Enzymatic and Molecular Characterization of Anti-Leishmania Molecules That Differently Target Leishmania and Mammalian eIF4A Proteins, LieIF4A and eIF4AMus.

Previous investigations of the Leishmania infantum eIF4A-like protein (LieIF4A) as a potential drug target delivered cholestanol derivatives inhibitors. Here, we investigated the mode of action of cholesterol derivatives as a novel scaffold structure of LieIF4A inhibitors on the RNA-dependent ATPase activity of LieIF4A and its mammalian ortholog (eIF4AI). We compared their biochemical effects on RNA-dependent ATPase activities of both proteins and investigated if rocaglamide, a known inhibitor of eIF4A, could affect LieIF4A as well. Kinetic measurements were conducted at different concentrations of ATP, of the compound and in the presence of saturating whole yeast RNA concentrations. Kinetic analyses showed different ATP binding affinities for the two enzymes as well as different sensitivities to 7-α-aminocholesterol and rocaglamide. The 7-α-aminocholesterol inhibited LieIF4A with a higher binding affinity relative to cholestanol analogs. Cholesterol, another tested sterol, had no effect on the ATPase activity of LieIF4A or eIF4AI. The 7-α-aminocholesterol demonstrated an anti-Leishmania activity on L. infantum promastigotes. Additionally, docking simulations explained the importance of the double bond between C5 and C6 in 7-α-aminocholesterol and the amino group in the C7 position. In conclusion, Leishmania and mammalian eIF4A proteins appeared to interact differently with effectors, thus making LieIF4A a potential drug against leishmaniases.

Leishmania infantum 5'-Methylthioadenosine Phosphorylase presents relevant structural divergence to constitute a potential drug target.

BACKGROUND: The 5'-methylthioadenosine phosphorylase (MTAP), an enzyme involved in purine and polyamine metabolism and in the methionine salvage pathway, is considered as a potential drug target against cancer and trypanosomiasis. In fact, Trypanosoma and Leishmania parasites lack de novo purine pathways and rely on purine salvage pathways to meet their requirements. Herein, we propose the first comprehensive bioinformatic and structural characterization of the putative Leishmania infantum MTAP (LiMTAP), using a comparative computational approach. RESULTS: Sequence analysis showed that LiMTAP shared higher identity rates with the Trypanosoma brucei (TbMTAP) and the human (huMTAP) homologs as compared to the human purine nucleoside phosphorylase (huPNP). Motifs search using MEME identified more common patterns and higher relatedness of the parasite proteins to the huMTAP than to the huPNP. The 3D structures of LiMTAP and TbMTAP were predicted by homology modeling and compared to the crystal structure of the huMTAP. These models presented conserved secondary structures compared to the huMTAP, with a similar topology corresponding to the Rossmann fold. This confirmed that both LiMTAP and TbMTAP are members of the NP-I family. In comparison to the huMTAP, the 3D model of LiMTAP showed an additional α-helix, at the C terminal extremity. One peptide located in this specific region was used to generate a specific antibody to LiMTAP. In comparison with the active site (AS) of huMTAP, the parasite ASs presented significant differences in the shape and the electrostatic potentials (EPs). Molecular docking of 5'-methylthioadenosine (MTA) and 5'-hydroxyethylthio-adenosine (HETA) on the ASs on the three proteins predicted differential binding modes and interactions when comparing the parasite proteins to the human orthologue. CONCLUSIONS: This study highlighted significant structural peculiarities, corresponding to functionally relevant sequence divergence in LiMTAP, making of it a potential drug target against Leishmania.

In silico analysis and in vitro evaluation of immunogenic and immunomodulatory properties of promiscuous peptides derived from Leishmania infantum eukaryotic initiation factor.

