In vitro toxicology evaluation of pharmaceuticals using Raman micro-spectroscopy

Raman micro-spectroscopy combined with multivariate analysis was employed to monitor real-time biochemical changes induced in living cells in vitro following exposure to a pharmaceutical. The cancer drug etoposide (topoisomerase II inhibitor) was used to induce double-strand DNA breaks in human type II pneumocyte-like cells (A549 cell-line). Raman spectra of A549 cells exposed to 100 microM etoposide were collected and classical least squares (CLS) analysis used to determine the relative concentrations of the main cellular components. It was found that the concentrations of DNA and RNA significantly (P < 0.05) decreased, whilst the concentration of lipids significantly (P < 0.05) increased with increasing etoposide exposure time as compared to control untreated A549 cells. The concentration of DNA decreased by 27.5 and 87.0% after 24 and 48 h exposure to etoposide respectively. Principal components analysis (PCA) successfully discriminated between treated and untreated cells, with the main variance between treatment groups attributed to changes in DNA and lipid. DNA fragmentation was confirmed by Western blot analysis of apoptosis regulator protein p53 and cell metabolic activity determined by MTT assay. The over-expression of p53 protein in the etoposide treated cells indicated a significant level of DNA fragmentation and apoptosis. MTT tests confirmed that cellular metabolic activity decreased following exposure to etoposide by 29.4 and 61.2% after 24 and 48 h, respectively. Raman micro-spectroscopy may find applications in the toxicology screening of other drugs, chemicals and new biomaterials, with a range of cell types.     

Simulation and estimation of gene number in a biological pathway using almost complete saturation mutagenesis screening of haploid mouse cells

Background

Genome-wide saturation mutagenesis and subsequent phenotype-driven screening has been central to a comprehensive understanding of complex biological processes in classical model organisms such as flies, nematodes, and plants. The degree of “saturation” (i.e., the fraction of possible target genes identified) has been shown to be a critical parameter in determining all relevant genes involved in a biological function, without prior knowledge of their products. In mammalian model systems, however, the relatively large scale and labor intensity of experiments have hampered the achievement of actual saturation mutagenesis, especially for recessive traits that require biallelic mutations to manifest detectable phenotypes.

Results

By exploiting the recently established haploid mouse embryonic stem cells (ESCs), we present an implementation of almost complete saturation mutagenesis in a mammalian system. The haploid ESCs were mutagenized with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and processed for the screening of mutants defective in various steps of the glycosylphosphatidylinositol-anchor biosynthetic pathway. The resulting 114 independent mutant clones were characterized by a functional complementation assay, and were shown to be defective in any of 20 genes among all 22 known genes essential for this well-characterized pathway. Ten mutants were further validated by whole-exome sequencing. The predominant generation of single-nucleotide substitutions by ENU resulted in a gene mutation rate proportional to the length of the coding sequence, which facilitated the experimental design of saturation mutagenesis screening with the aid of computational simulation.

Conclusions

Our study enables mammalian saturation mutagenesis to become a realistic proposition. Computational simulation, combined with a pilot mutagenesis experiment, could serve as a tool for the estimation of the number of genes essential for biological processes such as drug target pathways when a positive selection of mutants is available.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1016) contains supplementary material, which is available to authorized users.     

Knowledge about Cervical Cancer and Barriers of Screening Program among Women in Wufeng County,a High-Incidence Region of Cervical Cancer in China

Purpose

Cervical cancer screening is an effective method for reducing the incidence and mortality of cervical cancer, but the screening attendance rate in developing countries is far from satisfactory, especially in rural areas. Wufeng is a region of high cervical cancer incidence in China. This study aimed to investigate the issues that concern cervical cancer and screening and the factors that affect women’s willingness to undergo cervical cancer screening in the Wufeng area.

Participants and Methods

A cross-sectional survey of women was conducted to determine their knowledge about cervical cancer and screening, demographic characteristics and the barriers to screening.

Results

Women who were willing to undergo screenings had higher knowledge levels. “Anxious feeling once the disease was diagnosed” (47.6%), “No symptoms/discomfort” (34.1%) and “Do not know the benefits of cervical cancer screening” (13.4%) were the top three reasons for refusing cervical cancer screening. Women who were younger than 45 years old or who had lower incomes, positive family histories of cancer, secondary or higher levels of education, higher levels of knowledge and fewer barriers to screening were more willing to participate in cervical cancer screenings than women without these characteristics.

