Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397- transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.

An application of an extended theory of planned behaviour to understand drivers' compliance with the school zones speed limit in Australia and Malaysia

This research investigated individual and situational factors that influence driver compliance with the school zones speed limit in Australia and Malaysia. Despite all the countermeasures implemented in these two countries, driver compliance with the speed limit in school zones is still poor. The findings of the research provided support for the application of the Theory of Planned Behaviour to understand and therefore influence drivers' intention to comply with the school zones speed limit in both Australia and Malaysia. The research also revealed that mindfulness, a construct rarely used in road safety before, directly influenced Australian drivers' intention to comply, while habit influenced intention to comply in Malaysia. The research raised important theoretical and cross-cultural issues for future research and highlighted the need to increase the visibility of school zones with the use of more noticeable traffic control devices.

Inhibition of the JAK2/STAT3 pathway in ovarian cancer results in the loss of cancer stem cell-like characteristics and a reduced tumor burden

Background Current treatment of ovarian cancer patients with chemotherapy leaves behind a residual tumor which results in recurrent ovarian cancer within a short time frame. We have previously demonstrated that a single short-term treatment of ovarian cancer cells with chemotherapy in vitro resulted in a cancer stem cell (CSC)-like enriched residual population which generated significantly greater tumor burden compared to the tumor burden generated by control untreated cells. In this report we looked at the mechanisms of the enrichment of CSC-like residual cells in response to paclitaxel treatment. Methods The mechanism of survival of paclitaxel-treated residual cells at a growth inhibitory concentration of 50% (GI50) was determined on isolated tumor cells from the ascites of recurrent ovarian cancer patients and HEY ovarian cancer cell line by in vitro assays and in a mouse xenograft model. Results Treatment of isolated tumor cells from the ascites of ovarian cancer patients and HEY ovarian cancer cell line with paclitaxel resulted in a CSC-like residual population which coincided with the activation of Janus activated kinase 2 (JAK2) and signal transducer and activation of transcription 3 (STAT3) pathway in paclitaxel surviving cells. Both paclitaxel-induced JAK2/STAT3 activation and CSC-like characteristics were inhibited by a low dose JAK2-specific small molecule inhibitor CYT387 (1 μM) in vitro. Subsequent, in vivo transplantation of paclitaxel and CYT387-treated HEY cells in mice resulted in a significantly reduced tumor burden compared to that seen with paclitaxel only-treated transplanted cells. In vitro analysis of tumor xenografts at protein and mRNA levels demonstrated a loss of CSC-like markers and CA125 expression in paclitaxel and CYT387-treated cell-derived xenografts, compared to paclitaxel only-treated cell-derived xenografts. These results were consistent with significantly reduced activation of JAK2 and STAT3 in paclitaxel and CYT387-treated cell-derived xenografts compared to paclitaxel only-treated cell derived xenografts. Conclusions This proof of principle study demonstrates that inhibition of the JAK2/STAT3 pathway by the addition of CYT387 suppresses the ‘stemness’ profile in chemotherapy-treated residual cells in vitro, which is replicated in vivo, leading to a reduced tumor burden. These findings have important implications for ovarian cancer patients who are treated with taxane and/or platinum-based therapies. Keywords: Ovarian carcinoma, Cancer stem cell, Metastasis, Ascites, Chemoresistance, Recurrence, JAK2/STAT3 pathway

Dopamine transporter genotype predicts behavioural and neural measures of response inhibition

The ability to inhibit unwanted actions is a heritable executive function that may confer risk to disorders such as attention deficit hyperactivity disorder (ADHD). Converging evidence from pharmacology and cognitive neuroscience suggests that response inhibition is instantiated within frontostriatal circuits of the brain with patterns of activity that are modulated by the catecholamines dopamine and noradrenaline. A total of 405 healthy adult participants performed the stop-signal task, a paradigmatic measure of response inhibition that yields an index of the latency of inhibition, termed the stop-signal reaction time (SSRT). Using this phenotype, we tested for genetic association, performing high-density single-nucleotide polymorphism mapping across the full range of autosomal catecholamine genes. Fifty participants also underwent functional magnetic resonance imaging to establish the impact of associated alleles on brain and behaviour. Allelic variation in polymorphisms of the dopamine transporter gene (SLC6A3: rs37020; rs460000) predicted individual differences in SSRT, after corrections for multiple comparisons. Furthermore, activity in frontal regions (anterior frontal, superior frontal and superior medial gyri) and caudate varied additively with the T-allele of rs37020. The influence of genetic variation in SLC6A3 on the development of frontostriatal inhibition networks may represent a key risk mechanism for disorders of behavioural inhibition.

