Analyzing magnetic bead QuantiGene® Plex 2.0 gene expression data in high throughput mode using QGprofiler.

BACKGROUND: The QuantiGene® Plex 2.0 platform (ThermoFisher Scientific) combines bDNA with the Luminex/xMAP magnetic bead capturing technology to assess differential gene expression in a compound exposure setting. This technology allows multiplexing in a single well of a 96 or 384 multi-well plate and can thus be used in high throughput drug discovery mode. Data interpretation follows a three-step normalization/transformation flow in which raw median fluorescent gene signals are transformed to fold change values with the use of proper housekeeping genes and negative controls. Clear instructions on how to assess the data quality and tools to perform this analysis in high throughput mode are, however, currently lacking. RESULTS: In this paper we introduce QGprofiler, an open source R based shiny application. QGprofiler allows for proper QuantiGene® Plex 2.0 assay optimization, choice of housekeeping genes and data pre-processing up to fold change, including appropriate QC metrics. In addition, QGprofiler allows for an Akaike information criterion based dose response fold change model selection and has a built-in tool to detect the cytotoxic potential of compounds evaluated in a high throughput screening campaign. CONCLUSION: QGprofiler is a user friendly, open source available R based shiny application, which is developed to support drug discovery campaigns. In this context, entire compound libraries/series can be tested in dose response against a gene signature of choice in search for new disease relevant chemical entities. QGprofiler is available at: https://qgprofiler.openanalytics.eu/app/QGprofiler.

A genetics-led approach defines the drug target landscape of 30 immune-related traits.

Most candidate drugs currently fail later-stage clinical trials, largely due to poor prediction of efficacy on early target selection1. Drug targets with genetic support are more likely to be therapeutically valid2,3, but the translational use of genome-scale data such as from genome-wide association studies for drug target discovery in complex diseases remains challenging4-6. Here, we show that integration of functional genomic and immune-related annotations, together with knowledge of network connectivity, maximizes the informativeness of genetics for target validation, defining the target prioritization landscape for 30 immune traits at the gene and pathway level. We demonstrate how our genetics-led drug target prioritization approach (the priority index) successfully identifies current therapeutics, predicts activity in high-throughput cellular screens (including L1000, CRISPR, mutagenesis and patient-derived cell assays), enables prioritization of under-explored targets and allows for determination of target-level trait relationships. The priority index is an open-access, scalable system accelerating early-stage drug target selection for immune-mediated disease.

High-Throughput Gene Expression Profiles to Define Drug Similarity and Predict Compound Activity.

By adding biological information, beyond the chemical properties and desired effect of a compound, uncharted compound areas and connections can be explored. In this study, we add transcriptional information for 31K compounds of Janssen's primary screening deck, using the HT L1000 platform and assess (a) the transcriptional connection score for generating compound similarities, (b) machine learning algorithms for generating target activity predictions, and (c) the scaffold hopping potential of the resulting hits. We demonstrate that the transcriptional connection score is best computed from the significant genes only and should be interpreted within its confidence interval for which we provide the stats. These guidelines help to reduce noise, increase reproducibility, and enable the separation of specific and promiscuous compounds. The added value of machine learning is demonstrated for the NR3C1 and HSP90 targets. Support Vector Machine models yielded balanced accuracy values ≥80% when the expression values from DDIT4 & SERPINE1 and TMEM97 & SPR were used to predict the NR3C1 and HSP90 activity, respectively. Combining both models resulted in 22 new and confirmed HSP90-independent NR3C1 inhibitors, providing two scaffolds (i.e., pyrimidine and pyrazolo-pyrimidine), which could potentially be of interest in the treatment of depression (i.e., inhibiting the glucocorticoid receptor (i.e., NR3C1), while leaving its chaperone, HSP90, unaffected). As such, the initial hit rate increased by a factor 300, as less, but more specific chemistry could be screened, based on the upfront computed activity predictions.

Transcriptional Characterization of Compounds: Lessons Learned from the Public LINCS Data.

