Multiple conformational states of the HPK1 kinase domain in complex with sunitinib reveal the structural changes accompanying HPK1 trans-regulation.

Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) is a Ser/Thr kinase that operates via the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways to dampen the T-cell response and antitumor immunity. Accordingly, selective HPK1 inhibition is considered a means to enhance antitumor immunity. Sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for the management of gastrointestinal stromal tumors (GISTs), renal cell carcinoma (RCC), and pancreatic cancer, has been reported to inhibit HPK1 in vitro In this report, we describe the crystal structures of the native HPK1 kinase domain in both nonphosphorylated and doubly phosphorylated states, in addition to a double phosphomimetic mutant (T165E,S171E), each complexed with sunitinib at 2.17-3.00-Å resolutions. The native nonphosphorylated cocrystal structure revealed an inactive dimer in which the activation loop of each monomer partially occupies the ATP- and substrate-binding sites of the partner monomer. In contrast, the structure of the protein with a doubly phosphorylated activation loop exhibited an active kinase conformation with a greatly reduced monomer-monomer interface. Conversely, the phosphomimetic mutant cocrystal structure disclosed an alternative arrangement in which the activation loops are in an extended domain-swapped configuration. These structural results indicate that HPK1 is a highly dynamic kinase that undergoes trans-regulation via dimer formation and extensive intramolecular and intermolecular remodeling of the activation segment.

A CD3-bispecific molecule targeting P-cadherin demonstrates T cell-mediated regression of established solid tumors in mice.

Strong evidence exists supporting the important role T cells play in the immune response against tumors. Still, the ability to initiate tumor-specific immune responses remains a challenge. Recent clinical trials suggest that bispecific antibody-mediated retargeted T cells are a promising therapeutic approach to eliminate hematopoietic tumors. However, this approach has not been validated in solid tumors. PF-06671008 is a dual-affinity retargeting (DART®)-bispecific protein engineered with enhanced pharmacokinetic properties to extend in vivo half-life, and designed to engage and activate endogenous polyclonal T cell populations via the CD3 complex in the presence of solid tumors expressing P-cadherin. This bispecific molecule elicited potent P-cadherin expression-dependent cytotoxic T cell activity across a range of tumor indications in vitro, and in vivo in tumor-bearing mice. Regression of established tumors in vivo was observed in both cell line and patient-derived xenograft models engrafted with circulating human T lymphocytes. Measurement of in vivo pharmacodynamic markers demonstrates PF-06671008-mediated T cell activation, infiltration and killing as the mechanism of tumor inhibition.

Versatile technique for assessing thickness of 2D layered materials by XPS.

X-ray photoelectron spectroscopy (XPS) has been utilized as a versatile method for thickness characterization of various two-dimensional (2D) films. Accurate thickness can be measured simultaneously while acquiring XPS data for chemical characterization of 2D films having thickness up to approximately 10 nm. For validating the developed technique, thicknesses of few-layer graphene (FLG), MoS2 and amorphous boron nitride (a-BN) layer, produced by microwave plasma chemical vapor deposition (MPCVD), plasma enhanced chemical vapor deposition (PECVD), and pulsed laser deposition (PLD) respectively, were accurately measured. The intensity ratio between photoemission peaks recorded for the films (C 1s, Mo 3d, B 1s) and the substrates (Cu 2p, Al 2p, Si 2p) is the primary input parameter for thickness calculation, in addition to the atomic densities of the substrate and the film, and the corresponding electron attenuation length (EAL). The XPS data was used with a proposed model for thickness calculations, which was verified by cross-sectional transmission electron microscope (TEM) measurement of thickness for all the films. The XPS method determines thickness values averaged over an analysis area which is orders of magnitude larger than the typical area in cross-sectional TEM imaging, hence provides an advanced approach for thickness measurement over large areas of 2D materials. The study confirms that the versatile XPS method allows rapid and reliable assessment of the 2D material thickness and this method can facilitate in tailoring growth conditions for producing very thin 2D materials effectively over a large area. Furthermore, the XPS measurement for a typical 2D material is non-destructive and does not require special sample preparation. Therefore, after XPS analysis, exactly the same sample can undergo further processing or utilization.

Slow creep in soft granular packings.