Conclusion

Efforts are needed to increase women’s knowledge about cervical cancer, especially the screening methods, and to improve their perceptions of the screening process for early detection to reduce cervical cancer incidence and mortality rates.     

A phenome-guided drug repositioning through a latent variable model

Background

The phenome represents a distinct set of information in the human population. It has been explored particularly in its relationship with the genome to identify correlations for diseases. The phenome has been also explored for drug repositioning with efforts focusing on the search space for the most similar candidate drugs. For a comprehensive analysis of the phenome, we assumed that all phenotypes (indications and side effects) were inter-connected with a probabilistic distribution and this characteristic may offer an opportunity to identify new therapeutic indications for a given drug. Correspondingly, we employed Latent Dirichlet Allocation (LDA), which introduces latent variables (topics) to govern the phenome distribution.

Results

We developed our model on the phenome information in Side Effect Resource (SIDER). We first developed a LDA model optimized based on its recovery potential through perturbing the drug-phenotype matrix for each of the drug-indication pairs where each drug-indication relationship was switched to “unknown” one at the time and then recovered based on the remaining drug-phenotype pairs. Of the probabilistically significant pairs, 70% was successfully recovered. Next, we applied the model on the whole phenome to narrow down repositioning candidates and suggest alternative indications. We were able to retrieve approved indications of 6 drugs whose indications were not listed in SIDER. For 908 drugs that were present with their indication information, our model suggested alternative treatment options for further investigations. Several of the suggested new uses can be supported with information from the scientific literature.

Conclusions

The results demonstrated that the phenome can be further analyzed by a generative model, which can discover probabilistic associations between drugs and therapeutic uses. In this regard, LDA serves as an enrichment tool to explore new uses of existing drugs by narrowing down the search space.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-267) contains supplementary material, which is available to authorized users.     

Development and Validation of a Luminescence-based,Medium-Throughput Assay for Drug Screening in Schistosoma mansoni

Background

Schistosomiasis, one of the world’s greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs.

Methodology/Principal Findings

The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery.

Conclusions

The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies.     

Simple Field Assays to Check Quality of Current Artemisinin-Based Antimalarial Combination Formulations

Introduction

Malaria continues to be one of the major public health problems in Africa, Asia and Latin America. Artemisinin derivatives (ARTs; artesunate, artemether, and dihydroartemisinin) derived from the herb, Artemisia annua, are the most effective antimalarial drugs available providing rapid cures. The World Health Organisation (WHO) has recommended that all antimalarials must be combined with an artemisinin component (artemisinin-based combination therapy; ACT) for use as first line treatment against malaria. This class of drugs is now first-line policy in most malaria-endemic countries. Reports of ad hoc surveys from South East Asia show that up to 50% of the artesunate currently sold is counterfeit. Drug quality is rarely assessed in resource poor countries in part due to lack of dedicated laboratory facilities which are expensive to build, equip and maintain. With a view to address this unmet need we developed two novel colour reaction assays that can be used in the field to check the quality of ARTs.

Methods and Findings

Our assays utilise thin layer chromatography silica gel sheets and 2, 4 dinitrophenylhydrazine or 4-Benzoylamino-2, 5-dimethoxybenzenediazonium chloride hemi (zinc chloride) salt as the reagents showing a pink or blue product respectively only in the presence ARTs. We are able to detect as low as 10% of ARTs in ACTs (WINTHROP - artesunate/amodiaquine, Coartem^®-artemether/lumefantrine and Duocortexcin - dihydroartemisinin/piperaquine). The assays have been validated extensively by testing eighty readily accessible and widely used drugs in malaria endemic countries. None of the other antimalarial drugs or a range of commonly used excipients, antiretroviral drugs or other frequently used drugs from the WHO essential drugs list such as analgesics or antibiotics are detected with our assays.

Conclusions

Our two independent assays requiring no specialist training are specific, simple to use, rapid, robust, reproducible, inexpensive and, have successfully resulted in detecting two counterfeit drugs within a small scale screening survey of over 100 declared artemisinin-containing drugs collected from various Asian and African countries. These promising results indicate that the assays will provide a useful first test to assure the quality of the ACTs formulations in resource poor malaria endemic areas when there is an absence of dedicated medicines quality laboratory facilities.     

