Down syndrome: orofacial pain, masticatory muscle hypotonia, and sleep disorders.

The aim of the present study was to investigate orofacial pain in individuals with Down syndrome (DS) and determine possible associations with masticatory muscle hypotonia (MMH), maximum mouth opening (MMO), and sleep disorders. Twenty-three individuals with DS underwent a standardized clinical examination using Axis I of the Diagnostic Criteria for Temporomandibular Disorders, for the diagnosis of pain in the masseter and temporal muscles and temporomandibular joint (TMJ). MMH was investigated using electromyography of the temporal and masseter muscles and the measurement of maximum bite force (MBF). MMO was measured using an analog caliper. Sleep disorders (obstructive sleep apnea [OSA], snoring index [SI], and sleep bruxism index [SBI]) were investigated using type II polysomnography. Statistical analysis was performed. Nonsignificant differences were found in muscle and TMJ pain between the sexes. However, myalgia and referred myofascial pain in the left masseter muscle were more frequent in males (69%) than females (40%). Electrical activity of the temporal (left: p = .002; right: p = .004) and masseter (left: p = .008) muscles was significantly lower in males than in females. MBF range was lower in males than females, indicating the highest MMH among males. OSA, SI, and SBI were identified in both sexes, but with no statistically significant differences. We concluded that myalgia and referred myofascial pain were found in some individuals with DS, especially in males. Arthralgia was found mainly in females. Temporal and masseter myalgia may have exerted an influence on the severity of MMH in males, particularly on the left side.

Against the mainstream: exceptional evolutionary stability of ZW sex chromosomes across the fish families Triportheidae and Gasteropelecidae (Teleostei: Characiformes).

Teleost fishes exhibit a breath-taking diversity of sex determination and differentiation mechanisms. They encompass at least nine sex chromosome systems with often low degree of differentiation, high rate of inter- and intra-specific variability, and frequent turnovers. Nevertheless, several mainly female heterogametic systems at an advanced stage of genetic differentiation and high evolutionary stability have been also found across teleosts, especially among Neotropical characiforms. In this study, we aim to characterize the ZZ/ZW sex chromosome system in representatives of the Triportheidae family (Triportheus auritus, Agoniates halecinus, and the basal-most species Lignobrycon myersi) and its sister clade Gasteropelecidae (Carnegiella strigata, Gasteropelecus levis, and Thoracocharax stellatus). We applied both conventional and molecular cytogenetic approaches including chromosomal mapping of 5S and 18S ribosomal DNA clusters, cross-species chromosome painting (Zoo-FISH) with sex chromosome-derived probes and comparative genomic hybridization (CGH). We identified the ZW sex chromosome system for the first time in A. halecinus and G. levis and also in C. strigata formerly reported to lack sex chromosomes. We also brought evidence for possible mechanisms underlying the sex chromosome differentiation, including inversions, repetitive DNA accumulation, and exchange of genetic material. Our Zoo-FISH experiments further strongly indicated that the ZW sex chromosomes of Triportheidae and Gasteropelecidae are homeologous, suggesting their origin before the split of these lineages (approx. 40-70 million years ago). Such extent of sex chromosome stability is almost exceptional in teleosts, and hence, these lineages afford a special opportunity to scrutinize unique evolutionary forces and pressures shaping sex chromosome evolution in fishes and vertebrates in general.

Visceral Obesity and High Systolic Blood Pressure as the Substrate of Endothelial Dysfunction in Obese Adolescents. / Obesidade Visceral e Hipertensão Sistólica como Substratos da Disfunção Endotelial em Adolescentes Obesos.

BACKGROUND: Obesity affects adolescence and may lead to metabolic syndrome (MetS) and endothelial dysfunction, an early marker of cardiovascular risk. Albeit obesity is strongly associated with obstructive sleep apnea (OSA), it is not clear the role of OSA in endothelial function in adolescents with obesity. OBJECTIVE: To investigate whether obesity during adolescence leads to MetS and/or OSA; and causes endothelial dysfunction. In addition, we studied the possible association of MetS risk factors and apnea hypopnea index (AHI) with endothelial dysfunction. METHODS: We studied 20 sedentary obese adolescents (OA; 14.2±1.6 years, 100.9±20.3kg), and 10 normal-weight adolescents (NWA, 15.2±1.2 years, 54.4±5.3kg) paired for sex. We assessed MetS risk factors (International Diabetes Federation criteria), vascular function (Flow-Mediated Dilation, FMD), functional capacity (VO2peak) and the presence of OSA (AHI>1event/h, by polysomnography). We considered statistically significant a P<0.05. RESULTS: OA presented higher waist (WC), body fat, triglycerides, systolic (SBP) and diastolic blood pressure (DBP), LDL-c and lower HDL-c and VO2peak than NWA. MetS was presented in the 35% of OA, whereas OSA was present in 86.6% of OA and 50% of EA. There was no difference between groups in the AHI. The OA had lower FMD than NWA (6.17±2.72 vs. 9.37±2.20%, p=0.005). There was an association between FMD and WC (R=-0.506, p=0.008) and FMD and SBP (R=-0.493, p=0.006). CONCLUSION: In adolescents, obesity was associates with MetS and caused endothelial dysfunction. Increased WC and SBP could be involved in this alteration. OSA was observed in most adolescents, regardless of obesity. (Arq Bras Cardiol. 2021; 116(4):795-803).

