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Conventional approaches for studying and molecular typing of tumors include PCR, blotting, omics, immunocytochemistry, and immunohistochemistry. The last two methods are the most used, as they enable detecting both tumor protein markers and their localizations within the cells. In this study, we have investigated a possibility of using RNA aptamers, in particular, 2′-F-pyrimidyl-RNA aptamer ME07 (48 nucleotides long), specific to the receptor of epidermal growth factor (EGFR, ErbB1, Her1), as an alternative to monoclonal antibodies for aptacytochemistry and aptahistochemistry for human glioblastoma multiforme (GBM). A specificity of binding of FAM-ME07 to the receptor on the tumor cells has been demonstrated by flow cytometry; an apparent dissociation constant for the complex of aptamer – EGFR on the cell has been determined; a number of EGFR molecules has been semi-quantitatively estimated for the tumor cell lines having different amount of EGFR: A431 (106 copies per cell), U87 (104 copies per cell), MCF7 (103 copies per cell), and ROZH, primary GBM cell culture derived from patient (104 copies per cell). According to fluorescence microscopy, FAM-ME07 interacts directly with the receptors on A431 cells, followed by its internalization into the cytoplasm and translocation to the nucleolus; this finding opens a possibility of ME07 application as an escort aptamer for a delivery of therapeutic agents into tumor cells. FAM-ME07 efficiently stains sections of GBM clinical specimens, which enables an identification of EGFR-positive clones within a heterogeneous tumor; and providing a potential for further studying animal models of GBM.

While offering high-precision control of neural circuits, optogenetics is hampered by the necessity to implant fiber-optic waveguides in order to deliver photons to genetically engineered light-gated neurons in the brain. Unlike laser light, X-rays freely pass biological barriers. Here we show that radioluminescent Gd2(WO4)3:Eu nanoparticles, which absorb external X-rays energy and then downconvert it into optical photons with wavelengths of ∼610 nm, can be used for the transcranial stimulation of cortical neurons expressing red-shifted, ∼590-630 nm, channelrhodopsin ReaChR, thereby promoting optogenetic neural control to the practical implementation of minimally invasive wireless deep brain stimulation.

Zinc influx and efflux events are essential for meiotic progression in oocytes of several mammalian and amphibian species, but it is less clear whether this evolutionary conservation of zinc signals is also important in late-stage germline development in invertebrates. Using quantitative, single cell elemental mapping methods, we find that Caenorhabditis elegans oocytes undergo significant stage-dependent fluctuations in total zinc content, rising by over sevenfold from Prophase I through the beginning of mitotic divisions in the embryo. Live imaging of the rapid cell cycle progression in C. elegans enables us to follow changes in labile zinc pools across meiosis and mitosis in single embryo. We find a dynamic increase in labile zinc prior to fertilization that then decreases from Anaphase II through pronuclear fusion and relocalizes to the eggshell. Disruption of these zinc fluxes blocks extrusion of the second polar body, leading to a range of mitotic defects. We conclude that spatial temporal zinc fluxes are necessary for meiotic progression in C. elegans and are a conserved feature of germ cell development in a broad cross section of metazoa.

Phytoplankton iron contents (i.e., quotas) directly link biogeochemical cycles of iron and carbon and drive patterns of nutrient limitation, recycling, and export. Ocean biogeochemical models typically assume that iron quotas are either static or controlled by dissolved iron availability. We measured iron quotas in phytoplankton communities across nutrient gradients in the Pacific Ocean and found that quotas diverged significantly in taxon‐specific ways from laboratory‐derived predictions. Iron quotas varied 40‐fold across nutrient gradients, and nitrogen‐limitation allowed diatoms to accumulate fivefold more iron than co‐occurring flagellates even under low iron availability. Modeling indicates such “luxury” uptake is common in large regions of the low‐iron Pacific Ocean. Among diatoms, both pennate and centric genera accumulated luxury iron, but the cosmopolitan pennate genus Pseudo‐nitzschia maintained iron quotas 10‐fold higher than co‐occurring centric diatoms, likely due to enhanced iron storage. Biogeochemical models should account for taxonomic and macronutrient controls on phytoplankton iron quotas.

Oligonucleotide RA36 contains two G-quadruplex modules with thrombin binding aptamer sequence GGTTGGTGTGGTTGG. Each of the modules potentially can bind thrombin while differing in functional activity. Despite that, previously published studies report a single dissociation constant for the thrombin:RA36 complex, which value varies widely. Here we address this discrepancy using electrophoretic mobility shift assay. Our results reveal that the interaction between RA36 and thrombin is a two-stage process. The two modules have different affinities for thrombin, which explains the discrepancy in the published data.

Iron is essential to living cells, acting as a cofactor in a number of important enzymes in metabolism; however in the absence of correct storage iron forms dangerous oxygen radicals. In both yeast and plants, iron is stored in a membrane-bound vacuole through the action of a vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii, a pathogen of medical and veterinary importance. Here, we assess the role of VIT, and iron storage, in T. gondii. We show that iron is restricted to a compartment in the parasite that does not overlap with zinc. By deleting VIT we find a slight growth defect in vitro, however the absence of VIT leads to hypersensitivity to iron, confirming its essential role in iron detoxification in the parasite. This hypersensitivity can be rescued by scavenging of oxygen radicals. In the absence of VIT, parasites store less iron and are at a growth disadvantage when moving into an iron-depleted environment. We show parasite VIT expression is regulated by iron levels at both the transcript and protein level, and by altering the distribution of VIT within the cell. In the absence of VIT, we find that T. gondii responds by altering expression of genes with a role in iron metabolism and by increasing the activity of the antioxidant protein catalase. We also show that iron detoxification has an important role both in parasite survival within macrophages and in virulence in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.