Failure of amino acid homeostasis causes cell death following proteasome inhibition

The ubiquitin-proteasome system targets many cellular proteins for degradation and thereby controls most cellular processes. Although it is well established that proteasome inhibition is lethal, the underlying mechanism is unknown. Here, we show that proteasome inhibition results in a lethal amino acid shortage. In yeast, mammalian cells, and flies, the deleterious consequences of proteasome inhibition are rescued by amino acid supplementation. In all three systems, this rescuing effect occurs without noticeable changes in the levels of proteasome substrates. In mammalian cells, the amino acid scarcity resulting from proteasome inhibition is the signal that causes induction of both the integrated stress response and autophagy, in an unsuccessful attempt to replenish the pool of intracellular amino acids. These results reveal that cells can tolerate protein waste, but not the amino acid scarcity resulting from proteasome inhibition.

A Tobit model for analyzing speed limit compliance in work zones

Poor compliance with speed limits is a serious safety concern in work zones. Most studies of work zone speeds have focused on descriptive analyses and statistical testing without systematically capturing the effects of vehicle and traffic characteristics. Consequently, little is known about how the characteristics of surrounding traffic and platoons influence speeds. This paper develops a Tobit regression technique for innovatively modeling the probability and the magnitude of non-compliance with speed limits at various locations in work zones. Speed data is transformed into two groups—continuous for non-compliant and left-censored for compliant drivers—to model in a Tobit model framework. The modeling technique is illustrated using speed data from three long-term highway work zones in Queensland, Australia. Consistent and plausible model estimates across the three work zones support the appropriateness and validity of the technique. The results show that the probability and magnitude of speeding was higher for leaders of platoons with larger front gaps, during late afternoon and early morning, when traffic volumes were higher, and when higher proportions of surrounding vehicles were non-compliant. Light vehicles and their followers were also more likely to speed than others. Speeding was more common and greater in magnitude upstream than in the activity area, with higher compliance rates close to the end of the activity area and close to stop/slow traffic controllers. The modeling technique and results have great potential to assist in deployment of appropriate countermeasures by better identifying the traffic characteristics associated with speeding and the locations of lower compliance.

Offspring of mothers fed a high fat diet display hepatic cell cycle inhibition and associated changes in gene expression and DNA methylation

The association between an adverse early life environment and increased susceptibility to later-life metabolic disorders such as obesity, type 2 diabetes and cardiovascular disease is described by the developmental origins of health and disease hypothesis. Employing a rat model of maternal high fat (MHF) nutrition, we recently reported that offspring born to MHF mothers are small at birth and develop a postnatal phenotype that closely resembles that of the human metabolic syndrome. Livers of offspring born to MHF mothers also display a fatty phenotype reflecting hepatic steatosis and characteristics of non-alcoholic fatty liver disease. In the present study we hypothesised that a MHF diet leads to altered regulation of liver development in offspring; a derangement that may be detectable during early postnatal life. Livers were collected at postnatal days 2 (P2) and 27 (P27) from male offspring of control and MHF mothers (n = 8 per group). Cell cycle dynamics, measured by flow cytometry, revealed significant G0/G1 arrest in the livers of P2 offspring born to MHF mothers, associated with an increased expression of the hepatic cell cycle inhibitor Cdkn1a. In P2 livers, Cdkn1a was hypomethylated at specific CpG dinucleotides and first exon in offspring of MHF mothers and was shown to correlate with a demonstrable increase in mRNA expression levels. These modifications at P2 preceded observable reductions in liver weight and liver:brain weight ratio at P27, but there were no persistent changes in cell cycle dynamics or DNA methylation in MHF offspring at this time. Since Cdkn1a up-regulation has been associated with hepatocyte growth in pathologic states, our data may be suggestive of early hepatic dysfunction in neonates born to high fat fed mothers. It is likely that these offspring are predisposed to long-term hepatic dysfunction.