FUNDAMENTO: A obesidade afeta a adolescência, podendo levar à síndrome metabólica (SM) e disfunção endotelial, um marcador precoce de risco cardiovascular. Apesar de a obesidade ser fortemente associada à síndrome da apneia obstrutiva do sono (SAOS), ainda não está claro o papel da SAOS na função endotelial em adolescentes obesos. OBJETIVO: Investigar se a obesidade durante a adolescência leva à SM e/ou SAOS e causa disfunção endotelial nesses indivíduos. Além disso, estudamos a possível associação dos fatores de risco para SM e do índice de apneia e hipopneia (IAH) com disfunção endotelial. MÉTODOS: Estudamos 20 adolescentes obesos sedentários (AO; 14,2±1,6 anos, 100,9±20,3kg), e 10 adolescentes eutróficos (AE, 15,2±1,2 anos, 54,4±5,3kg) pareados por sexo. Avaliamos os fatores de risco para SM (critérios da Federação Internacional de Diabetes), função vascular (dilatação mediada pelo fluxo, DMF), capacidade funcional (VO2pico) e presença de SAOS (IAH > 1 evento/hora, pela polissonografia). Consideramos um p<0,05 como estatisticamente significativo. RESULTADOS: AO apresentaram maior circunferência da cintura (CC), gordura corporal, triglicerídeos, pressão arterial sistólica (PAS) e diastólica (PAD), maiores níveis de LDL e menores HDL e VO2pico em comparação a AE. Não houve diferença no IAH entre os grupos. AO apresentaram menor DMF que AE (6,17±2,72 vs. 9,37±2,20%, p=0,005). Observou-se uma associação entre DMF e CC (R=-0,506, p=0,008) e entre DMF e PAS (R=-0,493, p=0,006). CONCLUSÃO: Em adolescentes, a obesidade associou-se à SM e causou disfunção endotelial. CC e PAS aumentadas poderiam estar envolvidas nessa alteração. SAOS foi detectada na maioria dos adolescentes independentemente de obesidade. (Arq Bras Cardiol. 2021; 116(4):795-803).

Obesidade Visceral e Hipertensão Sistólica como Substratos da Disfunção Endotelial em Adolescentes Obesos / Visceral Obesity and High Systolic Blood Pressure as the Substrate of Endothelial Dysfunction in Obese Adolescents

Resumo Fundamento: A obesidade afeta a adolescência, podendo levar à síndrome metabólica (SM) e disfunção endotelial, um marcador precoce de risco cardiovascular. Apesar de a obesidade ser fortemente associada à síndrome da apneia obstrutiva do sono (SAOS), ainda não está claro o papel da SAOS na função endotelial em adolescentes obesos. Objetivo: Investigar se a obesidade durante a adolescência leva à SM e/ou SAOS e causa disfunção endotelial nesses indivíduos. Além disso, estudamos a possível associação dos fatores de risco para SM e do índice de apneia e hipopneia (IAH) com disfunção endotelial. Métodos: Estudamos 20 adolescentes obesos sedentários (AO; 14,2±1,6 anos, 100,9±20,3kg), e 10 adolescentes eutróficos (AE, 15,2±1,2 anos, 54,4±5,3kg) pareados por sexo. Avaliamos os fatores de risco para SM (critérios da Federação Internacional de Diabetes), função vascular (dilatação mediada pelo fluxo, DMF), capacidade funcional (VO2pico) e presença de SAOS (IAH > 1 evento/hora, pela polissonografia). Consideramos um p<0,05 como estatisticamente significativo. Resultados: AO apresentaram maior circunferência da cintura (CC), gordura corporal, triglicerídeos, pressão arterial sistólica (PAS) e diastólica (PAD), maiores níveis de LDL e menores HDL e VO2pico em comparação a AE. Não houve diferença no IAH entre os grupos. AO apresentaram menor DMF que AE (6,17±2,72 vs. 9,37±2,20%, p=0,005). Observou-se uma associação entre DMF e CC (R=-0,506, p=0,008) e entre DMF e PAS (R=-0,493, p=0,006). Conclusão: Em adolescentes, a obesidade associou-se à SM e causou disfunção endotelial. CC e PAS aumentadas poderiam estar envolvidas nessa alteração. SAOS foi detectada na maioria dos adolescentes independentemente de obesidade. (Arq Bras Cardiol. 2021; 116(4):795-803)

