Multiploidy occurrence in tomato calli from anther culture

Anther culture has long been used for the production of fully homozygous lines in order to produce, mainly, doubled haploid plants, which are of great interest in plant breeding. For tomato, a recalcitrant species for androgenesis production protocols have not been standardized. It is known that the genotype, anther size, the developmental stage of the microspore, and the medium composition are some factors that can influence the calli production. The present study aimed to adapt flow cytometry methodology to verify the microsporogenesis phases of anthers in order to assess the anther responsiveness of different tomato genotypes in an androgenesis-induction culture medium and to analyze the DNA ploidy level of calli produced by flow cytometry. Anthers from flower buds of length 1.0 to 5.9 mm, corresponding to the size range as analyzed by flow cytometry and cytogenetic methods, were inoculated into Murashige and Skoog (MS) basal medium containing the growth regulators 6-(y,ydimethylallylamino) purine and indole-3-acetic acid. The obtained calli were subsequently analyzed by flow cytometry to determine the DNA ploidy level. Surprisingly, despite no pretreatment with microtubule-depolymerizing agents, five classes of multiploid calli were observed, as follows: class I (2C-4C-8C-16C), class II (2C-4C-8C-16C-32C), class III (4C-8C), class IV (4C-8C-16C) and class V (8C-16C- 32C). Multiploid calli were identified in short-term (two month) culturing, suggesting that the variable culture duration did not directly influence the occurrence of endoreduplication. In this work, this type of somaclonal variation has been reported for the first time in tomato anther culture, and their possible origin has been discussed.Key words: Callogenesis, flow cytometry, polyploidy, Solanum lycopersicum, somaclonal variation

Understanding drought dynamics during dry season in Eastern Northeast Brazil

Eastern Northeast Brazil (ENEB) generally experiences a high variability in precipitation in the dry season, with amplitudes that can overcome 500mm. The understanding of this variability can help in mitigating the socio-economic issues related to the planning and management of water resources this region, which is highly vulnerable to drought. This work aims to assess spatio-temporal variability of precipitation during the dry season and investigate the relationships between climate phenomena and drought events in the ENEB, using univariate (Spearman correlation) and multivariate statistical techniques, such as Principal Component Analysis, Cluster Analysis, and Maximum Covariance Analysis. The results indicate that the variability of precipitation in the dry season can be explained mainly (62%) by local physical conditions and climate conditions have a secondary contribution. Further analysis of the larger anomalous events suggests that the state of Atlantic and Pacific oceans can govern the occurrence of those events, and the conditions of Atlantic Ocean can be considered a potential modulator of anomalous phenomena of precipitation in ENEB

Long-term tobacco exposure and immunosenescence: paradoxical effects on T-cells telomere length and telomerase activity

Immunosenescence are alterations on immune system that occurs throughout an individual life. The main characteristic of this process is replicative senescence, evaluated by telomere shortening. Several factors implicate on telomere shortening, such as smoking. In this study, we evaluated the influence of smoking and Chronic Obstructive Pulmonary Disease (COPD) on cytokines, telomere length and telomerase activity. Blood samples were collected from subjects aged over 60 years old: Healthy (never smokers), Smokers (smoking for over 30 years) and COPDs (ex-smokers for ≥15 years). A young group was included as control. PBMCs were cultured for assessment of telomerase activity using RT-PCR, and cytokines secretion flow cytometry. CD4+ and CD8+ purified lymphocytes were used to assess telomere length using FlowFISH. We observed that COPD patients have accelerated telomere shortening. Paradoxically, smokers without lung damage showed preserved telomere length, suggesting that tobacco smoking may affect regulatory mechanisms, such as telomerase. Telomerase activity showed diminished activity in COPDs, while Smokers showed increased activity compared to COPDs and Healthy groups. Extracellular environment reflected this unbalance, indicated by an anti-inflammatory profile in Smokers, while COPDs showed an inflammatory prone profile. Further studies focusing on telomeric maintenance may unveil mechanisms that are associated with cancer under long-term smoking