The NIH-funded LINCS program has been initiated to generate a library of integrated, network-based, cellular signatures (LINCS). A novel high-throughput gene-expression profiling assay known as L1000 was the main technology used to generate more than a million transcriptional profiles. The profiles are based on the treatment of 14 cell lines with one of many perturbation agents of interest at a single concentration for 6 and 24 hours duration. In this study, we focus on the chemical compound treatments within the LINCS data set. The experimental variables available include number of replicates, cell lines, and time points. Our study reveals that compound characterization based on three cell lines at two time points results in more genes being affected than six cell lines at a single time point. Based on the available LINCS data, we conclude that the most optimal experimental design to characterize a large set of compounds is to test them in duplicate in three different cell lines. Our conclusions are constrained by the fact that the compounds were profiled at a single, relative high concentration, and the longer time point is likely to result in phenotypic rather than mechanistic effects being recorded.

Evaluation of a proximity extension assay for the detection of H1 2009 pandemic influenza viruses.

The rapid influenza diagnostic tests (RIDTs) are widely distributed, simple to use, but often lack sensitivity as compared to gold standard methods (viral culture and nucleic acid detection technologies). Applying RIDTs outside of epidemic or pandemic infections results in large numbers of false negatives. Hence, a sensitive RIDT that would reduce the number of false negatives would result in an increased clinical value. We evaluated the potential of a proximity extension assay (PEA) for the detection of influenza A H1 viruses. This technology makes use of antibodies to capture the pathogen, followed by molecular detection. Forty-seven nasopharyngeal swab samples, all confirmed infections of the H1 2009 pandemic influenza virus, were evaluated. The performance of PEA was compared to the RIDT Quickvue Influenza A+B assay. The success rate of the comparative assays was modeled by means of a binary logistic response model. Both assays performed equally well within the current range of viral particles, expressed as log10 copies/ml. When the actual input of viral particles was taken into account, the 95% hitrate of PEA lies within the range of 4.60-7.02 log10 copies/reaction, which is an almost 2 log10 sensitivity improvement over the 95% hitrate of the Quickvue RIDT, ranging from 6.86 to 9.37 log10 copies/reaction. The PEA method holds promise to improve sensitive detection of influenza viruses in clinical samples.

HIV-1 phenotypic reverse transcriptase inhibitor drug resistance test interpretation is not dependent on the subtype of the virus backbone.

To date, the majority of HIV-1 phenotypic resistance testing has been performed with subtype B virus backbones (e.g. HXB2). However, the relevance of using this backbone to determine resistance in non-subtype B HIV-1 viruses still needs to be assessed. From 114 HIV-1 subtype C clinical samples (36 ARV-naïve, 78 ARV-exposed), pol amplicons were produced and analyzed for phenotypic resistance using both a subtype B- and C-backbone in which the pol fragment was deleted. Phenotypic resistance was assessed in resulting recombinant virus stocks (RVS) for a series of antiretroviral drugs (ARV's) and expressed as fold change (FC), yielding 1660 FC comparisons. These Antivirogram® derived FC values were categorized as having resistant or sensitive susceptibility based on biological cut-off values (BCOs). The concordance between resistance calls obtained for the same clinical sample but derived from two different backbones (i.e. B and C) accounted for 86.1% (1429/1660) of the FC comparisons. However, when taking the assay variability into account, 95.8% (1590/1660) of the phenotypic data could be considered as being concordant with respect to their resistance call. No difference in the capacity to detect resistance associated with M184V, K103N and V106M mutations was noted between the two backbones. The following was concluded: (i) A high level of concordance was shown between the two backbone phenotypic resistance profiles; (ii) Assay variability is largely responsible for discordant results (i.e. for FC values close to BCO); (iii) Confidence intervals should be given around the BCO's, when assessing resistance in HIV-1 subtype C; (iv) No systematic resistance under- or overcalling of subtype C amplicons in the B-backbone was observed; (v) Virus backbone subtype sequence variability outside the pol region does not contribute to phenotypic FC values. In conclusion the HXB2 virus backbone remains an acceptable vector for phenotyping HIV-1 subtype C pol amplicons.

Development and performance of conventional HIV-1 phenotyping (Antivirogram®) and genotype-based calculated phenotyping assay (virco®TYPE HIV-1) on protease and reverse transcriptase genes to evaluate drug resistance.

