Starch degradation in the bean fruit pericarp is characterized by an increase in maltose metabolism.

The bean fruit pericarp accumulates a significant amount of starch, which starts to be degraded 20 days after anthesis (DAA) when seed growth becomes exponential. This period is also characterized by the progressive senescence of the fruit pericarp. However, the chloroplasts maintained their integrity, indicating that starch degradation is a compartmentalized process. The process coincided with a transient increase in maltose and sucrose levels, suggesting that ß-amylase is responsible for starch degradation. Starch degradation in the bean fruit pericarp is also characterized by a large increase in starch phosphorylation, as well as in the activities of cytosolic disproportionating enzyme 2 (DPE2, EC 2.4.1.25) and glucan phosphorylase (PHO2, EC 2.4.1.1). This suggests that the rate of starch degradation in the bean fruit pericarp 20 DAA is dependent on the transformation of starch to a better substrate for ß-amylase and the increase in the rate of cytosolic metabolism of maltose.

Tratamiento de COVID-19 en Panamá. La historia del inicio, estudio PanaCovid2020

Antecedentes En Panamá, durante los primeros cinco meses de pandemia, se reportaron 65,256 casos y 1,421 fallecimientos por COVID-19. Los tratamientos utilizados en pacientes hospitalizados durante este periodo fueron variando en la medida que surgía evidencia científica. Metodología Presentamos un estudio multicéntrico observacional, descriptivo, de corte transversal y retrospectivo de la terapéutica administrada en pacientes hospitalizados con COVID-19 en siete centros estatales de la República de Panamá, entre el 1 de marzo al 31 de julio de 2020. Se analizó el resultado de recuperado o fallecido en relación al uso de hidroxicloroquina, heparinas y esteroides. Resultados Se revisaron 837 expedientes. 60.7% pacientes eran hombres y la mediana de edad fue 53.6 años. Los tratamientos más utilizados fueron heparinas 84.7%, esteroides sistémicos 76.5%, azitromicina 65.5% e hidroxicloroquina en 41.1%. La frecuencia de uso de oxigenoterapia fue de cánula nasal 73,2%, máscara facial con reservorio 39%, cánula de alto flujo 8.9%, ventilación mecánica no invasiva 11.1% y ventilación invasiva en el 15.3%. Los esteroides mostraron mayor tasa de recuperación (OR 1.72 [1.16-2.54). Conclusiones El reto de abordar una nueva enfermedad, sin tratamiento conocido, con evolución y desenlace variable, llevó a la comunidad médica a utilizar medicamentos empíricos con potencial terapéutico incierto. Destacamos que desde muy temprano se administró esteroides sistémicos, heparinas y tocilizumab; actualmente con beneficios para pacientes con requerimientos de hospitalización. Además, se empleó técnicas de oxigenación, como la cánula de alto flujo y ventilación mecánica no invasiva, con dudosa efectividad; pero que hoy ya tienen un rol estudiado en la falla respiratoria por COVID-19. (provisto por Infomedic International)

Background: In Panama, during the first five months of the pandemic, 65,256 cases and 1,421 deaths due to COVID-19 were reported. The treatments used in hospitalized patients during this period varied as scientific evidence emerged. Methodology: We present a retrospective, descriptive, cross-sectional, observational, descriptive, multicenter study of the therapeutics administered in hospitalized patients with COVID-19 in seven state centers in the Republic of Panama, between March 1 and July 31, 2020. The outcome of recovered or deceased was analyzed in relation to the use of hydroxychloroquine, heparins and steroids. Results: A total of 837 files were reviewed. 60.7% of patients were men and the median age was 53.6 years. The most commonly used treatments were heparins 84.7%, systemic steroids 76.5%, azithromycin 65.5% and hydroxychloroquine in 41.1%. The frequency of oxygen therapy use was nasal cannula 73.2%, face mask with reservoir 39%, high-flow cannula 8.9%, noninvasive mechanical ventilation 11.1% and invasive ventilation in 15.3%. Steroids showed higher recovery rate (OR 1.72 [1.16-2.54). Conclusions: The challenge of dealing with a new disease, without known treatment, with variable evolution and outcome, led the medical community to use empirical drugs with uncertain therapeutic potential. We highlight the early administration of systemic steroids, heparins and tocilizumab; currently with benefits for patients requiring hospitalization. In addition, oxygenation techniques were used, such as high-flow cannula and noninvasive mechanical ventilation, with doubtful effectiveness, but which today have a studied role in respiratory failure due to COVID-19.   (provided by Infomedic International)

Starch accumulation in bean fruit pericarp is mediated by the differentiation of chloroplasts into amyloplasts.

