Comparative cytogenetics of nine populations of the Rhinella genus (Anura, Bufonidae) with a highlight on their conservative karyotype.

The genus Rhinella is one of the most diverse groups of bufonid toads, currently composed by 93 valid species and naturally distributed throughout different Neotropical ecoregions. Here, we analyze nine Brazilian populations of toads representing species of the Rhinella margaritifera and Rhinella marina groups. These new data include the first description of the R. hoogmoedi and R. proboscidae karyotypes, as well as other taxonomically unresolved forms. Chromosomal analysis of the populations revealed pronounced chromosomal uniformity (2n=22), including the diploid number and chromosomal morphology. Three different NOR-bearing chromosomes were identified: in the subterminal region of pair 10q in R. hoogmoedi, Rhinella sp. 1 and Rhinella sp. 2, in subterminal region of 7p in R. proboscidae and Rhinella cf. margaritifera while in R. henseli and R. icterica was detected in interstitial region of 7p. Karyotypic uniformity of the genus permits the inference of interspecific chromosome homologies and evolutionary changes in the NOR-bearing chromosome may represent an informative character in species group level. The review of the cytogenetic data of the Rhinella species together with the new karyotypes reported here contributes to the understanding of the chromosomal evolution of these toads, which karyotypes are highly conserved despite the ample distribution of many forms.

The most relevant chromosomal abnormalities in the north of Rio Grande do Sul, Brazil: 26 years of cytogenetic analysis.

From 1992 to 2018, cytogenetic analyses were successfully performed to explore the chromosomal abnormalities of 729 patients, who utilised a pioneering counselling service in the city of Passo Fundo in the northern part of the Brazilian state of Rio Grande do Sul. This city is characterised by a large conglomerate of private and public hospitals. A classical cytogenetic analysis and G-banding were performed using the patient samples. Although normal karyotypes were observed for 562 of the cases, 167 individuals evidenced chromosomal alterations. Among those, 110 exhibited numerical alterations (65.86%), 41 demonstrated structural modifications (24.55%) and 16 showed both numerical and structural chromosomal changes (9.58%). This study describes the diversity of the chromosomal alterations in this region, which have not been previously examined. After 26 years of study, the findings are discussed herein in a self-critical form.

Unraveling the Sex Chromosome Heteromorphism of the Paradoxical Frog Pseudis tocantins.

The paradoxical frog Pseudis tocantins is the only species in the Hylidae family with known heteromorphic Z and W sex chromosomes. The Z chromosome is metacentric and presents an interstitial nucleolar organizer region (NOR) on the long arm that is adjacent to a pericentromeric heterochromatic band. In contrast, the submetacentric W chromosome carries a pericentromeric NOR on the long arm, which is adjacent to a clearly evident heterochromatic band that is larger than the band found on the Z chromosome and justify the size difference observed between these chromosomes. Here, we provide evidence that the non-centromeric heterochromatic bands in Zq and Wq differ not only in size and location but also in composition, based on comparative genomic hybridization (CGH) and an analysis of the anuran PcP190 satellite DNA. The finding of PcP190 sequences in P. tocantins extends the presence of this satellite DNA, which was previously detected among Leptodactylidae and Hylodidae, suggesting that this family of repetitive DNA is even older than it was formerly considered. Seven groups of PcP190 sequences were recognized in the genome of P. tocantins. PcP190 probes mapped to the heterochromatic band in Wq, and a Southern blot analysis indicated the accumulation of PcP190 in the female genome of P. tocantins, which suggests the involvement of this satellite DNA in the evolution of the sex chromosomes of this species.

Avaliação da atividade proliferativa celular das lesões bucais causadas pelo uso de próteses totais através da impregnação tecidual pela prata / Proliferative cellular activity evaluation of lesions caused by dentures through tissue silver impregnation

