An elevated level of interleukin-17A in a Senegalese malaria cohort is associated with rs8193038 IL-17A genetic variant.

Malaria infection is a multifactorial disease partly modulated by host immuno-genetic factors. Recent evidence has demonstrated the importance of Interleukin-17 family proinflammatory cytokines and their genetic variants in host immunity. However, limited knowledge exists about their role in parasitic infections such as malaria. We aimed to investigate IL-17A serum levels in patients with severe and uncomplicated malaria and gene polymorphism's influence on the IL-17A serum levels. In this research, 125 severe (SM) and uncomplicated (UM) malaria patients and 48 free malaria controls were enrolled. IL-17A serum levels were measured with ELISA. PCR and DNA sequencing were used to assess host genetic polymorphisms in IL-17A. We performed a multivariate regression to estimate the impact of human IL-17A variants on IL-17A serum levels and malaria outcomes. Elevated serum IL-17A levels accompanied by increased parasitemia were found in SM patients compared to UM and controls (P < 0.0001). Also, the IL-17A levels were lower in SM patients who were deceased than in those who survived. In addition, the minor allele frequencies (MAF) of two IL-17A polymorphisms (rs3819024 and rs3748067) were more prevalent in SM patients than UM patients, indicating an essential role in SM. Interestingly, the heterozygous rs8193038 AG genotype was significantly associated with higher levels of IL-17A than the homozygous wild type (AA). According to our results, it can be concluded that the IL-17A gene rs8193038 polymorphism significantly affects IL-17A gene expression. Our results fill a gap in the implication of IL-17A gene polymorphisms on the cytokine level in a malaria cohort. IL-17A gene polymorphisms also may influence cytokine production in response to Plasmodium infections and may contribute to the hyperinflammatory responses during severe malaria outcomes.

The CEPH aging cohort and biobank: a valuable collection of biological samples from exceptionally long-lived French individuals and their offspring for longevity studies.

The increasing aging of the human population is currently and for the coming decades a major public health issue in many countries, requiring the implementation of global public health policies promoting healthy and successful aging. Individuals are not equal in the face of aging and some can present exceptional healthspan and/or lifespan, which are notably influenced by both genetic and environmental factors. Research and studies on human aging, healthy aging and longevity should rely in particular on cohorts of long-lived individuals, also including biological samples allowing studies on the biology of aging and longevity. In this manuscript, we provide for the first time a complete description of the CEPH (Centre d'Etude du Polymophisme Humain) Aging cohort, an exceptional cohort recruited during the 90s to 2000s, including more than 1700 French long-lived individuals (≥ 90 years old) born between 1875 and 1916 as well as for some of them their siblings and offspring. Among the participants, 1265 were centenarians, including 255 semi-supercentenarians ([105-110] years old) and 25 supercentenarians (≥ 110 years old). The available anthropometric, epidemiologic and clinical data for the cohort participants are described and especially the collection of blood-derived biological samples associated with the cohort which includes DNA, cryopreserved cells and cell lines, plasma, and serum. This biological collection from the first cohort of centenarians in the world is an inestimable resource for ongoing and future molecular, cellular, and functional studies aimed at deciphering the mechanisms of human (successful) aging and longevity.

The HLA-B*57:01 allele corresponds to a very large MHC haploblock likely explaining its massive effect for HIV-1 elite control.

Introduction: We have reanalyzed the genomic data of the International Collaboration for the Genomics of HIV (ICGH), centering on HIV-1 Elite Controllers. Methods: We performed a genome-wide Association Study comparing 543 HIV Elite Controllers with 3,272 uninfected controls of European descent. Using the latest database for imputation, we analyzed 35,552 Single Nucleotide Polymorphisms (SNPs) within the Major Histocompatibility Complex (MHC) region. Results: Our analysis identified 2,626 SNPs significantly associated (p<5. 10-8) with elite control of HIV-1 infection, including well-established MHC signals such as the rs2395029-G allele which tags HLA-B*57:01. A thorough investigation of SNPs in linkage disequilibrium with rs2395029 revealed an extensive haploblock spanning 1.9 megabases in the MHC region tagging HLA-B*57:01, comprising 379 SNP alleles impacting 72 genes. This haploblock contains damaging variations in proteins like NOTCH4 and DXO and is also associated with a strong differential pattern of expression of multiple MHC genes such as HLA-B, MICB, and ZBTB12. The study was expanded to include two cohorts of seropositive African-American individuals, where a haploblock tagging the HLA-B*57:03 allele was similarly associated with control of viral load. The mRNA expression profile of this haploblock in African Americans closely mirrored that in the European cohort. Discussion: These findings suggest that additional molecular mechanisms beyond the conventional antigen-presenting role of class I HLA molecules may contribute to the observed influence of HLA-B*57:01/B*57:03 alleles on HIV-1 elite control. Overall, this study has uncovered a large haploblock associated with HLA-B*57 alleles, providing novel insights into their massive effect on HIV-1 elite control.

