The effect of storage temperature and time on total phenolics and enzymatic activity of sapodilla (Achras sapota L.)

The tropical fruits are sensitive to low storage temperatures, so optimal parameters have been searched for storage and transport for the purpose of maintaining its overall quality as long as possible to the consumer. The effect of different storage temperatures (6, 10, 15, 21 and 27 ºC) and storage durations (0 to 20 d) on total phenolics and enzymatic activity of peroxidase (POD), catalase (CAT), and polyphenol oxidase (PPO) on sapodilla (Achras sapota L.) fruit was investigated. The extraction and quantitation of protein and phenols from fruit was performed, then the enzymatic activity of PPO, POD and CAT was determined. The concentration of total phenolics decreased in the control fruit. POD activity was 3268.7 ± 1.4 U g-1 in ripening and senescence of sapodilla stored at 27 °C. CAT activity reached a peak of 34.0 ± 0.25 U g-1 in senescence in control fruit. PPO activity remained unchanged in the ripening stage and until consumption. The best storage temperatures to prolong the post-harvest life of the sapodilla fruit were 6 °C and 10 °C when storage was at low temperatures. POD activity was inactivated during sapodilla storage at low temperatures (6 and 10 °C) and after being transferred to 27 °C the activity was reactivated. Likewise of fruits stored at 21 °C after being transferred to 27 °C the POD activity was reactive with a maximum value of 46.3 ± 0.012 U g-1. Enzyme activity decreased at low temperatures, which contributed to the preservation of the fruit, showing that the cold retards the maturation processes

Involvement of RDR6 in short-range intercellular RNA silencing in Nicotiana benthamiana

In plants, non-cell autonomous RNA silencing spreads between cells and over long distances. Recent work has revealed insight on the genetic and molecular components essential for cell-to-cell movement of RNA silencing in Arabidopsis. Using a local RNA silencing assay, we report on a distinct mechanism that may govern the short-range (6–10 cell) trafficking of virus-induced RNA silencing from epidermal to neighbouring palisade and spongy parenchyma cells in Nicotiana benthamiana. This process involves a previously unrecognised function of the RNA-dependent RNA polymerase 6 (RDR6) gene. Our data suggest that plants may have evolved distinct genetic controls in intercellular RNA silencing among different types of cells

Adherencia, satisfacción al tratamiento y calidad de vida de pacientes con cáncer de mama en el Hospital Universitario del Caribe.(Cartagena, Colombia)

Objetivos: Determinar la adherencia, satisfacción al tratamiento y calidad de vida de pacientes con cáncer de mama. Materiales y métodos: Estudio descriptivo, prospectivo, trasversal,  desarrollado en el Hospital Universitario del Caribe, entre agosto de 2014 y  abril de 2015. La muestra fueron 23 pacientes, diagnosticadas de cáncer de mama. La adherencia, satisfacción al tratamiento y calidad de vida se determinaron con los instrumentos siguientes: Cuestionario simplified medication adherence questionnaire (SMAQ), registro de dispensación del hospital (RD), test de satisfacción ESTAR del Estudio ARPAS adaptado y el cuestionario WHOQOL BREF respectivamente. El cuestionario ESTAR fue  validado por expertos y mediante alfa de Cronbach.   Resultados: El 54,78% de las pacientes (según SMAQ), fueron no  adherentes a quimioterápicos, la satisfacción fue de 3,94 (rango 0-6). La calidad de vida estuvo en un promedio de 3.2 (rango 1-5), con valores de 14,5% y 7,14% en el nivel 5 del rango. La mayoría de dimensiones del cuestionario de calidad de vida guardan una correlación directamente  proporcional con el nivel total de satisfacción al tratamiento; arrojando un dato negativo (-0,3207) únicamente en la dimensión de satisfacción con la eficacia al tratamiento   Conclusiones: La calidad de vida fue media y baja, la adherencia y  satisfacción al tratamiento fueron bajas y se presentó una correlación inversa entre calidad de vida y la satisfacción al tratamiento, probablemente a causa de reacciones adversas indeseables que se constituyen en disminución de la calidad de vida

Adherencia, satisfacción al tratamiento y calidad de vida de pacientes con cáncer de mama en el Hospital Universitario del Caribe.(Cartagena, Colombia)