OBJECTIVES: A wide array of monitoring tests is commercially available to gauge HIV-1 disease progression and the overall health status of an HIV-1-infected patient. Viral load tests provide a picture of viral activity, while CD4 cell counts shed light on the immune status and can help physicians to prevent the development of opportunistic infections in patients. On the other hand, genotypic and phenotypic resistance testing and therapeutic drug monitoring help to optimize HIV-1 antiretroviral therapy. Resistance testing is currently recommended within the standard of care guidelines to aid the choice of new drug regimens following treatment failure(s). METHODS: Genotypic testing described here is based on the amplification and sequencing of an HIV-1 protease (PR) and reverse transcriptase (RT) region from a patient sample to identify resistance mutations associated with PR and RT inhibitor resistance. A genotypic test takes a week to perform and the results are reported as a list of detected mutations. The virco®TYPE HIV-1 report uses genotypic data to predict phenotypic susceptibility by linear regression modeling that uses a large correlative database of genotype-phenotype pairs. Phenotypic testing measures the ability of the virus to replicate in the presence of a drug and provides a direct measurement of drug susceptibility in vitro. Since phenotypic analysis is laborious and time consuming (28 days), genotypic resistance testing is currently the standard reference method used for HIV-1 resistance testing. However, a phenotypic test is important when a patient harbors virus with complex genetic patterns, or when the mutational resistance profile for a particular drug is not well-characterized. RESULTS AND CONCLUSIONS: Some of the currently used resistance tests are partially automated enabling laboratories to increase overall efficiency. However, maximum automation and standardization of the process, instruments and software that we have described here can overcome many of the problems encountered with current tests and aims at having a compliant, high-throughput, diagnostic laboratory, which can guarantee sample integrity from sample reception to result reporting. We also describe in detail the development and performance of virco®TYPE HIV-1 (genotype) and Antivirogram® (phenotype) assay on PR and RT genes to evaluate antiretroviral resistance.

HIV-1 nucleotide mixture detection in the virco(®)TYPE HIV-1 genotyping assay: a comparison between Sanger sequencing and 454 pyrosequencing.

HIV-1 Protease (PR) and Reverse Transcriptase (RT) genotyping is well established for the management of antiretroviral (ARV) drug therapy, as it is able to detect gene mutations encoding resistance to ARV compounds or drug classes, that are associated with reduced drug susceptibility (i.e. phenotype). A correct phenotypic interpretation from the derived PR-RT genotype (i.e. virtual phenotype), requires a well characterized geno-phenotype correlative database and appropriate statistical predictive models. The applicability of the virtual phenotype for the patient, will, however, not only depend on the accuracy of the statistical models and the database they rely on, but also depend largely on the sequence information that is provided. Since HIV-1 evolves as a complex of closely related but non-identical viral genomes (i.e. quasispecies) it is crucial that the sequencing method used, is able to characterize most of the genetic mixtures that make up the different quasispecies within a single patient. US regulatory agencies require that developers of HIV-1 genotyping assays, determine and report the HIV-1 mixture detection level of their assay. Hence, the mixture scoring sensitivity of the population-based Sanger sequencing method, along with the defined mixture scoring rules, used to drive the virco(®)TYPE HIV-1 virtual phenotype, was investigated by comparing it to the 454 pyrosequencing technique, which is able to generate the complete viral population sequence. To this end the PR-RT coding sequence of 20 clinical isolates was determined by both sequencing methodologies. The genotyping assay which feeds the virco(®)TYPE HIV-1 virtual phenotype was able to call automatically 97.5% (i.e. 268 mixtures) and 95.3% (i.e. 326 mixtures) of the mixtures that were present between 25 and 75% and between 20 and 80% in the viral population, as detected by 454. From the not called mixtures, all but one did present a mixture sequence in the Sanger DNA chromatograms, however, with a peak surface area for the second peak that was below the threshold setting for automatic mixture calling in the basecaller software (i.e. 25%). Viral loads ranged from 470 to 629,000 copies/mL and exerted no effect on the mixture calling relationship between both sequencing methodologies (R(2)=0.92). In some occasions (i.e. 55 mixtures) the genotyping assay would detect automatically mixtures that were present below 20% in the viral population, when measured by 454. Hence, the mixture scoring sensitivity of the automated high throughput virco(®)TYPE HIV-1 genotyping assay is currently set at 97.5% and 95.3%, for mixtures present at 25 and 20% in the viral population and may identify occasionally mutations that are present at lower frequencies. These findings were not influenced by the viral load of the examined samples.