A human leukocyte antigen imputation study uncovers possible genetic interplay between gut inflammatory processes and autism spectrum disorders.

Autism spectrum disorders (ASD) are neurodevelopmental conditions that are for subsets of individuals, underpinned by dysregulated immune processes, including inflammation, autoimmunity, and dysbiosis. Consequently, the major histocompatibility complex (MHC)-hosted human leukocyte antigen (HLA) has been implicated in ASD risk, although seldom investigated. By utilizing a GWAS performed by the EU-AIMS consortium (LEAP cohort), we compared HLA and MHC genetic variants, single nucleotide polymorphisms (SNP), and haplotypes in ASD individuals, versus typically developing controls. We uncovered six SNPs, namely rs9268528, rs9268542, rs9268556, rs14004, rs9268557, and rs8084 that crossed the Bonferroni threshold, which form the underpinnings of 3 independent genetic pathways/blocks that differentially associate with ASD. Block 1 (rs9268528-G, rs9268542-G, rs9268556-C, and rs14004-A) afforded protection against ASD development, whilst the two remaining blocks, namely rs9268557-T, and rs8084-A, associated with heightened risk. rs8084 and rs14004 mapped to the HLA-DRA gene, whilst the four other SNPs located in the BTNL2 locus. Different combinations amongst BTNL2 SNPs and HLA amino acid variants or classical alleles were found either to afford protection from or contribute to ASD risk, indicating a genetic interplay between BTNL2 and HLA. Interestingly, the detected variants had transcriptional and/or quantitative traits loci implications. As BTNL2 modulates gastrointestinal homeostasis and the identified HLA alleles regulate the gastrointestinal tract in celiac disease, it is proposed that the data on ASD risk may be linked to genetically regulated gut inflammatory processes. These findings might have implications for the prevention and treatment of ASD, via the targeting of gut-related processes.

Gene expression profiling of peripheral blood mononuclear cells from women with cervical lesions reveals new markers of cancer.

Cervical cancer (CC) is a multifactorial disease of which human papillomavirus (HPV) is the main etiological agent. Despite cervical Pap smear screening and anti­HPV vaccination, CC remains a major public health issue. Identification of specific gene expression signatures in the blood could allow better insight into the immune response of CC and could provide valuable information for the development of novel biomarkers. The present study performed a transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) from Senegalese patients with CC (n=31), low­grade cervical intraepithelial neoplasia (CIN1; n=27) and from healthy control (CTR) subjects (n=29). Individuals in the CIN1 and CTR groups exhibited similar patterns in gene expression. A total of 182 genes were revealed to be differentially expressed in patients with CC compared with individuals in the CIN1 and CTR groups. The IL1R2, IL18R1, MMP9 and FKBP5 genes were the most upregulated, whereas the T­cell receptor α gene TRA was the most downregulated in the CC group compared with in the CIN1 and CTR groups. The pathway enrichment analysis of the differentially expressed genes revealed pathways directly and indirectly linked to inflammation. To the best of our knowledge, the present study is the first large transcriptomic study on CC performed using PBMCs from African women; the results revealed the involvement of genes and pathways related to inflammation, most notably the IL­1 pathway, and the involvement of downregulation of the T­cell receptor α, a key component of the immune response. Several of the stated genes have already been reported in other cancer studies as putative blood biomarkers, thus reinforcing the requirement for deeper investigation. These findings may aid in the development of innovative clinical biomarkers for CC prevention and should be further replicated in other populations.

Linear epitope mapping of the humoral response against SARS-CoV-2 in two independent African cohorts.

