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Parallel logic simulation is an important method for verifying the correctness of complex VLSI system designs. Synchronous simulation technique has low overhead but has poor load balancing and frequent synchronization cost. We derive a performance evaluation formula for the synchronous simulation. The formula considers factors including load balancing, the ratio between computation and communication and number of simulation cycles. The formula reveals that for simulations with very fine computation granularity, the reduction in simulation cycles is one of the most important keys to improve the performance. We propose an optimistic synchronous algorithm that is targeted to reduce the number of synchronization steps. The experimental results on ISCAS89 and ISCAS85 circuits show that the proposed algorithm performs significantly better than synchronous simulation. For non-unit delay models, the proposed algorithm performs almost as good as unit delay models.

Liver cells isolated from intact tissue can reaggregate to form three-dimensional, multicellular spheroids in vitro. During this process, cells undergo a histological and environmental change. How cells respond biochemically to this change has not been studied in detail previously. We have investigated some biochemical changes in rat liver cells during the formation and maintenance of spheroids. Liver cells were isolated from male Sprague rats and spheroids cultured by a gyrotatory-mediated method. Liver cells were shown to respond to the isolation procedure and the formation of spheroids triggered histological environmental changes that increased arginine uptake, nitric oxide (NO) and urea syntheses, as well as raised levels of GSH, GSSG, glutamic acid and aspartic acid secretion within the first couple of days after cell isolation. Levels were maintained at a relatively stable level in the mature spheroids (>5 days) over the 3 week period of observation. P450 1A1 activity was lost in the first 2 days and gradually recovered thereafter. This study, for the first time, shows that liver cells after isolation and during spheroid formation actively uptake arginine and increase NO and urea syntheses. A high level of NO is likely to play an important role in modulating a series of biochemical changes in liver cells. It is considered that liver cells actively respond to the 'challenge' induced by the isolation procedure and subsequent histological environmental changes, and biochemical modulation and instability result. The stable cell-cell contacts and histological environment in mature spheroids permit and support functional recovery and maintenance in vitro. This period of stability permits the use of spheroids in toxicity studies to establish acute and chronic paradigms.

Multi-cellular spheroids are increasingly employed as in vitro sensors of toxicants and a single spheroid can be used as a test object. An ultrasonic standing wave trap (USWT) can hold small particles in a medium-flowing system. This study investigated the conditions for holding HepG2 spheroids in an USWT and its relevance to use in toxicity testing. It can take many hours to reach a detectable end point of cell damage in a standard cellular in vitro toxicant assay and the process might be accelerated through increased sample flow past the spheroid. A USWT was employed here to levitate and hold HepG2 spheroids stationary against a flow of 3 mm s−1 when the acoustic pressure amplitude is 1.9 MPa. The ultrasonic drive frequency was 1.64 MHz. Acoustic microstreaming in the standing wave chamber generated 1 mm s−1 flow past a levitated spheroid-scale (80 μm diameter) latex particle in the absence of sample through-flow. The conditions required to form aggregates of cells of a HepG2 cell line in a single half wavelength ultrasonic standing wave mini-chambers are also described here. It is argued that the manipulation capabilities demonstrated may have potential in increasing the efficiency of in vitro toxicant detection by spheroids. Preliminary, visual (unquantified) fluorescence microscopy observations of spheroids levitated in the standing wave in the presence of the toxicant dl -propranolol do suggest accelerated loss of viability compared with controls.