Effect of metal accumulation on metallothionein level and condition of the periwinkle Littorina littorea along the Scheldt estuary (the Netherlands).

Metal (i.e. Ag, As, Ca, Cd, Co, Cu, Mn, Pb and Zn) and metallothionein (MT) concentrations in the soft tissue of Littorina littorea were measured along the heavily polluted Western Scheldt (WS) and relatively clean Eastern Scheldt (ES) estuary. Along the WS metal and MT levels in periwinkles reflected the known downstream decreasing pollution gradient. Surprisingly in ES animals As, Mn and Zn concentrations decreased from east to west reflecting past pollution. Compared to the WS metal concentrations of ES periwinkles were significantly lower and both estuaries were maximally discriminated from each other based on their Cd soft tissue concentration using a canonical discriminant analysis. Furthermore, no overall difference was found in MT levels among animals from both estuaries. Using previously obtained condition data (i.e. dry/wet weight ratio and lipid content) the relation between soft tissue metal concentration (i.e. Cd, Cu and Zn) and fitness indicators (i.e. MT and condition data) was examined using a canonical correlation analysis. Periwinkles with a high metal load (i.e. Cd and Zn) also had high MT levels but were in a relatively poor condition.

Historical metal pollution in natural gudgeon populations: Inferences from allozyme, microsatellite and condition factor analysis.

This study presents the results of a microsatellite and allozyme analysis on natural populations of the gudgeon (Gobio gobio) located in a pollution gradient of cadmium and zinc. Differences among contaminated and reference populations were observed at 2 allozyme loci, as well as a relationship between the fish condition factor and glucose-6-phosphate dehydrogenase genotypes, the locus that showed the largest difference in allele frequencies. The microsatellite data partly confirmed the differentiation pattern that was revealed by the allozyme survey. Our data further suggest that at least 2 microsatellite loci may be affected by natural selection. We thus illustrate that both microsatellite and allozyme loci do not necessarily behave as selectively neutral markers in polluted populations. Estimates of population differentiation can therefore be significantly different depending on which loci are being studied. Finally, these results are discussed in the light of the conservation unit concept, because microsatellites are often used to assess genetic variation in endangered natural populations and to propose measures for conservation or management.

Comparative assessment of reproductive impairment in the gastropod mollusc Littorina littorea along the Belgian North Sea coast.

In this study we present the results of an intersex survey of Littorina littorea along the Belgian coast. Levels of female intersex and sterility were determined to assess TBT related adverse effects. In addition, we determined the levels of male penis shedding and trematode infestation and investigated the morphology of the shell. Significant differences were found for all these variables which clearly differentiated periwinkles from Zeebrugge (B2) from those at other locations. Intersex index (ISI) values were relatively low (i.e. 0.00-0.39), except at B2 where they ranged up to 3.52, the highest value ever reported in literature. Consequently, female reproductive impairment at B2 was severe. Indeed, up to 95% of female periwinkles were sterile at B2. In addition, 61% of the male periwinkles had shed their penis. Furthermore, no trematode infestation could be detected at B2 and specimens from this location had the largest and heaviest shells, which may be related to population demography and/or a different use of energy budgets.

Effects of environmental stress on the condition of Littorina littorea along the Scheldt estuary (The Netherlands).

The condition of the periwinkle Littorina littorea, expressed in terms of its shell morphology, reproductive impairment (i.e. female sterility/intersex, male penis shedding), trematode infestation load, lipid reserves and dry/wet weight ratio, was determined in function of environmental stress along the polluted Western and relatively clean Eastern Scheldt estuary (The Netherlands). The upstream increasing pollution and decreasing salinity levels along the Western Scheldt estuary (Fig. 1) are reflected in the dry/wet weight ratio and lipid content of the periwinkles. Compared to the Eastern Scheldt, female intersex (i.e. indicator of TBT pollution) and sterility occurred more frequently in the Western Scheldt estuary, while male penis shedding was even restricted to the latter estuary. The highest population intersex and sterility incidence was found near the harbour of Vlissingen and reflects potential nautical activities. The number of trematode infested periwinkles did not differ between both estuaries, although local sampling site differences were detected within each estuary, reflecting the complex interactions that exist among parasites, hosts and the local environment. Finally, both estuaries were maximally discriminated from each other based on the shell weight of the periwinkles using a canonical discriminant analysis. Periwinkles with the heaviest shells were found in the Western Scheldt estuary and may reflect growth rate or structural population differences caused by the less favourable living conditions in the Western Scheldt estuary.