Waiting for Godot? Welfare Attitudes in Portugal before and after the Financial Crisis

Do attitudes towards the welfare state change in response to economic crises? Addressing this question is sometimes difficult because of the lack of longitudinal data. This article deals with this empirical challenge using survey data from the 2008 European Social Survey and from our own follow-up survey of Spring 2013 to track welfare attitudes at the brink and at the peak of the socio-economic crisis in one of the hardest hit countries: Portugal. The literature on social policy preferences predicts an increased polarisation in opinions towards the welfare state between different groups within society – in particular between labour market insiders and outsiders. However, the prediction has scarcely been tested empirically. A notoriously dualised country, Portugal provides a critical setting in which to test this hypothesis. The results show attitudinal change, and this varies according to labour market vulnerability. However, we observe no polarisation and advance alternative explanations for why this is so. This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by SAGE

The terrestrial evolution of metabolism and life – by the numbers

<p>Abstract</p> <p>Background</p> <p>Allometric scaling relating body mass to metabolic rate by an exponent of the former (<it>Kleiber's Law</it>), commonly known as quarter-power scaling (QPS), is controversial for claims made on its behalf, especially that of its universality for all life. As originally formulated, Kleiber was based upon the study of heat; metabolic rate is quantified in watts (or calories per unit time). Techniques and technology for metabolic energy measurement have been refined but the math has not. QPS is susceptible to increasing deviations from theoretical predictions to data, suggesting that there is no single, universal exponent relevant to all of life. QPS's major proponents continue to fail to make good on hints of the power of the equation for understanding aging.</p> <p>Essentialist-deductivist view</p> <p>If the equation includes a term for efficiency in the exponent, thereby ruling out thermogenesis as part of metabolism, its heuristic power is greatly amplified, and testable deductive inferences are generated. If metabolic rate is measured in watts and metabolic efficiency is a redox-coupling ratio, then the equation is essentially about the energy storage capacity of organic molecules. The equation is entirely about the essentials of all life: water, salt, organic molecules, and energy. The water and salt provide an electrochemical salt bridge for the transmission of energy into and through the organic components. The equation, when graphed, treats the organic structure as battery-like, and relates its recharge rate and electrical properties to its longevity.</p> <p>Conclusion</p> <p>The equation models the longevity-extending effects of caloric restriction, and shows where those effects wane. It models the immortality of some types of cells, and supports the argument for the origin of life being at submarine volcanic vents and black smokers. It clarifies how early life had to change to survive drifting to the surface, and what drove mutations in its ascent. It does not deal with cause and effect; it deals with variables in the essentials of all life, and treats life as an epiphenomenon of those variables. The equation describes how battery discharge into the body can increase muscle mass, promote fitness, and extend life span, among other issues.</p

Contribuições da Pesquisa para o Beneficiamento da Castanha-de-Cutia (Couepia edulis Prance) e Aproveitamento de seus Resíduos.

bitstream/CNPDIA/10481/1/DOC15_2005.pd

The Mini‐Organo: A rapid high‐throughput 3D coculture organotypic assay for oncology screening and drug development

Background: The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine-tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three-dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease. Aim: The development of the 3D coculture collagen contraction and invasion assay, the "organotypic assay," has been widely adopted as a powerful approach to bridge the gap between standard two-dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer-associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low-throughput, time-consuming (up to several weeks), and work-intensive with often limited scalability. Our aim was to develop a fast, high-throughput, scalable 3D organotypic assay for use in oncology screening and drug development. Methods and results Here, we describe a modified 96-well organotypic assay, the "Mini-Organo," which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions. Conclusions: The Mini-Organo high-throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time-efficient manner

Free Rhodium (II) citrate and rhodium (II) citrate magnetic carriers as potential strategies for breast cancer therapy

<p>Abstract</p> <p>Background</p> <p>Rhodium (II) citrate (Rh₂(H₂cit)₄) has significant antitumor, cytotoxic, and cytostatic activity on Ehrlich ascite tumor. Although toxic to normal cells, its lower toxicity when compared to carboxylate analogues of rhodium (II) indicates Rh₂(H₂cit)₄as a promising agent for chemotherapy. Nevertheless, few studies have been performed to explore this potential. Superparamagnetic particles of iron oxide (SPIOs) represent an attractive platform as carriers in drug delivery systems (DDS) because they can present greater specificity to tumor cells than normal cells. Thus, the association between Rh₂(H₂cit)₄and SPIOs can represent a strategy to enhance the former's therapeutic action. In this work, we report the cytotoxicity of free rhodium (II) citrate (Rh₂(H₂cit)₄) and rhodium (II) citrate-loaded maghemite nanoparticles or magnetoliposomes, used as drug delivery systems, on both normal and carcinoma breast cell cultures.</p> <p>Results</p> <p>Treatment with free Rh₂(H₂cit)₄induced cytotoxicity that was dependent on dose, time, and cell line. The IC₅₀values showed that this effect was more intense on breast normal cells (MCF-10A) than on breast carcinoma cells (MCF-7 and 4T1). However, the treatment with 50 μM Rh₂(H₂cit)₄-loaded maghemite nanoparticles (Magh-Rh₂(H₂cit)₄) and Rh₂(H₂cit)₄-loaded magnetoliposomes (Lip-Magh-Rh₂(H₂cit)₄) induced a higher cytotoxicity on MCF-7 and 4T1 than on MCF-10A (p < 0.05). These treatments enhanced cytotoxicity up to 4.6 times. These cytotoxic effects, induced by free Rh₂(H₂cit)₄, were evidenced by morphological alterations such as nuclear fragmentation, membrane blebbing and phosphatidylserine exposure, reduction of actin filaments, mitochondrial condensation and an increase in number of vacuoles, suggesting that Rh₂(H₂cit)₄induces cell death by apoptosis.</p> <p>Conclusions</p> <p>The treatment with rhodium (II) citrate-loaded maghemite nanoparticles and magnetoliposomes induced more specific cytotoxicity on breast carcinoma cells than on breast normal cells, which is the opposite of the results observed with free Rh₂(H₂cit)₄treatment. Thus, magnetic nanoparticles represent an attractive platform as carriers in Rh₂(H₂cit)₄delivery systems, since they can act preferentially in tumor cells. Therefore, these nanopaticulate systems may be explored as a potential tool for chemotherapy drug development.</p

DETERMINATION OF VIRAL TROPISM BY GENOTYPING AND PHENOTYPING ASSAYS IN BRAZILIAN HIV-1-INFECTED PATIENTS

The clinical application of CCR5 antagonists involves first determining the coreceptor usage by the infecting viral strain. Bioinformatics programs that predict coreceptor usage could provide an alternative method to screen candidates for treatment with CCR5 antagonists, particularly in countries with limited financial resources. Thus, the present study aims to identify the best approach using bioinformatics tools for determining HIV-1 coreceptor usage in clinical practice. Proviral DNA sequences and Trofile results from 99 HIV-1-infected subjects under clinical monitoring were analyzed in this study. Based on the Trofile results, the viral variants present were 81.1% R5, 21.4% R5X4 and 1.8% X4. Determination of tropism using a Geno2pheno[coreceptor] analysis with a false positive rate of 10% gave the most suitable performance in this sampling: the R5 and X4 strains were found at frequencies of 78.5% and 28.4%, respectively, and there was 78.6% concordance between the phenotypic and genotypic results. Further studies are needed to clarify how genetic diversity amongst virus strains affects bioinformatics-driven approaches for determining tropism. Although this strategy could be useful for screening patients in developing countries, some limitations remain that restrict the wider application of coreceptor usage tests in clinical practice