Heat exposure and hypothyroid conditions decrease hydrogen peroxide generation in liver mitochondria

Exposure of rats to heat (39 +/- 1 degree C) decreased H2O2 generation in mitochondria of the liver, but not of the kidney or the heart. The effect was obtained with three substrates, succinate, glycerol 1-phosphate and choline, with a decrease to 50% in the first 2-3 days of exposure, and a further decrease on longer exposure. The dehydrogenase activity with only glycerol 1-phosphate decreased, which is indicative of the hypothyroid condition, whereas choline dehydrogenase activity remained unchanged and that of succinate dehydrogenase decreased on long exposure. The serum concentration of thyroxine decreased in heat-exposed rats. Thyroxine treatment of rats increased H2O2 generation. Hypothyroid conditions obtained by treatment with propylthiouracil or thyroidectomy caused a decrease in H2O2 generation and changes in dehydrogenase activities similar to those with heat exposure. Treatment of heat-exposed or thyroidectomized rats with thyroxine stimulated H2O2 generation by a mechanism apparently involving fresh protein synthesis. The results indicate that H2O2 generation in mitochondria of heat-exposed animals is determined by thyroid status.

Effects of Isothermal and Adiabatic Thermal Loadings on Size and Strain Rate Dependence of Copper Nanowire

In the present paper, the size and strain rate effects on ultra-thin < 100 >/{100} Cu nanowires at an initial temperature of 10 K have been discussed. Extensive molecular dynamics (MD) simulations have been performed using Embedded atom method (EAM) to investigate the structural behaviours and properties under high strain rate. Velocity-Verlet algorithm has been used to solve the equation of motions. Two different thermal loading cases have been considered: (i) Isothermal loading, in which Nose-Hoover thermostat is used to maintain the constant system temperature, and (ii) Adiabatic loading, i.e., without any thermostat. Five different wire cross-sections were considered ranging from 0.723 x 0.723 nm(2) to 2.169 x 2.169 nm(2) The strain rates used in the present study were 1 x 10(9) s(-1), 1 x 10(8) s(-1), and 1 x 10(7) s(-1). The effect of strain rate on the mechanical properties of copper nanowires was analysed, which shows that elastic properties are independent of thermal loading for a given strain rate and cross-sectional dimension of nanowire. It showed a decreasing yield stress and yield strain with decreasing strain rate for a given cross- section. Also, a decreasing yield stress and increasing yield strain were observed for a given strain rate with increasing cross-sectional area. Elastic modulus was found to be similar to 100 GPa, which was independent of processing temperature, strain rate, and size for a given initial temperature. Reorientation of < 100 >/{100} square cross-sectional copper nanowire into a series of stable ultra-thin Pentagon copper nanobridge structures with dia of similar to 1 nm at 10 K was observed under high strain rate tensile loading. The effect of isothermal and adiabatic loading on the formation of such pentagonal nanobridge structure has been discussed.

No evidence for genetic association with the let-7 microRNA-binding site or other common KRAS variants in risk of endometriosis

