Resolution of the chiral (1R,2S) enantiomer of cis-cyclohexane-1,2-dicarboxylic acid in the brucinium salt 2,3-dimethoxy-10-oxostrychnidinium (1R,2S)-2-carboxycyclohexane-1-carboxylate dihydrate

The structure of the 1:1 brucinium salt of cis-cyclohexane-1,2-dicarboxylic acid, 2,3-dimethoxy-10-oxostrychnidinium (1R,2S)-2-carboxycyclohexane-1-carboxylate dihydrate, has revealed the resolved (1R,2S) enantiomer of the acid. Crystals of the compound are orthorhombic, space group P212121, with unit cell dimensions a = 8.1955(3), b = 12.4034(3), c = 29.9073(9)Å, and Z = 4. The asymmetric unit comprises the brucinium cation, the hydrogen cis-cyclohexane-1,2-dicarboxylate cation, in which the carboxylate group is disordered over two sites (58, 42%), and two water molecules of solvation, one of which is occupies two 50% occupancy sites. The classic undulating brucinium cation substructures are present with the anion and the water molecules occupying the interstitial cavities and are hydrogen-bonded to them in a two-dimensional network structure.

Procaspase 8 overexpression in non-small-cell lung cancer promotes apoptosis induced by FLIP silencing

We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.

Prognostic and therapeutic relevance of FLIP and procaspase-8 overexpression in non-small cell lung cancer

Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.

Understanding QC and EQA : an authentic learning model for undergraduate students (Conference Abstract)

Introduction QC and EQA are integral to good pathology laboratory practice. Medical Laboratory Science students undertake a project exploring internal QC and EQA procedures used in chemical pathology laboratories. Each student represents an individual lab and the class group represents the peer group of labs performing the same assay using the same method. Methods Using a manual BCG assay for serum albumin, normal and abnormal controls are run with a patient sample over 7 weeks. The QC results are assessed each week using calculated z-scores and both 2S & 3S control rules to determine whether a run is ‘in control’. At the end of the 7 weeks a completed LJ chart is assessed using the Westgard Multirules. Students investigate causes of error and the implications for both lab practice and patient care if runs are not ‘in control’. Twice in the 7 weeks two EQA samples (with target values unknown) are assayed alongside the weekly QC and patient samples. Results from each student are collated and form the basis of an EQA program. ALP are provided and students complete a Youden Plot, which is used to analyse the performance of each ‘lab’ and the method to identify bias. Students explore the concept of possible clinical implications of a biased method and address the actions that should be taken if a lab is not in consensus with the peer group. Conclusion This project is a model of ‘real world’ practice in which student demonstrate an understanding of the importance of QC procedures in a pathology laboratory, apply and interpret statistics and QC rules and charts, apply critical thinking and analytical skills to quality performance data to make recommendations for further practice and improve their technical competence and confidence.

Effects of chronic intra-amniotic Ureaplasma parvum (serovar 3 and 6) colonization in the ovine fetus

Background: We have developed a sheep model of intrauterine ureaplasma infection. We aimed to examine the capability of ureaplasmas in the amniotic fluid to infect the fetus and alter fetal development...

The impact of co-locating regulatory and directional signs on driver performance

The use of intelligent transport systems is proliferating across the Australian road network, particularly on major freeways. New technology allows a greater range of signs and messages to be displayed to drivers. While there has been a long history of human factors analyses of signage, no evaluation has been conducted on this novel, sometimes dynamic, signage or potential interactions when co-located. The purpose of this driving simulator study was to investigate drivers’ behavioural changes and comprehension resulting from the co-location of Lane Use Management Systems with static signs and (Enhanced) Variable Message Signs on Queensland motorways. A section of motorway was simulated, and nine scenarios were developed which presented a combination of signage cases across levels of driving task complexity. Two higher-risk road user groups were targeted for this research on an advanced driving simulator: older (65+ years, N=21) and younger (18-22 years, N=20) drivers. Changes in sign co-location and task complexity had small effect on driver comprehension of the signs and vehicle dynamics variables, including difference with the posted speed limit, headway, standard deviation of lane keeping and brake jerks. However, increasing the amount of information provided to drivers at a given location (by co-locating several signs) increased participants’ gaze duration on the signs. With co-location of signs and without added task complexity, a single gaze was over 2s for more than half of the population tested for both groups, and up to 6 seconds for some individuals.

