Biochemical characterisation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the liver fluke, Fasciola hepatica

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses one of the two steps in glycolysis which generate the reduced coenzyme NADH. This reaction precedes the two ATP generating steps. Thus, inhibition of GAPDH will lead to substantially reduced energy generation. Consequently, there has been considerable interest in developing GAPDH inhibitors as anti-cancer and anti-parasitic agents. Here, we describe the biochemical characterisation of GAPDH from the common liver fluke Fasciola hepatica (FhGAPDH). The primary sequence of FhGAPDH is similar to that from other trematodes and the predicted structure shows high similarity to those from other animals including the mammalian hosts. FhGAPDH lacks a binding pocket which has been exploited in the design of novel antitrypanosomal compounds. The protein can be expressed in, and purified from Escherichia coli; the recombinant protein was active and showed no cooperativity towards glyceraldehyde 3-phosphate as a substrate. In the absence of ligands, FhGAPDH was a mixture of homodimers and tetramers, as judged by protein-protein crosslinking and analytical gel filtration. The addition of either NAD(+) or glyceraldehyde 3-phosphate shifted this equilibrium towards a compact dimer. Thermal scanning fluorimetry demonstrated that this form was considerably more stable than the unliganded one. These responses to ligand binding differ from those seen in mammalian enzymes. These differences could be exploited in the discovery of reagents which selectively disrupt the function of FhGAPDH.

PET-CT as a predictor of outcome in resectable colorectal liver metastases

Introduction
PET-computed tomography (PET-CT) is a useful staging imaging modality in colorectal liver metastases (CRLM). This study aimed to determine whether PET-CT parameters, standardized uptake value (SUV) and reconstructed tumour volume (RTV), are predictors of prognosis and survival.

Methods
A study of all resectable CRLM patients in the regional HPB unit from 2007–2009 was performed. Preoperative PET-CT scans were retrospectively reviewed; SUV, diameter and RTV for each lesion was recorded. Correlation analysis was performed with other pathological and biochemical parameters, by Pearson’s correlation analysis. Survival analysis was performed using Cox regression hazard model. A P value of less than 0.05 was considered statistically significant.

Results
A total of 79 patients were included. SUV moderately correlated with tumour diameter, both PET-CT (r=0.4927; P<0.0001) and histology (r=0.4513; P=0.0003); RTV (r=0.4489; P<0.001), preoperative carcinoembryonic antigen (CEA) (r=0.4977; P=0.0001), and postoperative CEA (r=0.3727; P=0.004). Multivariate analysis found that an independent predictor of SUVmax was preoperative CEA (P=0.03). RTV strongly correlated with preoperative CEA (r=0.9389; P<0.0001). SUV and RTV had a negative effect on survival.

Conclusion
PET-CT, in the setting of CRLM, may have a prognostic role in assessing survival. Although no definite conclusions can be drawn regarding the prognostic role of SUV and RTV, it acts to reinforce the need for further prospective studies to validate these findings.

β-Lapachone alleviates alcoholic fatty liver disease in rats

Alcohol-induced liver injury is the most common liver disease in which fatty acid metabolism is altered. It is thought that altered NAD+/NADH redox potential by alcohol in the liver causes fatty liver by inhibiting fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. β-Lapachone (βL), a naturally occurring quinone, has been shown to stimulate fatty acid oxidation in an obese mouse model by activating adenosine monophosphate-activated protein kinase (AMPK). In this report, we clearly show that βL reduced alcohol-induced hepatic steatosis and induced fatty acid oxidizing capacity in ethanol-fed rats. βL treatment markedly decreased hepatic lipids while serum levels of lipids and lipoproteins were increased in rats fed ethanol-containing liquid diets with βL administration. Furthermore, inhibition of lipolysis, enhancement of lipid mobilization to mitochondria and upregulation of mitochondrial β-oxidation activity in the soleus muscle were observed in ethanol/βL-treated animals compared to the ethanol-fed rats. In addition, the activity of alcohol dehydrogenase, but not aldehyde dehydrogenase, was significantly increased in rats fed βL diets. βL-mediated modulation of NAD+/NADH ratio led to the activation of AMPK signaling in these animals. Conclusion: Our results suggest that improvement of fatty liver by βL administration is mediated by the upregulation of apoB100 synthesis and lipid mobilization from the liver as well as the direct involvement of βL on NAD+/NADH ratio changes, resulting in the activation of AMPK signaling and PPARα-mediated β-oxidation. Therefore, βL-mediated alteration of NAD+/NADH redox potential may be of potential therapeutic benefit in the clinical setting.

Population pharmacokinetic and pharmacogenetic analysis of tacrolimus in paediatric liver transplant patients

Aims: To build a population pharmacokinetic model that describes the apparent clearance of tacrolimus and the potential demographic, clinical and genetically controlled factors that could lead to inter-patient pharmacokinetic variability within children following liver transplantation.

