Gene disruption of the DNA topoisomerase IB small subunit induces a non-viable phenotype in the hemoflagellate Leishmania major

<p>Abstract</p> <p>Background</p> <p>The unusual heterodimeric leishmanial DNA topoisomerase IB consists of a large subunit containing the phylogenetically conserved "core" domain, and a small subunit harboring the C-terminal region with the characteristic tyrosine residue in the active site. RNAi silencing of any of both protomers induces a non-viable phenotype in the hemoflagelate <it>Trypanosoma brucei</it>. Unfortunately, this approach is not suitable in <it>Leishmania </it>where gene replacement with an antibiotic marker is the only approach to generate lack-of-function mutants. In this work, we have successfully generated null mutants in the small subunit of the <it>L. major </it>DNA topoisomerase IB using two selection markers, each conferring resistance to hygromycin B and puromycin, respectively.</p> <p>Results</p> <p>We have successfully replaced both <it>topS </it>loci with two selection markers. However, to achieve the second transfection round, we have had to rescue the null-homozygous with an episomal vector carrying the <it>Leishmania major topS </it>gene. Phenotypic characterization of the <it>L. major </it>rescued strain and a <it>L. major </it>strain, which co-overexpresses both subunits, shows few differences in DNA relaxation and camptothecin cytotoxicity when it was compared to the wild-type strain. Studies on phosphatidylserine externalization show a poor incidence of camptothecin-induced programmed cell death in <it>L. major</it>, but an effective cell-cycle arrest occurs within the first 24 h. S-Phase delay and G₂/M reversible arrest was the main outcome at lower concentrations, but irreversible G₂arrest was detected at higher camptothecin pressure.</p> <p>Conclusion</p> <p>Results obtained in this work evidence the essentiality of the <it>topS </it>gene encoding the <it>L. major </it>DNA topoisomerase IB small subunit. Reversibility of the camptothecin effect points to the existence of effective checkpoint mechanisms in <it>Leishmania </it>parasites.</p

Trypanosomatids topoisomerase re-visited. New structural findings and role in drug discovery

AbstractThe Trypanosomatidae family, composed of unicellular parasites, causes severe vector-borne diseases that afflict human populations worldwide. Chagas disease, sleeping sickness, as well as different sorts of leishmaniases are amongst the most important infectious diseases produced by Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively. All these infections are closely related to weak health care services in low-income populations of less developed and least economically developed countries. Search for new therapeutic targets in order to hit these pathogens is of paramount priority, as no effective vaccine is currently in use against any of these parasites. Furthermore, present-day chemotherapy comprises old-fashioned drugs full of important side effects. Besides, they are prone to produce tolerance and resistance as a consequence of their continuous use for decades. DNA topoisomerases (Top) are ubiquitous enzymes responsible for solving the torsional tensions caused during replication and transcription processes, as well as in maintaining genomic stability during DNA recombination. As the inhibition of these enzymes produces cell arrest and triggers cell death, Top inhibitors are among the most effective and most widely used drugs in both cancer and antibacterial therapies. Top relaxation and decatenation activities, which are based on a common nicking–closing cycle involving one or both DNA strands, have been pointed as a promising drug target. Specific inhibitors that bind to the interface of DNA-Top complexes can stabilize Top-mediated transient DNA breaks. In addition, important structural differences have been found between Tops from the Trypanosomatidae family members and Tops from the host. Such dissimilarities make these proteins very interesting for drug design and molecular intervention. The present review is a critical update of the last findings regarding trypanosomatid’s Tops, their new structural features, their involvement both in the physiology and virulence of these parasites, as well as their use as promising targets for drug discovery

Hybrid Quinolinyl Phosphonates as Heterocyclic Carboxylate Isosteres: Synthesis and Biological Evaluation against Topoisomerase 1B (TOP1B)

This work describes, for the first time, the synthesis of dialkyl (2-arylquinolin-8-yl)phosphonate derivatives. The preparation was carried out through a direct and simple process as a multicomponent Povarov reaction of aminophenylphosphonates, aldehydes, and styrenes and subsequent oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or, alternatively, by a cycloaddition reaction between phosphonate aldimines and acetylenes. Based on phosphonate group structural characteristics, considered as phosphorous isosteres of carboxylic heterocycles, they may present interesting biological properties related to cell proliferation. In the current report, a new series of dialkyl (2-arylquinolin-8-yl)phosphonates have been synthesized and their antiproliferative effect evaluated on different human cancer and embryonic cells, as well as on Leishmania infantum parasites, a eukaryotic protist responsible for visceral leishmaniasis. Thereby, the antitumor effect was assessed in human lung adenocarcinoma cells (A549), human ovarian carcinoma cells (SKOV3), and human embryonic kidney cells (HEK293) versus the non-cancerous lung fibroblasts cell line (MRC5). On the other hand, the antileishmanial activity was tested against both stages of L. infantum cell cycle, namely free-living promastigotes and intramacrophage amastigotes, using a primary culture of Balb/c splenocytes to calculate the selectivity index. Besides the antiproliferative and antileishmanial capacities, their behavior as topoisomerase 1B inhibitors has been evaluated as a possible mechanism of action.This research was financially supported by Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) y Fondo Europeo de Desarrollo Regional (FEDER; RTI2018–101818-B-I00, UE) and by Gobierno Vasco, Universidad del País Vasco (GV, IT 992–16; UPV) is gratefully acknowledged. Technical and human support provided by IZO-SGI, SGIker (UPV/EHU, MICINN, GV/EJ, ERDF, and ESF) is gratefully acknowledged. AS thanks the Basque Government for a formation contract. This collaborative research was funded by MINECO; SAF2017–83575-R to RMR