Transient creep mechanisms in soft granular packings are studied numerically using a constant pressure and constant stress simulation method. Rapid compression followed by slow dilation is predicted on the basis of a logarithmic creep phenomenon. Characteristic scales of creep strain and time exhibit a power-law dependence on jamming pressure, and they diverge at the jamming point. Microscopic analysis indicates the existence of a correlation between rheology and nonaffine fluctuations. Localized regions of large strain appear during creep and grow in magnitude and size at short times. At long times, the spatial structure of highly correlated local deformation becomes time-invariant. Finally, a microscale connection between local rheology and local fluctuations is demonstrated in the form of a linear scaling between granular fluidity and nonaffine velocity.

Electroreflectance imaging of gold-H3PO4 supercapacitors. Part II: microsupercapacitor ageing characterization.

This microsupercapacitor ageing study demonstrates the usefulness of the electroreflectance technique by quantifying local charge accumulation. Two separate devices with interdigitated electrodes were evaulated over a period of 4.1 million charge/discharge cycles. The key results are spatial mapping of charge accumulation in the gold electrodes derived from variation in the observed electrode reflectance. The nominal device exhibited little change in spatial distribution throughout the ageing cycle and serves as a comparison for the test device, which exhibited some nonuniform charge accumulation behavior. Further, an accelerated ageing test was completed by applying increasing voltage pulses up to 1.46 V to the device. Visual evidence of electrode ageing emerged in the reflectance distribution. An equivalent circuit model was developed to assess the evolution of individual circuit elements that correlate to the physical causes of ageing.

Electroreflectance imaging of gold-H3PO4 supercapacitors. Part I: experimental methodology.

Electroreflectance microscopy is demonstrated as a high-resolution, non-contact method to image dynamic charge distribution in integrated microsupercapacitor structures during fast voltage cycling. Electroreflectance camera images of a gold electrode H3PO4 polymer electrolyte microsupercapacitor reveal time varying charge distribution with submicron spatial resolution, millisecond time resolution, and electroreflectance resolution on the order of 500 nC cm(-2). A model describing changes in the metal electrode's optical constants as a function of free electron concentration shows good agreement with measured electroreflectance. The proposed method can be used for sensitive, non-contact measurements of charge spatial distribution, and defect and performance characterization in electrode-electrolyte microdevices.

Engineering the electronic bandgaps and band edge positions in carbon-substituted 2D boron nitride: a first-principles investigation.

Modification of graphene to open a robust gap in its electronic spectrum is essential for its use in field effect transistors and photochemistry applications. Inspired by recent experimental success in the preparation of homogeneous alloys of graphene and boron nitride (BN), we consider here engineering the electronic structure and bandgap of C2xB1-xN1-x alloys via both compositional and configurational modification. We start from the BN end-member, which already has a large bandgap, and then show that (a) the bandgap can in principle be reduced to about 2 eV with moderate substitution of C (x < 0.25); and (b) the electronic structure of C2xB1-xN1-x can be further tuned not only with composition x, but also with the configuration adopted by C substituents in the BN matrix. Our analysis, based on accurate screened hybrid functional calculations, provides a clear understanding of the correlation found between the bandgap and the level of aggregation of C atoms: the bandgap decreases most when the C atoms are maximally isolated, and increases with aggregation of C atoms due to the formation of bonding and anti-bonding bands associated with hybridization of occupied and empty defect states. We determine the location of valence and conduction band edges relative to vacuum and discuss the implications on the potential use of 2D C2xB1-xN1-x alloys in photocatalytic applications. Finally, we assess the thermodynamic limitations on the formation of these alloys using a cluster expansion model derived from first-principles.

Sequential Bayesian-optimized graphene synthesis by direct solar-thermal chemical vapor deposition.

