A rotationally-driven dynamic solid phase sodium bisulfite conversion disc for forensic epigenetic sample preparation.

The approaches to forensic human identification (HID) are largely comparative in nature, relying upon the comparison of short tandem repeat profiles to known reference materials and/or database profiles. However, many profiles are generated from evidence materials that either do not have a reference material for comparison or do not produce a database hit. As an alternative to individualizing analysis for HID, researchers of forensic DNA have demonstrated that the human epigenome can provide a wealth of information. However, epigenetic analysis requires sodium b̲is̲ulfite c̲onversion (BSC), a sample preparation method that is time-consuming, labor-intensive, prone to contamination, and characterized by DNA loss and fragmentation. To provide an alternative method for BSC that is more amenable to integration with the forensic DNA workflow, we describe a rotationally-driven, microfluidic method for dynamic solid phase-BSC (dSP-BSC) that streamlines the sample preparation process in an automated format, capable of preparing up to four samples in parallel. The method permitted decreased incubation intervals by ∼36% and was assessed for relative DNA recovery and conversion efficiency and compared to gold-standard and enzymatic approaches.

Effect of nerve growth factor-ß administered at insemination for lactating Holstein dairy cows bred after timed-artificial insemination protocol.

The objectives of this study were to determine the effects of nerve growth factor-ß (NGF), purified from bulls' seminal plasma and administered at the time of artificial insemination (AI), on progesterone post-AI, interferon-stimulated genes (ISG), and pregnancy per AI (P/AI) for lactating Holstein dairy cows enrolled in a timed-AI protocol. We hypothesized that administration of NGF at the time of AI would increase plasma progesterone post-AI, upregulate relative abundance of ISG, and improve P/AI in lactating dairy cows. Holstein cows (n = 557) from a single commercial dairy farm were blocked by parity and randomly assigned to receive an intramuscular injection containing 296 µg of bovine purified NGF at the time of AI, diluted in 2 mL of phosphate-buffered saline (NGF: n = 275), or receive only the 2 mL of phosphate-buffered saline (control: n = 282). Plasma progesterone and corpus luteum size were assessed in a subset of cows (NGF: n = 32; control: n = 36) at d 7, 14, and 19 post-AI. Relative mRNA abundance of ISG (ISG15, MX1, MX2, and RTP4) was assessed in peripheral blood leukocytes on d 19 post-AI. Pregnancy diagnosis was performed at 37 and 65 d post-AI. There was an interaction effect between treatment and parity for plasma progesterone; however, plasma progesterone and ISG did not differ between treatments. There were no effects of NGF for P/AI at 37 d post-AI (NGF = 40.0% vs. control = 41.6%), 65 d post-AI (NGF = 36.0% vs. control = 38.1%), and for pregnancy loss (NGF = 8.4% vs. control = 7.7%). The current study revealed that effects to NGF in lactating Holstein cows were minor and contingent with parity for progesterone, and no improvement in ISG relative abundance and P/AI were observed.

Anthelmintic resistance in Creole horses in the South of Rio Grande do Sul, Brazil / [Resistência anti-helmíntica em equinos da raça Crioula no sul do Rio Grande do Sul, Brasil]

The objective of the study was to evaluate the antiparasitic resistance against horse nematodes in the South of Rio Grande do Sul, Brazil. The results concerning the tests of anthelmintic efficacy on horses, stored in the database of the Parasitic Diseases Study Group (GEEP) - Veterinary Faculty, at the Federal University of Pelotas (UFPel), were carried out in the laboratory from 2018 to 2019. Stool samples were received from farms with breeding of adult female and male Criollo horses naturally infected, located in municipalities in the country's southern region. The antiparasitic agents tested were Triclorfon + Fenbendazole, Closantel + Albendazole, Ivermectin + Praziquantel, Fenbendazole, Ivermectin, Doramectin, Mebendazole and Moxidectin. Techniques such as Gordon and Whitlock, Coproculture and Fecal Egg Count Reduction Test were performed. Of all the antiparasitic drugs tested, it was observed that only treatments with Ivermectin 2% showed desired values. The observed results indicate that resistance to macrocyclic lactones is usual in equine parasites in this Brazilian region, despite the results with isolated Ivermectin.(AU)

