Apn1 AP-endonuclease is essential for the repair of oxidatively damaged DNA bases in yeast frataxin-deficient cells

International audienceFrataxin deficiency results in mitochondrial dysfunction and oxidative stress and it is the cause of the hereditary neurodegenerative disease Friedreich ataxia (FA). Here, we present evidence that one of the pleiotrop-ic effects of oxidative stress in frataxin-deficient yeast cells (Dyfh1 mutant) is damage to nuclear DNA and that repair requires the Apn1 AP-endonuclease of the base excision repair pathway. Major phenotypes of Dyfh1 cells are respiratory deficit, disturbed iron homeostasis and sensitivity to oxidants. These phenotypes are weak or absent under anaerobiosis. We show here that exposure of anaerobically grown Dyfh1 cells to oxygen leads to down-regulation of antioxidant defenses, increase in reactive oxygen species, delay in G1-and S-phases of the cell cycle and damage to mitochondrial and nuclear DNA. Nuclear DNA lesions in Dyfh1 cells are primarily caused by oxidized bases and single-strand breaks that can be detected 15-30 min after oxygen exposition. The Apn1 enzyme is essential for the repair of the DNA lesions in Dyfh1 cells. Compared with Dyfh1, the double Dyfh1Dapn1 mutant shows growth impairment, increased mu-tagenesis and extreme sensitivity to H 2 O 2. On the contrary, overexpression of the APN1 gene in Dyfh1 cells decreases spontaneous and induced mutagenesis. Our results show that frataxin deficiency in yeast cells leads to increased DNA base oxidation and requirement of Apn1 for repair, suggesting that DNA damage and repair could be important features in FA disease progression

Clustering of strong replicators associated with active promoters is sufficient to establish an early-replicating domain

Vertebrate genomes replicate according to a precise temporal program strongly correlated with their organization into A/B compartments. Until now, the molecular mechanisms underlying the establishment of early-replicating domains remain largely unknown. We defined two minimal cis-element modules containing a strong replication origin and chromatin modifier binding sites capable of shifting a targeted mid-late-replicating region for earlier replication. The two origins overlap with a constitutive or a silent tissue-specific promoter. When inserted side-by-side, these modules advance replication timing over a 250 kb region through the cooperation with one endogenous origin located 30 kb away. Moreover, when inserted at two chromosomal sites separated by 30 kb, these two modules come into close physical proximity and form an early-replicating domain establishing more contacts with active A compartments. The synergy depends on the presence of the active promoter/origin. Our results show that clustering of strong origins located at active promoters can establish early-replicating domains

Rapid Species Diagnosis for Invasive Candidiasis Using Mass Spectrometry

BACKGROUND: Matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF-MS) allows the identification of most bacteria and an increasing number of fungi. The potential for the highest clinical benefit of such methods would be in severe acute infections that require prompt treatment adapted to the infecting species. Our objective was to determine whether yeasts could be identified directly from a positive blood culture, avoiding the 1-3 days subculture step currently required before any therapeutic adjustments can be made. METHODOLOGY/PRINCIPAL FINDINGS: Using human blood spiked with Candida albicans to simulate blood cultures, we optimized protocols to obtain MALDI TOF-MS fingerprints where signals from blood proteins are reduced. Simulated cultures elaborated using a set of 12 strains belonging to 6 different species were then tested. Quantifiable spectral differences in the 5000-7400 Da mass range allowed to discriminate between these species and to build a reference database. The validation of the method and the statistical approach to spectral analysis were conducted using individual simulated blood cultures of 36 additional strains (six for each species). Correct identification of the species of these strains was obtained. CONCLUSIONS/SIGNIFICANCE: Direct MALDI TOF-MS analysis of aliquots from positive blood cultures allowed rapid and accurate identification of the main Candida species, thus obviating the need for sub-culturing on specific media. Subsequent to this proof-of-principle demonstration, the method can be extended to other clinically relevant yeast species, and applied to an adequate number of clinical samples in order to establish its potential to improve antimicrobial management of patients with fungemia

Evaluation of the chagas western blot igg assay for the diagnosis of chagas disease