Profiling of the antibody responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) proteins in African populations is scarce. Here, we performed a detailed IgM and IgG epitope mapping study against 487 peptides covering SARS-CoV-2 wild-type structural proteins. A panel of 41 pre-pandemic and 82 COVID-19 RT-PCR confirmed sera from Madagascar and Senegal were used. We found that the main 36 immunodominant linear epitopes identified were (i) similar in both countries, (ii) distributed mainly in the Spike and the Nucleocapsid proteins, (iii) located outside the RBD and NTD regions where most of the reported SARS-CoV-2 variant mutations occur, and (iv) identical to those reported in European, North American, and Asian studies. Within the severe group, antibody levels were inversely correlated with the viral load. This first antibody epitope mapping study performed in patients from two African countries may be helpful to guide rational peptide-based diagnostic assays or vaccine development.

G6PD and HBB polymorphisms in the Senegalese population: prevalence, correlation with clinical malaria.

Background: Host genetic factors contribute to the variability of malaria phenotypes and can allow a better understanding of mechanisms involved in susceptibility and/or resistance to Plasmodium falciparum infection outcomes. Several genetic polymorphisms were reported to be prevalent among populations living in tropical malaria-endemic regions and induce protection against malaria. The present study aims to investigate the prevalence of HBB (chr11) and G6PD (chrX) deficiencies polymorphisms among Senegalese populations and their associations with the risk for severe Plasmodium falciparum malaria occurrence. Methods: We performed a retrospective study with 437 samples, 323 patients recruited in hospitals located in three different endemic areas where malaria episodes were confirmed and 114 free malaria controls. The patients enrolled were classified into two groups: severe malaria (SM) (153 patients) and uncomplicated malaria (UM) (170 patients). PCR and DNA sequencing assessed host genetic polymorphisms in HBB and G6PD. Using a multivariate regression and additive model, estimates of the impact of human HBB and G6PD polymorphisms on malaria incidence were performed. Results: Six frequent SNPs with minor allele frequencies (MAF) > 3% were detected in the HBB gene (rs7946748, rs7480526, rs10768683, rs35209591, HbS (rs334) and rs713040) and two in the G6PD gene (rs762515 and rs1050828 (G6PD-202 G > A). Analysis of selected HbS polymorphism showed significant association with protective effect against severe malaria with a significant p-value = 0.033 (OR 0.38, 95% CI [0.16-0.91]) for SM vs. UM comparison. Surprisingly, our study did not identify the protective effect of variant HbC polymorphism against severe malaria. Finally, we found some of the polymorphisms, like HbS (rs334), are associated with age and biological parameters like eosinophils, basophils, lymphocytes etc. Conclusion: Our data report HBB and G6PD polymorphisms in the Senegalese population and their correlation with severe/mild malaria and outcome. The G6PD and HBB deficiencies are widespread in West Africa endemic malaria regions such as The Gambia, Mali, and Burkina Faso. The study shows the critical role of genetic factors in malaria outcomes. Indeed, genetic markers could be good tools for malaria endemicity prognosis.

Identification of New Biological Pathways Involved in Skin Aging From the Analysis of French Women Genome-Wide Data.

Skin aging is an ineluctable process leading to the progressive loss of tissue integrity and is characterized by various outcomes such as wrinkling and sagging. Researchers have identified impacting environmental factors (sun exposure, smoking, etc.) and several molecular mechanisms leading to skin aging. We have previously performed genome-wide association studies (GWAS) in 502 very-well characterized French women, looking for associations with four major outcomes of skin aging, namely, photoaging, solar lentigines, wrinkling, and sagging, and this has led to new insights into the molecular mechanisms of skin aging. Since individual SNP associations in GWAS explain only a small fraction of the genetic impact in complex polygenic phenotypes, we have made the integration of these genotypes into the reference Kegg biological pathways and looked for associations by the gene set enrichment analysis (GSEA) approach. 106 pathways were tested for association with the four outcomes of skin aging. This biological pathway analysis revealed new relevant pathways and genes, some likely specific of skin aging such as the WNT7B and PRKCA genes in the "melanogenesis" pathway and some likely involved in global aging such as the DDB1 gene in the "nucleotide excision repair" pathway, not picked up in the previously published GWAS. Overall, our results suggest that the four outcomes of skin aging possess specific molecular mechanisms such as the "proteasome" and "mTOR signaling pathway" but may also share common molecular mechanisms such as "nucleotide excision repair."