Abstract Background: Obesity affects adolescence and may lead to metabolic syndrome (MetS) and endothelial dysfunction, an early marker of cardiovascular risk. Albeit obesity is strongly associated with obstructive sleep apnea (OSA), it is not clear the role of OSA in endothelial function in adolescents with obesity. Objective: To investigate whether obesity during adolescence leads to MetS and/or OSA; and causes endothelial dysfunction. In addition, we studied the possible association of MetS risk factors and apnea hypopnea index (AHI) with endothelial dysfunction. Methods: We studied 20 sedentary obese adolescents (OA; 14.2±1.6 years, 100.9±20.3kg), and 10 normal-weight adolescents (NWA, 15.2±1.2 years, 54.4±5.3kg) paired for sex. We assessed MetS risk factors (International Diabetes Federation criteria), vascular function (Flow-Mediated Dilation, FMD), functional capacity (VO2peak) and the presence of OSA (AHI>1event/h, by polysomnography). We considered statistically significant a P<0.05. Results: OA presented higher waist (WC), body fat, triglycerides, systolic (SBP) and diastolic blood pressure (DBP), LDL-c and lower HDL-c and VO2peak than NWA. MetS was presented in the 35% of OA, whereas OSA was present in 86.6% of OA and 50% of EA. There was no difference between groups in the AHI. The OA had lower FMD than NWA (6.17±2.72 vs. 9.37±2.20%, p=0.005). There was an association between FMD and WC (R=-0.506, p=0.008) and FMD and SBP (R=-0.493, p=0.006). Conclusion: In adolescents, obesity was associates with MetS and caused endothelial dysfunction. Increased WC and SBP could be involved in this alteration. OSA was observed in most adolescents, regardless of obesity. (Arq Bras Cardiol. 2021; 116(4):795-803)

High Genetic Diversity despite Conserved Karyotype Organization in the Giant Trahiras from Genus Hoplias (Characiformes, Erythrinidae).

In the fish genus Hoplias, two major general groups can be found, one of which is formed by the "common trahiras" (Hoplias malabaricus group) and the other by the "giant trahiras" (Hoplias lacerdae group, in addition to Hoplias aimara), which usually comprises specimens of larger body size. Previous investigations from the giant trahiras group recovered 2n = 50 meta/submetacentric chromosomes and no sex chromosome differentiation, indicating a probable conservative pattern for their karyotype organization. Here, we conducted comparative cytogenetic studies in six giant trahiras species, two of them for the first time. We employed standard and advanced molecular cytogenetics procedures, including comparative genomic hybridization (CGH), as well as genomic assessments of diversity levels and phylogenetic relationships among them. The results strongly suggest that the giant trahiras have a particular and differentiated evolutionary pathway inside the Hoplias genus. While these species share the same 2n and karyotypes, their congeneric species of the H. malabaricus group show a notable chromosomal diversity in number, morphology, and sex chromosome systems. However, at the same time, significant changes were characterized at their inner chromosomal level, as well as in their genetic diversity, highlighting their current relationships resulting from different evolutionary histories.

Comparative cytogenetic of six species of Amazonian Peacock bass (Cichla, Cichlinae): intrachromosomal variations and genetic introgression among sympatric species.

Cytogenetic data for the genus Cichla Bloch et Schneider, 1801 are still very limited, with only four karyotype descriptions to date. The sum of the available cytogenetic information for Cichla species, points to a maintenance of the diploid number of 48 acrocentric chromosomes, considered a typical ancestral feature in cichlids. In the current study, we performed molecular and classical cytogenetic analyses of the karyotype organization of six species of Cichla, the earliest-diverging genus of Neotropical cichlids. We cytogenetically analysed Cichla kelberi Kullander et Ferreira, 2006, Cichla monoculus Agassiz, 1831, Cichla piquiti Kullander et Ferreira, 2006, Cichla temensis Humboldt, 1821, Cichla vazzoleri Kullander et Ferreira, 2006 and Cichla pinima Kullander et Ferreira, 2006, including three individuals that showed mixed morphological characteristics, likely from different species, suggesting they were hybrid individuals. All individuals analysed showed 2n = 48 acrocentric chromosomes, with centromeric heterochromatic blocks on all chromosomes and a terminal heterochromatic region on the q arm of the 2nd pair. Mapping 18S rDNA gave hybridization signals, correlated with the nucleolus organizer regions, on the 2nd pair for all analyzed individuals. However, we found distinct patterns for 5S rDNA: interstitially at the proximal position on 6th pair of four species (C. kelberi, C. pinima, C. piquiti and C. vazzoleri), and on the distal of the 4th pair in two (C. monoculus and C. temensis). Accordingly, we present here new data for the genus and discuss the evolutionary trends in the karyotype of this group of fish. In addition, we provide data that supports the occurrence of hybrid individuals in the Uatumã River region, mainly based on 5S rDNA mapping.

