Cytokey: desenvolvimento de uma chave de identificação com dados citogenéticos para os vetores da doença de Chagas do estado da Paraíba, Brasil / Cytokey: development of an identification key with cytogenetic data for Chagas disease vectors from the state of Paraíba

Os triatomíneos são insetos hematófagos vetores do protozoário Trypanosoma cruzi, agente etiológico da doença de Chagas (DC). Embora existam diferentes formas de transmissão do parasito, a Organização Mundial de Saúde ressalta que a vetorial seja a principal via de transmissão do T. cruzi na América Latina. No Brasil, existe cerca de 4,6 milhões de pessoas vivendo com DC, sendo os casos mais recentes de contaminação por infecção oral, a partir de barbeiros infectados e/ou fezes contaminadas que foram processados com alimentos consumidos in natura. Diversos casos de infecção oral foram registrados na região Nordeste, associados a surtos causados pelo consumo de caldo de cana ou outros alimentos. Nos estados da Paraíba (PB) e do Rio Grande do Norte (RN), por exemplo, 26 óbitos foram associados com infecção por via oral e 18 casos positivos para DC e duas mortes foram notificadas, respectivamente. Por séculos, as principais chaves de classificação se fundamentaram em chaves dicotômicas baseadas em dados morfológicos. Apenas em 2019, chaves alternativas, com base em dados citogenéticos (CytoKeys), foram propostas. Recentemente, chaves foram desenvolvidas para os estados de PE e RN. Com base no exposto, uma chave dicotômica foi desenvolvida para auxiliar na identificação das espécies de triatomíneos da PB, a partir de dados citogenéticos. Com base na chave dicotômica desenvolvida com dados citogenéticos, todas as espécies de triatomíneos notificadas na PB puderam ser diferenciadas. O desenvolvimento de chaves alternativas mostra-se de grande importância para a comunidade científica e, principalmente, os agentes de saúde que atuam no controle de vetores, auxiliando-os na correta identificação de espécies que apresentam semelhanças morfológicas (como as espécies Triatoma brasilensis e T. petrocchiae notificadas na PB).

Triatomines are hematophagous insects vectors of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease (CD). Although there are different forms of transmission of the parasite, the World Health Organization emphasizes that vector transmission is the main route of transmission of T. cruzi in Latin America. In Brazil, there are about 4.6 million people living with CD, with the most recent cases of contamination by oral infection, from infected triatomines and/or contaminated feces that were processed with food consumed in natura. Several cases of oral infection were registered in the Northeast region, associated with outbreaks caused by the consumption of sugarcane juice or other foods. In the states of Paraíba (PB) and Rio Grande do Norte (RN), for example, 26 deaths were associated with oral infection and 18 positive cases for CD and two deaths were reported, respectively. For centuries, the main classification keys were based on dichotomous keys based on morphological data. Only in 2019, alternative keys, based on cytogenetic data (CytoKeys), were proposed. Recently, keys were developed for the states of PE and RN. Based on the above, a dichotomous key was developed to assist in the identification of BP triatomine species, based on cytogenetic data. Based on the dichotomous key developed with cytogenetic data, all triatomine species reported in BP could be differentiated. The development of alternative keys is of great importance for the scientific community and, mainly, for health agents who work in vector control, helping them in the correct identification of species that present morphological similarities (such as the species Triatoma brasilensis and T .petrocchiae notified in BP).

Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L) / Conteúdo de heterocromatina revela polimorfismos importantes para o melhoramento genético do alho (Allium sativum L)

Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n= 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the "Branco Mineiro Piauí" accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Allium sativum L. é uma erva da família Alliaceae com sabor e aroma específicos e propriedades medicinais e nutracêuticas amplamente comercializada em diversos países. O Brasil é um dos maiores importadores de alho do mundo, apesar da sua produção ser restrita e limitada ao consumo interno. Assim, explorar a diversidade genética do alho comercial conservado em bancos de germoplasma é essencial para fornecer informações genéticas adicionais acerca dessa cultura economicamente importante. Uma ferramenta adequada para esse fim é a caracterização citogenética desses acessos. Este estudo teve como objetivo caracterizar a diversidade citogenética entre sete acessos de alho de um Banco de Germoplasma no Brasil. Os cariótipos foram obtidos por coloração convencional e com os fluorocromos de cromomicina A3 (CMA) e 4,6-diamidino-2-fenilindol (DAPI). Todos os acessos analisados apresentaram número cromossômico 2n = 16, fórmula cariotípica 6M + 2SM, cariótipos simétricos, núcleos reticulados em intérfase e cromossomos com condensação uniforme da cromatina da prófase para a metáfase. A coloração com fluorocromos mostrou diferenças na quantidade e distribuição de heterocromatina ao longo dos cromossomos e entre os acessos estudados. Com base no padrão de distribuição desses pequenos polimorfismos, foi possível separar os sete acessos em três grupos. Também foi possível diferenciar individualmente alguns dos acessos. Um dos resultados obtidos mostrou distensão heteromórfica da região organizadora nucleolar observada nos pares dos cromossomos 6 ou 7 com características peculiares. Foi sugerido, por exemplo, que o bloco heteromórfico de heterocromatina (CMA +++ / DAPI-) no cromossomo 6 do acesso “Branco Mineiro Piauí” pode ser usado como um marcador para identificar esse genótipo ou pode estar associado a algum caráter de interesse econômico.

Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L. ) / Conteúdo de heterocromatina revela polimorfismos importantes para o melhoramento genético do alho (Allium sativum L. )

Abstract Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n = 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the "Branco Mineiro Piauí" accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Resumo Allium sativum L. é uma erva da família Alliaceae com sabor e aroma específicos e propriedades medicinais e nutracêuticas amplamente comercializada em diversos países. O Brasil é um dos maiores importadores de alho do mundo, apesar da sua produção ser restrita e limitada ao consumo interno. Assim, explorar a diversidade genética do alho comercial conservado em bancos de germoplasma é essencial para fornecer informações genéticas adicionais acerca dessa cultura economicamente importante. Uma ferramenta adequada para esse fim é a caracterização citogenética desses acessos. Este estudo teve como objetivo caracterizar a diversidade citogenética entre sete acessos de alho de um Banco de Germoplasma no Brasil. Os cariótipos foram obtidos por coloração convencional e com os fluorocromos de cromomicina A3 (CMA) e 4,6-diamidino-2-fenilindol (DAPI). Todos os acessos analisados ​​apresentaram número cromossômico 2n = 16, fórmula cariotípica 6M + 2SM, cariótipos simétricos, núcleos reticulados em intérfase e cromossomos com condensação uniforme da cromatina da prófase para a metáfase. A coloração com fluorocromos mostrou diferenças na quantidade e distribuição de heterocromatina ao longo dos cromossomos e entre os acessos estudados. Com base no padrão de distribuição desses pequenos polimorfismos, foi possível separar os sete acessos em três grupos. Também foi possível diferenciar individualmente alguns dos acessos. Um dos resultados obtidos mostrou distensão heteromórfica da região organizadora nucleolar observada nos pares dos cromossomos 6 ou 7 com características peculiares. Foi sugerido, por exemplo, que o bloco heteromórfico de heterocromatina (CMA +++ / DAPI-) no cromossomo 6 do acesso "Branco Mineiro Piauí" pode ser usado como um marcador para identificar esse genótipo ou pode estar associado a algum caráter de interesse econômico.

First constitutive heterochromatin characterization and Karyotype of white stork Ciconia ciconia (Aves: Ciconiidae) / Primeira caracterização constitutiva de heterocromatina e cariótipo de cegonha-branca Ciconia ciconia (Aves: Ciconiidae)

Abstract The karyotype and constitutive heterochromatin pattern of the white stork Ciconia ciconia samples obtained from Manzala lake, Dimiaat, Egypt was described. Somatic cells of Ciconia ciconia samples have diploid number 2n= 68 chromosomes. Out of 68 chromosomes, 11 pairs including sex chromosomes were macrochromosomes and the remaining pairs were microchromosomes. Of the 11 macrochromosome pairs, no.1, 2, 4 and 5 were submetacentric and pairs no. 6, 7 and 8 were described as metacentric. In addition, the autosome pair no.3 was subtelocentric, while autosome pair no.9 was acrocentric. Also, the sex chromosome Z represents the fourth one in size and it was classified as submetacentric while, W chromosome appeared as medium size and was acrocentric. Furthermore, C-banding pattern (constitutive heterochromatin) revealed variation in their sizes and occurrence between macrochromosomes. Pairs no. 7 and 8 of autosomes exhibited unusual distribution of heterochromatin, where they appeared as entirely heterochromatic. This may be related to the origin of sex chromosomes Z and W. However, there is no sufficient evidence illustrate the appearance of entirely heterochromatic autosomes. Therefore, there is no available cytogenetic literature that describes the C-banding and karyotype of Ciconia Ciconia, so the results herein are important and may assist in cytogenetic study and evolutionary pattern of Ciconiiformes.

Resumo O cariótipo e o padrão constitutivo de heterocromatina das amostras de cegonha-branca Ciconia ciconia obtidas no lago Manzala, Dimiaat, Egito, foram descritos. As células somáticas de amostras de Ciconia ciconia possuem número diploide 2n = 68 cromossomos. Dos 68 cromossomos, 11 pares incluindo cromossomos sexuais eram macrocromossomos e os pares restantes eram microcromossomos. Dos 11 pares de macrocromossomos, os nos 1, 2, 4 e 5 eram submetacêntricos, e os pares nos 6, 7 e 8 foram descritos como metacêntricos. Além disso, o par de autossomos no 3 era subtelocêntrico, enquanto o par de autossomos no 9 era acrocêntrico. Além disso, o cromossomo sexual Z representa o quarto em tamanho e foi classificado como submetacêntrico, enquanto o cromossomo W apareceu como de tamanho médio e acrocêntrico. Além disso, o padrão de bandamento C (heterocromatina constitutiva) revelou variação em seus tamanhos e ocorrência entre macrocromossomos. Pares nos 7 e 8 dos autossomos exibiram distribuição incomum de heterocromatina, onde apareceram como totalmente heterocromáticos. Isso pode estar relacionado à origem dos cromossomos sexuais Z e W. No entanto, não há evidências suficientes para ilustrar o aparecimento de autossomos totalmente heterocromáticos. Portanto, não há literatura citogenética disponível que descreva o bandamento C e o cariótipo de Ciconia ciconia, portanto os resultados aqui apresentados são importantes e podem auxiliar no estudo citogenético e no padrão evolutivo de Ciconiiformes.

First constitutive heterochromatin characterization and Karyotype of white stork Ciconia ciconia (Aves: Ciconiidae) / Primeira caracterização constitutiva de heterocromatina e cariótipo de cegonhabranca Ciconia ciconia (Aves: Ciconiidae)

The karyotype and constitutive heterochromatin pattern of the white stork Ciconia ciconia samples obtained from Manzala lake, Dimiaat, Egypt was described. Somatic cells of Ciconia ciconia samples have diploid number 2n= 68 chromosomes. Out of 68 chromosomes, 11 pairs including sex chromosomes were macrochromosomes and the remaining pairs were microchromosomes. Of the 11 macrochromosome pairs, no.1, 2, 4 and 5 were submetacentric and pairs no. 6, 7 and 8 were described as metacentric. In addition, the autosome pair no.3 was subtelocentric, while autosome pair no.9 was acrocentric. Also, the sex chromosome Z represents the fourth one in size and it was classified as submetacentric while, W chromosome appeared as medium size and was acrocentric. Furthermore, C-banding pattern (constitutive heterochromatin) revealed variation in their sizes and occurrence between macrochromosomes. Pairs no. 7 and 8 of autosomes exhibited unusual distribution of heterochromatin, where they appeared as entirely heterochromatic. This may be related to the origin of sex chromosomes Z and W. However, there is no sufficient evidence illustrate the appearance of entirely heterochromatic autosomes. Therefore, there is no available cytogenetic literature that describes the C-banding and karyotype of Ciconia Ciconia, so the results herein are important and may assist in cytogenetic study and evolutionary pattern of Ciconiiformes.