This work reports the use of a high-flux solar simulator that mimics the solar spectrum and a cold-wall CVD reactor to demonstrate the feasibility of utilizing a renewable energy resource in synthesizing graphene under various conditions. A parametric study of process parameters was conducted using a probabilistic approach. Gaussian process regression serves as a surrogate to establish a prior for Bayesian optimization, and an information acquisition function is employed to identify conditions that yield high-quality products. Backscattered electron images and Raman mapping were used to assess the effects of growth conditions on graphene characteristic sizes, film quality, and uniformity. We report the synthesis of high-quality single-layer graphene (SLG) and AB-stacked bilayer graphene films in a one-step, short-time process with [Formula: see text] ratios of 0.21 and 0.14, respectively. Electron diffraction analysis shows peak intensities that resemble SLG and AB-bilayer graphene with up to 5 and 20 [Formula: see text]m grain sizes, respectively. The optical transmissivities of SLG and AB-bilayer graphene fall between 0.959-0.977 and 0.929-0.953, whereas the sheet resistances measured by a 4-point probe with 1 mm spacing are 15.5 ± 4.6 and 3.4 ± 1.5 k[Formula: see text]/sq, respectively. Further scale-up of the optimized graphene growth area was achieved by flattening the insolation profile, leading to spatial uniformity up to 13 mm in radius. Direct solar capture for CVD synthesis enable a practical and sustainable option for synthesizing graphene films applicable for photonic and electronic applications.

Optical properties of ordered carbon nanotube arrays grown in porous anodic alumina templates.

We have synthesized ordered carbon nanotube (CNT) arrays in porous anodic alumina (PAA) matrix, and have characterized their total optical reflectance and bi-directional reflectance distribution function after each processing step of the microwave plasma chemical vapor deposition process (MPCVD). For a PAA sample without CNT growth, the reflectance shows an oscillating pattern with wavelength that agrees reasonably with a multilayer model. During the MPCVD process, heating the sample significantly reduces the reflectance by 30-40%, the plasma treatment reduces the reflectance by another 5-10%, and the CNT growth further reduces the reflectance by 2-3%. After an atomic layer deposition (ALD) process, the reflectance increases to the embedded CNT arrays. After etching and exposure of CNT tips, the reflectance almost returns to the original pattern with slightly higher reflectance. Bi-directional reflectance distribution function (BRDF) measurements show that the CNT-PAA surface is quite specular as indicated by a large lobe at the specular angle, while the secondary lobe can be attributed to surface roughness.

Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity.

4-1BB (CD137, TNFRSF9) is a costimulatory receptor expressed on several subsets of activated immune cells. Numerous studies of mouse and human T cells indicate that 4-1BB promotes cellular proliferation, survival, and cytokine production. 4-1BB agonist mAbs have demonstrated efficacy in prophylactic and therapeutic settings in both monotherapy and combination therapy tumor models and have established durable anti-tumor protective T-cell memory responses. PF-05082566 is a fully human IgG2 that binds to the extracellular domain of human 4-1BB with high affinity and specificity. In preclinical studies, this agonist antibody demonstrated its ability to activate NF-κB and induce downstream cytokine production, promote leukocyte proliferation, and inhibit tumor growth in a human PBMC xenograft tumor model. The mechanism of action and robust anti-tumor efficacy of PF-05082566 support its clinical development for the treatment of a broad spectrum of human malignancies.

Measurement of fractional synthetic rates of multiple protein analytes by triple quadrupole mass spectrometry.

BACKGROUND: Current approaches to measure protein turnover that use stable isotope-labeled tracers via GC-MS are limited to a small number of relatively abundant proteins. We developed a multiplexed liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM) assay to measure protein turnover and compared the fractional synthetic rates (FSRs) for 2 proteins, VLDL apolipoprotein B100 (VLDL apoB100) and HDL apoA-I, measured by both methods. We applied this technique to other proteins for which kinetics are not readily measured with GC-MS. METHODS: Subjects were given a primed-constant infusion of [5,5,5-D(3)]-leucine (D(3)-leucine) for 15 h with blood samples collected at selected time points. Apolipoproteins isolated by SDS-PAGE from lipoprotein fractions were analyzed by GC-MS or an LC-SRM assay designed to measure the M+3/M+0 ratio at >1% D(3)-leucine incorporation. We calculated the FSR for each apolipoprotein by curve fitting the tracer incorporation data from each subject. RESULTS: The LC-SRM method was linear over the range of tracer enrichment values tested and highly correlated with GC-MS (R(2) > 0.9). The FSRs determined from both methods were similar for HDL apoA-I and VLDL apoB100. We were able to apply the LC-SRM approach to determine the tracer enrichment of multiple proteins from a single sample as well as proteins isolated from plasma after immunoprecipitation. CONCLUSIONS: The LC-SRM method provides a new technique for measuring the enrichment of proteins labeled with stable isotopes. LC-SRM is amenable to a multiplexed format to provide a relatively rapid and inexpensive means to measure turnover of multiple proteins simultaneously.