Multiple Sex Chromosomes and Evolutionary Relationships in Amazonian Catfishes: The Outstanding Model of the Genus Harttia (Siluriformes: Loricariidae).

The armored Harttia catfishes present great species diversity and remarkable cytogenetic variation, including different sex chromosome systems. Here we analyzed three new species, H. duriventris, H. villasboas and H. rondoni, using both conventional and molecular cytogenetic techniques (Giemsa-staining and C-banding), including the mapping of repetitive DNAs using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) experiments. Both H. duriventris and H. villasboas have 2n = ♀56/♂55 chromosomes, and an X1X1X2X2 /X1X2Y sex chromosome system, while a proto or neo-XY system is proposed for H. rondoni (2n = 54♀♂). Single motifs of 5S and 18S rDNA occur in all three species, with the latter being also mapped in the sex chromosomes. The results confirm the general evolutionary trend that has been noticed for the genus: an extensive variation on their chromosome number, single sites of rDNA sequences and the occurrence of multiple sex chromosomes. Comparative genomic analyses with another congeneric species, H. punctata, reveal that the X1X2Y sex chromosomes of these species share the genomic contents, indicating a probable common origin. The remarkable karyotypic variation, including sex chromosomes systems, makes Harttia a suitable model for evolutionary studies focusing on karyotype differentiation and sex chromosome evolution among lower vertebrates.

Evaluation of the masticatory biomechanical function in Down syndrome and its Influence on sleep disorders, body adiposity and salivary parameters.

OBJECTIVE: To evaluate the phenotypic features of the masticatory biomechanics in atypical subjects with Down syndrome (DS). Its influence was analysed on sleep disorders, body adiposity and its risks, and some physicochemical properties of saliva. METHODS: Seventy subjects were enrolled to assess masticatory biomechanical function and divided into two groups: DS and control groups. Electrical activities of the masseter and temporal muscles (at rest and in maximum voluntary clench-MVC), maximum bite force-MBF and maximum mouth opening-MMO were investigated. Among the atypical subjects, just 24 participants underwent the anthropometry, the polysomnography II and the saliva testing (salivary flow rate-SFR, buffer capacity-BC and salivary cortisol levels, morning/SC-AM and night/SC-PM). RESULTS: MVC and MBF values showed high statistical significance in the control group (P < .001) than in the DS group of 35. MMO values were slightly increased in the DS group in relation to the control group. Overweight and obesity were found in both genders. Atypical women showed higher risk to develop cardiovascular-metabolic diseases than in atypical men. OSA severe was 20% for atypical women and 42.8% for atypical men, whereas snoring index was present in all genders. SFR was reduced in 100% of atypical subjects (hyposalivation in 10% women and 28.5% men). Furthermore, 100% BC, 66.6% SC-AM and 91.6% SC-PM showed normal patterns. CONCLUSION: Masseter and temporal muscle hypotonia was found in all atypical subjects with DS. This muscle dysfunction strongly was related to overweight/obesity, risks for development of cardiovascular/metabolic diseases, OSA severity, successive snoring episodes and salivary flow reduction in DS.

An Insight into the Chromosomal Evolution of Lebiasinidae (Teleostei, Characiformes).

Lebiasinidae fishes have been historically neglected by cytogenetical studies. Here we present a genomic comparison in eleven Lebiasinidae species, in addition to a review of the ribosomal DNA sequences distribution in this family. With that, we develop ten sets of experiments in order to hybridize the genomic DNA of representative species from the genus Copeina, Copella, Nannostomus, and Pyrrhulina in metaphase plates of Lebiasina melanoguttata. Two major pathways on the chromosomal evolution of these species can be recognized: (i) conservation of 2n = 36 bi-armed chromosomes in Lebiasininae, as a basal condition, and (ii) high numeric and structural chromosomal rearrangements in Pyrrhulininae, with a notable tendency towards acrocentrization. The ribosomal DNA (rDNA) distribution also revealed a marked differentiation during the chromosomal evolution of Lebiasinidae, since both single and multiple sites, in addition to a wide range of chromosomal locations can be found. With some few exceptions, the terminal position of 18S rDNA appears as a common feature in Lebiasinidae-analyzed species. Altogether with Ctenoluciidae, this pattern can be considered a symplesiomorphism for both families. In addition to the specific repetitive DNA content that characterizes the genome of each particular species, Lebiasina also keeps inter-specific repetitive sequences, thus reinforcing its proposed basal condition in Lebiasinidae.

Centric Fusions behind the Karyotype Evolution of Neotropical Nannostomus Pencilfishes (Characiforme, Lebiasinidae): First Insights from a Molecular Cytogenetic Perspective.

