Quantum dot based immunosensor using 3D circular microchannels fabricated in PDMS

Microchannel is basic functional component of microfluidic chip and every step-forward of its construction technique has been receiving concern all over the world. The present work describes a novel, rapid and simple fabrication technique for building 3D microchannels in poly(dimethyl siloxane) (PDMS) elastomer. These microchannels were used for rapid detection of antigens (E. coli) by quantum dot (QD) based approach. Luminescent QD (CdTe) were synthesized by aqueous method and characterized using high resolution transmission electron microscopy (HRTEM), fluorescence spectroscopy and X-ray diffraction (XRD). The QDs were functionalized with anti-E. coli antibodies for immuno-detection. The reported process allowed easier and faster method of fabrication of circular 3D micochannels and demonstrated their potential use in an immuno-biosensor device.     

Discovery of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 2: Structure–activity relationships of the 4-, 5-, 6-, 7- and 8-positions

As a continuation of our efforts to discover and develop the apoptosis inducing 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as potential anticancer agents, we explored substitutions at the 4-, 5-, 6-, 7- and 8-positions of pyrrolo[1,2-a]quinoline. SAR studies showed that substitution at the 6-position by a small group such as Cl resulted in potent compounds. Substitutions at the 5- and 8-positions were tolerated while substitutions at the 4- and 7-position led to inactive compounds. Several compounds, including 2c, 3a, 3b and 3f, were found to be highly active against human breast cancer cells T47D with EC₅₀ values of 0.053–0.080 μM, but much less active against human colon cancer cells HCT116 and hepatocellular carcinoma cancer cells SNU398 in the caspase activation assay. Compound 3f also was found to be highly active with a GI₅₀ value of 0.018 μM against T47D cells in a growth inhibition assay.     

Exploration of click reaction for the synthesis of modified nucleosides as chitin synthase inhibitors

Click reaction approach toward the synthesis of two sets of novel 1,2,3-triazolyl linked uridine derivatives 19a–19g and 21a–21g was achieved by Cu(I)-catalyzed 1,3-dipolar cycloaddition of 5′-azido-5′-deoxy-2′,3′-O-(1-methylethylidene)uridine (17) with propargylated ether of phenols 18a–18g and propargylated esters 20a–20g. Structure of one of the representative compound 19d was unambiguously confirmed by X-ray crystallography. Chitin synthase inhibition study of all these compounds 19a–19g and 21a–21g was carried out to develop antifungal strategy. Compounds 19d, 19e, 19f, and 21f were identified as potent chitin synthase inhibitors by comparing with nikkomycin. Compounds 19a, 19b, 19c, 19d, 21a, and 21b showed good antifungal activity against human and plant pathogens. Compounds 19a, 19b, 19f, 21c, 21f, and 21g were identified as lead chitin synthase inhibitors for further modifications by comparing results of inhibition of growth, % germ tube formation and chitin synthase activity.     

Diet‐induced Obese Mice Are Leptin Insufficient After Weight Reduction

Behavioral therapies aimed at reducing excess body fat result in limited fat loss after dieting. To understand the causes for maintenance of adiposity, high‐fat (HF) diet–induced obese (DIO) mice were switched to a low‐fat chow diet, and the effects of chow on histological and molecular alterations of adipose tissue and metabolic parameters were examined. DIO mice reduced and stabilized their body weights after being switched to chow (HF‐chow), but retained a greater amount of adiposity than chow‐fed mice. Reduction in adipocyte volume, not number, caused a decrease in fat mass. HF‐chow mice showed normalized circulating insulin and leptin levels, improved glucose tolerance, and reduced inflammatory status in white adipose tissue (WAT). Circulating leptin levels corrected for fat mass were lower in HF‐chow mice. Leptin administration was used to test whether reduced leptin level of HF‐chow mice inhibited further fat loss. Leptin treatment led to an additional reduction in adiposity. Finally, HF‐HF mice had lower mRNA levels of β₃ adrenergic receptor (β₃‐AR) in epididymal WAT (EWAT) compared to chow‐fed mice, and diet change led to an increase in the WAT β₃‐AR mRNA levels that were similar to the levels of chow‐fed mice, suggesting an elevation in sympathetic activation of WAT during diet switch relative to HF‐HF mice leading to the reduced leptin level and proinflammatory cytokine content. In summary, HF‐chow mice were resistant to further fat loss due to leptin insufficiency. Diet alteration from HF to low fat improved metabolic state of DIO mice, although their adiposity was defended at a higher level.     