STUDY QUESTION Is there a contribution of the minor allele at the KRAS single nucleotide polymorphism (SNP) rs61764370 in the let-7 microRNA-binding site to endometriosis risk? SUMMARY ANSWER We found no evidence for association between endometriosis risk and rs61764370 or any other SNPs in KRAS. WHAT IS KNOWN ALREADY The rs61764370 SNP in the 3' untranslated region of the KRAS gene is predicted to disrupt a complementary binding site (LCS6) for the let-7 microRNA, and was recently reported to be at a high frequency (31%) in 132 women of varying ancestry with endometriosis compared with frequencies in a database of population controls (up to 7.6% depending on ancestry), suggesting a strong effect of this KRAS SNP in the aetiology of endometriosis. STUDY DESIGN, SIZE AND DURATION This was a case-control study with a total of 11 206 subjects. The study was performed between February 2012 and July 2012. PARTICIPANTS/MATERIALS, SETTINGAND METHODS We first investigated a possible association between common markers in KRAS and endometriosis risk from our genome-wide association (GWA) data in 3194 surgically confirmed endometriosis cases and 7060 controls of European ancestry. Although rs61764370 was not genotyped on the GWA arrays, five SNPs typed in the study were highly correlated with this variant. The rs61764370 and two SNPs highly correlated with rs61764370 were then genotyped in 933 endometriosis cases and 952 controls using the Sequenom MassARRAY platform. MAIN RESULTS AND THE ROLE OF CHANCE There was no evidence for an association between rs61764370 and endometriosis risk P = 0.411 and odds ratio = 1.10 (95% confidence intervals: 0.88-1.36). We also found no evidence for an association between the highly correlated SNP rs17387019 and endometriosis. Their minor allele frequencies in cases and controls were of 0.087-0.091 similar to the population frequency reported previously for this variant in controls. Analyses of endometriosis cases with revised American Fertility Society stage III/IV disease also showed no evidence for an association between these SNPs and endometriosis risk. LIMITATIONS AND REASONS FOR CAUTION The GWA and genotyped data sets were not independent since individuals and cases from some families overlap. Controls in our GWA study were not screened for endometriosis. WIDER IMPLICATIONS OF THE FINDINGS The key SNP, rs61764370, was genotyped in a subset of samples. Our results do not support the suggestion that carrying the minor allele at rs61764370 contributes to a significant number of endometriosis cases and rs61764370 is, therefore, unlikely to be a useful marker of endometriosis risk. STUDY FUNDING/COMPETING INTEREST(S) The research was funded by grants from the Australian National Health and Medical Research Council and Wellcome Trust. None of the authors has competing interests for the study.

Australian pharmacists' self-perceptions in relation to the 'Advanced Pharmacy Practice Framework'

Background The Australian Pharmacy Practice Framework was developed by the Advanced Pharmacy Practice Steering Committee and endorsed by the Pharmacy Board of Australia in October 2012. The Steering Committee conducted a study that found practice portfolios to be the preferred method to assess and credential Advanced Pharmacy Practitioner, which is currently being piloted by the Australian Pharmacy Council. Credentialing is predicted to open to all pharmacists practising in Australia by November 2015. Objective To explore how Australian pharmacists self-perceived being advanced in practice and how they related their level of practice to the Australian Advanced Pharmacy Practice Framework. Method This was an explorative, cross-sectional study with mixed methods analysis. Advanced Pharmacy Practice Framework, a review of the recent explorative study on Advanced Practice conducted by the Advanced Pharmacy Practice Framework Steering Committee and semi-structured interviews (n = 10) were utilized to create, refine and pilot the questionnaire. The questionnaire was advertised across pharmacy-organizational websites via a purposive sampling method. The target population were pharmacists currently registered in Australia. Results Seventy-two participants responded to the questionnaire. The participants were mostly female (56.9%) and in the 30–40 age group (26.4%). The pharmacists self-perceived their levels of practice as either entry, transition, consolidation or advanced, with the majority selecting the consolidation level (38.9%). Although nearly half (43.1%) of the participants had not seen the Framework beforehand, they defined Advanced Pharmacy Practice similarly to the definition outlined in the Framework, but also added specialization as a requirement. Pharmacists explained why they were practising at their level of practice, stating that not having more years of practice, lacking experience, or postgraduate/post-registration qualifications, and more involvement and recognition in practice were the main reasons for not considering themselves as an Advanced Pharmacy Practitioner. To be considered advanced by the Framework, pharmacists would need to fulfill at least 70% of the Advanced Practice competency standards at an advanced level. More than half of the pharmacists (64.7%) that self-perceived as being advanced managed to fulfill 70% or more of these Advanced Practice competency standards at the advanced level. However, none of the self-perceived entry level pharmacists managed to match at least 70% of the competencies at the entry level. Conclusion Participants' self-perception of the term Advanced Practice was similar to the definition in the Advanced Pharmacy Practice Framework. Pharmacists working at an advanced level were largely able to demonstrate and justify their reasons for being advanced practitioners. However, pharmacists practising at the other levels of practice (entry, transition, consolidation) require further guidance regarding their advancement in practice.