Introdução: As próteses totais visam conservar a função do sistema estomatognático do paciente totalmente edêntulo. Porém, na mucosa bucal podem aparecer manifestações cuja principal causa são as próteses totais mal adaptadas. Objetivo: o presente estudo objetiva investigar a proliferação tecidual das lesões causadas por próteses totais removíveis através do método de impregnação pela prata (AgNOR), com isso facilitando o tratamento e a determinação do prognóstico das lesões a serem estudadas. Metodologia: foram selecionados todos os casos das lesões bucais mais comumente associadas à utilização de próteses totais registradas no Serviço de Diagnóstico Histopatológico do ICB­UPF nos anos de 2012 e 2013, tendo sido encontrados 5 casos de granuloma piogênico, 5 casos de hiperplasia de fundo de sulco, 5 casos de fibroma de irritação e 2 casos de fibroma ossificante periférico. Os cortes histopatológicos das lesões foram impregnados pela prata (método AgNOR), tendo sido obtido, com auxílio do programa Image Tool®, o número de NORs de 100 células de cada caso, resultando numa média de NORs em cada grupo de lesões. Resultados: os resultados obtidos foram tabulados em planilha eletrônica e a comparação do número médio de NORs de cada grupo foi realizado por meio do teste estatístico ANOVA, 5% de significância. Resultados: o grupo das hiperplasias de fundo de sulco mostrou média de 2,41 NORs por núcleo, o grupo dos granulo mas piogênicos mostrou 2,44, o fibroma de irritação 2,22, e o fibroma ossificante periférico mostrou média de 1,89 NORs por núcleo celular, diferindo estatisticamente esta lesão das anteriormente mencionadas (p = 0,002). Conclusão: o fibroma ossificante periférico mostrou ser a lesão causada por prótese total removível com a menor atividade proliferativa celular. Tal estudo precisa ser complementado por futuros estudos clínicos.

Introduction: the total dentures are aimed at preserving the function of the stomatognathic system of the fully edentulous patient. However, in the oral mucosa may appear manifestations whose main cause are the totally unsuitable dentures. Objective: this study aims to investigate the proliferation of tissue lesions caused by removable dentures by impregnation method for silver (AgNOR), thereby facilitating the treatment and determining the prognosis of the lesions to be studied. Methodology: we selected all cases of oral lesions most commonly associated with the use of dentures recorded in Histopathological Diagnostic Service ICB-UPF in the years 2012 and 2013, having been found 5 cases of pyogenic granuloma, 5 cases of hyperplasia, 5 cases of irritation fibroma and 2 cases of peripheral ossifying fibroma. Histopathological lesions cuts were impregnated by silver (AgNOR method), having been obtained with the aid of the program Image Tool®, the number of NOR cells 100 in each case, resulting in an average NORs in every group of lesions. Results: the results were tabulated in a spreadsheet and comparing the average number of NORs of each group was conducted through ANOVA, 5% significance level. Results: The group of hyperplasias showed average of 2.41 NORs per nucleus, the group of pyogenic granulomas showed 2.44, the irritation fibroma 2.22, and peripheral ossifying fibroma showed average of 1.89 NORs for cell nucleus, differing significantly from that of the aforementioned lesions (p = 0.002). Conclusion: the peripheral ossifying fibroma proved the injury caused by removable dentures with lower cell proliferative activity. This study needs to be complemented by future clinical studies.

Avaliação da atividade proliferativacelular da leucoplasia bucal por meio do método AgNOR / Assessment of cell proliferative activity of oral leukoplakia through the AgNOR method

A leucoplasia é considerada uma importante lesão bucal cancerizável. Objetivo: realizar um estudo epidemiológico dos casos de leucoplasia bucal registrados no Serviço de Diagnóstico Histopatológico do Instituto de Ciências Biológicas da Universidade de Passo Fundo (SDH/ICB-UPF) entre 1989 e 2012, aplicando a tais casos o método AgNOR para avaliar a atividade proliferativa celular desses. Materiais e método: o trabalho consistiu num estudo epidemiológico-histoquímico transversal de 44 casos de leucoplasia. Após a confirmação diagnóstica e a tabulação dos dados clínicos e histopatológicos dos casos de leucoplasia, novas secções histológicas (3 µm) das lesões foram impregnadas pela prata por meio do método AgNOR. As lâminas obtidas foram fotografadas em imersão, tendo sido contabilizadas as NORs de 100 células de cada caso. Tal procedimento foi realizado com o auxílio do software Image Tool®. Os resultados foram comparados levando-se em conta as diferentes características clínicas e histopatológicas dos casos de leucoplasia. Resultados: a faixa etária mais frequente foi de 61 a 76 anos (19 casos - 43,2%), 22 pacientes pertenciam ao sexo masculino (50%) e a etnia predominante foi a leucoderma (36 casos - 81,8%). No que diz respeito aos hábitos nocivos, 23 pacientes (52,3%) informaram ser tabagistas. Ao se comparar o número médio de NORs dos casos de leucoplasia a diferentes características clínicas das lesões, não foi observada uma relação estatisticamente significativa. Conclusão: conclui-se que a leucoplasia acomete principalmente homens leucodermas, fumantes e com mais de 60 anos de idade. Além disso, na amostra ora estudada, a relação da atividade proliferativa celular da leucoplasia bucal com as características clínicas/ histopatológicas das lesões não foi estatisticamente significativa.

