Polymer based biosensor for rapid electrochemical detection of virus infection of human cells

The demand in the field of medical diagnostics for simple, cost efficient and disposable devices is growing. Here, we present a label free, all-polymer electrochemical biosensor for detection of acute viral disease. The dynamics of a viral infection in human cell culture was investigated in a micro fluidic system on conductive polymer PEDOT:TsO microelectrodes by electrochemical impedance spectroscopy and video time lapse microscopy. Employing this sensitive, real time electrochemical technique, we could measure the immediate cell response to cytomegalovirus, and detect an infection within 3h, which is several hours before the cytopathic effect is apparent with conventional imaging techniques. Atomic force microscopy and scanning ion conductance microscopy imaging consolidate the electrochemical measurements by demonstrating early virus induced changes in cell morphology of apparent programmed cell death.     

Assessment of in vivo and in vitro cytotoxic activity of hydrolysable tannin extracted from Rhizophora apiculata barks

World Journal of Microbiology and Biotechnology - Rhizophora apiculata is a common mangrove tree in Malaysia. The bark of this tree has been reported to contain a chemical constituent such as...     

Oxidative stress, insulin signaling, and diabetes

Oxidative stress has been implicated as a contributor to both the onset and the progression of diabetes and its associated complications. Some of the consequences of an oxidative environment are the development of insulin resistance, β-cell dysfunction, impaired glucose tolerance, and mitochondrial dysfunction, which can lead ultimately to the diabetic disease state. Experimental and clinical data suggest an inverse association between insulin sensitivity and ROS levels. Oxidative stress can arise from a number of different sources, whether disease state or lifestyle, including episodes of ketosis, sleep restriction, and excessive nutrient intake. Oxidative stress activates a series of stress pathways involving a family of serine/threonine kinases, which in turn have a negative effect on insulin signaling. More experimental evidence is needed to pinpoint the mechanisms contributing to insulin resistance in both type 1 diabetics and nondiabetic individuals. Oxidative stress can be reduced by controlling hyperglycemia and calorie intake. Overall, this review outlines various mechanisms that lead to the development of oxidative stress. Intervention and therapy that alter or disrupt these mechanisms may serve to reduce the risk of insulin resistance and the development of diabetes.     

Discovery of inhibitors of plasminogen activator inhibitor-1: structure-activity study of 5-nitro-2-phenoxybenzoic acid derivatives

Two novel series of 5-nitro-2-phenoxybenzoic acid derivatives are designed as potent PAI-1 inhibitors using hybridization and conformational restriction strategy in the tiplaxtinin and piperazine chemo types. The lead compounds 5a, 6c, and 6e exhibited potent PAI-1 inhibitory activity and favorable oral bioavailability in the rodents.     

Complete genome sequence of Corynebacterium pseudotuberculosis strain CIP 52.97, isolated from a horse in Kenya

In this work, we report the whole-genome sequence of Corynebacterium pseudotuberculosis bv. equi strain CIP 52.97 (Collection Institut Pasteur), isolated in 1952 from a case of ulcerative lymphangitis in a Kenyan horse, which has evidently caused significant losses to agribusiness. Therefore, obtaining this genome will allow the detection of important targets for postgenomic studies, with the aim of minimizing problems caused by this microorganism.     

Hydrogen sulfide and L-cysteine increase phosphatidylinositol 3,4,5-trisphosphate (PIP3) and glucose utilization by inhibiting phosphatase and tensin homolog (PTEN) protein and activating phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (AKT)/protein kinase Cζ/λ (PKCζ/λ) in 3T3l1 adipocytes

This work examined the novel hypothesis that reduced levels of H(2)S or L-cysteine (LC) play a role in the impaired glucose metabolism seen in diabetes. 3T3L1 adipocytes were treated with high glucose (HG, 25 mM) in the presence or absence of LC or H(2)S. Both LC and H(2)S treatments caused an increase in phosphatidylinositol-3,4,5 trisphosphate (PIP3), AKT phosphorylation, and glucose utilization in HG-treated cells. The effect of LC on PIP3 and glucose utilization was prevented by propargylglycine, an inhibitor of cystathionine γ-lyase that catalyzes H(2)S formation from LC. This demonstrates that H(2)S mediates the effect of LC on increased PIP3 and glucose utilization. H(2)S and LC caused phosphatidylinositol 3-kinase activation and PTEN inhibition. Treatment with LC, H(2)S, or PIP3 increased the phosphorylation of IRS1, AKT, and PKCζ/λ as well as GLUT4 activation and glucose utilization in HG-treated cells. This provides evidence that PIP3 is involved in the increased glucose utilization observed in cells supplemented with LC or H(2)S. Comparative signal silencing studies with siAKT2 or siPKCζ revealed that PKCζ phosphorylation is more effective for the GLUT4 activation and glucose utilization in LC-, H(2)S-, or PIP3-treated cells exposed to HG. This is the first report to demonstrate that H(2)S or LC can increase cellular levels of PIP3, a positive regulator of glucose metabolism. The PIP3 increase is mediated by PI3K activation and inhibition of PTEN but not of SHIP2. This study provides evidence for a molecular mechanism by which H(2)S or LC can up-regulate the insulin-signaling pathways essential for maintenance of glucose metabolism.     