Lebiasinidae is a Neotropical freshwater family widely distributed throughout South and Central America. Due to their often very small body size, Lebiasinidae species are cytogenetically challenging and hence largely underexplored. However, the available but limited karyotype data already suggested a high interspecific variability in the diploid chromosome number (2n), which is pronounced in the speciose genus Nannostomus, a popular taxon in ornamental fish trade due to its remarkable body coloration. Aiming to more deeply examine the karyotype diversification in Nannostomus, we combined conventional cytogenetics (Giemsa-staining and C-banding) with the chromosomal mapping of tandemly repeated 5S and 18S rDNA clusters and with interspecific comparative genomic hybridization (CGH) to investigate genomes of four representative Nannostomus species: N. beckfordi, N. eques, N. marginatus, and N. unifasciatus. Our data showed a remarkable variability in 2n, ranging from 2n = 22 in N. unifasciatus (karyotype composed exclusively of metacentrics/submetacentrics) to 2n = 44 in N. beckfordi (karyotype composed entirely of acrocentrics). On the other hand, patterns of 18S and 5S rDNA distribution in the analyzed karyotypes remained rather conservative, with only two 18S and two to four 5S rDNA sites. In view of the mostly unchanged number of chromosome arms (FN = 44) in all but one species (N. eques; FN = 36), and with respect to the current phylogenetic hypothesis, we propose Robertsonian translocations to be a significant contributor to the karyotype differentiation in (at least herein studied) Nannostomus species. Interspecific comparative genome hybridization (CGH) using whole genomic DNAs mapped against the chromosome background of N. beckfordi found a moderate divergence in the repetitive DNA content among the species' genomes. Collectively, our data suggest that the karyotype differentiation in Nannostomus has been largely driven by major structural rearrangements, accompanied by only low to moderate dynamics of repetitive DNA at the sub-chromosomal level. Possible mechanisms and factors behind the elevated tolerance to such a rate of karyotype change in Nannostomus are discussed.

Comparative cytogenetic survey of the giant bonytongue Arapaima fish (Osteoglossiformes: Arapaimidae), across different Amazonian and Tocantins/Araguaia River basins

The South American giant fishes of the genus Arapaima, commonly known as pirarucu, are one of the most iconic among Osteoglossiformes. Previously cytogenetic studies have identified their karyotype characteristics; however, characterization of cytotaxonomic differentiation across their distribution range remains unknown. In this study, we compared chromosomal characteristics using conventional and molecular cytogenetic protocols in pirarucu populations from the Amazon and Tocantins-Araguaia river basins to verify if there is differentiation among representatives of this genus. Our data revealed that individuals from all populations present the same diploid chromosome number 2n=56 and karyotype composed of 14 pairs of meta- to submetacentric and 14 pairs of subtelo- to acrocentric chromosomes. The minor and major rDNA sites are in separate chromosomal pairs, in which major rDNA sites corresponds to large heterochromatic blocks. Comparative genomic hybridizations (CGH) showed that the genome of these populations shared a great portion of repetitive elements, due to a lack of substantial specific signals. Our comparative cytogenetic data analysis of pirarucu suggested that, although significant genetic differences occur among populations, their general karyotype patterns remain conserved.(AU)

Os peixes gigantes da América do Sul do gêneroArapaima, comumente conhecidos como pirarucus, são um dos mais icônicos de Osteoglossiformes. Estudos citogenéticos prévios identificaram suas características cariotípicas, entretanto a caracterização da diferenciação citotaxonômica através de suas distribuições geográficas ainda são desconhecidas. Nesse estudo, nós comparamos características cromossômicas utilizando técnicas de citogenética clássica e molecular em populações das bacias dos rios Amazonas e Tocantins-Araguaia, a fim de verificar se há alguma diferenciação entre representantes desse gênero. Nossos dados revelaram que indivíduos de todas as populações apresentam número diploide de 2n=56 cromossomos e que seus cariótipos são compostos de 14 pares de cromossomos meta- e submetacêntricos e 14 pares de subtelo- e acrocêntricos. Os sítios maiores e menores de rDNA estão localizados em pares cromossômicos separados, onde os sítios maiores de rDNA correspondem a grandes blocos heterocromáticos. Hibridizações genômicas comparativas (CGH) mostraram que o genoma dos espécimes dessas populações é amplamente compartilhado, devido à falta de sinais substanciais específicos. Nossos dados de citogenética comparativa do pirarucu sugerem que embora diferenças genéticas significativas ocorram entre populações, os padrões cariotípicos gerais se mantêm conservados.(AU)

Cytogenetics of the small-sized fish, Copeina guttata (Characiformes, Lebiasinidae): Novel insights into the karyotype differentiation of the family.