O cariótipo e o padrão constitutivo de heterocromatina das amostras de cegonha-branca Ciconia ciconia obtidas no lago Manzala, Dimiaat, Egito, foram descritos. As células somáticas de amostras de Ciconia ciconia possuem número diploide 2n = 68 cromossomos. Dos 68 cromossomos, 11 pares incluindo cromossomos sexuais eram macrocromossomos e os pares restantes eram microcromossomos. Dos 11 pares de macrocromossomos, os nos 1, 2, 4 e 5 eram submetacêntricos, e os pares nos 6, 7 e 8 foram descritos como metacêntricos. Além disso, o par de autossomos no 3 era subtelocêntrico, enquanto o par de autossomos no 9 era acrocêntrico. Além disso, o cromossomo sexual Z representa o quarto em tamanho e foi classificado como submetacêntrico, enquanto o cromossomo W apareceu como de tamanho médio e acrocêntrico. Além disso, o padrão de bandamento C (heterocromatina constitutiva) revelou variação em seus tamanhos e ocorrência entre macrocromossomos. Pares nºs 7 e 8 dos autossomos exibiram distribuição incomum de heterocromatina, onde apareceram como totalmente heterocromáticos. Isso pode estar relacionado à origem dos cromossomos sexuais Z e W. No entanto, não há evidências suficientes para ilustrar o aparecimento de autossomos totalmente heterocromáticos. Portanto, não há literatura citogenética disponível que descreva o bandamento C e o cariótipo de Ciconia ciconia, portanto os resultados aqui apresentados são importantes e podem auxiliar no estudo citogenético e no padrão evolutivo de Ciconiiformes.

Alteraciones citogenéticas en pacientes con diagnóstico de infertilidad en Lima, Perú / Cytogenetic abnormalities in patients with infertility diagnosis in Lima, Peru

INTRODUCCIÓN: La infertilidad es una enfermedad multicausal y el componente genético representa uno de sus principales eventos. Si bien la distribución de la infertilidad puede variar entre poblaciones, las parejas de los países con bajos y medianos ingresos pueden verse más afectadas por la infertilidad, con una proporción de alteraciones citogenéticas aún no esclarecidas. OBJETIVO: Evaluar la frecuencia de alteraciones citogenéticas y su correlación con el número de abortos en pacientes peruanas con diagnóstico de infertilidad. MÉTODO: Se realizó un estudio de corte transversal en 400 pacientes de 18 a 60 años, de ambos sexos, con diagnóstico de infertilidad. Se registraron las características clínicas disponibles durante el examen genético y el análisis citogenético convencional fue con bandeo GTG en muestras de sangre periférica. El análisis de correlación se realizó con la prueba de Spearman. RESULTADOS: Del total, 389 pacientes cumplieron los criterios de inclusión, y de estos, 169 (43,44%) tuvieron reportes de abortos (promedio: 2,25, rango: 1-7). Hallamos una correlación significativa ente el número de abortos y las alteraciones citogenéticas (p < 0,000). Reportamos 25/289 (6,43%) alteraciones cromosómicas, de las que 11/25 (44%) fueron heterocromatinas constitutivas y 6/25 (24%) fueron translocaciones reciprocas. Las alteraciones citogenéticas más frecuentes fueron 16qh+ y 9qh+ (ambas con un 16%), y afectaron a 17 (68%) varones. CONCLUSIONES: Existe una moderada frecuencia de alteraciones citogenéticas en pacientes peruanos con diagnóstico de infertilidad, y las alteraciones más frecuentes fueron heterocromatina constitutivas. Además, evidenciamos una correlación significativa ente el número de abortos y las alteraciones citogenéticas.

INTRODUCTION: Infertility is a multicausal disease and the genetic component represents one of its main events. Although the distribution of infertility may vary between populations, couples in low-and-middle-income countries may be more affected by infertility with a proportion of cytogenetic alterations still unclear. OBJECTIVE: To evaluate the frequency of cytogenetic alterations and their correlation with the number of abortions in Peruvian patients with a diagnosis of infertility. METHOD: A cross-sectional study was carried out in 400 patients between 18 and 60 years-old, of both genders with a diagnosis of infertility. The clinical characteristics available during the genetic examination were recorded and the conventional cytogenetic analysis was with GTG banding in peripheral blood samples. The correlation analysis was performed with the Spearman test. RESULTS: Of the total 389 patients who met the inclusion criteria, of these 169 (43.44%) patients had reports of abortions (mean: 2.25, range: 1-7). We found a significant correlation between the number of abortions and cytogenetic alterations (p < 0.000). We report 25/289 (6.43%) chromosomal alterations, where 11/25 (44%) were constitutive heterochromatin, and 6/25 (24%) were reciprocal translocations. The most frequent cytogenetic alterations were 16qh + and 9qh + (both 16%), and affected 17 (68%) men. CONCLUSIONS: There is a moderate frequency of cytogenetic alterations in Peruvian patients diagnosed with infertility, where the most frequent alterations were constitutive heterochromatin. Furthermore, we evidenced a significant correlation between the number of abortions and cytogenetic alterations.

Chromosome analysis in Saccodon wagneri (Characiformes) and insights into the karyotype evolution of Parodontidae

Parodontidae is a relatively small group of Neotropical characiform fishes consisting of three genera (Apareiodon, Parodon, and Saccodon) with 32 valid species. A vast cytogenetic literature is available on Apareiodon and Parodon, but to date, there is no cytogenetic data about Saccodon, a genus that contains only three species with a trans-Andean distribution. In the present study the karyotype of S. wagneri was described, based on both conventional (Giemsa staining, Ag-NOR, C-bands) and molecular (repetitive DNA mapping by fluorescent in situ hybridization) methods. A diploid chromosome number of 2n = 54 was observed in both sexes, and the presence of heteromorphic sex chromosomes of the ZZ/ZW type was detected. The W chromosome has a terminal heterochromatin band that occupies approximately half of the long arm, being this band approximately half the size of the Z chromosome. The FISH assay showed a synteny of the 18S-rDNA and 5S-rDNA genes in the chromosome pair 14, and the absence of interstitial telomeric sites. Our data reinforce the hypothesis of a conservative karyotype structure in Parodontidae and suggest an ancient origin of the sex chromosomes in the fishes of this family.(AU)

Parodontidae é um grupo relativamente pequeno de peixes caraciformes neotropicais que consiste em três gêneros (Apareiodon, Parodon e Saccodon) com 32 espécies válidas. Uma vasta literatura citogenética está disponível sobre Apareiodon e Parodon, mas até o momento não há dados citogenéticos sobre Saccodon, um gênero que contém apenas três espécies com distribuição transandina. No presente estudo foi descrito o cariótipo de S. wagneri, baseado em métodos convencionais (coloração de Giemsa, Ag-NOR, bandas C) e moleculares (mapeamento de DNA repetitivo por hibridização fluorescente in situ). Um número cromossômico diplóide de 2n = 54 foi observado, e a presença de cromossomos sexuais heteromórficos do tipo ZZ/ZW foi revelada. O cromossomo W possui uma banda terminal heterocromática que ocupa aproximadamente metade do braço longo, sendo esta banda aproximadamente a metade do tamanho do cromossomo Z. O ensaio FISH mostrou uma sintenia dos genes 18S-rDNA e 5S-rDNA no par de cromossomos 14, e a ausência de sítios teloméricos intersticiais. Nossos dados reforçam a hipótese de uma estrutura cariotípica conservadora em Parodontidae e sugerem uma origem ancestral dos cromossomos sexuais nos peixes desta família.(AU)

Occurrence of euchromatic B chromosomes in natural populations of Moenkhausia bonita and M. forestii (Pisces: Characidae)

Moenkhausia is a highly specious genus among the Characidae, composed of 96 valid species. Only twelve species have a known karyotype. Thus, here are presented the first cytogenetic data of two allopatric populations of Moenkhausia bonita and one of M. forestii, both belonging to the upper Paraná River basin (PR) with discussion on the evolutionary and cytotaxonomic aspects of the genus. The two species presented 2n = 50 chromosomes but different karyotype formulas and occurrence of 1-2 B chromosomes. These elements are small metacentrics in M. bonita and small acrocentrics in M. forestii. In both species, B chromosomes were euchromatic. Ag-NOR sites were found in pair 3 (metacentric), coinciding with fluorescent in situ hybridization (FISH) by the 18S rDNA probe in both species. However, the species differed in terms of the number and position of 5S rDNA sites. Heterochromatic blocks, mapped in M. bonita showed the least amount of heterochromatin in the terminal and pericentromeric regions, while the M. forestii karyotype revealed a greater amount of interstitial heterochromatic blocks. The karyotype distinctions between the two species, including the morphology of B chromosomes, may contribute as a reference in the taxonomic studies in this group.(AU)

Moenkhausia é um gênero altamente especioso dentre os Characidae, composto por 96 espécies válidas, mas apenas doze espécies têm seus cariótipos conhecidos. Portanto, são apresentados aqui os primeiros dados citogenéticos de duas populações alopátricas de Moenkhausia bonita e uma de M. forestii, ambas pertencentes à bacia do alto rio Paraná (PR), com uma ampla discussão sobre os aspectos evolutivos e citotaxonômicos do gênero. As duas espécies apresentaram 2n = 50 cromossomos, mas diferentes fórmulas cariotípicas e ocorrência de 1-2 cromossomos B. Esses elementos são pequenos metacêntricos em M. bonita e acrocêntricos pequenos em M. forestii. Em ambas as espécies, os cromossomos B apresentaram-se eucromáticos. Sítios Ag-NOR foram encontrados no par 3 (metacêntrico), coincidindo com a hibridização fluorescente in situ (FISH) pela sonda 18S rDNA em ambas as espécies. No entanto, as espécies diferiram em termos de número e posição dos sítios de 5S rDNA. Blocos heterocromáticos mapeados em M. bonita revelaram pequena quantidade de heterocromatina nas regiões terminal e pericentromérica, enquanto o cariótipo de M. forestii revelou uma maior quantidade de blocos heterocromáticos intersticiais. As distinções cariotípicas entre as duas espécies, incluindo a morfologia dos cromossomos B, podem contribuir como uma referência em estudos taxonômicos neste grupo.(AU)

Targeted Downregulation of kdm4a Ameliorates Tau-engendered Defects in Drosophila melanogaster

BACKGROUND: Tauopathies, a class of neurodegenerative diseases that includes Alzheimer's disease (AD), are characterized by the deposition of neurofibrillary tangles composed of hyperphosphorylated tau protein in the human brain. As abnormal alterations in histone acetylation and methylation show a cause and effect relationship with AD, we investigated the role of several Jumonji domain-containing histone demethylase (JHDM) genes, which have yet to be studied in AD pathology. METHODS: To examine alterations of several JHDM genes in AD pathology, we performed bioinformatics analyses of JHDM gene expression profiles in brain tissue samples from deceased AD patients. Furthermore, to investigate the possible relationship between alterations in JHDM gene expression profiles and AD pathology in vivo, we examined whether tissue-specific downregulation of JHDM Drosophila homologs (kdm) can affect tauR406W-induced neurotoxicity using transgenic flies containing the UAS-Gal4 binary system. RESULTS: The expression levels of JHDM1A, JHDM2A/2B, and JHDM3A/3B were significantly higher in postmortem brain tissue from patients with AD than from non-demented controls, whereas JHDM1B mRNA levels were downregulated in the brains of patients with AD. Using transgenic flies, we revealed that knockdown of kdm2 (homolog to human JHDM1), kdm3 (homolog to human JHDM2), kdm4a (homolog to human JHDM3A), or kdm4b (homolog to human JHDM3B) genes in the eye ameliorated the tauR406W-engendered defects, resulting in less severe phenotypes. However, kdm4a knockdown in the central nervous system uniquely ameliorated tauR406W-induced locomotion defects by restoring heterochromatin. CONCLUSION: Our results suggest that downregulation of kdm4a expression may be a potential therapeutic target in AD.

Olaparib induced senescence under P16 or P53 dependent manner in ovarian cancer / 부인종양

OBJECTIVE: Poly (ADP-ribose) polymerase (PARP) is an important molecule in the early stress response of DNA damage, which is involved in DNA damage repair and cellular senescence. Olaparib, as PARP inhibitor, has an anti-tumor effect on high grade serous ovarian cancer, but its effects on cellular senescence have not been reported. This study intends to explore the role of olaparib in the regulation of senescence in ovarian cancer cells. METHODS: The effects of olaparib on the senescence of ovarian cancer cells were detected by using the senescence-associated β-galactosidase (SA-β-Gal) and senescence-associated heterochromatin aggregation (SAHF). Quantitative real-time polymerase chain reaction was used to analyze the senescence-associated secretory phenotype (SASP). Cell cycle and apoptosis were detected by flow cytometry. The effect of olaparib on tumor growth was analyzed in a nude mouse xenograft transplantation model. RESULTS: Long-term (6 days) treatment with olaparib (5 μM) significantly inhibited the growth of ovarian cancer cells, leading to arrest the cell cycle at G0/G1 phase, significant increase the number of positive SA-β-Gal stained cells and positive SAHF cells. The expression of P16 and retinoblastoma protein (p-RB) were significantly enhanced in SKOV3 cells under olaparib treated, meanwhile, the expression of P53 and p-RB were upregulated in A2780 cells. In OVCAR-3 cells, the expression of P53 was downregulated and p-RB was upregulated. Mice with SKOV3 xenograft transplantation was given olaparib (10 mg/kg/day) via abdominal cavity administration, the tumor volume was reduced (p < 0.01). CONCLUSION: Continuous low dosage administration of olaparib induced senescence under P16 or P53 dependent manner in ovarian cancer.

Suppressor of Variegation 3–9 Homolog 2, a Novel Binding Protein of Translationally Controlled Tumor Protein, Regulates Cancer Cell Proliferation

Suppressor of Variegation 3–9 Homolog 2 (SUV39H2) methylates the lysine 9 residue of histone H3 and induces heterochromatin formation, resulting in transcriptional repression or silencing of target genes. SUV39H1 and SUV39H2 have a role in embryonic development, and SUV39H1 was shown to suppress cell cycle progression associated with Rb. However, the function of human SUV39H2 has not been extensively studied. We observed that forced expression of SUV39H2 decreased cell proliferation by inducing G1 cell cycle arrest. In addition, SUV39H2 was degraded through the ubiquitin-proteasomal pathway. Using yeast two-hybrid screening to address the degradation mechanism and function of SUV39H2, we identified translationally controlled tumor protein (TCTP) as an SUV39H2-interacting molecule. Mapping of the interacting regions indicated that the N-terminal 60 amino acids (aa) of full-length SUV39H2 and the C-terminus of TCTP (120–172 aa) were critical for binding. The interaction of SUV39H2 and TCTP was further confirmed by co-immunoprecipitation and immunofluorescence staining for colocalization. Moreover, depletion of TCTP by RNAi led to up-regulation of SUV39H2 protein, while TCTP overexpression reduced SUV39H2 protein level. The half-life of SUV39H2 protein was significantly extended upon TCTP depletion. These results clearly indicate that TCTP negatively regulates the expression of SUV39H2 post-translationally. Furthermore, SUV39H2 induced apoptotic cell death in TCTP-knockdown cells. Taken together, we identified SUV39H2, as a novel target protein of TCTP and demonstrated that SUV39H2 regulates cell proliferation of lung cancer cells.

Contributions to the systematic of Pimelodidae (Osteichthyes, Siluriformes): basic and molecular cytogenetics on seven species of Pimelodus from three Brazilian hydrographic systems

Pimelodidae harbors several species and is widely distributed throughout the Neotropical region. Pimelodus is the genus with the largest number of species, however it is a polyphyletic group. Cytogenetic analyzes of the valid species still covers less than half of them. Herein, seven Pimelodus species from three Brazilian hydrographic systems were analyzed through basic (Giemsa, AgNORs and C banding) and molecular (5S and 18S rDNA-FISH) cytogenetic methods. All species had 2n=56 chromosomes with different karyotype formulas observed among the species. AgNORs were corresponding to 18S rDNA and localized on long arm of one chromosome pair in all species. Heterochromatin distribution follows the pattern commonly verified in the family and allows to identify each one of the studied species. 5S rDNA marker was interspecifically variable in number and position of cistrons. Pimelodus ortmanni had B chromosomes varying intra and inter-individually. We performed a discussion on our own and available cytogenetic data for Pimelodidae, and the associating of them with available phylogeny enable us identifying features that distinguish subgroups within Pimelodidae, such as NORs location (terminal/long arm for species belonging to "Iheringichthys-Parapimelodus" and "Pimelodus maculatus" subclades) and location of 5S rDNA sites (pericentromeric/interstitial/ long arm for species belonging to Pimelodus group).(AU)

Pimelodidae abriga várias espécies e é amplamente distribuída ao longo da região Neotropical. Pimelodus é o gênero com o maior número de espécies, porém é um grupo polifilético. Análises citogenéticas foram realizadas em menos da metade das espécies válidas. Aqui, sete espécies de Pimelodus de três sistemas hidrográficos brasileiros foram estudadas através das técnicas citogenéticas básicas (Giemsa, AgRONs e banda C) e moleculares (FISH-DNAr 5S e 18S). Todas as espécies apresentaram 2n=56 cromossomos, sendo observadas variações na fórmula cariotípica entre algumas espécies. As AgRONs correspondentes ao DNAr 18S foram localizadas no braço longo de um par de cromossomos em todas as espécies. A heterocromatina segue o padrão comumente observado na família e permite identificar cada uma das espécies estudadas. O DNAr 5S apresentou variação interespecífica em número e na posição dos cístrons. Cromossomos B foram evidenciados em P. ortmanni com variação intra e interindividual. Nós discutimos os nossos resultados com os dados citogenéticos válidos para Pimelodidae, e a associação desses dados com a filogenia válida nos permitiu identificar características que distinguem subgrupos dentro de Pimelodidae, tais como a localização das RONs (terminal/braço longo para espécies pertencentes aos subclados "Iheringichthys-Parapimelodus" e "Pimelodus maculatus") e localização dos sítios de DNAr 5S (pericentromérico/intersticial no braço longo para espécies pertencentes ao grupo Pimelodus).(AU)

Karyotype analysis in Bignonieae (Bignoniaceae): chromosome numbers and heterochromatin

ABSTRACT Chromosome numbers and heterochromatin banding pattern variability have been shown to be useful for taxonomic and evolutionary studies of different plant taxa. Bignonieae is the largest tribe of Bignoniaceae, composed mostly by woody climber species whose taxonomies are quite complicated. We reviewed and added new data concerning chromosome numbers in Bignonieae and performed the first analyses of heterochromatin banding patterns in that tribe based on the fluorochromes chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI). We confirmed the predominant diploid number 2n = 40, as well as variations reported in the literature (dysploidy in Mansoa [2n = 38] and polyploidy in Dolichandra ungis-cati [2n = 80] and Pyrostegia venusta [2n = 80]). We also found a new cytotype for the genus Anemopaegma (Anemopaegma citrinum, 2n = 60) and provide the first chromosome counts for five species (Adenocalymma divaricatum, Amphilophium scabriusculum, Fridericia limae, F. subverticillata, and Xylophragma myrianthum). Heterochromatin analyses revealed only GC-rich regions, with six different arrangements of those bands. The A-type (one large and distal telomeric band) were the most common, although the presence and combinations of the other types appear to be the most promising for taxonomic studies.

Variation of the interphase heterochromatin in Artemia (Crustacea, Anostraca) of the Americas is related to changes in nuclear size and ionic composition of hipersaline habitats / Variação da heterocromatina interfásica em Artemia (Crustacea, Anostraca) das Américas está relacionada a mudanças no tamanho nuclear e composição iônica em habitats hipersalinos

Abstract The populations of Artemia (or brine shrimp) from the Americas exhibit a wide variation in the amount of interphase heterochromatin. There is interest in understanding how this variation affects different parameters, from the cellular to the organismal levels. This should help to clarify the ability of this organism to tolerate brine habitats regularly subject to strong abiotic changes. In this study, we assessed the amount of interphase heterochromatin per nucleus based on chromocenter number (N-CHR) and relative area of chromocenter (R-CHR) in two species of Artemia, A. franciscana (Kellog, 1906) (n=9 populations) and A. persimilis (Piccinelli and Prosdocimi, 1968) (n=3 populations), to investigate the effect on nuclear size (S-NUC). The relationship of the R-CHR parameter with the ionic composition (IC) of brine habitats was also analysed. Our results indicate a significant variation in the amount of heterochromatin both within and between species (ANOVA, p<0.001). The heterochromatin varied from 0.81 ± 1.17 to 12.58 ± 3.78 and from 0.19 ± 0.34% to 11.78 ± 3.71% across all populations, for N-CHR and R-CHR parameters, respectively. N-CHR showed less variation than R-CHR (variation index 15.5-fold vs. 62-fold). At least five populations showed a significant association (p<0.05) between R-CHR and S-NUC, either with negative (four populations, r= from -0.643 to -0.443), or positive (one population, r= 0.367) values.Within each species, there were no significant associations between both parameters (p>0.05). The R-CHR and IC parameters were associated significantly for the magnesium ion (r= 0.496, p<0.05) and also for the chloride, sodium and calcium ions (r = from -0.705 to -0.478, p<0.05). At species level, a significant association between both parameters was also found in A. franciscana populations, for the sulphate and calcium ions, in contrast to A. persimilis. These findings suggest that the amount of interphase heterochromatin modifies the nuclear size in Artemia. Our data also indicate that change in the amount of interphase heterochromatin is in line with the ionic composition of brines. This would be a species-specific phenomenon, whose occurrence may be involved in the ability of this organism to survive in these environments.

Resumo As populações de Artemia (ou camarão de salinas) das Américas apresentam uma grande variação na quantidade de heterocromatina interfásica. Há interesse em compreender como esta variação afeta diferentes parâmetros, desde o nível celular até os organismos. Isso deve ajudar a esclarecer a capacidade destes organismos tolerarem habitats extremos de água hipersalinas, que normalmente são submetidos a fortes mudanças abióticas. Neste estudo, avaliou-se a quantidade heterocromatina interfásica por núcleo através do número de cromocentros (N-CHR) e a área relativa de cromocentros (R-CHR) em duas espécies de Artemia, A. franciscana (Kellog, 1906) (n=9 populações) e A. persimilis (Piccinelli e Prosdocimi, 1968) (n=3 populações), para investigar o seu efeito no tamanho nuclear (S-NUC). Também foi analisada a relação de R-CHR com a composição iónica (CI) dos habitats hipersalinos. Nossos resultados indicam uma variação significativa na quantidade de heterocromatina dentro e entre espécies (ANOVA, p<0,001). Em todas as populações, a heterocromatina variou de 0,81 ± 1,17 para 12,58 ± 3,78 e de 0,19 ± 0,34% para 11,78 ± 3,71% para os parâmetros R-CHR e N-CHR, respectivamente. N-CHR apresentou menor variação do que R-CHR (amplitude de variação de 15,5 vezes vs. 62 vezes). Pelo menos cinco populações apresentaram uma associação significativa (p<0,05) entre R-CHR e S-NUC, seja com valores negativos (quatro populações, r = -0,643 a -0,443) ou positivo (uma população, r = 0,367). Os parâmetros R-CHR e CI foram associados significativamente para o íon de magnésio (r = 0,496, p<0,05) e também para os íons cloreto, sódio e cálcio (r = -0,705 a -0,478, p<0,05). Ao nível de espécie, foi também encontrada uma associação significativa entre esses dois parâmetros em populações de A. franciscana para os íons de sulfato e de cálcio, em contraste com A. persimilis. Estes achados sugerem que a quantidade heterocromatina interfásica modifica o tamanho nuclear em Artemia. Os nossos dados também indicam que a mudança na quantidade de heterocromatina interfásica está associada com a composição iónica das salinas. Este seria um fenómeno específico da espécie, cuja ocorrência pode estar envolvida na capacidade deste organismo sobreviver em tais ambientes.

Synaptic configuration of quadrivalents and their association with the XY bivalent in spermatocytes of Robertsonian heterozygotes of Mus domesticus

BACKGROUND: The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains and consequently is prone to modification by chromosomal rearrangements. We have shown that nuclear architecture is modified in spermatocytes of Robertsonian (Rb) homozygotes of Mus domesticus. In this study we analyse the synaptic configuration of the quadrivalents formed in the meiotic pro- phase of spermatocytes of mice double heterozygotes for the dependent Rb chromosomes: Rbs 11.16 and 16.17. RESULTS: Electron microscope spreads of 60 pachytene spermatocytes from four animals of Mus domesticus 2n = 38 were studied and their respective quadrivalents analysed in detail. Normal synaptonemal complex was found between arms 16 of the Rb metacentric chromosomes, telocentrics 11 and 17 and homologous arms of the Rb metacentric chromosomes. About 43% of the quadrivalents formed a synaptonemal complex between the heterologous short arms of chromosomes 11 and 17. This synaptonemal complex is bound to the nuclear envelope through a fourth synapsed telomere, thus dragging the entire quadrivalent to the nuclear envelope. About 57% of quadrivalents showed unsynapsed single axes in the short arms of the telocentric chromosomes. About 90% of these unsynapsed quadrivalents also showed a telomere-to-telomere association between one of the single axes of the telocentric chromosome 11 or 17 and the X chromosome single axis, which was otherwise normally paired with the Y chromosome. Nucleolar material was associated with two bivalents and with the quadrivalent. CONCLUSIONS: The spermatocytes of heterozygotes for dependent Rb chromosomes formed a quadrivalent where four chromosomes are synapsed together and bound to the nuclear envelope through four telomeres. The nuclear configuration is determined by the fourth shortest telomere, which drags the centromere regions and heterochromatin of all the chromosomes towards the nuclear envelope, favouring the reiterated encounter and eventual rearrangement between the heterologous chromosomes. The unsynapsed regions of quadrivalents are frequently bound to the single axis of the X chromosome, possibly perturbing chromatin condensation and gene expression.

Prenatal diagnosis of interchromosomal insertion of Y chromosome heterochromatin in a family

Interchromosomal insertion of Y chromosome heterochromatin in an autosome was identified in a fetus and a family. A fetal karyotype was analyzed as 46,XX,dup(7)(?q22q21.1) in a referred amniocentesis at 16 weeks of gestation for advanced maternal age. In the familial karyotype analyses for identification of der(7), the mother, the first daughter and the maternal grandmother showed the same der(7) as the fetus's. CBG-banding was positive at 7q22 region of der(7) that indicated inserted material was originated from heterochromatin. The origin of heterochromatic insertion region in der(7) of the fetus and the mother was found in Yq12 region by fluorescent in situ hybridization with a DYZ1 probe. In the specific analysis of Y chromosomal heterochromatic region of ins(7;Y) of the mother, 15 sequence tagged sites from Yp11.3 region including SRY to Yq11.223 region was not detected. Final karyotypes of the mother, the first daughter and the maternal grandmother were reported as 46,XX,der(7)ins(7;Y)(q21.3;q12q12). All female carriers of ins(7;Y) in the family showed normal phenotype and the mother and the maternal grandmother were fertile. A healthy girl was born at term. We report a rare case of familial interchromosomal insertion of Y chromosome heterochromatin detected only in female family members with normal phenotype that was diagnosed prenatally.