Genetic variation in two land snails, Cepaea nemoralis and Succinea putris (Gastropoda, Pulmonata), from sites differing in heavy metal content.

Allozyme variation was determined in two land snail species (Cepaea nemoralis and Succinea putris) from four localities in northern Belgium. In each locality we selected a polluted and a nearby, less-polluted, reference plot. We examined whether (i) genetic variability differed between the polluted and reference plots, (ii) populations from polluted plots experienced recent bottlenecks, and (iii) certain allele or genotype frequencies were associated with the pollution. Our results suggest that (i) about 13% of the genetic differentiation in C. nemoralis and 5% in S. putris was due to differences among polluted and reference plots, (ii) polluted and reference plots had comparable levels of genetic variation, but in C. nemoralis observed heterozygosities were higher in polluted plots, (iii) most plots showed significant evidence for recent bottlenecks, irrespective of the degree of pollution, so that bottlenecks seem poor indicators of pollution-induced stress in land snails, and (iv) mutagenic or pollution-induced modifications did not seem to account for new allozyme variants in polluted sites. The observed patterns of genetic variation may be explained by the action of genetic drift, pollution-mediated selection, restricted gene flow, or a combination of these processes.

Genotoxicity in wood mice (Apodemus sylvaticus) along a pollution gradient: exposure-, age-, and gender-related effects.

We investigated the effects of environmental pollution on genetic damage in wood mice (Apodemus sylvaticus) by means of the comet assay, with special attention to the role of age and gender as potential confounding variables. The present study was carried out at four sites along a pollution gradient in the vicinity of Antwerp (Belgium), with a nonferrous smelter as the main pollution source. We measured the concentration of heavy metals (Cd, Co, Cr, Cu, Fe, Mn, Pb, and Zn) in mouse liver and kidney and the concentration of organochlorine compounds (polychlorinated biphenyls and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) in mouse muscle tissue to assess individual exposure. Cadmium exposure was very high at the sites closest to the smelter, and exposure to this metal decreased with increasing distance from the smelter. Exposure to the other pollutants was low to moderate at the different sites. Genetic damage was higher in mice from populations in the vicinity of the nonferrous smelter compared with that in the control populations. A significant increase in genetic damage with age was observed at the most polluted sites, but not at the control sites. Genetic damage was higher in male mice than in female mice at the most polluted site, but not at the other areas. Yet, no obvious relationship was found between individual pollutant levels and individual genetic damage levels. We conclude that the comet assay can be used to compare genotoxicity at the population level if the confounding variables of gender and age are taken into account. However, its use for individual health risk assessment remains questionable.

Associations between shell strength, shell morphology and heavy metals in the land snail Cepaea nemoralis (Gastropoda, Helicidae).

In snails there is an intimate relation between shell size, thickness, strength and calcium content that may be influenced by environmental factors such as predation and heavy metal pollution. The snail Cepaea nemoralis shows variability for shell colour and banding pattern, and frequencies of colour morphs are highly variable in natural populations. We used C. nemoralis to investigate (i) the relations between shell morphology, shell Ca and heavy metal content (Cd, Cr, Pb, Zn), and shell strength, (ii) differences in shell morphology and shell strength among localities and yellow and pink shells and (iii) whether snails from polluted sites show increased levels of heavy metals in their shell. Larger shells were heavier, thicker, needed a higher force to be crushed but did not have a higher Ca concentration. Cd and Zn concentrations were higher in shells from polluted plots compared to shells from unpolluted plots but Ca levels in the shell were comparable among plots. Zn concentration was negatively correlated with shell traits. Although there was substantial variation in shell strength, thickness and dry weight among localities, none of the shell traits differed between individuals from polluted and reference plots nor between colour morphs. Our results suggest that the effect of heavy metal pollution on shell strength and morphology is limited in the investigated populations.

