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Recent studies from our lab and others have shown that complement components can enhance cell-intrinsic inflammatory signaling pathways. Specifically, we have found that activation of complement receptor C3aR enhances TLR4 signaling in lipopolysaccharide (LPS)-treated macrophages via enhanced p-38 phosphorylation. We found C3aR−/− mice treated with systemic LPS have decreased inflammatory cytokines (IL-6 and TNF) compared to wild-type (WT) mice. Our results corroborate previous studies showing C3aR−/− mice display decreased systemic cytokines (IL-6 and TNF) and increased bacterial burden during Gram-negative (GN) infection compared to WT mice. These results suggest C3aR is a critical regulator of TLR4 signaling. TLR4-TRIF signaling leads to activation of IRF3 and expression of interferon-regulated genes. We find that primary bone marrow-derived macrophages (BMDMs) from C3aR−/− mice show significantly reduced TRIF-dependent ifnb expression and IFNb secretion following 24h LPS exposure. Additionally, WT BMDMs treated with a C3aR antagonist (C3aRi) show a similar reduction in ifnb expression after LPS-treatment. These data demonstrate C3aR is critical for TRIF-dependent signaling downstream of TLR4 activation. Here we find WT BMDMs treated with C3aRi and LPS show increased intracellular cAMP. These findings demonstrate C3aR activation leads to depleted cAMP, which is essential for TRIF-mediated signaling downstream of TLR4. We hypothesize that C3aR is working as an inhibitory GPCR to regulate inflammation via intracellular cAMP and enhancing TRIF signaling in LPS-treated macrophages. Our findings are the first to suggest C3aR reduces cAMP to control TRIF-mediated inflammation in activated macrophages.

Microelectrode Array (MEA) devices are well established platforms for biosensing applications, however, limitations in electrode materials and cell coupling to electrodes still exist. Signal-to-noise ratio (SNR), a fabrication limitation, can be improved by reducing the impedance of the microelectrodes without increasing their physical size using conductive nanomaterials. Platinum is a well-studied conductive material that has been determined to be compatible with MEA devices, contains high electronic conductivity, is non-toxic to cells and has excellent catalytic activity. Nano-porous platinum, as compared to pure platinum, has larger surface area enabled by nano-scale definition, and as a result can lower the impedance of microelectrodes. Therefore, in this study nano-porous platinum was electroplated onto thin film gold electrodes to increase SNR in a single-well MEA device. Numerous commercial and research grade MEAs are available and have been subject to for various applications such as disease modeling and pharmaceutical screening. Although high densities of cells can increase the probability of cell-electrode coupling, only signals near electrodes will be detected. Our approach introduces a method of precise patterning of cells on electrodes to increase the probability of cell-electrode coupling and reducing inaccuracies and expenses associated with cell plating. The single-well MEA glass die was designed and fabricated to contain 64 microelectrodes (30 µm diameter with a pitch of 200 µm) and 4 internal on-chip reference electrodes. The MEA glass die fabrication was performed on a 4-inch, 500 µm thick glass wafer. First, metal traces composed of titanium and gold were deposited using lift-off lithography. Subsequently, 5 µm of SU-8 was spin coated and defined as an insulation layer. A separate printed circuit board (PCB) was designed and fabricated to contain 72 contact pads and routing traces. The biocompatible epoxy 353ND was used to package the glass die and PCB in specific orientations determined by alignment markers. Ball bonding with 20 µm diameter gold wire was used to wirebond the glass die to the PCB. A packaging technique was developed that would allow the characterization of key properties of the MEA. This technique involved using copper wire, 100 µm in diameter, and attaching it to the contact pads on the backside of PCBs using a conductive epoxy. Finally, a polystyrene culture well and lid were attached to the MEA using the epoxy, 353ND. Nano-Porous platinum electroplating solution was prepared with a combination of chloroplantinic acid, HCl and lead acetate, all diluted in DI water. Fully packaged devices containing approximately 3 mL of electrolyte were placed within the Axion BioSystems Muse for nano-porous platinum electrodeposition, utilizing the custom Application Specific Integrated Circuit (ASIC) in the system at room temperature. The nano-porous platinum electroplated devices were subsequently characterized using electrical impedance spectroscopy and cyclic voltammetry against the control devices, which contained gold electrodes. The impedance of each device was measured across a scanned frequency range of 10 Hz-100 kHz between the microelectrode, the internal reference electrode and cellular conductive media. The impedance of the nano-porous platinum devices is an order of magnitude lower than the devices with gold electrodes. Cyclic voltammograms were obtained at a scan rate of 20 mV/s in the range from -0.28 to 1.2 V vs. Ag/AgCl. RMS noise comparison between nano-porous platinum electroplated devices and control devices (gold electrodes) was determined using the Muse system and Axion’s Integrated Studio (AxIS) software. Biosensing applications for the devices were explored using electrogenic cells in combination with a precise plating technique. As depicted in Figure 1, cells were positioned in a pattern on and around electrodes to increase the probability of cell-electrode coupling. The electrical response was obtained and analyzed using the Muse system and AxIS software. SNR of action potentials recorded from cells growing on devices enhanced with nano-porous platinum electrodeposition and cells on control devices with gold electrodes were compared. Activity within neuronal networks / beat frequencies of cell patches on electrodes were monitored over the course of 2-3 weeks. The methods explored in this study not only decreases impedance of microelectrodes to increase SNR but also increases cell-electrode coupling by warranting the placement of cells next or on the electrodes by precision patterning. Future studies will aim to optimize electrode sensitivity using novel electrode materials, the porosity of different materials, and characterization using emerging analytical methods. Eventually these devices will provide in vitro systems to monitor both acute and chronic effects of drugs and toxins as well as to perform functional studies under physiological or induced pathophysiological conditions that mimic in vivo damages. Figure 1: Electrogenic cells patterned near nano-porous platinum electrode Figure 1

