Genome-wide analysis of the chromatin sites targeted by HEX 70a storage protein in the honeybee brain and fat body.

Hexamerins, the proteins massively stored in the larval haemolymph of insects, are gradually used throughout metamorphosis as a source of raw material and energy for the development of adult tissues. Such behaviour defined hexamerins as storage proteins. Immunofluorescence experiments coupled with confocal microscopy show a hexamerin, HEX 70a, in the nucleus of the brain and fat body cells from honeybee workers, an unexpected localization for a storage protein. HEX 70a colocalizes with fibrillarin, a nucleolar-specific protein and H3 histone, thus suggesting a potential role as a chromatin-binding protein. This was investigated through chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq). The significant HEX 70a-DNA binding sites were mainly localized at the intergenic, promoter and intronic regions. HEX 70a targeted DNA stretches mapped to the genomic regions encompassing genes with relevant functional attributes. Several HEX 70a targeted genes were associated with H3K27ac or/and H3K27me3, known as active and repressive histone marks. Brain and fat body tissues shared a fraction of the HEX 70 targeted genes, and tissue-specific targets were also detected. The presence of overrepresented DNA motifs in the binding sites is consistent with specific HEX 70a-chromatin association. In addition, a search for HEX 70a targets in RNA-seq public libraries of fat bodies from nurses and foragers revealed differentially expressed targets displaying hex 70a-correlated developmental expression, thus supporting a regulatory activity for HEX 70a. Our results support the premise that HEX 70a is a moonlighting protein that binds chromatin and has roles in the brain and fat body cell nuclei, apart from its canonical role as a storage protein.

N,N'-Di-cyclo-hexyl-N-(phthaloylglyc-yl)urea.

The mol-ecular structure of the title compound {systematic name: 1,3-di-cyclo-hexyl-1-[2-(1,3-dioxo-2,3-di-hydro-1H-isoindol-2-yl)acet-yl]urea}, C23H29N3O4, derived from N,N'-di-cyclo-hexyl-urea, shows that the tertiary N atom substituted by a cyclo-hexyl and phthaloylglycyl groups adopts a perfectly planar geometry (bond-angle sum = 360.0°). In the same way as for N,N'-di-cyclo-hexyl-urea, the extended structure of the title compound features N-H⋯O hydrogen bonds, which generate chains of mol-ecules running in the [001] direction.

Comparative performances of seven quantitative Reverse-Transcription Polymerase Chain Reaction assays (RT-qPCR) for detecting SARS-CoV-2 infection in samples from individuals suspected of COVID-19 in São Paulo, Brazil.

Introduction: Brazil is the second largest country with COVID-19 positive cases worldwide. Due to the potent spread of the virus and the scarcity of kits and supplies, the Brazilian Ministry of Health has granted authorization for the use of kits available during this emergency, without an accurate evaluation of their performance. This study compared the performance and cost-effectiveness of seven molecular assays/kits available in São Paulo, Brazil, for SARS-CoV-2 diagnosis. Materials and methods: A total of 205 nasopharyngeal/oropharyngeal samples from suspected cases of COVID-19, were tested using the following assays: (i) GeneFinder COVID-19 plus RealAmp kit; (ii) 2019-nCoV RNA PCR-Fluorescence Probing, Da An Gene Co.; (iii) in-house RT-qPCR SARS-CoV-2 IAL; (iv) 2019-nCoV kit, IDT; (v) molecular SARS-CoV-2 (E) kit, Bio-Manguinhos; (vi) Allplex 2019-nCoV modified Assay, Seegene Inc, and (vii) Biomol one-step COVID-19 kit, IBMP. The criteria for determining a SARS-CoV-2 true positive result included the cycle threshold cut-off values, the characteristics of exponential/linear curves, the gene target diversity, and a positive result in at least two assays. Results: The overall sensitivity of the assays listed were GeneFinder 83.6%, Da An Gene 100.0%, IAL 90.4%, IDT 94.6%, Bio-Manguinhos 87.7%, Allplex 97.3%, and IBMP 87.7%. The minor sensitive gene target was RdRP. Although all assays had a Cohen's Kappa index ≥0.893, the best tests used multiplex assays identifying N-gene and/or E-gene targets. Conclusion: All assays tested accurate for diagnosis, but considering cost-effectiveness (cost, time consumption, number of samples tested, and performance), the in-house IAL assay was ideal for COVID-19 diagnosis in São Paulo, Brazil.

