Automated single-cell motility analysis on a chip using lensfree microscopy.

Quantitative cell motility studies are necessary for understanding biophysical processes, developing models for cell locomotion and for drug discovery. Such studies are typically performed by controlling environmental conditions around a lens-based microscope, requiring costly instruments while still remaining limited in field-of-view. Here we present a compact cell monitoring platform utilizing a wide-field (24 mm(2)) lensless holographic microscope that enables automated single-cell tracking of large populations that is compatible with a standard laboratory incubator. We used this platform to track NIH 3T3 cells on polyacrylamide gels over 20 hrs. We report that, over an order of magnitude of stiffness values, collagen IV surfaces lead to enhanced motility compared to fibronectin, in agreement with biological uses of these structural proteins. The increased throughput associated with lensfree on-chip imaging enables higher statistical significance in observed cell behavior and may facilitate rapid screening of drugs and genes that affect cell motility

The non-genomic effects of the PPARβγ agonist GW0742 on streptozotocin treated rat aorta

Copyright© Bentham Science Publishers; For any queries, please email at [email protected]: The ubiquitous nuclear receptor PPARβ/δ is increasingly being studied in regards to numerous diseases including diabetes following on the finding that PPARβ/δ agonist GW0742 controls Type 1 Diabetes in rats. Studies have shown that GW0742 has off target, non- PPARβ/δ effects in the cell although there are some key questions that remain to be addressed in respect to the significance of this control on vascular tone. Methods: Using isometric organ baths, rat aorta rings were exposed to ROCK inhibitors and the changes in contraction and dilation measured. Results: Our data shows that the PPARβ/δ agonist GW0742 (10 -7M) inhibits contractile responses to U46619 and phenylephrine, and that these responses are similar in normal and Streptozotocin (STZ) diabetic rat aorta. ROCK inhibitors Fasudil and Y27632 significantly reduced GW0742 mediated dilation of naïve rat aorta, but Fasudil had no effect on GW0742 dilation in STZ diabetic rat aorta. In contrast, STZ diabetic rat aorta pre-contracted with high [K +] Krebs lacked a dilatory response to GW0742, which taken together indicates that the mechanism of action of GW0742 mediated dilation changes in the diabetic state compared to non-diabetic state. Conclusion: This is the first direct evidence demonstrating the non- PPARβ/δ effect of GW0742 on contraction is irrespective to the diabetic state, and that GW0742 has the potential to induce vasodilation via multiple off-target mechanisms.Peer reviewedFinal Accepted Versio

136 HIV-1 Nef regulates activity of endoplasmic reticulum chaperone calnexin

HIV-1 Nef promotes viral replication by downmodulating a number of cell surface transmembrane proteins, such as CD4, MHC-I and MHC-II, which are targeted by Nef to various degradation pathways. Nef is also responsible for downregulation of cellular cholesterol transporter ABCA1, and this effect contributes to development of atherosclerosis in HIV infected patients. Surprisingly, in contrast to CD4 and MHC I, to which Nef has to bind to exert downregulation, binding to ABCA1 turned out to be unnecessary for inactivation of ABCA1 by Nef. Here, we identified a novel mechanism by which Nef influences activity of host cell and viral proteins. We show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which is essential for folding and maturation of glycosylated proteins. Nef disrupts calnexin interaction with ABCA1, thus impairing functionality of this protein, but increases affinity and enhances interaction of calnexin with gp160, promoting maturation and functionality of viral Env proteins. Knock-down of calnexin lead to reduced fusion activity of HIV-1 envelope and reduced virion infectivity, as well as to defective cholesterol efflux, which is mediated by ABCA1. However, gp160 and ABCA1 interacted with calnexin differently: while gp160 binding to calnexin was dependent on glycosylation, interaction of ABCA1 with calnexin was glycosylation-independent. Therefore, Nef binds to calnexin and stimulates interaction between calnexin and gp160 at the expense of ABCA1 and probably other ER proteins. These results provide a mechanistic explanation for previously unexplained effect of Nef on functionality of ABCA1, and suggest a mechanism for upregulation of HIV infectivity by Nef through stimulation of Env maturation

Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.

HIV infection has a profound effect on "bystander" cells causing metabolic co-morbidities. This may be mediated by exosomes secreted by HIV-infected cells and containing viral factors. Here we show that exosomes containing HIV-1 protein Nef (exNef) are rapidly taken up by macrophages releasing Nef into the cell interior. This caused down-regulation of ABCA1, reduction of cholesterol efflux and sharp elevation of the abundance of lipid rafts through reduced activation of small GTPase Cdc42 and decreased actin polymerization. Changes in rafts led to re-localization of TLR4 and TREM-1 to rafts, phosphorylation of ERK1/2, activation of NLRP3 inflammasome, and increased secretion of pro-inflammatory cytokines. The effects of exNef on lipid rafts and on inflammation were reversed by overexpression of a constitutively active mutant of Cdc42. Similar effects were observed in macrophages treated with exosomes produced by HIV-infected cells or isolated from plasma of HIV-infected subjects, but not with exosomes from cells and subjects infected with ΔNef-HIV or uninfected subjects. Mice injected with exNef exhibited monocytosis, reduced ABCA1 in macrophages, increased raft abundance in monocytes and augmented inflammation. Thus, Nef-containing exosomes potentiated pro-inflammatory response by inducing changes in cholesterol metabolism and reorganizing lipid rafts. These mechanisms may contribute to HIV-associated metabolic co-morbidities