Plant‐produced human growth hormone shows biological activity in a rat model

Plants have been shown to be efficient systems for expressing a wide range of recombinant proteins from various origins. Here, using a plant virus‐based expression vector to produce human growth hormone (hGH) in Nicotiana benthamiana plants, we demonstrate, for the first time, that the plant‐produced hGH (pphGH) is biologically active in a hypophysectomized rat model. We observed an average weight gain of ～17 g per animal in a group of 10 animals that were injected subcutaneously with pphGH with 60 μg/dose for 10 days. With the increasing demand for hGH, accompanied with the need to make this recombinant protein available to a wider population at a more reasonable cost, plants provide a feasible alternative to current production platforms. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based HTS assay. Part 5: modifications of the 2- and 3-positions

As a continuation of our efforts to discover and develop apoptosis inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored modifications at the 2- and 3-positions. It was found that replacement of the 3-cyano group by an ester, including methyl and ethyl ester, resulted in >200-fold reduction of activity. Conversion of the 2-amino group into an amide or urea resulted in 4- to 10-fold drop of activity. Similarly, converting the 2-amino group into a hydrogen resulted in 4- to 10-fold reduction of activity. Compound 3d was highly active with an EC(50) value of 29 nM and a GI(50) value of 6 nM in T47D cells. Importantly, the 2-H analog 3d was found to be much more stable under acidic conditions compared to the 2-NH(2) analog 3b, suggesting that 2-H analogs might have better bioavailability than the 2-NH(2) analogs.     

Friend virus utilizes the BMP4-dependent stress erythropoiesis pathway to induce erythroleukemia

More than 50 years of genetic analysis has identified a number of host genes that are required for the expansion of infected cells during the progression of Friend-virus-induced erythroleukemia. In this report, we show that Friend virus induces the bone morphogenetic protein 4 (BMP4)-dependent stress erythropoiesis pathway in the spleen, which rapidly amplifies target cells, propagating their infection and resulting in acute splenomegaly. This mechanism mimics the response to acute anemia, in which BMP4 expressed in the spleen drives the expansion of a specialized population of stress erythroid progenitors. Previously we demonstrated that these progenitors, termed stress BFU-E, are targets for Friend virus in the spleen (A. Subramanian, H. E. Teal, P. H. Correll, and R. F. Paulson, J. Virol. 79:14586-14594, 2005). Here, we extend those findings by showing that Friend virus infects two distinct populations of bone marrow cells. One population, when infected, differentiates into mature erythrocytes in an Epo-independent manner, while a second population migrates to the spleen after infection, where it induces BMP4 expression and acts as a reservoir of virus. The activation of the stress erythropoiesis pathway in the spleen by Friend virus results in the rapid expansion of stress BFU-E, providing abundant target cells for viral infection. These observations suggest a novel mechanism by which a virus induces a stress response pathway that amplifies target cells for the virus, leading to acute expansion of infected cells.     