Scanning X-ray fluorescence (XRF) tomography provides powerful characterization capabilities in evaluating elemental distribution and differentiating their inter- and intra-cellular interactions in a three-dimensional (3D) space. Scanning XRF tomography encounters practical challenges from the sample itself, where the range of rotation angles is limited by geometric constraints, involving sample substrates or nearby features either blocking or converging into the field of view. This study aims to develop a reliable and efficient workflow that can (1) expand the experimental window for nanoscale tomographic analysis of local areas of interest within a laterally extended specimen, and (2) bridge 3D analysis at micrometer and nanoscales on the same specimen. We demonstrate the workflow using a specimen of HeLa cells exposed to iron oxide core and titanium dioxide shell (Fe3O4/TiO2) nanocomposites. The workflow utilizes iterative and multiscale XRF data collection with intermediate sample processing by focused ion beam (FIB) sample preparation between measurements at different length scales. Initial assessment combined with precise sample manipulation via FIB allows direct removal of sample regions that are obstacles to both incident X-ray beam and outgoing XRF signals, which considerably improves the subsequent nanoscale tomography analysis. This multiscale analysis workflow has advanced bio-nanotechnology studies by providing deep insights into the interaction between nanocomposites and single cells at a subcellular level as well as statistical assessments from measuring a population of cells.

The new APS-U 2-ID Microprobe instrument will provide high-throughput and versatile sample analysis capabilities and will bridge the gap to higher resolution APS-U nanoprobes. Available techniques include XRF microscopy, tomography, µXANES, µSAXS, ptychography, and laminography. A very high flux of more than 1013 photon/s is delivered across a wide range of x-ray energies from 7-20 keV. KB mirror focusing optics enable a small 50nm spot size for detailed resolution, while also providing a long working distance of 50 mm to accommodate large samples of up to 10x10 mm. The Microprobe will accommodate samples in an inert helium environment, with controlled flow of fluids, electrical biasing, and sample heating & cooling. High throughput is enabled by fast and smooth fly-scanning stages, a high-accuracy metrology system, new detectors, and upgrades in the multi-bend achromat storage ring, insertion device, 2-ID beamline, and monochromator. All these instrument upgrades will empower advancements in biological, materials and environmental sciences.

Tomography using diffracted x-rays produces reconstructions mapping quantities including crystal lattice parameter(s), crystallite size and crystallographic texture; this information is quite different from that obtained with absorption contrast or phase contrast. Diffraction tomography can be performed using energy dispersive diffraction (EDD) and polychromatic synchrotron x-radiation. In EDD, different, properly-oriented Bragg planes diffract different x-ray energies; these intensities are measured by an energy sensitive detector. A pencil beam defines the irradiated volume, and a collimator before the energy sensitive detector selects which portion of the irradiated column is sampled at any one time. A 3D map is assembled by translating the specimen along X, Y and Z axes. This paper reports results of 3D mapping of the integrated intensity of several reflections from the bioapatite in the mineralized cartilage centrum of a blue shark. The multiple detector EDD system at 6-BM-B, the Advanced Photon Source was used to map an entire blue shark centrum. The shark centrum consists of a double cone structure (corpora calcerea) supported by the intermedialia consisting of four wedges. The integrated intensities of the c-axis reflection and of a reflection with no c-axis component reveals the bioapatite within the cone wall is oriented with its c-axes lateral, i.e., perpendicular to the axis of the backbone, whereas the bioapatite within the wedges is oriented with its c-axes axial. Results of absorption microCT (laboratory and synchrotron) and x-ray excited x-ray fluorescence mapping are included to provide higher resolution data of the structures underlying the EDD maps. Application of EDD tomography to 3D mapping of large specimens promises to add to the understanding of other mineralized tissue samples which cannot be sectioned.

Simple Summary Metal-oxide nanomaterials enter cancer and normal cells even when not specifically targeted, and often interact with specific cellular structures and biological molecules solely due to their innate physical-chemical properties. This raises concerns for the use of nanoparticles, which can be alleviated only with rigorous studies of nanoparticle–cell interactions, studies independent of post-interaction labeling of nanomaterials. X-ray fluorescence microscopy is an imaging technique that quantifies and maps all chemical elements from the periodic table solely based on their native fluorescence excited by the incoming X-ray. We used two different instruments to interrogate the same sample in 3D at two different resolutions and determine heterogeneity of cell-to-cell interactions with nanomaterials, as well as subcellular nanoparticle distribution. This is the first example of multi-scale 3D X-ray fluorescence imaging. This work begins a new era of study on how nanoparticle-based therapies can be developed to be more predictable and safer for use. Abstract Research in cancer nanotechnology is entering its third decade, and the need to study interactions between nanomaterials and cells remains urgent. Heterogeneity of nanoparticle uptake by different cells and subcellular compartments represent the greatest obstacles to a full understanding of the entire spectrum of nanomaterials’ effects. In this work, we used flow cytometry to evaluate changes in cell cycle associated with non-targeted nanocomposite uptake by individual cells and cell populations. Analogous single cell and cell population changes in nanocomposite uptake were explored by X-ray fluorescence microscopy (XFM). Very few nanoparticles are visible by optical imaging without labeling, but labeling increases nanoparticle complexity and the risk of modified cellular uptake. XFM can be used to evaluate heterogeneity of nanocomposite uptake by directly imaging the metal atoms present in the metal-oxide nanocomposites under investigation. While XFM mapping has been performed iteratively in 2D with the same sample at different resolutions, this study is the first example of serial tomographic imaging at two different resolutions. A cluster of cells exposed to non-targeted nanocomposites was imaged with a micron-sized beam in 3D. Next, the sample was sectioned for immunohistochemistry as well as a high resolution “zoomed in” X-ray fluorescence (XRF) tomography with 80 nm beam spot size. Multiscale XRF tomography will revolutionize our ability to explore cell-to-cell differences in nanomaterial uptake.

