Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process

International audienceBACKGROUND:The FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage.METHODS:We performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot.RESULTS:Computational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA.CONCLUSIONS:Our results show that FA cells, despite extensive DNA damage, do not loss the capacity to express the transcriptional and protein components of checkpoint recovery that might eventually allow their division with unrepaired DNA damage. This might allow cell survival but increases the genomic instability inherent to FA individuals and promotes cancer

Impact of SPECT corrections on 3D-dosimetry for TARE

Purpose: Many centers aim to plan liver transarterial radioembolization (TARE) with dosimetry, even without CT-based attenuation correction (AC), or with unoptimized scatter correction (SC) methods. This work investigates the impact of presence vs absence of such corrections, and limited spatial resolution, on 3D dosimetry for TARE. Methods: Three voxelized phantoms were derived from CT images of real patients with different body sizes. Simulations of 99mTc-SPECT projections were performed with the SIMIND code, assuming three activity distributions in the liver: uniform, inside a "liver's segment," or distributing multiple uptaking nodules ("nonuniform liver"), with a tumoral liver/healthy parenchyma ratio of 5:1. Projection data were reconstructed by a commercial workstation, with OSEM protocol not specifically optimized for dosimetry (spatial resolution of 12.6 mm), with/without SC (optimized, or with parameters predefined by the manufacturer; dual energy window), and with/without AC. Activity in voxels was calculated by a relative calibration, assuming identical microspheres and 99mTc-SPECT counts spatial distribution. 3D dose distributions were calculated by convolution with 90Y voxel S-values, assuming permanent trapping of microspheres. Cumulative dose-volume histograms in lesions and healthy parenchyma from different reconstructions were compared with those obtained from the reference biodistribution (the "gold standard," GS), assessing differences for D95%, D70%, and D50% (i.e., minimum value of the absorbed dose to a percentage of the irradiated volume). γ tool analysis with tolerance of 3%/13 mm was used to evaluate the agreement between GS and simulated cases. The influence of deep-breathing was studied, blurring the reference biodistributions with a 3D anisotropic gaussian kernel, and performing the simulations once again. Results: Differences of the dosimetric indicators were noticeable in some cases, always negative for lesions and distributed around zero for parenchyma. Application of AC and SC reduced systematically the differences for lesions by 5%–14% for a liver segment, and by 7%–12% for a nonuniform liver. For parenchyma, the data trend was less clear, but the overall range of variability passed from −10%/40% for a liver segment, and −10%/20% for a nonuniform liver, to −13%/6% in both cases. Applying AC, SC with preset parameters gave similar results to optimized SC, as confirmed by γ tool analysis. Moreover, γ analysis confirmed that solely AC and SC are not sufficient to obtain accurate 3D dose distribution. With breathing, the accuracy worsened severely for all dosimetric indicators, above all for lesions: with AC and optimized SC, −38%/−13% in liver's segment, −61%/−40% in the nonuniform liver. For parenchyma, D50% resulted always less sensitive to breathing and sub-optimal correction methods (difference overall range: −7%/13%). Conclusions: Reconstruction protocol optimization, AC, SC, PVE and respiratory motion corrections should be implemented to obtain the best possible dosimetric accuracy. On the other side, thanks to the relative calibration, D50% inaccuracy for the healthy parenchyma from absence of AC was less than expected, while the optimization of SC was scarcely influent. The relative calibration therefore allows to perform TARE planning, basing on D50% for the healthy parenchyma, even without AC or with suboptimal corrections, rather than rely on nondosimetric methods

Testicular Morphological and Ultrasonographic Characterization of Male Gray Brocket Deers (Mazama gouazoubira) in Different Reproductive Status