Columnar order in jammed LiFePO4 cathodes: ion transport catastrophe and its mitigation.

The high-rate, high-capacity potential of LiFePO4-based lithium-ion battery cathodes has motivated numerous experimental and theoretical studies aiming to realize such performance through nano-sizing, tailoring of particle shape through synthesis conditions, and doping. Here, a granular mechanics study of microstructures formed by dense jammed packings of experimentally and theoretically inspired LiFePO4 particle shapes is presented. A strong dependence of the resultant packing structures on particle shapes is observed, in which columnar structures aligned with the [010] direction inhibit diffusion along [010] in anisotropic LiFePO4. Transport limitations are induced by [010] columnar order and lead to catastrophic performance degradation in anisotropic LiFePO4 electrodes. Further, judicious mixing of nanoplatelets with additive nanoparticles can frustrate columnar ordering and thereby enhance the rate capability of LiFePO4 electrodes by nearly an order of magnitude.

Indirect inverse flux mapping of a concentrated solar source using infrared imaging.

With the growing interest in high-flux solar sources, a need exists for simple, accurate, and inexpensive strategies to characterize their output radiative flux. In this paper, the irradiation output from a 10 kWe xenon lamp solar simulator is characterized by an inverse mapping technique that uses a custom radiometer and infrared camera, validated by a direct characterization method (heat flux gauge). The heat flux distribution is determined in a vacuum chamber using an easily obtainable graphite target and an inverse heat transfer model. The solar simulator produces peak fluxes in the range of 1.5-4.5 MW/m2 as measured directly by a heat flux gauge, and its output can be controlled using a variable power supply. Spectral measurements indicate that minor variations in the simulator's output with respect to its current supply occur in the spectral range of 450-800 nm. The radiometer presented in this work allows for characterizing solar irradiation under practical conditions (e.g., inside a solar reactor) and thus accounts for deviations due to additional components, such as viewport effects. Additionally, it provides an inexpensive and efficient means of monitoring any deterioration in the performance of solar sources over time without the need for complex recalibration.

Utomilumab in Patients With Immune Checkpoint Inhibitor-Refractory Melanoma and Non-Small-Cell Lung Cancer.

Section Head: Clinical/translational cancer immunotherapy. Background: The goal of this study was to estimate the objective response rate for utomilumab in adults with immune checkpoint inhibitor (ICI)-refractory melanoma and non-small-cell lung cancer (NSCLC). Methods: Utomilumab was dosed intravenously every 4 weeks (Q4W) and adverse events (AEs) monitored. Tumor responses by RECIST1.1 were assessed by baseline and on-treatment scans. Tumor biopsies were collected for detection of programmed cell death ligand 1, CD8, 4-1BB, perforin, and granzyme B, and gene expression analyzed by next-generation sequencing. CD8+ T cells from healthy donors were stimulated with anti-CD3 ± utomilumab and compared with control. Results: Patients with melanoma (n=43) and NSCLC (n=20) received utomilumab 0.24 mg/kg (n=36), 1.2 mg/kg (n=26), or 10 mg/kg (n=1). Treatment-emergent AEs (TEAEs) occurred in 55 (87.3%) patients and serious TEAEs in 18 (28.6%). Five (7.9%) patients discontinued owing to TEAEs. Thirty-two (50.8%) patients experienced treatment-related AEs, mostly grade 1-2. Objective response rate: 2.3% in patients with melanoma; no confirmed responses for patients with NSCLC. Ten patients each with melanoma (23.3%) or NSCLC (50%) had stable disease; respective median (95% confidence interval, CI) progression-free survival was 1.8 (1.7-1.9) and 3.6 (1.6-6.5) months. Utomilumab exposure increased with dose. The incidences of antidrug and neutralizing antibodies were 46.3% and 19.4%, respectively. Efficacy was associated with immune-active tumor microenvironments, and pharmacodynamic activity appeared to be blunted at higher doses. Conclusions: Utomilumab was well tolerated, but antitumor activity was low in patients who previously progressed on ICIs. The potential of 4-1BB agonists requires additional study to optimize efficacy while maintaining the tolerable safety profile.

