Zebrafish as a genetic model in pre-clinical drug testing and screening

The traditional drug discovery pipeline for the identification and development of compounds that selectively target specific molecules to ameliorate disease remains a major focus for medical research. However, the zebrafish is increasingly providing alternative strategies for various components of this pipeline. Zebrafish and their embryos are small, easily accessible and relatively low cost, making them applicable to high-throughput, small molecule screening. Zebrafish can also be manipulated by a range of forward and reverse genetics techniques to facilitate gene discovery and functional studies. Moreover, their physiological and developmental complexity provides accurate models of human disease to underpin mechanism of action and in vivo validation studies. Finally, several of these biological characteristics make zebrafish eminently suitable for toxicity testing, including eco-toxicology. Here we review the application of zebrafish to preclinical drug development and toxicity testing, including recent advances in mutant generation, drug screening and toxicology that serve to further enhance the capabilities of this valuable model organism in drug discovery.

Medulloblastoma Down Under 2013 : a report from the third annual meeting of the International Medulloblastoma Working Group.

Medulloblastoma is curable in approximately 70% of patients. Over the past decade, progress in improving survival using conventional therapies has stalled, resulting in reduced quality of life due to treatment-related side effects, which are a major concern in survivors. The vast amount of genomic and molecular data generated over the last 5-10 years encourages optimism that improved risk stratification and new molecular targets will improve outcomes. It is now clear that medulloblastoma is not a single-disease entity, but instead consists of at least four distinct molecular subgroups: WNT/Wingless, Sonic Hedgehog, Group 3, and Group 4. The Medulloblastoma Down Under 2013 meeting, which convened at Bunker Bay, Australia, brought together 50 leading clinicians and scientists. The 2-day agenda included focused sessions on pathology and molecular stratification, genomics and mouse models, high-throughput drug screening, and clinical trial design. The meeting established a global action plan to translate novel biologic insights and drug targeting into treatment regimens to improve outcomes. A consensus was reached in several key areas, with the most important being that a novel classification scheme for medulloblastoma based on the four molecular subgroups, as well as histopathologic features, should be presented for consideration in the upcoming fifth edition of the World Health Organization's classification of tumours of the central nervous system. Three other notable areas of agreement were as follows: (1) to establish a central repository of annotated mouse models that are readily accessible and freely available to the international research community; (2) to institute common eligibility criteria between the Children's Oncology Group and the International Society of Paediatric Oncology Europe and initiate joint or parallel clinical trials; (3) to share preliminary high-throughput screening data across discovery labs to hasten the development of novel therapeutics. Medulloblastoma Down Under 2013 was an effective forum for meaningful discussion, which resulted in enhancing international collaborative clinical and translational research of this rare disease. This template could be applied to other fields to devise global action plans addressing all aspects of a disease, from improved disease classification, treatment stratification, and drug targeting to superior treatment regimens to be assessed in cooperative international clinical trials.

A rapid test for heroin (3,6-Diacetylmorphine) based on two chemiluminescence reactions

A rapid method for screening drug seizure samples for 3,6-diacetylmorphine (heroin), which consists of a simple hydrolysis procedure and flow-injection analysis with two chemiluminescence reagents, is described. Before hydrolysis, 3,6-diacetylmorphine evokes an intense response with a tris(2,2'-bipyridyl)ruthenium(III) reagent (prepared by dissolving the perchlorate salt in acetonitrile), and a relatively weak chemiluminescence response with a second reagent: potassium permanganate in an aqueous acidic polyphosphate solution. However, the permanganate reagent is extremely sensitive toward the hydrolysis products of 3,6-diacetylmorphine (i.e., 6-monoacetylmorphine and morphine). Some compounds commonly found in drug laboratories may cause false positives with tris(2,2'-bipyridyl)ruthenium(III), but do not produce the markedly increased response with the permanganate reagent after the hydrolysis procedure. The combination of these two tests therefore provides an effective presumptive test for the presence of 3,6-diacetylmorphine, which we have verified with 14 samples obtained from a forensic science laboratory.

Apoptosis goes on a chip : advances in the microfluidic analysis of programmed cell death

Recent years have brought enormous progress in cell-based lab-on-a-chip technologies, allowing dynamic studies of cell death with an unprecedented accuracy. As interest in the microfabricated technologies for cell-based bioassays is rapidly gaining momentum, we highlight the most promising technologies that provide a new outlook for the rapid assessment of programmed and accidental cell death and are applicable in drug discovery, high-content drug screening, and personalized clinical diagnostics.

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.