The impact of interface modality on police officers' visual behaviour when using an in-vehicle system

Background Standard operating procedures state that police officers should not drive while interacting with their mobile data terminal (MDT) which provides in-vehicle information essential to police work. Such interactions do however occur in practice and represent a potential source of driver distraction. The MDT comprises visual output with manual input via touch screen and keyboard. This study investigated the potential for alternative input and output methods to mitigate driver distraction with specific focus on eye movements. Method Nineteen experienced drivers of police vehicles (one female) from the NSW Police Force completed four simulated urban drives. Three drives included a concurrent secondary task: imitation licence plate search using an emulated MDT. Three different interface methods were examined: Visual-Manual, Visual-Voice, and Audio-Voice (“Visual” and “Audio” = output modality; “Manual” and “Voice” = input modality). During each drive, eye movements were recorded using FaceLAB™ (Seeing Machines Ltd, Canberra, ACT). Gaze direction and glances on the MDT were assessed. Results The Visual-Voice and Visual-Manual interfaces resulted in a significantly greater number of glances towards the MDT than Audio-Voice or Baseline. The Visual-Manual and Visual-Voice interfaces resulted in significantly more glances to the display than Audio-Voice or Baseline. For longer duration glances (>2s and 1-2s) the Visual-Manual interface resulted in significantly more fixations than Baseline or Audio-Voice. The short duration glances (<1s) were significantly greater for both Visual-Voice and Visual-Manual compared with Baseline and Audio-Voice. There were no significant differences between Baseline and Audio-Voice. Conclusion An Audio-Voice interface has the greatest potential to decrease visual distraction to police drivers. However, it is acknowledged that an audio output may have limitations for information presentation compared with visual output. The Visual-Voice interface offers an environment where the capacity to present information is sustained, whilst distraction to the driver is reduced (compared to Visual-Manual) by enabling adaptation of fixation behaviour.

Role of amine oxidase expression to maintain putrescine homeostasis in Rhodococcus opacus

While applications of amine oxidases are increasing, few have been characterised and our understanding of their biological role and strategies for bacteria exploitation are limited. By altering the nitrogen source (NH4Cl, putrescine and cadaverine (diamines) and butylamine (monoamine)) and concentration, we have identified a constitutive flavin dependent oxidase (EC 1.4.3.10) within Rhodococcus opacus. The activity of this oxidase can be increased by over two orders of magnitude in the presence of aliphatic diamines. In addition, the expression of a copper dependent diamine oxidase (EC 1.4.3.22) was observed at diamine concentrations>1mM or when cells were grown with butylamine, which acts to inhibit the flavin oxidase. A Michaelis-Menten kinetic treatment of the flavin oxidase delivered a Michaelis constant (KM)=190μM and maximum rate (kcat)=21.8s(-1) for the oxidative deamination of putrescine with a lower KM (=60μM) and comparable kcat (=18.2s(-1)) for the copper oxidase. MALDI-TOF and genomic analyses have indicated a metabolic clustering of functionally related genes. From a consideration of amine oxidase specificity and sequence homology, we propose a putrescine degradation pathway within Rhodococcus that utilises oxidases in tandem with subsequent dehydrogenase and transaminase enzymes. The implications of PUT homeostasis through the action of the two oxidases are discussed with respect to stressors, evolution and application in microbe-assisted phytoremediation or bio-augmentation.

Modular green roof systems in mid-rise multifamily residential units

This paper presents the results of a full-scale research project undertaken to assess scour losses/gains for modular tray green roof specimens placed on a mock-up building, and identify important factors to consider for wind design criteria. Visual assessment of the experimental results showed that usage of vegetation, parapet height, wind direction, and test duration were the predominant factors affecting scour resistance of the growth media in tested specimens. Statistical analysis results indicated that the differences in soil losses measured among Phase 2’s test trials were more significant than those in Phase 1. This was attributed to the lack of parapet, cornering wind conditions, and longer test duration found in Phase 2. Findings presented in this paper constitute a benchmark for future research to improve the knowledge gap that exists in green roof wind design.

Delivering crushed tablets using thickened fluids : is drug diffusion into gastric fluids restricted?