Methods: The present study retrospectively examined tacrolimus whole blood pre-dose concentrations (n = 628) of 43 children during their first year post-liver transplantation. Population pharmacokinetic analysis was performed using the non-linear mixed effects modelling program (nonmem) to determine the population mean parameter estimate of clearance and influential covariates.

Results: The final model identified time post-transplantation and CYP3A5*1 allele as influential covariates on tacrolimus apparent clearance according to the following equation:

TVCL=12.9×(Weight /13.2)^0.75×EXP(-0.00158×TPT)×EXP(0.428×CYP3A5)

where TVCL is the typical value for apparent clearance, TPT is time post-transplantation in days and the CYP3A5 is 1 where*1 allele is present and 0 otherwise. The population estimate and inter-individual variability (%CV) of tacrolimus apparent clearance were found to be 0.977 l h kg (95% CI 0.958, 0.996) and 40.0%, respectively, while the residual variability between the observed and predicted concentrations was 35.4%.

Conclusion: Tacrolimus apparent clearance was influenced by time post-transplantation and CYP3A5 genotypes. The results of this study, once confirmed by a large scale prospective study, can be used in conjunction with therapeutic drug monitoring to recommend tacrolimus dose adjustments that take into account not only body weight but also genetic and time-related changes in tacrolimus clearance. © 2013 The British Pharmacological Society.

Loss-of-Mains Protection System by Application of Phasor Measurement Unit Technology with Experimentally Assessed Threshold Settings

Loss-of-mains protection is an important component of the protection systems of embedded generation. The role of loss-of-mains is to disconnect the embedded generator from the utility grid in the event that connection to utility dispatched generation is lost. This is necessary for a number of reasons, including the safety of personnel during fault restoration and the protection of plant against out-of-synchronism reclosure to the mains supply. The incumbent methods of loss-of-mains protection were designed when the installed capacity of embedded generation was low, and known problems with nuisance tripping of the devices were considered acceptable because of the insignificant consequence to system operation. With the dramatic increase in the installed capacity of embedded generation over the last decade, the limitations of current islanding detection methods are no longer acceptable. This study describes a new method of loss-of-mains protection based on phasor measurement unit (PMU) technology, specifically using a low cost PMU device of the authors' design which has been developed for distribution network applications. The proposed method addresses the limitations of the incumbent methods, providing a solution that is free of nuisance tripping and has a zero non-detection zone. This system has been tested experimentally and is shown to be practical, feasible and effective. Threshold settings for the new method are recommended based on data acquired from both the Great Britain and Ireland power systems.

An Enchanted Tool? Humanitarian Assistance and the ILC Draft Articles on the Protection of Persons in the Event of Disasters

Despite previous attempts at codification of international law regarding international responses to natural and human-made disasters, there is currently no binding international legal framework to regulate the provision of humanitarian assistance outside armed conflicts. Nevertheless, since the International Law Commission (ILC) included the protection of persons in the event of disasters on its programme of work in 2006, it has provisionally adopted eleven draft articles that have the potential to create binding obligations on states and humanitarian actors in disaster settings. Draft articles adopted include the definition of ‘a disaster’, the relationship of the draft articles to the international humanitarian law of armed conflict, recognition of the inherent dignity of the human person, and the duty of international cooperation. However, the final form of the draft articles has not been agreed. The Codification Division of the UN Office of Legal Affairs has proposed a framework convention format, which has seen support in the ILC and the UN General Assembly Sixth Committee. The overall aim of this article is to provide an analysis of the potential forms of international regulation open to the ILC and states in the context of humanitarian responses to disasters. However to avoid enchanting the ILC draft articles with unwarranted power, any examination of form requires an understanding of the substantive subject matter of the planned international regulation. The article therefore provides an overview of the international legal regulation of humanitarian assistance following natural and human-made disasters, and the ILC’s work to date on the topic. It then examines two key issues that remain to be addressed by the ILC and representatives of states in the UN General Assembly Sixth Committee. Drawing on the UN Guiding Principles on Internal Displacement, the development and implications of binding and non-binding international texts are examined, followed by an analysis of the suggested framework convention approach identified by the Special Rapporteur as a potential outcome of the ILC work.

Minority Rights and Minority Protection in Europe

In order to gain access to the EU, nations must be seen to implement formal instruments that protect the rights of minorities. This book examines the ways in which these tools have worked in a number of post-communist states, and explores the interaction of domestic and international structures that determine the application of these policies.

Πολιτικες ασύλου της ΕΕ- A critical review of the European policies of asylum: The re-positioning of asylum seekers from refugees/victims in need of protection to criminals (illegal immigrants)

This publication traces how asylum seekers are repositioned in the existing European asylum legislation from asylum seekers as victims in need of protection, to criminals . It is argued that this is due to the European legislation concerning the area of freedom, security and justice. The latest asylum legislation seems to undermine the refugee status which -as it is widely known- is safeguarded by the 1951 Geneva Convention relating to the Status of Refugees and its relevant 1967 Protocol. Additionally, in this paper the role of social workers and other social scientists to protect the rights of asylum seekers and question the existing legislation is presented.

