Elucidating Zinc Distribution In Cancerous Prostate Cells Using Novel FRET Sensors

Zinc (Zn2+) is an important trace element that is found throughout the human body and plays essential roles in proteins to ensure structural integrity and catalytic activity. It is the second most abundant transition metal in the body. Transition metals are required for numerous enzymes, proteins, and cellular processes. It is important for cells to regulate distribution of metals because numerous enzymes and cellular processes depend on transition metals, yet metal imbalance leads to a wide range of diseases. Prostate tissue has the highest Zn2+ concentration in the body compared to other tissues but Zn2+ is significantly depleted upon onset of malignancy. Numerous studies have revealed that cancerous prostate tissue exhibits dramatically reduced levels of Zn2+ and that the levels of Zn2+ appear to correlate with progression from benign to invasive and metastatic cancer. The mechanistic correlation between malignancy and Zn2+ levels is not well understood and in particular, it is not clear whether Zn2+ depletion contributes to or is a consequence of disease progression. Although the majority of Zn2+ found in cells in bound by proteins, enzymes, and cellular ligands, pools of free Zn2+ have been identified in pancreatic islet cells, brain, and prostate. In this current study we used normal and cancerous prostate cells as a model system to map free Zn2+ levels in the nucleus, cytosol, endoplasmic reticulum, and mitochondria to identify the differences in Zn2+ at the subcellular level. This was accomplished by using novel Zn2+ sensors based on fluorescence resonance energy transfer (FRET) targeted to each organelle compartment and carrying out live cell imaging experiments. FRET sensors are powerful tools to monitor Zn2+ dynamics in cells. This type of probes gives us the ability to monitor Zn2+ in multiple cellular comparments or simultaneously track two cellular signals using orthogonal FRET sensors. Such studies allow us to precisely correlate the timing of two interdependent cellular events or to track the movement of ions or molecules from one compartment to another. We discovered that the free Zn2+ pool in the nucleus and cytosol is higher in our prostate cancer cell line models compared to normal cell line. In the ER we observed that the free Zn2+ level in normal prostate cells is about 4 times higher than all three prostate cancer cell lines models used. Similar results were observed in mitochondria, revealing 3-4 times higher Zn2+ in the normal cells compared to all three prostate cancer cell lines. Although all three prostate cancer cell lines used in this study were characterized by over 50% reduction in total cellular Zn2+, surprisingly we discovered this reduction did not translate into across-the-board deficiency in intracellular Zn2+ stores, but rather there was substantial redistribution of Zn2+ between subcellular locations. In an effort to define how and why Zn2+ pools are redistributed in prostate cancer, we measured the changes in expression levels of key Zn2+ regulatory proteins such as transporters (hZIP1, hZIP2, hZIP3, ZnT1, ZnT2, ZnT4, and ZnT7). Our results suggest that there is a dysregulation of the Zn2+ transporters in cancerous prostate cells and there is some heterogeneity between the prostate carcinoma cell lines. To complement existing tools, we developed a suite of sensors using alternately colored FRET pairs using tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE that were used simultaneously with CFP-YFP sensors. Using these combinations of FRET sensors we were able to monitor Zn2+ uptake simultaneously in two compartments, revealing that nuclear Zn2+ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn2+ more slowly and with a delay of 600-700 sec. These new green-red sensors provided the starting point for developing vesicle-targeted sensors. In summary, this thesis details efforts to develop new fluorescent sensors for defining free Zn2+ in cells and applies these sensors to quantify free Zn2+ in normal prostate and cancerous cells at the subcellular level. We discovered unprecedented redistribution of Zn2+ stores in all three cancer cell lines and discovered this correlates, at least in part, to alteration of Zn2+ transporters. This work not only provides the first quantitative maps of Zn2+ distribution in cells, it also provides a critical glimpse of how Zn2+ is altered with disease progression

In vitro activity effects of combinations of cephalothin, dicloxacillin, imipenem, vancomycin and amikacin against methicillin-resistant Staphylococcus spp. strains

