Design and implementation of wire tension measurement system for MWPCs used in the STAR iTPC upgrade

The STAR experiment at RHIC is planning to upgrade the Time Projection Chamber which lies at the heart of the detector. We have designed an instrument to measure the tension of the wires in the multi-wire proportional chambers (MWPCs) which will be used in the TPC upgrade. The wire tension measurement system causes the wires to vibrate and then it measures the fundamental frequency of the oscillation via a laser based optical platform. The platform can scan the entire wire plane, automatically, in a single run and obtain the wire tension on each wire with high precision. In this paper, the details about the measurement method and the system setup will be described. In addition, the test results for a prototype MWPC to be used in the STAR-iTPC upgrade will be presented.Comment: 6 pages, 10 figues, to appear in NIM

Platelet-derived growth factor CC-mediated neuroprotection against HIV Tat involves TRPC-mediated inactivation of GSK 3beta.

Platelet-derived growth factor-CC (PDGF-CC) is the third member of the PDGF family, and has been implicated both in embryogenesis and development of the CNS. The biological function of this isoform however, remains largely unexplored in the context of HIV-associated dementia (HAD). In the present study, we demonstrate that exposure of human neuroblastoma cells SH-SY5Y to HIV transactivator protein Tat resulted in decreased intrinsic expression of PDGF-CC as evidenced by RT-PCR and western blot assays. Reciprocally, pretreatment of SH-SY5Y cells with PDGF-CC abrogated Tat-mediated neurotoxicity by mitigating apoptosis and neurite & MAP-2 loss. Using pharmacological and loss of function approaches we identified the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in PDGF-CC-mediated neuroprotection. We report herein a novel role about the involvement of transient receptor potential canonical (TRPC) channel 1 in modulation of calcium transients in PDGF-CC-mediated neuroprotection. Furthermore we also demonstrated PDGF-CC-mediated inactivation of the downstream mediator--glycogen synthase kinase 3β (GSK3β) evidenced by its phosphorylation at Ser-9. This was further validated by gain and loss of function studies using cells transfected with either the wild type or mutant GSK3β constructs. Intriguingly, pretreatment of cells with either the PI3K inhibitor or TRPC blocker resulted in failure of PDGF-CC to inactivate GSK3β, thereby suggesting the intersection of PI3K and TRPC signaling at GSK3β. Taken together our findings lead to the suggestion that PDGF-CC could be developed as a therapeutic target to reverse Tat-mediated neurotoxicity with implications for HAD

TRPC 1 channel is critical for PDGF-CC-mediated neuroprotection.

<p>(<b>A</b>) SH-SY5Y cells were labeled with Fluo-4 prior to treatment with PDGF-CC followed by recording of the fluorescence density representing the level of intracellular Ca²⁺. A representative change of fluorescence intensity is shown in the lower panel. The arrow indicates the time of addition of PDGF-CC. Number in upper panel shows the recording time. (<b>B</b>) Change in intracellular Ca²⁺ induced by PDGF-CC (50 ng/ml) in the presence of absence of pharmacological inhibitors (STI-571, 1 µM; EGTA, 2 mM; SKF96365, 20 µM; 2APB, 100 µM; U73122, 1 µM). All the data in these figures are presented as mean ± SEM of three individual experiments. ***p<0.001 vs control; ##p<0.01 vs PDGF-CC-treated group. (<b>C</b>) Cell viability in SH-SY5Y cells pretreated with SKF96365 (20 µM) by MTT assay. All the data in these figures are presented as mean ± SEM of three individual experiments. *p<0.05 vs control, #p<0.05 vs Tat-treated group. (<b>D</b>) SH-SY5Y cells were transfected with 100 nM siRNA targeting TRPC1, 5 or 6. After 24 h cells were lysed, mRNA was isolated and subjected to RT-PCR. Arrow indicates the RT-PCR product bands. (<b>E</b>) SH-SY5Y cells seeded in 96-well plate were transfected with TRPC 1, 5 or 6 siRNAs (100 nM) for 24 h later followed by treatment of cells with PDGF-CC and/or Tat. MTT assay was conducted to detect cell viability. All the data in these figures are presented as mean ± SEM of three individual experiments. *p<0.05 vs control; ##p<0.01 vs Tat-treated group.</p

PDGF receptor is involved in PDGF-CC neuroprotection.