Background: Gray brocket deer (Mazama gouazoubira) populations have been declining due to human intervention. Yet, only a few studies have assessed ultrasonographic testicular characteristics in cervids. Considering the relevance of monitoring testicular size, blood flow, and parenchyma, the present study aims to establish baseline information on scrotal circumference, testicular volume, and spectral Doppler parameters, to describe differences among adult male gray brocket deer in different reproductive status, and to correlate ultrasound parameters with testes size measurements.Materials, Methods & Results: Six adult male gray brocket deers were used in the study. Scrotal circumference and testicular volume were measured. B mode ultrasound images of testes (longitudinal and cross-sectional views) and epididymes were subjected to computer-assisted analysis, obtaining the numerical pixel values (NPV) and pixel standard deviation (PSD). Using spectral Doppler, supratesticular artery blood flow velocities (peak systolic velocity - PSV, end diastolic velocity - EDV, time-average maximum velocity - TAMAX, resistivity - RI and pulsatility indices - PI) were obtained. Semen was analyzed through total motility, vigor, and concentration tests. Three animals were normospermic (F+ group) and three were oligo/azoospermic (F- group). Groups were compared using were compared using a one-way ANOVA or Kruskal-Wallis followed by Student-Newman-Keuls (SNK) test. Ultrasound parameters were correlated to testes size parameters using Pearson’s correlation for parametric variables and Spearman’s correlation for non-parametric variables. F+ group presented significantly higher scrotal circumference (14.57 ± 1.19 cm), testicular volume (26.18 ± 4.94 cm3), and testes cross-sectional NPV (69.88 ± 24.00) and PSD (10.78 ± 3.42) than group F- (NPV: 28.26 ± 13.75, PSD: 6.70 ± 1.84). No significant differences were observed between the groups regarding the spectral Doppler ultrasound parameters. Significant correlations were observed between scrotal circumference and longitudinal (r = 0.76) and cross-sectional testes NPV (r = 0.89), and testicular volume was correlated with longitudinal (r = 0.78) and cross-sectional testes NPV (r = 0.91) and with cross-sectional testes PSD (ρ = 0.82).Discussion: Increased testicular echogenicity (higher NPV) has been positively associated with improved testicular growth, cell population expansion, inner and outer seminiferous tubules diameter, spermatids percentages and testis weight. In addition, more heterogenous testes (higher PSD) were associated with higher sperm output. It was suggested that the animals in group F- had compromised testicular development and spermatogenesis. The correlation observed between testes NPV and scrotal circumference was proposed to be associated with seminiferous tubules impairment. The F- group showed lower testicular volume, NPVs and PSDs in cross-sectional testicular images, suggesting higher protein levels and lower lipid contents were present in their parenchyma, influencing in testicular echogenicity and echotexture. No differences in spectral Doppler parameters were observed between the two groups. Also observed in stallions. However, PSV, EDV, TAMAX could be potential infertility indicators in other mammalians. These different results may be due to different locations of the evaluated vessel, species and techniques, age, ambient temperature, pathological conditions, and anaesthesia. Thus, it is suggested that scrotal circumference, testicular volume, and testes NPV are good indicators of male reproductive health in gray brocket deer and may help with better male selection in the species

REPRESENTAÇÕES SOCIAIS DA ALIMENTAÇÃO SAUDÁVEL PARA PESSOA IDOSA: UMA REVISÃO INTEGRATIVA

Objetivo: conhecer as evidências científicas das representações sociais sobre a alimentação saudável no comportamento alimentar de idosos. Método: trata-se de uma revisão integrativa da literatura, realizando buscas em portais e bases de dados: Biblioteca Virtual em Saúde, SciELO, Web of Science, SCOPUS e PUBMED. Foram incluídos os estudos disponibilizados em português, inglês ou espanhol com população idosa (idade ≥60 anos), sem filtros para ano de publicação e estar diretamente relacionado ao tema. Foram excluídos publicações não disponíveis eletronicamente na íntegra e que não atenderam à pergunta da pesquisa. Extraíram-se as informações referentes em autor(es)/ano de publicação, tipo de estudo/ amostra, objetivo, e os principais resultados. A qualidade metodológica dos estudos foi avaliada pelos instrumentos Critical Appraisal Skill Programme e Agency for Health care and Research and Quality. Para o delineamento da seleção dos artigos utilizou-se o fluxograma do Preferred Reporting Items for Systematic Reviews and Meta-analyses, além da participação de dois pesquisadores de forma simultânea e independente. Resultados: Os principais resultados “denunciam” a escassez de estudos que abordem as representações sociais da alimentação, exclusivamente para pessoas idosas. Foram classificados três grupos para descrição das representações sociais: às relacionadas ao alimento; as vinculadas à doença que emanaram a preocupação com a alimentação; e a alimentação representada socialmente como preocupação com a saúde e bem estar. Conclusão: as representações sociais sobre a alimentação saudável no comportamento alimentar de idosos é um tema complexo, limitado e pouco discutido, no entanto, de forma indireta foi possível  vislumbrar representações sociais relacionadas à temática

Especiarias e sua importância na gastronomia / Spices and their importance in gastronomy

As especiarias fazem parte da história por serem utilizadas desde o período de a.C. como remédios e fabricação de perfumes. Junto com o surgimento das comunidades e povos, bem como a criação do hábito de sentar-se à mesa para compartilhar os alimentos entre famílias, a gastronomia foi desenvolvendo sua principal característica; adaptar-se de acordo com os hábitos e culturas de cada povo e região, sofrendo adaptações culturais na forma de preparo dos alimentos, transmitindo dessa forma, através das gerações, receitas e temperos tradicionais. No início do século XIV, as especiarias passaram a ser de grande importância tendo uma contribuição significativa no comércio internacional, gerando concorrência entre as grandes potências marítimas que disputavam seu monopólio para a venda gerando riquezas para seus países. As especiarias então, estão relacionadas com a cultura de cada povo, região e o desenvolvimento e avanço de diversos continentes, possibilitando a relação comercial entre países.  Sabendo que as especiarias, são muito utilizadas na gastronomia, a questão motivadora desse estudo é: Qual a importância das especiarias na gastronomia, considerando desde os tempos primórdios aos dias atuais? Nesse sentido, o objetivo desse estudo é apresentar a história e a importância das especiarias na gastronomia

