Replication origin location might contribute to genetic variability in Trypanosoma cruzi

Background: DNA replication in trypanosomatids operates in a uniquely challenging environment, since most of their genomes are constitutively transcribed. Trypanosoma cruzi, the etiological agent of Chagas disease, presents high variability in both chromosomes size and copy number among strains, though the underlying mechanisms are unknown. Results: Here we have mapped sites of DNA replication initiation across the T. cruzi genome using Marker Frequency Analysis, which has previously only been deployed in two related trypanosomatids. The putative origins identified in T. cruzi show a notable enrichment of GC content, a preferential position at subtelomeric regions, coinciding with genes transcribed towards the telomeres, and a pronounced enrichment within coding DNA sequences, most notably in genes from the Dispersed Gene Family 1 (DGF-1). Conclusions: These findings suggest a scenario where collisions between DNA replication and transcription are frequent, leading to increased genetic variability, as seen by the increase SNP levels at chromosome subtelomeres and in DGF-1 genes containing putative origins

Educomunicação e suas áreas de intervenção: Novos paradigmas para o diálogo intercultural

oai:omp.abpeducom.org.br:publicationFormat/1O material aqui divulgado representa, em essência, a contribuição do VII Encontro Brasileiro de Educomunicação ao V Global MIL Week, da UNESCO, ocorrido na ECA/USP, entre 3 e 5 de novembro de 2016. Estamos diante de um conjunto de 104 papers executivos, com uma média de entre 7 e 10 páginas, cada um. Com este rico e abundante material, chegamos ao sétimo e-book publicado pela ABPEducom, em seus seis primeiros anos de existência. A especificidade desta obra é a de trazer as “Áreas de Intervenção” do campo da Educomunicação, colocando-as a serviço de uma meta essencial ao agir educomunicativo: o diálogo intercultural, trabalhado na linha do tema geral do evento internacional: Media and Information Literacy: New Paradigms for Intercultural Dialogue

A novel intracellular peptide derived from cyclin D2 induces cell death.

Peptídeos intracelulares são constantemente produzidos pelo sistema ubiquitina proteassomo e muitos são provavelmente funcionais. Aqui, um nonapeptídio derivado da ciclina-D2, específica da transição G1/S, chamado \"pep5\" mostrou um aumento específico durante a fase S do ciclo celular em células HeLa. O pep5 (50-100 μM) induziu a morte celular em células HeLa e em várias outras células tumorais, mas isso só ocorreu quando o pep5 foi sintetizado acoplado ao peptídeo penetrador de células (pep5-cpp). In vivo, o pep5-cpp reduziu o volume do glioblastoma C6 de ratos Wistar em cerca de 50%. A acetilação reduziu a potência do pep5-cpp, enquanto substituições Leu-Ala aboliram totalmente a atividade deste peptídeo. Os resultados de caracterização inicial do mecanismo de morte celular indizida pelo pep5 incluem ativação de caspases 3/7 e 9, inibição da fosforilação Akt2 e inibição da atividade do proteassomo. Esses dados colaboram com a hipótese da função de peptídeos intracelulares na sinalização.Intracellular peptides are constantly produced by the ubiquitinproteasome system and many are probably functional. Here, a nonapeptide of G1/S-specific cyclin-D2 named pep5 showed a specific increase during the S phase of HeLa cell cycle. Pep5 (50-100 μM) induced cell death in HeLa and in several other tumor cells, only when it was fused to a cell penetrating peptide (pep5-cpp). In vivo, the pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. Acetylation reduced the potency of the pep5-cpp while Leu-Ala substitutions totally abolished the pep5 activity. Findings from the initial characterization of the cell death mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation and inhibition of proteasome activity. These data further support the intracellular function of peptides

Replication Protein A presents canonical functions and is also involved in the differentiation capacity of Trypanosoma cruzi

