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This file contains supplemental materials and methods describing protein calibrator preparation and pre-analytical variability

This file contains supplemental Figure S2: Temperature stability of analytes

Purpose: To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosis-targeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane-associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors.Experimental Design: The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax–Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL–Bak heterodimer, Mcl1–Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis.Results: Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dose-dependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies.Conclusions: The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials. Clin Cancer Res; 22(4); 1000–10. ©2015 AACR.

This file contains supplemental Table S2A: Analytical validation- interlaboratory precision, and Table S2B: Fitness-for-purpose clinical utility- intertumoral biological variability

Scanning transmission electron microscopy (STEM) allows for imaging, diffraction, and spectroscopy of materials on length scales ranging from microns to atoms. By using a high-speed, direct electron detector, it is now possible to record a full two-dimensional (2D) image of the diffracted electron beam at each probe position, typically a 2D grid of probe positions. These 4D-STEM datasets are rich in information, including signatures of the local structure, orientation, deformation, electromagnetic fields, and other sample-dependent properties. However, extracting this information requires complex analysis pipelines that include data wrangling, calibration, analysis, and visualization, all while maintaining robustness against imaging distortions and artifacts. In this paper, we present py4DSTEM, an analysis toolkit for measuring material properties from 4D-STEM datasets, written in the Python language and released with an open-source license. We describe the algorithmic steps for dataset calibration and various 4D-STEM property measurements in detail and present results from several experimental datasets. We also implement a simple and universal file format appropriate for electron microscopy data in py4DSTEM, which uses the open-source HDF5 standard. We hope this tool will benefit the research community and help improve the standards for data and computational methods in electron microscopy, and we invite the community to contribute to this ongoing project.

ConspectusScanning electron nanobeam diffraction, or 4D-STEM (four-dimensional scanning transmission electron microscopy), is a flexible and powerful approach to elucidate structure from "soft" materials that are challenging to image in the transmission electron microscope because their structure is easily damaged by the electron beam. In a 4D-STEM experiment, a converged electron beam is scanned across the sample, and a pixelated camera records a diffraction pattern at each scan position. This four-dimensional data set can be mined for various analyses, producing maps of local crystal orientation, structural distortions, crystallinity, or different structural classes. Holding the sample at cryogenic temperatures minimizes diffusion of radicals and the resulting damage and disorder caused by the electron beam. The total fluence of incident electrons can easily be controlled during 4D-STEM experiments by careful use of the beam blanker, steering of the localized electron dose, and by minimizing the fluence in the convergent beam thus minimizing beam damage. This technique can be applied to both organic and inorganic materials that are known to be beam-sensitive; they can be highly crystalline, semicrystalline, mixed phase, or amorphous.One common example is the case for many organic materials that have a π-π stacking of polymer chains or rings on the order of 3.4-4.2 Å separation. If these chains or rings are aligned in some regions, they will produce distinct diffraction spots (as would other crystalline spacings in this range), though they may be weak or diffuse for disordered or weakly scattering materials. We can reconstruct the orientation of the π-π stacking, the degree of π-π stacking in the sample, and the domain size of the aligned regions. This Account summarizes illumination conditions and experimental parameters for 4D-STEM experiments with the goal of producing images of structural features for materials that are beam-sensitive. We will discuss experimental parameters including sample cooling, probe size and shape, fluence, and cameras. 4D-STEM has been applied to a variety of materials, not only as an advanced technique for model systems, but as a technique for the beginning microscopist to answer materials science questions. It is noteworthy that the experimental data acquisition does not require an aberration-corrected TEM but can be produced on a variety of instruments with the right attention to experimental parameters.

