A83-01 and DMH1 effects in the zebrafish spermatogonial niche: Unraveling the roles of TGF-ß and BMP signaling in the Fsh-mediated spermatogonial fate.

Transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signaling has fundamental roles in the regulation of the stem cell niche for both embryonic and adult stem cells. In zebrafish, male germ stem cell niche is regulated by follicle-stimulating hormone (Fsh) through different members of the TGF-ß superfamily. On the other hand, the specific roles of TGF-ß and BMP signaling pathways are unknown in the zebrafish male germ stem cell niche. Considering this lack of information, the present study aimed to investigate the pharmacological inhibition of TGF-ß (A83-01) and BMP (DMH1) signaling pathways in the presence of recombinant zebrafish Fsh using testicular explants. We also reanalyzed single cell-RNA sequencing (sc-RNA-seq) dataset from adult zebrafish testes to identify the testicular cellular sites of smad expression, and to understand the physiological significance of the changes in smad transcript levels after inhibition of TGF-ß or BMP pathways. Our results showed that A83-01 potentiated the pro-stimulatory effects of Fsh on spermatogonial differentiation leading to an increase in the proportion area occupied by differentiated spermatogonia with concomitant reduction of type A undifferentiated (Aund) spermatogonia. In agreement, expression analysis showed lower mRNA levels for the pluripotency gene pou5f3, and increased expression of dazl (marker of type B spermatogonia and spermatocyte) and igf3 (pro-stimulatory growth factor) following the co-treatment with TGF-ß inhibitor and Fsh. Contrariwise, the inhibition of BMP signaling nullified the pro-stimulatory effects of Fsh, resulting in a reduction of differentiated spermatogonia and increased proportion area occupied by type Aund spermatogonia. Supporting this evidence, BMP signaling inhibition increased the mRNA levels of pluripotency genes nanog and pou5f3, and decreased dazl levels when compared to control. The sc-RNA-seq data unveiled a distinctive pattern of smad expression among testicular cells, primarily observed in spermatogonia (smad 2, 3a, 3b, 8), spermatocytes (smad 2, 3a, 8), Sertoli cells (smad 1, 3a, 3b), and Leydig cells (smad 1, 2). This finding supports the notion that inhibition of TGF-ß and BMP signaling pathways may predominantly impact cellular components within the spermatogonial niche, namely spermatogonia, Sertoli, and Leydig cells. In conclusion, our study demonstrated that TGF-ß and BMP signaling pathways exert antagonistic roles in the zebrafish germ stem cell niche. The members of the TGF-ß subfamily are mainly involved in maintaining the undifferentiated state of spermatogonia, while the BMP subfamily promotes spermatogonial differentiation. Therefore, in the complex regulation of the germ stem cell niche by Fsh, members of the BMP subfamily (pro-differentiation) should be more predominant in the niche than those belonging to the TGF-ß (anti-differentiation). Overall, these findings are not only relevant for understanding the regulation of germ stem cell niche but may also be useful for expanding in vitro the number of undifferentiated spermatogonia more efficiently than using recombinant hormones or growth factors.

Thyroid axis participates in high-temperature-induced male sex reversal through its activation by the stress response.

Environmental changes alter the sex fate in about 15% of vertebrate orders, mainly in ectotherms such as fish and reptiles. However, the effects of temperature changes on the endocrine and molecular processes controlling gonadal sex determination are not fully understood. Here, we provide evidence that thyroid hormones (THs) act as co-players in heat-induced masculinization through interactions with the stress axis to promote testicular development. We first demonstrated that the thyroid axis (through thyroid-related genes and T3 levels) is highly active in males during the gonadal development in medaka (Oryzias latipes). Similarly, T3 treatments promoted female-to-male sex reversal in XX embryos. Subsequently, embryonic exposure to temperature-induced stress up-regulated the genes related to the thyroid and stress axes with a final increase in T3 levels. In this context, we show that blocking the stress axis response by the loss of function of the corticotropin-releasing hormone receptors suppresses thyroid-stimulating hormone expression, therefore, heat-induced activation of the thyroid axis. Thus, our data showed that early activation of the stress axis and, in consequence, the TH axis, too, leaves us with that both being important endocrine players in inducing female-to-male reversal, which can help predict possible upcoming physiological impacts of global warming on fish populations.

