POLθ prevents MRE11-NBS1-CtIP-dependent fork breakage in the absence of BRCA2/RAD51 by filling lagging-strand gaps.

POLθ promotes repair of DNA double-strand breaks (DSBs) resulting from collapsed forks in homologous recombination (HR) defective tumors. Inactivation of POLθ results in synthetic lethality with the loss of HR genes BRCA1/2, which induces under-replicated DNA accumulation. However, it is unclear whether POLθ-dependent DNA replication prevents HR-deficiency-associated lethality. Here, we isolated Xenopus laevis POLθ and showed that it processes stalled Okazaki fragments, directly visualized by electron microscopy, thereby suppressing ssDNA gaps accumulating on lagging strands in the absence of RAD51 and preventing fork reversal. Inhibition of POLθ DNA polymerase activity leaves fork gaps unprotected, enabling their cleavage by the MRE11-NBS1-CtIP endonuclease, which produces broken forks with asymmetric single-ended DSBs, hampering BRCA2-defective cell survival. These results reveal a POLθ-dependent genome protection function preventing stalled forks rupture and highlight possible resistance mechanisms to POLθ inhibitors.

POLθ processes ssDNA gaps and promotes replication fork progression in BRCA1-deficient cells.

Polymerase theta (POLθ) is an error-prone DNA polymerase whose loss is synthetically lethal in cancer cells bearing breast cancer susceptibility proteins 1 and 2 (BRCA1/2) mutations. To investigate the basis of this genetic interaction, we utilized a small-molecule inhibitor targeting the POLθ polymerase domain. We found that POLθ processes single-stranded DNA (ssDNA) gaps that emerge in the absence of BRCA1, thus promoting unperturbed replication fork progression and survival of BRCA1 mutant cells. A genome-scale CRISPR-Cas9 knockout screen uncovered suppressors of the functional interaction between POLθ and BRCA1, including NBN, a component of the MRN complex, and cell-cycle regulators such as CDK6. While the MRN complex nucleolytically processes ssDNA gaps, CDK6 promotes cell-cycle progression, thereby exacerbating replication stress, a feature of BRCA1-deficient cells that lack POLθ activity. Thus, ssDNA gap formation, modulated by cell-cycle regulators and MRN complex activity, underlies the synthetic lethality between POLθ and BRCA1, an important insight for clinical trials with POLθ inhibitors.

Uncovering the effects of kaolin on balancing berry phytohormones and quality attributes of Vitis vinifera grown in warm-temperate climate regions.

BACKGROUND: The application of kaolin particle film is considered a short-term strategy against several environmental stresses in areas with a Mediterranean-like climate. However, it is known that temperature fluctuations and water availability over the season can jeopardize kaolin efficiency in many Mediterranean crops. Hence, this study aims to evaluate the effects of kaolin foliar application on berry phytohormones, antioxidant defence, and oenological parameters at veraison and harvest stages of Touriga-Franca (TF) and Touriga-Nacional (TN) grapevines in two growing seasons (2017 and 2018). The 2017 growing season was considered the driest (-147.1 dryness index) and the warmest (2705 °C growing degree days) of the study. RESULTS: In 2017, TF kaolin-treated berries showed lower salicylic acid (-26.6% compared with unsprayed vines) and abscisic acid (ABA) (-10.5%) accumulation at veraison, whereas salicylic acid increased up to 28.8% at harvest. In a less hot season, TN and TF kaolin-treated grapevines showed a twofold in ABA content and a threefold increase in the indole-3-acetic acid content at veraison and lower ABA levels (83.8%) compared with unsprayed vines at harvest. Treated berries showed a decreased sugar content, without compromising malic and tartaric acid levels, and reactive oxygen species accumulation throughout berry ripening. CONCLUSION: The results suggest kaolin exerts a delaying effect in triggering ripening-related processes under severe summer stress conditions. Treated berries responded with improved antioxidant defence and phytohormone balance, showing significant interactions between kaolin treatment, variety, and developmental stage in both assessed years. © 2021 Society of Chemical Industry.

