Rapid in vitro production of single-stranded DNA.

There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ∼40-50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 µl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.

Multilayer DNA Origami with Terminal Interfaces That Are Flat and Wide-Area.

DNA origami is a popular nanofabrication strategy that employs self-assembly of a long single scaffold strand, typically less than 10 kilobases in length, with hundreds of shorter staple strands into a desired shape. In particular, origami arranged as a single-layer rectangle has proven popular as flat pegboards that can display functionalities at staple-strand breakpoints, off the sides of the constituent double helices, with a ∼5.3 nm rhombic-lattice spacing. For applications that demand tighter spacing, functionalities can be displayed instead on the termini of helices of multilayer DNA origami. However, pegboards with the greatest addressable surface area are often found to be the most versatile. Given the practical limitations of the length of the scaffold that can be easily realized, designs that minimize the length of each helix would have advantages for maximizing the number of helices and therefore the number of addressable pixels on each terminal surface. Here we present an architecture for multilayer DNA origami displaying flush terminal interfaces from over 200 helices that each are only 5.3 turns in length. We characterize an example using cryo-EM imaging paired with single-particle analysis for further analysis of the global structure.

1.2†Šresolution crystal structure of Escherichia coli WrbA holoprotein.

The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2 Šresolution, the highest yet reported. Faster-growing deep yellow crystals formed with FMN display an atypical shape, but diffract to only ∼1.6 Šresolution and are not analysed further here. The 1.2 Šresolution structure detailed here revealed only FMN in the active site and no electron density that can accommodate the missing parts of FAD. The very high resolution supports the modelling of the FMN isoalloxazine with a small but distinct propeller twist, apparently the first experimental observation of this predicted conformation, which appears to be enforced by the protein through a network of hydrogen bonds. Comparison of the electron density of the twisted isoalloxazine ring with the results of QM/MM simulations is compatible with the oxidized redox state. The very high resolution also supports the unique refinement of Met10 as the sulfoxide, confirmed by mass spectrometry. Bond lengths, intramolecular distances, and the pattern of hydrogen-bond donors and acceptors suggest the cofactor may interact with Met10. Slow incorporation of FMN, which is present as a trace contaminant in stocks of FAD, into growing crystals may be responsible for the near-atomic resolution, but a direct effect of the conformation of FMN and/or Met10 sulfoxide cannot be ruled out.

Extrusion of RNA from a DNA-Origami-Based Nanofactory.

Cells often spatially organize biomolecules to regulate biological interactions. Synthetic mimicry of complex spatial organization may provide a route to similar levels of control for artificial systems. As a proof-of-principle, we constructed an RNA-extruding nanofactory using a DNA-origami barrel with an outer diameter of 60 nm as a chassis for integrated rolling-circle transcription and processing of RNA through spatial organization of DNA templates, RNA polymerases, and RNA endonucleases. The incorporation efficiency of molecular components was quantified to be roughly 50% on designed sites within the DNA-origami chassis. Each integrated nanofactory with RNA-producing units, composed of DNA templates and RNA polymerases, produced 100 copies of target RNA in 30 min on average. Further integration of RNA endonucleases that cleave rolling-circle transcripts from concatemers into monomers resulted in 30% processing efficiency. Disabling spatial organization of molecular components on DNA origami resulted in suppression of RNA production as well as processing.

The posterior shift anticipatory postural adjustment in choice reaction step initiation.

The ability to step quickly in response to a perturbation has been shown to be critical for prevention of falls. The cognitive processing, weight shifting, and locomotion must be well timed to execute a successful step. The purpose of this study was to compare the response preparation and response execution processes between a simple (SRST) and a choice reaction stepping task (CRST). Nine healthy young subjects were recruited to participate in this study. Subjects were required to stand on a forceplate and maintain their balance, and step forward on a second forceplate with either the left or right foot after hearing an auditory tone. The center of pressure (COP) was analyzed to determine the types of anticipatory postural adjustment (APA) prior to a step. The APA phase and stepping phase timing was calculated based on the COP trajectory. Findings showed that reaction time (RT), APA phase and overall stepping latency were slower for CRST than for SRST. We also identified an intermediate type of APA response (posterior shift APA) in addition to the correct and error APA response, and found the posterior shift APA response had the fastest execution time for CRST, and may be beneficial for falls prevention.