Introduction: TheraSphere® microspheres containing yttrium 90Y are among many radioembolization agents used clinically to reduce liver tumor burden, and their effects on cancer volume reduction are well-established. At the same time, concerns about off target tissue injury often limit their use. Deeper investigation into tissue distribution and long-term impact of these microspheres could inform us about additional ways to use them in practice.Methods: Healthy rat liver and rabbit liver tumor samples from animals treated with TheraSpheres were sectioned and their elemental maps were generated by X-ray fluorescence microscopy (XFM) at the Advanced Photon Source (APS) synchrotron at Argonne National Laboratory (ANL).Results: Elemental imaging allowed us to identify the presence and distribution of TheraSpheres in animal tissues without the need for additional sample manipulation or staining. Ionizing radiation produced by 90Y radioactive contaminants present in these microspheres makes processing TheraSphere treated samples complex. Accumulation of microspheres in macrophages was observed.Conclusions: This is the first study that used XFM to evaluate the location of microspheres and radionuclides in animal liver and tumor samples introduced through radioembolization. XFM has shown promise in expanding our understanding of radioembolization and could be used for investigation of human patient samples in the future.

Objective: Copper (Cu) is essential micronutrient, and its dysregulation is implicated in aortic aneurysm (AA) development. The Cu exporter ATP7A (copper-transporting P-type ATPase/Menkes ATPase) delivers Cu via the Cu chaperone Atox1 (antioxidant 1) to secretory Cu enzymes, such as lysyl oxidase, and excludes excess Cu. Lysyl oxidase is shown to protect against AA formation. However, the role and mechanism of ATP7A in AA pathogenesis remain unknown. Approach and Results: Here, we show that Cu chelator markedly inhibited Ang II (angiotensin II)–induced abdominal AA (AAA) in which ATP7A expression was markedly downregulated. Transgenic ATP7A overexpression prevented Ang II–induced AAA formation. Conversely, Cu transport dysfunctional ATP7A mut/+ /ApoE −/− mice exhibited robust AAA formation and dissection, excess aortic Cu accumulation as assessed by X-ray fluorescence microscopy, and reduced lysyl oxidase activity. In contrast, AAA formation was not observed in Atox1 −/− /ApoE −/− mice, suggesting that decreased lysyl oxidase activity, which depends on both ATP7A and Atox1, was not sufficient to develop AAA. Bone marrow transplantation suggested importance of ATP7A in vascular cells, not bone marrow cells, in AAA development. MicroRNA (miR) array identified miR-125b as a highly upregulated miR in AAA from ATP7A mut/+ /ApoE −/− mice. Furthermore, miR-125b target genes (histone methyltransferase Suv39h1 and the NF-κB negative regulator TNFAIP3 [tumor necrosis factor alpha induced protein 3]) were downregulated, which resulted in increased proinflammatory cytokine expression, aortic macrophage recruitment, MMP (matrix metalloproteinase)-2/9 activity, elastin fragmentation, and vascular smooth muscle cell loss in ATP7A mut/+ /ApoE −/− mice and reversed by locked nucleic acid-anti-miR-125b infusion. Conclusions: ATP7A downregulation/dysfunction promotes AAA formation via upregulating miR-125b, which augments proinflammatory signaling in a Cu-dependent manner. Thus, ATP7A is a potential therapeutic target for inflammatory vascular disease.

The Indian Ocean accounts for about one fifth of global ocean net primary production but remains undersampled relative to other major ocean basins. The eastern tropical Indian Ocean is characterized by extremely low concentrations of both macronutrients and the micronutrient iron. We measured concentrations of dissolved and particulate trace metals (Fe, Mn, Zn, Pb) in the upper ocean along the GO-SHIP IO9N transect (28˚S to 17˚N, mostly along the 95˚E meridian) during a cruise in April 2016. Cellular quotas (metal/C) of Fe, Mn, Co, Ni, Cu, and Zn were measured in small eukaryotic flagellates (2–7 μm). Deckboard bottle incubation experiments were conducted at one station in each of three putative biogeochemical regions: southern Indian Ocean gyre (SIO, 28-10˚S); equatorial Indian Ocean (EqIO, 10˚S - 5˚N); Bay of Bengal (BoB, 5-17˚N). Nitrate and phosphate were below detection limits in surface waters across the transect. Dissolved and particulate Fe were

