the native tetrameric structure of EcAII, while being essential for activity, is not required for antibody recognition because monomeric EcAII is equally antigenic. By modulating the mode of immobilization, we observed that low-density surface coverage obtained upon covalent immobilization allowed each tetrameric EcAII to bind up to two antibody molecules, whereas high-density surface coverage arising from metal chelation by N- or C-terminal histidine-tag reduced the sensing efficiency to less than one antibody molecule per tetramer. Nonetheless, immobilization of EcAII by metal chelation procured up to 10-fold greater surface coverage, thus resulting in increased SPR sensitivity and allowing reliable detection of lower analyte concentrations. Importantly, only metal chelation achieved highly reproducible immobilization of EcAII, providing the sensing reproducibility that is required for plasmonic sensing in clinical samples. This report sheds light on the impact of multiple factors that need to be considered to optimize the practical applications of plasmonic sensors

The colonic epithelium plays an active role in promoting colitis by shaping the tissue cytokine profile

Inflammatory bowel disease (IBD) is a chronic condition driven by loss of homeostasis between the mucosal immune system, the commensal gut microbiota, and the intestinal epithelium. Our goal is to understand how these components of the intestinal ecosystem cooperate to control homeostasis. By combining quantitative measures of epithelial hyperplasia and immune infiltration with multivariate analysis of inter- and intracellular signaling, we identified epithelial mammalian target of rapamycin (mTOR) signaling as a potential driver of inflammation in a mouse model of colitis. A kinetic analysis of mTOR inhibition revealed that the pathway regulates epithelial differentiation, which in turn controls the cytokine milieu of the colon. Consistent with our in vivo analysis, we found that cytokine expression of organoids grown ex vivo, in the absence of bacteria and immune cells, was dependent on differentiation state. Our study suggests that proper differentiation of epithelial cells is an important feature of colonic homeostasis because of its effect on the secretion of inflammatory cytokines

Real-time measurement of small molecules directly in awake, ambulatory animals

The development of a technology capable of tracking the levels of drugs, metabolites, and biomarkers in the body continuously and in real time would advance our understanding of health and our ability to detect and treat disease. It would, for example, enable therapies guided by high-resolution, patient-specific pharmacokinetics (including feedback-controlled drug delivery), opening new dimensions in personalized medicine. In response, we demonstrate here the ability of electrochemical aptamer-based (E-AB) sensors to support continuous, real-time, multihour measurements when emplaced directly in the circulatory systems of living animals. Specifically, we have used E-AB sensors to perform the multihour, real-time measurement of four drugs in the bloodstream of even awake, ambulatory rats, achieving precise molecular measurements at clinically relevant detection limits and high (3 s) temporal resolution, attributes suggesting that the approach could provide an important window into the study of physiology and pharmacokinetics

Epithelial differentiation reduces inflammatory cytokine expression in colonic organoids.

<p>(A) RNA markers of stem cells (<i>Lgr5</i>), enterocytes (<i>Alpi</i>), and goblet cells (<i>Muc2</i>) and enteroendocrine cells <i>(Chga</i>) were measured by quantitative polymerase chain reaction (qPCR) and normalized to standard culture condition organoid expression levels. Conditions for enrichment of stem cells or specific differentiated lineages can be found in the Materials and methods section. Horizontal and vertical lines represent mean +/− standard error. Significance was determined by ANOVA with Tukey-Kramer post-test. * represents <i>p</i> ≤ 0.05; *** represents <i>p</i> ≤ 0.001. (B) Representative cytokines, chemokines, and growth factors were measured by Luminex and represented in pg/ml. Horizontal and vertical lines represent mean +/− standard error. Significance was determined by ANOVA with Tukey-Kramer post-test. * represents <i>p</i> ≤ 0.05; ** represents <i>p</i> ≤ 0.01. (C) A partial least squares regression (PLSR) model built on cytokine, chemokine, and growth factor measurements from the mouse experiment described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002417#pbio.2002417.g001" target="_blank">Fig 1</a> with organoid protein expression data projected. Differentiated organoids project with focal inflammation and control groups, while proliferative, stem-like organoids are projected with the continuous inflammation group. Original samples used to generate the model project into the gray rectangle (control), blue rectangle (focal inflammation), and red rectangle (continuous inflammation). Underlying numerical values for panels A–C are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002417#pbio.2002417.s001" target="_blank">S1 Data</a>. MCP-1, monocyte chemoattractant protein 1; MIG, mitokine induced by gamma interferon; MIP-1α, macrophage inflammatory protein 1-alpha.</p

Goblet cell differentiation leads to reduced cytokine expression and innate immune cell infiltration.