Weighted correlation network analysis (WGCNA) applied to the tomato fruit metabolome

Background

Advances in “omics” technologies have revolutionized the collection of biological data. A matching revolution in our understanding of biological systems, however, will only be realized when similar advances are made in informatic analysis of the resulting “big data.” Here, we compare the capabilities of three conventional and novel statistical approaches to summarize and decipher the tomato metabolome.

Methodology

Principal component analysis (PCA), batch learning self-organizing maps (BL-SOM) and weighted gene co-expression network analysis (WGCNA) were applied to a multivariate NMR dataset collected from developmentally staged tomato fruits belonging to several genotypes. While PCA and BL-SOM are appropriate and commonly used methods, WGCNA holds several advantages in the analysis of highly multivariate, complex data.

Conclusions

PCA separated the two major genetic backgrounds (AC and NC), but provided little further information. Both BL-SOM and WGCNA clustered metabolites by expression, but WGCNA additionally defined “modules” of co-expressed metabolites explicitly and provided additional network statistics that described the systems properties of the tomato metabolic network. Our first application of WGCNA to tomato metabolomics data identified three major modules of metabolites that were associated with ripening-related traits and genetic background.     

Predicted protein-protein interactions in the moss Physcomitrella patens: a new bioinformatic resource

Background

Physcomitrella patens, a haploid dominant plant, is fast becoming a useful molecular genetics and bioinformatics tool due to its key phylogenetic position as a bryophyte in the post-genomic era. Genome sequences from select reference species were compared bioinformatically to Physcomitrella patens using reciprocal blasts with the InParanoid software package. A reference protein interaction database assembled using MySQL by compiling BioGrid, BIND, DIP, and Intact databases was queried for moss orthologs existing for both interacting partners. This method has been used to successfully predict interactions for a number of angiosperm plants.

Results

The first predicted protein-protein interactome for a bryophyte based on the interolog method contains 67,740 unique interactions from 5,695 different Physcomitrella patens proteins. Most conserved interactions among proteins were those associated with metabolic processes. Over-represented Gene Ontology categories are reported here.

Conclusion

Addition of moss, a plant representative 200 million years diverged from angiosperms to interactomic research greatly expands the possibility of conducting comparative analyses giving tremendous insight into network evolution of land plants. This work helps demonstrate the utility of “guilt-by-association” models for predicting protein interactions, providing provisional roadmaps that can be explored using experimental approaches. Included with this dataset is a method for characterizing subnetworks and investigating specific processes, such as the Calvin-Benson-Bassham cycle.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0524-1) contains supplementary material, which is available to authorized users.     

Efficacy of a Contact Lens Sensor for Monitoring 24-H Intraocular Pressure Related Patterns

Purpose

To study performance of a contact lens sensor (CLS) for 24-hour monitoring of IOP-related short-term patterns and compare with IOP obtained by pneumatonometry.

Methods

Prospective clinical trial. Thirty-one healthy volunteers and 2 glaucoma patients were housed for 24 hours in a sleep laboratory. One randomly selected eye was fitted with a CLS (Triggerfish, Sensimed, Switzerland), which measures changes in ocular circumference. In the contralateral eye, IOP measurements were taken using a pneumatonometer every two hours with subjects in the habitual body positions. Heart rate (HR) was measured 3 times during the night for periods of 6 minutes separated by 2 hours. Performance of CLS was defined in two ways: 1) recording the known pattern of IOP increase going from awake (sitting position) to sleep (recumbent), defined as the wake/sleep (W/S) slope and 2) accuracy of the ocular pulse frequency (OPF) concurrent to that of the HR interval. Strength of association between overall CLS and pneumatonometer curves was assessed using coefficients of determination (R²).

Results

The W/S slope was statistically significantly positive in both eyes of each subject (CLS, 57.0 ± 40.5 mVeq/h, p<0.001 and 1.6 ± 0.9 mmHg/h, p<0.05 in the contralateral eye). In all, 87 CLS plots concurrent to the HR interval were evaluated. Graders agreed on evaluability for OPF in 83.9% of CLS plots. Accuracy of the CLS to detect the OPF was 86.5%. Coefficient of correlation between CLS and pneumatonometer for the mean 24-h curve was R² = 0.914.

Conclusions

CLS measurements compare well to the pneumatonometer and may be of practical use for detection of sleep-induced IOP changes. The CLS also is able to detect ocular pulsations with good accuracy in a majority of eyes.