The lack of precision therapies combined with limited therapeutic access to intracranial tumor sites due to the presence of the blood-brain/blood-tumor barriers have contributed to glioblastoma (GBM) being one of the most difficult cancers to effectively treat. We have developed a novel precision medicine approach for GBM treatment that involves the use of brain-penetrant RNA interference (RNAi)-based spherical nucleic acids (SNAs). In this study, we used SNAs consisting of gold nanoparticle cores covalently conjugated with radially oriented and densely packed siRNA oligonucleotides. Based upon previous preclinical evaluation, we conducted toxicology and toxicokinetic studies in non-human primates, and a single-arm, open-label phase 0 first-in-human trial (NCT03020017) to determine safety, pharmacokinetics, intratumoral accumulation and gene-suppressive activity of systemically administered SNAs carrying siRNA specific for the GBM oncogene Bcl2Like12 (Bcl2L12). Patients with recurrent GBM were treated with intravenous administration of siBcl2L12-SNAs (drug moniker: NU-0129), at a dose corresponding to 1/50th of the no-observed-adverse-event-level (NOAEL), followed by tumor resection. Safety assessment revealed no significant treatment-related toxicities. Inductively coupled plasma mass spectrometry, X-ray fluorescence microscopy, and silver staining of resected GBM tissue demonstrated that intravenously administered SNAs reached patient tumors, with gold (Au) enrichment observed in the tumor-associated endothelium, macrophages and tumor cells. NU-0129 uptake into glioma cells correlated with significant reduction in tumor-associated Bcl2L12 protein expression, as indicated by comparison of NU-0129-treated recurrent vs. matched primary (i.e., untreated) tumor. Our results establish SNA nanoconjugates as a brain-penetrant precision medicine approach for the systemic treatment of GBM.

Summary X-ray fluorescence microscopy (XFM) is a powerful tool for mapping and quantifying the spatial distribution of elemental composition of biological samples. Recently, it was reported that transition metal fluctuations occur during Drosophila reproduction, analogous to what is seen in mammals and nematodes, and may contribute to Drosophila female fertility. To further support XFM studies on Drosophila reproduction, we describe procedures for isolating oocytes and activated eggs and examining their elemental composition by XFM scanning and analysis. For complete details on the use and execution of this protocol, please refer to Hu et al. (2020)., Graphical abstract, Highlights • Preparing Drosophila oocytes and activated eggs for X-ray fluorescence microscopy scans • XFM scanning to reveal the elemental contents and distribution of samples • Analysis and interpretation of XFM data on Drosophila oocyte and egg samples, X-ray fluorescence microscopy (XFM) is a powerful tool for mapping and quantifying the spatial distribution of elemental composition of biological samples. Recently, it was reported that transition metal fluctuations occur during Drosophila reproduction, analogous to what is seen in mammals and nematodes, and may contribute to Drosophila female fertility. To further support XFM studies on Drosophila reproduction, we describe procedures for isolating oocytes and activated eggs and examining their elemental composition by XFM scanning and analysis.

Phosphorus is one of the essential nutrients for plant growth, but it may be relatively unavailable to plants because of its chemistry. In soil, the majority of phosphorus is present in the form of a phosphate, usually as metal complexes making it bound to minerals or organic matter. Therefore, inorganic phosphate solubilization is an important process of plant growth promotion by plant associated bacteria and fungi. Non-nodulating plant species have been shown to thrive in low-nutrient environments, in some instances by relying on plant associated microorganisms called endophytes. These microorganisms live within the plant and help supply nutrients for the plant. Despite their potential enormous environmental importance, there are a limited number of studies looking at the direct molecular impact of phosphate solubilizing endophytic bacteria on the host plant. In this work, we studied the impact of two endophyte strains of wild poplar (Populus trichocarpa) that solubilize phosphate. Using a combination of x-ray imaging, spectroscopy methods, and proteomics, we report direct evidence of endophyte-promoted phosphorus uptake in poplar. We found that the solubilized phosphate may react and become insoluble once inside plant tissue, suggesting that endophytes may aid in the re-release of phosphate. Using synchrotron x-ray fluorescence spectromicroscopy, we visualized the nutrient phosphorus inside poplar roots inoculated by the selected endophytes and found the phosphorus in both forms of organic and inorganic phosphates inside the root. Tomography-based root imaging revealed a markedly different root biomass and root architecture for poplar samples inoculated with the phosphate solubilizing bacteria strains. Proteomics characterization on poplar roots coupled with protein network analysis revealed novel proteins and metabolic pathways with possible involvement in endophyte enriched phosphorus uptake. These findings suggest an important role of endophytes for phosphorus acquisition and provide a deeper understanding of the critical symbiotic associations between poplar and the endophytic bacteria.

Collagen type I is a major constituent of animal bodies. It is found in large quantities in tendon, bone, skin, cartilage, blood vessels, bronchi, and the lung interstitium. It is also produced and accumulates in large amounts in response to certain inflammations such as lung fibrosis. Our understanding of the molecular organization of fibrillar collagen and cellular interaction motifs, such as those involved with immune-associated molecules, continues to be refined. In this study, antibodies raised against type I collagen were used to label intact D-periodic type I collagen fibrils and observed with atomic force microscopy (AFM), and X-ray diffraction (XRD) and immunolabeling positions were observed with both methods. The antibodies bind close to the C-terminal telopeptide which verifies the location and accessibility of both the major histocompatibility complex (MHC) class I (MHCI) binding domain and C-terminal telopeptide on the outside of the collagen fibril. The close proximity of the C-telopeptide and the MHC1 domain of type I collagen to fibronectin, discoidin domain receptor (DDR), and collagenase cleavage domains likely facilitate the interaction of ligands and receptors related to cellular immunity and the collagen-based Extracellular Matrix.