Solid medications are often crushed and mixed with food or thickened water to aid drug delivery for those who cannot or prefer not to swallow whole tablets or capsules. Dysphagic patients have the added problem of being unable to safely swallow thin fluids so water thickened with polysaccharides is used to deliver crushed medications and ensure safe swallowing. It is postulated that these polysaccharide systems may restrict drug release by reducing the diffusion of the drug into gastric fluids. METHODS By using a vertical diffusion cell separated with a synthetic membrane, the diffusion of a model drug (atenolol) was studied from a donor system containing the drug dispersed into thickened water with xanthan gum (concentration range from 0.005%-2.2%) into a receptor system containing simulated gastric fluid (SGF) at 37°C. The amount of drug transferred was measured over 8 hours and diffusion coefficients estimated using the Higuchi model approach. RESULTS Atenolol diffusion decreased with increasing xanthan gum concentration up to 1.0%, above which diffusion remained around 300 μ2s-1. The rheological measurements captured the influence of the structure and conformation of the polysaccharide in water on the movement and availability of the drug in SGF. DISCUSSION Dose form administration for dysphagic patients’ needs special attention from general practitioners, pharmacist and patients. Improving drug release of crushed tablets from thickening agents requires a reduction in the diffusion pathway (e.g. by decreasing drop size radius). This approach could make the drug available in SGF in a short time without compromising the mechanical aspects of thickening agents that guarantee safe swallowing.

Crystal structures and hydrogen bonding in the proton transfer salts of nicotine with 3,5-dinitrosalicylic acid and 5-sulfosalicylic acid

The structures of the 1:1 anhydrous salts of nicotine (NIC) with 3,5-dinitrosalicylic acid (DNSA) and 5-sulfosalicylic acid (5-SSA), namely (1R,2S)-1-methyl-2-(3-pyridyl)-1H-pyrrolidin-1-ium 2-carboxy-4,6-dinitrophenolate, C10H15N2+ C7H3N2O7-, (I) and (1R,2S)-1-methyl-2-(3-pyridyl)-1H-pyrrolidin-1-ium 3-carboxy-4-hydroxybenzenesulfonate, C10H15N2+ C7H5O6S-, (II) are reported. The asymmetric units of both (I) and (II) comprise two independent nicotinium cations (C and D) and either two DNSA or two 5-SSA anions (A and B), respectively. One of the DNSA anions shows a 25% rotational disorder in the benzene ring system. In the crystal of (I), inter-unit pyrrolidinium N-H...N(pyridine) hydrogen bonds generate zigzag NIC cation chains which extend along a while the DNSA anions are not involved in any formal inter-species hydrogen bonding but instead form pi--pi associated stacks which parallel the NIC chains along a [ring centroid separation, 3.857(2)A]. Weak C-H...O interactions between chain substructures give an overall three-dimensional structure. With (II), A and B anions form independent zigzag chains with C and D cations, respectively, through carboxylic acid O-H...N(pyridine) hydrogen bonds. These chains, which extend along b are pseudo-centrosymmetrically related and give pi--pi interactions between the benzene rings of anions A and B and the pyridine rings of the NIC cations C and D, respectively [ring centroid separations, 3.6422(19) and 3.7117(19)A]. Present also are weak intermolecular C-H...O hydrogen-bonding interactions between the chains, giving an overall three-dimensional structure.

On the interaction between radon progeny and particles generated by electronic and traditional cigarettes

During their entire lives, people are exposed to the pollutants present in indoor air. Recently, Electronic Nicotine Delivery Systems, mainly known as electronic cigarettes, have been widely commercialized: they deliver particles into the lungs of the users but a “second-hand smoke” has yet to be associated to this indoor source. On the other hand, the naturally-occurring radioactive gas, i.e. radon, represents a significant risk for lung cancer, and the cumulative action of these two agents could be worse than the agents separately would. In order to deepen the interaction between radon progeny and second-hand aerosol from different types of cigarettes, a designed experimental study was carried out by generating aerosol from e-cigarette vaping as well as from second-hand traditional smoke inside a walk-in radon chamber at the National Institute of Ionizing Radiation Metrology (INMRI) of Italy. In this chamber, the radon present in air comes naturally from the floor and ambient conditions are controlled. To characterize the sidestream smoke emitted by cigarettes, condensation particle counters and scanning mobility particle sizer were used. Radon concentration in the air was measured through an Alphaguard ionization chamber, whereas the measurement of radon decay product in the air was performed with the Tracelab BWLM Plus-2S Radon daughter Monitor. It was found an increase of the Potential Alpha-Energy Concentration (PAEC) due to the radon decay products attached to aerosol for higher particle number concentrations. This varied from 7.47 ± 0.34 MeV L−1 to 12.6 ± 0.26 MeV L−1 (69%) for the e-cigarette. In the case of traditional cigarette and at the same radon concentration, the increase was from 14.1 ± 0.43 MeV L−1 to 18.6 ± 0.19 MeV L−1 (31%). The equilibrium factor increases, varying from 23.4% ± 1.11% to 29.5% ± 0.26% and from 30.9% ± 1.0% to 38.1 ± 0.88 for the e-cigarette and traditional cigarette, respectively. These growths still continue for long time after the combustion, by increasing the exposure risk.

