High temperature ion channels and pores

The present invention includes an apparatus, system and method for stochastic sensing of an analyte to a protein pore. The protein pore may be an engineer protein pore, such as an ion channel at temperatures above 55.degree. C. and even as high as near 100.degree. C. The analyte may be any reactive analyte, including chemical weapons, environmental toxins and pharmaceuticals. The analyte covalently bonds to the sensor element to produce a detectable electrical current signal. Possible signals include change in electrical current. Detection of the signal allows identification of the analyte and determination of its concentration in a sample solution. Multiple analytes present in the same solution may also be detected

Unzipping Kinetics of Double-Stranded DNA in a Nanopore

We studied the unzipping kinetics of single molecules of double-stranded DNA by pulling one of their two strands through a narrow protein pore. PCR analysis yielded the first direct proof of DNA unzipping in such a system. The time to unzip each molecule was inferred from the ionic current signature of DNA traversal. The distribution of times to unzip under various experimental conditions fit a simple kinetic model. Using this model, we estimated the enthalpy barriers to unzipping and the effective charge of a nucleotide in the pore, which was considerably smaller than previously assumed.Comment: 10 pages, 5 figures, Accepted: Physics Review Letter

Carga laboral y notificación de eventos adversos en un centro hospitalario, Chimbote 2022

La investigación tenía como objetivo determinar la relación entre la carga laboral y la notificación de eventos adversos por el personal de salud de un Centro Hospitalario, Chimbote – 2022, para lo cual fue necesario el uso de la investigación aplicada y el diseño no experimental, siendo la población censal de 40 trabajadores, la técnica y el instrumento usado para el análisis de ambas variables fue la encuesta y el cuestionario, se halló que la dimensión de carga laboral y notificación de eventos adversos es percibida como regular (50%), finalmente se halló que el índice de relación fue de 0.722, por lo mencionado se determina que las variables se encuentran relacionadas

Prolonged Residence Time of a Noncovalent Molecular Adapter, β-Cyclodextrin, within the Lumen of Mutant α-Hemolysin Pores

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the α-hemolysin (αHL) pore, thereby offering a basis for the detection of a variety of organic molecules with αHL as a sensor element. β-Cyclodextrin (βCD) resides in the wild-type αHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric αHL pores that are capable of accommodating βCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane β barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind βCD ∼10(4)-fold more avidly than the remaining αHL pores, including WT-αHL. The lower K (d) values of these mutants are dominated by reduced values of k(off). The major effect of the mutations is most likely a remodeling of the binding site for βCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral βCD molecule by electroosmotic flow. The mutant pores for which the dwell time of βCD is prolonged can serve as improved components for stochastic sensors

Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry

Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric alpha-hemolysin pore (alpha HL) has been engineered to form a nanoreactor to study covalent chemistry at the single -molecule level. Previous work has been confined largely to the chemistry of cysteine side chains or, in one instance, to an irreversible reaction of an unnatural amino acid side chain bearing a terminal alkyne. Here, we present four different alpha HL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic alpha HL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side chain at the single -molecule level

Motricidad fina en los niños de 5 años de la Institución Educativa N° 208

En la Institución Educativa 208 María Inmaculada – Trujillo, en un estudio del año 2023 titulado Motricidad Fina en niños de cinco años, el objetivo general fue determinar la motricidad fina de los niños de cinco años de la institución educativa 208 María Inmaculada – Trujillo. El tipo de investigación es descriptiva; el diseño no experimental, descriptivo y los métodos son cuantitativos, diecisiete niños de la Institución Educativa Inicial 208 María Inmaculada conformaron la muestra El método de recolección de datos utilizado fue una ficha de observación como herramienta. La validez de la herramienta se basa en el juicio de expertos y su confiabilidad mediante el Alfa de Cronbach. El valor es igual a 954. Las habilidades motoras finas son altas para fines generales. Esta información se refleja en la tabla, en la que se destaca que de 15 niños en el mismo 60%, 10 tenían un nivel alto de motricidad fina (42,6%). Por lo tanto, el estudio es estadísticamente consistente con la muestra de prueba. Fuentes elaboradas por los propios investigadores de educación inicial - Trujillo 2023

Prolonged Residence Time of a Noncovalent Molecular Adapter, β-Cyclodextrin, within the Lumen of Mutant α-Hemolysin Pores

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the α-hemolysin (αHL) pore, thereby offering a basis for the detection of a variety of organic molecules with αHL as a sensor element. β-Cyclodextrin (βCD) resides in the wild-type αHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric αHL pores that are capable of accommodating βCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane β barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind βCD ∼10(4)-fold more avidly than the remaining αHL pores, including WT-αHL. The lower K (d) values of these mutants are dominated by reduced values of k(off). The major effect of the mutations is most likely a remodeling of the binding site for βCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral βCD molecule by electroosmotic flow. The mutant pores for which the dwell time of βCD is prolonged can serve as improved components for stochastic sensors

Location of a Constriction in the Lumen of a Transmembrane Pore by Targeted Covalent Attachment of Polymer Molecules

Few methods exist for obtaining the internal dimensions of transmembrane pores for which 3-D structures are lacking or for showing that structures determined by crystallography reflect the internal dimensions of pores in lipid bilayers. Several approaches, involving polymer penetration and transport, have revealed limiting diameters for various pores. But, in general, these approaches do not indicate the locations of constrictions in the channel lumen. Here, we combine cysteine mutagenesis and chemical modification with sulfhydryl-reactive polymers to locate the constriction in the lumen of the staphylococcal alpha-hemolysin pore, a model protein of known structure. The rates of reaction of each of four polymeric reagents (MePEG-OPSS) of different masses towards individual single cysteine mutants, comprising a set with cysteines distributed over the length of the lumen of the pore, were determined by macroscopic current recording. The rates for the three larger polymers (1.8, 2.5, and 5.0 kD) were normalized with respect to the rates of reaction with a 1.0-kD polymer for each of the seven positions in the lumen. The rate of reaction of the 5.0-kD polymer dropped dramatically at the centrally located Cys-111 residue and positions distal to Cys-111, whether the reagent was applied from the trans or the cis side of the bilayer. This semi-quantitative analysis sufficed to demonstrate that a constriction is located at the midpoint of the pore lumen, as predicted by the crystal structure, and although the constriction allows a 2.5-kD polymer to pass, transport of a 5.0-kD molecule is greatly restricted. In addition, PEG chains gave greater reductions in pore conductance when covalently attached to the narrower regions of the lumen, permitting further definition of the interior of the pore. The procedures described here should be applicable to other pores and to related structures such as the vestibules of ion channels