Stable expression of shRNA for the control of recombinant adenovirus replication

Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.

Cateterismo posterior do plexo braquial em cão: estudo radiológico e avaliação de três protocolos anestésicos na duração do bloqueio motor e sensitivo / Posterior catheterization of the brachial plexus: radiological study and evaluation of three anesthetic protocols in motor and sensory block duration

Avaliou-se a eficiência do cateterismo posterior do plexo braquial em cães para promover bloqueio motor e sensitivo, por meio de três protocolos anestésicos. Foram utilizados nove cães, machos e fêmeas, sem distinção de raça e idade, com peso variando de 6 a 15kg, distribuídos em três grupos de três animais por grupo. Após a confirmação do correto posicionamento do cateter pela via posterior do plexo braquial por meio do exame radiográfico, foram aplicadas as medicações de acordo com os grupos. No grupo 1, a solução anestésica de bupivacaína 0,5% sem vasoconstrictor, na dose de 2mg.kg-1, foi usada isoladamente. No grupo 2, a solução anestésica de bupivacaína foi associada ao butorfanol na dose de 0,25mg.kg-1 . No grupo 3, o fentanil, na dose de 0,005mg.kg¹, foi associado à solução anestésica de bupivacaína. Não houve diferença estatística significante entre os grupos. Observou-se que a duração dos bloqueios motor e sensitivo foi clinicamente maior no grupo 2. O cateterismo posterior do plexo braquial permite a aplicação de fármacos mais próximos do plexo nervoso, promovendo analgesia complementar nos membros anteriores.

We evaluated the efficiency of the posterior brachial plexus catheterization in dogs to promote motor and sensory block using three anesthetic protocols. We used nine dogs, males and females, without distinction of race and age, weighing 6 to 15kg which were divided into three groups of three animals per group. After confirming the correct positioning of the catheter through the posterior brachial plexus through radiographic examination, the medications were administered according to the groups. In group 1 the anesthetic bupivacaine 0.5% without a vasoconstrictor dose of 2mg.kg-1 was used alone. In group 2 the anesthetic bupivacaine was associated with butorphanol at a dose of 0.25mg.kg-1. In group 3, the fentanyl dose was associated with 0.005mg.kg-1 anesthetic bupivacaine. There was no statistically significant difference between groups. It was observed that the duration of sensory and motor block was clinically higher in group 2. The posterior brachial plexus catheterization allows the application of drugs closer to the nerve plexus promoting additional analgesia in the forelimbs.

Impairment of locomotor activity induced by the novel N-acylhydrazone derivatives LASSBio-785 and LASSBio-786 in mice

The N-acylhydrazone (NAH) analogues N-methyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-785) and N-benzyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-786) were prepared from 2-thienylidene 3,4-methylenedioxybenzoylhydrazine (LASSBio-294). The ability of LASSBio-785 and LASSBio-786 to decrease central nervous system activity was investigated in male Swiss mice. LASSBio-785 or LASSBio-786 (30 mg/kg, ip) reduced locomotor activity from 209 ± 26 (control) to 140 ± 18 (P < 0.05) or 146 ± 15 crossings/min (P < 0.05), respectively. LASSBio-785 (15 or 30 mg/kg, iv) also reduced locomotor activity from 200 ± 15 to 116 ± 29 (P < 0.05) or 60 ± 16 crossings/min (P < 0.01), respectively. Likewise, LASSBio-786 (15 or 30 mg/kg, iv) reduced locomotor activity from 200 ± 15 to 127 ± 10 (P < 0.01) or 96 ± 14 crossings/min (P < 0.01), respectively. Pretreatment with flumazenil (20 mg/kg, ip) prevented the locomotor impairment induced by NAH analogues (15 mg/kg, iv), providing evidence that the benzodiazepine (BDZ) receptor is involved. This finding was supported by the structural similarity of NAH analogues to midazolam. However, LASSBio-785 showed weak binding to the BDZ receptor. LASSBio-785 or LASSBio-786 (30 mg/kg, ip, n = 10) increased pentobarbital-induced sleeping time from 42 ± 5 (DMSO) to 66 ± 6 (P < 0.05) or 75 ± 4 min (P < 0.05), respectively. The dose required to achieve 50% hypnosis (HD50) following iv injection of LASSBio-785 or LASSBio-786 was 15.8 or 9.5 mg/kg, respectively. These data suggest that both NAH analogues might be useful for the development of new neuroactive drugs for the treatment of insomnia or for use in conjunction with general anesthesia.

DNA-lipid systems. A physical chemistry study

It is well known that the interaction of polyelectrolytes with oppositely charged surfactants leads to an associative phase separation; however, the phase behavior of DNA and oppositely charged surfactants is more strongly associative than observed in other systems. A precipitate is formed with very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. Techniques like phase diagram determinations, fluorescence microscopy, and ellipsometry were used to study these systems. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants) was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of an anionic surfactant can release DNA back into solution from a compact globular complex between DNA and the cationic surfactant. Finally, DNA interactions with polycations, chitosans with different chain lengths, were studied by fluorescence microscopy, in vivo transfection assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection