Micromechanical Study of Hyperacetylated Nucleosomes Using Single Molecule Transverse Magnetic Tweezers.

Nucleosomes are stable complexes of DNA and histone proteins that are essential for the proper functioning of the genome. These structures must be unwrapped and disassembled for processes such as gene expression, replication, and repair. Histone post-translational modifications (PTMs) are known to play a significant role in regulating the structural changes of nucleosomes. However, the underlying mechanisms by which these modifications function remain unclear. In this study, we report the results of single molecule micromanipulation experiments on DNA-protein complexes composed of hyperacetylated histone proteins using transverse magnetic tweezers. The experiments were conducted by pre-extending λ-DNA with a force less than 4 pN before introducing hyperacetylated histones into the sample chamber. The DNA shortened as the histones formed complexes with it and the nucleosome arrays were then exposed to increasing tension, resulting in quantized changes in the DNA's extension with step sizes of (integral multiples of) ~50 nm. We also compared results of experiments using PTM histones and native histones with data collected for both types of histones for the same force ranges (2-80 pN) and loading rates. Our data show that hyperacetylated nucleosomes require an unbinding force of around ~2.5 pN, which is similar to that required for native histones. Moreover, we identified clear differences between the step-size distributions of native and hyperacetylated histones and found that in contrast to tethers reconstituted with native histones, the majority of nucleosomes in tethers compacted with hyperacetylated histones underwent disassembly at forces significantly lower than 6 pN.

Magnetic tweezers: development and use in single-molecule research.

The use of magnetic tweezers for single-molecule micromanipulation has evolved rapidly since its introduction approximately 30 years ago. Magnetic tweezers have provided important insights into the dynamic activity of DNA-processing enzymes, as well as detailed, high-resolution information on the mechanical properties of DNA. These successes have been enabled by major advancements in the hardware and software components of these devices. These developments now allow for a much richer mechanistic understanding of the functions and mechanisms of DNA-binding enzymes. In this review, the authors briefly discuss the fundamental principles of magnetic tweezers and describe the advancements that have made it a superlative tool for investigating, at the single-molecule level, DNA and its interactions with DNA-binding proteins.

A Horizontal Magnetic Tweezers for Studying Single DNA Molecules and DNA-Binding Proteins.

We report data from single molecule studies on the interaction between single DNA molecules and core histones using custom-designed horizontal magnetic tweezers. The DNA-core histone complexes were formed using λ-DNA tethers, core histones, and NAP1 and were exposed to forces ranging from ~2 pN to ~74 pN. During the assembly events, we observed the length of the DNA decrease in approximate integer multiples of ~50 nm, suggesting the binding of the histone octamers to the DNA tether. During the mechanically induced disassembly events, we observed disruption lengths in approximate integer multiples of ~50 nm, suggesting the unbinding of one or more octamers from the DNA tether. We also observed histone octamer unbinding events at forces as low as ~2 pN. Our horizontal magnetic tweezers yielded high-resolution, low-noise data on force-mediated DNA-core histone assembly and disassembly processes.

A Horizontal Magnetic Tweezers and Its Use for Studying Single DNA Molecules.

We report the development of a magnetic tweezers that can be used to micromanipulate single DNA molecules by applying picoNewton (pN)-scale forces in the horizontal plane. The resulting force⁻extension data from our experiments show high-resolution detection of changes in the DNA tether's extension: ~0.5 pN in the force and <10 nm change in extension. We calibrate our instrument using multiple orthogonal techniques including the well-characterized DNA overstretching transition. We also quantify the repeatability of force and extension measurements, and present data on the behavior of the overstretching transition under varying salt conditions. The design and experimental protocols are described in detail, which should enable straightforward reproduction of the tweezers.

Reporte de nuestra experiencia en la identificación del ganglio centinela en cáncer de mama: comparación entre la técnica de azul patente y la técnica combinada (azul patente + tecnecio 99) / Diagnostic accuracy of sentinel lymph node biopsy (sln) in the management of breast cancer at Hospital Privado Universitario de Córdoba: comparison of the detection rate between blue dye (patent blue) technique and combined technique (blue dye + radioactive coloid Tc99)

Objetivo Determinar la capacidad diagnóstica de la técnica del ganglio centinela (gc) en cáncer de mama temprano en el Hospital Privado Universitario de Córdoba. Comparar la tasa de detección entre la técnica de azul patente y la técnica combinada (azul patente + Tecnecio 99). Material y método Se estudiaron 640 pacientes con cáncer de mama en las que se realizó la biopsia del gc entre los años 2008 y 2016. Fueron divididas en: Grupo 1 (565 pacientes), en el que se utilizó solo azul patente como técnica para su identificación; y Grupo 2 (75 pacientes), en el que se empleó la técnica combinada. El estudio del gc se llevó a cabo con impronta citológica para el examen intraoperatorio y con hematoxilina y eosina (h/e) para el examen diferido. Se concretó la linfadenectomía axilar (la) en aquellos casos donde el gc fue positivo (por congelación o diferido) o bien en los que no pudo ser identificado. Resultados La tasa de detección global del gc fue del 94% (587), siendo del 91% (514) cuando se utilizó solo azul patente y de 97% (73) con la técnica combinada. El 18,6% (109) del total de los gc fueron positivos. La sensibilidad de la impronta citológica del gc fue del 69,7%. Conclusiones La biopsia del gc es el método de elección para la estadificación axilar en el cáncer mama inicial, ya que ha demostrado ser seguro, eficaz y asociarse a una baja morbilidad. Los porcentajes de identificación del gc ascendieron del 91% al 97% con la incorporación del Tc 99. Al comparar con series internacionales y nacionales, no hubo diferencias significativas en cuanto a la sensibilidad y tasa de falsos negativos del método intraoperatorio.

Objectives To determine the diagnostic accuracy of sentinel lymph node biopsy (sln) in the management of breast cancer at Hospital Privado Universitario de Córdoba. To compare the detection rate between blue dye (patent blue) technique and combined technique (blue dye + radioactive coloid Tc99). Materials and method 640 breast cancer patients treated with sln were studied between 2008 and 2016. They were divided in 2: Group 1, where snl was identified using blue dye; and Group 2, where a combined technique was used. The imprint cytology was used for intraoperative examination, and the evaluation with hematoxylin and eosin (h&e) was used for the deferred examination. An axillary lymph node dissection (alnd) was performed in positive sln cases and when the sln wasn´t founded. Results Overall detection rate of sln was 94% (587), while only 91% (514) when using patent blue, and 97% (73) when using blue dye and radioactive tracer combined. The 18,6% (109) of all sln were positive. The overall sensitivity of imprint cytology of sln was 69,7%. Conclusions sln biopsy is an accurate and safe technique to assess the axillary stage in our population. The identification percentage rose from 91% to 97% with the introduction of Tc 99. It was no difference in sensitivity and false negative rate between our results and national and international data.