Aedes albopictus diversity and relationships in south-western Europe and Brazil by rDNA/mtDNA and phenotypic analyses: ITS-2, a useful marker for spread studies

Background: Aedes albopictus is a very invasive mosquito, which has recently colonized tropical and temperate regions worldwide. Of concern is its role in the spread of emerging or re-emerging mosquito-borne diseases. Ae. albopictus from south-western Europe and Brazil were studied to infer genetic and phenetic diversity at intra-individual, intra-population and inter-population levels, and to analyse its spread. Methods: Genotyping was made by rDNA 5.8S-ITS-2 and mtDNA cox1 sequencing to assess haplotype and nucleotide diversity, genetic distances and phylogenetic networks. Male and female phenotyping included combined landmark-and outlined-based geometric morphometrics of wing size and shape. Results: Specimens from seven populations from Spain, France and Brazil provided 12 cox1 and 162 5.8S-ITS-2 haplotypes, with great genetic variability difference between both markers (0.9% vs 31.2%). Five cox1 haplotypes were shared with other countries, mainly Italy, USA and China, but none was shared between Europe and Brazil. The 5.8S-ITS-2 showed 2–7 intra-individual (mean 4.7) and 16–34 intra-/inter-population haplotypes (24.7), including haplotypes shared between Spain, France and Brazil. A 4.3% of ITS-2 haplotypes were shared, mainly with Italy, USA and Thailand, evidencing worldwide spread and introductions from areas where recent outbreaks of Ae. albopictus-transmitted pathogens occurred. Wing size showed sex differences. Wing shape distinguished between Brazilian and European specimens. Both genetic and morphometric markers showed differences between insular Spain and continental Spain, France and Brazil. Conclusions: ITS-2 proves to be a useful marker to assess Ae. albopictus spread, providing pronouncedly more information than cox1, including intra-individual, intra-population and inter-population levels, furnishing a complete overview of the evolutionary exchanges followed by this mosquito. Wing morphometry proves to be a useful phenotyping marker, allowing to distinguish different populations at the level of both male and female specimens. Results indicate the need for periodic surveillance monitorings to verify that no Ae. albopictus with high virus transmission capacity is introduced into Europe. Graphic Abstract: [Figure not available: see fulltext.] © 2021, The Author(s)

Gel-Free Tools for Quick and Simple Screening of Anti-Topoisomerase 1 Compounds

With the increasing need for effective compounds against cancer or pathogen-borne diseases, the development of new tools to investigate the enzymatic activity of biomarkers is necessary. Among these biomarkers are DNA topoisomerases, which are key enzymes that modify DNA and regulate DNA topology during cellular processes. Over the years, libraries of natural and synthetic small-molecule compounds have been extensively investigated as potential anti-cancer, anti-bacterial, or anti-parasitic drugs targeting topoisomerases. However, the current tools for measuring the potential inhibition of topoisomerase activity are time consuming and not easily adaptable outside specialized laboratories. Here, we present rolling circle amplification-based methods that provide fast and easy readouts for screening of compounds against type 1 topoisomerases. Specific assays for the investigation of the potential inhibition of eukaryotic, viral, or bacterial type 1 topoisomerase activity were developed, using human topoisomerase 1, Leishmania donovani topoisomerase 1, monkeypox virus topoisomerase 1, and Mycobacterium smegmatis topoisomerase 1 as model enzymes. The presented tools proved to be sensitive and directly quantitative, paving the way for new diagnostic and drug screening protocols in research and clinical settings.This research was funded in part by the Ministerio de Ciencia e Innovación, Spain (PID2021-122558OB-I00, UE) and by Gobierno Vasco, Universidad del País Vasco (GV, IT1701-22; UPV)

Simple and Fast DNA Based Sensor System for Screening of Small-Molecule Compounds Targeting Eukaryotic Topoisomerase 1

