RESUMEN
OBJECTIVE: The authors developed a negative-pressure, patient face-mounted antechamber and tested its efficacy as a tool for sequestering aerated particles and improving the safety of endonasal surgical procedures. METHODS: Antechamber prototyping was performed with 3D printing and silicone-elastomer molding. The lowest vacuum settings needed to meet specifications for class I biosafety cabinets (flow rate ≥ 0.38 m/sec) were determined using an anemometer. A cross-validation approach with two different techniques, optical particle sizing and high-speed videography/shadowgraphy, was used to identify the minimum pressures required to sequester aerosolized materials. At the minimum vacuum settings identified, physical parameters were quantified, including flow rate, antechamber pressure, and time to clearance. RESULTS: The minimum tube pressures needed to meet specifications for class I biosafety cabinets were -1.0 and -14.5 mm Hg for the surgical chambers with ("closed face") and without ("open face") the silicone diaphragm covering the operative port, respectively. Optical particle sizing did not detect aerosol generation from surgical drilling at these vacuum settings; however, videography estimated higher thresholds required to contain aerosols, at -6 and -35 mm Hg. Simulation of surgical movement disrupted aerosol containment visualized by shadowgraphy in the open-faced but not the closed-faced version of the mask; however, the closed-face version of the mask required increased negative pressure (-15 mm Hg) to contain aerosols during surgical simulation. CONCLUSIONS: Portable, negative-pressure surgical compartments can contain aerosols from surgical drilling with pressures attainable by standard hospital and clinic vacuums. Future studies are needed to carefully consider the reliability of different techniques for detecting aerosols.
RESUMEN
Although chromosomal deletions and inversions are important in cancer, conventional methods for detecting DNA rearrangements require laborious indirect assays. Here we develop fluorescent reporters to rapidly quantify CRISPR/Cas9-mediated deletions and inversions. We find that inversion depends on the non-homologous end-joining enzyme LIG4. We also engineer deletions and inversions for a 50 kb Pten genomic region in mouse liver. We discover diverse yet sequence-specific indels at the rearrangement fusion sites. Moreover, we detect Cas9 cleavage at the fourth nucleotide on the non-complementary strand, leading to staggered instead of blunt DNA breaks. These reporters allow mechanisms of chromosomal rearrangements to be investigated.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Reordenamiento Génico , Animales , Deleción Cromosómica , Inversión Cromosómica , Femenino , Genómica/métodos , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación INDEL , Hígado/metabolismo , Ratones , Ratones Endogámicos , PlásmidosRESUMEN
Nanoparticles are used for delivering therapeutics into cells. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell-specific internalization, excretion, toxicity and efficacy. A variety of materials have been explored for delivering small interfering RNAs (siRNAs)--a therapeutic agent that suppresses the expression of targeted genes. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, a lack of tissue specificity and potential toxicity. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer-targeting ligands (such as peptides and folate) on the nanoparticle surface can be controlled precisely. We show that at least three folate molecules per nanoparticle are required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t(1/2) ≈ 24.2 min) than the parent siRNA (t(1/2) ≈ 6 min).