System level DSP synthesis using voltage overscaling, unequal error protection & adaptive quality tuning

In this paper, we propose a system level design approach considering voltage over-scaling (VOS) that achieves error resiliency using unequal error protection of different computation elements, while incurring minor quality degradation. Depending on user specifications and severity of process variations/channel noise, the degree of VOS in each block of the system is adaptively tuned to ensure minimum system power while providing "just-the-right" amount of quality and robustness. This is achieved, by taking into consideration system level interactions and ensuring that under any change of operating conditions only the "lesscrucial" computations, that contribute less to block/system output quality, are affected. The design methodology applied to a DCT/IDCT system shows large power benefits (up to 69%) at reasonable image quality while tolerating errors induced by varying operating conditions (VOS, process variations, channel noise). Interestingly, the proposed IDCT scheme conceals channel noise at scaled voltages. ©2009 IEEE.

Logic and Memory Design Based on Unequal Error Protection for Voltage-scalable, Robust and Adaptive DSP Systems

In this paper, we propose a system level design approach considering voltage over-scaling (VOS) that achieves error resiliency using unequal error protection of different computation elements, while incurring minor quality degradation. Depending on user specifications and severity of process variations/channel noise, the degree of VOS in each block of the system is adaptively tuned to ensure minimum system power while providing "just-the-right" amount of quality and robustness. This is achieved, by taking into consideration block level interactions and ensuring that under any change of operating conditions, only the "less-crucial" computations, that contribute less to block/system output quality, are affected. The proposed approach applies unequal error protection to various blocks of a system-logic and memory-and spans multiple layers of design hierarchy-algorithm, architecture and circuit. The design methodology when applied to a multimedia subsystem shows large power benefits ( up to 69% improvement in power consumption) at reasonable image quality while tolerating errors introduced due to VOS, process variations, and channel noise.

Protection of DNA against low-energy electrons by amino acids: A first-principles molecular dynamics study

Using first-principles molecular dynamics simulations, we have investigated the notion that amino acids can play a protective role when DNA is exposed to excess electrons produced by ionizing radiation. In this study we focus on the interaction of glycine with the DNA nucleobase thymine. We studied thymine-glycine dimers and a condensed phase model consisting of one thymine molecule solvated in amorphous glycine. Our results show that the amino acid acts as a protective agent for the nucleobase in two ways. If the excess electron is initially captured by the thymine, then a proton is transferred in a barrier-less way from a neighboring hydrogen-bonded glycine. This stabilizes the excess electron by reducing the net partial charge on the thymine. In the second mechanism the excess electron is captured by a glycine, which acts as a electron scavenger that prevents electron localization in DNA. Both these mechanisms introduce obstacles to further reactions of the excess electron within a DNA strand, e.g. by raising the free energy barrier associated with strand breaks.

RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro

Background: Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).

Methodology/Principal Findings: Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.

Conclusions/Significance: In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.

Resilience and Burnout in Child protection social work: Individual and organisational themes from a systematic literature review

Child protection social work is acknowledged as a very stressful occupation, with high turnover and poor retention of staff being a major concern. This paper highlights themes that emerged from findings of sixty-five articles that were included as part of a systematic literature review. The review focused on the evaluation of research findings, which considered individual and organisational factors associated with resilience or burnout in child protection social work staff. The results identified a range of individual and organisational themes for staff in child protection social work. Nine themes were identified in total. These are categorised under ‘Individual’ and ‘Organisational’ themes. Themes categorised as individual included personal history of maltreatment, training and preparation for child welfare, coping, secondary traumatic stress, compassion fatigue and compassion satisfaction. Those classified as organisational included workload, social support and supervision, organisational culture and climate, organisational and professional commitment, and job satisfaction or dissatisfaction. The range of factors is discussed with recommendations and areas for future research are highlighted.

Pitfalls in Protection: How Theory Can Enrich Our Understanding of Regulatory Compliance Problems in Planning Practice

This paper responds to demands for greater academic investigation into environmental protection, specifically the practical and structural problems which underpin regulatory compliance in the planning system. It critiques traditional theories of regulation and answers calls for the development of a thematic lens to facilitate the scrutiny of not only operational practice, but also the broader institutional regime. An empirical investigation builds upon the construct of really responsive regulation to study planning control and it becomes apparent that not only are there significant procedural planning difficulties facing regulatory compliance, but also that a much wider raft of issues must be considered if the complex equation is to be solved. The findings demonstrate how theory can be applied to enrich our rudimentary understanding of deep-seated problems and foster insights into areas of structural importance which are relevant to both planning and the wider regulatory arena.

Clozapine-induced liver injury and pleural effusion

Clozapine, whilst associated commonly with a transient and benign increase in liver enzymes, has also been associated with varying presentations of hepatitis in existing case reports. This report describes what we believe to be the first documented case of acute liver injury and pleural effusion associated with clozapine, resolving after cessation of the agent. The case supports existing literature in advocating a high index of suspicion, particularly in the 4-5 weeks following clozapine initiation, when considering nonspecific clinical symptoms and signs.