Biochemical characterization of recombinant human telomerase

Résumé Les télomères sont les structures ADN-protéines des extrémités des chromosomes des eucaryotes. L'ADN télomérique est constitué de courtes séquences répétitives. L'intégrité des télomères est essentielle pour protéger les extrémités des chromosomes contre les systèmes de dégradations et pour les distinguer des cassures de l'ADN double brin. Parce que la machinerie de la réplication de l'ADN n'est pas capable de répliquer l'extrémité des chromosomes, les télomères raccourcissent au fur et à mesure des cycles de réplication. Dès que les télomères atteignent une longueur critique, leur structure protectrice est perdue. Cela induit un signal de dommage de l'ADN et l'arrêt du cycle cellulaire. Pour contrebalancer le raccourcissement des télomères, les cellules qui s'auto régénèrent, dont les cellules de la moelle osseuse, les lymphocytes activés et 80-90% des cellules cancéreuses, expriment la télomérase. C'est une ribonucléoprotéine qui a la capacité de synthétiser des séquences télomériques par transcription inverse d'une courte séquence contenue dans sa propre sous-unité ARN avec laquelle elle est associée. La télomérase humaine est une enzyme processive au niveau de l'addition des nucléotides et aussi des répétitions télomériques. La télomérase de levure et la télomérase humaine sont toutes deux dimériques et il a été montré que la télomérase humaine recombinante contient deux ARN qui coopèrent pour fonctionner ainsi que deux sous-unités catalytiques. Cependant, il n'a pas encore été montré quel est le rôle de la dimérisation dans l'activité de la télomérase. Afin d'élucider ce rôle, nous avons exprimé, reconstitué et purifié la télomérase humaine dimérique recombinante. Et pour étudier l'effet d'ARN mutants sur l'activité de la télomérase, nous avons développé une méthode pour reconstituer et enrichir en hétérodimères de télomérase. Les hétérodimères contiennent une sous-unité ARN sauvage et une sous-unité ARN mutée au niveau de la séquence de la matrice. Sur l'ARN muté nous avons introduit une étiquette aptamer ARN-S1 puis nous avons purifié la télomérase via l'etiquette Si. Nous avons montré que la dimérisation est essentielle pour l'activité de la télomérase. Nos données indiquent que chaque télomérase du dimère allonge leur substrat, l'ADN télomérique, indépendamment l'une de l'autre à chaque cycle d'élongation mais que l'addition itérative de répétitions télomériques nécessite une coopération entre les deux télomérases du dimère. Nous proposons donc un modèle dans lequel les deux télomérases du dimères se lient et allongent deux substrats télomères et que pendant l'élongation processive les deux enzymes subissent un changement de conformation de manière coordonnée, ce changement va permettre le repositionnement des substrats pour d'autres cycles d'additions de répétitions télomériques. Dyskeratosis congenita est une maladie mortelle due majoritairement au disfonctionnement de la moelle osseuse. Dans la forme autosomale de la maladie, l'ARN de la télomérase contient des mutations. En utilisant notre système de reconstitution, nous avons montré que ces ARN mutés, qui ont perdu leur activité enzymatique dans le cas d'un homodimère de mutants, sont dominant négatifs quand ils sont présents dans les hétérodimères sauvage/mutant. Cet effet trans-dominant négatif pourrait contribuer à la progression de la maladie. Abstract Telomeres are protein-DNA structures at the ends of linear eukaryotic chromosomes. The telomeric DNA consists of tandemly repeated sequences. Telomeric integrity is essential to protect chromosomal ends from nucleolytic degradation and to prevent their recognition as DNA double strand breaks. Due to the inability of the conventional DNA replication machinery to replicate terminal DNA stretches, telomeres shorten with continuous rounds of DNA replication. As soon as telomeres reach a critical length, their protective structure is lost and the deprotected telomeres will induce a DNA damage response leading to cell cycle arrest. To counteract telomere shortening, self-renewing cells, including bone marrow cells, activated lymphocytes and 80-90% of cancer cells express the cellular reverse transcriptase telomerase, which has the capacity to synthesize telomeric repeats by reverse transcription of a short template sequence encoded by its stably associated RNA subunit. Human telomerase is a processive enzyme for nucleotide as well as repeat addition. Both yeast and human telomerase are dimeric enzymes and recombinant human telomerase has been shown to contain two functionally cooperating RNAs and most probably also two protein subunits. However, it has remained unclear how dimerization may contribute to telomerase activity. To study the role of dimerization, we expressed, reconstituted and purified recombinant human telomerase. We also developed a new method to reconstitute and enrich for telomerase heterodimers containing wild-type (wt) and mutant telomerase RNA subunits. To this end we introduced an S1-RNA-aptamer tag into telomerase RNA and purified telomerase reconstituted with a mixture of untagged and tagged RNA via the S1-tag. Using this experimental system, we introduced template mutations in the tagged RNA subunit and examined the effect of mutant RNAs on wt telomerase activity in wt/mutant heterodimers. We obtained evidence that dimerization is essential for telomerase activity. Our data indicate that the two subunits elongate telomere substrates independently of each other during single rounds of elongation, but that iterative addition of telomeric repeats requires cooperation between the two subunits. We suggest a model, in which dimeric telomerases bind and elongate two telomere substrates and that the two subunits undergo coordinated conformational changes during processive elongation that enable repositioning the substrates for subsequent rounds of repeat addition. Dyskeratosis congenita is a multisystemic disease with bone marrow failure as the major cause of death. The autosomal form of this disease was found to harbor mutations in the telomerase RNA. Using our reconstitution system, we tested whether mutant dyskeratosis telomerase RNAs behaved in a dominant negative manner. We observed that dyskeratosis telomerase RNA mutants, which lacked enzymatic activity were dominant negative, when present in wt/ mutant heterodimers. The transdominant negative effect of these mutants may contribute to disease progression.

