Phallus eversion sexing in Phrynops geoffroanus (Testudines: Chelidae): a new non-invasive approach

Abstract The sexing of species is an important tool for population management. In tortoises and turtles, sexing is usually done according to secondary characteristics, such as plastron morphology or phallus eversion techniques. In this paper, we present the phallus eversion method and compare its efficiency to other existing techniques. This new method aims at shortening the management time and reducing the physical aftereffects on the animals, fostering the clinical and reproductive management of Geoffroys side-necked turtle (Phrynops geoffroanus) and related species. A sample of fifty individuals was taken in the central region of Piauí state, Northeastern Brazil. The proposed method consisted of moving the head from its resting position. The methods achieving a success rate higher than 80% were compared to each other as for the stimulation time required for phallus eversion. The method as here proposed achieved 100% efficiency in the sex determination of specimens with a linear carapace length between 10 and 34 cm. Also, when compared to the second most efficient method, it substantially reduced the time needed for managing the specimens (91%), avoiding possible sequelae in the individuals handled.

Resumo A sexagem das espécies é uma ferramenta importante para o manejo populacional. Em tartarugas, cágados e jabutis, a sexagem geralmente é feita de acordo com características secundárias, como morfologia do plastrão ou técnicas de eversão do falo. Neste artigo, apresentamos o método de eversão do falo e comparamos sua eficiência com outras técnicas existentes. Este novo método visa encurtar o tempo de manejo e reduzir os efeitos colaterais físicos nos animais, promovendo o manejo clínico e reprodutivo do cágado-de-barbicha (Phrynops geoffroanus) e espécies relacionadas. Uma amostra de cinquenta indivíduos foi coletada na região central do estado do Piauí, Nordeste do Brasil. O método proposto consiste em remover a cabeça de sua posição de repouso. Os métodos que obtiveram índice de sucesso superior a 80% foram comparados entre si quanto ao tempo de estimulação necessário para a eversão do falo. O método aqui proposto alcançou 100% de eficiência na determinação do sexo de espécimes com comprimento de carapaça linear entre 10 e 34 cm. Além disso, quando comparado ao segundo método mais eficiente, reduziu substancialmente o tempo necessário para o manejo dos espécimes (91%), evitando possíveis sequelas nos indivíduos manuseados.

Phallus eversion sexing in Phrynops geoffroanus (Testudines: Chelidae): a new non-invasive approach / Sexagem por eversão do falo em Phrynops geoffroanus (Testudines: Chelidae): uma nova abordagem não invasiva

Abstract The sexing of species is an important tool for population management. In tortoises and turtles, sexing is usually done according to secondary characteristics, such as plastron morphology or phallus eversion techniques. In this paper, we present the phallus eversion method and compare its efficiency to other existing techniques. This new method aims at shortening the management time and reducing the physical aftereffects on the animals, fostering the clinical and reproductive management of Geoffroy's side-necked turtle (Phrynops geoffroanus) and related species. A sample of fifty individuals was taken in the central region of Piauí state, Northeastern Brazil. The proposed method consisted of moving the head from its resting position. The methods achieving a success rate higher than 80% were compared to each other as for the stimulation time required for phallus eversion. The method as here proposed achieved 100% efficiency in the sex determination of specimens with a linear carapace length between 10 and 34 cm. Also, when compared to the second most efficient method, it substantially reduced the time needed for managing the specimens (91%), avoiding possible sequelae in the individuals handled.

Resumo A sexagem das espécies é uma ferramenta importante para o manejo populacional. Em tartarugas, cágados e jabutis, a sexagem geralmente é feita de acordo com características secundárias, como morfologia do plastrão ou técnicas de eversão do falo. Neste artigo, apresentamos o método de eversão do falo e comparamos sua eficiência com outras técnicas existentes. Este novo método visa encurtar o tempo de manejo e reduzir os efeitos colaterais físicos nos animais, promovendo o manejo clínico e reprodutivo do cágado-de-barbicha (Phrynops geoffroanus) e espécies relacionadas. Uma amostra de cinquenta indivíduos foi coletada na região central do estado do Piauí, Nordeste do Brasil. O método proposto consiste em remover a cabeça de sua posição de repouso. Os métodos que obtiveram índice de sucesso superior a 80% foram comparados entre si quanto ao tempo de estimulação necessário para a eversão do falo. O método aqui proposto alcançou 100% de eficiência na determinação do sexo de espécimes com comprimento de carapaça linear entre 10 e 34 cm. Além disso, quando comparado ao segundo método mais eficiente, reduziu substancialmente o tempo necessário para o manejo dos espécimes (91%), evitando possíveis sequelas nos indivíduos manuseados.