It is generally considered as imperative the ability to control leishmaniasis through the development of a protective vaccine capable of inducing long-lasting and protective cell-mediated immune responses. In this current study, we demonstrated potential epitopes that bind to H2 MHC class I and II molecules by conducting the in silico analysis of Leishmania infantum eukaryotic Initiation Factor (LieIF) protein, using online available algorithms. Moreover, we synthesized five peptides (16-18 amino acids long) which are part of the N-terminal portion of LieIF and contain promising MHC class I and II-restricted epitopes and afterwards, their predicted immunogenicity was evaluated in vitro by monitoring peptide-specific T-cell responses. Additionally, the immunomodulatory properties of these peptides were investigated in vitro by exploring their potential of inducing phenotypic maturation and functional differentiation of murine Bone-Marrow derived Dendritic Cells (BM-DCs). It was revealed by our data that all the synthetic peptides predicted for H2 alleles; present the property of immunogenicity. Among the synthetic peptides which contained T-cell epitopes, the peptide 52-68 aa (LieIF_2) exhibited immunomodulatory properties with the larger potential. LieIF_2-pulsed BM-DCs up-regulated the expression of the co-stimulatory surface molecules CD80 and CD86, as well as the production of the proinflammatory cytokine TNF-α and of the Th1-polarizing cytokines IL-12 and IFN-γ. The aforementioned data suggest that selected parts of LieIF could be used to develop innovative subunit protective vaccines able to induce effective immunity mediated by MHC class I-restricted as well as class II-restricted T-cell responses.

Identification of binding sites and favorable ligand binding moieties by virtual screening and self-organizing map analysis.

BACKGROUND: Identifying druggable cavities on a protein surface is a crucial step in structure based drug design. The cavities have to present suitable size and shape, as well as appropriate chemical complementarity with ligands. RESULTS: We present a novel cavity prediction method that analyzes results of virtual screening of specific ligands or fragment libraries by means of Self-Organizing Maps. We demonstrate the method with two thoroughly studied proteins where it successfully identified their active sites (AS) and relevant secondary binding sites (BS). Moreover, known active ligands mapped the AS better than inactive ones. Interestingly, docking a naive fragment library brought even more insight. We then systematically applied the method to the 102 targets from the DUD-E database, where it showed a 90% identification rate of the AS among the first three consensual clusters of the SOM, and in 82% of the cases as the first one. Further analysis by chemical decomposition of the fragments improved BS prediction. Chemical substructures that are representative of the active ligands preferentially mapped in the AS. CONCLUSION: The new approach provides valuable information both on relevant BSs and on chemical features promoting bioactivity.

[Screening of cervical HPV infection at the reproductive health centre of ariana]. / Détection de i'infection à papillomavirus humain chez des consultants d'un centre tunisien de recherché de la santé de la reproduction.

BACKGROUND: Cervical cancer is the leading cause of cancer mortality among women in developing countries. Over 99 per cent of cases are linked to genital infection with Human Papillomaviruses (HPVs), which are the most common sexually-transmitted viruses worldwide and infect an estimated 660 million people. In light of the advances of the development of HPV vaccines in the world, the challenges to introducing and sustaining such vaccines in the public sector are faced by our countries, generating the need of understanding the local epidemiology of the disease. aim : This study aims to obtain some relevant data on local disease burden in a big health care centre of Tunis. method : In this study, 133 healthy Tunisian women are screened for HPV infection with detection of 37 genotypes. Samples are collected on dry cotton swabs and genotyping use PCR and linear array assay. results : 7,8 % of women were infected and HPV 16 was the only High-risk genotype encountered. 62,5% of infected women doesn't show any cytological abnormality on pap smears. Low and High grade epithelial lesions were associated with HPV 16. Conclusion : There is currently few and sparse data in Tunisia. This work represents to our knowledge the first HPV cervical infection screening study in Tunisia in a basic health centre. The most relevant data is the High frequency of High risk HPV infections related to HPV 16. More than a half of cervical smears in infected women does not show any cytological abnormality and thus, pap smear screening is not appropriated to detect this infection.

Handheld Ultra-Fast Duplex Polymerase Chain Reaction Assays and Lateral Flow Detection and Identification of Leishmania Parasites for Cutaneous Leishmaniases Diagnosis.

Early and accurate detection of infectious diseases is a key step for surveillance, epidemiology and control, notably timely disease diagnosis, patient management and follow-up. In this study, we aimed to develop handheld ultra-fast duplex PCR assays coupled to amplicon detection by lateral flow (LF) immunoassay to deliver a rapid and simple molecular diagnostic test for concomitant detection and identification of the main Leishmania parasites encountered in Tunisia. We selected two DNA targets to amplify L. major/L. tropica and L. infantum/L. tropica groups of species DNAs, respectively. We optimized the experimental conditions of a duplex ultra-fast PCR. The amplification is performed using a portable Palm convection PCR machine within 18 min, and the products are detected using an LF cassette within 10 min. The test allows the identification of the infecting species according to the position and number of test lines revealed. Tested on a selection of DNAs of representative Leishmania strains of the three studied species (N = 37), the ultra-fast duplex PCR-LF showed consistent, stable and reproducible results. The analytical limit of detection of the test was 0.4 pg for L. major, 4 pg for L. infantum and 40 pg for L. tropica.