Evaluation of Cellular Proliferative Activity in Patients with Oral Lichen Planus and Hepatitis C through AgNOR Method

The objective of this study was to evaluate the cellular proliferative potential of oral lichen planus (OLP) lesions from patients without hepatitis C virus (HCV) by means of AgNOR method, as well as the cellular proliferative potential of the normal oral mucosa from patients with HCV, treated or untreated by interferon and ribavirin. A cross-sectional study was developed to investigate four groups: 10 HCV+ patients without clinical signs of OLP who had never been treated for HCV infection - Group 1; 10 HCV+ patients that were under interferon and ribavirin treatment - Group 2; 15 patients with reticular OLP lesions histopathologically confirmed, without HCV - Group 3; and 15 blood donors without HCV infection and no clinical signs of OLP GROUP 4 Control Group. The cytological material of all groups was collected by the liquid-based cytology technique. Then, the sedimented material from each patient was filled with the Nucleolar Organizer Regions impregnation by silver method (AgNOR). The count of NORs was performed on 100 epithelial cell nuclei per patient using the Image Tool(tm) software. The Tukey HSD test was used to compare the median value of NORs among the groups and showed that the oral mucosa of HCV+ patients previously treated with anti-HCV drugs (GROUP 2), presented a higher average number of NORs in relation to others (p<0.05). The anti-HCV treatment may be related to increased cell proliferation of oral mucosa, indicating a possible relationship between OLP and HCV+ patients treated with interferon and ribavirin.

O propósito deste estudo foi avaliar o potencial proliferativo celular das lesões de líquen plano bucal (LPB) de pacientes sem vírus da hepatite C (VHC) por meio do método AgNOR, comparando-o ao potencial proliferativo celular da mucosa bucal normal de portadores de VHC, tratados ou não com interferon e ribavirina. Um estudo transversal foi realizado para investigar 4 grupos: 10 pacientes VHC+ sem sinais clínicos de LPB que nunca haviam sido tratados para a infecção por VHC - Grupo 1; 10 pacientes VHC+ que estavam sob tratamento com interferon e ribavirina - Grupo 2; 15 pacientes com LPB reticular histopatologicamente confirmado, sem VHC - Grupo 3; e 15 doadores de sangue sem infecção por VHC e sem sinais clínicos de LPB (Grupo 4 - Grupo de Controle). O material celular de todos os grupos foi coletado pela técnica da citologia em base líquida. Então, o material sedimentado de cada paciente foi submetido ao método da impregnação das regiões organizadoras nucleolares pela prata (AgNOR). A contagem das NORs foi realizada em 100 núcleos celulares epiteliais por paciente por meio do programa Image Tool(r). O teste Tukey HSD foi utilizado para comparar o valor médio de NORs entre os grupos e mostrou que a mucosa bucal dos pacientes VHC+ previamente tratados com fármacos anti-VHC (Grupo 2) apresentou maior número médio de NORs por núcleo em relação aos outros (p<0,05). O tratamento anti-VHC pode estar relacionado ao aumento da atividade proliferativa celular da mucosa bucal, aventando uma possível relação entre LPB e pacientes VHC+ tratados com interferon e ribavirina.

Evaluation of cellular proliferative activity in patients with oral lichen planus and hepatitis C through AgNOR method.