In the process of isolated single liver cells coming together to form three-dimensional spheroids, cells undergo dramatic environmental changes. How liver cells respond to these changes has not been well studied before. This study characterized the functional and biochemical changes during liver spheroid formation and maintenance. Spheroids were prepared in 6-well plates from freshly isolated liver cells from male Sprague rats by a gyrotatory-mediated method. Morphological formation, and functional and biochemical parameters of liver spheroids were evaluated over a period of 21 days in culture. Liver spheroid formation was divided into two stages, immature (1-5 days) and mature (>5 days), according to their size and shape, and changes in their functionality. Galactose and pyruvate consumption was maintained at a relatively stable level throughout the period of observation. However, glucose secretion and cellular GPT and GOT activities were higher in immature spheroids, decreased upto day 5 and remained stable thereafter. Cellular gamma-glutamyltransferase (gamma-GT) and lactate dehydrogenase (LDH) activities were initially undetectable or low and increased as spheroids matured. Albumin secretion decreased rapidly within the first 2 days and increased as spheroids matured. It is concluded that cells undergo functional and biochemical changes during spheroid formation following isolation of liver cells from intact tissue. Functionality and biochemical properties recovered and were maintained in mature spheroids. A relatively stable period (6-15 days) of functionality in mature spheroids was identified and is recommended for applications of the model.

Cytotoxicity endpoints, spontaneous glucose secretion/consumption and LDH and gamma-GT release, were characterised in rat liver and HepG2 spheroids as in vitro models for toxicology studies. Preprepared rat liver spheroids and HepG2 spheroids cultured in a six-well plate format were exposed to varying concentrations of galactosamine, propranolol, diclofenac, and paracetamol. All four model toxins significantly affected glucose secretion, which agreed well with LDH and/or gamma-GT release in rat liver spheroids. These toxins also significantly increased LDH and/or gamma-GT release in HepG2 spheroids. Whereas glucose consumption in HepG2 spheroids did not show conclusive results, LDH activities in both types of spheroids were similar and their levels were relatively high. Accordingly, the level of LDH leakage in both types of spheroids was much higher than gamma-GT after exposure to the toxins. In contrast, gamma-GT activity in HepG2 spheroids was sixfold higher than that in rat liver spheroids. This study revealed that galactosamine interfered with the gamma-GT assay and paracetamol interfered with the LDH assay. It demonstrated, for the first time, that glucose secretion by liver spheroids can be used as a functional indicator of cytotoxicity. Test compounds may interfere with enzymatic assays as indicated by LDH and gamma-GT release in this study. Combining functional parameters together with two or more indicators of enzyme releases can provide a reliable cytotoxicity evaluation. Liver and HepG2 spheroids as in vitro models showed good predictions in chemical-induced hepatic cytotoxicity.

Cells in liver spheroids and Hep G2 spheroids transferred from gyrotatory culture conditions and maintained in normal static culture conditions will spread out at the edges. Based on this observation, we developed a new test called the Spheroid Cell Spreading Inhibition Test (SCSIT) to screen hepatic cytotoxicity of xenobiotics and determine the spheroid cell spreading inhibition concentration (SCSIC) of test chemicals. Four model hepatoxicants, D-galactosamine, propranolol, diclofenac, and paracetamol, were studied with SCSIT in both rat liver and HepG2 spheroids. Both liver and HepG2 spheroids were prepared under gyrotatory culture conditions and used at 6 days in vitro. The results showed that all four hepatotoxicants tested inhibited cell spreading in liver spheroids (D-galactosamine at 20 mM, propranolol at 125 microM, diclofenac at 500 microM, and paracetamol at 25 mM) and HepG2 spheroids (D-galactosamine at 16 mM, propranolol at 125 microM, diclofenac at 500 microM, and paracetamol at 25 mM). The SCSIT results agreed with the conventional cytotoxic indicators, release of LDH and/or gamma-GT and the inhibition of glucose secretion from rat liver spheroids. In conclusion, this study, for the first time, described the biological characteristics of liver and HepG2 spheroid cell spreading and demonstrates its application in hepatic cytotoxicity studies. This method may be used in testing in vitro "acute" toxicity, comparing relative cytotoxicity and generating reference concentrations for subsequent studies. Therefore, SCSIT could be a useful tool for screening hepatotoxicity relevant to preclinical lead optimization and compound library screening.

Piglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV) are born severely immunocompromised. In this article we more closely examine the effects of in utero PRRSV infection on circulating and thymic T cell populations. Numbers of CD4+, CD8+, and dual-positive lymphocytes were quantitated in circulation and in the thymus during the 2 weeks following birth. At birth we found that the number of circulating lymphocytes was suppressed by 60%. Lymphocyte numbers were also suppressed by 42% at day 7, but by day 14 the number of lymphocytes had rebounded and was actually 47% greater than controls. At birth and day 7, a drop in the number of CD4+ cells could partially explain the suppression we observed, while the rebound in total lymphocyte numbers seen at day 14 was due to a nearly fourfold increase in the number of circulating CD8+ cells. As a result, the normal CD4+:CD8+ ratio of between 1.4 and 2.2 for neonatal pigs was reduced to 0.1–0.5. The thymuses of infected piglets were found to be 50% smaller than those of control pigs and were characterized by cortical involution and severe cortical depletion of thymocytes. Analysis of the population of thymocytes revealed that double-positive thymocytes were suppressed to a greater degree than either single positive subpopulation. In addition, we show that the number of thymocytes undergoing apoptosis was increased twofold in piglets infected with PRRSV. Taken together, these results help explain the dramatic immunosuppression observed in neonatal animals infected in utero with PRRSV.

1. This study has investigated absorption, distribution, metabolism and excretion of p-bromophenylacetylurea (BPAU) in the F344 female rat. BPAU and its metabolites were determined by HPLC. 2. Following a single p.o. dose of 150 mg/kg BPAU, the absorbed fraction of dosed BPAU was 65.9% and its half-life in the blood was 9.4 h. The relative distribution of BPAU (tissue/serum ratio) at 6 h (peak time point) after a single i.p. dose of 150 mg/kg BPAU was spinal cord (4.6+/-0.2) > liver (3.7+/-0.1) > brain (2.9+/-0.1) (mean+/-SD, n = 5), and they were significantly different from each other (p < 0.05). BPAU in spinal cord reached the highest level. 3. Absorbed BPAU was metabolized in vivo into three major metabolites. N'-hydroxy-p-bromophenylacetylurea (M1) was a dominant metabolite in tissues, whereas 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione (M2) reached a high concentration in blood. N'-methyl-p-bromophenylacetylurea (M3) was mainly found in the urine. All three metabolites were excreted via the urine and together accounted for 87% of absorbed BPAU. 4. This study provides a basic understanding of BPAU absorption, distribution, metabolism and elimination in rat.

m-Dinitrobenzene is a multitarget toxicant. This study presents a concentration-time threshold model in m-dinitrobenzene (m-DNB)-induced neurotoxicity in F344 rats based on pharmacokinetic modeling and variable duration infusions with neuropathological end points. Pharmacokinetic parameters for m-DNB were determined after giving a single i.v. dose of 10 mg/kg m-DNB. Time dependency of the brain lesions was studied by either giving a single bolus i.v. dose of 30 mg/kg m-DNB or infusing this dose over 6, 12, or 24 h, or 2, 4, 6, 8, or 14 days. The results show that the 6-day infusion, in which the theoretical steady-state blood concentration was 2.0 microM, caused brain damage, whereas the 8- and 14-day infusions, in which the steady-state blood concentrations were 1.5 and 0.8 microM, respectively, did not induce apparent brain damage. When this dose was infused over 6 h, the peak blood concentration of m-DNB was 35 microM and the time (T(m)) for which m-DNB exceeded the 2-microM concentration threshold was 18.8 h, but no brain damage was observed. However, when the same total dosage was infused over periods of either 12 or 24 h, or 2, 4, or 6 days, the theoretical blood concentrations were from 21.9 to 2.0 microM and the T(m) was from 22. 7 to 144 h, and brain damage was produced. Hence a T(m) of 22.7 h was considered to be the time threshold for m-DNB-induced brain damage. It is concluded that a high concentration alone does not result in m-DNB-induced neurotoxicity and that in addition to a concentration threshold, there also exists a time threshold. Both apparently need to be exceeded before neurotoxicity is seen.

