Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production

Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors

Nitrogen fertilization associated with cold storage and its impacts on the maintenance of peach quality.

Nitrogen, which is considered the most important nutrient for peach trees, may interfere in both quantitative production characteristics and quality of fruits. This study aimed at evaluating the effect of the combination of doses of N fertilization and different periods of cold storage on physico-chemical and phytochemical characteristics of peaches in post-harvest. The experiment had a randomized complete block design in a 4x3 factorial scheme, i. e., four doses of fertilization (0, 60, 120 and 180 Kg N ha-1) and three periods of storage (on the harvest day, on both the 15th and the 30th storage days at 1±1ºC, each followed by a day of simulated commercialization at 20±1ºC). The following aspects were evaluated in fruits yielded by peach trees of the genotype Cascata 1067: fruit color, soluble solid content, titratable acidity, pulp firmness, mass loss, total phenolic compounds and antioxidant activity. Different periods of cold storage and doses of N fertilization were found to affect epidermis luminosity, pulp firmness and titratable acidity of fruits. Peaches may be stored at low temperatures for 15+1 days. After that, loss of fruit firmness increases. N fertilization affects neither the soluble solid content nor the epidermis color of peaches, but both parameters are influenced by storage. Values of phenolic compounds and antioxidant activity decrease when there is N increment in the soil and when longer storage is carried out. Results suggest that peach composition may be affected by cultural practices, such as N fertilization, in harvest and after storage

Perfil hormonal em mudas Bordô/VR043-43 pós-forçagem a partir de estacas com diferentes épocas de coleta e tempos de armazenagem.

Híbridos Vitis rotundifolia (ex.: VR043-43) têm sido empregados como porta-enxertos em vinhedos com problemas de estresses bióticos, principalmente no estado de Santa Catarina. Entretanto, a produção de mudas é dificultada com estacas lignificadas, pela restrição em calogênese de enxertia e enraizamento desses genótipos.CLAUDIO DE ANDRADE BARROS, CNPUV, 29742

Qualidade de mudas de videira em pós-forçagem submetidas a aplicação de etefom, diferentes épocas de coleta e tempo de câmara fria.

A procura por mudas de videira com qualidade superior tem se intensificado ultimamente, exigindo aprimoramento no processo de produção pelos viveiristas. A cultivar Bordô (B) enxertada nos porta-enxertos P1103 (P) e VR043-43 (VR) são as combinações tradicionais utilizadas no Rio Grande do Sul e em Santa Catarina, respectivamente.Daniel Antunes Souza, 333734, CNPU

Dark chocolate modulates platelet function with a mechanism mediated by flavan-3-ol metabolites

Cocoa is a rich source bioactive compounds, i.e., flavan-3-ols, and its consumption has been associated with several beneficial effects, such as the positive modulation of the hemostasis targeted by the platelet function. However, these phenolic compounds have a very low bioavailability and extensively undergo phase I and II metabolism, with the appearing into the bloodstream of (epi) catechin conjugates and phenyl-g-valerolactones and their conjugates, at different times. The aims of this study were to explore the effect of dark chocolate on platelet function and to investigate the relationship between this interplay and flavan-3-ol derived metabolites. Eighteen healthy male volunteers ingested 50 g of 90% cocoa chocolate within 5 minutes. Blood samples were collected immediately before chocolate ingestion (T0) and 4 hours afterwards (T1). Platelet function analyzer (PFA)-100 closure time was assessed using collagen/adenosine-50-diphosphate (COL/ADP) and collagen/epinephrine (COL/EPI) cartridges. Plasma flavan-3-ol metabolites were identified and quantified by means of liquid chromatography coupled to a triple quadrupole mass spectrometer (UHPLC-ESI-MS/MS). Results evidenced a significant increase of COL/ADP-induced PFA-100 closure time, but not COL/EPI, 4 hours after ingestion of dark chocolate. Total plasma structurally-related (epi)catechin metabolite (SREM) concentration significantly increased at T1, together with 4 out of the 6 detected metabolites. Total phenyl-g-valerolactone concentrations remained unchanged. Spearman correlations evidenced a strong correlation between COL/ADP closure time and SREMs, mainly led by (epi)catechin-sulfate isomers. These data confirm that the potential beneficial effect of dark chocolate on primary hemostasis may be mediated by flavan-3-ol circulating metabolites

Allele-specific editing ameliorates dominant retinitis pigmentosa in a transgenic mouse model

Retinitis pigmentosa (RP) is a group of progressive retinal degenerations of mostly monogenic inheritance, which cause blindness in about 1:3,500 individuals worldwide. Heterozygous variants in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP). Among these, missense variants at C-terminal proline 347, such as p.Pro347Ser, cause severe adRP recurrently in European affected individuals. Here, for the first time, we use CRISPR/Cas9 to selectively target the p.Pro347Ser variant while preserving the wild-type RHO allele in vitro and in a mouse model of adRP. Detailed in vitro, genomic, and biochemical characterization of the rhodopsin C-terminal editing demonstrates a safe downregulation of p.Pro347Ser expression leading to partial recovery of photoreceptor function in a transgenic mouse model treated with adeno-associated viral vectors. This study supports the safety and efficacy of CRISPR/Cas9-mediated allele-specific editing and paves the way for a permanent and precise correction of heterozygous variants in dominantly inherited retinal diseases