The objective of this study was to evaluate the cellular proliferative potential of oral lichen planus (OLP) lesions from patients without hepatitis C virus (HCV) by means of AgNOR method, as well as the cellular proliferative potential of the normal oral mucosa from patients with HCV, treated or untreated by interferon and ribavirin. A cross-sectional study was developed to investigate four groups: 10 HCV+ patients without clinical signs of OLP who had never been treated for HCV infection - Group 1; 10 HCV+ patients that were under interferon and ribavirin treatment - Group 2; 15 patients with reticular OLP lesions histopathologically confirmed, without HCV - Group 3; and 15 blood donors without HCV infection and no clinical signs of OLP GROUP 4 Control Group. The cytological material of all groups was collected by the liquid-based cytology technique. Then, the sedimented material from each patient was filled with the Nucleolar Organizer Regions impregnation by silver method (AgNOR). The count of NORs was performed on 100 epithelial cell nuclei per patient using the Image Tool(tm) software. The Tukey HSD test was used to compare the median value of NORs among the groups and showed that the oral mucosa of HCV+ patients previously treated with anti-HCV drugs (GROUP 2), presented a higher average number of NORs in relation to others (p<0.05). The anti-HCV treatment may be related to increased cell proliferation of oral mucosa, indicating a possible relationship between OLP and HCV+ patients treated with interferon and ribavirin.

Evaluation of the taxonomic status of populations assigned to Phyllomedusa hypochondrialis (Anura, Hylidae, Phyllomedusinae) based on molecular, chromosomal, and morphological approach.

BACKGROUND: The taxonomic and phylogenetic relationships of the genus Phyllomedusa have been amply discussed. The marked morphological similarities among some species hamper the reliable identification of specimens and may often lead to their incorrect taxonomic classification on the sole basis of morphological traits. Phenotypic variation was observed among populations assigned to either P. azurea or P. hypochondrialis. In order to evaluate whether the variation observed in populations assigned to P. hypochondrialis is related to that in genotypes, a cytogenetic analysis was combined with phylogenetic inferences based on mitochondrial and nuclear sequences. RESULTS: The inter- and intra-population variation in the external morphology observed among the specimens analyzed in the present study do not reflect the phylogenetic relationships among populations. A monophyletic clade was recovered, grouping all the specimens identified as P. hypochondrialis and specimens assigned P. azurea from Minas Gerais state. This clade is characterized by conserved chromosomal morphology and a common C-banding pattern. Extensive variation in the nucleolar organizing region (NOR) was observed among populations, with four distinct NOR positions being recognized in the karyotypes. Intra-population polymorphism of the additional rDNA clusters observed in specimens from Barreiras, Bahia state, also highlights the marked genomic instability of the rDNA in the genome of this group. Based on the topology obtained in the phylogenetic analyses, the re-evaluation of the taxonomic status of the specimens from the southernmost population known in Brazil is recommended. CONCLUSIONS: The results of this study support the need for a thorough revision of the phenotypic features used to discriminate P. azurea and P. hypochondrialis. The phylogenetic data presented here also contribute to an extension of the geographic range of P. hypochondrialis, which is known to occur in the Amazon basin and neighboring areas of the Cerrado savanna, where it may be sympatric with P. azurea, within contact zones. The misidentification of specimens may have led to inconsistencies in the original definition of the geographic range of P. azurea. The variability observed in the NOR of P. hypochondrialis reinforces the conclusion that these sites represent hotspots of rearrangement. Intraspecific variation in the location of these sites is the result of constant rearrangements that are not detected by classical cytogenetic methods or are traits of an ancestral, polymorphic karyotype, which would not be phylogenetically informative for this group.

Cytogenetic analysis of two species in the Phyllomedusa hypochondrialis group (Anura, Hylidae).

The genus Phyllomedusa has been the target of regular taxonomic investigations. The species Phyllomedusa nordestina was recently separated from P. hypochondrialis. Morphological variations in the P. rohdei interpopulation have already been reported, suggesting the existence of more than one taxon under that name. In the present study, we have cytogenetically characterized two populations of P. nordestina and one of P. rohdei. Both species displayed 2n = 26 chromosomes with 12 metacentric, 12 submetacentric and 2 subtelocentric chromosomes. The C-banding analyses revealed discrete differences in the quantity of centromeric heterochromatin between the two species. The nucleolus organizer region (NOR) was detected in pair 9 of both species, but is located in the pericentromeric region of the short arm in P. nordestina and in the long arm subtelomeric region of P. rohdei. Chromosomal data from this study indicate karyotypic homeology between the two groups of P. hypochondrialis species and suggest the existence of more than one taxon under the P. rohdei name.