Wasl is crucial to maintain microglial core activities during glioblastoma initiation stages.

Microglia actively promotes the growth of high-grade gliomas. Within the glioma microenvironment an amoeboid microglial morphology has been observed, however the underlying causes and the related impact on microglia functions and their tumor promoting activities is unclear. Using the advantages of the larval zebrafish model, we identified the underlying mechanism and show that microglial morphology and functions are already impaired during glioma initiation stages. The presence of pre-neoplastic HRasV12 expressing cells induces an amoeboid morphology of microglia, increases microglial numbers and decreases their motility and phagocytic activity. RNA sequencing analysis revealed lower expression levels of the actin nucleation promoting factor wasla in microglia. Importantly, a microglia specific rescue of wasla expression restores microglial morphology and functions. This results in increased phagocytosis of pre-neoplastic cells and slows down tumor progression. In conclusion, we identified a mechanism that de-activates core microglial functions within the emerging glioma microenvironment. Restoration of this mechanism might provide a way to impair glioma growth.

New contributions to the drug profile of TNFα inhibitor SPD304: Affinity, selectivity and ADMET considerations.

Tumor necrosis factor alpha (TNFα) is a relevant clinical target for the treatment of chronic inflammatory diseases. Currently, only few small molecules are known as direct inhibitors of TNFα. To date, none of these molecules has shown both an efficient activity and a low toxicity to be considered for clinical trials. The SPD304 is considered as a reference of direct inhibitors of TNFα because of its well demonstrated mechanism (He et al., 2005). Herein, we provide new insights regarding the drug profile, selectivity and absorption, distribution, metabolism, excretion and toxicity (ADMET) considerations of SPD304 to evaluate its potential as a hit for the structure-based design of novel TNFα inhibitors. ELISA experiments confirmed the inhibition of TNFα/TNF receptor 1 binding (IC50 = 12 µM). Cellular-based assays highlighted the cytotoxicity of SPD304, as well as its ability to inhibit TNFα signaling pathways at non-cytotoxic concentrations. A surface acoustic wave (SAW) experiment highlighted only one binding site with a dissociation constant of 6.1 ± 4.7 nM. SPD304 inhibited the binding of the cytokines like interleukins (IL)-4 and IL-13 to their receptors and showed no direct inhibition on proteins involved in the TNFα pathway. Finally, the thermodynamic solubility and Caco-2 cells permeability of SPD304 were experimentally evaluated and ADMET in silico predictions are also discussed. The physicochemical, pharmacological and ADMET studies of SPD304 have shown that is not an ideal hit for a drug optimization program based on its chemical structure.

Gene expression profiling reveals a conserved microglia signature in larval zebrafish.

Microglia are the resident macrophages of the brain. Over the past decade, our understanding of the function of these cells has significantly improved. Microglia do not only play important roles in the healthy brain but are involved in almost every brain pathology. Gene expression profiling allowed to distinguish microglia from other macrophages and revealed that the full microglia signature can only be observed in vivo. Thus, animal models are irreplaceable to understand the function of these cells. One of the popular models to study microglia is the zebrafish larva. Due to their optical transparency and genetic accessibility, zebrafish larvae have been employed to understand a variety of microglia functions in the living brain. Here, we performed RNA sequencing of larval zebrafish microglia at different developmental time points: 3, 5, and 7 days post fertilization (dpf). Our analysis reveals that larval zebrafish microglia rapidly acquire the core microglia signature and many typical microglia genes are expressed from 3 dpf onwards. The majority of changes in gene expression happened between 3 and 5 dpf, suggesting that differentiation mainly takes place during these days. Furthermore, we compared the larval microglia transcriptome to published data sets of adult zebrafish microglia, mouse microglia, and human microglia. Larval microglia shared a significant number of expressed genes with their adult counterparts in zebrafish as well as with mouse and human microglia. In conclusion, our results show that larval zebrafish microglia mature rapidly and express the core microglia gene signature that seems to be conserved across species.

Accurate, scalable and integrative haplotype estimation.

The number of human genomes being genotyped or sequenced increases exponentially and efficient haplotype estimation methods able to handle this amount of data are now required. Here we present a method, SHAPEIT4, which substantially improves upon other methods to process large genotype and high coverage sequencing datasets. It notably exhibits sub-linear running times with sample size, provides highly accurate haplotypes and allows integrating external phasing information such as large reference panels of haplotypes, collections of pre-phased variants and long sequencing reads. We provide SHAPEIT4 in an open source format and demonstrate its performance in terms of accuracy and running times on two gold standard datasets: the UK Biobank data and the Genome In A Bottle.