Submitted by Luciane Willcox ([email protected]) on 2017-04-19T13:31:24Z No. of bitstreams: 1 Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi.pdf: 2837768 bytes, checksum: 4bd66cfcd5f9128f11a7dd42e4e9ed77 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2017-07-26T11:56:07Z (GMT) No. of bitstreams: 1 Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi.pdf: 2837768 bytes, checksum: 4bd66cfcd5f9128f11a7dd42e4e9ed77 (MD5)Made available in DSpace on 2017-07-26T11:56:07Z (GMT). No. of bitstreams: 1 Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi.pdf: 2837768 bytes, checksum: 4bd66cfcd5f9128f11a7dd42e4e9ed77 (MD5) Previous issue date: 2016Instituto Butantan. Laboratório Especial de Ciclo Celular. São Paulo, SP, Brasil. / Instituto Butantan. Center of Toxins. Immune Response and Cell Signaling—CeTICS. São Paulo, SP, Brazil.Instituto Butantan. Laboratório Especial de Ciclo Celular. São Paulo, SP, Brasil. / Instituto Butantan. Center of Toxins. Immune Response and Cell Signaling—CeTICS. São Paulo, SP, Brazil.Universidade Estadual Paulista Júlio de Mesquita Filho. Instituto de Biociências. Departamento de Física e Biofísica. Botucatu, SP, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Instituto Butantan. Laboratório Especial de Ciclo Celular. São Paulo, SP, Brasil. / Instituto Butantan. Center of Toxins. Immune Response and Cell Signaling—CeTICS. São Paulo, SP, Brazil.Universidade Estadual Paulista Júlio de Mesquita Filho. Instituto de Biociências. Departamento de Física e Biofísica. Botucatu, SP, Brasil.Instituto Butantan. Center of Toxins. Immune Response and Cell Signaling—CeTICS. São Paulo, SP, Brazil / Instituto Butantan. Laboratório de Imunoquímica. São Paulo, SP, Brasil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brasil.Universidade Estadual Paulista Julio Mesquita Filho. Instituto de Biociências. Departamento de Genética. Botucatu, SP, Brasil.Replication Protein A (RPA), the major single stranded DNA binding protein in eukaryotes, is composed of three subunits and is a fundamental player in DNA metabolism, participating in replication, transcription, repair, and the DNA damage response. In human pathogenic trypanosomatids, only limited studies have been performed on RPA-1 from Leishmania. Here, we performed in silico, in vitro and in vivo analysis of Trypanosoma cruzi RPA-1 and RPA-2 subunits. Although computational analysis suggests similarities in DNA binding and Ob-fold structures of RPA from T. cruzi compared with mammalian and fungi RPA, the predicted tridimensional structures of T. cruzi RPA-1 and RPA-2 indicated that these molecules present a more flexible tertiary structure, suggesting that T. cruzi RPA could be involved in additional responses. Here, we demonstrate experimentally that the T. cruzi RPA complex interacts with DNA via RPA-1 and is directly related to canonical functions, such as DNA replication and DNA damage response. Accordingly, a reduction of TcRPA-2 expression by generating heterozygous knockout cells impaired cell growth, slowing down S-phase progression. Moreover, heterozygous knockout cells presented a better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms and metacyclic trypomastigote infection. Taken together, these findings indicate the involvement of TcRPA in the metacyclogenesis process and suggest that a delay in cell cycle progression could be linked with differentiation in T. cruzi

Generation of heterozygous knockout cells expressing reduced levels of TcRPA-2.