The sucrose supply to bean fruits remains almost constant during seed development, and the early stages of this process are characterized by a significant amount of starch and soluble sugars (glucose, fructose and sucrose) accumulated in the pericarp. Bean fruits are photosynthetically active; however, our results indicated that starch synthesis in the pericarp was largely dependent on the photosynthetic activity of the leaves. The photosynthetic activity and the amount of the Rubisco large subunit were gradually reduced in the fruit pericarp, and a large increase in the amount of the ADP-glucose pyrophosphorylase small subunit (AGPase SS) was observed. These changes suggested differentiation of chloroplasts into amyloplasts. Pericarp chloroplasts imported glucose 1-P to support starch synthesis, and their differentiation into amyloplasts allowed the surplus sucrose to be used in the synthesis of starch, which was later degraded to meet the needs of fast-growing seeds. Starch stored in the bean fruit pericarp was not degraded in response to drought stress, but it was rapidly used under severe nutrient restriction. Together, this work indicated that starch accumulation in the pericarp of bean fruits is important to adjust the needs of developing seeds to the amount of sucrose that is provided to fruits.

Human Metapneumovirus: Laboratory Methods for Isolation, Propagation, and Plaque Titration.

The human metapneumovirus (hMPV) is an important viral agent associated with severe infections of the upper and lower airways, especially in young children and immunosuppressed subjects. Nevertheless, in vitro studies of hMPV are very difficult due to the little knowledge we have on its laboratory manipulation. OBJECTIVE: The aim of this study was to isolate and propagate hMPV from patients, and to establish a method to quantify the virus by plaque assay. METHOD: As part of a Latin American respiratory virus surveillance study, 12 nasal secretion samples - hMPV-positive by direct fluorescence - were inoculated on LLC-MK2 cells to isolate the virus. The supernatants were re-inoculated and the cytopathic effect and syncytium formation were evaluated daily; the infection was confirmed by immunofluorescence and RT-PCR. A protocol to titrate the harvested virus was established inoculating serial dilutions on LLC-MK2 cells, and agarose was then added as an overlay. After different time periods, the monolayers were fixed and stained with Naphthol blue/black or crystal violet and finally the viral titer was obtained. RESULTS: Eight out of 12 hMPV-positive respiratory samples were positive for the isolation and confirmed by RT-PCR and immunofluorescence, but the cytopathic effect and syncytium formation were observed only in 5 cultures. One out of 8 viral isolates was used for propagation and plaque assay standardization. We found that incubation for 7 days in the semisolid overlay yielded plaques with appropriate size and shape to be counted, although crystal violet staining showed slightly larger plaques than those seen with Naphthol blue/black staining. CONCLUSIONS: The isolation and propagation from patient-derived hMPV and the standardization of a practical, reliable, and inexpensive method of detection and quantification of hMPV were carried out, without the additional use of antibodies that had not been reported previously. These results offer some important insights for future studies of cellular and molecular biology of hMPV.

Aislamiento de metapneumovirus humano (HMPV) a partir de muestras clínicas / Human metapneumovirus isolation using clinical respiratory samples

Objetivo: El objetivo de este trabajo fue establecer una metodología para el aislamiento de una cepa de hMPV a partir de hisopados nasales provenientes de pacientes con sintomatología de enfermedad respiratoria aguda. Materiales y métodos: A partir de 12 muestras positivas por fluorescencia para hMPV, se hizo la inoculación en células LLCMK2 (epiteliales de riñón de mono Rhesus) con un medio de mantenimiento que contenía tripsina como proteasa. Se evaluó el efecto citopático diario por 10 días y se tomaron células infectadas que se utilizaron para la prueba de fluorescencia y para confirmar la infección. Se utilizó RT-PCR para corroborar la presencia del virus. Resultados: En 8 de las 12 muestras procesadas, se logró aislar el hMPV, confirmándose la infección por fluorescencia y RT-PCR. Conclusiones: A pesar de ser un virus de crecimiento lento y difícil de aislar según los reportes, en este trabajo se logró aislar el hMPV a partir de muestras clínicas frescas, lo cual resulta de gran utilidad en futuros estudios sobre la patogénesis de la infección respiratoria.

Objective: The aim of this work was to establish a laboratory protocol to isolate human metapneumovirus (hMPV) using as a source confirmed respiratory secretion samples from symptomatic patients. Methods: Twelve respiratory secretion samples confirmed for hMPV by immunofluorescence were inoculated on LLCMK2 primate cells using trypsin to improve the virus binding. The cytopathic effect was evaluated during 10 days, then they were stained with an hMPV specific antibody and culture supernatants were used to detect viral RNA by RT-PCR. Results: We isolated hMPV in 8 out of 12 samples. Monkey cells were susceptible to infection and showed viral replication in both immunocytochemistry and molecular tests and even syncytia formation. Conclusion: Despite the hMPV has low replication rates in culture, causing difficulties to isolate it, in this work we accomplish the viral isolation in eight samples that had been previously confirmed containing hMPV. These clinical isolates are a useful tool to study the respiratory infection pathogenesis.

Aislamiento de metapneumovirus humano (HMPV) a partir de muestras clínicas / Human metapneumovirus isolation using clinical respiratory samples

Objetivo: El objetivo de este trabajo fue establecer una metodología para el aislamiento de una cepa de hMPV a partir de hisopados nasales provenientes de pacientes con sintomatología de enfermedad respiratoria aguda. Materiales y métodos: A partir de 12 muestras positivas por fluorescencia para hMPV, se hizo la inoculación en células LLCMK2 (epiteliales de riñón de mono Rhesus) con un medio de mantenimiento que contenía tripsina como proteasa. Se evaluó el efecto citopático diario por 10 días y se tomaron células infectadas que se utilizaron para la prueba de fluorescencia y para confirmar la infección. Se utilizó RT-PCR para corroborar la presencia del virus. Resultados: En 8 de las 12 muestras procesadas, se logró aislar el hMPV, confirmándose la infección por fluorescencia y RT-PCR. Conclusiones: A pesar de ser un virus de crecimiento lento y difícil de aislar según los reportes, en este trabajo se logró aislar el hMPV a partir de muestras clínicas frescas, lo cual resulta de gran utilidad en futuros estudios sobre la patogénesis de la infección respiratoria.(AU)

Objective: The aim of this work was to establish a laboratory protocol to isolate human metapneumovirus (hMPV) using as a source confirmed respiratory secretion samples from symptomatic patients. Methods: Twelve respiratory secretion samples confirmed for hMPV by immunofluorescence were inoculated on LLCMK2 primate cells using trypsin to improve the virus binding. The cytopathic effect was evaluated during 10 days, then they were stained with an hMPV specific antibody and culture supernatants were used to detect viral RNA by RT-PCR. Results: We isolated hMPV in 8 out of 12 samples. Monkey cells were susceptible to infection and showed viral replication in both immunocytochemistry and molecular tests and even syncytia formation. Conclusion: Despite the hMPV has low replication rates in culture, causing difficulties to isolate it, in this work we accomplish the viral isolation in eight samples that had been previously confirmed containing hMPV. These clinical isolates are a useful tool to study the respiratory infection pathogenesis.(AU)

Possible role played by R1 protein in starch accumulation in bean (Phaseolus vulgaris) seedlings under phosphate deficiency.

The effect of phosphate (Pi) deficiency on starch accumulation was studied in bean (Phaseolus vulgaris). After 3 weeks of Pi deprivation total Pi concentration in root and shoot was reduced by 68% and 42%, respectively; however, only shoot growth was affected. In leaves, Pi deprivation induced glucose, fructose and starch accumulation. Pi deficiency did not affect starch synthesis, but it reduced its mobilization during the dark period. At the same time, starch produced by Pi deficient plants have fewer Pi bound and was also less susceptible to beta-amylase hydrolysis. R1 protein is the protein responsible of phosphorylating C3 and C6 glucosyl residues of the polyglucan, increasing the hydration capacity and the interaction with amylolytic enzymes. Pi deprivation did not change the amount of R1 protein detected in total extracts but decreased its association with starch granules.