Differences in the Behavior of Luteinizing Hormones of Various Species at the Rat Gonadal Cell Receptor Site

The ability of different LH-like hormones, such as hCG, PMSG/equine (e) CG, ovine (o) LH, eLH, and rat (r) LH, to bind to and stimulate steroidogenesis in two types of rat gonadal cells was studied under the same experimental conditions. In both Leydig and granulosa cells, the maximal steroidogenic responses elicited by optimal doses of different LHs present during a 2-h incubation were comparable. However, if the cells were exposed to the different LHs for a brief period and then subjected to interference with hormone action by removing the unbound hormone from the medium by washing or adding specific antisera, differences were observed in the amount of steroid produced during subsequent incubation in hormone-free medium. Thus, in the case of hCG, either of these procedures carried out at 15 or 30 min of incubation had little inhibitory effect on the amount of steroid produced at 2 h, the latter being similar to that produced by cells incubated in the continued presence of hCG for 2 h. With eCG and rLH, the effect was dramatic, in that there was a total inhibition of subsequent steroidogenic response. In cells exposed to eLH and oLH, inhibition of subsequent steroidogenesis due to either removal of the free-hormone or addition of specific antisera at 15 or 30 min was only partial. Although all of the antisera used were equally effective in inhibiting the steroidogenic response to respective gonadotropins when added along with hormones at the beginning of incubation, differences were observed in the degree of inhibition of this response when the same antisera were added at later times of incubation. Thus, when antisera were added 60 min after the hormone, the inhibition of steroidogenesis was total (100%) for eCG, partial (10–40%) for eLH and oLH, and totally lacking in cells treated with hCG. From this, it appears that hCG bound to the receptor probably becomes unavailable for binding to its antibody with time, while in the case of eCG and other LHs used, the antibody can still inhibit the biological activity of the hormone. Studies with 125I-labeled hormones further supported the conclusion that hCG differs from all other LHs in being most tightly bound and, hence, least dissociable, while eCG and rLH dissociate most readily; oLH and eLH can be placed in between these hormones in the extent of their dissociability. (Endocrinology 116: 597–603,1985)

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of similar to 50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (similar to 100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.

Molecular pathogenesis of human parechovirus 1 infection

The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members. We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3 UTR RNA. It was found to bind also duplex RNA containing 3 UTR(+)-3 UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic. Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.

Binding of polyphenols to plant cell wall analogues - Part 1: Anthocyanins

Anthocyanins are located within the vacuole of plant cells, and are released following cell rupture during eating or processing at which time they first come into contact with the plant cell wall. The extent of anthocyanin-cell wall interaction was investigated by monitoring the rate of anthocyanin depletion in the presence of pure cellulose or cellulose-pectin composites as cell wall models. It was found that anthocyanins interact with both cellulose and pectin over a two-stage process with initially (mins-hours) 13 similar to 18% of anthocyanins binding to cellulose or cellulose/pectincomposites. With prolonged exposure (days-weeks), a gradual increase in anthocyanin binding occurs, possibly due to anthocyanins stacking on top of a base layer. Binding of acylated and non-acylated anthocyanins followed a similar pattern with slightly more (5-10%) binding of the acylated forms. Composites with the highest pectin content had the greatest anthocyanin binding suggesting the existence of both ionic interactions (with pectin) and hydrophobic interactions (with cellulose) of anthocyanin with plant cell walls.