Hundreds of millions of people worldwide are exposed to unacceptable levels of carcinogenic inorganic arsenic. Animal models have shown that selenium and arsenic are mutually protective through the formation and elimination of the seleno-bis(S-glutathionyl) arsinium ion [(GS)2AsSe]−. Consistent with this, human selenium deficiency in arsenic-endemic regions is associated with arsenic-induced disease, leading to the initiation of human selenium supplementation trials. In contrast to the protective effect observed in vivo, in vitro studies have suggested that selenite increases arsenite cellular retention and toxicity. This difference might be explained by the rapid conversion of selenite to selenide in vivo. In the current study, selenite did not protect the human hepatoma (HepG2) cell line against the toxicity of arsenite at equimolar concentrations, however selenide increased the IC50 by 2.3-fold. Cytotoxicity assays of arsenite + selenite and arsenite + selenide at different molar ratios revealed higher overall mutual antagonism of arsenite + selenide toxicity than arsenite + selenite. Despite this protective effect, in comparison to 75Se-selenite, HepG2 cells in suspension were at least 3-fold more efficient at accumulating selenium from reduced 75Se-selenide, and its accumulation was further increased by arsenite. X-ray fluorescence imaging of HepG2 cells also showed that arsenic accumulation, in the presence of selenide, was higher than in the presence of selenite. These results are consistent with a greater intracellular availability of selenide relative to selenite for protection against arsenite, and the formation and retention of a less toxic product, possibly [(GS)2AsSe]−.

Bud dormancy allows deciduous perennial plants to rapidly grow following seasonal cold conditions. Although many studies have examined the hormonal regulation of bud growth, the role of nutrients remains unclear. Insufficient accumulation of the key micronutrient zinc (Zn) in dormant buds affects the vegetative and reproductive growth of perennial plants during the subsequent year, requiring the application of Zn fertilizers in orchard management to avoid growth defects in fruit trees. However, the mechanisms of seasonal Zn homeostasis in perennial plants remain poorly understood. Here, we provide new insights into Zn distribution and speciation within reproductive and vegetative buds of apple (Malus domestica) and four other deciduous fruit trees (peach [Amygdalus persica], grape [Vitis vinifera], pistachio [Pistacia vera], and blueberry [Vaccinium spp.]) using microscopic and spectroscopic characterization techniques comprising synchrotron-based x-ray fluorescence and x-ray absorption near-edge-structure analyses. By establishing a link between bud development and Zn distribution, we identified the following important steps of Zn storage and use in deciduous plants: Zn is preferentially deposited in the stem nodes subtending apical and axillary buds; Zn may then be sequestered as Zn-phytate prior to dormancy; in spring, Zn effectively releases for use during budbreak and subsequent meristematic growth. The mechanisms of Zn homeostasis during the seasonal cycles of plant growth and dormancy described here will contribute to improving orchard management, and to selection and breeding of deciduous perennial species.

Nucleic acid aptamers have been proven to be a useful tool in many applications. Particularly, aptamers to epidermal growth factor receptor (EGFR) have been successfully used for the recognition of EGFR-expressing cells, the inhibition of EGFR-dependent pathways, and targeted drug delivery into EGFR-positive cells. Several aptamers are able to discriminate wild-type EGFR from its mutant form, EGFRvIII. Aptamers to EGFR have hairpin-like secondary structures with several possible folding variations. Here, an aptamer, previously selected to EGFRvIII, was chosen as a lead compound for extensive post-SELEX maturation. The aptamer was 1.5-fold truncated, the ends of the hairpin stem were appended with GC-pairs to increase thermal stability, and single pyrene modification was introduced into the aptamer to increase affinity to the target protein. Pyrene modification was selected from extensive computer docking studies of a library of thousands of chemicals to EGFR near the EGF-binding interface. The resulting aptamers bound extracellular domains of both variants of EGFR: EGFRwt and EGFRvIII with subnanomolar apparent dissociation constants. Compared with the initial aptamer, affinity to EGFRwt was increased up to 7.5-fold, whereas affinity to EGFRvIII was increased up to 4-fold.

Temporal fluctuations in zinc concentration are emerging as essential signaling events in the regulation of meiosis during oogenesis and early embryogenesis. For example, accumulation and release of zinc in mammalian gametes are required for oocyte maturation and egg activation, respectively. Here, we report that significant zinc fluctuations occur in Drosophila melanogaster oocytes and activated eggs, and that zinc is required for female fertility. Using synchrotron-based X-ray fluorescence microscopy (XFM), we mapped the subcellular localization of essential transition metals during fly oogenesis and egg activation. Quantitative analysis of the XFM data reveals that zinc is the most abundant transition metal in oocytes and that its levels increase during oocyte maturation. This increase is accompanied by appearance of zinc-enriched intracellular granules in the oocyte. The zinc transporter Znt35C is required for maintenance of these zinc-enriched granules in mature oocytes. During egg activation, the process that follows oocyte maturation and mediates the transition of the egg to an embryo, oocyte zinc levels decrease significantly, accompanied by a decrease in the number of zinc-enriched granules. This essential pattern of zinc dynamics in Drosophila oocytes follows a similar trajectory to that in mammals, rising during oogenesis and decreasing during egg activation. This further extends the parallels in female gamete processes between Drosophila and mammals, suggesting that the former may provide an important genetically tractable model for studying the role of zinc in female reproduction.

The analysis of the dynamic infrastructure of the social virtuum as an increasing variety of social spheres and dimensions has been conducted. It was shown that to build its logistics in accordance with the cyclical, linear and rhizome changes that from radically re-equip is quite problematic; and therefore, society as a virtuum was explained largely as an abstraction, which, however, can exist as a material formation in space and time. On this basis, the main purpose of the research was conceptualization of the primary mechanisms for managing the social virtuum as a singular space-time continuum based on phenomenological divergence and convergence, as well as an analysis of its conditional logistics and the complexity of its virtual topology. The phenomenological method as the theoretical basis of this research showed the social virtuum as an interconnected unity of various principles of existence in its economic, political, cultural, civilizational, virtual, spiritual dimensions and as a set of events experienced by individuals as continuous primary experiences. The divergent and convergent approaches depicted social processes in continuous interaction, providing progressive movement, stable development and functioning of all existing subsystems of a complexsociety.