Distribution of high-risk human papillomavirus in women with ASC-US or LSIL in Tunisian women: Place of HPV testing for women triage in Tunisia.

The present study aimed to evaluate the distribution of oncogenic HPVs in Tunisian women diagnosed with ASC-US or LSIL in order to highlight the importance of HPV testing in the management of women with minor cytological lesions. The study involved 213 cervical samples from women aged from 18 to 82 years and diagnosed with ASC-US or LSIL. HPV detection and genotyping was performed by nested PCR followed by reverse Line Blotting. HPV DNA was identified in 161 cases (76.3%). Oncogenic HPV genotypes were detected in 53.1% of cases. The most frequent high-risk genotypes in this study were HPV16 (28.8%) followed by: HPV51 (9.6%), HPV18, HPV31 HPV56 (7.1%) and HPV45 (5.1%). Thus, 24 % of studied women were not infected by HPV and about 47% of infections are without oncogenic HPV. These results highlight the value of HPV testing in the decision algorithm of management of minor abnormalities lesions.

Genetic Diversity and Population Structure of Leishmania infantum in Morocco as Revealed by Multilocus Sequence Typing (MLST) Approach.

Leishmania infantum is endemic in Morocco, and it causes both visceral (VL) and cutaneous leishmaniasis (CL). In this study, the multilocus sequence typing (MLST) approach was used to investigate the phylogeny and population structure of Leishmania infantum strains isolated from CL and VL patients and the canine reservoir in different leishmaniasis endemic foci in Morocco. For this purpose, eight loci (pgm, alat, me, fh, g6pd, pgd, gpi and cytb) were amplified in 40 samples, out of which 31 were successfully sequenced. The genetic diversity analysis detected a high degree of intraspecific genetic variability among the studied strains. The phylogenetic and the haplotype analyses showed that most of the strains from the same geographical areas clustered together. The recombination among Leishmania infantum strains was revealed through a splits tree analysis and the number of recombination events. Moreover, the assessment of the gene flow between Leishmania infantum and Leishmania tropica through phylogenetic analysis and haplotype diversity in two endemic foci where the two species were sympatric showed no genetic exchange between the two species.

The E6 gene polymorphism of Human papillomavirus 16 in relation to the risk of cervical cancer in Tunisian women.

Human papillomavirus type 16 (HPV-16) is the most prevalent HPV type worldwide and in Tunisia and the major carcinogenic HPV type found in cervical precancers and cancers. Previous studies have reported that genetic diversity of HPV16-E6 oncoprotein might be associated with cervical intraepithelial neoplasia progression. In this study we aimed to investigate the prevalence of HPV-16 E6 variants in precancerous lesions in Tunisian population to assess potential correlation with disease severity. Positive HPV cervical samples were obtained from the Laboratory of Anatomy Pathology of Pasteur Institute of Tunis. Cytological study was performed to identify cervical precancerous lesions. HPVs were typed using Reverse Line Hybridization. Only samples with HPV-16 single infection were selected for HP16-E6 genetic diversity investigation. HPV-16 E6 gene amplification was performed by PCR using specific primers and sequenced by Sanger Sequencing. The multiple alignment of generated sequences was performed using MEGAX software. Phylogenetic tree was constructed using Maximum Likehood method. The ternary complex of E6, E6AP and p53 core domain was used to perform in silico point mutations and thermodynamic calculations to assess stability and binding affinity. Genetic analysis of Tunisian E6-HPV16 sequences showed the presence of three lineages: European (A), African (C) and Asian American (D). Interestingly, the EUR variants were identified as the dominant lineage of HPV-16 and HPV-16 E6 350 G (L83V) was the most detected mutation in precancerous lesions. Modelling data showed that African variants induced the largest destabilizing effect on E6 structure and decreasing thereby in the affinity toward E6AP. Therefore, women infected with European variants are associated with low and high intraepithelial lesions. The findings give useful information for personalized decision algorithms of intra-epithelial cervical neoplasia in Tunisian women.