Evaluation of proliferative activity in oral squamous cell carcinoma by the AgNOR staining method / Avaliação da atividade proliferativa do carcinoma espinocelular pelo método AgNOR

The objective of this study was to evaluate the cellular proliferative activity of argyrophilic nucleolar organizer regions (AgNORs) in a series of oral squamous cell carcinoma (OSCC) cases and to correlate this activity with the histopathologic grade of the tumor defined by the World Health Organization (WHO).Methodology: twenty cases of OSCC obtained from the Diagnostic Histopathology Service of UPF database were studied. Age, gender and ethnic origin of the patients and oral site affected by the tumor were obtained directly from the histopathologic reports. The AgNOR technique was used for histochemical analysis of the tumors.Results: there was a predominance of OSCC in male patients in the sixth decade of life. The lip was the most affected oral site and WHO grade I was the predominant histologic grade. The mean number of NORs per nucleus in the sample was 2.00 (standard deviation: 0.58). Pearson’s correlation test showed that the number of NORs in OSCC was directly correlated with histologic tumor grade (p = 0.021).Conclusion: the AgNOR method was useful as a diagnostic tool and prognostic indicator of OSCC when combined with conventional histopathologic analysis...

Avaliar a atividade proliferativa celular das regiões organizadoras nucleolares (AgNORs) em uma série de casos de carcinoma espinocelular da cavidade bucal (CECB) e correlacionar esta atividade com o grau histológico do tumor, definida pela Organização Mundial da Saúde (OMS). Metodologia: vinte casos de CECB estudados foram obtidos a partir de diagnóstico do Serviço de Diagnóstico Histopatológico da UPF. Dados relativos à idade, sexo e origem étnica dos pacientes, além da localização da lesão na cavidade bucal, foram obtidos a partir dos relatórios histopatológicos. A técnica de AgNOR foi utilizada para análise histoquímica dos tumores.Resultados: houve predomínio de CECB em pacientes do sexo masculino na sexta década de vida. O lábio foi o local mais afetado e o grau histológico I foi o predominante. O número médio de NORs por núcleo na amostra foi de 2,00 (desvio padrão: 0,58). O teste de correlação de Pearson mostrou que o número de NORs no CECB foi diretamente correlacionado com o grau histológico do tumor (p = 0,021). Conclusão: o método AgNOR foi útil como uma ferramenta de diagnóstico e indicador de prognóstico do CECB, quando combinado com a análise histopatológica convencional...

Estudo comparativo da atividade proliferativa celular do fibroma ossificante periférico e da lesão de células gigantes periférica / Comparative study of the proliferative activity of the peripheral giant cells lesion and of peripheral ossifying fibroma

Foi realizado um estudo clínico retrospectivo descritivo e histoquímico de casos do fibroma ossificante periférico (FOP) e da lesão de células gigantes periférica (LCGP), coletados aleatoriamente no arquivo do Serviço de Diagnóstico Histopatológico da Universidade de Passo Fundo. O objetivo do trabalho foi comparar as atividades proliferativas celulares dessas lesões, visando o estabelecimento de uma conduta terapêutica adequada para cada uma das enfermidades. Os dados referentes ao sexo, idade, raça, localização da lesão e ocorrência de recidiva foram analisados e, o número de regiões organizadoras nucleolares (NORs) por núcleo de células ovóides foi determinado por meio do método de impregnação pela prata (Ag-NOR). Os resultados obtidos para o FOP foram de prevalência da lesão em mulheres (70%) da raça branca (60%) com média de idade de 28,11 anos, maior ocorrência da lesão na maxila anterior e índice de recidiva de 30%. O número médio de NORs foi de 1,72 para cada núcleo. Já para a LCGP, o sexo feminino também atingiu 70%, porém a média de idade foi de 42,90 anos e 100% dos pacientes pertenciam à raça branca. A ocorrência da lesão foi maior na região mandibular anterior e apresentou um índice de 20% de recidiva. Nesta lesão, o número médio de NORs foi de 1,93 por núcleo. Contudo, nos testes de correlação, nenhuma das características clínicas apresentou associação com o número médio de NORs por núcleo e a comparação desse número médio por núcleo nas LCGPs e nos FOPs não mostrou diferença significativa entre os grupos.