BACKGROUND: combinations of drugs has been proposed as an alternative for oxacillin-resistant staphylococci infections, however, limited information about in vitro combinations are available for multi-resistant strains. The objective of this study was to describe the interaction of beta-lactams in combination with vancomycin or amikacin against 26 oxacillin and amikacin-resistant nosocomial Staphylococcus spp. isolates. METHODS: activity of dicloxacillin plus amikacin, cephalothin plus amikacin, cephalothin plus vancomycin, imipenem plus vancomycin and vancomycin plus amikacin was evaluated by checkerboard synergy tests and the fractional inhibitory concentration index (FIC) was calculated. Results: dicloxacillin plus amikacin, and cephalothin plus amikacin were synergistic or partially synergistic in 84.6% and 100% respectively. For nearly half of the isolates the mean concentrations of dicloxacillin, cephalothin and amikacin at which FIC indexes were calculated were achievable therapeutically. Vancomycin plus amikacin had synergistic effect only against two isolates, and partially synergistic in 38.6%. For the combinations vancomycin plus cephalothin and vancomycin plus imipenem the effect was additive in 76.9% and 80.7% respectively. CONCLUSION: in this study the checkerboard analysis showed that amikacin in combination with cephalothin or dicloxacillin was synergistic against most of the resistant strains of S. aureus and coagulase-negative Staphylococcus. Vancomycin in combination with a beta-lactam (cephalothin or imipenem) showed additivity. An indifferent effect predominated for the combination vancomycin plus amikacin. Even though a synergistic effect is expected when using a beta-lactam plus amikacin combination, it is possible that the effect cannot be clinically achievable. Careful selection of antimicrobial combinations and initial MICs are mandatory for future evaluations

Electrochemical Properties of Al2O3-Fe/Si Composites Prepared by High-Energy Mechanical Milling

open accessThe growing demand in the manufacture of advanced materials with desired and unique properties (e.g. high mechanical strength, durability, good corrosion resistance and low cost of maintenance/replacing) is one of reasons to motivate the researchers to pay special attention in Ceramics as high performance materials for industrial applications. This is because conventional materials cannot meet the engineering requirements during their service that is why the need for advanced ceramic materials to achieve these industrial requirements. In the present work a study was made on Al2O3-matrix ceramic composites reinforced with 2%wt. or 5% wt. of Fe/Si particulates that were produced using a mechanical ball milling at high-energy condition. The electrochemical behavior of these ceramics was investigated by anodic polarization and Electrochemical Impedance Spectroscopy (EIS) measurements in a solution containing 0.5N NaCl, whereas the morphology and microstructural features were examined by optical or Scanning Electron Microscopy (SEM)

RELACIÓN DE LAS TECNOLOGÍAS DE LA INFORMACIÓN Y COMUNICACIÓN CON EL TURISMO RURAL, CASO SIERRA DE SONORA

Con el uso ampliado, continuo y variado del desarrollo de tecnologías en el sector turismo se encuentra en constante cambio, de tal forma que en los últimos años se ha estado presentando un proceso de reorganización y cambio en su forma de operación. Con relación a lo anterior, la presente investigación pretende determinar el uso de las TIC´s con el turismo rural en la sierra de Sonora, así como el uso que los turistas hacen de la tecnología, ya que basta con tener acceso internet por medio de una computadora, tableta o teléfono celular para poder planificar un viaje; y dado que mediante una encuesta personal se arrojó información que conecte con los turistas que visitaron parte de la región serrana de Sonora, durante el período vacacional de semana Santa 2017. Dentro de los resultados más importantes derivados de la presente investigación es que la procedencia de los turistas fue de la capital de Sonora, de entre los 18 a 31 años de edad y es precisamente en esta edad que le dan mayor importancia al uso de las TIC´s en el turismo. Además, el uso del celular es el que mayormente es utilizado para no solo mantenerse comunicado con familia o amigos, sino que es la principal herramienta por la cual consultan, reservan y se informan de los acontecimientos turísticos. Conjuntando la información se estará en posibilidad de incrementar el uso de recursos para mantener la conexión de las TIC´s con el turismo, para que no sea solo una herramienta de viaje, sino de uso de recursos del área donde el turista visite

INTRODUCCIÓN A LA INGENIERÍA DE MATERIALES

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