Applied Machine Learning Toward Drug Discovery Enhancement: Leishmaniases as a Case Study.

Drug discovery (DD) research is a complex field with a high attrition rate. Machine learning (ML) approaches combined to chemoinformatics are of valuable input to this field. We, herein, focused on implementing multiple ML algorithms that shall learn from different molecular fingerprints (FPs) of 65 057 molecules that have been identified as active or inactive against Leishmania major promastigotes. We sought to build a classifier able to predict whether a given molecule has the potential of being anti-leishmanial or not. Using the RDkit library, we calculated 5 molecular FPs of the molecules. Then, we implemented 4 ML algorithms that we trained and tested for their ability to classify the molecules into active/inactive classes based on their chemical structure, encoded by the molecular FPs. Best performers were random forest (RF) and support vector machine (SVM), while atom-pair and topology torsion FPs were the best embedding functions. Both models were further assessed on different stratification levels of the dataset and showed stable performances. At last, we used them to predict the potential of molecules within the Food and Drug Administration (FDA)-approved drugs collection to present anti-Leishmania effects. We ranked these drugs according to their anti-Leishmanial probability and obtained in total seven anti-Leishmania agents, previously described in the literature, within the top 10 of each model. This validates the robustness of the approach, the algorithms, and FPs choices as well as the importance of the dataset size and content. We further engaged these molecules into reverse docking experiments on 3D crystal structures of seven well-studied Leishmania drug targets and could predict the molecular targets for 4 drugs. The results bring novel insights into anti-Leishmania compounds.

High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia.

BACKGROUND: Leishmaniasis is endemic in Tunisia and presents with different clinical forms, caused by the species Leishmania infantum, Leishmania major, and Leishmania tropica. The life cycle of Leishmania is complex and involves several phlebotomine sand fly vectors and mammalian reservoir hosts. The aim of this work is the development and evaluation of a high-resolution melting PCR (PCR-HRM) tool to detect and identify Leishmania parasites in wild and domestic hosts, constituting confirmed (dogs and Meriones rodents) or potential (hedgehogs) reservoirs in Tunisia. METHODS: Using in vitro-cultured Leishmania isolates, PCR-HRM reactions were developed targeting the 7SL RNA and HSP70 genes. Animals were captured or sampled in El Kef Governorate, North West Tunisia. DNA was extracted from the liver, spleen, kidney, and heart from hedgehogs (Atelerix algirus) (n = 3) and rodents (Meriones shawi) (n = 7) and from whole blood of dogs (n = 12) that did not present any symptoms of canine leishmaniasis. In total, 52 DNA samples were processed by PCR-HRM using both pairs of primers. RESULTS: The results showed melting curves enabling discrimination of the three Leishmania species present in Tunisia, and were further confirmed by Sanger sequencing. Application of PCR-HRM assays on reservoir host samples showed that overall among the examined samples, 45 were positive, while seven were negative, with no Leishmania infection. Meriones shawi were found infected with L. major, while dogs were infected with L. infantum. However, co-infections with L. major/L. infantum species were detected in four Meriones specimens and in all tested hedgehogs. In addition, multiple infections with the three Leishmania species were found in one hedgehog specimen. Sequence analyses of PCR-HRM products corroborated the Leishmania species found in analyzed samples. CONCLUSIONS: The results of PCR-HRM assays applied to field specimens further support the possibility of hedgehogs as reservoir hosts of Leishmania. In addition, we showed their usefulness in the diagnosis of canine leishmaniasis, specifically in asymptomatic dogs, which will ensure a better evaluation of infection extent, thus improving elaboration of control programs. This PCR-HRM method is a robust and reliable tool for molecular detection and identification of Leishmania and can be easily implemented in epidemiological surveys in endemic regions.

The Prevalence, Genotype Distribution and Risk Factors of Human Papillomavirus in Tunisia: A National-Based Study.