Matrix-independent activation of phosphatidylinositol 3-kinase,Stat3, and cyclin A-associated Cdk2 Is essential for anchorage-independent growth of v-Ros-transformed chicken embryo fibroblasts

The question remains open whether the signaling pathways shown to be important for growth and transformation in adherent cultures proceed similarly and play similar roles for cells grown under anchorage-independent conditions. Chicken embryo fibroblasts (CEF) infected with the avian sarcoma virus UR2, encoding the oncogenic receptor protein-tyrosine kinase (RPTK) v-Ros, or with two of its transformation-impaired mutants were grown in nonadherent conditions in methylcellulose (MC)-containing medium, and the signaling functions essential for Ros-induced anchorage-independent growth were analyzed. We found that the overall tyrosine phosphorylation of cellular proteins in CEF transformed by v-Ros or by two oncogenic nonreceptor protein-tyrosine kinases (PTKs), v-Src and v-Yes, was dramatically reduced in nonadherent conditions compared with that in adherent conditions, indicating that cell adhesion to the extracellular matrix plays an important role in efficient substrate phosphorylation by these constitutively activated PTKs. The UR2 transformation-defective mutants were differentially impaired compared with UR2 in the activation of phosphatidylinositol 3-kinase (PI 3-kinase) and Stat3 in nonadherent conditions. Consistently, the constitutively activated mutants of PI 3-kinase and Stat3 rescued the ability of the UR2 mutants to promote anchorage-independent growth. Conversely, dominant negative mutants of PI 3-kinase and Stat3 inhibited UR2-induced anchorage-independent growth. UR2-infected CEF grown in nonadherent conditions displayed faster cell cycle progression than the control or the UR2 mutant-infected cells, and this appeared to correlate with a PI 3-kinase-dependent increase in cyclin A-associated Cdk2 activity. Treatment of UR2-infected cells with Cdk2 inhibitors led to the loss of the anchorage-independent growth-promoting activity of UR2. In conclusion, we have adopted an experimental system enabling us to study the signaling pathways in cells grown under anchorage-independent conditions and have identified matrix-independent activation of PI 3-kinase and Stat3 signaling functions, as well as the PI 3-kinase-dependent increase of cyclin A-associated Cdk2 kinase activity, to be critical for the Ros-PTK-induced anchorage-independent growth.     

Heat and mass transfer scale-up issues during freeze-drying,I: Atypical radiation and the edge vial effect

The aim of this study is to determine whether radiation heat transfer is responsible for the position dependence of heat transfer known as the edge vial effect. Freeze drying was performed on a laboratory-scale freeze dryer using pure water with vials that were fully stoppered but had precision cut metal tubes inserted in them to ensure uniformity in resistance to vapor flow. Sublimation rates were determined gravimetrically. Vials were sputter-coated with gold and placed at selected positions on the shelf. Average sublimation rates were determined for vials located at the front, side, and center of an array of vials. Sublimation rates were also determined with and without the use of aluminum foil as a radiation shield. The effect of the guardrail material and its contribution to the edge vial effect by conduction heat transfer was studied by replacing the stainless steel band with a low-thermal conductivity material (styrofoam). The emissivities (ε) of relevant surfaces were measured using an infrared thermometer. Sublimation rate experiments were also conducted with vials suspended off the shelf to study the role of convection heat transfer. It was found that sublimation rates were significantly higher for vials located in the front compared to vials in the center. Additional radiation shields in the form of aluminum foil on the inside door resulted in a decrease in sublimation rates for the front vials and to a lesser extent, the center vials. There was a significant decrease in sublimation rate for goldcoated vials (ε≈0.4) placed at the front of an array when compared to that of clear vials (ε≈0.9). In the case of experiments with vials suspended off the shelf, the heat transfer coefficient was found to be independent of chamber pressure, indicating that pure convection plays no significant role in heat transfer. Higher sublimation rates were observed when the steel band was used instead of Styrofoam while the highest sublimation rates were obtained in the absence of the guardrail, indicating that the metal band can act as a thermal shield but also transmits some heat from the shelf via conduction and radiation. Atypical radiation heat transfer is responsible for higher sublimation rates for vials located at the front and side of an array. However, the guardrail contributes a little to heat transfer by conduction.