Assessing Maturity for e-Government Services

Part 11: e-GovernanceInternational audienceE-service development as an integral part of e-government is growing area so the assessment of maturity of these services is becoming increasingly relevant. This paper presents more precise method for the evaluation of e-service maturity. It is based on stage model, the service division into the components – operations and the statistics of intensity of their usage in traditional and electronic space. The method is illustrated by data sample for the driver license e-service maturity evaluation. It can be applied to both the online service compared to the same at different stages of its evolution, or installed in different organizations (e.g. municipalities) or even in different countries, as well as comparing maturity among different e-services

Interferon-γ suppresses intestinal epithelial aquaporin-1 expression via Janus kinase and STAT3 activation.

Inflammatory bowel diseases are associated with dysregulated electrolyte and water transport and resultant diarrhea. Aquaporins are transmembrane proteins that function as water channels in intestinal epithelial cells. We investigated the effect of the inflammatory cytokine, interferon-γ, which is a major player in inflammatory bowel diseases, on aquaporin-1 expression in a mouse colonic epithelial cell line, CMT93. CMT93 monolayers were exposed to 10 ng/mL interferon-γ and aquaporin-1 mRNA and protein expressions were measured by real-time PCR and western blot, respectively. In other experiments, CMT93 cells were pretreated with inhibitors or were transfected with siRNA to block the effects of Janus kinases, STATs 1 and 3, or interferon regulatory factor 2, prior to treatment with interferon-γ. Interferon-γ decreased aquaporin-1 expression in mouse intestinal epithelial cells in a manner that did not depend on the classical STAT1/JAK2/IRF-1 pathway, but rather, on an alternate Janus kinase (likely JAK1) as well as on STAT3. The pro-inflammatory cytokine, interferon-γ may contribute to diarrhea associated with intestinal inflammation in part through regulation of the epithelial aquaporin-1 water channel via a non-classical JAK/STAT receptor signalling pathway

Signaling pathways induced by serine proteases to increase intestinal epithelial barrier function.

Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn's disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted epithelial barrier function

A pan-JAK inhibitor prevents the decrease in AQP1 expression following treatment with IFNγ.

<p>(A) Immunoblots showing expression of phosphorylated and total STAT1, phosphorylated and total STAT3, AQP1 and actin in CMT93 cells pretreated with a pan-JAK inhibitor (20 μM for 2 hr) or DMSO vehicle followed by treatment with or without IFNγ (10 ng/mL for 24 hr). (B) Densitometry graph of phospho-STAT1 normalized to total (t) STAT1 expression. (C,D) Densitometry graph of phospho-STAT3α and phospho-STAT3β each normalized to total (t) STAT3 expression. (E) Densitometry graph of AQP1 normalized to actin expression. *p < 0.05, **p < 0.01, ***p < 0.001 vs. DMSO + IFNγ; ^$$$p < 0.001 vs. DMSO alone and pan-JAK Inhibitor alone; ^#p < 0.05 vs. DMSO alone. Blots are representative of 4 separate experiments.</p

Inhibition of CK2 completely prevents the serine protease induced increase in TER.

<p>SCBN cells were mounted in Ussing chambers and treated apically with 50 or 100 μM TBCA for 30 minutes prior to apical challenge with 45 BAU/mL trypsin. A representative tracing is shown in <b>A</b>. The peak change in TER post trypsin was determined and TBCA at 100 μM significantly reduces the change in TER in response to trypsin (<b>B</b>). * p<0.05 as assessed by ANOVA with Dunnett’s post hoc test compared to DMSO control. N = 5–8.</p

Knockdown of STAT3 partially restores the IFNγ-induced decrease in AQP1 expression.

<p>(A) Immunoblots of phospho-STAT3, total STAT3, AQP1 and actin levels in CMT93 cells pretreated with STAT3 or scrambled siRNA (80 nM for 24 hr) or with transfection medium alone (Lipofectamine Control) in the presence or absence of IFNγ (10 ng/mL for 24 hr) (B) Densitometry graph of total STAT3 normalized to actin expression. (C) Densitometry graph of AQP1 normalized to actin expression. **p < 0.01 vs. scrambled siRNA + media; ^##p < 0.01 vs. scrambled siRNA + IFNγ blots are representative of 3 separate experiments.</p

Epithelial AQP1 expression is decreased following treatment of CMT93 epithelial cells with IFNγ.

<p>Confocal immunocytochemistry was performed to detect AQP1 in CMT93 cells treated with either vehicle or IFNγ (10 ng/mL for 24 hr). Constitutive AQP1 immunoreactivity was observed apically and laterally in control cells, which co-localized with E-cadherin. An overall decrease in AQP1 expression in the cell monolayers was observed after treatment with IFNγ. Much of the remaining AQP1 appeared to be re-localized from cell membranes to vesicular cytosolic structures. Images are representative of 4 monolayers per group.</p