RESUMO
A strategy to increase the transfection efficiency of chitosan-based nanoparticles for gene therapy is by adding nuclear localization signals through karyophilic peptides. Here, the effect of the length and sequence of these peptides and their interaction with different plasmids on the physical characteristics and biological functionality of nanoparticles is reported. The karyophilic peptides (P1 or P2) were used to assemble nanoparticles by complex coacervation with pEGFP-N1, pQBI25 or pSelect-Zeo-HSV1-tk plasmids, and chitosan. Size, polydispersity index, zeta potential, and morphology, as well as in vitro nucleus internalization and transfection capability of nanoparticles were determined. The P2 nanoparticles resulted smaller compared to the ones without peptides or P1 for the three plasmids. In general, the addition of either P1 or P2 did not have a significant impact on the polydispersity index and the zeta potential. P1 and P2 nanoparticles were localized in the nucleus after 30 min of exposure to HeLa cells. Nevertheless, the presence of P2 in pEGFP-N1 and pQBI25 nanoparticles raised their capability to transfect and express the green fluorescent protein. Thus, karyophilic peptides are an efficient tool for the optimization of nonviral vectors for gene delivery; however, the sequence and length of peptides have an impact on characteristics and functionality of nanoparticles.
RESUMO
Propolis is a resin that honey bees (Apis mellifera) produce by mixing wax, exudates collected from tree shoots, pollen, and enzymes. It has been used for its biological properties against pathogenic microorganisms including those of viral origin. In the present study, we demonstrate the antiviral effect of Mexican propolis, as well as of the three commercial flavonoids (quercetin, naringenin, and pinocembrin) present in its composition, in cell cultures infected with Canine Distemper Virus. The treatments were carried out with propolis, flavonoids individually, and a mixture of the three flavonoids at three different times. Antiviral activity was evaluated by the inhibition of the relative expression of the virus nucleoprotein gene (Real-Time qPCR) and by the determination of cellular viability (MTT assay). Propolis applied before infection decreased viral expression (0.72 versus 1.0, 1.65, and 1.75 relative expressions) and correlated with increased cell viability (0.314 versus 0.215, 0.259, and 0.237 absorbance units (AU)). The administration of a flavonoid mixture containing the three commercial flavonoids before infection induces a slight decrease in viral expression (0.93 versus 1, 1.42, and 1.82 relative expressions); however, it does not improve cellular viability (0.255 versus 0.247, 0.282, and 0.245 AU). Quercetin administrated at the same time of infection decreases viral expression (0.90 versus 1.0, 3.25, and 1.02 relative expressions) and improves cellular viability (0.294 versus 0.240, 0.250, and 0.245 AU). Pinocembrin and naringenin individually did not show any antiviral activity at the administration times evaluated in this study. The present work is the first in vitro study of the effect of propolis in Canine Distemper Virus and demonstrated the antiviral activity of Mexican propolis, in addition to the synergy that exists between the three flavonoids on cell viability and the expression of the nucleoprotein virus gene.
RESUMO
Maternal and social behavior in multiparous Pelibuey ewes was assessed under intensive production system. The social behavior was recorded several days before and during birth. Mother-young behavior during the first hour after birth was recorded. A selective maternal test was performed at 2 hours postpartum. Eight hours after birth, a double choice test was done in mothers to asses their ability to discriminate between the own or alien lamb. While a similar double choice test was performed in 12 hours-old lambs. The agitation index when the ewes were tested without companions was lower in parturient ewes than in145 days pregnant ewes and non pregnant ewes [parturient ewes: (-0.59 ± 0.9), 145 days pregnant ewes: (2.6 ± 0.7) and non pregnant ewes (4.2 ± 0.9) P < 0.05]. During the first hour after birth the latency to stand up, to begin to look for the udder and to suck was shorter in male than female lambs, also the male lambs sucked for longer time than females (P < 0.05). During this time mothers emitted more high-pitched bleats than low-pitched bleats (P < 0.05). Besides, the ewes got shorter latencies to lick the area of head and thorax of the lamb than its tail, but they licked more frequently the lamb's tail and thorax (P < 0.03). Two hours postpartum all mothers were selective, they showed clear preference to accept to the udder their own lamb and reject actively the alien ones (P < 0.05). At eight or 12 hours, mothers and lambs spent longer time watching and staying with their own familiar animal than with the alien ones (P < 0.05). It is concluded that Pelibuey ewes show an adequate maternal temperament, and the behavior of the newborn lamb could be affected because of its sex.
Se evaluó el comportamiento materno en ovejas multíparas de raza Pelibuey en estabulación, así como la conducta social, antes y durante el parto. Se evaluó el comportamiento madre-cría durante la primera hora posparto y se realizó una prueba de selectividad a dos horas posparto. A las ocho horas se realizó una prueba de elección entre su cordero y uno ajeno. A las 12 h, a los corderos se les hizo una prueba de elección entre la madre propia y una ajena. El índice de agitación sin compañeras fue menor al parto que en ovejas a 145 días de gestación y en ovejas vacías [al parto: (-0.59 ± 0.9), con 145 días (2.6 ± 0.7) y a vacías (4.2 ± 0.9) P < 0.05]. En la primera hora posparto, los corderos machos se incorporaron, iniciaron la búsqueda de la ubre y se amantaron más rápido y por más tiempo que las hembras (P < 0.05). Las madres emitieron más balidos bajos que altos (P < 0.05); limpiaron más rápido a la cría en la cabeza y el tórax que en la cola (P < 0.05), pero limpiaron más veces la cola que el tórax (P < 0.03). A dos horas posparto, las madres fueron selectivas y rechazaron a la cría ajena (P < 0.05). A las ocho y 12 horas, tanto madres como corderos se miraron y permanecieron más tiempo con su co-familiar que con el ajeno (P < 0.05). Se concluyó que las ovejas de la raza Pelibuey muestran buena aptitud maternal, y que la conducta de la cría varía según su sexo.