Cannabidiol improves Nile tilapia cichlid fish welfare.

Cannabidiol (CBD) is a substance derived from Cannabis sativa, widely studied in medicine for controlling neural diseases in humans. Besides the positive effects on humans, it also presents anxiolytic proprieties and decreases aggressiveness and stress in mammals. Therefore, CBD has the potential to increase welfare in reared animals, as it seems to reduce negative states commonly experienced in artificial environments. Here, we tested the effect of different CBD doses (0, 1, 10 and 20 mg/kg) on aggressiveness, stress and reproductive development of the Nile tilapia (Oreochromis niloticus) a fish reared worldwide for farming and research purposes. CBD mixed with fish food was offered to isolated fish for 5 weeks. The 10 mg/kg dose decreased fish's aggressiveness over time, whereas 20 mg/kg attenuated non-social stress. Both doses decreased the baseline cortisol level of fish and increased the gonadosomatic index. However, CBD 1 and 10 mg/kg doses decreased the spermatozoa number. No CBD dose affected feeding ingestion and growth variables, showing that it is not harmful to meat production amount. Despite the effect on spermatozoa, CBD supplementation exhibits high potential to benefit animals' lives in artificial environments. Therefore, we showed for the first time that CBD could be used as a tool to increase non-mammal welfare, presenting a great potential to be explored in other husbandry and captivity species.

Thyroid Hormones Deficiency Impairs Male Germ Cell Development: A Cross Talk Between Hypothalamic-Pituitary-Thyroid, and-Gonadal Axes in Zebrafish.

In vertebrates, thyroid hormones are critical players in controlling different physiological processes such as development, growth, metabolism among others. There is evidence in mammals that thyroid hormones are also an important component of the hormonal system that controls reproduction, although studies in fish remain poorly investigated. Here, we tested this hypothesis by investigating the effects of methimazole-induced hypothyroidism on the testicular function in adult zebrafish. Treatment of fish with methimazole, in vivo, significantly altered zebrafish spermatogenesis by inhibiting cell differentiation and meiosis, as well as decreasing the relative number of spermatozoa. The observed impairment of spermatogenesis by methimazole was correlated with significant changes in transcript levels for several genes implicated in the control of reproduction. Using an in vitro approach, we also demonstrated that in addition to affecting the components of the brain-pituitary-peripheral axis, T3 (triiodothyronine) also exerts direct action on the testis. These results reinforce the hypothesis that thyroid hormones are an essential element of multifactorial control of reproduction and testicular function in zebrafish and possibly other vertebrate species.

Environmentally-induced sex reversal in fish with chromosomal vs. polygenic sex determination.

Sex ratio depends on sex determination mechanisms and is a key demographic parameter determining population viability and resilience to natural and anthropogenic stressors. There is increasing evidence that the environment can alter sex ratio even in genetically sex-determined species (GSD), as elevated temperature can cause female-to-male sex reversal (neomales). Alarmingly, neomales are being discovered in natural populations of several fish, amphibian and reptile species worldwide. Understanding the basis of neomale development is important for conservation biology. Among GSD species, it is unknown whether those with chromosomal sex determination (CSD), the most common system, will better resist the influence of high temperature than those with polygenic sex determination (PSD). Here, we compared the effects of elevated temperature in two wild zebrafish strains, Nadia (NA) and Ekkwill (EKW), which have CSD with a ZZ/ZW system, against the AB laboratory strain, which has PSD. First, we uncovered novel sex genotypes and the results showed that, at control temperature, the masculinization rate roughly doubled with the addition of each Z chromosome, while some ZW and WW fish of the wild strains became neomales. Surprisingly, we found that at elevated temperatures WW fish were just as likely as ZW fish to become neomales and that all strains were equally susceptible to masculinization. These results demonstrate that the Z chromosome is not essential for male development and that the dose of W buffers masculinization at the control temperature but not at elevated temperature. Furthermore, at the elevated temperature the testes of neomales, but not of normal males, contained more spermatozoa than at the control temperature. Our results show in an unprecedented way that, in a global warming scenario, CSD species may not necessarily be better protected against the masculinizing effect of elevated temperature than PSD species, and reveal genotype-by-temperature interactions in male sex determination and spermatogenesis.

NAToRA, a relatedness-pruning method to minimize the loss of dataset size in genetic and omics analyses.

