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Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers

G. D. Calvey, A. M. Katz, C. B. Schaffer, and L. Pollack,

Structural dynamics (2016)

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Knowledge of protein structure provides essential insight into function, enhancing our understanding of diseases and enabling new treatment development. X-ray crystallography has been used to solve the structures of more than 100 000 proteins; however, the vast majority represent long-lived states that do not capture the functional motions of these molecular machines. Reactions triggered by the addition of a ligand can be the most challenging to detect with crystallography because of the difficulty of synchronizing reactions to create detectable quantities of transient states. The development of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) enables new approaches for solving protein structures following the rapid diffusion of ligands into micron sized protein crystals. Conformational changes occurring on millisecond timescales can be detected and time-resolved. Here, we describe a new XFEL injector which incorporates a microfluidic mixer to rapidly combine reactant and sample milliseconds before the sample reaches the X-ray beam. The mixing injector consists of bonded, concentric glass capillaries. The fabrication process, employing custom laser cut centering spacers and UV curable epoxy, ensures precise alignment of capillaries for repeatable, centered sample flow and dependable mixing. Crystal delivery capillaries are 50 or 75 μm in diameter and can contain an integrated filter depending on the demands of the experiment. Reaction times can be varied from submillisecond to several hundred milliseconds. The injector features rapid and uniform mixing, low sample dilution, and high hit rates. It is fully compatible with existing SFX beamlines.

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The origin and implementation of the Broadening Experiences in Scientific Training programs: an NIH common fund initiative

F. J. Meyers, A. Mathur, C. N. Fuhrmann, T. C. O’Brien, I. Wefes, P. A. Labosky, D. S. Duncan, A. August, A. Feig, K. L. Gould, M. J. Friedlaner, C. B. Schaffer, A. Van Wart, R. Chalkley

FASEB Journal (2016)

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Recent national reports and commentaries on the current status and needs of the U.S. biomedical research workforce have highlighted the limited career development opportunities for predoctoral and postdoctoral trainees in academia, yet little attention is paid to preparation for career pathways outside of the traditional faculty path. Recognizing this issue, in 2013, the U.S. National Institutes of Health (NIH) Common Fund issued a request for application titled "NIH Director's Biomedical Research Workforce Innovation Award: Broadening Experiences in Scientific Training (BEST)." These 5-yr 1-time grants, awarded to 17 single or partnering institutions, were designed to develop sustainable approaches to broaden graduate and postgraduate training, aimed at creating training programs that reflect the range of career options that trainees may ultimately pursue. These institutions have formed a consortium in order to work together to develop, evaluate, share, and disseminate best practices and challenges. This is a first report on the early experiences of the consortium and the scope of participating BEST programs. In this report, we describe the state of the U.S. biomedical workforce and development of the BEST award, variations of programmatic approaches to assist with program design without BEST funding, and novel approaches to engage faculty in career development programs. To test the effectiveness of these BEST programs, external evaluators will assess their outcomes not only over the 5 yr grant period but also for an additional 10 yr beyond award completion.

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TRAIL-coated leukocytes that prevent the bloodborne metastasis of prostate cancer,”

W.C. Wayne, S. Chandrasekaran, M. J. Mitchell, M. F. Chan, R. E. Lee, C. B. Schaffer, M. R. King

Journal of Controlled Release (2016)

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Prostate cancer, once it has progressed from its local to metastatic form, is a disease with poor prognosis and limited treatment options. Here we demonstrate an approach using nanoscale liposomes conjugated with E-selectin adhesion protein and Apo2L/TRAIL (TNF-related apoptosis-inducing ligand) apoptosis ligand that attach to the surface of leukocytes and rapidly clear viable cancer cells from circulating blood in the living mouse. For the first time, it is shown that such an approach can be used to prevent the spontaneous formation and growth of metastatic tumors in an orthotopic xenograft model of prostate cancer, by greatly reducing the number of circulating tumor cells. We conclude that the use of circulating leukocytes as a carrier for the anti-cancer protein TRAIL could be an effective tool to directly target circulating tumor cells for the prevention of prostate cancer metastasis, and potentially other cancers that spread through the bloodstream.