Trial Registration

ClinicalTrials.gov NCT01390779     

A simple peak detection and label-free quantitation algorithm for chromatography-mass spectrometry

Background

Label-free quantitation of mass spectrometric data is one of the simplest and least expensive methods for differential expression profiling of proteins and metabolites. The need for high accuracy and performance computational label-free quantitation methods is still high in the biomarker and drug discovery research field. However, recent most advanced types of LC-MS generate huge amounts of analytical data with high scan speed, high accuracy and resolution, which is often impossible to interpret manually. Moreover, there are still issues to be improved for recent label-free methods, such as how to reduce false positive/negatives of the candidate peaks, how to expand scalability and how to enhance and automate data processing. AB3D (A simple label-free quantitation algorithm for Biomarker Discovery in Diagnostics and Drug discovery using LC-MS) has addressed these issues and has the capability to perform label-free quantitation using MS1 for proteomics study.

Results

We developed an algorithm called AB3D, a label free peak detection and quantitative algorithm using MS1 spectral data. To test our algorithm, practical applications of AB3D for LC-MS data sets were evaluated using 3 datasets. Comparisons were then carried out between widely used software tools such as MZmine 2, MSight, SuperHirn, OpenMS and our algorithm AB3D, using the same LC-MS datasets. All quantitative results were confirmed manually, and we found that AB3D could properly identify and quantify known peptides with fewer false positives and false negatives compared to four other existing software tools using either the standard peptide mixture or the real complex biological samples of Bartonella quintana (strain JK31). Moreover, AB3D showed the best reliability by comparing the variability between two technical replicates using a complex peptide mixture of HeLa and BSA samples. For performance, the AB3D algorithm is about 1.2 - 15 times faster than the four other existing software tools.

Conclusions

AB3D is a simple and fast algorithm for label-free quantitation using MS1 mass spectrometry data for large scale LC-MS data analysis with higher true positive and reasonable false positive rates. Furthermore, AB3D demonstrated the best reproducibility and is about 1.2- 15 times faster than those of existing 4 software tools.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0376-0) contains supplementary material, which is available to authorized users.     

Laboratory evaluation of twelve portable devices for medicine quality screening

BackgroundPost-market surveillance is a key regulatory function to prevent substandard and falsified (SF) medicines from being consumed by patients. Field deployable technologies offer the potential for rapid objective screening for SF medicines.Methods and findingsWe evaluated twelve devices: three near infrared spectrometers (MicroPHAZIR RX, NIR-S-G1, Neospectra 2.5), two Raman spectrometers (Progeny, TruScan RM), one mid-infrared spectrometer (4500a), one disposable colorimetric assay (Paper Analytical Devices, PAD), one disposable immunoassay (Rapid Diagnostic Test, RDT), one portable liquid chromatograph (C-Vue), one microfluidic system (PharmaChk), one mass spectrometer (QDa), and one thin layer chromatography kit (GPHF-Minilab). Each device was tested with a series of field collected medicines (FCM) along with simulated medicines (SIM) formulated in a laboratory. The FCM and SIM ranged from samples with good quality active pharmaceutical ingredient (API) concentrations, reduced concentrations of API (80% and 50% of the API), no API, and the wrong API. All the devices had high sensitivities (91.5 to 100.0%) detecting medicines with no API or the wrong API. However, the sensitivities of each device towards samples with 50% and 80% API varied greatly, from 0% to 100%. The infrared and Raman spectrometers had variable sensitivities for detecting samples with 50% and 80% API (from 5.6% to 50.0%). The devices with the ability to quantitate API (C-Vue, PharmaChk, QDa) had sensitivities ranging from 91.7% to 100% to detect all poor quality samples. The specificity was lower for the quantitative C-Vue, PharmaChk, & QDa (50.0% to 91.7%) than for all the other devices in this study (95.5% to 100%).ConclusionsThe twelve devices evaluated could detect medicines with the wrong or none of the APIs, consistent with falsified medicines, with high accuracy. However, API quantitation to detect formulations similar to those commonly found in substandards proved more difficult, requiring further technological innovation.     

Detection of copy number variations and their effects in Chinese bulls

Background

Copy number variations (CNVs) are a main source of genomic structural variations underlying animal evolution and production traits. Here, with one pure-blooded Angus bull as reference, we describe a genome-wide analysis of CNVs based on comparative genomic hybridization arrays in 29 Chinese domesticated bulls and examined their effects on gene expression and cattle growth traits.