Anurans from the upper basins of the Rio Grande and Rio Preto: eastern portion of the Serra da Mantiqueira Environmental Protection Area, state of Minas Gerais, Brazil.

The Neotropical zone stands out for having the greatest richness of amphibians in the world. About 625 species of anurans are known on Atlantic Forest. The southeastern mountainous regions of Brazil called Serra do Mar and Serra da Mantiqueira, are very important areas for the diversity of anurans in the country because they shelter remaining strata of the Atlantic Forest that have several endemic species. The present study aims to expand the knowledge about the Serra da Mantiqueira anurans, specifically from the Rio Preto and Rio Grande upper basins. The study area comprised 10 sample centers. We realized 38 campaigns, lasting two days each. During this period, we used active search as visual and auditory cues. The passive search was comprised of pitfall traps. Bioacoustic analyses were used to determine the cryptic species. The sufficiency of the sampling effort was estimated through the accumulation curve using Bootstrap and Jackknife-1 as estimators. During the study 55 species were found, 40 of them in the basin of the Rio Preto, and 44 in the basin of the Rio Grande. Twenty-nine species were found in both areas. The Hylidae family was predominant, followed by Leptodactylidae. In addition to species of the Atlantic Forest, the place houses typical components of the Cerrado biome and altitudinal swamps with unexpected sympatries. This inventory expands the area of occurrence of Scinax duartei, a species considered threatened. Although adjacent to the oldest national park in Brazil, the composition of anuran species of this area was unknown until then.

Phallus eversion sexing in Phrynops geoffroanus (Testudines: Chelidae): a new non-invasive approach.

The sexing of species is an important tool for population management. In tortoises and turtles, sexing is usually done according to secondary characteristics, such as plastron morphology or phallus eversion techniques. In this paper, we present the phallus eversion method and compare its efficiency to other existing techniques. This new method aims at shortening the management time and reducing the physical aftereffects on the animals, fostering the clinical and reproductive management of Geoffroy's side-necked turtle (Phrynops geoffroanus) and related species. A sample of fifty individuals was taken in the central region of Piauí state, Northeastern Brazil. The proposed method consisted of moving the head from its resting position. The methods achieving a success rate higher than 80% were compared to each other as for the stimulation time required for phallus eversion. The method as here proposed achieved 100% efficiency in the sex determination of specimens with a linear carapace length between 10 and 34 cm. Also, when compared to the second most efficient method, it substantially reduced the time needed for managing the specimens (91%), avoiding possible sequelae in the individuals handled.

Ultrasound-guided percutaneous biopsy of thoracic lesions: high diagnostic yield and low complication rate.

AIM: To investigate the use of ultrasound (US)-guided biopsy of thoracic lesions aiming to determine diagnostic success and complication rates and to identify factors that may affect sample adequacy and safety. MATERIAL AND METHODS: This was a retrospective study of consecutive percutaneous US-guided biopsy over 10-year period, including 147 procedures in 146 patients (66 ± 7 years, 83 men, 63 women) with lesions located in the lung (67/147), chest wall (54/147), mediastinum (14/147) and pleura (12/147). Overall diagnostic success, yield for benign and malignant diagnoses and diagnostic success according to lesion location, biopsy type (fine-need aspiration [FNA] or core-needle biopsy [CNB]) and number of specimens were calculated. Presence of complications and effect of age, lesion location, biopsy type, and number of specimens were measured. RESULTS: The overall diagnostic success rate was 90.5% and was similar for malignant (90.6%) and benign (87.5%) diagnoses. Specimen adequacy was similar for FNA and CNB (91.2% and 88.9%, p=0.66); number of specimens did not affect yield. Diagnostic success was highest for mediastinal and chest wall lesions (92.9% and 94.4%) and lowest for pleural lesions (75%), albeit not statistically significant (p=0.45). Complications occurred in 4/147(2.7%) cases, exclusively in lung lesions, and were not associated with any covariates analysed. CONCLUSIONS: US-guided biopsy is an effective and safe technique for diagnosis of thoracic lesions, with high diagnostic yield and low complication rate. In the presence of an adequate acoustic window, US guidance can be valuable for diagnosis of peripheral lung and mediastinal lesions. Radiologists performing thoracic biopsy should be encouraged to implement or expand the use of US guidance in their practice.