Anti-staphylococcal activity of C-methyl flavanones from propolis of Australian stingless bees (Tetragonula carbonaria) and fruit resins of Corymbia torelliana (Myrtaceae)

Propolis of Australian stingless bees (Tetragonula carbonaria, Meliponini) originating from Corymbia torelliana (Myrtaceae) fruit resins was tested for its antimicrobial activities as well as its flavonoid contents. This study aimed at the isolation, structural elucidation and antibacterial testing of flavanones of C. torelliana fruit resins that are incorporated into stingless bee propolis. Flavanones of this study were elucidated by spectroscopic and spectrometric methods including UV, 1D and 2D NMR, EI-MS, ESI-MS and HR-MS. The results indicated known C-methylated flavanones namely, 1 (2S)-cryptostrobin, its regioisomer 2 (2S)- stroboponin, 3 (2S)- cryptostrobin 7-methyl ether, and 6 (2S)- desmethoxymatteucinol, and known flavanones 4 (2S)- pinostrobin and 5 (2S)- pinocembrin as markers for C. torelliana fruit resins and one propolis type. Ethanolic preparations of propolis were shown to be active against Staphylococcus aureus (ATCC 25923) and to a lesser extent against Pseudomonas aeruginosa (ATCC 27853). C. torelliana flavanones inhibited the growth of S. aureus therefore contributing to the antibacterial effects observed for Australian stingless bee propolis extracts.

The Post-Illumination Pupil Response (PIPR)

Purpose The post-illumination pupil response (PIPR) has been quantified using four metrics, but the spectral sensitivity of only one is known; here we determine the other three. To optimize the human PIPR measurement, we determine the protocol producing the largest PIPR, the duration of the PIPR, and the metric(s) with the lowest coefficient of variation. Methods The consensual pupil light reflex (PLR) was measured with a Maxwellian view pupillometer. - Experiment 1: Spectral sensitivity of four PIPR metrics [plateau, 6 s, area under curve (AUC) early and late recovery] was determined from a criterion PIPR to a 1s pulse and fitted with Vitamin A1 nomogram (λmax = 482nm). - Experiment 2: The PLR was measured as a function of three stimulus durations (1s, 10s, 30s), five irradiances spanning low to high melanopsin excitation levels (retinal irradiance: 9.8 to 14.8 log quanta.cm-2.s-1), and two wavelengths, one with high (465nm) and one with low (637nm) melanopsin excitation. Intra and inter-individual coefficients of variation (CV) were calculated. Results The melanopsin (opn4) photopigment nomogram adequately describes the spectral sensitivity of all four PIPR metrics. The PIPR amplitude was largest with 1s short wavelength pulses (≥ 12.8 log quanta.cm-2.s-1). The plateau and 6s PIPR showed the least intra and inter-individual CV (≤ 0.2). The maximum duration of the sustained PIPR was 83.0±48.0s (mean±SD) for 1s pulses and 180.1±106.2s for 30s pulses (465nm; 14.8 log quanta.cm-2.s-1). Conclusions All current PIPR metrics provide a direct measure of the intrinsic melanopsin photoresponse. To measure progressive changes in melanopsin function in disease, we recommend that the PIPR be measured using short duration pulses (e.g., ≤ 1s) with high melanopsin excitation and analyzed with plateau and/or 6s metrics. Our PIPR duration data provide a baseline for the selection of inter-stimulus intervals between consecutive pupil testing sequences.