We have used differential display to identify genes whose expression is altered in type 2 diabetes thus contributing to its pathogenesis. One mRNA is overexpressed in fibroblasts from type 2 diabetics compared with non-diabetic individuals, as well as in skeletal muscle and adipose tissues, two major sites of insulin resistance in type 2 diabetes. The levels of the protein encoded by this mRNA are also elevated in type 2 diabetic tissues; thus, we named it PED for phosphoprotein enriched in diabetes. PED cloning shows that it encodes a 15 kDa phosphoprotein identical to the protein kinase C (PKC) substrate PEA-15. The PED gene maps on human chromosome 1q21-22. Transfection of PED/PEA-15 in differentiating L6 skeletal muscle cells increases the content of Glut1 transporters on the plasma membrane and inhibits insulin-stimulated glucose transport and cell-surface recruitment of Glut4, the major insulin-sensitive glucose transporter. These effects of PED overexpression are reversed by blocking PKC activity. Overexpression of the PED/ PEA-15 gene may contribute to insulin resistance in glucose uptake in type 2 diabetes.

This paper presents an updated and comprehensive review on the different methods used for detection and quantification of viruses in wastewater treatment systems. The analysis of viability of viruses in wastewater and sludge is another thrust of this review. Recent studies have mostly focused on determining the abundance and diversity of viruses in wastewater influents, in samples from primary, secondary, and tertiary treatment stages, and in final effluents. A few studies have also examined the occurrence and diversity of viruses in raw and digested sludge samples. Recent efforts to improve efficiency of virus detection and quantification methods in the complex wastewater and sludge matrices are highlighted in this review. A summary and a detailed comparison of the pre-treatment methods that have been utilized for wastewater and sludge samples are also presented. The role of metagenomics or sequencing analysis in monitoring wastewater systems to predict disease outbreaks, to conduct public health surveillance, to assess the efficiency of existing treatment systems in virus removal, and to re-evaluate current regulations regarding pathogenic viruses in wastewater is discussed in this paper. Challenges and future perspectives in the detection of viruses, including emerging and newly emerged viruses such as the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), in wastewater systems are discussed in this review.

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PED/PEA-15 is a recently cloned 15 kDa protein possessing a death eector domain (DED). In MCF-7 and HeLa cells, a ®vefold overexpression of PED/PEA-15 blocked FasL and TNFa apoptotic eects. This eect of PED overexpression was blocked by inhibition of PKC activity. In MCF-7 and HeLa cell lysates, PED/ PEA-15 co-precipitated with both FADD and FLICE. PED/PEA-15-FLICE association was inhibited by overexpression of the wild-type but not of a DED-deletion mutant of FADD. Simultaneous overexpression of PED/ PEA-15 with FADD and FLICE inhibited FADD-FLICE co-precipitation by threefold. Based on cleavage of the FLICE substrate PARP, this inhibitory eect was paralleled by a threefold decline in FLICE activation in response to TNF-a. TNFa, in turn, reduces PED association with the endogenous FADD and FLICE of the cells. Thus, PED/PEA-15 is an endogenous protein inhibiting FAS and TNFR1-mediated apoptosis. At least in part, this function may involve displacement of FADD-FLICE binding through the death eector domain of PED/PEA-15.

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Overexpression of the ped/pea-15 gene is a common feature of type 2 diabetes. In the present work, we show that transgenic mice ubiquitously overexpressing ped/pea-15 exhibited mildly elevated random-fed blood glucose levels and decreased glucose tolerance. Treatment with a 60% fat diet led ped/pea-15 transgenic mice to develop diabetes. Consistent with insulin resistance in these mice, insulin administration reduced glucose levels by only 35% after 45 min, compared to 70% in control mice. In vivo, insulin-stimulated glucose uptake was decreased by almost 50% in fat and muscle tissues of the ped/pea-15 transgenic mice, accompanied by protein kinase C␣ activation and block of insulin induction of protein kinase C. These changes persisted in isolated adipocytes from the transgenic mice and were rescued by the protein kinase C inhibitor bisindolylmaleimide. In addition to insulin resistance, ped/pea-15 transgenic mice showed a 70% reduction in insulin response to glucose loading. Stable overexpression of ped/pea-15 in the glucose-responsive MIN6 beta-cell line also caused protein kinase C␣ activation and a marked decline in glucose-stimulated insulin secretion. Antisense block of endogenous ped/pea-15 increased glucose sensitivity by 2.5-fold in these cells. Thus, in vivo, overexpression of ped/pea-15 may lead to diabetes by impairing insulin secretion in addition to insulin action.

Overexpression of the PED/PEA-15 protein in muscle and adipose cells increases glucose transport and impairs further insulin induction. Like glucose transport, protein kinase C (PKC)-α and -β are also constitutively activated and are not further stimulatable by insulin in L6 skeletal muscle cells overexpressing PED (L6PED). PKC-ζ features no basal change but completely loses insulin sensitivity in L6PED. In these cells, blockage of PKC-α and -β additively returns 2-deoxy-d-glucose (2-DG) uptake to the levels of cells expressing only endogenous PED (L6WT). Blockage of PKC-α and -β also restores insulin activation of PKC-ζ in L6PED cells, with that of PKC-α sixfold more effective than PKC-β. Similar effects on 2-DG uptake and PKC-ζ were also achieved by 50-fold overexpression of PKC-ζ in L6PED. In L6WT, fivefold overexpression of PKC-α or -β increases basal 2-DG uptake and impairs further insulin induction with no effect on insulin receptor or insulin receptor substrate phosphorylation. In these cells, overexpression of PKC-α blocks insulin induction of PKC-ζ activity. PKC-β is 10-fold less effective than PKC-α in inhibiting PKC-ζ stimulation. Expression of the dominant-negative K281→W PKC-ζ mutant simultaneously inhibits insulin activation of PKC-ζ and 2-DG uptake in the L6WT cells. We conclude that activation of classic PKCs, mainly PKC-α, inhibits PKC-ζ and may mediate the action of PED on glucose uptake in L6 skeletal muscle cells.

In L6 skeletal muscle cells and immortalized hepatocytes, insulin induced a 2-fold increase in the activity of the pyruvate dehydrogenase (PDH) complex. This effect was almost completely blocked by the protein kinase C (PKC) ␦ inhibitor Rottlerin and by PKC␦ antisense oligonucleotides. At variance, overexpression of wild-type PKC␦ or of an active PKC␦ mutant induced PDH complex activity in both L6 and liver cells. Insulin stimulation of the activity of the PDH complex was accompanied by a 2.5-fold increase in PDH phosphatases 1 and 2 (PDP1/2) activity with no change in the activity of PDH kinase. PKC␦ antisense blocked insulin activation of PDP1/2, the same as with PDH. In insulin-exposed cells, PDP1/2 activation was paralleled by activation and mitochondrial translocation of PKC␦, as revealed by cell subfractionation and confocal microscopy studies. The mitochondrial translocation of PKC␦, like its activation, was prevented by Rottlerin. In extracts from insulinstimulated cells, PKC␦ co-precipitated with PDP1/2. PKC␦ also bound to PDP1/2 in overlay blots, suggesting that direct PKC␦-PDP interaction may occur in vivo as well. In intact cells, insulin exposure determined PDP1/2 phosphorylation, which was specifically prevented by PKC␦ antisense. PKC␦ also phosphorylated PDP in vitro, followed by PDP1/2 activation. Thus, in muscle and liver cells, insulin causes activation and mitochondrial translocation of PKC␦, accompanied by PDP phosphorylation and activation. These events are necessary for insulin activation of the PDH complex in these cells.