Acto-myosin cross-bridge kinetics are important for beat-to-beat regulation of cardiac contractility; however, physiological and pathophysiological mechanisms for regulation of contractile kinetics are incompletely understood. Here we explored whether thin filament-mediated Ca(2+) sensitization influences cross-bridge kinetics in permeabilized, osmotically compressed cardiac muscle preparations. We used a murine model of hypertrophic cardiomyopathy (HCM) harboring a cardiac troponin C (cTnC) Ca(2+)-sensitizing mutation, Ala8Val in the regulatory N-domain. We also treated wild-type murine muscle with bepridil, a cTnC-targeting Ca(2+) sensitizer. Our findings suggest that both methods of increasing myofilament Ca(2+) sensitivity increase cross-bridge cycling rate measured by the rate of tension redevelopment (k(TR)); force per cross-bridge was also enhanced as measured by sinusoidal stiffness and I(1,1)/I(1,0) ratio from X-ray diffraction. Computational modeling suggests that Ca(2+) sensitization through this cTnC mutation or bepridil accelerates k(TR) primarily by promoting faster cross-bridge detachment. To elucidate if myofilament structural rearrangements are associated with changes in k(TR), we used small angle X-ray diffraction to simultaneously measure myofilament lattice spacing and isometric force during steady-state Ca(2+) activations. Within in vivo lattice dimensions, lattice spacing and steady-state isometric force increased significantly at submaximal activation. We conclude that the cTnC N-domain controls force by modulating both the number and rate of cycling cross-bridges, and that the both methods of Ca(2+) sensitization may act through stabilization of cTnC’s D-helix. Furthermore, we propose that the transient expansion of the myofilament lattice during Ca(2+) activation may be an additional factor that could increase the rate of cross-bridge cycling in cardiac muscle. These findings may have implications for the pathophysiology of HCM.

Aptamers are widely used as molecular recognition elements for detecting and blocking functional biological molecules. Since the common "alphabet" of DNA and RNA consists of only four letters, the chemical diversity of aptamers is less than the diversity of protein recognition elements built of 20 amino acids. Chemical modification of nucleotides enlarges the potential of DNA/RNA aptamers. This review describes the latest achievements in a variety of approaches to aptamers selection with an extended genetic alphabet.

Results from experiments conducted for the parameter Residual Organic Solvents during the course of developing a draft regulation for the drug substance of the innovative pharmacological agent thrombaptanib are reported. The structure of the compound is given. Its pharmacological action is described. A list of potential residual organic solvents was compiled by analyzing the synthetic scheme and manufacturing technology. A separate part of the work describes in detail a developed GC-MS procedure for determining residual organic solvents. Validation results of the procedure for Specificity, Linearity, Accuracy, Precision, and Reproducibility are presented. The validation protocol for the procedure at the time of the studies followed domestic guides for validation that were written based on existing international documents because there was no existing GPM for validation in the current edition of the SP RF. A test sample of thrombaptanib was analyzed using the developed procedure for Residual Organic Solvents. The developed procedure was included in the Residual Organic Solvents section of the draft regulation.

Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is biologically distinct from HPV-negative HNSCC. Outside of HPV-status, few tumor-intrinsic variables have been identified that correlate to improved survival. As part of exploratory analysis into the trace elemental composition of oropharyngeal squamous cell carcinoma (OPSCC), we performed elemental quanitification by X-ray fluorescence microscopy (XFM) on a small cohort (n = 32) of patients with HPV-positive and -negative OPSCC and identified in HPV-positive cases increased zinc (Zn) concentrations in tumor tissue relative to normal tissue. Subsequent immunohistochemistry of six Zn-binding proteins—zinc-α2-glycoprotein (AZGP1), Lipocalin-1, Albumin, S100A7, S100A8 and S100A9—revealed that only AZGP1 expression significantly correlated to HPV-status (p AZGP1 mRNA levels correlated to improved overall survival (p = 0.023). By showing for the first time that HPV-positive OPSCC patients have increased intratumoral Zn levels and AZGP1 expression, we identify possible positive prognostic biomarkers in HNSCC as well as possible mechanisms of increased sensitivity to chemoradiation in HPV-positive OPSCC.

Arsenic contamination is a major environmental issue, as it may lead to serious health hazard. The reduced trivalent form of inorganic arsenic, arsenite, is in general more toxic to plants compared with the fully oxidized pentavalent arsenate. The uptake of arsenite in plants has been shown to be mediated through a large subfamily of plant aquaglyceroporins, nodulin 26-like intrinsic proteins (NIPs). However, the efflux mechanisms, as well as the mechanism of arsenite-induced root growth inhibition, remain poorly understood. Using molecular physiology, synchrotron imaging, and root transport assay approaches, we show that the cellular transport of trivalent arsenicals in Arabidopsis thaliana is strongly modulated by PIN FORMED 2 (PIN2) auxin efflux transporter. Root transport assay using radioactive arsenite, X-ray fluorescence imaging (XFI) coupled with X-ray absorption spectroscopy (XAS), and inductively coupled plasma mass spectrometry analysis revealed that pin2 plants accumulate higher concentrations of arsenite in roots compared with the wild-type. At the cellular level, arsenite specifically targets intracellular sorting of PIN2 and thereby alters the cellular auxin homeostasis. Consistently, loss of PIN2 function results in arsenite hypersensitivity in roots. XFI coupled with XAS further revealed that loss of PIN2 function results in specific accumulation of arsenical species, but not the other metals such as iron, zinc, or calcium in the root tip. Collectively, these results suggest that PIN2 likely functions as an arsenite efflux transporter for the distribution of arsenical species in planta., The cellular efflux mechanism of arsenite, the more toxic form of arsenic, remains elusive. Using molecular and cellular physiology, root transport assay, and synchrotron imaging, this study identified and characterized PIN Formed 2 (PIN2) as a new, putative arsenite efflux facilitator.