Decomposing the mutational landscape of cancer genomes with RepairSig.

Mutational signatures are the outcomes of mutagenic processes that occur prior to, and during, tumorigenesis as a result of DNA damage, DNA repair, and DNA replication. In this issue of Cell Systems, Wojtowicz et al. introduce a new computational model aimed at deconstructing the mutational processes that shape cancer genomes.

Particle film technology modulates xanthophyll cycle and photochemical dynamics of grapevines grown in the Douro Valley.

Field-grown grapevines are often exposed to multiple environmental stresses, which challenges wine-growers to develop sustainable measures to sustain vine growth, yield, and quality. Under field conditions this task is demanding, due to differences in the magnitudes of stresses and associated plant responses. In this study we explored the hypothesis that kaolin-particle film application improves grapevine photoprotection through the regulation of xanthophyll cycle genes, limiting the thermal dissipation of excess energy under harsh environmental conditions. Hence, we selected two grapevine varieties, Touriga-Nacional (TN) and Touriga-Franca (TF), grown in the Douro Demarcated Region, and evaluated changes in light dissipation mechanisms, xanthophyll cycle components, and the expression of xanthophyll cycle genes during the 2017 summer season. The results showed that, from veraison to ripening, kaolin triggered the up-regulation of violaxanthin de-epoxidase (VvVDE1) and zeaxanthin epoxidase (VvZEP1) genes, indicating optimised regulation of the xanthophyll cycle. Kaolin treatment also decreased chlorophyll (Chla, Chlb, Chl(a+b)) and carotenoid (Car) accumulation under increasing summer stress conditions in both varieties and lowered the non-photochemical quenching (NPQ) of grapevines on ripening, suggesting a long-term response to summer stress. In addition, kaolin-treated grapevines showed increased Chla/Chlb and lower Chl(a+b)/Car ratios, displaying some features of high light adapted leaves. Overall, this study suggests that kaolin application enabled grapevines to benefit from fluctuating periods of summer stress by managing chlorophyll and carotenoid content and limiting down-regulation of both photochemistry and photoinhibition processes. Under Mediterranean field conditions, kaolin application can be considered an efficient method of minimising summer stress impact on grapevines.

Notch inhibition overcomes resistance to tyrosine kinase inhibitors in EGFR-driven lung adenocarcinoma.

EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFRT790M and EGFRC797S. It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (p-STAT3) in EGFRT790M and EGFRC797S tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a p-STAT3-dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor-resistant tumors, with EGFRT790M and EGFRC797S mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib or osimertinib, respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib-treated patients after disease progression.

Aggregation effects on the magnetic properties of iron oxide colloids.

Magnetic nanoparticles (MNPs), and in particular iron oxide nanoparticles (mainly magnetite and maghemite), are being widely used in the form of aqueous colloids for biomedical applications. In such colloids, nanoparticles tend to form assemblies, either aggregates, if the union is permanent, or agglomerates, if it is reversible. These clustering processes have a strong impact on the MNPs' properties that are often not well understood. In this review, the causes and consequences of MNPs aggregation/agglomeration are reviewed and discussed. Special attention has been paid to the impact of the MNPs aggregation/agglomeration on their magnetic properties and heating properties, when exposed to an alternating magnetic field in the frame of magnetic hyperthermia. In addition, a model system with MNPs of two different sizes coated with three different molecules oleic acid, meso-2, 3-dimercaptosuccinic acid and poly(maleic anhydride-alt-1-octadecene) has been characterized and the results used to support the ideas reviewed.

Kaolin particle film application stimulates photoassimilate synthesis and modifies the primary metabolome of grape leaves.