Results

We identified 486 copy number variable regions (CNVRs), covering 2.45% of the bovine genome, in 24 taurine (Bos taurus), together with 161 ones in 2 yaks (Bos grunniens) and 163 ones in 3 buffaloes (Bubalus bubalis). Totally, we discovered 605 integrated CNVRs, with more “loss” events than both “gain” and “both” ones, and clearly clustered them into three cattle groups. Interestingly, we confirmed their uneven distributions across chromosomes, and the differences of mitochondrion DNA copy number (gain: taurine, loss: yak & buffalo). Furthermore, we confirmed approximately 41.8% (253/605) and 70.6% (427/605) CNVRs span cattle genes and quantitative trait loci (QTLs), respectively. Finally, we confirmed 6 CNVRs in 9 chosen ones by using quantitative PCR, and further demonstrated that CNVR22 had significantly negative effects on expression of PLA2G2D gene, and both CNVR22 and CNVR310 were associated with body measurements in Chinese cattle, suggesting their key effects on gene expression and cattle traits.

Conclusions

The results advanced our understanding of CNV as an important genomic structural variation in taurine, yak and buffalo. This study provides a highly valuable resource for Chinese cattle’s evolution and breeding researches.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-480) contains supplementary material, which is available to authorized users.     

Distance-based assessment of the localization of functional annotations in 3D genome reconstructions

Background

Recent studies used the contact data or three-dimensional (3D) genome reconstructions from Hi-C (chromosome conformation capture with next-generation sequencing) to assess the co-localization of functional genomic annotations in the nucleus. These analyses dichotomized data point pairs belonging to a functional annotation as “close” or “far” based on some threshold and then tested for enrichment of “close” pairs. We propose an alternative approach that avoids dichotomization of the data and instead directly estimates the significance of distances within the 3D reconstruction.

Results

We applied this approach to 3D genome reconstructions for Plasmodium falciparum, the causative agent of malaria, and Saccharomyces cerevisiae and compared the results to previous approaches. We found significant 3D co-localization of centromeres, telomeres, virulence genes, and several sets of genes with developmentally regulated expression in P. falciparum; and significant 3D co-localization of centromeres and long terminal repeats in S. cerevisiae. Additionally, we tested the experimental observation that telomeres form three to seven clusters in P. falciparum and S. cerevisiae. Applying affinity propagation clustering to telomere coordinates in the 3D reconstructions yielded six telomere clusters for both organisms.

Conclusions

Distance-based assessment replicated key findings, while avoiding dichotomization of the data (which previously yielded threshold-sensitive results).

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-992) contains supplementary material, which is available to authorized users.     

Stakeholders’ views on the ethical challenges of pragmatic trials investigating pharmaceutical drugs

BackgroundWe explored the views of key stakeholders to identify the ethical challenges of pragmatic trials investigating pharmaceutical drugs. A secondary aim was to capture stakeholders’ attitudes towards the implementation of pragmatic trials in the drug development process.MethodsWe conducted semistructured, in-depth interviews among individuals from different key stakeholder groups (academia and independent research institutions, the pharmaceutical industry, regulators, Health Technology Assessment (HTA) agencies and patients’ organizations) through telephone or face-to-face sessions. Interviews were structured around the question “what challenges were experienced or perceived during the design, conduct and/or review of pragmatic trials.” Respondents were additionally asked about their views on implementation of pragmatic trials in the drug development process. Thematic analysis was used to identify the ethically relevant features across data sets.ResultsWe interviewed 34 stakeholders in 25 individual sessions and four group sessions. The four perceived challenges of ethical relevance were: (1) less controlled conditions creating safety concerns, (2) comparison with usual care potentially compromising clinical equipoise, (3) tailored or waivers of informed consent affecting patient autonomy, and (4) minimal interference with “real-world” practice reducing the knowledge value of trial results.ConclusionsWe identified stakeholder concerns regarding risk assessment, use of suboptimal usual care as a comparator, tailoring of informed consent procedures and ensuring the social value of pragmatic trials. These concerns increased when respondents were asked about pragmatic trials conducted before market authorization.

Electronic supplementary material

The online version of this article (doi:10.1186/s13063-016-1546-3) contains supplementary material, which is available to authorized users.     