Genetic and omics analyses frequently require independent observations, which is not guaranteed in real datasets. When relatedness cannot be accounted for, solutions involve removing related individuals (or observations) and, consequently, a reduction of available data. We developed a network-based relatedness-pruning method that minimizes dataset reduction while removing unwanted relationships in a dataset. It uses node degree centrality metric to identify highly connected nodes (or individuals) and implements heuristics that approximate the minimal reduction of a dataset to allow its application to complex datasets. When compared with two other popular population genetics methodologies (PLINK and KING), NAToRA shows the best combination of removing all relatives while keeping the largest possible number of individuals in all datasets tested and also, with similar effects on the allele frequency spectrum and Principal Component Analysis than PLINK and KING. NAToRA is freely available, both as a standalone tool that can be easily incorporated as part of a pipeline, and as a graphical web tool that allows visualization of the relatedness networks. NAToRA also accepts a variety of relationship metrics as input, which facilitates its use. We also release a genealogies simulator software used for different tests performed in this study.

Gdnf Acts as a Germ Cell-Derived Growth Factor and Regulates the Zebrafish Germ Stem Cell Niche in Autocrine- and Paracrine-Dependent Manners.

Glial cell line-derived neurotrophic factor (GDNF) and its receptor (GDNF Family Receptor α1-GFRα1) are well known to mediate spermatogonial stem cell (SSC) proliferation and survival in mammalian testes. In nonmammalian species, Gdnf and Gfrα1 orthologs have been found but their functions remain poorly investigated in the testes. Considering this background, this study aimed to understand the roles of the Gdnf-Gfrα1 signaling pathway in zebrafish testes by combining in vivo, in silico and ex vivo approaches. Our analysis showed that zebrafish exhibit two paralogs for Gndf (gdnfa and gdnfb) and its receptor, Gfrα1 (gfrα1a and gfrα1b), in accordance with a teleost-specific third round of whole genome duplication. Expression analysis further revealed that both ligands and receptors were expressed in zebrafish adult testes. Subsequently, we demonstrated that gdnfa is expressed in the germ cells, while Gfrα1a/Gfrα1b was detected in early spermatogonia (mainly in types Aund and Adiff) and Sertoli cells. Functional ex vivo analysis showed that Gdnf promoted the creation of new available niches by stimulating the proliferation of both type Aund spermatogonia and their surrounding Sertoli cells but without changing pou5f3 mRNA levels. Strikingly, Gdnf also inhibited late spermatogonial differentiation, as shown by the decrease in type B spermatogonia and down-regulation of dazl in a co-treatment with Fsh. Altogether, our data revealed that a germ cell-derived factor is involved in maintaining germ cell stemness through the creation of new available niches, supporting the development of spermatogonial cysts and inhibiting late spermatogonial differentiation in autocrine- and paracrine-dependent manners.

Toxicity of spike fragments SARS-CoV-2 S protein for zebrafish: A tool to study its hazardous for human health?

Despite the significant increase in the generation of SARS-CoV-2 contaminated domestic and hospital wastewater, little is known about the ecotoxicological effects of the virus or its structural components in freshwater vertebrates. In this context, this study evaluated the deleterious effects caused by SARS-CoV-2 Spike protein on the health of Danio rerio, zebrafish. We demonstrated, for the first time, that zebrafish injected with fragment 16 to 165 (rSpike), which corresponds to the N-terminal portion of the protein, presented mortalities and adverse effects on liver, kidney, ovary and brain tissues. The conserved genetic homology between zebrafish and humans might be one of the reasons for the intense toxic effects followed inflammatory reaction from the immune system of zebrafish to rSpike which provoked damage to organs in a similar pattern as happen in severe cases of COVID-19 in humans, and, resulted in 78,6% of survival rate in female adults during the first seven days. The application of spike protein in zebrafish was highly toxic that is suitable for future studies to gather valuable information about ecotoxicological impacts, as well as vaccine responses and therapeutic approaches in human medicine. Therefore, besides representing an important tool to assess the harmful effects of SARS-CoV-2 in the aquatic environment, we present the zebrafish as an animal model for translational COVID-19 research.

Barriers to enrollment in pulmonary rehabilitation: medical knowledge analysis.