Long-term changes of serum chemokine levels in vaccinated military personnel

BACKGROUND: Members of the United States Armed Forces receive a series of vaccinations during their course of service. To investigate the influence of multiple vaccinations on innate immunity, we measured concentrations of a panel of immunomodulatory and pro-inflammatory cytokines in serum samples from a group of such individuals. RESULTS: Significantly increased levels of macrophage inflammatory protein 1α (MIP-1α), MIP-1β and interleukin 8 (IL-8) were detected. Since these cytokines are known to have anti-human immunodeficiency virus (HIV) activity, we tested the effect of serum from these individuals on HIV-1 infectivity and susceptibility of their peripheral blood mononuclear cells (PBMCs) to HIV-1 infection in vitro. Sera from vaccinated military personnel inhibited, and their PBMCs were partially resistant to, infection by HIV-1 strains tropic to CCR5 (R5), but not to CXCR4 (X4), chemokine receptor. CONCLUSION: These findings demonstrate that increased anti-HIV chemokines can be detected in vaccine recipients up to 68 weeks following immunization

The level of CD147 expression correlates with cyclophilin-induced signalling and chemotaxis

<p>Abstract</p> <p>Background</p> <p>Previous studies identified CD147 as the chemotactic receptor on inflammatory leukocytes for extracellular cyclophilins (eCyp). However, CD147 is not known to associate with signal transducing molecules, so other transmembrane proteins, such as proteoglycans, integrins, and CD98, were suggested as receptor or co-receptor for eCyp. CD147 is ubiquitously expressed on many cell types, but relationship between the level of CD147 expression and cellular responses to eCyp has never been analyzed. Given the role of eCyp in pathogenesis of many diseases, it is important to know whether cellular responses to eCyp are regulated at the level of CD147 expression.</p> <p>Results</p> <p>Here, we manipulated CD147 expression levels on HeLa cells using RNAi and investigated the signalling and chemotactic responses to eCypA. Both Erk activation and chemotaxis correlated with the level of CD147 expression, with cells exhibiting low level expression being practically unresponsive to eCypA.</p> <p>Conclusions</p> <p>Our results provide the first demonstration of a chemotactic response of HeLa cells to eCypA, establish a correlation between the level of CD147 expression and the magnitude of cellular responses to eCypA, and indicate that CD147 may be a limiting factor in the receptor complex determining cyclophilin-induced Erk activation and cell migration.</p

HIV-1 Replication through hHR23A-Mediated Interaction of Vpr with 26S Proteasome

HIV-1 Vpr is a virion-associated protein. Its activities link to viral pathogenesis and disease progression of HIV-infected patients. In vitro, Vpr moderately activates HIV-1 replication in proliferating T cells, but it is required for efficient viral infection and replication in vivo in non-dividing cells such as macrophages. How exactly Vpr contributes to viral replication remains elusive. We show here that Vpr stimulates HIV-1 replication at least in part through its interaction with hHR23A, a protein that binds to 19S subunit of the 26S proteasome and shuttles ubiquitinated proteins to the proteasome for degradation. The Vpr-proteasome interaction was initially discovered in fission yeast, where Vpr was shown to associate with Mts4 and Mts2, two 19S-associated proteins. The interaction of Vpr with the 19S subunit of the proteasome was further confirmed in mammalian cells where Vpr associates with the mammalian orthologues of fission yeast Mts4 and S5a. Consistently, depletion of hHR23A interrupts interaction of Vpr with proteasome in mammalian cells. Furthermore, Vpr promotes hHR23A-mediated protein-ubiquitination, and down-regulation of hHR23A using RNAi significantly reduced viral replication in non-proliferating MAGI-CCR5 cells and primary macrophages. These findings suggest that Vpr-proteasome interaction might counteract certain host restriction factor(s) to stimulate viral replication in non-dividing cells

Small-Size Resonant Photoacoustic Cell of Inclined Geometry for Gas Detection

A photoacoustic cell intended for laser detection of trace gases is represented. The cell is adapted so as to enhance the gas-detection performance and, simultaneously, to reduce the cell size. The cell design provides an efficient cancellation of the window background (a parasite response due to absorption of laser beam in the cell windows) and acoustic isolation from the environment for an acoustic resonance of the cell. The useful photoacoustic response from a detected gas, window background and noise are analyzed in demonstration experiments as functions of the modulation frequency for a prototype cell with the internal volume ~ 0.5 cm^3. The minimal detectable absorption for the prototype is estimated to be ~ 1.2 10^{-8} cm^{-1} W Hz^{-1/2}.Comment: 11 pages, 5 figure