<p>A. Schematic representation of the TcRPA-2 locus (central panel) or the locus generated after replacement of the RPA-2 gene by gene targeting (bottom panel) with the recombination cassette carrying the hygromycin resistance gene (top panel). Arrows indicate the regions where the primers used in the experiments presented in B anneal. B. DNA extracted from RPA-2 heterozygous knockout cells was amplified using the primers presented in A. 1- Hyg_f (2) + Hyg_r (3) (1.026 bp); 2- Hyg_f (2) + EXT5’_r (4) (2.113 bp); 3- EXT5’_f (1) + Hyg_r (3) (1.994 bp); 4- EXT5’_f (1) + EXT3’_r (4). 5–1 kb plus DNA ladder marker (Invitrogen). The primers EXT5’_f and EXT3’_r anneal in regions outside of the recombination site. Thus, the two bands observed in lane 4 correspond to the amplified regions from the wild-type (2.070 bp) and targeted RPA-2 alleles (3.081 bp), which are present in the RPA-2+/- parasites. C. Cell extracts from control and RPA-2+/- cells were subjected to SDS-PAGE, transferred onto nitrocellulose membranes and incubated with anti-rTcRPA-2 or anti-GAPDH, which was used as a loading control. D. The intensity of the bands obtained with anti-rTcRPA-2 presented on C were normalized using the intensity of the GAPDH bands. Graph shows average and standard deviation of three independents experiments.</p

The pattern of TcRPA distribution changes after DNA damage.

<p>Cells were treated with HU (A) or irradiated with UV (B). Cells were fixed, permeabilized and incubated with anti-rTcRPA-1 (anti-RPA-1), anti-rTcRPA-2 (anti-RPA-2) and anti-CPD and stained with DAPI. In A, the percentage of cells found with each pattern of RPA distribution is indicated.</p

TcRPA-1 and TcRPA-2 form a complex <i>in vivo</i> and localize at replication foci.

<p>A. Cell extracts were immunoprecipitated with an anti-rTcRPA-2 antibody (anti-RPA2) or with pre-immune serum as a control. Samples eluted from the resin were submitted to SDS-PAGE, transferred onto nitrocellulose membranes and incubated with anti-rTcRPA-1 (anti-RPA-1) or anti-rTcRPA-2 (anti-RPA-2) antibodies. B. Cells were incubated with EdU, fixed, incubated with anti-rTcRPA-1 (anti-RPA-1) or anti-rTcRPA-2 (anti-RPA-2) and stained with DAPI. N-nucleus, k-kinetoplast.</p

TcRPA-1 is able to bind ssDNA by OBF1.

<p>A and B. Circular dichroism (CD) spectra of (A) rTcRPA-1 and (B) rTcRPA-2 in the absence (black line) or presence of single stranded DNA of 24 bp (ssDNA24; blue line). A digoxin-labeled single stranded DNA (ssDNA) was maintained alone or in the presence (+) of recombinant TcRPA-1 (rRPA-1) (C) or of increasing concentrations of recombinant TcRPA-2 (rRPA-2) (D). The samples were analyzed by EMSA. E. Schematic representation of entire TcRPA-1 and three truncated mutants corresponding to each OBF domain. F. A digoxigenin-labeled ssDNA was maintained alone (-) or in the presence of recombinant OBF1, OBF2, and OBF3.</p

RPA-2 heterozygous knockout cells present growth and DNA replication impairment and S-phase progression delay.

<p>A. Growth curve of RPA-2+/- and wild type (wt) cells. * indicates the day during the growth curve that samples were taken to perform assays presented in B and C. B. Control and RPA-2+/- cells were maintained in the presence of EdU for 5 minutes and the percentage of labeled cells was determined. Graph shows average and standard deviation of three independents experiments. C. FACS analysis of wild type (wt) and RPA-2+/- cells labeled with propidium iodide. D. Growth culture of wild type (wt) and RPA-2+/- cells after treatment with 75μM of cisplatin or with no treatment (no drug). Statistical analyses were performed using a Student t-test, ** means p < 0.001; n.s. means p > 0.8.</p

RPA-2+/- cells present a higher capacity of differentiation and infection compared with wild type cells.

<p>(A) The same amount of wild type (wt) and heterozygous knockout RPA-2+/- cells were submitted to metacyclogenesis and obtained metacyclic trypomastigotes were quantified. B. The same amount of wild type (wt) and heterozygous knockout RPA-2+/- metacyclic trypomastigotes were used to infect cells. After 24 h, the percentage of infected cells was determined. Statistical analyses were performed using a Student t-test, * means p < 0.01.</p