Estrogenic and chemopreventive activities of xanthones and flavones of Syngonanthus (Eriocaulaceae).

The possible benefits of some bioactive flavones and xanthones present in plants of the genus Syngonanthus prompted us to screen them for estrogenic activity. However, scientific research has shown that such substances may have undesirable properties, such as mutagenicity, carcinogenicity and toxicity, which restrict their use as therapeutic agents. Hence, the aim of this study was to assess the estrogenicity and mutagenic and antimutagenic properties. We used recombinant yeast assay (RYA), with the strain BY4741 of Saccharomyces cerevisiae, and Ames test, with strains TA100, TA98, TA97a and TA102 of Salmonella typhimirium, to evaluate estrogenicity, mutagenicity and antimutagenicity of methanolic extracts of Syngonanthus dealbatus (S.d.), Syngonanthus macrolepsis (S.m.), Syngonanthus nitens (S.n.) and Syngonanthus suberosus (S.s.), and of 9 compounds isolated from them (1=luteolin, 2=mix of A-1,3,6-trihydroxy-2-methoxyxanthone and B-1,3,6-trihydroxy-2,5-dimethoxyxanthone, 3=1,5,7-trihydroxy-3,6-dimethoxyxanthone, 4=1,3,6,8-tetrahydroxy-2,5-dimethoxyxanthone, 5=1,3,6,8-tetrahydroxy-5-methoxyxanthone, 6=7-methoxyluteolin-8-C-ß-glucopyranoside, 7=7-methoxyluteolin-6-C-ß-glucopyranoside, 8=7,3'-dimethoxyluteolin-6-C-ß-glucopyranoside and 9=6-hydroxyluteolin). The results indicated the estrogenic potential of the S. nitens methanol extract and four of its isolated xanthones, which exhibited, respectively, 14.74±1.63 nM; 19.54±6.61; 7.20±0.37; 6.71±1.02 e 10.01±4.26 nM of estradiol-equivalents (EEQ). None of the extracts or isolated compounds showed mutagenicity in any of the test strains and all of them showed antimutagenic potential, in particular preventing mutations caused by aflatoxin B1 (AFB1) and benzo[a]pyrene (B[a]P). The results show that the xanthones, only isolated from the methanol extract of S. nitens capitula, probably were the responsible for its estrogenic activity and could be useful as phytoestrogens, providing a new opportunity to develop hormonal agents. In addition, flavones and xanthones could also be used as a new antimutagenic agent. Since, the mutagens are involved in the initiation and promotion of several human diseases, including cancer, the significance of novel bioactive phytocompounds in counteracting these pro-mutagenic and carcinogenic effects is now gaining credence.

Phytochemical study guided by the myorelaxant activity of the crude extract, fractions and constituent from stem bark of Hymenaea courbaril L.

ETHNOPHARMACOLOGICAL RELEVANCE: Hymenaea courbaril L. (Caesalpinoideae) is used in Brazilian folk medicine to treat anemia, kidney problems, sore throat and other dysfunctions of the respiratory system, such as bronchitis and asthma, although such properties are yet to be scientifically validated. AIM OF THE STUDY: In order to give a scientific basis to support the traditional use of Hymenaea courbaril, this study was designed to evaluate antioxidant, myorelaxant and anti-inflammatory properties of the ethanol extract from stem bark and its fractions. The myorelaxant effect of astilbin, a flavonoid isolated from the bioactive ethyl acetate fraction (EAF), has also been evaluated. MATERIAL AND METHODS: In the present study ethanol extract from stem bark (EEHC) and fractions were analyzed using bioassay-guided fractionation. The following activities were investigated: antioxidant by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, myorelaxant on rat tracheal smooth muscle, and anti-inflammatory using ovalbumin-induced leukocytosis and airway hyperresponsiveness in rats. RESULTS: The results of the present investigation show that the whole extract of Hymenaea courbaril and some of its fractions strongly scavenged DPPH radical. The extract showed myorelaxant activity on rat trachea, being EAF its highest efficient fraction. Bio-guided study allowed the isolation of astilbin, a well-known flavonoid. The activity induced by this compound indicates that it may be partly responsible for the myorelaxant effect of EAF. EAF reduced contractions that depended on divalent cation inflow through voltage-operated Ca(2+) channels (VOCCs) or receptor-operated Ca(2+) channels (ROCCs), but it was more potent to inhibit VOCC- than ROCC-dependent contraction induced by Ca(2+) addition in ACh-enriched Ca(2+)-free medium. Oral pretreatment of antigen-challenged animals with EAF prevented airway hyperresponsiveness on KCl-induced contraction and reduced the number of total white cells, particularly eosinophils and neutrophils in bronchoalveolar lavage. CONCLUSIONS: This study provided scientific basis that Hymenaea courbaril presents potential antioxidant, myorelaxant and anti-inflammatory actions, which support its use in folk medicine to treat inflammatory airway diseases.