<p>(A) Alcian Blue/Periodic Acid Schiff (AB/PAS) stain for mucin shows loss of goblet cells in inflamed animals that is restored by treatment with the Notch pathway inhibitor dibenzazepine (DBZ). (B) Immune infiltrate, as measured by flow cytometry expressed as a percentage of total cells, shows a reduction in macrophages and neutrophils following 1-week treatment with DBZ. Significance determined by unpaired <i>t</i> test, with <i>p</i>-values indicated above each cell type. <i>N</i> = 2–3 animals per condition. Bar graphs represent mean +/− standard error. (C) Inflammation-associated cytokine and chemokine expression was measured by Luminex and displayed as log2-fold changes versus control animals. These proteins represent the top 20 loadings from the partial least squares regression (PLSR) model described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002417#pbio.2002417.g005" target="_blank">Fig 5D</a>. Data for individual animals are presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002417#pbio.2002417.s009" target="_blank">S8 Fig</a>. (D) DBZ- and vehicle (Veh.)-treated cytokine and chemokine measurements were projected onto a PLSR model built from non-drug-treated animals. DBZ-treated animals cluster with focal inflamed and noninflamed animals. Original samples used to generate the model project into the gray rectangle (control), blue rectangle (focal inflammation), and red rectangle (continuous inflammation). Underlying numerical values for panels B–D are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002417#pbio.2002417.s001" target="_blank">S1 Data</a>. Avg, average; IFNγ, interferon gamma; IL, interleukin; LIF, leukemia inhibitory factor; LV1, latent variable 1; MCP-1, monocyte chemoattractant protein 1; MIG, mitokine induced by gamma interferon; MIP-1α/β, macrophage inflammatory protein 1-alpha/beta; M-CSF, macrophage colony-stimulating factor; RANTES, regulated on activation, normal T cell expressed and secreted; VEGF, vascular endothelial growth factor.</p

Expansion of transit amplifying cells in animals with colitis.

<p>(A) Markers of goblet cells, enterocytes (Ent.), enteroendocrine cells (EE), and stem cells measured by RNA microarray. Each gene was mean centered, and the means of inflamed and noninflamed groups are indicated in red and gray, respectively. Horizontal and vertical lines represent mean +/− standard error. Significance was determined by unpaired <i>t</i> test. * represents <i>p</i> ≤ 0.05, ** represents <i>p</i> ≤ 0.01, *** represents <i>p</i> ≤ 0.001. (B) Average fold change between inflamed and noninflamed groups for each of the genes in panel A. (C) Enrichment plots for experimentally derived gene sets for secretory progenitors (Secpro), stem cells, enterocytes (Ent), goblet cell-deficient epithelium (Atoh null), and transit-amplifying cell (TAC) epithelium. (D) Normalized enrichment scores for the gene sets indicated in panel C; false discovery rate (FDR) values are indicated above or below each bar. (E) Mean-centered expression values of the 4 chemokines found in the TAC gene set. The means of inflamed and noninflamed groups are indicated in red and gray, respectively. Horizontal and vertical lines represent mean +/− standard error. All genes showed a <i>p</i> < 0.001 in a <i>t</i> test, as indicated by ***. Underlying numerical values for panels A and C–E are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002417#pbio.2002417.s001" target="_blank">S1 Data</a>.</p

Multivariate molecular analysis of murine colitis.

<p>(A) Principal component analysis (PCA) based on Luminex measurements from distal colons identifies clusters of the control, focal inflammation, and continuous inflammation classes on principal component 1 (PC1). (B) Correlation between PCA and crypt height. For each animal, the score on PC1 was plotted against the average crypt height in the distal colon. This indicates that there is a strong correlation (R² = 0.8) between PC1 and epithelial measures of inflammation. (C) Loadings on PC1 represented as single dots for each protein and grouped and color-coded by pathway or category. Signals on the right are positively correlated with inflammation. (D) Immunohistochemistry for p-S6 ribosomal protein (p-S6RP), a readout of the mammalian target of rapamycin (mTOR) pathway. p-S6RP was strongly induced in inflamed animals and was found primarily in epithelial cells. Underlying numerical values for panels A and B are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002417#pbio.2002417.s001" target="_blank">S1 Data</a>. MAPK, mitogen-activated protein kinase.</p