BACKGROUND G-quadruplex folding oligodeoxyribonucleotides (G4 ODNs) are considers to be potential anticancer agents, because G4 interferes with some functions of cell, like survival, proliferation, ets, by interacting with G4 binding regulatory proteins. There are some experimental data on anti-proliferative activities of G4: for telomerase complex, aptamers for STAT3, nucleolin, TOP1, SP1, VEGF, and SHP-2, but there is no molecular mechanism yet. Because ODNs are supposed to block regulatory proteins, like cryptic aptamers do, they could be potentially therapeutics, like monoclonal antibodies. The only current approach to find proper model for G4 plus reference cell line, is to screen sets of G4 for sets of cancer cell lines. To avoid selection from large number of G4 structures, we have initiated anti-proliferative study of minimal DNA nano-construct biHD1, built from two minimal active G4 15-mer, by testing 7 human cancer cell lines, including glioblastoma (GBM): U87 and primary cultures from GBM surgery samples. MATERIAL AND METHODS ODN were from Evrogen (Russia). ODN were folded: heated to 950C, slowly cooled to 250C. biHD1, GGTTGGTGTGGTTGG TGGTTGGTGTGGTTGG, and derivatives with G4 structure variations. CD in saline phosphate, pH 7.5; CHIRASCAN (UK) and MARK-5 (France); melting curves at 294 nm. 7 human cancer cell lines: colon, HCT116; prostate, PC3; lung, RL-67; breast adenocarcinoma, MCF7; melanoma, mS; HeLa; GBM, U87 and primary cultures from surgery samples; and human embryonic fibroblast, hEF, as control. MTT for cell viability by colorimetric measurements of metabolic activity (Tecan plate reader, Switzerland). ODN: 0.10 μM, 1.0 μM, 10 μM for 3 days. RESULTS biHD1 is dose-dependent toxic for several human cancer cell lines, most active for lung cancer RL-67 and GBM U87, survival at 10 μM ODN 35% and 45%, respectively. According to data for biHD1 derivatives with deviations from G4 conformation: length of linker joining modules, light distortions of loops, strong distortions of G4, freezing of G4 conformation, it is G4 fold which is responsible for toxicity for U87 cell line. CONCLUSION Cell survival after treatment with G4 minimal bimodular DNA oligonucleotide biHD1 and its derivatives have been tested for 7 human cancer cell lines, including CNS, U87. biHD1 is toxic for cancer cells, but not for embryonic fibroblast, as control; activity depends on G4 fold. Therefore, it is possible to manipulate with minimal G4 as module, and build G4 nano-construct being toxic for cancer cells, including GBM, U87. Research is supported by RFBR grants 17-00-00157, 18-29-01047

Arsenic contamination is a major environmental issue as it may lead to serious health hazard. Reduced trivalent form of inorganic arsenic, arsenite, is in general more toxic to plants compared with the fully oxidized pentavalent arsenate. The uptake of arsenite in plants has been shown to be mediated through a large subfamily of plant aquaglyceroporins, nodulin 26-like intrinsic proteins (NIPs). However, the efflux mechanisms, as well as the mechanism of arsenite-induced root growth inhibition, remain poorly understood. Using molecular physiology, synchrotron imaging, and root transport assay approaches, we show that the cellular transport of trivalent arsenicals inArabidopsis thalianais strongly modulated by PIN FORMED 2 (PIN2) auxin efflux transporter. Direct transport assay using radioactive arsenite, X-ray fluorescence imaging (XFI) coupled with X-ray absorption spectroscopy (XAS), and ICP-MS analysis revealed thatpin2plants accumulate higher concentrations of arsenite in root compared to wild-type. At the cellular level, arsenite specifically targets intracellular cycling of PIN2 and thereby alters the cellular auxin homeostasis. Consistently, loss of PIN2 results in aresenite hypersensitivity in root. XFI coupled with XAS further revealed that loss of PIN2 results in specific accumulation of arsenical species, but not the other metals like iron, zinc or calcium in the root tip. Collectively, these results demonstrate that PIN2 serves as a putative transporter of arsenical speciesin planta.