<p>(<b>A</b>) Expression of PDGF-αR and βR in SH-SY5Y cells by RT-PCR. (<b>B</b>) SH-SY5Y cells were exposed to PDGF-CC (50 ng/ml) for 30 min in presence or absence of tyrosine kinase inhibitor STI-571 (1 µM). Phosphorylation of PDGF-αR & βR was detected by co-immunoprecipitation. (<b>C</b>) Cell viability of SH-SY5Y cells exposed to PDGF-CC and/or Tat in presence or absence of tyrosine STI-571 was assessed by MTT assay. Figure is a representative of three independent experiments. All data in these figures are presented as mean ± SEM of three individual experiments. *p<0.05 vs control, #p<0.05 vs Tat-treated group, %p<0.05 vs PDGF-CC plus Tat-treated group.</p

TRPC channel and Akt are involved in PDGF-CC mediated inactivation of GSK3β.

<p>(<b>A</b>) SH-SY5Y cells pretreated with or without the PI3K inhibitor PI-103 (0.2 µM), were exposed to PDGF-CC and/or Tat for 30 min. Cells were lysed, proteins were isolated and subject to WB to detect the phosphorylation of GSK3β. Data are presented as mean ± SEM of three individual experiments. **p<0.01 vs control, #p<0.05 vs PDGF-CC plus Tat-treated group. (<b>B</b>) WB demonstrating that blocking TRPC channel with SKF96365 resulted in loss of PDGF-CC mediated inactivation of GSK3β inactivation. Data were presented as mean ± SEM of three individual experiments. **p<0.01, ***p<0.001 vs control, ###p<0.001 vs PDGF-CC-treated group. (<b>C</b>) Treatment of cells with TRPC channel inhibitor SKF96365 (20 µM) resulted in mitigation of PDGF-CC-mediated activation of Akt. Data were presented as mean ± SEM of three individual experiments. ***p<0.001 vs control, ###p<0.05 vs PDGF-CC-treated group. (<b>D</b>) SH-SY5Y cells were treated with PDGF-CC (50 ng/ml) and/or Tat (14 nM) for the indicated times, and subjected to cytoplasm and nuclear protein extraction. WB analysis demonstrated PDGF-CC mediated accumulation of β catenin in nucleus. Figure is a representative of three independent experiments. **p<0.01 vs control.</p

PDGF-CC exerts neuroprotection against Tat toxicity.

<p>SH-SY5Y cells (<b>A</b>) or Rat primary cortical neurons (<b>B</b>) were exposed to Tat (14 nM) or heat-inactivated Tat (14 nM) with or without pretreatment with PDGF-CC (50 ng/ml), and were then subjected to MTT assay. (<b>C</b>) SH-SY5Y cells were treated with Tat in presence or absence of PDGF-CC for 5 days and examined phase-contrast microscopy. (<b>D</b>) Densitometric scan of neuritis from panel C expressed as a ratio of neurite length/neuron. (<b>E</b>) Immunostaining of SH-SY5Y cells treated with PDGF-CC and/or Tat for 3 days with anti-MAP-2 antibody. (<b>F</b>) MAP-2 ELISA was done on SH-SY5Y cells treated as described in panel E. (<b>G</b>) SH-SY5Y cells were treated with 14 nM Tat and/or 50 ng/ml PDGF-CC for 24 h and were then subjected to TUNEL assay. The data (<b>H</b>) were expressed as ratio of TUNEL positive to total cell number in the same view area. For all images, scale bars represent 20 µm. Figure is a representative of three independent experiments. All data in these figures are presented as mean ± SEM of three individual experiments. *p<0.05, **p<0.01 vs control; #p<0.05, ##p<0.01 vs Tat-treated group.</p

Down-regulation of PDGF-CC in neurons exposed to Tat.

<p>SH-SY5Y cells or rat primary neurons were exposed to 14 nM Tat for the indicated times, followed by RNA isolation and assessment of PDGF-CC using real-time PCR (<b>A</b>), RT-PCR (<b>B</b>), or western blot (<b>C</b>). In panels B and C, SH-SY5Y cells were treated for 24 h and monitored for PDGF-CC RNA and protein. Figure shown is a representative of three independent experiments. All data in these figures are presented as mean ± SEM of three individual experiments. *p<0.05, **p<0.01, ***p<0.001 vs control.</p

Schematic illustration demonstrating putative signaling pathways involved in PDGF-CC-mediated neuroprotection.

<p>PDGF-CC-mediated engagement of the PDGF receptor stimulates the PLC/PI3R pathway, which in turn, activates TRPC channels resulting in elevation of [Ca²⁺] transient. The elevation of [Ca²⁺] is required for PDGF induced PI3K/Akt pathway leading to inactivation of GSK3β signal. The inactivated GSK3β fails to induce the degradation of β-catenin resulting in accumulation of β-catenin in the cytoplasm, followed by its translocation into nucleus and subsequent induction of expression of genes associated with the cell survival.</p