Paradoxical enhancement of fear extinction memory and synaptic plasticity by inhibition of the histone acetyltransferase p300

It is well established that the coordinated regulation of activity-dependent gene expression by the histone acetyltransferase (HAT) family of transcriptional coactivators is crucial for the formation of contextual fear and spatial memory, and for hippocampal synaptic plasticity. However, no studies have examined the role of this epigenetic mechanism within the infralimbic prefrontal cortex (ILPFC), an area of the brain that is essential for the formation and consolidation of fear extinction memory. Here we report that a postextinction training infusion of a combined p300/CBP inhibitor (Lys-CoA-Tat), directly into the ILPFC, enhances fear extinction memory in mice. Our results also demonstrate that the HAT p300 is highly expressed within pyramidal neurons of the ILPFC and that the small-molecule p300-specific inhibitor (C646) infused into the ILPFC immediately after weak extinction training enhances the consolidation of fear extinction memory. C646 infused 6 h after extinction had no effect on fear extinction memory, nor did an immediate postextinction training infusion into the prelimbic prefrontal cortex. Consistent with the behavioral findings, inhibition of p300 activity within the ILPFC facilitated long-term potentiation (LTP) under stimulation conditions that do not evoke long-lasting LTP. These data suggest that one function of p300 activity within the ILPFC is to constrain synaptic plasticity, and that a reduction in the function of this HAT is required for the formation of fear extinction memory.

Proactive safety assessment in roadwork zones : a synthesis of surrogate measures of safety

Work zone safety studies have traditionally relied on historical crash records—an approach which is reactive in nature as it requires crashes to accumulate first before taking any preventive actions. However, detailed and accurate data on work zone crashes are often not available, as is the case for Australian road work zones. The lack of reliable safety records and the reactive nature of the crash-based safety analysis approach motivated this research to seek alternative and proactive measures of safety. Various surrogate measures of safety have been developed in the traffic safety literature including time to collision, time to accident, gap time, post encroachment time, required deceleration rate, proportion of stopping distances, lateral distance to departure, and time to departure. These measures express how close road-user(s) are from a potential crash by analysing their movement trajectories. A review of this fast-growing literature is presented in this paper from the viewpoint of applying the measures to untangle work zone safety issues. The review revealed that the use of the surrogate measures is very limited for analysing work zone safety, although numerous studies have used these measures for analysing safety in other parts of the road network, such as intersections and motorway ramps. There exist great opportunities for adopting this proactive safety assessment approach to transform work zone safety for both roadworkers and motorists.

Stop-signal response inhibition in schizophrenia : Behavioural, event-related potential and functional neuroimaging data

Inhibitory control deficits are well documented in schizophrenia, supported by impairment in an established measure of response inhibition, the stop-signal reaction time (SSRT). We investigated the neural basis of this impairment by comparing schizophrenia patients and controls matched for age, sex and education on behavioural, functional magnetic resonance imaging (fMRI) and event-related potential (ERP) indices of stop-signal task performance. Compared to controls, patients exhibited slower SSRT and reduced right inferior frontal gyrus (rIFG) activation, but rIFG activation correlated with SSRT in both groups. Go stimulus and stop-signal ERP components (N1/P3) were smaller in patients, but the peak latencies of stop-signal N1 and P3 were also delayed in patients, indicating impairment early in stop-signal processing. Additionally, response-locked lateralised readiness potentials indicated response preparation was prolonged in patients. An inability to engage rIFG may predicate slowed inhibition in patients, however multiple spatiotemporal irregularities in the networks underpinning stop-signal task performance may contribute to this deficit.

Common hazards and their mitigating measures in work zones : a qualitative study of worker perceptions

Road construction and maintenance activities present challenges for ensuring the safety of workers and the traveling public alike. Hazards in work zones are typically studied using historical crash records but the current study took a qualitative approach by interviewing 66 workers from various work zones in Queensland, Australia. This supplemented and enhanced the limited available data regarding the frequency and nature of work zone crashes in Australia, provided worker insights into contributing factors, and assessed their opinions on the likely effectiveness of current or future approaches to hazard mitigation. Workers may not be aware of objective data regarding effectiveness, but their attitudes and consequent levels of compliance can influence both the likelihood of implementation and the outcomes of safety measures. Despite the potential importance of worker perceptions, they have not been studied comprehensively to date, and thus this study fills a significant gap in the literature. Excessive vehicle speeds, driver distraction and aggression towards roadworkers, working in wet weather, at night and close to traffic stream were among the most common hazards noted by workers. The safety measures perceived to be most effective included police presence, active enforcement, and improving driver awareness and education about work zones. Worker perceptions differed according to their level of exposure to hazards.