Nuclear Immunolocalization of Hexamerins in the Fat Body of Metamorphosing Honey Bees.

Hexamerins are storage proteins with primordial functions in insect metamorphosis. They are actively secreted by the larval fat body and stored in the hemolymph. During metamorphosis, they return to the fat body to be processed. For decades, these proteins were thought to exclusively function as an amino acid source for tissue reconstruction during the non-feeding pupal and pharate adult stages and, in some species, for egg production. Recently, new findings have linked the hexamerins to caste polyphenism and gonad development in social insects. To explore the roles of hexamerins during the honey bee metamorphosis, we used specific antibodies in expression analysis by western blot, in situ immunolocalization by confocal laser-scanning microscopy and in vivo injections to lower their endogenous levels. Our expression analysis highlighted the changing expression patterns in the fat body and hemolymph during development, which is consistent with the temporal dynamics of hexamerin secretion, storage and depletion. Confocal microscopy showed hexamerin expression in the cytoplasm of both types of fat body cells, trophocytes and oenocytes. Notably, hexamerin foci were also found in the nuclei of these cells, thus confirming our western blot analysis of fat body nuclear-enriched fractions. We also observed that the decrease in soluble hexamerins in antibody-treated pharate adults led to a precocious adult ecdysis, perhaps in response to the lack (or decrease) in hexamerin-derived amino acids. Taken together, these findings indicate that hexamerins have other functions in addition to their well-established role as amino acid sources for development.

Avaliação in vitro da variação do torque de remoção de parafusos para fixação de pilares protéticos submetidos a ciclos de parafusamento e desparafusamento / Torque removal evaluation of prosthetic screws after cycles of tightening and loosening: an in vitro study

O afrouxamento dos parafusos protéticos é descrito na literatura como uma das complicações mais frequentes das próteses sobre implantes. Durante sua confecção, os profissionais sentem necessidade de remover várias vezes as próteses e/ou componentes protéticos, soltando e re-apertando os parafusos repetidamente. O principal objetivo deste trabalho foi avaliar a variação do torque de remoção de parafusos de fixação de pilares protéticos a implantes osteointegráveis após sucessivos ciclos de parafusamento e desparafusamento. Outro objetivo foi avaliar a influência do hexágono da base do pilar no torque de remoção dos parafusos. Para isso, foram utilizados 20 implantes de plataforma regular com hexágono externo e 20 pilares protéticos sextavados, que foram parafusados aos implantes com um parafuso de titânio, aplicando-se a este um torque de 32Ncm, por meio de um torquímetro digital. Os conjuntos implante/pilar/parafuso foram divididos em dois grupos: (1) pilares cujo hexágono da base foram removidos e (2) pilares convencionais, com hexágono na base. Cada conjunto recebeu uma restauração provisória e foi submetido a ciclagem mecânica por 15 minutos. Depois, os parafusos foram removidos, medindo-se o torque de remoção. Esta sequência foi repetida dez vezes e então o parafuso foi trocado por outro sem uso, e mais um ciclo foi realizado. Uma análise de regressão linear demonstrou nos dois grupos uma queda do torque de remoção do parafuso ao longo dos repetidos ciclos de inserção/remoção. A comparação entre os coeficientes da regressão nos dois grupos não revelou diferença entre eles. Também não houve diferença entre as médias das 5 últimas repetições e o 11° ciclo, com o parafuso novo. Concluiu-se que (1) repetidos parafusamentos e desparafusamentos promoveram a diminuição progressiva do torque de remoção dos parafusos, (2) a troca do parafuso por outro sem uso após dez ciclos de inserção/remoção não aumentou sua resistência ao afrouxamento, e (3) a remoção ...

