A Three-Component Gene Expression System and Its Application for Inducible Flavonoid Overproduction in Transgenic Arabidopsis thaliana

Inducible gene expression is a powerful tool to study and engineer genes whose overexpression could be detrimental for the host organisms. However, only limited systems have been adopted in plant biotechnology. We have developed an osmotically inducible system using three components of plant origin, RD29a (Responsive to Dehydration 29A) promoter, CBF3 (C-repeat Binding Factor 3) transcription factor and cpl1-2 (CTD phosphatase-like 1) mutation. The osmotic stress responsible RD29a promoter contains the CBF3 binding sites and thus RD29A-CBF3 feedforward cassette enhances induction of RD29a promoter under stress. The cpl1-2 mutation in a host repressor CPL1 promotes stress responsible RD29a promoter expression. The efficacy of this system was tested using PAP1 (Production of Anthocyanin Pigment 1) transgene, a model transcription factor that regulates the anthocyanin pathway in Arabidopsis. While transgenic plants with only one or two of three components did not reproducibly accumulate anthocyanin pigments above the control level, transgenic cpl1 plants containing homozygous RD29a-PAP1 and RD29a-CBF3 transgenes produced 30-fold higher level of total anthocyanins than control plants upon cold treatment. Growth retardation and phytochemical production of transgenic plants were minimum under normal conditions. The flavonoid profile in cold-induced transgenic plants was determined by LC/MS/MS, which resembled that of previously reported pap1-D plants but enriched for kaempferol derivatives. These results establish the functionality of the inducible three-component gene expression system in plant metabolic engineering. Furthermore, we show that PAP1 and environmental signals synergistically regulate the flavonoid pathway to produce a unique flavonoid blend that has not been produced by PAP1 overexpression or cold treatment alone

Uso de ácido ascórbico e cloreto de cálcio na qualidade de repolho minimamente processado

The objective of this trial was to evaluate the effect of ascorbic acid (AA) and calcium chloride (CaCl2) applied by immersion at temperatures of 20 and 40 degrees C on the physicochemical and sensory characteristics of minimally processed cabbage, stored under refrigeration. Cabbages were processed in an industrial food processing equipment to be cut in slices with thickness of 3 mm. Slices were immersed in sodium hypochlorite (50 ppm) during 5 minutes for sanitization. After, the following treatments were carried out T1= control (immersion in water during 5 minutes at 20 degrees C); T2= immersion in 1% AA solution, during 5 minutes at 20 degrees C; T3= immersion in 2% AA solution, during 5 minutes at 20 degrees C; T4= immersion in 1% CaCl2 solution during 5 minutes at 20 degrees C; T5= immersion in 2% CaCl2 solution during 5 minutes at 20 degrees C, T6= immersion in 1% CaCl2 solution during 5 minutes at 40 degrees C; and T7= immersion in 2% CaCl2 solution during 5 minutes at 40 degrees C; with four replications each one. After application of treatments, cabbage was centrifuged during one minute, wrapped with polyvinyl chloride, 20 mu m, in trays of expanded polystyrene and maintained in refrigerated environment, at 6 +/- 1 degrees C and 85-90% of relative humidity, during eight days. Little increasing was observed in pH and titratable acidity values and reduction in soluble solids during conservation period on all treatments. Treatment with AA did not differ from control for color and general appearance, while treatment with 2% CaCl2 at 20 degrees C maintained the best quality, with less intensity of browning, best general appearance and purchase intent and least strange odor at the end of evaluation period.O objetivo deste trabalho foi avaliar o efeito do ácido ascórbico (AA) e do cloreto de cálcio (CaCl2) aplicado em imersão nas temperaturas de 20 e 40ºC, sobre as características físico-químicas e sensoriais do repolho minimamente processado e mantido sob refrigeração. Os repolhos foram cortados em fatias com espessura de 3 mm. As fatias foram imersas em hipoclorito de sódio (50 ppm) por 5 minutos para higienização. Foram realizados os tratamentos T1= testemunha (imersão em água por 5 min a 20ºC); T2= imersão em solução contendo AA a 1%, por 5 min a 20ºC; T3= imersão em solução contendo AA a 2%, por 5 min a 20ºC; T4= imersão em solução contendo CaCl2 a 1%, por 5 min a 20oC; T5= imersão em solução contendo CaCl2 a 2%, por 5 min a 20oC; T6= imersão em solução contendo CaCl2 a 1%, por 5 min a 40oC; T7= imersão em solução contendo CaCl2 a 2%, por 5 min a 40oC, com quatro repetições cada. Os produtos foram centrifugados por 1 minuto, embalados em bandejas de poliestireno expandido, recobertos com policloreto de vinila de 20 µm e mantidos a 6±1oC e UR de 85-90%, por oito dias. Observou-se pequena elevação dos valores de pH e acidez titulável e diminuição nos teores de sólidos solúveis durante o período de conservação, independente do tratamento. O tratamento com AA não diferiu da testemunha quanto a cor e aparência geral, enquanto que o tratamento com CaCl2 2% a 20oC foi o que melhor manteve a qualidade, com menor escurecimento, as melhores notas para aparência geral e intenção de compra e menor odor estranho no final do período de conservaçãoUNESP FCA, Dept Hort, BR-18610307 Botucatu, SP, BrazilUNESP FCA, Dept Hort, BR-18610307 Botucatu, SP, Brazi

Neurological manifestations of COVID-19 in adults and children

Different neurological manifestations of coronavirus disease 2019 (COVID-19) in adults and children and their impact have not been well characterized. We aimed to determine the prevalence of neurological manifestations and in-hospital complications among hospitalized COVID-19 patients and ascertain differences between adults and children. We conducted a prospective multicentre observational study using the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC) cohort across 1507 sites worldwide from 30 January 2020 to 25 May 2021. Analyses of neurological manifestations and neurological complications considered unadjusted prevalence estimates for predefined patient subgroups, and adjusted estimates as a function of patient age and time of hospitalization using generalized linear models. Overall, 161 239 patients (158 267 adults; 2972 children) hospitalized with COVID-19 and assessed for neurological manifestations and complications were included. In adults and children, the most frequent neurological manifestations at admission were fatigue (adults: 37.4%; children: 20.4%), altered consciousness (20.9%; 6.8%), myalgia (16.9%; 7.6%), dysgeusia (7.4%; 1.9%), anosmia (6.0%; 2.2%) and seizure (1.1%; 5.2%). In adults, the most frequent in-hospital neurological complications were stroke (1.5%), seizure (1%) and CNS infection (0.2%). Each occurred more frequently in intensive care unit (ICU) than in non-ICU patients. In children, seizure was the only neurological complication to occur more frequently in ICU versus non-ICU (7.1% versus 2.3%, P < 0.001). Stroke prevalence increased with increasing age, while CNS infection and seizure steadily decreased with age. There was a dramatic decrease in stroke over time during the pandemic. Hypertension, chronic neurological disease and the use of extracorporeal membrane oxygenation were associated with increased risk of stroke. Altered consciousness was associated with CNS infection, seizure and stroke. All in-hospital neurological complications were associated with increased odds of death. The likelihood of death rose with increasing age, especially after 25 years of age. In conclusion, adults and children have different neurological manifestations and in-hospital complications associated with COVID-19. Stroke risk increased with increasing age, while CNS infection and seizure risk decreased with age