Acute myeloid leukaemia of donor cell origin developing 17 years after allogenic hematopoietic cell transplantation for acute promyelocytic leukaemia.

Donor cell leukaemia (DCL) is a rare complication of allogenic hematopoietic cell transplantation (HCT). We report the case of a female patient with acute promyelocytic leukaemia (APL), FAB type M3, who developed acute myeloid leukaemia (AML) type M5 of donor origin 17 years after allogenic bone marrow transplantation (BMT) from her HLA-matched sister. Morphology and immunophenotyping showed differences with the initial leukaemia, and short tandem repeat (STR) analysis confirmed donor-type haematopoiesis. Interphase fluorescence in situ hybridisation (FISH) showed an 11q23 deletion. Given that the latency period between transplant and development of leukaemia was the longest reported to date, we discuss the mechanisms underlying delayed leukaemia onset.

Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus.Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts inR. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.

Are there positive emotions in short-term dynamic psychotherapy or is it all Freude-less ?

Psychodynamic therapists are often suspicious of positive emotions and consider them to be nothing more than a form of denial or of another defense aiming to diminish painful or difficult affects. Positive emotions seem to exist only through the absence of negative emotions or as something that may happen outside of therapy. On the other hand, clinicians also agree that psychoanalytic work could not be successful without such positive emotions as interest, pleasure, surprise and creativity. Contemporary psychoanalytic thinking and new research findings in the area of relationship regulation are likely to give positive emotions an increasingly prominent place in dynamically oriented therapies. With today's emphasis on the therapeutic relationship and intersubjectivity, the time appears right to integrate positive emotions more formally into psychodynamic clinical theories.

Inferences about the global scenario of human T-cell lymphotropic virus type 1 infection using data mining of viral sequences

Human T-cell lymphotropic virus type 1 (HTLV-1) is mainly associated with two diseases: tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM) and adult T-cell leukaemia/lymphoma. This retrovirus infects five-10 million individuals throughout the world. Previously, we developed a database that annotates sequence data from GenBank and the present study aimed to describe the clinical, molecular and epidemiological scenarios of HTLV-1 infection through the stored sequences in this database. A total of 2,545 registered complete and partial sequences of HTLV-1 were collected and 1,967 (77.3%) of those sequences represented unique isolates. Among these isolates, 93% contained geographic origin information and only 39% were related to any clinical status. A total of 1,091 sequences contained information about the geographic origin and viral subtype and 93% of these sequences were identified as subtype “a”. Ethnicity data are very scarce. Regarding clinical status data, 29% of the sequences were generated from TSP/HAM and 67.8% from healthy carrier individuals. Although the data mining enabled some inferences about specific aspects of HTLV-1 infection to be made, due to the relative scarcity of data of available sequences, it was not possible to delineate a global scenario of HTLV-1 infection.

Morphology of the larvae, male genitalia and DNA sequences of Anopheles (Kerteszia) pholidotus (Diptera: Culicidae) from Colombia

Since 1984, Anopheles (Kerteszia) lepidotus has been considered a mosquito species that is involved in the transmission of malaria in Colombia, after having been incriminated as such with epidemiological evidence from a malaria outbreak in Cunday-Villarrica, Tolima. Subsequent morphological analyses of females captured in the same place and at the time of the outbreak showed that the species responsible for the transmission was not An. lepidotus, but rather Anopheles pholidotus. However, the associated morphological stages and DNA sequences of An. pholidotus from the foci of Cunday-Villarrica had not been analysed. Using samples that were caught recently from the outbreak region, the purpose of this study was to provide updated and additional information by analysing the morphology of female mosquitoes, the genitalia of male mosquitoes and fourth instar larvae of An. pholidotus, which was confirmed with DNA sequences of cytochrome oxidase I and rDNA internal transcribed spacer. A total of 1,596 adult females were collected in addition to 37 larval collections in bromeliads. Furthermore, 141 adult females, which were captured from the same area in the years 1981-1982, were analysed morphologically. Ninety-five DNA sequences were analysed for this study. Morphological and molecular analyses showed that the species present in this region corresponds to An. pholidotus. Given the absence of An. lepidotus, even in recent years, we consider that the species of mosquitoes that was previously incriminated as the malaria vector during the outbreak was indeed An. pholidotus, thus ending the controversy.

On spectral approximation, Folner sequences and crossed products

In this article we review first some of the possibilities in which the notions of Fo lner sequences and quasidiagonality have been applied to spectral approximation problems. We construct then a canonical Fo lner sequence for the crossed product of a concrete C* -algebra and a discrete amenable group. We apply our results to the rotation algebra (which contains interesting operators like almost Mathieu operators or periodic magnetic Schrödinger operators on graphs) and the C* -algebra generated by bounded Jacobi operators.

Ecoepidemiology, short history and control of Chagas disease in the endemic countries and the new challenge for non-endemic countries

Chagas disease is maintained in nature through the interchange of three cycles: the wild, peridomestic and domestic cycles. The wild cycle, which is enzootic, has existed for millions of years maintained between triatomines and wild mammals. Human infection was only detected in mummies from 4,000-9,000 years ago, before the discovery of the disease by Carlos Chagas in 1909. With the beginning of deforestation in the Americas, two-three centuries ago for the expansion of agriculture and livestock rearing, wild mammals, which had been the food source for triatomines, were removed and new food sources started to appear in peridomestic areas: chicken coops, corrals and pigsties. Some accidental human cases could also have occurred prior to the triatomines in peridomestic areas. Thus, triatomines progressively penetrated households and formed the domestic cycle of Chagas disease. A new epidemiological, economic and social problem has been created through the globalisation of Chagas disease, due to legal and illegal migration of individuals infected by Trypanosoma cruzi or presenting Chagas disease in its varied clinical forms, from endemic countries in Latin America to non-endemic countries in North America, Europe, Asia and Oceania, particularly to the United States of America and Spain. The main objective of the present paper was to present a general view of the interchanges between the wild, peridomestic and domestic cycles of the disease, the development of T. cruzi among triatomine, their domiciliation and control initiatives, the characteristics of the disease in countries in the Americas and the problem of migration to non-endemic countries.

Folner sequences and finite operators

This article analyzes Folner sequences of projections for bounded linear operators and their relationship to the class of finite operators introduced by Williams in the 70ies. We prove that each essentially hyponormal operator has a proper Folner sequence (i.e. a Folner sequence of projections strongly converging to 1). In particular, any quasinormal, any subnormal, any hyponormal and any essentially normal operator has a proper Folner sequence. Moreover, we show that an operator is finite if and only if it has a proper Folner sequence or if it has a non-trivial finite dimensional reducing subspace. We also analyze the structure of operators which have no Folner sequence and give examples of them. For this analysis we introduce the notion of strongly non-Folner operators, which are far from finite block reducible operators, in some uniform sense, and show that this class coincides with the class of non-finite operators.

The N-terminal DNA-binding 'zinc finger' of the oestrogen and glucocorticoid receptors determines target gene specificity.

Steroid hormone receptors activate specific gene transcription by binding as hormone-receptor complexes to short DNA enhancer-like elements termed hormone response elements (HREs). We have shown previously that a highly conserved 66 amino acid region of the oestrogen (ER) and glucocorticoid (GR) receptors, which corresponds to part of the receptor DNA binding domain (region C) is responsible for determining the specificity of target gene activation. This region contains two sub-regions (CI and CII) analogous to the 'zinc-fingers' of the transcription factor TFIIIA. We show here that CI and CII appear to be separate domains both involved in DNA binding. Furthermore, using chimaeric ERs in which either the first (N-terminal) (CI) or second (CII) 'zinc finger' region has been exchanged with that of the GR, indicates that it is the first 'zinc finger' which largely determines target gene specificity. We suggest that receptor recognition of the HRE is analogous to that of the helix-turn-helix DNA binding motif in that the receptor binds to DNA as a dimer with the first 'zinc finger' lying in the major groove recognizing one half of the palindromic HRE, and that protein-DNA interaction is stabilized through non-specific DNA binding and dimer interactions contributed by the second 'zinc finger'.