There are limited national population-based studies on HPV genotypes distribution in Tunisia, thus making difficult an assessment of the burden of vaccine-preventable cervical cancer. In this context, we conducted a national survey to determine the HPV prevalence and genotypes distribution and the risk factors for HPV infections in Tunisian women. This is a cross-sectional study performed between December 2012 and December 2014. A liquid-based Pap smear sample was obtained from all women and samples' DNAs were extracted. Only women with betaglobin-positive PCR were further analysed for HPV detection and typing by a nested-PCR of the L1 region followed by next-generation sequencing. A multiple logistic regression model was used for the analysis of associations between the variables. A total of 1517 women were enrolled in this study, and 1229 out of the 1517 cervical samples were positive for the betaglobin control PCR and tested for HPV. Overall HPV infection prevalence was measured to be 7.8% (96/1229), with significant differences between the grand regions, ranging from 2% in the North to 13.1% in Grand Tunis. High-risk HPV genotypes accounted for 5% of the infections. The most prevalent genotypes were HPV 31 (1%), 16 (0.9%), 59 (0.7%). HPV18 was detected only in four cases of the study population. Potential risk factors were living in Grand Tunis region (OR: 7.94 [2.74-22.99]), married status (OR: 2.74 [1.23-6.13]), smoking habit (OR: 2.73 [1.35-5.51]), occupation (OR: 1.81 [1.09-3.01]) and women with multiple sexual partners (OR: 1.91 [1.07-3.39]). These findings underscore the need to evaluate the cost effectiveness of HPV vaccine implementation, contribute to the evidence on the burden of HPV infections, the critical role of sexual behaviour and socioeconomic status, and call for increased support to the preventive program of cervical cancer in Tunisia.

Molecular Epidemiology of SARS-CoV-2 in Tunisia (North Africa) through Several Successive Waves of COVID-19.

Documenting the circulation dynamics of SARS-CoV-2 variants in different regions of the world is crucial for monitoring virus transmission worldwide and contributing to global efforts towards combating the pandemic. Tunisia has experienced several waves of COVID-19 with a significant number of infections and deaths. The present study provides genetic information on the different lineages of SARS-CoV-2 that circulated in Tunisia over 17 months. Lineages were assigned for 1359 samples using whole-genome sequencing, partial S gene sequencing and variant-specific real-time RT-PCR tests. Forty-eight different lineages of SARS-CoV-2 were identified, including variants of concern (VOCs), variants of interest (VOIs) and variants under monitoring (VUMs), particularly Alpha, Beta, Delta, A.27, Zeta and Eta. The first wave, limited to imported and import-related cases, was characterized by a small number of positive samples and lineages. During the second wave, a large number of lineages were detected; the third wave was marked by the predominance of the Alpha VOC, and the fourth wave was characterized by the predominance of the Delta VOC. This study adds new genomic data to the global context of COVID-19, particularly from the North African region, and highlights the importance of the timely molecular characterization of circulating strains.

Distribution of human papillomavirus in precancerous and cancerous cervical neoplasia in Tunisian women.

BACKGROUND: High-risk human papillomavirus (HR-HPV) are responsible for cervical cancer (CC) which represents the second most prevalent gynecological cancer among Tunisian women. Preventive strategies against CC are based on prophylactic vaccines that have not yet been implemented into the national vaccination program of Tunisia. Therefore, the present study aimed to investigate the HPV genotypes distribution in cervical neoplasia in Tunisian women in order to predict the impact of using current HPV vaccines on cancer prevention in Tunisia. METHODS: A total of 200 formalin-fixed paraffin embedded biopsies were collected in our study. DNA was extracted using Qiagen Mini prep kit. DNA quality was controlled by Beta Globin PCR. Only positive samples for Beta Globin test were used. HPV detection was performed by a nested PCR using PYGMY and GP5+/6+ primers. Genotyping was performed by Reverse Line hybridization using 31 probes. RESULTS: The mean age of participants was 38.97 years and 75% were over 30 years. Cervical neoplasia distribution according to age showed that CINII/CINIII was observed among women over 30 years old. All samples were positive for Beta Globin PCR. Overall HPV prevalence in cervical lesions was 83% (166/200). HPV was present in 65% of CINI, 82% of CINII/CINIII and 85% of CC. HR-HPV was statistically significantly associated with cervical intraepithelial neoplasia (p < 10-3). HR-HPV distribution according to lesion grade and cervical cancer showed that HPV16 and HPV18 were present in all lesions. For CINII/CINIII, HPV 35 (37.5%) was the most detected type, followed by HPV18 (33.3%) HPV 45 (28.5%) and HPV 16 (18.9%). HPV 45(57.5%), HPV 18 (53.3%) were the most detected in CC. HPV58, 59, 68 were only detected in CC and associated with HPV45, 18 and HPV16. HPV39, 31, 33, 52, 56 and HPV70 was associated only with CINI. CONCLUSIONS: Our findings can give useful information for vaccine implementation by helping the health policymakers to choose the most appropriate vaccine type in Tunisia.