Hits Discovery on the Androgen Receptor: In Silico Approaches to Identify Agonist Compounds.

The androgen receptor (AR) is a transcription factor that plays a key role in sexual phenotype and neuromuscular development. AR can be modulated by exogenous compounds such as pharmaceuticals or chemicals present in the environment, and particularly by AR agonist compounds that mimic the action of endogenous agonist ligands and whether restore or alter the AR endocrine system functions. The activation of AR must be correctly balanced and identifying potent AR agonist compounds is of high interest to both propose treatments for certain diseases, or to predict the risk related to agonist chemicals exposure. The development of in silico approaches and the publication of structural, affinity and activity data provide a good framework to develop rational AR hits prediction models. Herein, we present a docking and a pharmacophore modeling strategy to help identifying AR agonist compounds. All models were trained on the NR-DBIND that provides high quality binding data on AR and tested on AR-agonist activity assays from the Tox21 initiative. Both methods display high performance on the NR-DBIND set and could serve as starting point for biologists and toxicologists. Yet, the pharmacophore models still need data feeding to be used as large scope undesired effect prediction models.

Large-Scale "OMICS" Studies to Explore the Physiopatholgy of HIV-1 Infection.

In this review, we present the main large-scale experimental studies that have been performed in the HIV/AIDS field. These "omics" studies are based on several technologies including genotyping, RNA interference, and transcriptome or epigenome analysis. Due to the direct connection with disease evolution, there has been a large focus on genotyping cohorts of well-characterized patients through genome-wide association studies (GWASs), but there have also been several invitro studies such as small interfering RNA (siRNA) interference or transcriptome analyses of HIV-1-infected cells. After describing the major results obtained with these omics technologies-including some with a high relevance for HIV-1 treatment-we discuss the next steps that the community needs to embrace in order to derive new actionable therapeutic or diagnostic targets. Only integrative approaches that combine all big data results and consider their complex interactions will allow us to capture the global picture of HIV molecular pathogenesis. This novel challenge will require large collaborative efforts and represents a huge open field for innovative bioinformatics approaches.

Genome-wide scan identified genetic variants associated with skin aging in a Chinese female population.

BACKGROUND: The progression of human skin aging has a strong genetic basis. However, recent studies have mainly focused on Caucasian populations and we have thus performed a genetic association study on skin aging signs in Han Chinese population. OBJECTIVE: To investigate genetic risk factors in skin aging in Han Chinese female, we performed a genome-wide association study. METHODS: We collected genotype data from 1534 Han Chinese female from Taizhou cohort and evaluated 15 skin aging phenotypes by using the validated skin aging SCINEXA™ score. Genetic associations were tested by linear and logistic regression analyses and adjusted for potential confounders. RESULTS: Six genomic regions significantly associated with a risk for skin aging were revealed : 6q24.2 (rs3804540, P=4.6×10-9, additive model) with size of pigmented spots on forehead, 10q26.13 (rs4962295, P=1.9 ×10-8, additive model) with wrinkles under eyes, 15q21.1 (rs28392847, P=1.6×10-8, additive model) with crow's feet, 2p25.1 (rs191497052, P=5.5×10-9, dominant model) with telangiectasia, 13q34 (rs3825460, P=3.7×10-8, dominant model) with size of pigmented spots on cheeks and 16p13.11(rs76053540, P=5.0×10-9, dominant model) with nasolabialfold. The signal on 15q21.1 was replicated in the meta-analysis with two independent Caucasian cohorts (P=8.6×10-10). We have also successfully replicated in our cohort an association between SNP rs1048943 of gene CYP1A1 (P=7.1 × 10-4) and pigmented spots on cheeks previously described in Caucasian cohort. CONCLUSIONS: Our study has identified new genetic risk factors for signs of skin aging in the Han Chinese. This study suggests there are differences in genetic susceptibility to skin aging between Caucasians and the Han Chinese.

A genome wide association study identifies new genes potentially associated with eyelid sagging.