Objetivos: Determinar la adherencia, satisfacción al tratamiento y calidad de vida de pacientes con cáncer de mama. Materiales y métodos: Estudio descriptivo, prospectivo, trasversal,  desarrollado en el Hospital Universitario del Caribe, entre agosto de 2014 y  abril de 2015. La muestra fueron 23 pacientes, diagnosticadas de cáncer de mama. La adherencia, satisfacción al tratamiento y calidad de vida se determinaron con los instrumentos siguientes: Cuestionario simplified medication adherence questionnaire (SMAQ), registro de dispensación del hospital (RD), test de satisfacción ESTAR del Estudio ARPAS adaptado y el cuestionario WHOQOL BREF respectivamente. El cuestionario ESTAR fue  validado por expertos y mediante alfa de Cronbach.   Resultados: El 54,78% de las pacientes (según SMAQ), fueron no  adherentes a quimioterápicos, la satisfacción fue de 3,94 (rango 0-6). La calidad de vida estuvo en un promedio de 3.2 (rango 1-5), con valores de 14,5% y 7,14% en el nivel 5 del rango. La mayoría de dimensiones del cuestionario de calidad de vida guardan una correlación directamente  proporcional con el nivel total de satisfacción al tratamiento; arrojando un dato negativo (-0,3207) únicamente en la dimensión de satisfacción con la eficacia al tratamiento   Conclusiones: La calidad de vida fue media y baja, la adherencia y  satisfacción al tratamiento fueron bajas y se presentó una correlación inversa entre calidad de vida y la satisfacción al tratamiento, probablemente a causa de reacciones adversas indeseables que se constituyen en disminución de la calidad de vida

Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production

Influence of viral genes on the cell-to-cell spread of RNA silencing

The turnip crinkle virus-based vector TCV–GFPΔCP had been devised previously to study cell-to-cell and long-distance spread of virus-induced RNA silencing. TCV–GFPΔCP, which had been constructed by replacing the coat protein (CP) gene with a green fluorescent protein (GFP) coding sequence, was able to induce RNA silencing in single epidermal cells, from which RNA silencing spread from cell-to-cell. Using this unique local silencing assay together with mutagenesis analysis, two TCV genes, p8 and p9, which were involved in the intercellular spread of virus-induced RNA silencing, were identified. TCV–GFPΔCP and its p8- or p9-mutated derivatives, TCVmp8–GFPΔCP and TCVmp9–GFPΔCP, replicated efficiently but were restricted to single Nicotiana benthamiana epidermal cells. TCV–GFPΔCP, TCVmp8–GFPΔCP, or TCVmp9–GFPΔCP was able to initiate RNA silencing that targeted and degraded recombinant viral RNAs in inoculated leaves of the GFP-expressing N. benthamiana line 16c. However, cell-to-cell spread of silencing to form silencing foci was triggered only by TCV–GFPΔCP. Non-replicating TCVmp88–GFPΔCP and TCVmp28mp88–GFPΔCP with dysfunctional replicase genes, and single-stranded gfp RNA did not induce RNA silencing. Transient expression of the TCV p9 protein could effectively complement TCVmp9–GFPΔCP to facilitate intercellular spread of silencing. These data suggest that the plant cellular trafficking machinery could hijack functional viral proteins to permit cell-to-cell movement of RNA silencing

APC/C-Mediated Degradation of dsRNA-Binding Protein 4 (DRB4) Involved in RNA Silencing

Background: Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. Methodology/Principal Findings: In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed

RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration

Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB) transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi) machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions. To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU) after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system

Graft-Transmitted siRNA Signal from the Root Induces Visual Manifestation of Endogenous Post-Transcriptional Gene Silencing in the Scion

In plants, post-transcriptional gene silencing (PTGS) spreads systemically, being transmitted from the silenced stock to the scion expressing the corresponding transgene. It has been reported that a graft-transmitted siRNA signal can also induce PTGS of an endogenous gene, but this was done by top-grafting using silenced stock. In the present study involving grafting of Nicotiana benthamiana, we found that PTGS of an endogenous gene, glutamate-1-semialdehyde aminotransferase (GSA), which acts as a visible marker of RNAi via inhibition of chlorophyll synthesis, was manifested along the veins of newly developed leaves in the wild-type scion by the siRNA signal synthesized only in companion cells of the rootstock