There is pressure on primary care trusts, and therefore on GPs, to reach specific levels of use of low-cost statins as a proportion of total statin prescribing. This simple study looks at some markers of the quality of the results achieved. A correlation is found between a higher proportion of low-cost statin prescribing and lower achievement raising questions as to whether financial savings may be offset by poorer results.

This paper reports on the computational simulation and modeling of an in vitro alveolar construct system along the optical coherence microscopy (OCM) methods for visualizing engineered tissue. The optical imaging methods will be compared to immunohistochemical light microscopy samples of engineered alveolar constructs. Results show depth images of the alveolar tissue construct for a bilayer construct, as well as predictions of the gas exchange process in a simple model of a bio-reactor hosting the construct.

EDITOR,—Colin Douglas suggests that general practitioners should be labelled in the way that …

Four Ss with histories of reported spontaneous ESP occurrence participated in 8 sessions per S. Each session consisted of 3 trials in which the contents (art prints) of sealed envelopes were assessed by clairvoyant means. One trial took place while S was in his ordinary waking state, one while S was in a self-induced altered conscious state, and one while S was in an altered state induced by sensory-bombardment techniques. Written transcripts of each S‘s statements were given to 3 outside evaluators who ranked each transcript against the 4 possible art prints in the target pool randomly selected for that session. Using median ranks of 1 and 2 as “hits” and ranks of 3 and 4 as “misses,” the 4 Ss were awarded a total of 58 “hits” and 38 “misses” ( CR = 2.04, p = .041, two-tailed). S-judgings produced a total of 61 “hits” and 35 “misses” ( CR = 2.65, p = .008, two-tailed).

Plasma concentrations of 14 hormones or hormone metabolites and urinary excretion of 15 hormones or hormone metabolites were studied in four rigorously selected groups: 13 men who had recovered well from prior myocardial infarction (at least 6 months earlier), 35 clinically normal men, 25 men with severe coronary artery disease diagnosed by coronary arteriograms but no history or signs of myocardial infarction, and 44 men with normal coronary arteriograms. Aside from a minor finding that a subset of the post-infarction patients (3 out of 12) showed subnormal plasma T3 levels (? the non-specific low-T3 syndrome), there were four major findings: post-infarction patients had significantly higher 24-hour mean plasma concentrations of estrone (80 vs 49 pg/ml, P 0.0001), dehydroisoandrosterone (444 vs 298 ng/dl, P 0.025), and dehydroisoandrosterone sulfate (112 vs 72 micro g/dl, P = 0.05) than clinically normal men, while men with positive coronary arteriograms but no infarction had the same levels as men with negative arteriograms; men with positive arteriograms but no infarction had significantly lower urinary excretion of androsterone glucuronide (2.6 vs 3.2 mg/g creatinine, P 0.05) than men with negative arteriograms, while post-infarction patients excreted normal amounts of this steroid. In the case of all three differing plasma steroids, nearly all the post-infarction patients had values at or below the upper 95% confidence limit of the normal range; the significant elevation of the post-infarction patients' mean value was due in each case to clustering of the patients' values in the upper half of the normal range.