A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake.

PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.

Carbon nanowalls amplify the surface-enhanced Raman scattering from Ag nanoparticles.

We report surface-enhanced Raman scattering (SERS) from Ag nanoparticles decorated on thin carbon nanowalls (CNWs) grown by microwave plasma chemical vapor deposition. The Ag morphology is controlled by exposing the CNWs to oxygen plasma and through the electrodeposition process by varying the number of deposition cycles. The SERS substrates are capable of detecting low concentrations of rhodamine 6G and bovine serum albumin, showing much higher Raman enhancement than ordinary planar HOPG with Ag decoration. The major factors contributing to this behavior include: high density of Ag nanoparticles, large surface area, high surface roughness, and the underlying presence of vertically oriented CNWs. The relatively simple procedure of substrate preparation and nanoparticle decoration suggests that this is a promising approach for fabricating ultrasensitive SERS substrates for biological and chemical detection at the single-molecule level, while also enabling the study of fundamental SERS phenomena.

Charge storage in mesoscopic graphitic islands fabricated using AFM bias lithography.

Electrochemical oxidation and etching of highly oriented pyrolytic graphite (HOPG) has been achieved using biased atomic force microscopy (AFM) lithography, allowing patterns of varying complexity to be written into the top layers of HOPG. The graphitic oxidation process and the trench geometry after writing were monitored using intermittent contact mode AFM. Electrostatic force microscopy reveals that the isolated mesoscopic islands formed during the AFM lithography process become positively charged, suggesting that they are laterally isolated from the surrounding HOPG substrate. The electrical transport studies of these laterally isolated finite-layer graphitic islands enable detailed characterization of electrical conduction along the c-direction and reveal an unexpected stability of the charged state. Utilizing conducting-atomic force microscopy, the measured I(V) characteristics revealed significant non-linearities. Micro-Raman studies confirm the presence of oxy functional groups formed during the lithography process.

PCSK9 is not involved in the degradation of LDL receptors and BACE1 in the adult mouse brain.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates hepatic low-density lipoprotein receptor (LDLR) levels in humans. PCSK9 has also been shown to regulate the levels of additional membrane-bound proteins in vitro, including the very low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (ApoER2) and the beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), which are all highly expressed in the CNS and have been implicated in Alzheimer's disease. To better understand the role of PCSK9 in regulating these additional target proteins in vivo, their steady-state levels were measured in the brain of wild-type, PCSK9-deficient, and human PCSK9 overexpressing transgenic mice. We found that while PCSK9 directly bound to recombinant LDLR, VLDLR, and apoER2 protein in vitro, changes in PCSK9 expression did not alter the level of these receptors in the mouse brain. In addition, we found no evidence that PCSK9 regulates BACE1 levels or APP processing in the mouse brain. In conclusion, our results suggest that while PCSK9 plays an important role in regulating circulating LDL cholesterol levels by reducing the number of hepatic LDLRs, it does not appear to modulate the levels of LDLR and other membrane-bound proteins in the adult mouse brain.

Biochemical characterization of cholesteryl ester transfer protein inhibitors.

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.

Optical properties of ordered vertical arrays of multi-walled carbon nanotubes from FDTD simulations.

A finite-difference time-domain (FDTD) method is used to model thermal radiative properties of vertical arrays of multi-walled carbon nanotubes (MWCNT). Individual CNTs are treated as solid circular cylinders with an effective dielectric tensor. Consistent with experiments, the results confirm that CNT arrays are highly absorptive. Compared with the commonly used Maxwell-Garnett theory, the FDTD calculations generally predict larger reflectance and absorbance, and smaller transmittance, which are attributed to the diffraction and scattering within the cylinder array structure. The effects of volume fraction, tube length, tube distance, and incident angle on radiative properties are investigated systematically. Low volume fraction and long tubes are more favorable to achieve low reflectance and high absorbance. For a fixed volume fraction and finite tube length, larger periodicity results in larger reflectance and absorbance. The angular dependence studies reveal an optimum incident angle at which the reflectance can be minimized. The results also suggest that an even darker material could be achieved by using CNTs with good alignment on the top surface.