Screw loosening is one of the most common problems of implant-retained prostheses. During their confection, dentists need to remove and re-install them several times, loosening and re-tightening the screws repeatedly. The first purpose of this study was to evaluate the effects of repeated insertion/removal cycles on the removal torque of retaining prosthetic screws. The second purpose was to evaluate the influence of the hexagon of the abutment on the removal torque. For that, 20 regular external hex implants were used and 20 hexagonal abutments were tightened to the implants with a titanium alloy screw, with an insertion torque of 32Ncm, given by a digital torque gauge. The implant/abutment/screw assemblies were divided into two groups: (1) abutments with no hexagon on the base and (2) abutments with an hexagon on the base. Each assembly received a provisional restoration and was submitted to mechanical loading cycles for 15 minutes. After this, the screws were removed and the removal torque measured. This sequence was repeated ten times and then, the screw was changed for a new one, and another cycle was done. Linear regression analysis was performed and showed that removal torque values tended to decrease as the number of insertion/removal cycles increased, for both groups. Comparisons of the slopes and the intercepts between the two groups showed no statistic difference between them. There was also no difference between the mean values of last 5 cycles and the 11th cycle. It was concluded that (1) repeated insertion/removal cycles promoted gradual reduction in removal torque of screws, (2) changing screw by a new one after ten insertion/removal cycles did not increase resistance to loosening, and (3) removal of the hexagon of the abutment base had no effect on the removal torque of the screw.

Avaliação das medidas lineares dos hexágonos de abutments unitários em sistemas de implantes com hexágono externo / Evaluation of linear measurements of hexagons of single abutments in external hex implant systems

O sucesso da osseointegração está intimamente relacionado ao ajuste passivo entre os componentes utilizados e à precisão de adaptação da prótese instalada sobre os implantes. Quando os sistemas de implantes possuem um bom grau de padronização de usinagem existe a possibilidade de intercambiar seus componentes, de acordo com a necessidade, sem prejuízo para a conexão protética. O objetivo desta investigação foi avaliar o grau de liberdade entre os hexágonos dos implantes e abutments das empresas 3i, Implamed e Nobel Biocare, analisando: 1) as diferenças entre as medidas lineares das faces opostas dos implantes e abutments; 2) as diferenças entre as medidas lineares dos ângulos opostos dos implantes e abutments nos sistemas avaliados. A metodologia envolveu imagens obtidas através da microscopia eletrônica de varredura analisadas por um programa computadorizado específico. Através da análise de variância com nível de significância de 5% e teste de Tuckey para comparações múltiplas pôde-se observar que: 1) para as diferenças entre as medidas lineares das faces opostas dos hexágonos dos implantes e dos abutments houve diferença estatisticamente significante entre os grupos Nobel e Implamed, tendo o grupo Nobel os melhores resultados; 2) para as diferenças entre as medidas lineares dos ângulos opostos dos hexágonos dos implantes e dos abutments, o grupo Implamed diferiu dos demais, com os piores resultados.

The success of osseointegration is closely related to passive fit among the components employed and accurate fit of the implant-supported prosthesis. Implant systems with good machining standardization allow exchange of components according to the needs, without damage to the prosthetic connection. This study aimed to evaluate the degree of freedom among hexagons of implants and abutments fabricated by 3i, Implamed and Nobel Biocare, by analysis of: 1)differences among linear measurements of opposite aspects of implants and abutments 2) differences among linear measurements of opposite angles of implants and abutments in these systems. The metodology comprised analysis of scanning electron microscopy images by a specific computer software. Analysis of variance and the Tukey test for multiple comparisons at a significance level of 5% revealed the following: 1) there were statistically significant differences in linear measurements of opposite aspects of hexagons of implants and abutments between Nobel and implamed, with better results for the Nobel system; 2) concerning the differences among linear measurements of opposite angles of hexagons of implants and abutments, the Implamed system was different from the others, presenting the worst results.