Genetics and molecular study in group of patients with malformations of cerebral cortex

OBJECTIVES: Malformations of cerebral cortex (MCC) are an important cause of epilepsy. Our main goals were: to search for mutations in genes responsible for MCC (FLN1, LIS1, DCX and EMX2), to map the locus for familial perisylvian polymicrogyria and to investigate the molecular mechanisms of the mutations identified. Methods: Mutation screening was performed by PCR, DHPLC and sequencing. HUMARA and Real Time PCR were performed to study the molecular mechanisms of mutations. Linkage analysis was carried out by PCR, Fragment profiler® and MLINK® software. RESULTS: Deleterious mutations were identified in 3/108 patients. We found a G987C splicing mutation in the FLN1 in two related patients with periventricular nodular heterotopia. Skewed X-chromosome inactivation was detected as the possible mechanism responsible for clinical differences observed in the two patients. An A1385C transversion (H277P) in LIS1 was identified in one patient with lisencephaly. Only neutral variants were identified in DCX and EMX2. Linkage analysis has detected a locus in Xq27.2-Xq27.3 for familial polymicrogyria. CONCLUSION: We believe that the low frequency of mutations identified may be due to mosaicism, mutations in non-coding regions, deletions and patients with atypical neuroimaging findings. Deleterious mutations in EMX2 were not found in patients with schizencephaly and polymicrogyria. We found a locus for familial perisylvian polymicrogyria in Xq27.2-Xq27.3.OBJETIVOS: As malformações do córtex cerebral (MCC) são uma causa importante de epilepsia. Nossas metas foram: triagem de mutações em genes associados às MCC (FLN1, LIS1, DCX e EMX2), investigar funcionalmente as mutações e mapear o locus para polimicrogiria perisylviana familiar. MÉTODOS: A triagem de mutações foi realizada por PCR, DHPLC e sequênciamento. Estudo funcional foi realizado por RT-PCR, PCR em tempo real e HUMARA. O estudo de ligação foi realizado por PCR e análise com programas Fragment Profiler® e MLINK®. RESULTADOS: Mutações deletérias foram identificadas em 3/108 pacientes. Uma mutação de splicing (G987C) em FLN1 foi identificada em duas pacientes aparentadas com heterotopia nodular periventricular. Mudança no padrão de inativação do cromossomo X é responsável pelas diferenças clínicas entre as pacientes. Uma substituição A1385C (H277P) foi identificada em LIS1 em um indivíduo com lissencefalia. Alterações neutras foram identificadas em DCX e EMX2. A análise de ligação identificou um locus em Xq27.2-Xq27.3 para polimicrogiria familiar. CONCLUSÃO: Mosaicismo, mutações em regiões não codificantes, deleções, rearranjos e casos atípicos podem estar contribuindo para a baixa freqüência de mutações identificadas. Esquizencefalia e polimicrogiria parecem não ter base genética relacionada com o gene EMX2. Um novo locus candidato em Xq27.2-Xq27.3 foi identificado para polimicrogiria perisylviana familiar.101105Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Modeling the Dynamic Transmission of Dengue Fever: Investigating Disease Persistence

Dengue is the most rapidly spreading mosquito-borne viral disease in the world and approximately 2.5 billion people live in dengue endemic countries. In Brazil it is mainly transmitted by Aedes aegypti mosquitoes. The wide clinical spectrum ranges from asymptomatic infections or mild illness, to the more severe forms of infection such as dengue hemorrhagic fever or dengue shock syndrome. The spread and dramatic increase in the occurrence of dengue cases in tropical and subtropical countries has been blamed on uncontrolled urbanization, population growth and international traveling. Vaccines are under development and the only current disease control strategy is trying to keep the vector quantity at the lowest possible levels. Mathematical models have been developed to help understand the disease's epidemiology. These models aim not only to predict epidemics but also to expand the capacity of phenomena explanation. We developed a spatially explicit model to simulate the dengue transmission in a densely populated area. The model involves the dynamic interactions between humans and mosquitoes and takes into account human mobility as an important factor of disease spread. We investigated the importance of human population size, human renewal rate, household infestation and ratio of vectors per person in the maintenance of sustained viral circulation

Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data