A retrospective clinical descritive and histochemical study envolving cases of periferic bone fibroma and of periferic giant cell lesion, randomly collected from the archives of Histophatologic Diagnostic Service of University of Passo Fundo was made. The objective of this work was to compare the proliferating cellular activities of these lesions, aiming the establishment of an adequate therapeutic conduct to each one of the disorders. The datas referring to the gender, age, race, position of the lesion and recurrence occurrence were analized and considered and the NORs’ numbers by egg-shaped cells’ nucleus were analized through the impregnation of silver method (Ag-NOR), getting as a result of periferic bone fibroma, 70% women, with an age average of 28.11 years old, where 60% of the pacients were white, and with the lesion’s position more often being on the front upper jaw, with the recurrence’s index up to 30%. The NORs’ average number was of 1.72 to each nucleus. On the periphery giant cell lesion, the female gender had also achieved 70%, however the age’s average was of 42.90 and 100% were white. The occurrence of the lesion was higher on the front inferior jaw and had showed a recurrence’s index of 20%. The NORs’ average of this lesion was 1.93 to each nucleus. Nevertheless, on the correlation’s tests none of these clinic characteristics showed a connection with the higher average’s number of NORs by nucleus and the comparison of NORs average’s number by nucleus on periphery bone fibroma and periphery giant cell lesion did not show a significant difference between the groups.

Cytogenetic analysis of four species of Pseudis (Anura, Hylidae), with the description of ZZ/ZW sex chromosomes in P. tocantins.

Pseudis paradoxa paradoxa, P. p. platensis, P. bolbodactyla, P. fusca and P. tocantins were analyzed cytogenetically by conventional chromosomal staining, C-banding, silver staining and fluorescent in situ hybridization with an rDNA probe. Pseudis tocantins chromosomes were also stained with distamycin A/DAPI. All of the species had a diploid number of 2n = 24 chromosomes and the nucleolar organizer region (NOR) was located on pair 7. However, the karyotypes could be differentiated based on the morphology of chromosomal pairs 2 and 8, the region that the NORs occupied on the long arms of the homologous of pair 7, and the pattern of heterochromatin distribution. The subspecies P. p. paradoxa and P. p. platensis had identical karyotypes. Heteromorphism in NOR size was seen in P. p. paradoxa, P. p. platensis, P. bolbodactyla and P. fusca. Heteromorphic sex chromosomes (ZZ/ZW) were identified in P. tocantins. The W chromosome was subtelocentric and larger than the metacentric Z chromosomes. The differences observed in the C-banding pattern and in the position of the NOR on the sex chromosomes suggested that inversions and heterochromatinization were responsible for the morphological differentiation of these chromosomes.

Chromosomal differentiation of populations of Lysapsus limellus limellus, L. l. bolivianus, and of Lysapsus caraya (Hylinae, Hylidae).

Cytogenetic analysis were done on specimens from two populations of Lysapsus limellus limellus, three of L. l. bolivianus and of one of Lysapsus caraya. All animals showed a diploid chromosomal number of 2n=24. The karyotypes of the two L. limellus subspecies were very similar, differing only by the larger amount of telomeric heterochromatin and a small pericentromeric C-band on the short arms of pair 2 in L. l. limellus specimens. The karyotype of L. caraya differed from those of the two L. limellus subspecies in terms of chromosomal morphology, C-banding pattern and location of the main NOR on chromosomes 7 and 6, respectively. The karyotype of the L. l. bolivianus population from Guajará-Mirim/RO differed from those of the other populations of the same subspecies in morphology and heterochromatin pattern of chromosomes 7 and 8. Additional NORs were detected by silver staining and confirmed by FISH in one of the homologues of pairs 1 and 8 in L. l. bolivianus and in pair 7 in L. caraya. These results suggest that a reassessment of the taxonomic status of L. limellus subspecies, especially of the L. l. bolivianus populations, may be necessary.