Sagging eyelid is considered as an outward of skin ageing and may cause medical issues. However, little is known about the factors involved in sagging eyelid. The study, which aims at determining genetic risk factors for eyelid sagging, was conducted in a cohort of 502 unrelated Caucasian women living in the Paris region. All included participants were aged between 44 and 70 years old (mean age, 57.6 years old). The severity of sagging eyelid was graded in 6 categories by a dermatologist using standardized photographs of the face. A genome wide association study adjusted on potential risk factors (including age and smoking habits) was conducted to identify genetic associations. Two single nucleotide polymorphisms in total linkage disequilibrium on chromosome 10, rs16927253 (P = 7.07 × 10-10 ) and rs4746957 (P = 1.06 × 10-8 ), were significantly associated with eyelid sagging severity. The rs16927253-T and rs4746957-A alleles showed a dominant protective effect towards eyelid sagging. These polymorphisms are located in intronic parts of the H2AFY2 gene which encodes a member of the H2A histone family and very close to the AIFM2 gene that induces apoptosis. Additionally, single nucleotide polymorphisms with a false discovery rate below 0.25 were located nearby the type XIII collagen COL13A1 gene on chromosome 10 and in the ADAMTS18 gene on chromosome 16. Several relevant genes were identified by the genome wide association study for their potential role in the sagging eyelid severity.

Nuclear Receptors Database Including Negative Data (NR-DBIND): A Database Dedicated to Nuclear Receptors Binding Data Including Negative Data and Pharmacological Profile.

Nuclear receptors (NRs) are transcription factors that regulate gene expression in various physiological processes through their interactions with small hydrophobic molecules. They constitute an important class of targets for drugs and endocrine disruptors and are widely studied for both health and environment concerns. Since the integration of negative data can be critical for accurate modeling of ligand activity profiles, we manually collected and annotated NRs interaction data (positive and negative) through a sharp review of the corresponding literature. 15 116 positive and negative interactions data are provided for 28 NRs together with 593 PDB structures in the freely available Nuclear Receptors Database Including Negative Data ( http://nr-dbind.drugdesign.fr ). The NR-DBIND contains the most extensive information about interaction data on NRs, which should bring valuable information to chemists, biologists, pharmacologists and toxicologists.

CoDE-seq, an augmented whole-exome sequencing, enables the accurate detection of CNVs and mutations in Mendelian obesity and intellectual disability.

OBJECTIVE: The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step. METHODS: CoDE-seq is based on an augmented WES method, using probes distributed uniformly throughout the genome. CoDE-seq was validated in 40 patients for whom chromosomal DNA microarray was available. CNVs and mutations were assessed in 82 children/young adults with suspected Mendelian obesity and/or intellectual disability and in their parents when available (ntotal = 145). RESULTS: CoDE-seq not only detected all of the 97 CNVs identified by chromosomal DNA microarrays but also found 84 additional CNVs, due to a better resolution. When compared to CoDE-seq and chromosomal DNA microarrays, WES failed to detect 37% and 14% of CNVs, respectively. In the 82 patients, a likely molecular diagnosis was achieved in >30% of the patients. Half of the genetic diagnoses were explained by CNVs while the other half by mutations. CONCLUSIONS: CoDE-seq has proven cost-efficient and highly effective as it avoids the sequential genetic screening approaches currently used in clinical practice for the accurate detection of CNVs and point mutations.

Decoys Selection in Benchmarking Datasets: Overview and Perspectives.

Virtual Screening (VS) is designed to prospectively help identifying potential hits, i.e., compounds capable of interacting with a given target and potentially modulate its activity, out of large compound collections. Among the variety of methodologies, it is crucial to select the protocol that is the most adapted to the query/target system under study and that yields the most reliable output. To this aim, the performance of VS methods is commonly evaluated and compared by computing their ability to retrieve active compounds in benchmarking datasets. The benchmarking datasets contain a subset of known active compounds together with a subset of decoys, i.e., assumed non-active molecules. The composition of both the active and the decoy compounds subsets is critical to limit the biases in the evaluation of the VS methods. In this review, we focus on the selection of decoy compounds that has considerably changed over the years, from randomly selected compounds to highly customized or experimentally validated negative compounds. We first outline the evolution of decoys selection in benchmarking databases as well as current benchmarking databases that tend to minimize the introduction of biases, and secondly, we propose recommendations for the selection and the design of benchmarking datasets.