O objetivo deste estudo é avaliar a resistência antiparasitária contra nematodeos de equinos no sul do Rio Grande do Sul, Brasil. Os resultados referentes aos testes de eficácia anti-helmíntica em cavalos, armazenados no banco de dados do Grupo de Estudos de Doenças Parasitárias (GEEP) - Faculdade de Veterinária, da Universidade Federal de Pelotas (UFPel), foram realizados em laboratório, no período de 2018 a 2019. Amostras de fezes foram recebidas de fazendas com criação de cavalos Crioulos adultos fêmeas e machos naturalmente infectados, localizadas em municípios da região Sul do país. Os agentes antiparasitários testados foram triclorfon + fenbendazol, closantel + ivermectina + praziquantel, fenbendazol, ivermectina, doramectina, mebendazol e moxidectina. Técnicas como Gordon e Whitlock, coprocultura e teste de redução da contagem de ovos fecais foram realizadas. De todos os antiparasitários testados, observou-se que apenas os tratamentos com ivermectina 2% apresentaram os valores desejados. Os resultados indicam que a resistência às lactonas macrocíclicas é comum em parasitas equinos nessa região brasileira, apesar dos resultados com ivermectina isolada.(AU)

Episodic angioedema with eosinophilia (Gleich's syndrome) associated with urticarial vasculitis: a coincidence or a novel clinical entity?

Episodic angioedema with eosinophilia (EAE) is a rare condition characterized by recurrent attacks of angioedema and urticaria accompanied by a marked elevation of peripheral eosinophil count. We report the case of a young female patient diagnosed with EAE associated with urticarial vasculitis. A 40-year-old female patient was admitted to our institution due to recurrent episodes of cheek and eyelid angioedema in the previous year. Episodes of facial angioedema lasted for two months with spontaneous remission afterwards. In addition, she presented pruritic and painful skin eruptions of erythematous circles, which persisted for longer than 24 h, that were palpable, somewhat purplish, and more pronounced on the face, arms, and trunk. Laboratory investigation showed a sustained elevation of white cell counts with marked eosinophilia. Serum IgM, IgE, and IgA were normal; IgG was slightly elevated. C1-esterase inhibitor and tryptase test were normal. Reverse transcriptase-polymerase chain reaction was performed for detection of FIP1L1-PDGFRA and BCR-ABL rearrangements. None of these alterations were found. Skin biopsies were suggestive of urticarial vasculitis. The patient was submitted to esophagogastroduodenoscopy, which showed mild chronic gastritis, with no eosinophilic infiltration. Cardiac dimensions and function were normal. Abdominal ultrasound and total body CT-scan failed to show lymphadenopathy, organomegaly, and tumors. We report the first case of association between episodic angioedema with eosinophilia and urticarial vasculitis. It is possible that both conditions share a physiopathological mechanism, suggesting that it is not just a chance association.

Characterization of pregnancy-associated glycoproteins and progesterone as a predictor of twins and conceptus loss in high-risk-pregnancy Holstein cows.

The objective of this study was to characterize plasma concentrations of pregnancy-associated glycoprotein (PAG) and progesterone (P4) as predictors of twins and pregnancy loss in Holstein cows with high-risk pregnancies. High-risk pregnancy was characterized using transrectal ultrasonography 37 d after artificial insemination (AI) based on the following criteria: small embryo size <15 mm, n = 10), slow heartbeat (<60 beats per minute, n = 11), and extra-amniotic membrane (additional amniotic membrane, n = 3). A cohort of twins (n = 41) diagnosed at d 37 post-AI was also enrolled. Each high-risk and twin pregnancy cow was paired with a cow of the same parity carrying a normal singleton at d 37 post-AI (control, n = 65). Blood samples were collected to measure PAG and P4 at d 37, 44, and 51 post-AI. Statistical analysis was performed using ANOVA, logistic regression, and receiver operator characteristic (ROC) curve with JMP software (SAS Institute Inc., Cary, NC). Pregnancy loss at d 51 post-AI was greater in high-risk pregnancies than in controls and twins (control = 1.5%; high-risk = 87.5%; twins = 12.2%). Concentration of PAG at d 37 post-AI did not differ among groups (control = 5.3 ± 0.7; high-risk = 4.8 ± 1.2; twins = 4.0 ± 0.9 ng/mL). Concentration of P4 at d 37 post-AI was greater in twins than in high-risk pregnancies and control, and lesser in high-risk pregnancies than in controls (control = 7.0 ± 0.3; high-risk = 5.9 ± 0.4; twins = 8.4 ± 0.3 ng/mL). Regression and ROC analysis for PAG at d 37 post-AI did not find a threshold to predict pregnancy loss or twins. Regression and ROC analysis for P4 at d 37 post-AI found that a threshold of 6.5 ng/mL predicted pregnancy loss with an area under the curve (AUC) of 0.64, and a threshold of 7.2 ng/mL predicted twins with AUC of 0.71. In summary, pregnancy loss and twins were predicted with only moderate accuracy by P4 concentration at d 37 post-AI, and the variability in PAG concentrations at d 37 post-AI was not sufficient to generate a threshold to predict pregnancy loss and twins in Holstein lactating cows.