Water scarcity is associated with extreme temperatures and high irradiance, and significantly and increasingly affects grapevine yield and quality. In this context, the foliar application of kaolin, a chemically inert mineral that greatly reflects ultraviolet and infrared radiations, as well as, in part, photosynthetically active radiation, has recently been shown to decrease photoinhibition in mature leaves. Here, the influence of this particle film on grapevine leaf metabolome and carbohydrate metabolism was evaluated. Molecular mechanisms underlying photoassimilate synthesis, metabolism and transport capacity were assessed by targeted transcriptional analyses and enzymatic activity assays. Kaolin application increased sucrose concentration in leaves and sucrose transport/phloem loading capacity, as suggested by the stimulation of the transcription of sucrose transporters VvSUC12 and VvSUC27 in these source organs. While the biosynthesis of sucrose increased, as evidenced by higher sucrose content and sucrose phosphate synthase (SPS) activity in leaves, the concentration of transitory starch before the dark period remained unaltered, despite a higher total amylolytic activity in the leaves of kaolin-treated plants. Metabolomic analysis by GC-TOF-MS showed that the application of kaolin enhanced the amounts of simple sugars, including fructose, maltose, xylulose, xylose, sophorose, ribose and erythrose; sugars-phosphate, like mannose-6-Pi, hexose-6-Pi, glucose-6-Pi, glucose-1-Pi, glycerol-α-Pi and fructose-6-Pi; polyols, like xylitol, maltitol, lactitol, glycerol, galactinol and erythritol; organic acids and amino acids.

Independência/autonomia da pessoa em processos de transição de saúde / Independence/autonomy of the person in health transition processes

A dependência define-se pelo comprometimento da capacidade individual para o autocuidado e a sua avaliação determina a implementação de um plano terapêutico adequado e exequível, com base em metodologias de ensino, estratégias adaptativas e treino de capacidades/habilidades remanescentes. Apesar de existirem inúmeras escalas validadas nesta área, a caracterização da capacidade para o autocuidado é notoriamente exígua, o que se reflete numa maior dificuldade no exercício profissional dos enfermeiros. A problemática em estudo insere-se na linha de investigação da UICISA-E Bemestar, saúde e doença. A escolha da escala Care Dependency Scale for Rehabilitation (CDS-R) prende-se com o facto de esta escala permitir avaliar, além de aspetos físicos, os aspetos psicossociais, nomeadamente a adaptação da pessoa à sua situação de doença, uma vez que esta condiciona o seu processo de reabilitação. O objetivo geral do estudo foi validar culturalmente para a língua portuguesa a CDS-R, cuja autora é Juliane Eichhorn-Kissel, que validou esta escala em 2011, na população de um centro de reabilitação austríaco. A tradução e adaptação cultural para a língua portuguesa da CDS-R foram realizadas segundo as guidelines de Beaton, Bombardier, Guillemin & Ferraz (2000) em 2015, pela autora desta dissertação no âmbito do seu trabalho de investigação do Curso de Pós Licenciatura de Especialização em Enfermagem de Reabilitação. A validação para a língua portuguesa foi realizada segundo as guidelines de Vilelas (2009), com uma amostra não probabilística acidental constituída por 80 utentes internados na vertente de média duração e reabilitação, da Unidade de Cuidados Continuados Integrados (UCCI) de Leiria. A CDS-R é um instrumento válido e fiável, com o valor de alfa de Cronbach total obtido de 0,971 e a percentagem de variação explicada de 71,7%, que permite aos Enfermeiros Especialistas em Reabilitação avaliar o grau de independência/autonomia da pessoa em processos de transição de saúde e, assim, contribuir para uma melhor qualidade dos cuidados em Enfermagem de Reabilitação.

Kaolin Foliar Application Has a Stimulatory Effect on Phenylpropanoid and Flavonoid Pathways in Grape Berries.