Efeito do hipotireoidismo materno na expressão espaço-temporal de mediadores imunológicos e população de células natural killers na decídua e na glândula metrial de ratas / Effect of maternal hypothyroidism on the spatiotemporal expression of immunological mediators and population of Natural Killers cells in decidua and metrial gland of rats

Estudou-se o efeito do hipotireoidismo materno na expressão espaço-temporal de mediadores imunológicos e na população de células natural killers (NK) na decídua e na glândula metrial de ratas durante a gestação. Avaliou-se a detecção imunoistoquímica de interferon γ (IFNγ), do fator inibidor de migração (MIF), da interleucina 15 (IL15), do óxido nítrico sintase induzível (iNOS), a marcação com lectina DBA para evidenciação das células NK uterinas DBA+ e a expressão gênica de Ifnγ e Nos2. O hipotireoidismo aumentou o iNOS aos sete dias, a IL15 e o MIF aos 10 e 12 dias, o IFNγ e o MIF aos 14 DG e a expressão dos transcritos gênicos para iNos aos 12 e 19 dias e para Ifnγ aos 14 DG. O hipotireoidismo reduziu a imunomarcação de MIF e lectina DBA aos sete dias, lectina DBA aos 10 e 14 DG, IFNγ aos 12 dias, e a expressão de Ifnγ aos 10 e 19 DG e de iNOS aos 12, 14 e 19 DG, bem como reduziu seus transcritos gênicos aos 10 e 14 DG. Conclui-se que o hipotireoidismo compromete o perfil imunológico na interface materno-fetal ao longo da gestação, particularmente por reduzir o fator anti-inflamatório iNOS e a população de células uNK DBA+.(AU)

The goal of this study was to evaluate the effect of maternal hypothyroidism on the spatiotemporal expression of immunological mediators and population of Natural Killers cells in decidua and metrial gland of rats. Interferon gamma (IFNγ), migration inhibitory factor (MIF), interleukin 15 (IL15), inducible nitric oxide synthase (iNOS), and DBA-Lectin labeling for evidence of uNK DBA+ cells in decidua and genetic expression of Ifnγ and iNos by real-time RT-PCR were evaluated. Hypothyroidism increased protein expression of iNOS at 7 days, IL15 and MIF at 10 and 12 days, IFNγ and MIF at 14 DG in the decidua and/or metrial gland and the gene transcripts for iNOS at 12 and 19 days and for Inf at 14 DG. In addition, hypothyroidism reduced the protein expression of MIF and DBA-Lectin at 7 days, DBA-Lectin at 10 and 14 DG, IFNγ at 12 days, and the gene transcript to Ifnγ at 10 and 19 DGs. Hypothyroidism also reduced the protein expression of iNOS at 12, 14 and 19 DG and reduced its gene transcripts at 10 and 14 DGs. It is concluded that hypothyroidism compromises the immunology profile at the maternal-fetal interface throughout pregnancy, particularly by reducing the anti-inflammatory factor iNOS and population of uNK DBA+ cells.(AU)

Evidence-based best practices for EGFR T790M testing in lung cancer in Canada.

Epidermal growth factor receptor (egfr) tyrosine kinase inhibitors (tkis) are recommended as first-line systemic therapy for patients with non-small-cell lung cancer (nsclc) having mutations in the EGFR gene. Resistance to tkis eventually occurs in all nsclc patients treated with such drugs. In patients with resistance to tkis caused by the EGFR T790M mutation, the third-generation tki osimertinib is now the standard of care. For optimal patient management, accurate EGFR T790M testing is required. A multidisciplinary working group of pathologists, laboratory medicine specialists, medical oncologists, a respirologist, and a thoracic radiologist from across Canada was convened to discuss best practices for EGFR T790M mutation testing in Canada. The group made recommendations in the areas of the testing algorithm and the pre-analytic, analytic, and post-analytic aspects of clinical testing for both tissue testing and liquid biopsy circulating tumour dna testing. The recommendations aim to improve EGFR T790M testing in Canada and to thereby improve patient care.