Background Association studies have been employed to investigate the relationships between host single nucleotide polymorphisms (SNPs) and susceptibility to pulmonary Tuberculosis (PTB). However, such candidate genetic markers have not been widely studied in Chinese population, especially with respect to the disease development from latent M. tuberculosis infection (LTBI). Methods In this case–control study, 44 candidate SNPs were examined in a total of 600 participants (PTB patients, LTBI controls and healthy controls without M. tuberculosis infection) from Zhengzhou, China. The two groups of controls were frequency matched on gender and age with PTB patients. Genotyping was carried out by the Illumina Golden Gate assay. Results When comparing PTB patients with LTBI controls but not healthy controls without M. tuberculosis infection, significant associations with disease development were observed for TLR9 1174 A/G, TLR9 1635 A/G and IFNG 2109G/A. The two loci in TLR9 were in LD in our study population (r2=0.96, D’=1.00). A combined effect of the genotypes associated with increased risk of PTB (i.e. TLR9 1174G/G and IFNG 2109 A/A) was found when comparing PTB patients with LTBI controls (p=0.004) but not with healthy controls without infection (p=0.433). Conclusions Potential associations between TLR9 and IFN-γ genetic polymorphisms and PTB were observed in a Chinese population which supports further study of the roles played by TLR9/IFN-γ pathway during the development of PTB.

Cell-cell reconnection after isolation or during healing process after injury is a cell membrane function. It is assumed that each tissue has its own architecture and accordingly, cells in different tissues could have different adaptability to resume their tissue-specific architectures. How membranes adapt themselves to make cell-cell reconnections has not been elucidated. This study investigated how rat hepatic cell membranes adapt themselves after isolation to establish cell-cell reconnections, which is essential to liver tissue healing process. Hepatic cells were obtained from rat liver by collagenase perfusion. The cells were maintained in suspension using a gyratory-mediated cell spheroid culture method. F-actin, claudin-2 and connexin-32 on cell membranes were stained and observed by confocal microscopy. Cells and spheroids were also observed by Scanning Electron Microscopy. It was found that hepatic cells underwent adaptive change after enzymatic isolation by diminishing membrane F-actin residues and forming F-actin plaques. Once an F-actin plaque is formed (within 1-4 h), it is ready to make initial contact with other cells through plaque-plaque overlapping in a manner similar to ‘Velcro-like’ binding. A cell could form one or more F-actin plaques and establish plaque-plaque contacts with one or more other cells. When the cells had established plaque-plaque contacts, their contacts would further develop to make full cell-cell bindings. Claudin-2, a tight-junction molecule, and connexin-32, a gap-junction molecule, gradually diminished and lost their original distribution patterns within 4 h after isolation. They did not show involvement in establishing initial cell-cell connections. It is concluded that F-actin plaque formation is a characteristic change that precedes cell-cell reconnection. F-actin plaques are transitional microstructures that serve as a special device to facilitate cell-cell contacts in the early stage after cell isolation. F-actin can be used as a biomarker to study membrane adaptability and the healing process of hepatic cells.

Pseudorabies virus survival was compared using three different types of applicator swabs in Eagle's minimum essential medium held under shipping conditions (packed with frozen gel packs) for up to 96 hours. Virus titer decay rates for dacron-tipped applicators were not statistically different from those of controls. Titer decay rates were statistically different from controls for cotton- and calcium alginate-tipped applicators. With the lowest input virus titer, virus was detectable up to 96, 72, or 24 hours after inoculation for dacron-, cotton-, and calcium alginate-tipped applicators, respectively. Dacron-tipped applicators were chosen to evaluate pseudorabies virus survival on tonsil swabs collected from experimentally challenged or contact control pigs to simulate field sampling and shipping conditions. Virus was still detectable in 20 of 24 swab samples after 72 hours in cell culture medium under shipping conditions.