This paper explains the importance of education in the dying process and outlines a full curriculum to create a society more prepared for difficult end-of-life situations. This course is structured to be taught as a high school elective course in which the principles and approaches to death and dying are discussed in depth. For a high school student, death can be approached from diverse perspectives. Within these perspectives, there are several vital topics to understand in order to ensure a student’s proper education on death. The complexity of death is represented by the titles of the nine chapters of the course. Chapter One, titled “Definition of Death, Consciousness, and Persistent Vegetative State,” highlights the development of death throughout history, the general consensus about death today, and the morality of different levels of treatment for dying patients. Chapter Two, “End Stage Medical Conditions,” outlines the most common end-of-life health conditions along with their symptoms and possible treatments. In Chapter Three, titled “Bad News and Communication,” the course introduces patient-physician communication, the current shortcomings of medical education in this vital area, and the possible protocols for effectively conveying both truth and support to a patient. Chapter Four, labelled “Do Not Resuscitate Orders, Living Wills/Advance Directives, and Durable Powers of Attorney for Health Care,” presents the appropriate options available to prepare for a patient’s inability to make informed decisions autonomously. Chapter Five, “Ordinary vs. Extraordinary Means: The Issue of Tube Feedings,” discusses the distinction between different types of end-of-life treatment and when certain treatments can be morally excused. Chapter Six, titled “Palliative Care and Hospice: A Paradigm for End-of-Life Care,” gives effective options for terminally ill patients that ensure care is never stopped and a patient's dignity is never lost. Chapter Seven, “Pain Management,” highlights the difficulties and recommendations for establishing proper pain management. Chapter Eight, entitled “Medical Futility,” presents the debate surrounding patient autonomy with physician’s beneficence and nonmaleficence, forgoing and requesting treatments, and the values associated with this issue. Finally Chapter Nine, titled “Spirituality and End of Life Care,” emphasizes the inevitable linkage between healthcare, death, and spirituality and the benefits of proper support for a patient. This paper also highlights the ethical implications surrounding the death and dying process. The importance of this subject lies within the death-denying reality that society is today in which death is avoided and human dignity is increasingly sacrificed as a result. Discussions and education among society’s youngest adults can improve general knowledge about the dying process and lead to overall better preparation for handling this inevitable stage of humanity.

Understanding and visualizing the heterogeneous structure of immiscible semicrystalline polymer systems is critical for optimizing their morphology and microstructure. We demonstrate a cryogenic 4D-STEM technique using a combination of amorphous radial profile mapping and correlative crystalline growth processing methods to map both the crystalline and amorphous phase distribution in an isotactic polypropylene (iPP)/ethylene-octene copolymer (EO) multilayer film with 5-nm step size. The resulting map shows a very sharp interface between the amorphous iPP and EO with no preferential crystalline structure near or at the interface, reinforcing the expected incompatibility and immiscibility of iPP and EO, which is a short-chain branched polyethylene. This technique provides a method for direct observation of interfacial structure in an unstained semicrystalline complex multicomponent system with a single cryogenic 4D-STEM dataset.

LiVOPO4 is a promising cathode material for Li-ion batteries due to its ability to intercalate up to two electrons per vanadium redox center. However, LiVOPO4 exhibits polymorphism, forming either the αI, β, or e phase. A thorough comparison between the properties of these phases is difficult because they usually differ in synthesis methods. In this study, we synthesize all three polymorphs by annealing a single precursor, LiVOPO4·2H2O, thereby reducing the effect of synthesis on the properties of the materials. We show through in situ XRD with heating and DFT calculations that, in terms of stability, αI-LiVOPO4 ⋘ e-LiVOPO4 ≤ β-LiVOPO4. We also show experimentally and through DFT calculations that the tolerance to O-interstitials and O-vacancies can explain the differences in stability, morphology, and electrochemical performance between β- and e-LiVOPO4. β-LiVOPO4 is more stable in the presence of O-interstitials while e-LiVOPO4 is favored in the presence of O-vacancies. These defects affect the surface energies and morphology of the products formed, which are confirmed in the Wulff shape calculations and transmission electron microscopy images. These imply that β-LiVOPO4 has an improved rate performance under an oxidizing atmosphere due to the increased presence of facets with superior ion diffusion at the surface. This improved performance is seen by the improved rate capability and capacity of β-LiVOPO4 in the high-voltage region. In contrast, synthesis conditions have little effect on improving the rate performance of e-LiVOPO4.

Here we report a room-temperature sodium metal battery, where the sodium initially stored in a Na3V2(PO4)3 cathode is plated, upon charging, onto an aluminum current collector coated with a thin nucleation layer. To maximize the battery performance, conventional sodium-ion anode materials, including non-graphitized carbons and sodium-alloying metals, were evaluated as nucleation layers to facilitate stable electroplating of sodium metal. Among several materials studied, carbon black and bismuth showed the highest sodium plating–stripping coulombic efficiencies in half-cell testing, averaging 99.9% and 99.85%, respectively, over 50 cycles at 0.5 mA cm−2. Building on these findings, anode-free cells with Na3V2(PO4)3 cathodes were assembled in a discharged state, demonstrating energy densities up to 318 W h kg−1 at 0.25 mA cm−2 (∼C/6), a first-cycle coulombic efficiency up to 92%, a stable discharge voltage at 3.35 V, an average round-trip energy efficiency of 98%, and a capacity retention of 82.5% after 100 cycles at 0.5 mA cm−2 (∼C/3). With its unique performance highlighted in this work, the anode-free sodium battery emerges as a low-cost, high-performance option for stationary electric storage.