OBJECTIVE: To assess clinicians' knowledge about pulmonary rehabilitation, and identify the barriers faced when referring patients with health insurance to pulmonary rehabilitation. METHODS: This was a survey-based cross-sectional study conducted in 2019, at a private reference hospital in São Paulo, Brazil. Eligible participants were physicians registered with the following specialties: internal medicine, geriatrics, cardiology, pulmonology or thoracic surgery. RESULTS: We collected 72 responses, and 99% of participants recognized chronic obstructive pulmonary disease as a potential indication for pulmonary rehabilitation; less often (75%), they listed interstitial lung disease, bronchiectasis and pulmonary hypertension. Most participants (67%) incorrectly associated pulmonary rehabilitation with lung function improvement, while 28% of cardiologists and 35% of internists/geriatricians failed to recognize benefits on mood disorders. Notably, 18% of participants recommended pulmonary rehabilitation only to patients on supplemental oxygen and 14% prescribed only home physical therapy, patterns more commonly seen among non-respiratory physicians. The three most perceived barriers to referral and adherence were health insurance coverage (79%), transportation to pulmonary rehabilitation center (63%) and lack of social support (29%). CONCLUSION: Financial, logistic and social constraints pose challenges to pulmonary rehabilitation enrollment, even for patients with premium healthcare insurance. Moreover, physician knowledge gaps may be an additional barrier to pulmonary rehabilitation referral and adherence. Providing continued medical education, incorporating automatic reminders in electronic medical records, and using telerehabilitation tools may improve pulmonary rehabilitation referral, adherence, and ultimately, patient care.

Gonadal morphology and difference in reproductive development of two isolated populations of Astyanax rivularis (Teleostei, Characidae).

Individuals of the same species may present different reproductive tactics depending on the environment in which they develop and mature. The present study aimed to define the gonadal development phases of males and females of Astyanax rivularis and to carry out a comparative analysis of the reproductive development of specimens captured in two isolated environments of the São Francisco River basin in Serra da Canastra, Brazil (Point 1: low vegetation and river showing calm and crystalline waters with small well formations; Point 2: current waters, and well-established areas of arboreal vegetation). Thus, the gonads of A. rivularis specimens were collected, fixed and processed with techniques for light microscopy. Five maturation phases of the females' reproductive cycle were established: immature, developing, spawning capable, regressing and regenerating. Three maturation phases of the males' reproductive cycle were observed: spawning capable, regressing, and regenerating. There are differences in the phases of gonadal development of A. rivularis between the two sampling points so that, possibly, animals upstream of the waterfall demonstrate a delay in the reproductive cycle in relation to animals downstream.

Genome-Wide SNPs Clarify a Complex Radiation and Support Recognition of an Additional Cat Species.

Phylogenetic reconstruction and species delimitation are often challenging in the case of recent evolutionary radiations, especially when postspeciation gene flow is present. Leopardus is a Neotropical cat genus that has a long history of recalcitrant taxonomic problems, along with both ancient and current episodes of interspecies admixture. Here, we employ genome-wide SNP data from all presently recognized Leopardus species, including several individuals from the tigrina complex (representing Leopardus guttulus and two distinct populations of Leopardus tigrinus), to investigate the evolutionary history of this genus. Our results reveal that the tigrina complex is paraphyletic, containing at least three distinct species. While one can be assigned to L. guttulus, the other two remain uncertain regarding their taxonomic assignment. Our findings indicate that the "tigrina" morphology may be plesiomorphic within this group, which has led to a longstanding taxonomic trend of lumping these poorly known felids into a single species.

Interaction between thyroid hormones and gonadotropin inhibitory hormone in ex vivo culture of zebrafish testis: An approach to study multifactorial control of spermatogenesis.