Real-time lane detection on suburban streets using visual cue integration

The detection of lane boundaries on suburban streets using images obtained from video constitutes a challenging task. This is mainly due to the difficulties associated with estimating the complex geometric structure of lane boundaries, the quality of lane markings as a result of wear, occlusions by traffic, and shadows caused by road-side trees and structures. Most of the existing techniques for lane boundary detection employ a single visual cue and will only work under certain conditions and where there are clear lane markings. Also, better results are achieved when there are no other onroad objects present. This paper extends our previous work and discusses a novel lane boundary detection algorithm specifically addressing the abovementioned issues through the integration of two visual cues. The first visual cue is based on stripe-like features found on lane lines extracted using a two-dimensional symmetric Gabor filter. The second visual cue is based on a texture characteristic determined using the entropy measure of the predefined neighbourhood around a lane boundary line. The visual cues are then integrated using a rulebased classifier which incorporates a modified sequential covering algorithm to improve robustness. To separate lane boundary lines from other similar features, a road mask is generated using road chromaticity values estimated from CIE L*a*b* colour transformation. Extraneous points around lane boundary lines are then removed by an outlier removal procedure based on studentized residuals. The lane boundary lines are then modelled with Bezier spline curves. To validate the algorithm, extensive experimental evaluation was carried out on suburban streets and the results are presented. 

Cooperative data scheduling in hybrid vehicular ad hoc networks: VANET as a software defined network

This paper presents the first study on scheduling for cooperative data dissemination in a hybrid infrastructure-to-vehicle (I2V) and vehicle-to-vehicle (V2V) communication environment. We formulate the novel problem of cooperative data scheduling (CDS). Each vehicle informs the road-side unit (RSU) the list of its current neighboring vehicles and the identifiers of the retrieved and newly requested data. The RSU then selects sender and receiver vehicles and corresponding data for V2V communication, while it simultaneously broadcasts a data item to vehicles that are instructed to tune into the I2V channel. The goal is to maximize the number of vehicles that retrieve their requested data. We prove that CDS is NP-hard by constructing a polynomial-time reduction from the Maximum Weighted Independent Set (MWIS) problem. Scheduling decisions are made by transforming CDS to MWIS and using a greedy method to approximately solve MWIS. We build a simulation model based on realistic traffic and communication characteristics and demonstrate the superiority and scalability of the proposed solution. The proposed model and solution, which are based on the centralized scheduler at the RSU, represent the first known vehicular ad hoc network (VANET) implementation of software defined network (SDN) concept.

A screening test for heroin based on sequential injection analysis with dual-reagent chemiluminescence detection

A sequential injection analysis procedure with dual-reagent chemiluminescence detection was applied to the screening of street drug seizure samples for the presence of heroin. The chemiluminescence reagents (acidic potassium permanganate and tris(2,2′-bipyridine)ruthenium(III)) were aspirated from either side of a sample aliquot that was sufficiently large to prevent interdispersion of the reagent zones, and therefore two different chemical reactions could be performed simultaneously at either end of the sample zone. The presence of heroin in seizure samples was indicated by a strong response with the tris(2,2′-bipyridine)ruthenium(III) reagent and confirmed by a significant increase in the response with the permanganate reagent when the sample was treated with sodium hydroxide to hydrolyse the heroin to morphine. Nicomorphine (a morphine-derived pharmaceutical) was synthesised and tested under the same conditions. The responses with the permanganate reagent were similar to those for heroin, which supports the proposed chemical basis for the test. However, the responses with tris(2,2′-bipyridine)ruthenium(III) were far lower for nicomorphine than heroin (approximately 5-fold for the samples that had not been hydrolysed).

Implementation and evaluation of brief depression and anxiety screening in clients contacting a drug and alcohol service

Introduction and Aims. High prevalence mental health (HPMH) comorbidity is common in clients seeking alcohol and other drug (AOD) treatment yet can remain undetected. Although research has reported on the introduction of screening into AOD services, little research has reported on the processes surrounding the introduction or evaluated its effectiveness.This study reports on the implementation and evaluation of brief anxiety and depression screening within a specialised, publicly funded AOD service in South-East Victoria.
Design and Methods. Study one examined the implementation of standardised HPMH screening with 114 adult clients (Mean age = 35.49, SD = 9.53; 64% male) telephoning an AOD service over a 5 week period. Measures included severity of HPMH problems,AOD use, care plans and referrals. Study two used semistructured interviews with nine staff/managers to evaluate the effectiveness of screening and its impact on service delivery.
Results. Ninety-four per cent of clients were identified at risk of anxiety or depression. Most care plans incorporated counselling, and concurrent referrals commonly involved a general practitioner. Staff and management found systematic screening increased identification and understanding of comorbid issues and enhanced client interaction but impacted on resource requirements.
Discussion and Conclusions. Most AOD treatment seekers were identified HPMH comorbid and care plans generally included counselling.Adjunctive referrals were more common for severely depressed clients. Screening was effective and enhanced client rapport.Evaluations revealed low confidence in treating HPMH issues in-house.Training may increase worker confidence in managing mental health interventions with subclinical cases, enhancing services’ ability to move towards dual diagnosis capability.

Screening and intervention for mental health problems in alcohol and other drug settings: Can training change practitioner behaviour?