Routine HIV screening in France: clinical impact and cost-effectiveness

Background

In France, roughly 40,000 HIV-infected persons are unaware of their HIV infection. Although previous studies have evaluated the cost-effectiveness of routine HIV screening in the United States, differences in both the epidemiology of infection and HIV testing behaviors warrant a setting-specific analysis for France.

Methods/Principal Findings

We estimated the life expectancy (LE), cost and cost-effectiveness of alternative HIV screening strategies in the French general population and high-risk sub-populations using a computer model of HIV detection and treatment, coupled with French national clinical and economic data. We compared risk-factor-based HIV testing (“current practice”) to universal routine, voluntary HIV screening in adults aged 18–69. Screening frequencies ranged from once to annually. Input data included mean age (42 years), undiagnosed HIV prevalence (0.10%), annual HIV incidence (0.01%), test acceptance (79%), linkage to care (75%) and cost/test (€43). We performed sensitivity analyses on HIV prevalence and incidence, cost estimates, and the transmission benefits of ART. “Current practice” produced LEs of 242.82 quality-adjusted life months (QALM) among HIV-infected persons and 268.77 QALM in the general population. Adding a one-time HIV screen increased LE by 0.01 QALM in the general population and increased costs by €50/person, for a cost-effectiveness ratio (CER) of €57,400 per quality-adjusted life year (QALY). More frequent screening in the general population increased survival, costs and CERs. Among injection drug users (prevalence 6.17%; incidence 0.17%/year) and in French Guyana (prevalence 0.41%; incidence 0.35%/year), annual screening compared to every five years produced CERs of €51,200 and €46,500/QALY.

Conclusions/Significance

One-time routine HIV screening in France improves survival compared to “current practice” and compares favorably to other screening interventions recommended in Western Europe. In higher-risk groups, more frequent screening is economically justifiable.     

A collaborative epidemiological investigation into the criminal fake artesunate trade in South East Asia

Background

Since 1998 the serious public health problem in South East Asia of counterfeit artesunate, containing no or subtherapeutic amounts of the active antimalarial ingredient, has led to deaths from untreated malaria, reduced confidence in this vital drug, large economic losses for the legitimate manufacturers, and concerns that artemisinin resistance might be engendered.

Methods and Findings

With evidence of a deteriorating situation, a group of police, criminal analysts, chemists, palynologists, and health workers collaborated to determine the source of these counterfeits under the auspices of the International Criminal Police Organization (INTERPOL) and the Western Pacific World Health Organization Regional Office. A total of 391 samples of genuine and counterfeit artesunate collected in Vietnam (75), Cambodia (48), Lao PDR (115), Myanmar (Burma) (137) and the Thai/Myanmar border (16), were available for analysis. Sixteen different fake hologram types were identified. High-performance liquid chromatography and/or mass spectrometry confirmed that all specimens thought to be counterfeit (195/391, 49.9%) on the basis of packaging contained no or small quantities of artesunate (up to 12 mg per tablet as opposed to ∼ 50 mg per genuine tablet). Chemical analysis demonstrated a wide diversity of wrong active ingredients, including banned pharmaceuticals, such as metamizole, and safrole, a carcinogen, and raw material for manufacture of methylenedioxymethamphetamine (‘ecstasy''). Evidence from chemical, mineralogical, biological, and packaging analysis suggested that at least some of the counterfeits were manufactured in southeast People''s Republic of China. This evidence prompted the Chinese Government to act quickly against the criminal traders with arrests and seizures.

Conclusions

An international multi-disciplinary group obtained evidence that some of the counterfeit artesunate was manufactured in China, and this prompted a criminal investigation. International cross-disciplinary collaborations may be appropriate in the investigation of other serious counterfeit medicine public health problems elsewhere, but strengthening of international collaborations and forensic and drug regulatory authority capacity will be required.     

Transcriptome analysis of cattle muscle identifies potential markers for skeletal muscle growth rate and major cell types

Background

This study aimed to identify markers for muscle growth rate and the different cellular contributors to cattle muscle and to link the muscle growth rate markers to specific cell types.