Reproduction is under multifactorial control of neurohormones, pituitary gonadotropins, as well as of local gonadal signaling systems including sex steroids, growth factors and non-coding RNAs. Among the factors, gonadotropin-inhibitory hormone (Gnih) is a novel RFamide neuropeptide which directly modulates gonadotropin synthesis and release from pituitary, and in the gonads, Gnih mediated inhibitory actions on gonadotropin response of zebrafish spermatogenesis. Thyroid hormones are peripheral hormones which are also known to interact with reproductive axis, in particular, regulating testicular development and function. This study investigated the interaction between Gnih and thyroid hormones in zebrafish spermatogenesis using in vivo and ex vivo approaches. Three experimental groups were established: "control" (non-treated fish), "methimazole" and "methimazole + T4". Fish were exposed to goitrogen methimazole for 3 weeks; T4 (100 µg/L) was added in the water from the second week only in the "reversal treatment" group. After exposure, testes were dissected out and immediately incubated in Leibovitz's L-15 culture medium containing hCG, Gnih or hCG + Gnih for 7 days. Germ cell cysts and haploid cell population were evaluated by histomorphometry and flow cytometry, respectively. Our results showed that hypothyroidism affected germ cell development in basal and gonadotropin-induced spermatogenesis, in particular, meiosis and spermiogenesis. Hypothyroid testes showed lower amount of spermatozoa, and decreased potency of hCG. We also showed that goitrogen treatment nullified the inhibitory actions of Gnih on the gonadotropin-induced spermatogenesis. This study provided evidences that thyroid hormones are important regulatory factors for hCG- and Gnih-mediated functions in zebrafish spermatogenesis.

Molecular characterization and expression analysis of anti-Müllerian hormone in common carp (Cyprinus carpio) adult testes.

Anti-Müllerian hormone (Amh) is a member of the transforming growth factor-ß (Tgf-ß) superfamily required in the regression of Müllerian ducts during gonadal sex differentiation of higher vertebrates. Teleost fish lack Müllerian ducts, but identified Amh orthologs have been shown to exert crucial functions during sex determination and differentiation of several species of teleosts. However, the function of Amh during gametogenesis in adult fish remains poorly investigated. Therefore, to expand present knowledge on the role of Amh in teleosts, the present study aimed to isolate and clone full-length amh cDNA in the common carp, Cyprinus carpio, and examine its expression levels throughout the male reproductive cycle and in response to different hormone treatments of testicular explants. Molecular cloning and characterization showed that the common carp Amh precursor amino acid sequence shared common features to other fish Amh precursors, including a conserved C-terminus (Tgf-ß domain) and a double proteolytic cleavage site (R-X-X-R-X-X-R) upstream to the Tgf-ß domain. Expression analysis showed amh dimorphic expression in the adult gonads with higher expression in the testes than ovaries. In testes, amh mRNA was detected in Sertoli cells contacting different types of germ cells, although the expression was greatest in Sertoli cells associated with type A undifferentiated spermatogonia. Expression analysis during the reproductive cycle showed that amh transcripts were down-regulated during the developing phase, which is characterized by an increased proliferation of type A undifferentiated spermatogonia and Sertoli cells and appearance of spermatocytes (meiosis) in the testes. Furthermore, ex vivo experiments showed that a 7 day exposure to Fsh or estrogens was required to decrease amh mRNA levels in common carp testicular explants. In summary, this study provided information on the molecular characterization and transcript abundance of amh in common carp adult testes. Altogether, these data will be useful for further investigations on sex determination and differentiation in this species, and also to improved strategies for improved carp aquaculture, such as inhibiting precocious maturation of males.

Admixture/fine-mapping in Brazilians reveals a West African associated potential regulatory variant (rs114066381) with a strong female-specific effect on body mass and fat mass indexes.

BACKGROUND/OBJECTIVES: Admixed populations are a resource to study the global genetic architecture of complex phenotypes, which is critical, considering that non-European populations are severely underrepresented in genomic studies. Here, we study the genetic architecture of BMI in children, young adults, and elderly individuals from the admixed population of Brazil. SUBJECTS/METHODS: Leveraging admixture in Brazilians, whose chromosomes are mosaics of fragments of Native American, European, and African origins, we used genome-wide data to perform admixture mapping/fine-mapping of body mass index (BMI) in three Brazilian population-based cohorts from Northeast (Salvador), Southeast (Bambuí), and South (Pelotas). RESULTS: We found significant associations with African-associated alleles in children from Salvador (PALD1 and ZMIZ1 genes), and in young adults from Pelotas (NOD2 and MTUS2 genes). More importantly, in Pelotas, rs114066381, mapped in a potential regulatory region, is significantly associated only in females (p = 2.76e-06). This variant is rare in Europeans but with frequencies of ~3% in West Africa and has a strong female-specific effect (95% CI: 2.32-5.65 kg/m2 per each A allele). We confirmed this sex-specific association and replicated its strong effect for an adjusted fat mass index in the same Pelotas cohort, and for BMI in another Brazilian cohort from São Paulo (Southeast Brazil). A meta-analysis confirmed the significant association. Remarkably, we observed that while the frequency of rs114066381-A allele ranges from 0.8 to 2.1% in the studied populations, it attains ~9% among women with morbid obesity from Pelotas, São Paulo, and Bambuí. The effect size of rs114066381 is at least five times higher than the FTO SNPs rs9939609 and rs1558902, already emblematic for their high effects. CONCLUSIONS: We identified six candidate SNPs associated with BMI. rs114066381 stands out for its high effect that was replicated and its high frequency in women with morbid obesity. We demonstrate how admixed populations are a source of new relevant phenotype-associated genetic variants.