Sensitivity to cadmium along a salinity gradient in populations of the periwinkle, Littorina littorea, using time-to-death analysis.

In this study, we assessed the combined effect of Cd concentration and salinity, on Cd uptake and mortality rate of Littorina littorea, collected along a salinity and pollution gradient in the Western Scheldt estuary (The Netherlands). Animals kept at their field salinity levels were exposed to three Cd concentrations (i.e. 10, 40 and 320 microM), while animals kept in 10 microM of Cd were subjected to five salinity treatments (i.e. 15, 20, 25, 30 and 35 per thousand). Mortality was recorded every 24h and Cd body burdens were measured with ICP-AES. Time-to-death data were analysed via Cox proportional hazard models, including the co-variates "site-Cd treatment" in the Cd experiment and "site-salinity treatment" in the salinity experiment. "Cd-treatment" and "field-salinity" affected mortality rates significantly in the Cd experiment, such that the mortality risk increased by 2.3 times when salinity was lowered from 35 to 15 per thousand, while it decreased by 19.7 times when Cd dropped from 320 to 10 microM. "Site" did not significantly affect the mortality risk in the salinity experiment but affected time-to-death via its interaction with the "salinity-treatment". Generally, mortality did not occur at a given threshold Cd tissue level, but changed over time and treatments, in function of the site. The results demonstrate the importance of the animals' environmental history and illustrate the usefulness of time-to-death analyses in ecotoxicological experiments.

The population genetic structure of Littorina littorea (Mollusca: Gastropoda) along a pollution gradient in the Scheldt estuary (The Netherlands) using RAPD analysis.

The population genetic structure of the periwinkle Littorina littorea was analysed using random amplified polymorphic DNA (RAPD). Three primers, coding for six putative polymorphic loci were surveyed to infer the genetic structure of seven populations located along the heavily polluted Western (i.e. in order of decreasing pollution load W1, W2, W3 and R1) and the relatively clean Eastern Scheldt (E1, E2 and E3) estuary (The Netherlands). A genetic distance based UPGMA (Unweighted pair group method with arithmetic mean) dendrogram revealed an estuary-related structuring, as Eastern and Western Scheldt sites formed two separate clusters. The Western Scheldt cluster was, however, much more heterogeneous, with three RAPD loci revealing a significant genetic heterogeneity compared to none when the Eastern Scheldt sites were compared. Overall mean heterozygosity levels were high, but did not reveal a difference between the estuaries. The current data (1) confirm the patterns of variation previously observed with electrophoretic analyses of esterases and (2) strongly support that these patterns of variation have a genetic basis, in the presence of intense gene flow. In addition, it is suggested that selection, rather than bottleneck effects, induced by the less favourable living conditions at W1, W2 and W3 are responsible for the genetic patterning.

The use of RAPD in ecotoxicology.

Toxic compounds may interfere with the genetic constitution of populations, either directly through mutagenic activity, or indirectly via population-mediated processes (i.e. selection, bottleneck). These processes are initiated when toxic compounds reduce the survival and/or fecundity of exposed organisms, either through the accumulation of unfavorable mutations or when they adversely affect the physiology of an organism and/or the environment in which it has to survive. In this review, we describe how the RAPD technique can be applied in an ecotoxicological context, providing information on all direct and indirect routes through which toxicants may affect the genetic structure of populations. Based on RAPD band intensity, gain/loss and band numbers, three major types of RAPD fingerprint analyses are discussed, yielding diagnostic, phenetic and genetic information. Ecotoxicological literature examples demonstrate that, under strictly standardized conditions, the RAPD technique can be a useful tool to preliminary assess toxicological population genetic effects, particularly since this technique is relatively inexpensive and yields information on a large number of loci without having to obtain sequence data for primer design. However, currently only a small fraction of its potential is used in ecotoxicology. Statistical tools and parameters, as used in other RAPD studies, should be applied in ecotoxicological research as well in order to fully exploit the potential of this technique. Finally, due to their random nature, RAPD data often must be considered as preliminary until they are further documented by cloning, sequencing and probing techniques.