Lebiasinidae is a small fish family composed by miniature to small-sized fishes with few cytogenetic data (most of them limited to descriptions of diploid chromosome numbers), thus preventing any evolutionary comparative studies at the chromosomal level. In the present study, we are providing, the first cytogenetic data for the red spotted tetra, Copeina guttata, including the standard karyotype, C-banding, repetitive DNA mapping by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH), providing chromosomal patterns and novel insights into the karyotype differentiation of the family. Males and females share diploid chromosome number 2n = 42 and karyotype composed of 2 metacentric (m), 4 submetacentric (sm) and 36 subtelocentric to acrocentric (st-a) chromosomes. Blocks of constitutive heterochromatin were observed in the centromeric and interstitial regions of several chromosomes, in addition to a remarkably large distal block, heteromorphic in size, which fully corresponded with the 18S rDNA sites in the fourth chromosomal pair. This overlap was confirmed by 5S/18S rDNA dual-color FISH. On the other hand, 5S rDNA clusters were situated in the long and short arms of the 2nd and 15th pairs, respectively. No sex-linked karyotype differences were revealed by male/female CGH experiments. The genomic probes from other two lebiasinid species, Lebiasina melanoguttata and Pyrrhulina brevis, showed positive hybridization signals only in the NOR region in the genome of C. guttata. We demonstrated that karyotype diversification in lebiasinids was accompanied by a series of structural and numeric chromosome rearrangements of different types, including particularly fusions and fissions.

Interspecific Genetic Differences and Historical Demography in South American Arowanas (Osteoglossiformes, Osteoglossidae, Osteoglossum).

The South American arowanas (Osteoglossiformes, Osteoglossidae, Osteoglossum) are emblematic species widely distributed in the Amazon and surrounding basins. Arowana species are under strong anthropogenic pressure as they are extensively exploited for ornamental and food purposes. Until now, limited genetic and cytogenetic information has been available, with only a few studies reporting to their genetic diversity and population structure. In the present study, cytogenetic and DArTseq-derived single nucleotide polymorphism (SNP) data were used to investigate the genetic diversity of the two Osteoglossum species, the silver arowana O. bicirrhosum, and the black arowana O. ferreirai. Both species differ in their 2n (with 2n = 54 and 56 for O. ferreirai and O. bicirrhosum, respectively) and in the composition and distribution of their repetitive DNA content, consistent with their taxonomic status as different species. Our genetic dataset was coupled with contemporary and paleogeographic niche modeling, to develop concurrent demographic models that were tested against each other with a deep learning approach in O. bicirrhosum. Our genetic results reveal that O. bicirrhosum colonized the Tocantins-Araguaia basin from the Amazon basin about one million years ago. In addition, we highlighted a higher genetic diversity of O. bicirrhosum in the Amazon populations in comparison to those from the Tocantins-Araguaia basin.

Deciphering the Evolutionary History of Arowana Fishes (Teleostei, Osteoglossiformes, Osteoglossidae): Insight from Comparative Cytogenomics.

Arowanas (Osteoglossinae) are charismatic freshwater fishes with six species and two genera (Osteoglossum and Scleropages) distributed in South America, Asia, and Australia. In an attempt to provide a better assessment of the processes shaping their evolution, we employed a set of cytogenetic and genomic approaches, including i) molecular cytogenetic analyses using C- and CMA3/DAPI staining, repetitive DNA mapping, comparative genomic hybridization (CGH), and Zoo-FISH, along with ii) the genotypic analyses of single nucleotide polymorphisms (SNPs) generated by diversity array technology sequencing (DArTseq). We observed diploid chromosome numbers of 2n = 56 and 54 in O. bicirrhosum and O. ferreirai, respectively, and 2n = 50 in S. formosus, while S. jardinii and S. leichardti presented 2n = 48 and 44, respectively. A time-calibrated phylogenetic tree revealed that Osteoglossum and Scleropages divergence occurred approximately 50 million years ago (MYA), at the time of the final separation of Australia and South America (with Antarctica). Asian S. formosus and Australian Scleropages diverged about 35.5 MYA, substantially after the latest terrestrial connection between Australia and Southeast Asia through the Indian plate movement. Our combined data provided a comprehensive perspective of the cytogenomic diversity and evolution of arowana species on a timescale.

Comparative Cytogenetics and Neo-Y Formation in Small-Sized Fish Species of the Genus Pyrrhulina (Characiformes, Lebiasinidae).