Estimation of a closed population size of tadpoles in temporary pond / Estimativa do tamanho da população fechada de girinos em lagoas temporárias

Abstract The practice of capture-recapture to estimate the diversity is well known to many animal groups, however this practice in the larval phase of anuran amphibians is incipient. We aimed at evaluating the Lincoln estimator, Venn diagram and Bayes theorem in the inference of population size of a larval phase anurocenose from lotic environment. The adherence of results was evaluated using the Kolmogorov-Smirnov test. The marking of tadpoles for later recapture and methods measurement was made with eosin methylene blue. When comparing the results of Lincoln-Petersen estimator corresponding to the Venn diagram and Bayes theorem, we detected percentage differences per sampling, i.e., the proportion of sampled anuran genera is kept among the three methods, although the values are numerically different. By submitting these results to the Kolmogorov-Smirnov test we have found no significant differences. Therefore, no matter the estimator, the measured value is adherent and estimates the total population. Together with the marking methodology, which did not change the behavior of tadpoles, the present study helps to fill the need of more studies on larval phase of amphibians in Brazil, especially in semi-arid northeast.

Resumo A prática de captura-recaptura para a estimação da diversidade é bem conhecida para diversos grupos animais, porém na fase larvar de anfíbios anuros essa prática é incipiente. Objetivamos avaliar os métodos do estimador de Lincoln, diagrama de Venn e o teorema de Bayes na inferência do tamanho populacional de uma anurocenose em fase larvar de ambiente lótico. A aderência dos resultados foi avaliada através do teste de Kolmogorov-Smirnov. A marcação dos girinos para posterior recaptura e aferição dos métodos foi feita com eosina de azul de metileno. Ao compararmos os resultados do estimador de Lincoln que corresponde com o do diagrama de Venn e com o teorema de Bayes detectamos diferenças percentuais por amostragem, isto é, a manutenção da proporção dos gêneros de anuros amostrados é mantida entre os três métodos, embora com valores numericamente diferentes. Ao submetermos esses resultados ao teste Kolmogorov-Smirnov não encontramos diferenças significativas. Logo, qualquer que seja o estimador o valor aferido é aderente e estima a população total. Aliado à metodologia de marcação que não alterou o comportamento dos girinos, o presente estudo ajuda a preencher a necessidade de mais estudos na fase larvar dos anfíbios no Brasil, em especial no semiárido nordestino.

Estimation of a closed population size of tadpoles in temporary pond.

The practice of capture-recapture to estimate the diversity is well known to many animal groups, however this practice in the larval phase of anuran amphibians is incipient. We aimed at evaluating the Lincoln estimator, Venn diagram and Bayes theorem in the inference of population size of a larval phase anurocenose from lotic environment. The adherence of results was evaluated using the Kolmogorov-Smirnov test. The marking of tadpoles for later recapture and methods measurement was made with eosin methylene blue. When comparing the results of Lincoln-Petersen estimator corresponding to the Venn diagram and Bayes theorem, we detected percentage differences per sampling, i.e., the proportion of sampled anuran genera is kept among the three methods, although the values are numerically different. By submitting these results to the Kolmogorov-Smirnov test we have found no significant differences. Therefore, no matter the estimator, the measured value is adherent and estimates the total population. Together with the marking methodology, which did not change the behavior of tadpoles, the present study helps to fill the need of more studies on larval phase of amphibians in Brazil, especially in semi-arid northeast.

Solitary fibrous tumour of pleura: CT differentiation of benign and malignant types.