Inhibition of form-deprivation myopia by a GABAAOr receptor antagonist, (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA), in guinea pigs

Purpose To investigate the effects of the relatively selective GABAAOr receptor antagonist (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) on form-deprivation myopia (FDM) in guinea pigs. Methods A diffuser was applied monocularly to 30 guinea pigs from day 10 to 21. The animals were randomized to one of five treatment groups. The deprived eye received daily sub-conjunctival injections of 100 μl TPMPA at a concentration of (i) 0.03 %, ( ii) 0.3 %, or (iii) 1 %, a fourth group (iv) received saline injections, and another (v) no injections. The fellow eye was left untreated. An additional group received no treatment to either eye. Prior to and at the end of the treatment period, refraction and ocular biometry were performed. Results Visual deprivation produced relative myopia in all groups (treated versus untreated eyes, P < 0.05). The amount of myopia was significantly affected by the drug treatment (one-way ANOVA, P < 0.0001); myopia was less in deprived eyes receiving either 0.3 % or 1 % TPMPA (saline = −4.38 ± 0.57D, 0.3 % TPMPA = −3.00 ± 0.48D, P < 0.01; 1 % TPMPA = −0.88 ± 0.51D, P < 0.001). The degree of axial elongation was correspondingly less (saline = 0.13 ± 0.02 mm, 0.3 % TPMPA = 0.09 ± 0.01 mm, P < 0.01, 1 % TPMPA = 0.02 ± 0.01 mm, P < 0.001) as was the VC elongation (saline = 0.08 ± 0.01 mm, 0.3 % TPMPA = 0.05 ± 0.01 mm, P < 0.01, 1 % TPMPA = 0.01 ± 0.01 mm; P < 0.001). ACD and LT were not affected (one-way ANOVA, P > 0.05). One percent TPMPA was more effective at inhibiting myopia than 0.3 % (P < 0.01), and 0.03 % did not appreciably inhibit the myopia (0.03 % TPMPA versus saline, P > 0.05). Conclusions Sub-conjunctival injections of TPMPA inhibit FDM in guinea pig models in a dose-dependent manner.

Primary and secondary neural networks of auditory prepulse inhibition: a functional magnetic resonance imaging study of sensorimotor gating of the human acoustic startle response

Feedforward inhibition deficits have been consistently demonstrated in a range of neuropsychiatric conditions using prepulse inhibition (PPI) of the acoustic startle eye-blink reflex when assessing sensorimotor gating. While PPI can be recorded in acutely decerebrated rats, behavioural, pharmacological and psychophysiological studies suggest the involvement of a complex neural network extending from brainstem nuclei to higher order cortical areas. The current functional magnetic resonance imaging study investigated the neural network underlying PPI and its association with electromyographically (EMG) recorded PPI of the acoustic startle eye-blink reflex in 16 healthy volunteers. A sparse imaging design was employed to model signal changes in blood oxygenation level-dependent (BOLD) responses to acoustic startle probes that were preceded by a prepulse at 120 ms or 480 ms stimulus onset asynchrony or without prepulse. Sensorimotor gating was EMG confirmed for the 120-ms prepulse condition, while startle responses in the 480-ms prepulse condition did not differ from startle alone. Multiple regression analysis of BOLD contrasts identified activation in pons, thalamus, caudate nuclei, left angular gyrus and bilaterally in anterior cingulate, associated with EMGrecorded sensorimotor gating. Planned contrasts confirmed increased pons activation for startle alone vs 120-ms prepulse condition, while increased anterior superior frontal gyrus activation was confirmed for the reverse contrast. Our findings are consistent with a primary pontine circuitry of sensorimotor gating that interconnects with inferior parietal, superior temporal, frontal and prefrontal cortices via thalamus and striatum. PPI processes in the prefrontal, frontal and superior temporal cortex were functionally distinct from sensorimotor gating.