Results

The expression of two groups of genes in the longissimus muscle (LM) of 48 Brahman steers of similar age, significantly enriched for “cell cycle” and “ECM (extracellular matrix) organization” Gene Ontology (GO) terms was correlated with average daily gain/kg liveweight (ADG/kg) of the animals. However, expression of the same genes was only partly related to growth rate across a time course of postnatal LM development in two cattle genotypes, Piedmontese x Hereford (high muscling) and Wagyu x Hereford (high marbling). The deposition of intramuscular fat (IMF) altered the relationship between the expression of these genes and growth rate. K-means clustering across the development time course with a large set of genes (5,596) with similar expression profiles to the ECM genes was undertaken. The locations in the clusters of published markers of different cell types in muscle were identified and used to link clusters of genes to the cell type most likely to be expressing them. Overall correspondence between published cell type expression of markers and predicted major cell types of expression in cattle LM was high. However, some exceptions were identified: expression of SOX8 previously attributed to muscle satellite cells was correlated with angiogenesis. Analysis of the clusters and cell types suggested that the “cell cycle” and “ECM” signals were from the fibro/adipogenic lineage. Significant contributions to these signals from the muscle satellite cells, angiogenic cells and adipocytes themselves were not as strongly supported. Based on the clusters and cell type markers, sets of five genes predicted to be representative of fibro/adipogenic precursors (FAPs) and endothelial cells, and/or ECM remodelling and angiogenesis were identified.

Conclusions

Gene sets and gene markers for the analysis of many of the major processes/cell populations contributing to muscle composition and growth have been proposed, enabling a consistent interpretation of gene expression datasets from cattle LM. The same gene sets are likely to be applicable in other cattle muscles and in other species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1403-x) contains supplementary material, which is available to authorized users.     

Human settlement history between Sunda and Sahul: a focus on East Timor (Timor-Leste) and the Pleistocenic mtDNA diversity

Background

Distinct, partly competing, “waves” have been proposed to explain human migration in(to) today’s Island Southeast Asia and Australia based on genetic (and other) evidence. The paucity of high quality and high resolution data has impeded insights so far. In this study, one of the first in a forensic environment, we used the Ion Torrent Personal Genome Machine (PGM) for generating complete mitogenome sequences via stand-alone massively parallel sequencing and describe a standard data validation practice.

Results

In this first representative investigation on the mitochondrial DNA (mtDNA) variation of East Timor (Timor-Leste) population including >300 individuals, we put special emphasis on the reconstruction of the initial settlement, in particular on the previously poorly resolved haplogroup P1, an indigenous lineage of the Southwest Pacific region. Our results suggest a colonization of southern Sahul (Australia) >37 kya, limited subsequent exchange, and a parallel incubation of initial settlers in northern Sahul (New Guinea) followed by westward migrations <28 kya.

Conclusions

The temporal proximity and possible coincidence of these latter dispersals, which encompassed autochthonous haplogroups, with the postulated “later” events of (South) East Asian origin pinpoints a highly dynamic migratory phase.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-014-1201-x) contains supplementary material, which is available to authorized users.     

Characteristics of replication-independent endogenous double-strand breaks in Saccharomyces cerevisiae

Background

Replication-independent endogenous double-strand breaks (RIND-EDSBs) occur in both humans and yeast in the absence of inductive agents and DNA replication. In human cells, RIND-EDSBs are hypermethylated, preferentially retained in the heterochromatin and unbound by γ-H2AX. In single gene deletion yeast strains, the RIND-EDSB levels are altered; the number of RIND-EDSBs is higher in strains with deletions of histone deacetylase, endonucleases, topoisomerase, or DNA repair regulators, but lower in strains with deletions of the high-mobility group box proteins or Sir2. In summary, RIND-EDSBs are different from pathologic DSBs in terms of their causes and consequences. In this study, we identified the nucleotide sequences surrounding RIND-EDSBs and investigated the features of these sequences as well as their break locations.

Results

In recent work, we detected RIND-EDSBs using ligation mediated PCR. In this study, we sequenced RIND-EDSB PCR products of resting state Saccharomyces cerevisiae using next-generation sequencing to analyze RIND-EDSB sequences. We found that the break locations are scattered across a number of chromosomes. The number of breaks correlated with the size of the chromosomes. Most importantly, the break occurrences had sequence pattern specificity. Specifically, the majority of the breaks occurred immediately after the sequence “ACGT” (P = 2.2E-156). Because the “ACGT” sequence does not occur primarily in the yeast genome, this specificity of the “ACGT” sequence cannot be attributed to chance.

Conclusions

RIND-EDSBs occur non-randomly; that is, they are produced and retained by specific mechanisms. Because these particular mechanisms regulate their generation and they possess potentially specific functions, RIND-EDSBs could be epigenetic marks.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-750) contains supplementary material, which is available to authorized users.