Synthesis and characterization of diethyltin-based three dimensional self-assemblies derived from sulfonate-phosphonate ligands

Diethyltin(methoxy)methanesulfonate reacts readily with equimolar amount of t-butylphosphonic acid (RT, 8-10 h) in moist dichloromethane and methanol to afford [(Et₂Sn)₆(O₃PBu^t)₄(OSO₂Me)₄·CH₂Cl₂]_n (1) and [(Et₂Sn)₆(O₃PBu^t)₄(OSO₂Me)₄·2H₂O]_n (2), respectively. The identity of these compounds has been established by IR, multinuclear (¹H, ¹³C, ³¹P and ¹¹⁹Sn) NMR as well as X-ray crystallographic studies. Despite apparent similarity in the composition, a marked difference in the structural motifs between 1 and 2 is evident in the solid state. For 1, the formation of three-dimensional self- assembly results from μ₃-phosphonate and μ₂, μ₃-sulfonate binding modes and exhibits continuous channel of voids which are occupied by disordered CH₂Cl₂ molecules. In contrast, the sulfonate ligands in 2 act exclusively in μ₂-fashion and hydrogen bonding interactions (O-H?O) between coordinated water molecules and methanesulfonate groups is significant in the construction of 3D supramolecular assembly.     

An HLA-presented fragment of macrophage migration inhibitory factor is a therapeutic target for invasive breast cancer

This report describes a novel HLA/peptide complex with potential prognostic and therapeutic roles for invasive breast cancer. Macrophage migration inhibitory factor (MIF) mediates inflammation and immunity, and MIF overexpression is observed in breast cancer. We hypothesized that the HLA class I of cancerous breast epithelial cells would present MIF-derived peptides. Consistent with this hypothesis, the peptide FLSELTQQL (MIF(19-27)) was eluted from the HLA-A*0201 (HLA-A2) of breast cancer cell lines. We posited that if this MIF(19-27)/HLA-A2 complex was exclusively found in invasive breast cancer, it could be a useful prognostic indicator. To assess the presentation of MIF peptides by the HLA of various cells and tissues, mice were immunized with the MIF(19-27)/HLA-A2 complex. The resulting mAb (RL21A) stained invasive ductal carcinoma (IDC) but not ductal carcinoma in situ, fibroadenoma, or normal breast tissues. RL21A did not stain WBCs (total WBCs) or normal tissues from deceased HLA-A2 donors, substantiating the tumor-specific nature of this MIF/HLA complex. As this MIF/HLA complex appeared specific to the surface of IDC, RL21A was tested as an immunotherapeutic for breast cancer in vitro and in vivo. In vitro, RL21A killed the MDA-MB-231 cell line via complement and induction of apoptosis. In an in vivo orthotopic mouse model, administration of RL21A reduced MDA-MB-231 and BT-20 tumor burden by 5-fold and by >2-fold, respectively. In summary, HLA-presented MIF peptides show promise as prognostic cell surface indicators for IDC and as targets for immunotherapeutic intervention.     

TCR mimic monoclonal antibodies induce apoptosis of tumor cells via immune effector-independent mechanisms

mAbs that recognize peptides presented on the cell surface by MHC class I molecules are potential therapeutic agents for cancer therapy. We have previously demonstrated that these Abs, which we termed TCR mimic mAbs (TCRm), reduce tumor growth in models of breast carcinoma. However, mechanisms of TCRm-mediated tumor growth reduction remain largely unknown. In this study, we report that these Abs, in contrast to several mAbs used currently in the clinic, destroy tumor cells independently of immune effector mechanisms such as Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We found that TCRm-mediated apoptosis of tumor cells was associated with selective and specific binding of these Abs to peptide/HLA class I complexes, which triggered the activation of JNK and intrinsic caspase pathways. This signaling was accompanied by the release of mitochondrial cytochrome c and apoptosis-inducing factor. TCRm-induced apoptosis in tumor cells was completely inhibited by soluble MHC tetramers loaded with relevant peptide as well as with inhibitors for JNK and caspases. Furthermore, mAbs targeting MHC class I, independent of the peptide bound by HLA, did not stimulate apoptosis, suggesting that the Ab-binding site on the MHC/peptide complex determines cytotoxicity. This study suggests the existence of mechanisms, in addition to ADCC and CDC, through which these therapeutic Abs destroy tumor cells. These mechanisms would appear to be of particular importance in severely immunocompromised patients with advanced neoplastic disease, since immune cell-mediated killing of tumor cells through ADCC and CDC is substantially limited in these individuals.     

Host-based ribavirin resistance influences hepatitis C virus replication and treatment response

Many individuals infected with hepatitis C virus (HCV) develop a chronic infection, and of those who are treated with pegylated interferon and ribavirin (RBV), many do not respond. While the nucleoside analog RBV improves treatment outcome, and will likely be an important component of therapy with next-generation viral inhibitors, RBV's mechanism is controversial. Most of RBV's proposed mechanisms require RBV import into cells. Therefore, we explored whether host-based RBV resistance develops through reduced cellular uptake, akin to chemotherapy resistance in some cancers. We examined the effect of host-based RBV resistance on HCV replication in cultured hepatoma Huh7.5 liver cells and whether RBV resistance develops in HCV patients. When Huh7.5 cells were exposed to RBV, resistance developed through reduced RBV uptake via the ENT1 nucleoside transporter and antiviral efficacy was reduced. The uptake defect in RBV-resistant cells was specific to RBV, since transport of another ENT1 substrate, cytidine, was unaffected. Importantly, RBV uptake significantly declined in HCV patient peripheral blood mononuclear cells (PBMCs) following 4 weeks of therapy. Furthermore, maintenance of RBV uptake correlated with rapid treatment response. Our results uncovered a novel form of antiviral drug resistance and suggest that host-based RBV resistance develops in HCV patients undergoing therapy and that maintenance of RBV uptake may contribute to rapid viral clearance.