Episodic angioedema with eosinophilia (Gleich's syndrome) associated with urticarial vasculitis: a coincidence or a novel clinical entity?

Episodic angioedema with eosinophilia (EAE) is a rare condition characterized by recurrent attacks of angioedema and urticaria accompanied by a marked elevation of peripheral eosinophil count. We report the case of a young female patient diagnosed with EAE associated with urticarial vasculitis. A 40-year-old female patient was admitted to our institution due to recurrent episodes of cheek and eyelid angioedema in the previous year. Episodes of facial angioedema lasted for two months with spontaneous remission afterwards. In addition, she presented pruritic and painful skin eruptions of erythematous circles, which persisted for longer than 24 h, that were palpable, somewhat purplish, and more pronounced on the face, arms, and trunk. Laboratory investigation showed a sustained elevation of white cell counts with marked eosinophilia. Serum IgM, IgE, and IgA were normal; IgG was slightly elevated. C1-esterase inhibitor and tryptase test were normal. Reverse transcriptase-polymerase chain reaction was performed for detection of FIP1L1-PDGFRA and BCR-ABL rearrangements. None of these alterations were found. Skin biopsies were suggestive of urticarial vasculitis. The patient was submitted to esophagogastroduodenoscopy, which showed mild chronic gastritis, with no eosinophilic infiltration. Cardiac dimensions and function were normal. Abdominal ultrasound and total body CT-scan failed to show lymphadenopathy, organomegaly, and tumors. We report the first case of association between episodic angioedema with eosinophilia and urticarial vasculitis. It is possible that both conditions share a physiopathological mechanism, suggesting that it is not just a chance association.

Investigation on the association between thyroid tumorigeneses and herpesviruses.

Herpesviruses have been associated with various human malignancies and with thyroid autoimmunity. Aiming to investigate the presence of these viruses in thyroid nodules, we analyzed serum and thyroid tissue from 183 patients (83 benign and 100 malignant thyroid nodules). We also obtained 104 normal thyroid tissues extracted from the contralateral lobe of these patients. We used ELISA to screen the serology of all patients and a real-time quantitative PCR to analyze thyroid tissue viral load in antibody-positive patients. In addition, the presence of herpesviruses was tested by histological analysis in 20 EBV-positive tissues using the expression of LMP-1 by immunohistochemistry (IHC) and EBER by in situ hybridization (ISH). There was no evidence of HSV-2 or CMV DNA, but we found EBV DNA sequences in 29 (16%) thyroid tissue samples. We also found 7 positive EBV cases out of 104 normal tissues. Viral load was higher in tumors than in their respective normal tissues (p = 0.0002). ISH analysis revealed EBER expression in 11 out of 20 (52%) EBV-positive tissues, mostly in malignant cases (8/11, 73%). The presence of high EBV copy numbers in thyroid tumors and the expression of EBER only in malignant cases suggest an association between EBV and thyroid malignancies. However, we did not find any association between the presence of EBV and/or its viral load and any clinical or pathological tumor feature. Further studies aiming to clarify the mechanisms of EBV infection in thyroid cells are necessary to support a possible role in the development of thyroid cancer.

The influence of the environment on the development of thyroid tumors: a new appraisal.

Most epidemiological studies concerning differentiated thyroid cancers (DTC) indicate an increasing incidence over the last two decades. This increase might be partially explained by the better access to health services worldwide, but clinicopathological analyses do not fully support this hypothesis, indicating that there are carcinogenetic factors behind this noticeable increasing incidence. Although we have undoubtedly understood the biology and molecular pathways underlying thyroid carcinogenesis in a better way, we have made very little progresses in identifying a risk profile for DTC, and our knowledge of risk factors is very similar to what we knew 30-40 years ago. In addition to ionizing radiation exposure, the most documented and established risk factor for DTC, we also investigated the role of other factors, including eating habits, tobacco smoking, living in a volcanic area, xenobiotics, and viruses, which could be involved in thyroid carcinogenesis, thus, contributing to the increase in DTC incidence rates observed.

Genes of detoxification are important modulators of hereditary medullary thyroid carcinoma risk.

CONTEXT: Different inherited profiles of genes involved in cellular mechanisms of activation and detoxification of carcinogenic products can provide specific protection or determine the risk for cancer. Low-penetrance polymorphic genes related to the biotransformation of environmental toxins have been associated with susceptibility to and the phenotype of, human tumours. OBJECTIVE: To investigate the role of germline inheritance of polymorphisms in CYP1A2*F, CYP1A1 m1, GSTP1, NAT2 and TP53 genes in hereditary medullary thyroid carcinoma (HMTC) patients. DESIGN: This study was developed in University of Campinas (Unicamp). PATIENTS: We studied 132 patients with HMTC, 88 first-degree relatives of HMTC patients and 575 control individuals. MEASUREMENTS: All patients with MTC and their relatives were sequenced for the RET gene and five genes were genotyped using TaqMan(®) system. RESULTS: We observed that the inheritance of CYP1A2*F (OR = 2·10; 95% CI = 1·11-3·97; P = 0·022), GSTP1 (OR = 4·41; 95% CI = 2·47-7·88; P < 0·001) and NAT2 (OR = 2·54; 95% CI = 1·16-5·58; P = 0·020) variants increased the risk for HMTC. In addition, multiple regression analysis showed that the inheritance of GSTP1 polymorphisms was associated with the diagnosis in older patients (B = 8·0229; 95% IC = ± 5·5735; P = 0·0054). Concerning the group of HTMC relatives, CYP1A2*F (OR = 2:40; 95% CI = 1·19-4·86; P = 0·015), CYP1A1 m1 (OR = 2·79; 95% CI = 1:04-7·51; P = 0·042), GSTP1 (OR = 2·86; 95% IC = 1·53-5·32; P < 0·001) and NAT2 (OR = 2·25; 95% IC = 1·20-4·22; P = 0·012) were associated with HMTC risk. CONCLUSIONS: We have demonstrated that the inheritance of specific genes determining the individual response to environmental toxins may contribute to the risk and phenotypic variability that exists in patients with HMTC. Moreover, we identified a group at risk in relatives of HMTC patients.

Chromatin changes in papillary thyroid carcinomas may predict patient outcome.

PURPOSE: New insights in prognostic predictions are urgently needed for papillary thyroid carcinoma (PTC). The present study aimed to investigate whether computerized analysis of chromatin texture allows the identification of PTC patients with a poor prognosis. METHODS: We randomnly selected paraffin-embedded blocks from surgical specimens of 103 classic cases of PTC. During follow-up, 68 of the patients were classified as free of disease, whereas 35 presented with recurrences. Characteristics of chromatin were obtained from digitized images of at least 100 randomly selected tumor nuclei per patient. An independent series of 30 goiters was used to validate our observations. RESULTS: Stage, age and distant metastases were found to serve as independent prognostic factors for survival. In addition, multivariate Cox regression confirmed variable cluster prominence as an independent prognostic factor. By comparing malignant and benign nodules, we found that the PTC lesions presented with higher nuclear perimeters, nuclear areas, Minkowski fractal dimensions, optical densities and nuclear longest chords. CONCLUSION: From our results we conclude that, in conjunction with clinical and histopathological data, morphometric data may provide relevant prognostic information in PTC patients.

Clinical and pathological implications of concurrent autoimmune thyroid disorders and papillary thyroid cancer.

Cooccurrences of chronic lymphocytic thyroiditis (CLT) and thyroid cancer (DTC) have been repeatedly reported. Both CLT and DTC, mainly papillary thyroid carcinoma (PTC), share some epidemiological and molecular features. In fact, thyroid lymphocytic inflammatory reaction has been observed in association with PTC at variable frequency, although the precise relationship between the two diseases is still debated. It also remains a matter of debate whether the association with a CLT or even an autoimmune disorder could influence the prognosis of PTC. A better understanding about clinical implications of autoimmunity in concurrent thyroid cancer could raise new insights of thyroid cancer immunotherapy. In addition, elucidating the molecular mechanisms involved in autoimmune disease and concurrent cancer allowed us to identify new therapeutic strategies against thyroid cancer. The objective of this article was to review recent literature on the association of these disorders and its potential significance.