Heterochromatin base pair composition and diversification in holocentric chromosomes of kissing bugs (Hemiptera, Reduviidae)

The subfamily Triatominae (Hemiptera, Reduviidae) includes 150 species of blood-sucking insects, vectors of Chagas disease or American trypanosomiasis. Karyotypic information reveals a striking stability in the number of autosomes. However, this group shows substantial variability in genome size, the amount and distribution of C-heterochromatin, and the chromosome positions of 45S rDNA clusters. Here, we analysed the karyotypes of 41 species from six different genera with C-fluorescence banding in order to evaluate the base-pair richness of heterochromatic regions. Our results show a high heterogeneity in the fluorescent staining of the heterochromatin in both autosomes and sex chromosomes, never reported before within an insect subfamily with holocentric chromosomes. This technique allows a clear discrimination of the heterochromatic regions classified as similar by C-banding, constituting a new chromosome marker with taxonomic and evolutionary significance. The diverse fluorescent patterns are likely due to the amplification of different repeated sequences, reflecting an unusual dynamic rearrangement in the genomes of this subfamily. Further, we discuss the evolution of these repeated sequences in both autosomes and sex chromosomes in species of Triatominae.

First cytogenetic record for a species of Otothyropsis Ribeiro, Carvalho & Melo, 2005 (Loricariidae, Hypoptopomatinae)

Hypoptopomatinae is a monophyletic subfamily that includes 147 species, distributed in 20 genera. Otothyropsis is a genus of Hypoptopomatinae, recently described. Here, we provided the first cytogenetic information of Otothyropsis . The specimens were collected from córrego Dourado, a small tributary of rio Iguatemi, which flows into rio Paraná. The specimens of Otothyropsis cf. polyodon were analyzed with respect to diploid number, C-Band and Ag-NOR patterns. The diploid number was 54 chromosomes, distributed in 18 metacentric, 28 submetacentric, and 8 subtelocentric chromosomes, with single Ag-NOR and conspicuous heterochromatic blocks on the short and long arms of the 24th pair of chromosomes. Our study highlights the conservation trend of the diploid number (2n=54) and fundamental number (FN = 108) among the species of Hypoptopomatinae. However, the karyotype formula (18m+28sm+8st) seems to be specific to O. cf. polyodon , considering the other Hypoptopomatinae species already analyzed.

Hypoptopomatinae é uma subfamília monofilética que inclui 147 espécies distribuídas em 20 gêneros, sendo Otothyropsis um gênero recentemente descrito. Aqui, fornecemos a primeira informação citogenética do gênero Otothyropsis . Espécimes foram coletados no córrego Dourado, um pequeno tributário do rio Iguatemi, o qual deságua no rio Paraná. Espécimes de Otothyropsis cf. polyodon foram analisados em relação ao número diploide e padrões de Banda-C e Ag-NOR. O número diploide foi de 54 cromossomos, distribuídos em 18 metacêntricos, 28 submetacêntricos e 8 subtelocêntricos, com Ag-NOR simples e blocos heterocromáticos evidentes no braços curto e longo do par de cromossomos 24. Nosso estudo destaca a tendência de conservação do número diploide (2n=54) e número fundamental (NF=108) entre as espécies de Hypoptopomatinae. Entretanto, a fórmula cariotípica (18m+28sm+8st) parece ser específica para O. cf. polyodon, considerando as outras espécies de Hypoptopomatinae já analisadas.

Vitamin C alleviates aging defects in a stem cell model for Werner syndrome

Werner syndrome (WS) is a premature aging disorder that mainly affects tissues derived from mesoderm. We have recently developed a novel human WS model using WRN-deficient human mesenchymal stem cells (MSCs). This model recapitulates many phenotypic features of WS. Based on a screen of a number of chemicals, here we found that Vitamin C exerts most efficient rescue for many features in premature aging as shown in WRN-deficient MSCs, including cell growth arrest, increased reactive oxygen species levels, telomere attrition, excessive secretion of inflammatory factors, as well as disorganization of nuclear lamina and heterochromatin. Moreover, Vitamin C restores in vivo viability of MSCs in a mouse model. RNA sequencing analysis indicates that Vitamin C alters the expression of a series of genes involved in chromatin condensation, cell cycle regulation, DNA replication, and DNA damage repair pathways in WRN-deficient MSCs. Our results identify Vitamin C as a rejuvenating factor for WS MSCs, which holds the potential of being applied as a novel type of treatment of WS.

Interactome Analysis Reveals that Heterochromatin Protein 1gamma (HP1gamma) Is Associated with the DNA Damage Response Pathway / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Heterochromatin protein 1gamma (HP1gamma) interacts with chromosomes by binding to lysine 9-methylated histone H3 or DNA/RNA. HP1gamma is involved in various biological processes. The purpose of this study is to gain an understanding of how HP1gamma functions in these processes by identifying HP1gamma-binding proteins using mass spectrometry. MATERIALS AND METHODS: We performed affinity purification of HP1gamma-binding proteins using G1/S phase or prometaphase HEK293T cell lysates that transiently express mock or FLAG-HP1gamma. Coomassie staining was performed for HP1gamma-binding complexes, using cell lysates prepared by affinity chromatography FLAG-agarose beads, and the bands were digested and then analyzed using a mass spectrometry. RESULTS: We identified 99 HP1gamma-binding proteins with diverse cellular functions, including spliceosome, regulation of the actin cytoskeleton, tight junction, pathogenic Escherichia coli infection, mammalian target of rapamycin signaling pathway, nucleotide excision repair, DNA replication, homologous recombination, and mismatch repair. CONCLUSION: Our results suggested that HP1gamma is functionally active in DNA damage response via protein-protein interaction.