Bovine cysticercosis-Development of a real-time PCR to enhance classification of suspect cysts identified at meat inspection

Laboratory confirmation methods are important in bovine cysticerosis diagnosis as other pathologies can result in morphologically similar lesions resulting in false identifications. We developed a probe-based real-time PCR assay to identify Taenia saginata in suspect cysts encountered at meat inspection and compared its use with the traditional method of identification, histology, as well as a published nested PCR. The assay simultaneously detects T. saginata DNA and a bovine internal control using the cytochrome c oxidase subunit 1 gene of each species and shows specificity against parasites causing lesions morphologically similar to those of T. saginata. The assay was sufficiently sensitive to detect 1 fg (Ct 35.09 +/- 0.95) of target DNA using serially-diluted plasmid DNA in reactions spiked with bovine DNA as well as in all viable and caseated positive control cysts. A loss in PCR sensitivity was observed with increasing cyst degeneration as seen in other molecular methods. In comparison to histology, the assay offered greater sensitivity and accuracy with 10/19 (53%) T. saginata positives detected by real-time PCR and none by histology. When the results were compared with the reference PCR, the assay was less sensitive but offered advantages of faster turnaround times and reduced contamination risk. Estimates of the assay's repeatability and reproducibility showed the assay is highly reliable with reliability coefficients greater than 0.94. Crown Copyright (C) 2013 Published by Elsevier B.V. All rights reserved.

Liveweight prediction from hip height, condition score, fetal age and breed in tropical female cattle

Hip height, body condition, subcutaneous fat, eye muscle area, percentage Bos taurus, fetal age and diet digestibility data were collected at 17 372 assessments on 2181 Brahman and tropical composite (average 28% Brahman) female cattle aged between 0.5 and 7.5 years of age at five sites across Queensland. The study validated the subtraction of previously published estimates of gravid uterine weight to correct liveweight to the non-pregnant status. Hip height and liveweight were linearly related (Brahman: P<0.001, R-2 = 58%; tropical composite P<0.001, R-2 = 67%). Liveweight varied by 12-14% per body condition score (5-point scale) as cows differed from moderate condition (P<0.01). Parallel effects were also found due to subcutaneous rump fat depth and eye muscle area, which were highly correlated with each other and body condition score (r = 0.7-0.8). Liveweight differed from average by 1.65-1.66% per mm of rump fat depth and 0.71-0.76% per cm(2) of eye muscle area (P<0.01). Estimated dry matter digestibility of pasture consumed had no consistent effect in predicting liveweight and was therefore excluded from final models. A method developed to estimate full liveweight of post-weaning age female beef cattle from the other measures taken predicted liveweight to within 10 and 23% of that recorded for 65 and 95% of cases, respectively. For a 95% chance of predicted group average liveweight (body condition score used) being within 5, 4, 3, 2 and 1% of actual group average liveweight required 23, 36, 62, 137 and 521 females, respectively, if precision and accuracy of measurements matches that used in the research. Non-pregnant Bos taurus female cattle were calculated to be 10-40% heavier than Brahmans at the same hip height and body condition, indicating a substantial conformational difference. The liveweight prediction method was applied to a validation population of 83 unrelated groups of cattle weighed in extensive commercial situations on 119 days over 18 months (20 917 assessments). Liveweight prediction in the validation population exceeded average recorded liveweight for weigh groups by an average of 19 kg (similar to 6%) demonstrating the difficulty of achieving accurate and precise animal measurements under extensive commercial grazing conditions.

Hanging drop: An in vitro air toxic exposure model using human lung cells in 2D and 3D structures

Using benzene as a candidate air toxicant and A549 cells as an in vitro cell model, we have developed and validated a hanging drop (HD) air exposure system that mimics an air liquid interface exposure to the lung for periods of 1 h to over 20 days. Dose response curves were highly reproducible for 2D cultures but more variable for 3D cultures. By comparing the HD exposure method with other classically used air exposure systems, we found that the HD exposure method is more sensitive, more reliable and cheaper to run than medium diffusion methods and the CULTEX (R) system. The concentration causing 50% of reduction of cell viability (EC50) for benzene, toluene, p-xylene, m-xylene and o-xylene to A549 cells for 1 h exposure in the HD system were similar to previous in vitro static air exposure. Not only cell viability could be assessed but also sub lethal biological endpoints such as DNA damage and interleukin expressions. An advantage of the HD exposure system is that bioavailability and cell concentrations can be derived from published physicochemical properties using a four compartment mass balance model. The modelled cellular effect concentrations EC50(cell) for 1 h exposure were very similar for benzene, toluene and three xylenes and ranged from 5 to 15 mmol/kg(dry weight) which corresponds to the intracellular concentration of narcotic chemicals in many aquatic species, confirming the high sensitivity of this exposure method. (C) 2013 Elsevier B.V. All rights reserved.