The influence of increased water temperature on the duration of spermatogenesis in a neotropical fish, Astyanax altiparanae (Characiformes, Characidae).

In view of the established climate change scenario and the consequent changes in global temperature, it is essential to study its effects on animal spermatogenesis. Therefore, the aim of this study was to verify the duration of spermatogenesis at different temperatures. For this purpose, 96 male and adult specimens of Astyanax altiparanae were kept in a closed circulation system with water temperature stabilized at 27 °C and 32 °C. Subsequently, the specimens received pulses of BrdU (bromodeoxyuridine) at a concentration of 100 mg/kg/day for 2 consecutive days, and the samples were collected daily for a period of 15 days. Their testes were removed, fixed, processed in historesin, and sectioned in 3 µm, submitted to hematoxylin/eosin staining and to bromodeoxyuridine immunodetection. Partial results of the optimum temperature experiments allowed the classification of A. altiparanae spermatogenic cells in Aund, Adiff, and type B spermatogonia, spermatocytes, spermatids, and spermatozoa. The duration of spermatogenesis was determined as approximately 6 days for animals at a temperature of 27 °C and 1 day for animals at 32 °C. The elevated temperature was also responsible for increasing cell proliferation, resulting in an increase in the number of spermatocytes, spermatids, spermatozoa, and cell death (cell pyknotic). The duration of spermatogenesis in A. altiparanae was directly affected by the elevated water temperature, causing a reduction in the estimated time of spermatogenesis.

Effects of gonadotropin-inhibitory hormone on early and late stages of spermatogenesis in ex-vivo culture of zebrafish testis.

Gonadotropin-inhibitory hormone (Gnih) is known to play a role in the regulation of reproduction in vertebrates by influencing gonadotropin release and synthesis. While the endocrine actions of Gnih have been identified in several species, its paracrine/autocrine effects in the control of spermatogenesis are less defined. We have used ex vivo culture of zebrafish testis to investigate the role of gonadal zebrafish Gnih (zGnih) in the regulation of the spermatogenic process. We used FACScan cell cycle analysis, morphometric quantifications, BrdU incorporation and caspase-3 activity assays as well as measuring 11-Ketotestosterone (11-KT) level in the culture media. FACScan analysis and morphometric quantification results demonstrated direct action of zGnih on basal and gonadotropin (Lh and Fsh)-induced spermatogenesis. Treatment with zGnih (10 nM) significantly decreased the number of G0/G1 cells after 7-days of culture while no significant changes were found in the proportion area of spermatogonia cell types. Investigation of DNA synthesis using BrdU (5-Bromo-2'-Deoxyuridine) labeling showed that treatment with zGnih (10 nM) significantly decreased proliferative activity of type A spermatogonia, while increased the mitotic activity of type B spermatogonia. We also showed that treatment with zGnih (100 nM) completely eliminated 11-KT release induced by 100 ng/ml Fsh. Treatment with zGnih (10 and 100 nM) also inhibited both hCG and Fsh-induced spermatogenesis. These results, plus our previous findings, demonstrate that zGnih produced locally in the testis is a component of a complex multifactorial system that regulates testicular function in zebrafish.