Although fishes have traditionally been the subject of comparative evolutionary studies, few reports have concentrated on the application of multipronged modern molecular cytogenetic techniques (such as comparative genomic hybridization = CGH and whole chromosome painting = WCP) to analyze deeper the karyotype evolution of specific groups, especially the historically neglected small-sized ones. Representatives of the family Lebiasinidae (Characiformes) are a notable example, where only a few cytogenetic investigations have been conducted thus far. Here, we aim to elucidate the evolutionary processes behind the karyotype differentiation of Pyrrhulina species on a finer-scale cytogenetic level. To achieve this, we applied C-banding, repetitive DNA mapping, CGH and WCP in Pyrrhulina semifasciata and P. brevis. Our results showed 2n = 42 in both sexes of P. brevis, while the difference in 2n between male and female in P. semifasciata (♂41/♀42) stands out due to the presence of a multiple X1X2Y sex chromosome system, until now undetected in this family. As a remarkable common feature, multiple 18S and 5S rDNA sites are present, with an occasional synteny or tandem-repeat amplification. Male-vs.-female CGH experiments in P. semifasciata highlighted the accumulation of male-enriched repetitive sequences in the pericentromeric region of the Y chromosome. Inter-specific CGH experiments evidenced a divergence between both species' genomes based on the presence of several species-specific signals, highlighting their inner genomic diversity. WCP with the P. semifasciata-derived Y (PSEMI-Y) probe painted not only the entire metacentric Y chromosome in males but also the X1 and X2 chromosomes in both male and female chromosomes of P. semifasciata. In the cross-species experiments, the PSEMI-Y probe painted four acrocentric chromosomes in both males and females of the other tested Pyrrhulina species. In summary, our results show that both intra- and interchromosomal rearrangements together with the dynamics of repetitive DNA significantly contributed to the karyotype divergence among Pyrrhulina species, possibly promoted by specific populational and ecological traits and accompanied in one species by the origin of neo-sex chromosomes. The present results suggest how particular evolutionary scenarios found in fish species can help to clarify several issues related to genome organization and the karyotype evolution of vertebrates in general.

Cytogenetics and DNA barcode reveal an undescribed Apareiodon species (Characiformes: Parodontidae).

Parodontidae is a small group of fish and some species are particularly difficult to identify due to the lack of sufficiently consistent morphological traits. Cytogenetically, the species possess 2n = 54 chromosomes and are either sex-homomorphic or sex-heteromorphic (regarding its chromosomes). We evaluated data on color, tooth morphology, cytogenetics, and mitochondrial markers (COI) in Apareiodon specimens from the Aripuanã River (Amazon basin) and the results were compared to other congeneric taxa. Morphological results show an overlap of body color and tooth morphology to other known Apareiodon. The cytogenetics data showed that the 2n = 54 chromosomes, 50 m/sm + 4 st and, a ZZ/ZW sex chromosome system in Apareiodon sp. are common to other species of the genus. However, the number and chromosomal localization of the 45S ribosomal and pPh2004 satellite DNA sites, in addition to W chromosome localization of the pPh2004 appear to be exclusive cytogenetic features in Apareiodon sp. Our phylogenetic tree revealed well-supported clades and confirmed, by barcode species delimitation analysis, a new Molecular Operational Taxonomic Unit (MOTU) for Apareiodon sp. (Aripuanã River). As a whole, the above features support the occurrence of a new species of the Apareiodon, thus far unknown for the Parodontidae.

Deciphering the Origin and Evolution of the X1X2Y System in Two Closely-Related Oplegnathus Species (Oplegnathidae and Centrarchiformes).

Oplegnathus fasciatus and O. punctatus (Teleostei: Centrarchiformes: Oplegnathidae), are commercially important rocky reef fishes, endemic to East Asia. Both species present an X1X2Y sex chromosome system. Here, we investigated the evolutionary forces behind the origin and differentiation of these sex chromosomes, with the aim to elucidate whether they had a single or convergent origin. To achieve this, conventional and molecular cytogenetic protocols, involving the mapping of repetitive DNA markers, comparative genomic hybridization (CGH), and whole chromosome painting (WCP) were applied. Both species presented similar 2n, karyotype structure and hybridization patterns of repetitive DNA classes. 5S rDNA loci, besides being placed on the autosomal pair 22, resided in the terminal region of the long arms of both X1 chromosomes in females, and on the X1 and Y chromosomes in males. Furthermore, WCP experiments with a probe derived from the Y chromosome of O. fasciatus (OFAS-Y) entirely painted the X1 and X2 chromosomes in females and the X1, X2, and Y chromosomes in males of both species. CGH failed to reveal any sign of sequence differentiation on the Y chromosome in both species, thereby suggesting the shared early stage of neo-Y chromosome differentiation. Altogether, the present findings confirmed the origin of the X1X2Y sex chromosomes via Y-autosome centric fusion and strongly suggested their common origin.

Chromosomal Evolution and Evolutionary Relationships of Lebiasina Species (Characiformes, Lebiasinidae).