This study presents a HPLC method for the separation and purification of p-bromophenylacetylurea (BPAU) and its metabolites. The method effectively separated and purified BPAU and its metabolites. Three metabolites of BPAU, M1, M2 and M3 were characterised by mass spectroscopy and nuclear magnetic resonance. They are named as N′-hydroxy-p-bromophenylacetylurea, 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione and N′-methyl-p-bromophenylacetylurea, respectively. The major metabolic pathways of BPAU were proposed. The establishment of the HPLC method and characterisation of BPAU metabolites make it possible for further pharmacokinetic studies to explore the mechanism of BPAU-induced delayed neuropathy.

Background Bioimpedance analysis (BIA) has been reported as helpful in identifying hypervolemia. Observation data showed that hypervolemic maintenance hemodialysis (MHD) patients identified using BIA methods have higher mortality risk. However, it is not known if BIA-guided fluid management can improve MHD patients’ survival. The objectives of the BOCOMO study are to evaluate the outcome of BIA guided fluid management compared with standard care. Methods This is a multicenter, prospective, randomized, controlled trial. More than 1300 participants from 16 clinical sites will be included in the study. The enrolment period will last 6 months, and minimum length of follow-up will be 36 months. MHD patients aged between 18 years and 80 years who have been on MHD for at least 3 months and meet eligibility criteria will be invited to participate in the study. Participants will be randomized to BIA arm or control arm in a 1:1 ratio. A portable whole body bioimpedance spectroscopy device (BCM—Fresenius Medical Care D GmbH) will be used for BIA measurement at baseline for both arms of the study. In the BIA arm, additional BCM measurements will be performed every 2 months. The primary intent-to-treat analysis will compare outcomes for a composite endpoint of death, acute myocardial infarction, stroke or incident peripheral arterial occlusive disease between groups. Secondary endpoints will include left ventricular wall thickness, blood pressure, medications, and incidence and length of hospitalization. Discussions Previous results regarding the benefit of strict fluid control are conflicting due to small sample sizes and unstable dry weight estimating methods. To our knowledge this is the first large-scale, multicentre, prospective, randomized controlled trial to assess whether BIS-guided volume management improves outcomes of MHD patients. The endpoints of the BOCOMO study are of utmost importance to health care providers. In order to obtain that aim, the study was designed with very careful important considerations related to the endpoints, sample size, inclusion criteria, exclusion criteria and so on. For example, annual mortality of Beijing MHD patients was around 10%. To reach statistical significance, the sample size will be very large. By using composite endpoint, the sample size becomes reasonable and feasible. Limiting inclusion to patients with urine volume less than 800 ml/day the day before dialysis session will limit confounding due to residual renal function effects on the measured parameters. Patients who had received BIS measurement within 3 months prior to enrolment are excluded as data from such measurements might lead to protocol violation. Although not all patients enrolled will be incident patients, we will record the vintage of dialysis in the multivariable analysis. Trial registration Current Controlled Trials NCT01509937

The cryopreservation of hen and rat brain spheroids was investigated. Brain spheroid cultures were prepared from 7-day-old hen embryos or 16-day-old rat embryos, by using a rotation-mediated culture system. The spheroids were cryopreserved in medium containing 5–15% dimethyl sulphoxide (DMSO) and stored in liquid nitrogen, by using a two-stage cooling procedure. The results show that the viability, as indicated by the total protein content of hen embryo brain spheroids at 24 hours, and at 3, 7 and 28 days after thawing, ranged from 45.5% to 64.2% of control values. It took 3 days for the post-thaw brain spheroids to stabilise, as indicated by their morphology and selected neural markers of functionality. These functions were maintained over a 28-day observation period. Spheroids cultured for 12–15 days in vitro before cryopreservation survived better than those that were cryopreserved after 5–7 days in vitro. The viability and biochemical functionality of spheroids after long-term (up to 6 months) storage were similar to those following short-term storage. The viability of rat brain spheroids cryopreserved in 15% DMSO, as indicated by total protein content, at 24 hours, and at 3 or 7 days after thawing, ranged from 23.1% to 32.1% of control values. This study shows for the first time that brain spheroids prepared from primary tissue can be successfully cryopreserved.