Background: Eukaryotic topoisomerase 1 is a potential target of anti-parasitic and anti-cancer drugs. Parasites require topoisomerase 1 activity for survival and, consequently, compounds that inhibit topoisomerase 1 activity may be of interest. All effective topoisomerase 1 drugs with anti-cancer activity act by inhibiting the ligation reaction of the enzyme. Screening for topoisomerase 1 targeting drugs, therefore, should involve the possibility of dissecting which step of topoisomerase 1 activity is affected. Methods: Here we present a novel DNA-based assay that allows for screening of the effect of small-molecule compounds targeting the binding/cleavage or the ligation steps of topoisomerase 1 catalysis. This novel assay is based on the detection of a rolling circle amplification product generated from a DNA circle resulting from topoisomerase 1 activity. Results: We show that the binding/cleavage and ligation reactions of topoisomerase 1 can be investigated separately in the presented assay termed REEAD (C|L) and demonstrate that the assay can be used to investigate, which of the individual steps of topoisomerase 1 catalysis are affected by small-molecule compounds. The assay is gel-free and the results can be detected by a simple colorimetric readout method using silver-on-gold precipitation rendering large equipment unnecessary. Conclusion: REEAD (C|L) allows for easy and quantitative investigations of topoisomerase 1 targeting compounds and can be performed in non-specialized laboratories.K.V. thanks Aase and Ejnar Danielsens Fond, Aage and Johanne Louis-Hansens Fond and Edel and Wilhelm Daubenmerkls Almenvelgørende Fond; A.S. thanks the Basque Government for a formation contract

Deletion Study of DNA Topoisomerase IB from Leishmania donovani: Searching for a Minimal Functional Heterodimer

The substantial differences between trypanosomal and leishmanial DNA topoisomerase IB concerning to their homologues in mammals have provided a new lead in the study of the structural determinants that can be effectively targeted. Leishmania donovani, the causative agent of visceral leishmaniasis, contains an unusual heterodimeric DNA topoisomerase IB. The catalytically active enzyme consists of a large subunit (LdTopIL), which contains the non-conserved N-terminal end and the phylogenetically conserved “core” domain, and of a small subunit (LdTopIS) which harbors the C-terminal region with the characteristic tyrosine residue in the active site. Heterologous co-expression of LdTopIL and LdTopIS genes in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme LdTopIL/S which can be used for structural studies. An approach by combinatorial cloning of deleted genes encoding for truncated versions of both subunits was used in order to find out structural insights involved in enzyme activity or protein-protein interaction. The role played by the non-conserved N-terminal extension of LdTopIL in both relaxation activity and CPT sensitivity has been examined co-expressing the full-length LdTopIS and a fully active LdTopIΔS deletion with several deletions of LdTopIL lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 26 amino acids placed at the N-terminal end and a variable region comprised between Ala548 to end of the C-terminal extension of LdTopIL were enzymatically dispensable. Altogether this combinatorial approach provides important structural insights of the regions involved in relaxation activity and for understanding the atypical structure of this heterodimeric enzyme

Research Group on Sedimentary Record of Climatic Changes– SERCC

[EN] SERCC was created at the end of 2021 in response to the recent incorporation of the IGME as a National Centre of the Spanish Research Council (CSIC) and the need to converge to CSIC´s research organization, which is structured on Research Groups as basic units. SERCC is composed of 11 persons (6 Staff Scientists – 3 of them recently promoted to Scientific Researchers, 4 Specialized Technicians and 1 Predoctoral Student), and focuses on the imprint of past climate changes on the characteristics and properties of the sedimentary record, accumulated both in marine and continental realms.Peer reviewe

Influence of the Cumulative Incidence of COVID-19 Cases on the Mental Health of the Spanish Out-of-Hospital Professionals

This study aimed to analyze the psychological affectation of health professionals (HPs) of Spanish Emergency Medical Services (EMSs) according to the cumulative incidence (CI) of COVID19 cases in the regions in which they worked. A cross-sectional descriptive study was designed, including all HPs working in any EMS of the Spanish geography between 1 February 2021 and 30 April 2021. Their level of stress, anxiety and depression (DASS-21) and the perception of self-efficacy (GSES) were the study’s main results. A 2-factor analysis of covariance was used to determine if the CI regions of COVID-19 cases determined the psychological impact on each of the studied variables. A total of 1710 HPs were included. A third presented psychological impairment classified as severe. The interaction of CI regions with the studied variables did not influence their levels of stress, anxiety, depression or self-efficacy. Women, younger HPs or those with less EMS work experience, emergency medical technicians (EMT), workers who had to modify their working conditions or those who lived with minors or dependents suffered a greater impact from the COVID-19 pandemic in certain regions. These HPs have shown high levels of stress, anxiety, depression and medium levels of self-efficacy, with similar data in the different geographical areas. Psychological support is essential to mitigate their suffering and teach them to react to adverse events.This research was funded by Fundación ASISA and Sociedad Española de Urgencias y Emergencias (SEMES)