Etiología e incidencia de retardo mental y epilepsia en pacientes de la sala de oligofrénicos del Hospital Psiquiatrico Julio Endara Conocoto-Quito

El presente, es un estudio retrospectivo, se revisan 43 historias clínicas (100 por ciento) de pacientes con retardo mental ingresados en la sala de Oligofrénicos del Hospital Psiquiatrico Julio Endara, desde enero de 1971 hasta enero de 1996; de entre ellos 15 pacientes (34.88 por ciento presentan retardo mental asociado a epilepsia generalizada, el sexo que predomina es el femenino con el 66.60 por ciento. En lo referente al grado de retardo mental y epilepsia obtuvimos los siguientes porcentajes, con el moderado el 20 porciento, con el grave el 73.33 por ciento, y con el profundo el 6.66 por ciento. La etiología puede ser uni (46.66 por ciento) o multifactorial (26.66 por ciento). Entre las causas genéticas la de mayor frecuencia es la relacionada con trastornos heredados con el 40 por ciento, en cuanto a las lesiones cerebrales perinatales la de mayor incidencia es la hipoxia con el 13.33 por ciento, y en lo referente a las causas por lesiones cerebrales postnatales es el traumatismo cráneo enfefálico la de mayor presentación con el 20 por ciento. El pronostico de retardo mental ensombrece cuando se asocia a la epilepsia ya que por sí sóla causa déficit intelectual, motor y trastornos de la conducta.

Causas de morbilidad en pacientes geriátricos. Centro Médico Perpetuo Socorro-Quito, 1996

Geriatría es la medicina de los ancianos, basada en la prevención y tratamiento de las enfermedades, durante el envejecimiento se produce atrofía del organismo y disminución de la eficacia funcional predisponiendo al anciano a enfermar de infecciones agudas, especialmente las del aparato respiratorio, con el aumento de los años se produce con mayor frecuencia la acumulación de enfermedades crónicas e incapacitantes citándose entre las principales a la artritis, los accidentes cerebrovasculares, los trastornos cardíacos y pulmonares crónicos, la disminución de la agudeza visual y de la audición, etc. Estas enfermedades conllevan a disminuir la calidad de vida del anciano e inciden en el incremento de la mortalidad...

Causas de morbilidad en pacientes con retardo mental: Hospital Psiquiátrico Julio Endara

Con el propósito de investigar las principales causas de morbilidad en pacientes con retardo mental, se realizó un estudio retrospectivo tomando para el efecto información de las historias clínicas en 1996. De un total de 42 pacientes, 16 son varones y 26 son mujeres; con una edad promedio de 29,5 años, el mayor lugar de procedencia es la provincia de Pichincha (13 pacientes); así como los de procedencia desconocida con igual porcentaje. En cuanto a las principales causas de morbilidad encontramos en orden de importancia: síndrome gripal (30.81 por ciento); parasitosis (16.86 por ciento); amigdalitis (13.37 por ciento); heridas cortantes (8.13 por ciento); EDA (5.23 por ciento); neumonía (4.65 por ciento); abscesos (4.06 por ciento)...

Terapia ocupacional en retardo mental: sala de oligofrénicos, Hospital Psiquiátrico Julio Endara, 1996 / Occupational Therapy in mental retardation: room of oligofrénicos, Psiquiatrico Hospital Julio Endara, 1996

Presenta al lector, la importancia que tiene la Terapia Ocupacional en pacientes con retardo mental, y por ende el trabajo realizado durante un año (1996) en la sala de oligofrénicos del Hospital Psiquiátrico Julio Endara. De un total de 42 pacientes, el 30,9xcto (16) son varones y el 66,6xcto (28) son mujeres con edad promedio de 29.5 años, Todos con el diagnóstico de retardo mental cuyo grado varía del moderado al profundo; se ha realizado el seguimiento de 15 pacientes integrados en el área de Terapia Ocupacional, siendo estos el 20xcto (3) hombres y el 80xcto (12) mujeres en ellos después de haber sido evaluados en lo referente a: Independencia en actividades de la vida diaria (AVD), función más Músculo Esquelética, Psico-Motrizidad, Destreza, se ha podido aplicar en varias actividades como es: armar juegos didácticos de encaje recíproco, manualidades, recreación, gimnasia con el fin de hacer más soportable su existencia hospitalaria y el desarrollo de destrezas y habilidades, brindando por consiguiente una rehabilitación acorde a sus necesidades, mejorando así su calidad de vida. Concluyéndose que la atención que requiere cada uno de los pacientes de la sala amerita esfuerzo, constancia, conocimiento real de Patología y la oportunidad que por derecho les corresponde como seres humanos, que a pesar de sus limitaciones tienen un valor singular, cambiando así la imagen del tratamiento y atención que anteriormente se venía realizando en este tipo de pacientes de la sala de Oligofrénicos del HPJE.