Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies

Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies.     

PIP3 but not PIP2 increases GLUT4 surface expression and glucose metabolism mediated by AKT/PKCζ/λ phosphorylation in 3T3L1 adipocytes

Phosphatidylinositol-3,4,5-triphosphate (PIP3) and phosphatidylinositol-4,5-biphosphate (PIP2) are two well-known membrane bound polyphosphoinositides. Diabetes is associated with impaired glucose metabolism. Using a 3T3L1 adipocyte cell model, this study investigated the role of PIP3 and PIP2 on insulin stimulated glucose metabolism in high glucose (HG) treated cells. Exogenous PIP3 supplementation (1, 5, or 10 nM) increased the phosphorylation of AKT and PKCζ/λ, which in turn upregulated GLUT4 total protein expression as well as its surface expression, glucose uptake, and glucose utilization in cells exposed to HG (25 mM); however, PIP2 had no effect. Comparative signal silencing studies with antisense AKT2 and antisense PKCζ revealed that phosphorylation of PKCζ/λ is more effective in PIP3 mediated GLUT4 activation and glucose utilization than in AKT phosphorylation. Supplementation with PIP3 in combination with insulin enhanced glucose uptake and glucose utilization compared to PIP2 with insulin, or insulin alone, in HG-treated adipocytes. This suggests that a decrease in cellular PIP3 levels may cause impaired insulin sensitivity in diabetes. PIP3 supplementation also prevented HG-induced MCP-1 and resistin secretion and lowered adiponectin levels. This study for the first time demonstrates that PIP3 but not PIP2 plays an important role in GLUT4 upregulation and glucose metabolism mediated by AKT/PKCζ/λ phosphorylation. Whether PIP3 levels in blood can be used as a biomarker of insulin resistance in diabetes needs further investigation.     

Quinone reduction via secondary B-branch electron transfer in mutant bacterial reaction centers

Symmetry-related branches of electron-transfer cofactors-initiating with a primary electron donor (P) and terminating in quinone acceptors (Q)-are common features of photosynthetic reaction centers (RC). Experimental observations show activity of only one of them-the A branch-in wild-type bacterial RCs. In a mutant RC, we now demonstrate that electron transfer can occur along the entire, normally inactive B-branch pathway to reduce the terminal acceptor Q(B) on the time scale of nanoseconds. The transmembrane charge-separated state P(+)Q(B)(-) is created in this manner in a Rhodobacter capsulatus RC containing the F(L181)Y-Y(M208)F-L(M212)H-W(M250)V mutations (YFHV). The W(M250)V mutation quantitatively blocks binding of Q(A), thereby eliminating Q(B) reduction via the normal A-branch pathway. Full occupancy of the Q(B) site by the native UQ(10) is ensured (without the necessity of reconstitution by exogenous quinone) by purification of RCs with the mild detergent, Deriphat 160-C. The lifetime of P(+)Q(B)(-) in the YFHV mutant RC is >6 s (at pH 8.0, 298 K). This charge-separated state is not formed upon addition of competitive inhibitors of Q(B) binding (terbutryn or stigmatellin). Furthermore, this lifetime is much longer than the value of approximately 1-1.5 s found when P(+)Q(B)(-) is produced in the wild-type RC by A-side activity alone. Collectively, these results demonstrate that P(+)Q(B)(-) is formed solely by activity of the B-branch carriers in the YFHV RC. In comparison, P(+)Q(B)(-) can form by either the A or B branches in the YFH RC, as indicated by the biexponential lifetimes of approximately 1 and approximately 6-10 s. These findings suggest that P(+)Q(B)(-) states formed via the two branches are distinct and that P(+)Q(B)(-) formed by the B side does not decay via the normal (indirect) pathway that utilizes the A-side cofactors when present. These differences may report on structural and energetic factors that further distinguish the functional asymmetry of the two cofactor branches.     

Biodegradable poly(vinyl alcohol)-polyethylenimine nanocomposites for enhanced gene expression in vitro and in vivo

Use of cationic polymers as nonviral gene vectors has several limitations such as low transfection efficiency, high toxicity, and inactivation by serum. In this study, varying amounts of low molecular weight branched polyethylenimine 1.8 kDa (bPEI 1.8) were introduced on to a neutral polymer, poly(vinyl alcohol) (PVA), to bring in cationic charge on the resulting PVA-PEI (PP) nanocomposites. We rationalized that by introducing bPEI 1.8, buffering and condensation properties of the proposed nanocomposites would result in improved gene transfer capability. A series of PVA-PEI (PP) nanocomposites was synthesized using well-established epoxide chemistry and characterized by IR and NMR. Particle size of the PP/DNA complexes ranged between 120 to 135 nm, as determined by dynamic light scattering (DLS), and DNA retardation assay revealed efficient binding capability of PP nanocomposites to negatively charged nucleic acids. In vitro transfection of PP/DNA complexes in HEK293, HeLa, and CHO cells revealed that the best working formulation in the synthesized series, PP-3/DNA complex, displayed ~2-50-fold higher transfection efficiency than bPEIs (1.8 and 25 kDa) and commercial transfection reagents. More importantly, the PP/DNA complexes were stable over a period of time, along with their superior transfection efficiency in the presence of serum compared to serum-free conditions, retaining the nontoxic property of low molecular weight bPEI. The in vivo administration of PP-3/DNA complex in Balb/c mice showed maximum gene expression in their spleen. The study demonstrates the potential of PP nanocomposites as promising nonviral gene vectors for in vivo applications.     

A novel CD4 T-cell epitope described from one of the cervical cancer patients vaccinated with HPV 16 or 18 E7-pulsed dendritic cells

Previously, safety and immunogenicity of human papillomavirus type 16 (HPV16) or 18 E7-pulsed dendritic cells (DC) vaccinations were demonstrated in a dose-escalation Phase I clinical trial which enrolled ten patients diagnosed with stage IB or IIA cervical cancer (nine HPV 16-positive, one HPV 18-positive). The goal of the study was to define the T-cell epitopes of HPV 16 or 18 E7 protein in these patients in order to develop new strategies for treating HPV-associated malignancies. This was accomplished through establishing T-cell lines by stimulating peripheral blood mononuclear cells with autologous mature DC pulsed with the HPV 16 or 18 E7 protein, examining the T-cell responses using ELISPOT assays, and isolating E7-specific T-cell clones based on IFN-γ secretion. Then, the epitope was characterized in terms of its core sequence and the restriction element. Twelve T-cell lines from eight subjects (seven HPV 16-positive, one HPV 18-positive) were evaluated. Positive T-cell responses were demonstrated in four subjects (all HPV 16-positive). All four were positive for the HPV 16 E7 46-70 (EPDRAHYNIVTFCCKCDSTLRLCVQ) region. T-cell clones specific for the E7 47–70 region were isolated from one of the subjects. Further analyses revealed a novel, naturally processed, CD4 T-cell epitope, E7 58–68 (CCKCDSTLRLC), restricted by the HLA-DR17 molecule. This work was supported by the National Institutes of Health (R21CA094507). An erratum to this article can be found at     

TRPC1-mediated inhibition of 1-methyl-4-phenylpyridinium ion neurotoxicity in human SH-SY5Y neuroblastoma cells

Mammalian homologues of the Drosophila canonical transient receptor potential (TRP) proteins have been implicated to function as plasma membrane Ca(2+) channels. This study examined the role of TRPC1 in human neuroblastoma (SH-SY5Y) cells. SH-SY5Y cells treated with an exogenous neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+)) significantly decreased TRPC1 protein levels. Confocal microscopy on SH-SY5Y cells treatment with MPP(+) showed decreased plasma membrane staining of TRPC1. Importantly, overexpression of TRPC1 reduced neurotoxicity induced by MPP(+). MPP(+)-induced alpha-synuclein expression was also suppressed by TRPC1 overexpression. Protection of SH-SY5Y cells against MPP(+) was significantly decreased upon the overexpression of antisense TRPC1 cDNA construct or the addition of a nonspecific transient receptor potential channel blocker lanthanum. Activation of TRPC1 by thapsigargin or carbachol decreased MPP(+) neurotoxicity, which was partially dependent on external Ca(2+). Staining of SH-SY5Y cells with an apoptotic marker (YO-PRO-1) showed that TRPC1 protects SH-SY5Y neuronal cells against apoptosis. Further, TRPC1 overexpression inhibited cytochrome c release and decreased Bax and Apaf-1 protein levels. Interpretation of the above data suggests that reduction in the cell surface expression of TRPC1 following MPP(+) treatment may be involved in dopaminergic neurodegeneration. Furthermore, TRPC1 may inhibit degenerative apoptotic signaling to provide neuroprotection against Parkinson's disease-inducing agents.     

Brevibacterium frigoritolerans a novel entomopathogen of Anomala dimidiata and Holotrichia longipennis (Scarabaeidae: Coleoptera)

We describe the isolation, biochemical characterization, phylogenetic analysis, and pathogenecity of a novel entomopathogenic bacterium Brevibacterium frigoritolerans to first instar larvae of Anomala dimidiata and Holotrichia longipennis. The almost full length 16S rRNA sequence of the bacterium has 99% identity with the type strain of B. frigoritolerans, while phylogenetic analysis revealed that the isolate formed a tightly linked branch with the type strain of B. frigoritolerans. Under in vitro bioassay conditions, the isolate infected and caused 89±5.4 and 74±7.7% mortality, in first instar larvae of A. dimidiata and H. longipennis, respectively. The infected larvae exhibited bacteremia like symptoms and mortality occurred between the second and fifth weeks after inoculation. This is an early report on the entomopathogenic potential of the hitherto lesser-known bacterium Brevibacterium frigoritolerans.     

A three-hybrid system to probe in vivo protein-protein interactions: application to the essential proteins of the RD1 complex of M. tuberculosis

Background

Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes.

Methodology/Principal Findings

The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3.

Conclusions/Significance

The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes.     

Alterations in brain metabolism induced by chronic morphine treatment: NMR studies in rat CNS

High-resolution (500 MHz) multiresonance/multinuclear proton (¹H) nuclear magnetic resonance (NMR) spectroscopy was used to detect metabolic changes and cellular injury in the rat brain stem and spinal cord following chronic morphine treatment. Compensatory changes were observed in glycine, glutamate, and inositols in the brain stem, but not the spinal cord, of chronic morphine-treated rats. In spinal cord, increases were detected in lactate and N-acetyl-aspartate (NAA), suggesting that there is anaerobic glycolysis, plasma membrane damage, and altered pH preferentially in the spinal cord of chronic morphine-treated rats.