Drought, elevated air temperature, and high evaporative demand are increasingly frequent during summer in grape growing areas like the Mediterranean basin, limiting grapevine productivity and berry quality. The foliar exogenous application of kaolin, a radiation-reflecting inert mineral, has proven effective in mitigating the negative impacts of these abiotic stresses in grapevine and other fruit crops, however, little is known about its influence on the composition of the grape berry and on key molecular mechanisms and metabolic pathways notably important for grape berry quality parameters. Here, we performed a thorough molecular and biochemical analysis to assess how foliar application of kaolin influences major secondary metabolism pathways associated with berry quality-traits, leading to biosynthesis of phenolics and anthocyanins, with a focus on the phenylpropanoid, flavonoid (both flavonol- and anthocyanin-biosynthetic) and stilbenoid pathways. In grape berries from different ripening stages, targeted transcriptional analysis by qPCR revealed that several genes involved in these pathways-VvPAL1, VvC4H1, VvSTSs, VvCHS1, VvFLS1, VvDFR, and VvUFGT-were more expressed in response to the foliar kaolin treatment, particularly in the latter maturation phases. In agreement, enzymatic activities of phenylalanine ammonia lyase (PAL), flavonol synthase (FLS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were about two-fold higher in mature or fully mature berries from kaolin-treated plants, suggesting regulation also at a transcriptional level. The expression of the glutathione S-transferase VvGST4, and of the tonoplast anthocyanin transporters VvMATE1 and VvABCC1 were also all significantly increased at véraison and in mature berries, thus, when anthocyanins start to accumulate in the vacuole, in agreement with previously observed higher total concentrations of phenolics and anthocyanins in berries from kaolin-treated plants, especially at full maturity stage. Metabolomic analysis by reverse phase LC-QTOF-MS confirmed several kaolin-induced modifications including a significant increase in the quantities of several secondary metabolites including flavonoids and anthocyanins in the latter ripening stages, probably resulting from the general stimulation of the phenylpropanoid and flavonoid pathways.

Aging With Rising Risk: Have Older Households Experienced Faster-Growing Risk Exposure Than Younger Ones Since the 1990s?

Financial markets have been characterized by boom and bust cycles since the 1980s, while the responsibility for managing retirement wealth has increasingly shifted onto individual households at the same time. Policy makers and experts have expressed concern over rising risk exposure among older householders, who appear to be increasingly exposed to the growing financial risks just as they near retirement. We consider household data from the Federal Reserve's Survey of Consumer Finances from 1989 to 2010 to analyze the correlation between age and risk exposure. We test whether older householders' risk exposure has indeed grown over time, whether it has increased more than that of younger householders, whether changes in the demographic composition of older householders have contributed to older households' rising risk exposure, and the degree to which increases in risk exposure can be traced to a growing concentration of household assets held in stocks and housing and to rising householder indebtedness. Our results indicate that risk exposure has grown more for older householders than for younger ones, that demographic changes among older householders have contributed to additional increases in older householders' risk exposure, and that the growth of older householders' risk exposure is driven more by rising risky asset concentration and less by greater indebtedness.

Variable delay-to-signal: a fast paradigm for assessment of aspects of impulsivity in rats.

Testing impulsive behavior in rodents is challenging and labor-intensive. We developed a new behavioral paradigm-the Variable Delay-to-Signal (VDS) test-that provides rapid and simultaneous assessment of response and decision impulsivity in rodents. Presentation of a light at variable delays signals the permission for action (nose poke) contingent with a reward. 2 blocks of 25 trials at 3 s delay flank a block of 70 trials in which light is presented with randomly selected 6 or 12 s delays. Exposure to such large delays boosts the rate of premature responses when the delay drops to 3 s in the final block, an effect that is blunted by an acute methamphetamine challenge and that correlates with the delay-discounting (DD) paradigm (choice impulsivity). Finally, as expected, treatment with the NMDA antagonist MK-801 caused a generalized response increase in all VDS blocks. The pharmacological validation, particularly with methamphetamine which has a well established dual effect on response and decision impulsivity, and the correlations between the impulsive behavior in the DD and VDS paradigms, suggests that the later is able to provide, in a single session, a multi-dimensional assessment of impulsive behavior.