Chagas disease is a debilitating and often fatal pathology resulting from infection by the protozoan parasite Trypanosoma cruzi. In its recommendations, the World Health Organization states that the diagnosis of T. cruzi infection is usually based on the detection of antibodies against T. cruzi antigens and performed with two methodologically different assays. An inconclusive result can be resolved with a third “confirmatory” assay. The objective of this article is to evaluate the effectiveness of the Chagas Western Blot IgG assay (LDBio Diagnostics, Lyon, France) as a confirmatory serologic test. The Chagas Western Blot IgG assay was performed with native antigens derived from a T. cruzi strain of the TcVI genotype. Retrospective sera were provided by two parasitology laboratories (France and Argentina). The sensitivity, specificity, positive predictive value and negative predictive value of the Chagas blot were all 100% in our sera collection. The Chagas blot is an easy and qualitative method for the diagnosis of Chagas disease, with results in less than 2 h. This immunoblot has potential as a supplemental test for the confirmation of the presence of antibodies against T. cruzi in serum specimens. Nonetheless, the very good initial results presented here will need to be confirmed in larger studies.Fil: Brossas, Jean Yves. Sorbonne Université Hôpital Pitié-Salpêtrière; FranciaFil: Ballering, Griselda Edith. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; ArgentinaFil: Bisio, Margarita María Catalina. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; ArgentinaFil: Guihenneuc, Jeremy. Sorbonne Université Hôpital Pitié-Salpêtrière; FranciaFil: Gulin, Julián Ernesto Nicolás. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Jauréguiberry, S.. Sorbonne Université Hôpital Pitié-Salpêtrière; FranciaFil: Lescure, François Xavier. Hôpitaux Universitaires Paris Nord val de Seine; FranciaFil: Fekkar, Arnaud. Sorbonne Université Hôpital Pitié-Salpêtrière; FranciaFil: Mazier, Dominique. Sorbonne University; FranciaFil: Altcheh, Jaime Marcelo. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; ArgentinaFil: Paris, Luc. Sorbonne Université Hôpital Pitié-Salpêtrière; Franci

Aspergillus PCR in Bronchoalveolar Lavage Fluid for the Diagnosis and Prognosis of Aspergillosis in Patients With Hematological and Non-hematological Conditions

Objectives: We evaluated the usefulness of an Aspergillus fumigatus quantitative PCR assay performed in bronchoalveolar lavage fluid (BAL) for the diagnosis and prognosis of both invasive and non-invasive aspergillosis.Methods: This 4-year retrospective study involved 613 at-risk patients who had either hematological disorders or other immunosuppressive conditions, notably solid organ transplants. Thirty-five patients had proven/probable aspergillosis and thirteen had chronic non-invasive aspergillosis. We compared PCR, galactomannan index and mycological analysis of BAL.Results: For invasive aspergillosis (IA), PCR performed in BAL yielded 88.6% sensitivity and 95.5% specificity. Comparatively, galactomannan index and mycological examination yielded only 56.3 and 63.6% sensitivity and 97.6 and 94.5% specificity, respectively. Considering the 13 chronic aspergillosis cases, PCR, galactomannan index and mycological examination yielded 76.9, 15.4, and 84.6% sensitivity and 92.2, 94.9, and 93% specificity, respectively. Fungal load in BAL evaluated by PCR was able to discriminate between aspergillosis and contamination, but not between invasive and non-invasive forms. Finally, fungal load was predictive of 90-day mortality, with 23.1% mortality for patients with less than 500 copies/mL versus 68.4% for patients above that cut-off (p < 0.05).Conclusion: Our results indicate that Aspergillus PCR in BAL is of particular interest for both the diagnosis and the prognosis of IA. It is likewise an interesting tool for the diagnosis of non-invasive forms

New 8-nitroquinolinone derivative displaying submicromolar in vitro activities against both Trypanosoma brucei and cruzi

International audienceAn antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L. infantum, T. brucei brucei, and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed, presenting EC50 values of 12 and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes respectively, in comparison with four reference drugs (30 nM ≤ EC50 ≤ 13 μM). Moreover, compound 12 was not genotoxic in the comet assay and showed high in vitro microsomal stability (half life >40 min) as well as favorable pharmacokinetic behavior in the mouse after oral administration. Finally, molecule 12 (E° = −0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, in both Leishmania and Trypanosoma, and appears to be a good candidate to search for novel antitrypanosomal lead compounds