Here we demonstrate a full-cell battery design that bridges the energy density and rate capability between that of supercapacitors or pseudocapacitors with that of traditional lithium-ion batteries. This is accomplished by pairing an anode that enables ultrafast ion co-intercalation, an open framework cathode that allows rapid ion diffusion, and linear ether based electrolyte that sustains cell-level stability and high rate performance. We show this platform to be suitable for both sodium and potassium batteries using graphite as the co-intercalation anode, and Prussian blue as the open framework cathode. Our devices exhibit active material energy densities >100 W h kg-1 with power density >1000 W kg-1 with cycling durability approaching ∼80% energy density retention over 2000 cycles. This work brings together state-of-the-art concepts for fast-charging batteries into a full-cell configuration.

Purpose: To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosis-targeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane-associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors. Experimental Design: The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax–Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL–Bak heterodimer, Mcl1–Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis. Results: Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dose-dependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies. Conclusions: The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials. Clin Cancer Res; 22(4); 1000–10. ©2015 AACR.

Background: The Division of Cancer Treatment and Diagnostics/National Cancer Institute has embarked on development of a multiplex panel of biomarkers of the apoptosis pathway to support proof-of-mechanism studies of investigational agents targeting apoptosis. The selected biomarkers provide quantitative estimate of the commitment, onset, and induction of apoptosis, especially the intrinsic apoptosis pathway. This study updates progress in development of the multiplex panel. Methods: The immunoassays are built on the Luminex® xMAP multiplex technology platform using magnetic bead capture. The current panel included caspase-3, Lamin-B, Survivin, and markers involved in mitochondrial apoptosis including oligomeric forms of BCL2 family proteins. Calibrators for oligomeric proteins, which are formed in vivo by the non-covalent association of BH3 domain proteins, are developed as synthetic recombinant fusion proteins. The panel is designed for analysis of biomarkers in fractionated cell lysates prepared from flash frozen tumor biopsies. Fit-for-purpose approach is utilized for validation of biomarkers. Results: We have completed development of 15 biomarkers in the panel. Highly specific and sensitive sandwich immunoassays were designed using capture & labeled antibody pairs and recombinant proteins as calibrators. To overcome complications arising from the multiple readout of some markers (due to protein-protein interactions), the multiplex is grouped in three smaller panels: Panel 1(BAK, BAX, Total Caspase-3, Lamin B(intact +45kDa), & SMAC); Panel 2 (BAD, BAX-BCL-2, BCL-xl, BIM& Mcl-1); and Panel 3 (Active Caspase-3, BCL-xl - BAK, MCL-1 - BAK, phospho-BAD, & Survivin). Specificity of oligomeric forms of biomarkers, BAK-MCL1, BAX-Bcl2, BAK-BCL-xl, and BAX-BAK was achieved by capturing BAK and BAX protein and probing with labeled antibodies specific to MCL-1, BCL-xl, BAK, or BAX. Lysates from tumor biopsy samples can be examined in as little as 50 μg of protein. Specificity of the oligomeric assays was also confirmed by immunoprecipitation and immunoblotting techniques. Fit-for-purpose validation studies are ongoing. Conclusions: The development phase of the multiplex panel is completed. The panel is now ready for transfer from the development laboratory to clinical laboratories for first-in-human use. In the next phase, the panel will be launched as commercial assay kits. One unique feature of the panel is the ability to measure novel oligomeric biomarkers that mimic alterations in protein-protein interactions. This dynamic readout of intrinsic pathway proteins could provide much needed direct evidence for activity of important classes of molecularly targeted drugs in early clinical trials. Funded by NCI Contract No HHSN261200800001E. First three authors contributed equally to the study. Citation Format: Apurva K. Srivastava, Soumya Jaganathan, Laurie L. Stephen, Melinda G. Hollingshead, Jason Scull, Robert J. Kinders, Eric Damour, Jennifer Donohue, Adam Layhee, James Mapes, Dominic Esposito, Joseph E. Tomaszewski, Ralph E. Parchment, James H. Doroshow. Progress on development of multiplex panel of 15 biomarkers to support development of anticancer drugs targeting apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3366. doi:10.1158/1538-7445.AM2013-3366