Rhipicephalus (Boophilus) microplus tick in vitro feeding methods for functional (dsRNA) and vaccine candidate (antibody) screening

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5–6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated (CPDA) and EDTA tubes. Although >30 mg semi-engorged ticks fed more reliably, ticks as small as 15 mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10 mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86 did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA. This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and treatments.

Strangers in a strange land: do life history traits differ for alien and native colonisers of novel environments?

Do alien invasive species exhibit life history characteristics that are similar to those of native species that have become pests in their continent of origin? We compared eucalypt specialists that have become pests in Australian plantations (natives) to those that have established overseas (aliens) using 13 life history traits and found that although traits that support rapid population build-up were shared, overall, aliens and native colonisers differed significantly. Distance from source (New Zealand vs. other) had no significant effect, but species that established more than 50 years ago exhibited different life history traits from those that established within the last 50 years, possibly because of more effective quarantine. Native and alien eucalypt insect invaders differed predominantly in traits that facilitate long-distance movement (pathway traits), compared to traits that facilitate establishment and spread. Aliens had longer adult flight seasons, were smaller and more closely host-associated (cryptic eggs and larvae), had lower incidence of diapause (i.e. were more seasonally plastic) and more generations per year than natives. Thus, studies of species invasive within their country of origin can shed light on alien invasions.

Elevated levels of PFOS and PFHxS in firefighters exposed to aqueous film forming foam (AFFF)

Exposure to aqueous film forming foam (AFFF) was evaluated in 149 firefighters working at AFFF training facilities in Australia by analysis of PFOS and related compounds in serum. A questionnaire was designed to capture information about basic demographic factors, lifestyle factors and potential occupational exposure (such as work history and self-reported skin contact with foam). The results showed that a number of factors were associated with PFAA serum concentrations. Blood donation was found to be linked to low PFAA levels, and the concentrations of PFOS and PFHxS were found to be positively associated with years of jobs with AFFF contact. The highest levels of PFOS and PFHxS were one order of magnitude higher compared to the general population in Australia and Canada. Study participants who had worked ten years or less had levels of PFOS that were similar to or only slightly above those of the general population. This coincides with the phase out of 3M AFFF from all training facilities in 2003, and suggests that the exposures to PFOS and PFHxS in AFFF have declined in recent years. Self-reporting of skin contact and frequency of contact were used as an index of exposure. Using this index, there was no relationship between PFOS levels and skin exposure. This index of exposure is limited as it relies on self-report and it only considers skin exposure to AFFF, and does not capture other routes of potential exposure. Possible associations between serum PFAA concentrations and five biochemical outcomes were assessed. The outcomes were serum cholesterol, triglycerides, high-density lipoproteins, low density lipoproteins, and uric acid. No statistical associations between any of these endpoints and serum PFAA concentrations were observed.

Fungus-feeding phlaeothripine Thysanoptera in the genus Holothrips from Australia and New Caledonia, with a structurally similar new genus, Holoengythrips

Ten species of Holothrips, including seven new species, are recognized from Australia, with one further new species from New Caledonia. A new genus, Holoengythrips, is described from Australia, with nine new species that look similar to Holothrips species in having elongate maxillary stylets that are close together medially for the full length of the head. In contrast to species of Holothrips, the species of Holoengythrips are strongly sexually dimorphic, with antennal segment VIII separated from VII and the maxillary stylets more slender, and the males have a pore plate on the eighth sternite. Holoengythrips is therefore considered to be more closely related to Hoplandrothrips.