AIM: To analyse and compare the computed tomography (CT) features of benign and malignant types of histopathologically proven cases of solitary fibrous tumours of pleura (SFTP). MATERIALS AND METHODS: Retrospective analysis of preoperative CT images of 28 cases of histopathologically proven and classified SFTP from three participating institutions was performed. Patient demographics and lesion characteristics including size, borders, presence of a pedicle, extension into the fissure, attenuation, enhancement, pleural effusion, and calcifications were recorded and correlated with the final histopathological diagnosis. Type and results of preoperative biopsy were also recorded. Follow-up imaging and the clinical charts were reviewed to identify recurrence. RESULTS: Out of 28 cases (15 women and 13 men), 18 were proven to be benign and 10 were malignant. The mean age of patients was 58.1±15.9 and 66.5±11.8 years (p=0.1564) for benign and malignant tumours, respectively. The median (interquartile range) diameter was 6.05 (3.2-10.9) cm for benign and 15.7 (7.1-17.5) cm for malignant type tumours (p=0.0291). Tumours had lobulate borders in 28% (5/18) of benign cases and in 80% (8/10) of malignant cases (p=0.0163). Extension into adjacent fissure was seen in 22% (4/18) of benign lesions and 40% (4/10) of malignant lesions (p=0.40). A pedicle was present in 17% (3/18) of benign and 10% (1/10) of malignant lesions (p=1). Heterogeneous attenuation was present in 61% (11/18) of benign and 90% (9/10) of malignant lesions (p=0.19). Calcification was present in 17% (3/18) of benign tumours and in 70% (7/10) of malignant tumours (p=0.0113). Pleural effusion was present in 6% (1/18) of benign and 40% (4/10) of malignant lesions (p=0.04). Only 1/13 preoperative fine-needle aspirates yielded diagnosis of SFTP. Preoperative diagnosis of SFTP was made in all cases (11/11) with core biopsies. At follow-up (1-10 years, mean 3 years), local recurrence occurred in 3/6 (50%) patients with malignant SFTP and in none of the 10 patients with benign SFTP. CONCLUSION: No definite imaging feature to differentiate benign from malignant SFTP was found. Large size, lobulate borders, presence of calcification, and ipsilateral pleural effusion were the only CT features predictive of malignancy. In suspected cases, core biopsies should be performed rather than fine-needle aspiration.

Validation of a reaction volume reduction protocol for analysis of Y chromosome haplotypes targeting DNA databases.

The use of Y chromosome haplotypes, important for the detection of sexual crimes in forensics, has gained prominence with the use of databases that incorporate these genetic profiles in their system. Here, we optimized and validated an amplification protocol for Y chromosome profile retrieval in reference samples using lesser materials than those in commercial kits. FTA® cards (Flinders Technology Associates) were used to support the oral cells of male individuals, which were amplified directly using the SwabSolution reagent (Promega). First, we optimized and validated the process to define the volume and cycling conditions. Three reference samples and nineteen 1.2 mm-diameter perforated discs were used per sample. Amplification of one or two discs (samples) with the PowerPlex® Y23 kit (Promega) was performed using 25, 26, and 27 thermal cycles. Twenty percent, 32%, and 100% reagent volumes, one disc, and 26 cycles were used for the control per sample. Thereafter, all samples (N = 270) were amplified using 27 cycles, one disc, and 32% reagents (optimized conditions). Data was analyzed using a study of equilibrium values between fluorophore colors. In the samples analyzed with 20% volume, an imbalance was observed in peak heights, both inside and in-between each dye. In samples amplified with 32% reagents, the values obtained for the intra-color and inter-color standard balance calculations for verification of the quality of the analyzed peaks were similar to those of samples amplified with 100% of the recommended volume. The quality of the profiles obtained with 32% reagents was suitable for insertion into databases.

Human autosomal DNA and X chromosome STR profiles obtained from Chrysomya albiceps (Diptera: Calliphoridae) larvae used as a biological trace.