Multilocus sequence analysis provides new insight into population structure and genetic diversity of Leishmania tropica in Morocco.

Cutaneous leishmaniasis (CL) is one of the most neglected tropical diseases, caused by different Leishmania species. Despite its high incidence in Morocco, CL due to Leishmania tropica is poorly understood in terms of its epidemiological status and population structure. In this study, we used multilocus sequence typing (MLST) in order to explore the genetic heterogeneity of L. tropica strains. Samples (N = 48) were collected from CL patients in two localities in Morocco (Foum Jamaa in the Azilal province and Imintanoute in Chichaoua province). PCR-sequencing of 18 strains was carried out for six housekeeping genes (cytb, me, fh, g6pd, pgd and gpi), Genetic diversity indices showed a high population genetic differentiation between and among populations. There was no shared haplotypes between the two localities studied. Our results reveal a considerable degree of differentiation through the relatively high FST value (> 0.4) and remarkable intraspecific polymorphism (S = 29). Imintanoute strains have more polymorphisms (S = 22) than the Foum Jamaa strains despite their small sample size. These results provide crucial background information of epidemiology in Imintanoute which raises questions about animal involvement in L. tropica transmission cycle.

Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples.

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/µl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

Deep Learning Algorithms Achieved Satisfactory Predictions When Trained on a Novel Collection of Anticoronavirus Molecules.

Drug discovery and repurposing against COVID-19 is a highly relevant topic with huge efforts dedicated to delivering novel therapeutics targeting SARS-CoV-2. In this context, computer-aided drug discovery is of interest in orienting the early high throughput screenings and in optimizing the hit identification rate. We herein propose a pipeline for Ligand-Based Drug Discovery (LBDD) against SARS-CoV-2. Through an extensive search of the literature and multiple steps of filtering, we integrated information on 2,610 molecules having a validated effect against SARS-CoV and/or SARS-CoV-2. The chemical structures of these molecules were encoded through multiple systems to be readily useful as input to conventional machine learning (ML) algorithms or deep learning (DL) architectures. We assessed the performances of seven ML algorithms and four DL algorithms in achieving molecule classification into two classes: active and inactive. The Random Forests (RF), Graph Convolutional Network (GCN), and Directed Acyclic Graph (DAG) models achieved the best performances. These models were further optimized through hyperparameter tuning and achieved ROC-AUC scores through cross-validation of 85, 83, and 79% for RF, GCN, and DAG models, respectively. An external validation step on the FDA-approved drugs collection revealed a superior potential of DL algorithms to achieve drug repurposing against SARS-CoV-2 based on the dataset herein presented. Namely, GCN and DAG achieved more than 50% of the true positive rate assessed on the confirmed hits of a PubChem bioassay.

The In Silico Identification of Potential Members of the Ded1/DDX3 Subfamily of DEAD-Box RNA Helicases from the Protozoan Parasite Leishmania infantum and Their Analyses in Yeast.

DEAD-box RNA helicases are ubiquitous proteins found in all kingdoms of life and that are associated with all processes involving RNA. Their central roles in biology make these proteins potential targets for therapeutic or prophylactic drugs. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest because of their important role(s) in translation. In this paper, we identified and aligned the protein sequences of 28 different DEAD-box proteins from the kinetoplast-protozoan parasite Leishmania infantum, which is the cause of the visceral form of leishmaniasis that is often lethal if left untreated, and compared them with the consensus sequence derived from DEAD-box proteins in general, and from the Ded1/DDX3 subfamily in particular, from a wide variety of other organisms. We identified three potential homologs of the Ded1/DDX3 subfamily and the equivalent proteins from the related protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness. We subsequently tested these proteins for their ability to complement a yeast strain deleted for the essential DED1 gene. We found that the DEAD-box proteins from Trypanosomatids are highly divergent from other eukaryotes, and consequently they are suitable targets for protein-specific drugs.