Background Efficacy and safety of netakimab (NTK), a humanized anti-IL17A antibody, was established in phase 2 clinical trials in patients (pts) with radiographic axial spondyloarthritis (r-axSpA)1 and psoriasis2. Objectives The abstract presents 16-week data from ongoing ASTERA study (NCT03447704) in pts with active r-axSpA. Methods ASTERA is a phase 3 international double-blind placebo (PBO)-controlled study. 228 adult pts with r-axSpA, active (BASDAI ≥ 4) despite the standard NSAIDs, were randomly assigned (1:1) to receive 120 mg NTK or PBO SC at Week (Wk) 0,1,2 and then q2w through Wk 16. After Wk 16 all pts will start to receive NTK up to Wk 52. Primary endpoint was ASAS40 rate at Wk 16. Results The mean age at baseline was 39.14±9.9 years, the mean symptoms duration was 4.3±4.5 years. 76.8% of pts were naive to biological treatment. At Wk 16 ASAS40 rate was higher in NTK arm compared to PBO: 40.35% versus (vs.) 2.63% pts (95% CI for the difference in ASAS40 rate was [27.37%; 48.07%] (p Conclusion NTK at a dose 120 mg is well-tolerated drug with favorable safety profile that leads to decline in r-axSpA activity, function improvement and axial mobility in 16 Wks. References [1] Mazurov V, et al. Ann Rheum Dis 2018;77:A64. 2Samtsov A, et al. Vestn Dermatol Venerol 2017;0(5):52-63. Disclosure of Interests Inna Gaydukova Grant/research support from: JSC BIOCAD, Speakers bureau: paiment from Pfizer, Novartis, Abbvie, Biocad, Selgene, MSD, Sanofy does not exceed 10 000 euros, V Mazurov Grant/research support from: JSC BIOCAD, Shandor Erdes Grant/research support from: JSC BIOCAD, Speakers bureau: JSC BIOCAD, Tatiana Dubinina: None declared, Olga Nesmeyanova Grant/research support from: JSC BIOCAD, Elena Ilivanova Grant/research support from: JSC BIOCAD, Alena Kundzer: None declared, Nikolaj Soroka: None declared, Aleksander Kastanayan Grant/research support from: JSC BIOCAD, Tatyana Povarova Grant/research support from: JSC BIOCAD, Elena Zhugrova Grant/research support from: JSC BIOCAD, Tatyana Plaksina Grant/research support from: JSC BIOCAD, Pavel Shesternya Grant/research support from: JSC BIOCAD, Tatyana Kropotina Grant/research support from: JSC BIOCAD, Olga Antipova Grant/research support from: JSC BIOCAD, Elena Smolyarchuk Grant/research support from: JSC BIOCAD, Oksana Tciupa Grant/research support from: JSC BIOCAD, Diana Abdulganieva: None declared, Diana Kretchikova Grant/research support from: JSC BIOCAD, Ivan Gordeev Grant/research support from: JSC BIOCAD, Vadim Tyrenko Grant/research support from: JSC BIOCAD, Aleksandra Strelkova Grant/research support from: JSC BIOCAD, Anna Eremeeva Grant/research support from: JSC BIOCAD, Ekaterina Chernyaeva Employee of: JSC BIOCAD, Roman Ivanov Employee of: JSC BIOCAD

Metal fasteners are used to hold wood structures together. In outdoor applications, these fasteners are subject to corrosion when the wood is treated with certain preservative treatments. Typically, these treatments contain copper. Prior work has hypothesized that the mechanism of corrosion in treated wood involves reduction of copper ions from the wood treatments. However, copper was rarely detected in the corrosion products of metals embedded in treated wood, which contradicts the hypothesized mechanism. This present work utilizes synchrotron based X-ray fluorescence microscopy (XFM) and X-ray absorption near edge spectroscopy to examine the corrosion mechanism in treated wood by looking at the spatial distribution and oxidation states of copper in the treated wood near the fastener and in the corrosion products removed from the fastener surface. The samples were obtained after a 1-year corrosion test. In the wood cell walls, the oxidation state of the copper treatment did not change in the immediate vicinity of the fastener, although there was a depletion of copper near the fastener. Furthermore, copper was detected in the corrosion products in trace amounts using XFM. Together, these techniques confirm that the corrosion mechanism involves transport of the cupric ions to the fastener surface, where they are reduced and suggest that previous attempts to detect copper were unsuccessful because the concentration of copper in the corrosion products was below the level of detection of the previously used techniques.

The fabrication and performance evaluation of single-crystal diamond refractive X-ray lenses of which the surfaces are paraboloids of revolution for focusing X-rays in two dimensions simultaneously are reported. The lenses were manufactured using a femtosecond laser micromachining process and tested using X-ray synchrotron radiation. Such lenses were stacked together to form a standard compound refractive lens (CRL). Owing to the superior physical properties of the material, diamond CRLs could become indispensable wavefront-preserving primary focusing optics for X-ray free-electron lasers and the next-generation synchrotron storage rings. They can be used for highly efficient refocusing of the extremely bright X-ray sources for secondary optical schemes with limited aperture such as nanofocusing Fresnel zone plates and multilayer Laue lenses.

Summary Temporal fluctuations in zinc concentration are essential signals, including during oogenesis and early embryogenesis. In mammals, zinc accumulation and release are required for oocyte maturation and egg activation, respectively. Here, we demonstrate that zinc flux occurs in Drosophila oocytes and activated eggs, and that zinc is required for female fertility. Our synchrotron-based X-ray fluorescence microscopy reveals zinc as the most abundant transition metal in Drosophila oocytes. Its levels increase during oocyte maturation, accompanied by the appearance of zinc-enriched intracellular granules in the oocyte, which depend on transporters. Subsequently, in egg activation, which mediates the transition from oocyte to embryo, oocyte zinc levels decrease significantly, as does the number of zinc-enriched granules. This pattern of zinc dynamics in Drosophila oocytes follows a similar trajectory to that in mammals, extending the parallels in female gamete processes between Drosophila and mammals and establishing Drosophila as a model for dissecting reproductive roles of zinc., Graphical Abstract, Highlights • Dietary zinc is required for Drosophila female fertility • Oocyte zinc levels increase over oogenesis and decrease during egg activation • Zinc is enriched in the oocyte and forms aggregated granules in mature oocytes • Transporters are required for zinc retention and zinc-enriched granules in oocytes, Evolutionary Developmental Biology; Biotechnology; Embryology