Barriers to enrollment in pulmonary rehabilitation: medical knowledge analysis / Barreiras à inclusão na reabilitação pulmonar: análise do conhecimento médico

ABSTRACT Objective To assess clinicians' knowledge about pulmonary rehabilitation, and identify the barriers faced when referring patients with health insurance to pulmonary rehabilitation. Methods This was a survey-based cross-sectional study conducted in 2019, at a private reference hospital in São Paulo, Brazil. Eligible participants were physicians registered with the following specialties: internal medicine, geriatrics, cardiology, pulmonology or thoracic surgery. Results We collected 72 responses, and 99% of participants recognized chronic obstructive pulmonary disease as a potential indication for pulmonary rehabilitation; less often (75%), they listed interstitial lung disease, bronchiectasis and pulmonary hypertension. Most participants (67%) incorrectly associated pulmonary rehabilitation with lung function improvement, while 28% of cardiologists and 35% of internists/geriatricians failed to recognize benefits on mood disorders. Notably, 18% of participants recommended pulmonary rehabilitation only to patients on supplemental oxygen and 14% prescribed only home physical therapy, patterns more commonly seen among non-respiratory physicians. The three most perceived barriers to referral and adherence were health insurance coverage (79%), transportation to pulmonary rehabilitation center (63%) and lack of social support (29%). Conclusion Financial, logistic and social constraints pose challenges to pulmonary rehabilitation enrollment, even for patients with premium healthcare insurance. Moreover, physician knowledge gaps may be an additional barrier to pulmonary rehabilitation referral and adherence. Providing continued medical education, incorporating automatic reminders in electronic medical records, and using telerehabilitation tools may improve pulmonary rehabilitation referral, adherence, and ultimately, patient care.

RESUMO Objetivo Avaliar o conhecimento dos médicos sobre reabilitação pulmonar e identificar as barreiras que eles encontram ao encaminhar pacientes com seguro saúde para reabilitação pulmonar. Métodos Trata-se de estudo transversal com uso de questionário, realizado em 2019, em um hospital privado de referência em São Paulo. Os participantes elegíveis eram médicos registrados nas seguintes especialidades: clínica médica, geriatria, cardiologia, pneumologia ou cirurgia torácica. Resultados Foram coletadas 72 respostas, e 99% dos participantes reconheceram doença pulmonar obstrutiva crônica como possível indicação para reabilitação pulmonar; com menor frequência (75%), listaram doença pulmonar intersticial, bronquiectasia e hipertensão pulmonar. A maioria dos participantes (67%) associou incorretamente a reabilitação pulmonar à melhora da função pulmonar, ao passo que 28% dos cardiologistas e 35% dos clínicos/geriatras deixaram de reconhecer os benefícios nos transtornos de humor. Notavelmente, 18% dos participantes somente recomendaram reabilitação pulmonar para pacientes em uso de oxigênio suplementar, e 14% prescreveram apenas fisioterapia domiciliar, padrão mais comumente visto entre médicos que não são especialistas em transtornos respiratórios. As três barreiras mais percebidas para encaminhamento e adesão foram cobertura de seguro saúde (79%), transporte para centro de reabilitação pulmonar (63%) e falta de apoio social (29%). Conclusão Restrições financeiras, logísticas e sociais representam desafios para a inclusão na reabilitação pulmonar, mesmo para pacientes com planos de seguro de saúde da categoria premium. Além disso, as lacunas de conhecimento dos médicos podem ser uma barreira adicional para o encaminhamento e a aceitação da reabilitação pulmonar. Educação médica continuada, lembretes automáticos nos prontuários eletrônicos e ferramentas de telerreabilitação podem melhorar o encaminhamento para reabilitação pulmonar, a adesão e, por fim, o atendimento ao paciente.

The Brazilian Initiative on Precision Medicine (BIPMed): fostering genomic data-sharing of underrepresented populations.

The development of precision medicine strategies requires prior knowledge of the genetic background of the target population. However, despite the availability of data from admixed Americans within large reference population databases, we cannot use these data as a surrogate for that of the Brazilian population. This lack of transferability is mainly due to differences between ancestry proportions of Brazilian and other admixed American populations. To address the issue, a coalition of research centres created the Brazilian Initiative on Precision Medicine (BIPMed). In this study, we aim to characterise two datasets obtained from 358 individuals from the BIPMed using two different platforms: whole-exome sequencing (WES) and a single nucleotide polymorphism (SNP) array. We estimated allele frequencies and variant pathogenicity values from the two datasets and compared our results using the BIPMed dataset with other public databases. Here, we show that the BIPMed WES dataset contains variants not included in dbSNP, including 6480 variants that have alternative allele frequencies (AAFs) >1%. Furthermore, after merging BIPMed WES and SNP array data, we identified 809,589 variants (47.5%) not present within the 1000 Genomes dataset. Our results demonstrate that, through the incorporation of Brazilian individuals into public genomic databases, BIPMed not only was able to provide valuable knowledge needed for the implementation of precision medicine but may also enhance our understanding of human genome variability and the relationship between genetic variation and disease predisposition.

