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The Woarani of the Pastaza Province in Amazonian Ecuador face complex pressures both externally and internally to develop. While there are both positive and negative impacts associated with this transition, it remains a problem that development solutions designed external to the system they’re meant to impact risk imposing asymmetries on that system. This research explores the case of a culturally and historically embedded boundary organization operating at the nexus between Western academics and two communities of Woarani in the Pastaza Province of Ecuador. Through unstructured and semi-structured interviews, field- observation and social and historical research, this study seeks to understand: 1) the pathways to development for the Waorani, what and why they choose what they do and, 2) in the context of the I-W boundary at the field school site, the elements of development that are critical for sustainable scaling. Key findings include eight elements that explain development at the field school and situate the school as a boundary organization. Another important finding is the support for sustainable scaling, that in this context, sustainable scaling is modeled by organic growth along known networks of relations.
Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.
X-ray free electron lasers are used in measuring diffraction patterns from nanocrystals in the 'diffract-before-destroy' mode by outrunning radiation damage. The finite-sized nanocrystals provide an opportunity to recover intensity between Bragg spots by removing the modulating function that depends on crystal shape, i.e. the transform of the crystal shape. This shape-transform dividing-out scheme for solving the phase problem has been tested using simulated examples with cubic crystals. It provides a phasing method which does not require atomic resolution data, chemical modification to the sample, or modelling based on the protein databases. It is common to find multiple structural units (e.g. molecules, in symmetry-related positions) within a single unit cell, therefore incomplete unit cells (e.g. one additional molecule) can be observed at surface layers of crystals. In this work, the effects of such incomplete unit cells on the 'dividing-out' phasing algorithm are investigated using 2D crystals within the projection approximation. It is found that the incomplete unit cells do not hinder the recovery of the scattering pattern from a single unit cell (after dividing out the shape transforms from data merged from many nanocrystals of different sizes), assuming that certain unit-cell types are preferred. The results also suggest that the dynamic range of the data is a critical issue to be resolved in order to apply the shape transform method practically.
X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.
Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.
Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.
Methodology
We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.
Principal Findings
We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.
Conclusions
Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.