Human Bocavirus Infection in Children with Gastroenteritis, Brazil

Human bocavirus (HBoV) was detected in 14 (2%) of 705 fecal specimens from Brazilian children with gastroenteritis. Coinfection with rotavirus, adenovirus, or norovirus was found in 3 (21.4%) HBoV-positive specimens. None of the HBoV-positive patients had respiratory symptoms

Digital dermoscopic changes during follow-up of de-novo and nevus-associated melanoma: a cohort study

Background: Nevus-associated melanoma (NAM) has been regarded as a distinct biological entity from de-novo melanoma (DNM); however, static dermoscopy often fails in differentiating these entities. Digital dermoscopic monitoring allows to identify dynamic changes occurring during follow-up; this may improve diagnostic accuracy and potentially our knowledge on NAM biology. We aimed to define main independent factors associated with NAM diagnosis and those influencing follow-up time in a population of melanomas excised at follow-up. Methods: A cohort of melanomas excised at follow-up was retrospectively and consecutively selected. NAMs and DNMs were compared according to baseline features and main dermoscopic changes occurring during follow-up. Univariate and multivariable logistic and Cox's regression analysis were performed to respectively define factors associated with NAM diagnosis and those influencing the risk for excision. Results: Eighty-six melanomas were enrolled, of which 21 (24.4%) were nevus-associated. During follow-up NAMs mainly underwent atypical network modifications (47.6%), followed by inverse network (28.6%) and dermoscopic island (23.8%) worsening or appearance. DNMs were also mainly characterized by atypical network modifications (47.7%), however, a significant proportion of cases underwent irregular pigmentation/dots/globules or regression changes (29.2%), which were rarely seen among NAMs. Furthermore, both multivariable logistic and Cox's regression analysis demonstrated a significant association between NAM and a longer follow-up. Conclusions: We demonstrated that among melanomas excised at follow-up, different patterns of dermoscopic changes may be found between NAMs and DNMs. This finding, together with the association of NAM with a longer follow-up time, supports the hypothesis of different biological behavior of these two entities

Effect of salicylic acid in post-harvest quality of "BRS Kampai" peaches submitted to different cold storage periods.

Fast ripening of fruits is one of the main hindrances to peach preservation. Even though the use of cold storage has extended commercialization periods, fruit quality is affected during storage. This study aimed at evaluating the influence of application of salicylic acid (SA) during cold storage, followed by commercialization simulation, regarding physical and chemical characteristics of ?BRS Kampai? peaches, which were harvested in the 2017 crop. The experiment was conducted in a completely randomized factorial design (4×3: SA concentrations × storage days), with four replications of ten fruits each. SA was applied at the following concentrations: 0, 2, 4 and 6 mM. Afterwards, fruits were submitted to cold storage at 1±0.5ºC and 80-85% relative humidity. Analyses were carried out on harvest day, on the 10th cold storage day, followed by a day at room temperature (20ºC) and on the 20th cold storage day, followed by a day at room temperature. Fruits were evaluated in terms of their mass loss, epidermis color, chromatic hue, pulp firmness, soluble solids (SS), pH and titratable acidity (TA). Both application of SA and cold storage contributed to decrease mass loss and TA, besides increasing the SS/TA relation. Fruits which were stored for 20 days and kept at room temperature for a day exhibited the highest values of SS and the lowest values of both brightness and pulp firmness. SA was efficient to maintain the quality of ?BRS Kampai? peaches submitted to cold storage for 10 and 20 days and an extra day of commercialization simulation

Intermittent temperatures in the conservation of "BRS Kampai" peaches.

This study aimed at evaluating the quality of peaches borne by the cultivar BRS Kampai when cooled at intermittent temperatures throughout storage. They were stored at 1°C and relative humidity between 80% and 90% in a cold storage chamber. The following treatments were applied: T1 - fruit stored at 1±0.5°C; T2 - fruit stored at 1±0.5°C and increase in temperature every five days to 25°C; and T3 - fruit stored at 1±0.5°C and increase in temperature every seven days to 25°C. Fruit were submitted to increase in temperature when they were withdrawn from the cold storage chamber and kept in a heated room. Evaluations were conducted when fruit were harvested, after 10 cold storage days and 2 days of commercialization simulation at 25ºC (10+2) and after 20 cold storage days and 2 days of commercialization simulation at 25ºC (20+2). Peaches were evaluated in terms of mass loss, pulp firmness, soluble solids, titratable acidity, pH and epidermis color. T1 exhibited higher mass loss, higher soluble solid content and higher pulp firmness after 20 cold storage days, by comparison with the other treatments. T2 led to lower mass loss after 20 cold storage days than the ones of the other treatments. However, T2 also showed higher loss of pulp firmness in the same period. The use of intermittent temperatures throughout storage of ?BRS Kampai? peaches may be an alternative to extend their post-harvest period