Whole-genome sequences of influenza A(H3N2) viruses isolated from Brazilian patients with mild illness during the 2014 season

The influenza A(H3N2) virus has circulated worldwide for almost five decades and is the dominant subtype in most seasonal influenza epidemics, as occurred in the 2014 season in South America. In this study we evaluate five whole genome sequences of influenza A(H3N2) viruses detected in patients with mild illness collected from January-March 2014. To sequence the genomes, a new generation sequencing (NGS) protocol was performed using the Ion Torrent PGM platform. In addition to analysing the common genes, haemagglutinin, neuraminidase and matrix, our work also comprised internal genes. This was the first report of a whole genome analysis with Brazilian influenza A(H3N2) samples. Considerable amino acid variability was encountered in all gene segments, demonstrating the importance of studying the internal genes. NGS of whole genomes in this study will facilitate deeper virus characterisation, contributing to the improvement of influenza strain surveillance in Brazil.

Full-genome sequences of hepatitis B virus subgenotype D3 isolates from the Brazilian Amazon Region

The Brazilian Amazon Region is a highly endemic area for hepatitis B virus (HBV). However, little is known regarding the genetic variability of the strains circulating in this geographical region. Here, we describe the first full-length genomes of HBV isolated in the Brazilian Amazon Region; these genomes are also the first complete HBV subgenotype D3 genomes reported for Brazil. The genomes of the five Brazilian isolates were all 3,182 base pairs in length and the isolates were classified as belonging to subgenotype D3, subtypes ayw2 (n = 3) and ayw3 (n = 2). Phylogenetic analysis suggested that the Brazilian sequences are not likely to be closely related to European D3 sequences. Such results will contribute to further epidemiological and evolutionary studies of HBV.

Large-scale analysis of orthologs and paralogs under covarion-like and constant-but-different models of amino acid evolution.

Functional divergence between homologous proteins is expected to affect amino acid sequences in two main ways, which can be considered as proxies of biochemical divergence: a "covarion-like" pattern of correlated changes in evolutionary rates, and switches in conserved residues ("conserved but different"). Although these patterns have been used in case studies, a large-scale analysis is needed to estimate their frequency and distribution. We use a phylogenomic framework of animal genes to answer three questions: 1) What is the prevalence of such patterns? 2) Can we link such patterns at the amino acid level with selection inferred at the codon level? 3) Are patterns different between paralogs and orthologs? We find that covarion-like patterns are more frequently detected than "constant but different," but that only the latter are correlated with signal for positive selection. Finally, there is no obvious difference in patterns between orthologs and paralogs.

Draft genome sequences of three NDM-1-producing Enterobacteriaceae species isolated from Brazil

The emergence of multidrug-resistant Enterobacteriaceae strains producing carbapenemases, such as NDM-1, has become a major public health issue due to a high dissemination capacity and limited treatment options. Here we describe the draft genome of three NDM-1-producing isolates: Providencia rettgeri(CCBH11880), Enterobacter hormaecheisubsp. oharae(CCBH10892) and Klebsiella pneumoniae(CCBH13327), isolated in Brazil. BesidesblaNDM-1, resistance genes to aminoglycosides [aadA1, aadA2,aac(6’)-Ib-cr] and quinolones (qnrA1,qnrB4) were observed which contributed to the multidrug resistance profile. The element ISAba125 was found associated to theblaNDM-1 gene in all strains.

Effect of length and location of heterologous sequences on RecA-mediated strand exchange.

We systematically investigated the effect of heterology on RecA-mediated strand exchange between double-stranded linear and single-stranded circular DNA. Strand exchange took place through heterologies of up to 150-200 base pairs when the insertion was at the proximal (initiating) end of the duplex DNA but was completely blocked by an insert of only 22 base pairs placed at the distal end of the duplex. In the case of medial heterology created by insertion either in the duplex or the single-stranded DNA, the ability of RecA to exchange strands decreased as the heterology was shifted toward the distal end of the duplex. These results suggest that two different strand exchange mechanisms operate in the proximal and distal portions of the duplex substrate.