Aims: The comorbidity of substance use and mental health problems poses a significant challenge for alcohol and other drug (AOD) treatment services. In many cases, AOD practitioners do not have experience or training in identifying or managing mental health conditions. Methods: This project examined the implementation of screening and intervention practices for mental health disorders among AOD clients. Training and supervision was provided to 20 AOD practitioners across five sites in four agencies with a focus on enhancing skills in detection of, and intervention for, mental health conditions among their clients. A package developed for this purpose, known as PsyCheck, was used. A random file audit was undertaken to examine changes in detection of mental health conditions. Findings: There were significant improvements in detection after training and supervision, with detection rates almost doubling in this time. Conclusions: Training and supervision using the PsyCheck package appears to have the potential to improve mental health detection and intervention in AOD services. This study shows promise for the implementation of mental health intervention in AOD services.

Wrong side of the road

On its release in 1981, Ned Lander's 'Wrong Side of the Road' won that year's Jury Prize at the Australian Film Institute Awards. Emphasising the film's status as a pre-eminent Australian production, the theorist and critic Sylvia Lawson, writing in 2013, called Wrong Side of the Road 'the best Australian film of 1981 and indeed of many other years'. Progressing at a restrained pace, it breaks 'Hollywood conventions (which most Australian films obediently copy) about what constitutes a proper narrative focus', with the result that the film stands as 'something of an event in Australian cinema'. In this way, Wrong Side of the Road is the 'first narrative feature to take the experience of a contemporary Aboriginal group [of characters] as its theme instead of using them as contrasts or complements to the main action'.

'What if you live on top of a bakery and you like cakes?'- Drug use and harm trajectories before, during and after the emergence of Silk Road

BACKGROUND: Cryptomarkets are digital platforms that use anonymising software (e.g. Tor) and cryptocurrencies (e.g. Bitcoin) to facilitate peer-to-peer (P2P) trade of goods and services. Their emergence has facilitated access to a wide range of high-quality psychoactive substances, according to surveys of users. In this paper, we ask the question 'How does changing access to drugs through cryptomarkets affect the drug use and harm trajectories of their users?'

METHODS: We conducted a digital ethnography spanning 2012-2014, a period that included the seizure of the original Silk Road marketplace and forum by law enforcement. Using encrypted online chat, we interviewed 17 people who reported using Silk Road to purchase illicit drugs. The interviews were in-depth and unstructured, and also involved the use of life history timelines to trace trajectories. Transcripts were analysed thematically using NVivo.

RESULTS: For some, Silk Road facilitated initiation into drug use or a return to drug use after cessation. Typically, participants reported experiencing a glut of drug consumption in their first months using Silk Road, described by one participant as akin to 'kids in a candy store'. There was evidence that very high availability reduced the need for drug hoarding which helped some respondents to moderate use and feel more in control of purchases made online. Cryptomarket access also appeared to affect solitary and social drug users differently. Most participants described using other cryptomarkets after the closure of Silk Road, albeit with less confidence.

CONCLUSION: In the context of high levels of drug access, supply and diversity occurring within a community regulated environment online, the impacts of cryptomarkets upon drug use trajectories are complex, often posing new challenges for self-control, yet not always leading to harmful outcomes. A major policy challenge is how to provide support for harm reduction in these highly volatile settings.

Role of the ATP-binding cassette drug transporters in the intestinal absorption of tanshinone IIB, one of the major active diterpenoids from the root of Salvia miltiorrhiza

There is an increasing use of herbal medicines worldwide, and the extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we investigated the mechanism for the intestinal absorption of tanshinone IIB (TSB), a major constituent of S. miltiorrhiza. The oral bioavailability of TSB was about 3% in rats with less proportional increase in its maximum plasma concentration (C_max) and area under the plasma concentration-time curve (AUC) with increasing dosage. The time to C_max (T_max) was prolonged at higher oral dosage. In a single pass rat intestinal perfusion model, the permeability coefficients (P_app) based on TSB disappearance from the lumen (P_lumen) were 6.2- to 7.2-fold higher (p < 0.01) than those based on drug appearance in mesenteric venous blood (P_blood). The uptake and efflux of TSB in Caco-2 cells were also significantly altered in the presence of an inhibitor for P-glycoprotein (PgP) or for multi-drug resistance associated protein (MRP1/2). TSB transport from the apical (AP) to basolateral (BL) side in Caco-2 monolayers was 3.3- to 5.7-fold lower than that from BL to AP side, but this polarized transport was attenuated by co-incubation of PgP or MRP1/2 inhibitors. The P_app values of TSB in the BL-AP direction were significantly higher in MDCKII cells over-expressing MDR1 or MRP1, but not in cells over-expressing MRP2-5, as compared with the wild-type cells. The plasma AUC_0-24hr in mdr1a and mrp1 gene-deficient mice was 10.2- to 1.7-fold higher than that in the wild-type mice. Furthermore, TSB significantly inhibited the uptake of digoxin and vinblastine in membrane vesicles containing PgP or MRP1. TSB also moderately stimulated PgP ATPase activity. Taken collectively, our findings indicate that TSB is a substrate for PgP and MRP1 and that drug resistance to TSB therapy and drug interactions may occur through PgP and MRP1 modulation.