New diagnostic approaches to American Trypanosomiasis

La maladie de Chagas affecte six à huit personnes dans le monde. Depuis quelques décennies, cette pathologie se diffuse hors de sa zone d’endémie, l’Amérique latine. Cette infection présente de grandes difficultés tant pour le diagnostic que pour le traitement. Malgré les avancées importantes de ces dernières années, l'amélioration du diagnostic reste un défi majeur dans la prise en charge des patients atteints de Trypanosomiase Américaine. Dans une première partie, nous présentons nos études sur le développement d'un nouveau test de diagnostic basé sur l'identification directe d'antigènes parasitaires présents dans le sérum. Nous avons analysé le surnageant de culture de cellules infectées par T. cruzi par spectrométrie de masse et nous avons identifié une protéase parasitaire, la Chagasin, qui pourrait être un marqueur sérique de l’infection à T. cruzi. Dans une deuxième partie, nous présentons une étude permettant d’interpréter et d’évaluer un nouveau test diagnostic "Western Chagas IgG" fabriqué, à partir d’extraits protéique de T. cruzi, conjointement par la société LDBIO Diagnostics (Lyon, France) et le laboratoire de parasitologie et de mycologie de l'hôpital Pitié Salpétrière. Les résultats de cette étude montrent que les valeurs de sensibilité et de spécificité de ce test sont proches de 100%. Nous considérons que l'immunoblot "Western Chagas IgG"est un excellent test diagnostic de confirmation de la maladie de Chagas et destiné à la vérification de résultats équivoques obtenus par le biais de tests de dépistage plus classiques.Six to eight people are affected by Chagas disease worldwide. Most of the patients are found in South America; however, more and more countries in the north are confronted with this infection that presents great difficulties both in the diagnosis and in the treatment. Despite significant technological advances in recent years, improved diagnosis is a major challenge in the management of patients with Chagas disease. In a first part, we present our studies about the development of a new diagnostic test based on the direct identification of parasitic antigens present in the serum. We analyzed the culture supernatant of cells infected with T.cruzi by mass spectrometry and we identified soluble parasitic proteins. Subsequently, we showed that Chagasin, a parasitic protease, which could be good serum marker of T. cruzi infection.In second part, we present a study that allow the interpretation and evaluation of a new immunoblot assay "Western Chagas IgG" (CE mark) by LDBIO Diagnostics (Lyon, France). This assay based on trypomastigote and amastigote extract manufactured by the laboratory of parasitology and mycology of Pitié Salpétrière hospital. These results show that this assay has sensitivity and specificity values close to 100% and is considered an excellent serological assay intended for confirmatory testing of a positive or equivocal result obtained through classic screening tests

Evolution of replication origins in vertebrate genomes: rapid turnover despite selective constraints

The replication program of vertebrate genomes is driven by the chromosomal distribution and timing of activation of tens of thousands of replication origins. Genome-wide studies have shown the association of origins with promoters and CpG islands, and their enrichment in G-quadruplex motifs (G4). However, the genetic determinants driving their activity remain poorly understood. To gain insight on the constraints operating on origins, we conducted the first evolutionary comparison of origins across vertebrates. We generated a genome-wide map of chicken origins (the first of a bird genome), and performed a comparison with human and mouse maps. The analysis of intra-species polymorphism revealed a strong depletion of genetic diversity at the core of replication initiation loci. This depletion is not linked to the presence of G4 motifs, promoters or CpG islands. In contrast, we show that origins experienced a rapid turnover during vertebrate evolution, since pairwise comparisons of origin maps revealed that <24% of them are conserved among vertebrates. This study unravels the existence of a novel determinant of origins, the precise functional role of which remains to be determined. Despite the importance of replication initiation for the fitness of organisms, the distribution of origins along vertebrate chromosomes is highly flexible.Association pour la Recherche sur le Cancer [Labellisation PGA120150202272 to M.-N.P., F.P.]; Agence Nationale de la Recherche [ANR-15-CE12-0004, OriMolMech to F.M., M.-N.P., F.P.]; Spanish Ministry of Economy and Competitiveness [BFU2016-78849-P, co-financed by the European UnionFEDERfunds to J.M.F.-J., M.G.