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Ultra-large field-of-view two-photon microscopy

P. S. Tsai, C. Mateo, J. J. Field, C. B. Schaffer, M. E. Anderson, and D. Kleinfeld

Optics Express (2015)

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We present a two-photon microscope that images the full extent of murine cortex with an objective-limited spatial resolution across an 8 mm by 10 mm field. The lateral resolution is approximately 1 µm and the maximum scan speed is 5 mm/ms. The scan pathway employs large diameter compound lenses to minimize aberrations and performs near theoretical limits. We demonstrate the special utility of the microscope by recording resting-state vasomotion across both hemispheres of the murine brain through a transcranial window and by imaging histological sections without the need to stitch.

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Vascular contributions to cognitive impairment and dementia including Alzheimer’s disease

H. M. Snyder, R. A. Corriveau, S. Craft, J. E. Faber, S. Greenberg, D. Knopman, B. T. Lamb, T. J. Montine, M. Nedergaard, C. B. Schaffer, J. A. Schneider, C. Wellington, D. M. Wilcock, G. J. Zipfel, B. Zlokovic, L. J. Bain, F. Bosetti, Z. S. Galis, W. Koroshetz, M. C. Carrillo

Alzheimer’s and Dementia (2015)

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Scientific evidence continues to demonstrate the linkage of vascular contributions to cognitive impairment and dementia such as Alzheimer's disease. In December, 2013, the Alzheimer's Association, with scientific input from the National Institute of Neurological Disorders and Stroke and the National Heart, Lung and Blood Institute from the National Institutes of Health, convened scientific experts to discuss the research gaps in our understanding of how vascular factors contribute to Alzheimer's disease and related dementia. This manuscript summarizes the meeting and the resultant discussion, including an outline of next steps needed to move this area of research forward.

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Constitutively active Notch4 receptor elicits brain arteriovenous malformations through enlargement of capillary-like vessels

Patrick A. Murphya, Tyson N. Kima, Lawrence Huanga, Corinne M. Nielsena , Michael T. Lawtonb , Ralf H. Adamsc , Chris B. Schafferd , and Rong A. Wanga,

Proceedings of the National Academy of Sciences (2014)

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Arteriovenous (AV) malformation (AVM) is a devastating condition characterized by focal lesions of enlarged, tangled vessels that shunt blood from arteries directly to veins. AVMs can form anywhere in the body and can cause debilitating ischemia and life-threatening hemorrhagic stroke. The mechanisms that underlie AVM formation remain poorly understood. Here, we examined the cellular and hemodynamic changes at the earliest stages of brain AVM formation by time-lapse two-photon imaging through cranial windows of mice expressing constitutively active Notch4 (Notch4*). AVMs arose from enlargement of preexisting microvessels with capillary diameter and blood flow and no smooth muscle cell coverage. AV shunting began promptly after Notch4* expression in endothelial cells (ECs), accompanied by increased individual EC areas, rather than increased EC number or proliferation. Alterations in Notch signaling in ECs of all vessels, but not arteries alone, affected AVM formation, suggesting that Notch functions in the microvasculature and/or veins to induce AVM. Increased Notch signaling interfered with the normal biological control of hemodynamics, permitting a positive feedback loop of increasing blood flow and vessel diameter and driving focal AVM growth from AV connections with higher blood velocity at the expense of adjacent AV connections with lower velocity. Endothelial expression of constitutively active Notch1 also led to brain AVMs in mice. Our data shed light on cellular and hemodynamic mechanisms underlying AVM pathogenesis elicited by increased Notch signaling in the endothelium.

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 Supplement 2

TRAIL-coated leukocytes that kill cancer cells in the circulation

Michael J. Mitchell, Elizabeth Wayne, Kuldeepsinh Rana, Chris B. Schaffer, and Michael R. King

Proceedings of the National Academy of Sciences USA (2014)

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Metastasis through the bloodstream contributes to poor prognosis in many types of cancer. Mounting evidence implicates selectin- based adhesive interactions between cancer cells and the blood vessel wall as facilitating this process, in a manner similar to leukocyte trafficking during inflammation. Here, we describe a unique approach to target and kill colon and prostate cancer cells in the blood that causes circulating leukocytes to present the cancer-specific TNF-related apoptosis inducing ligand (TRAIL) on their surface along with E-selectin adhesion receptor. This approach, demonstrated in vitro with human blood and also in mice, mimics the cytotoxic activity of natural killer cells and increases the sur- face area available for delivery of the receptor-mediated signal. The resulting “unnatural killer cells” hold promise as an effective means to neutralize circulating tumor cells that enter blood with the potential to form new metastases.