The use of insects to answer questions in criminal investigations, as well as a combination of forensic genetic techniques to obtain human DNA from the organisms, especially necrophagous dipterians, have gained ground in recent decades among researchers and professionals in this area. The objective of our study was to evaluate and compare two methods of human DNA extraction, commonly used for forensic samples, to obtain human autosomal DNA and X chromosome short tandem repeat profiles from the digestive tract of Chrysomya albiceps (Diptera: Calliphoridae) larvae. Immature specimens were collected from corpses at the Institute of Forensic Medicine of Pernambuco and raised in bovine ground meat to allow stabilization of the colony. Groups of larvae in the third instar were provided with bovine ground meat plus human blood for 48 h, dissected, and then subjected to DNA extraction. DNA was extracted using two methods: a DNA IQ™ kit and a phenol-chloroform method. Genomic DNA was amplified using AmpFℓSTR® Identifiler® Plus PCR and Argus-X-12® kits, and samples were sequenced to determine if the two extraction techniques generated reliable profiles that were compatible with a reference sample. The existence of comparable profiles from both techniques demonstrates the usefulness of dipteran larvae for obtaining human DNA from corpses, which can be further used to correlate genetic profiles in a crime scene when other traces are not available. However, several variables still require revision; thus, the technique should be further investigated for its validity, security, and, in particular, its reproducibility.

Detecting multiple DNA human profile from a mosquito blood meal.

Criminal traces commonly found at crime scenes may present mixtures from two or more individuals. The scene of the crime is important for the collection of various types of traces in order to find the perpetrator of the crime. Thus, we propose that hematophagous mosquitoes found at crime scenes can be used to perform genetic testing of human blood and aid in suspect investigation. The aim of the study was to obtain a single Aedes aegypti mosquito profile from a human DNA mixture containing genetic materials of four individuals. We also determined the effect of blood acquisition time by setting time intervals of 24, 48, and 72 h after the blood meal. STR loci and amelogenin were analyzed, and the results showed that human DNA profiles could be obtained from hematophagous mosquitos at 24 h following the blood meal. It is possible that hematophagous mosquitoes can be used as biological remains at the scene of the crime, and can be used to detect human DNA profiles of up to four individuals.

PCDD, PCDF, dl-PCB and organochlorine pesticides monitoring in São Paulo City using passive air sampler as part of the Global Monitoring Plan.

The persistent organic pollutants (POPs), such as organochlorine pesticides and PCBs, are ordinarily monitored in the aquatic environment or in soil in the environmental quality monitoring programs in São Paulo, Brazil. One of the core matrices proposed in the POPs Global Monitoring Plan (GMP) from the Stockholm Convention list is the ambient air, which is not a usual matrix for POPs monitoring in the country. In this study POP levels were evaluated in the air samples from an urban site in São Paulo City over five years, starting in 2010 as a capacity building project for Latin America and the Caribbean region for POP monitoring in ambient air using passive samplers. Furthermore, after the end of the Project in 2012, the monitoring continued in the same sampling site as means to improving the analytical capacity building and contribute to the GMP data. The POPs monitored were 17 congeners of 2,3,7,8 chloro-substituted PCDDs and PCDFs, dioxin-like PCBs, indicator PCBs, organochlorine pesticides and toxaphene. The results show a slight decrease in PCDD/F, dl-PCBs and indicator PCBs levels along the five years. The organochlorine pesticide endosulfan was present at its highest concentration at the beginning of the monitoring period, but it was below detection level in the last year of the monitoring. Some other organochlorine pesticides were detected close to or below quantitation limits. The compounds identified were dieldrin, chlordane, α-HCH, γ-HCH, heptachlor, heptachlor epoxide, hexachlorobenzene and DDTs. Toxaphene congeners were not detected. These results have confirmed the efficacy of passive sampling for POP monitoring and the capacity building for POP analysis and monitoring was established. However more needs to be done, including expansion of sampling sites, new POPs and studies on sampling rates to be considered in calculating the concentration of POPs in ambient air using a passive sampler.

Pulmonary Langerhans Cell Histiocytosis and Diabetes Insipidus in a Young Smoker.

Langerhans cell histiocytosis is characterized by the abnormal nodular proliferation of histiocytes in various organ systems. Pulmonary involvement seen in young adults is nearly always seen in the context of past or current cigarette smoking. Although it tends to be a single-system disease, extrapulmonary manifestations involving the skin, bone, and hypothalamic-pituitary-axis are possible. High resolution CT (HRCT) of the thorax findings includes centrilobular nodules and cysts that are bizarre in shape, variable in size, and thin-walled. Often the diagnosis can be made based on the appropriate clinical presentation and typical imaging findings. Treatment includes smoking cessation and the potential use of glucocorticoids or cytotoxic agents depending on the severity of disease and multisystem involvement.