We present the first cytogenetic data for Lebiasina bimaculata and L. melanoguttata with the aim of (1) investigating evolutionary events within Lebiasina and their relationships with other Lebiasinidae genera and (2) checking the evolutionary relationships between Lebiasinidae and Ctenoluciidae. Both species have a diploid number 2n = 36 with similar karyotypes and microsatellite distribution patterns but present contrasting C-positive heterochromatin and CMA3+ banding patterns. The remarkable interstitial series of C-positive heterochromatin occurring in L. melanoguttata is absent in L. bimaculata. Accordingly, L. bimaculata shows the ribosomal DNA sites as the only GC-rich (CMA3+) regions, while L. melanoguttata shows evidence of a clear intercalated CMA3+ banding pattern. In addition, the multiple 5S and 18S rDNA sites in L. melanogutatta contrast with single sites present in L. bimaculata. Comparative genomic hybridization (CGH) experiments also revealed a high level of genomic differentiation between both species. A polymorphic state of a conspicuous C-positive, CMA3+, and (CGG)n band was found only to occur in L. bimaculata females, and its possible relationship with a nascent sex chromosome system is discussed. Whole chromosome painting (WCP) and CGH experiments indicate that the Lebiasina species examined and Boulengerella maculata share similar chromosomal sequences, thus supporting the relatedness between them and the evolutionary relationships between the Lebiasinidae and Ctenoluciidae families.

Evaluation of the masticatory muscle function, physiological sleep variables, and salivary parameters after electromechanical therapeutic approaches in adult patients with Down syndrome: a randomized controlled clinical trial.

BACKGROUND: There are many comorbidities associated with Down syndrome (DS), including obstructive sleep apnea (OSA) and masticatory muscle alteration. Muscular hypotonia, in particular, of the masticatory and oropharyngeal muscles is one of the main characteristics of individuals with DS, resulting in impairments of speech, swallowing, and mastication in these individuals. In addition, total or partial obstruction of the airways during sleep can occur due to pharyngeal hypotonia, leading to snoring and to OSA. This progressive respiratory disorder is associated with a high risk of morbidity and mortality in individuals with DS. The aim of this research is to assess the therapeutic effects of surface neuromuscular electrical stimulation (NMES), the mastication apparatus (MA), and a mandibular advancement oral appliance (OAm) with an embedded thermosensitive microchip on the functions of masticatory muscles (bilateral masseter and temporal muscles), physiological sleep variables, and salivary parameters in adult patients with DS. METHODS: The patients with DS will be randomly selected and divided into three groups (DS-NMES, DS-MA, and DS-OAm) with a minimum of 10 patients in each group. A thermosensitive microchip will be embedded in the OAm to record its compliance. The therapeutic effects on masticatory muscle function will be investigated through electromyography, a caliper, and a force-transducer device; the sleep variables, in turn, will be evaluated by means of polysomnography. The physicochemical and microbiological properties of the saliva will also be analyzed, including the salivary flow, viscosity, buffer capacity, cortisol levels (susceptibility to psychological and/or physical stress), and Pseudomonas aeruginosa levels (risk of aspiration pneumonia) in these patients. The methods determined for this study will be carried out prior to and after 2 months of the recommended therapies. DISCUSSION: The primary outcomes would be the improvement and/or reestablishment of the function of masticatory muscles and the physiological sleep variables in this target public since individuals with DS commonly present generalized muscular hypotonia and dysfunction of the oropharyngeal musculature. As a secondary outcome indicator, the impact of the applied therapies (NMES, MA, and OAm) on the salivary microbiological and physicochemical properties in DS individuals will also be assessed. Furthermore, the compliance of OAm usage will be measured through a thermosensitive microchip. TRIAL REGISTRATION: Registro Brasileiro de Ensaios Clínicos, RBR-3qp5np . Registered on 20 February 2018.

Cytogenetics, genomics and biodiversity of the South American and African Arapaimidae fish family (Teleostei, Osteoglossiformes).

Osteoglossiformes represents one of the most ancestral teleost lineages, currently widespread over almost all continents, except for Antarctica. However, data involving advanced molecular cytogenetics or comparative genomics are yet largely limited for this fish group. Therefore, the present investigations focus on the osteoglossiform family Arapaimidae, studying a unique fish model group with advanced molecular cytogenetic genomic tools. The aim is to better explore and clarify certain events and factors that had impact on evolutionary history of this fish group. For that, both South American and African representatives of Arapaimidae, namely Arapaima gigas and Heterotis niloticus, were examined. Both species differed markedly by diploid chromosome numbers, with 2n = 56 found in A. gigas and 2n = 40 exhibited by H. niloticus. Conventional cytogenetics along with fluorescence in situ hybridization revealed some general trends shared by most osteoglossiform species analyzed thus far, such as the presence of only one chromosome pair bearing 18S and 5S rDNA sites and karyotypes dominated by acrocentric chromosomes, resembling thus the patterns of hypothetical ancestral teleost karyotype. Furthermore, the genomes of A. gigas and H. niloticus display remarkable divergence in terms of repetitive DNA content and distribution, as revealed by comparative genomic hybridization (CGH). On the other hand, genomic diversity of single copy sequences studied through principal component analyses (PCA) based on SNP alleles genotyped by the DArT seq procedure demonstrated a very low genetic distance between the South American and African Arapaimidae species; this pattern contrasts sharply with the scenario found in other osteoglossiform species. Underlying evolutionary mechanisms potentially explaining the obtained data have been suggested and discussed.