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In vivo three-photon microscopy of subcortical structures within an intact mouse brain

Nicholas G. Horton, Ke Wang, Demirhan Kobat, Catharine G. Clark, Frank W. Wise, Chris B. Schaffer and Chris Xu

Nature Photonics 7, 205 (2013)

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Two-photon fluorescence microscopy enables scientists in various fields including neuroscience, embryology and oncology to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of two-photon fluorescence microscopy to the cortical layer within mouse brain, and imaging subcortical structures currently requires the removal of overlying brain tissue or the insertion of optical probes. Here, we demon- strate non-invasive, high-resolution, in vivo imaging of subcor- tical structures within an intact mouse brain using three-photon fluorescence microscopy at a spectral excitation window of 1,700nm. Vascular structures as well as red fluorescent protein-labelled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher-order nonlinear excitation overcomes the limit- ations of two-photon fluorescence microscopy, enabling biological investigations to take place at a greater depth within tissue.

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 Supplement 1

Intracerebral haemorrhage associated with antithrombotic treatment: translational insights from experimental studies

Arne Lauer, Waltraud Pfeilschifter, Chris B Schaffer, Eng H Lo, Christian Foerch

Lancet Neurology 12, 394 (2013)

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Little is known about the pathophysiology of intracerebral haemorrhage that occurs during anticoagulant treatment. In observational studies, investigators have reported larger haematoma volumes and worse functional outcome in these patients than in those with intracerebral haemorrhage and a normal coagulation status. The need to prevent extensive haematoma enlargement by rapid reversal of the anticoagulation seems intuitive, although no evidence is available from randomised clinical trials. New oral anticoagulants, such as the direct thrombin inhibitor dabigatran and the factor Xa inhibitor rivaroxaban, have been approved recently; however, intracerebral haemorrhage during dabigatran or rivaroxaban anticoagulation has not been characterised, and whether anticoagulation reversal can be beneficial in this scenario is unknown. In a translational approach, new experimental models have been developed to study anticoagulation-associated intracerebral haemorrhage in more detail and to test treatment strategies. Vitamin k antagonists enlarge haematoma volumes and worsen functional outcome in animal models. Rapid reversal of anticoagulation in the experimental setting prevents prolonged haematoma expansion and improves outcome. The new oral anticoagulants increase intracerbral haemorrhage volumes less than does warfarin. Haemostatic approaches that have been used for vitamin k-associated intracerebral haemorrhage also seem to be effective in intracerebral haemorrhage associated with the new anticoagulants. These experimental studies are valuable for filling gaps in knowledge, but the results need careful translation into routine clinical practice.

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Intracerebral haemorrhage associated with antithrombotic treatment: translational insights from experimental studies.

A. Lauer, W. Pfeilschifter, C. B. Schaffer, E. H. Lo, and C. Foerch

Lancet Neurology (2013)

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Little is known about the pathophysiology of intracerebral haemorrhage that occurs during anticoagulant treatment. In observational studies, investigators have reported larger haematoma volumes and worse functional outcome in these patients than in those with intracerebral haemorrhage and a normal coagulation status. The need to prevent extensive haematoma enlargement by rapid reversal of the anticoagulation seems intuitive, although no evidence is available from randomised clinical trials. New oral anticoagulants, such as the direct thrombin inhibitor dabigatran and the factor Xa inhibitor rivaroxaban, have been approved recently; however, intracerebral haemorrhage during dabigatran or rivaroxaban anticoagulation has not been characterised, and whether anticoagulation reversal can be beneficial in this scenario is unknown. In a translational approach, new experimental models have been developed to study anticoagulation-associated intracerebral haemorrhage in more detail and to test treatment strategies. Vitamin k antagonists enlarge haematoma volumes and worsen functional outcome in animal models. Rapid reversal of anticoagulation in the experimental setting prevents prolonged haematoma expansion and improves outcome. The new oral anticoagulants increase intracerbral haemorrhage volumes less than does warfarin. Haemostatic approaches that have been used for vitamin k-associated intracerebral haemorrhage also seem to be effective in intracerebral haemorrhage associated with the new anticoagulants. These experimental studies are valuable for filling gaps in knowledge, but the results need careful translation into routine clinical practice.

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