Origins, Admixture Dynamics, and Homogenization of the African Gene Pool in the Americas.

The Transatlantic Slave Trade transported more than 9 million Africans to the Americas between the early 16th and the mid-19th centuries. We performed a genome-wide analysis using 6,267 individuals from 25 populations to infer how different African groups contributed to North-, South-American, and Caribbean populations, in the context of geographic and geopolitical factors, and compared genetic data with demographic history records of the Transatlantic Slave Trade. We observed that West-Central Africa and Western Africa-associated ancestry clusters are more prevalent in northern latitudes of the Americas, whereas the South/East Africa-associated ancestry cluster is more prevalent in southern latitudes of the Americas. This pattern results from geographic and geopolitical factors leading to population differentiation. However, there is a substantial decrease in the between-population differentiation of the African gene pool within the Americas, when compared with the regions of origin from Africa, underscoring the importance of historical factors favoring admixture between individuals with different African origins in the New World. This between-population homogenization in the Americas is consistent with the excess of West-Central Africa ancestry (the most prevalent in the Americas) in the United States and Southeast-Brazil, with respect to historical-demography expectations. We also inferred that in most of the Americas, intercontinental admixture intensification occurred between 1750 and 1850, which correlates strongly with the peak of arrivals from Africa. This study contributes with a population genetics perspective to the ongoing social, cultural, and political debate regarding ancestry, admixture, and the mestizaje process in the Americas.

Cortisol Directly Stimulates Spermatogonial Differentiation, Meiosis, and Spermiogenesis in Zebrafish (Danio rerio) Testicular Explants.

Cortisol is the major endocrine factor mediating the inhibitory effects of stress on vertebrate reproduction. It is well known that cortisol affects reproduction by interacting with the hypothalamic-pituitary-gonads axis, leading to downstream inhibitory and stimulatory effects on gonads. However, the mechanisms are not fully understood. In this study, we provide novel data demonstrating the stimulatory effects of cortisol on spermatogenesis using an ex vivo organ culture system. The results revealed that cortisol treatment did not modulate basal androgen production, but it influenced transcript levels of a selected number of genes involved in the zebrafish testicular function ar (androgen receptor), star (steroidogenic acute regulatory), cyp17a1 (17α-hydroxylase/17,20 lyase/17,20 desmolase), cyp11a2 (cytochrome P450, family 11, subfamily A, polypeptide 2), hsd11b2 (11-beta hydroxysteroid dehydrogenase), cyp2k22 (cytochrome P450, family 2, subfamily K, polypeptide 22), fkbp5 (FKBP prolyl isomerase 5), grα (glucocorticoid receptor alpha), and grß (glucocorticoid receptor beta) in a short-term culture. We also showed that cortisol stimulates spermatogonial proliferation and differentiation in an androgen independent manner as well as promoting meiosis and spermiogenesis by increasing the number of spermatozoa in the testes. Moreover, we demonstrated that concomitant treatment with RU 486, a potent glucocorticoid receptor (Gr) antagonist, did not affect the cortisol effects on spermatogonial differentiation but blocked the induced effects on meiosis and spermiogenesis. Supporting the Gr-mediated effects, RU 486 nullified the cortisol-induced expression of sycp3l (synaptonemal complex protein 3), a marker for the meiotic prophase that encodes a component of the synaptonemal complex. This is consistent with in silico analysis that found 10 putative GREs (glucocorticoid response elements) upstream of the zebrafish sycp3l. Finally, we also showed that grα mRNA is expressed in Sertoli and Leydig cells, but also in several types of germ cells, including spermatogonia and spermatocytes. Altogether, this evidence indicates that cortisol exerts paracrine roles in the zebrafish testicular function and spermatogenesis, highlighting its effects on spermatogonial differentiation, meiosis, and spermiogenesis.