Atelerix algirus, the North African Hedgehog: Suitable Wild Host for Infected Ticks and Fleas and Reservoir of Vector-Borne Pathogens in Tunisia.

Small wild mammals are an important element in the emergence and transmission of vector-borne pathogens (VBPs). Among these species, hedgehogs have been found to be a reservoir of VBPs and host of arthropod vectors. Surveillance of VBPs in wildlife and their arthropods are crucial in a one health context. We conducted an exploratory study to screen Atelerix algirus hedgehogs and their infesting ticks and fleas for VBPs using a high throughput microfluidic real-time PCR system. Tested biopsies from hedgehogs were found to be naturally infected by Theileria youngi, Hepatozoon sp., Ehrlichia ewingii, Coxiella burnetii, and Candidatus Ehrlichia shimanensis. Similarly, Haemaphysalis erinacei and Rhipicephalus sanguineus tick species were infected by Ehrlichia ewingii, Rickettsia spp., Rickettsia massiliae, Borrelia sp., Coxiella burnetii, Rickettsia lusitaniae and Anaplasma sp. Archaeopsylla erinacei fleas were infected by Rickettsia asembonensis, Coxiella burnetii, and Rickettsia massiliae. Co-infections by two and three pathogens were detected in hedgehogs and infesting ticks and fleas. The microfluidic real-time PCR system enabled us not only to detect new and unexpected pathogens, but also to identify co-infections in hedgehogs, ticks, and fleas. We suggest that hedgehogs may play a reservoir role for VBPs in Tunisia and contribute to maintaining enzootic pathogen cycles via arthropod vectors.

Dipeptidyl peptidase III as a DNA marker to investigate epidemiology and taxonomy of Old World Leishmania species.

BACKGROUND: Dipeptidyl peptidase III (DPPIII) member of M49 peptidase family is a zinc-dependent metallopeptidase that cleaves dipeptides sequentially from the N-terminus of its substrates. In Leishmania, DPPIII, was reported with other peptidases to play a significant role in parasites' growth and survival. In a previous study, we used a coding sequence annotated as DPPIII to develop and evaluate a PCR assay that is specific to dermotropic Old World (OW) Leishmania species. Thus, our objective was to further assess use of this gene for Leishmania species identification and for phylogeny, and thus for diagnostic and molecular epidemiology studies of Old World Leishmania species. METHODOLOGY: Orthologous DDPIII genes were searched in all Leishmania genomes and aligned to design PCR primers and identify relevant restriction enzymes. A PCR assays was developed and seventy-two Leishmania fragment sequences were analyzed using MEGA X genetics software to infer evolution and phylogenetic relationships of studied species and strains. A PCR-RFLP scheme was also designed and tested on 58 OW Leishmania strains belonging to 8 Leishmania species and evaluated on 75 human clinical skin samples. FINDINGS: Sequence analysis showed 478 variable sites (302 being parsimony informative). Test of natural selection (dN-dS) (-0.164, SE = 0.013) inferred a negative selection, characteristic of essential genes, corroborating the DPPIII importance for parasite survival. Inter- and intra-specific genetic diversity was used to develop universal amplification of a 662bp fragment. Sequence analyses and phylogenies confirmed occurrence of 6 clusters congruent to L. major, L. tropica, L. aethiopica, L. arabica, L. turanica, L. tarentolae species, and one to the L. infantum and L. donovani species complex. A PCR-RFLP algorithm for Leishmania species identification was designed using double digestions with HaeIII and KpnI and with SacI and PvuII endonucleases. Overall, this PCR-RFLP yielded distinct profiles for each of the species L. major, L. tropica, L. aethiopica, L. arabica and L. turanica and the L. (Sauroleishmania) L. tarentolae. The species L. donovani, and L. infantum shared the same profile except for strains of Indian origin. When tested on clinical samples, the DPPIII PCR showed sensitivities of 82.22% when compared to direct examination and was able to identify 84.78% of the positive samples. CONCLUSION: The study demonstrates that DPPIII gene is suitable to detect and identify Leishmania species and to complement other molecular methods for leishmaniases diagnosis and epidemiology. Thus, it can contribute to evidence-based disease control and surveillance.