Global alteration of T-lymphocyte metabolism by PD-L1 checkpoint involves a block of de novo nucleoside phosphate synthesis.

Metabolic obstacles of the tumor microenvironment remain a challenge to T-cell-mediated cancer immunotherapies. To better understand the interplay of immune checkpoint signaling and immune metabolism, this study developed and used an optimized metabolite extraction protocol for non-adherent primary human T-cells, to broadly profile in vitro metabolic changes effected by PD-1 signaling by mass spectrometry-based metabolomics and isotopomer analysis. Inhibitory signaling reduced aerobic glycolysis and glutaminolysis. A general scarcity across the panel of metabolites measured supported widespread metabolic regulation by PD-1. Glucose carbon fate analysis supported tricarboxylic acid cycle reliance on pyruvate carboxylation, catabolic-state fluxes into acetyl-CoA and succinyl-CoA, and a block in de novo nucleoside phosphate synthesis that was accompanied by reduced mTORC1 signaling. Nonetheless, exogenous administration of nucleosides was not sufficient to ameliorate proliferation of T-cells in the context of multiple metabolic insufficiencies due to PD-L1 treatment. Carbon fate analysis did not support the use of primarily glucose-derived carbons to fuel fatty acid beta oxidation, in contrast to reports on T-memory cells. These findings add to our understanding of metabolic dysregulation by PD-1 signaling and inform the effort to rationally develop metabolic interventions coupled with immune-checkpoint blockade for increased treatment efficacy.

Reconfigurable mosaic annular arrays.

Mosaic annular arrays (MAA) based on reconfigurable array (RA) transducer electronics assemblies are presented as a potential solution for future highly integrated ultrasonic transducer subsystems. Advantages of MAAs include excellent beam quality and depth of field resulting from superior elevational focus compared with 1-D electronically scanned arrays, as well as potentially reduced cost, size, and power consumption resulting from the use of a limited number of beamforming channels for processing a large number of subelements. Specific design tradeoffs for these highly integrated arrays are discussed in terms of array specifications for center frequency, element pitch, and electronic switch-on resistance. Large-area RAs essentially function as RC delay lines. Efficient architectures which take into account RC delay effects are presented. Architectures for integration of the transducer and electronics layers of large-area array implementations are reviewed.

Packaging and modular assembly of large-area and fine-pitch 2-D ultrasonic transducer arrays.

A promising transducer architecture for largearea arrays employs 2-D capacitive micromachined ultrasound transducer (CMUT) devices with backside trench-frame pillar interconnects. Reconfigurable array (RA) application-specified integrated circuits (ASICs) can provide efficient interfacing between these high-element-count transducer arrays and standard ultrasound systems. Standard electronic assembly techniques such as flip-chip and ball grid array (BGA) attachment, along with organic laminate substrate carriers, can be leveraged to create large-area arrays composed of tiled modules of CMUT chips and interface ASICs. A large-scale, fully populated and integrated 2-D CMUT array with 32 by 192 elements was developed and demonstrates the feasibility of these techniques to yield future large-area arrays. This study demonstrates a flexible and reliable integration approach by successfully combining a simple under-bump metallization (UBM) process and a stacked CMUT/interposer/ASIC module architecture. The results show high shear strength of the UBM (26.5 g for 70-µm balls), high interconnect yield, and excellent CMUT resonance uniformity (s = 0.02 MHz). A multi-row linear array was constructed using the new CMUT/interposer/ASIC process using acoustically active trench-frame CMUT devices and mechanical/ nonfunctional Si backside ASICs. Imaging results with the completed probe assembly demonstrate a functioning device based on the modular assembly architecture.

Carbohydrate modified catanionic vesicles: probing multivalent binding at the bilayer interface.

This article reports on the synthesis, characterization, and binding studies of surface-functionalized, negatively charged catanionic vesicles. These studies demonstrate that the distribution of glycoconjugates in the membrane leaflet can be controlled by small alterations of the chemical structure of the conjugate. The ability to control the glycoconjugate concentration in the membrane provides a method to explore the relationship between ligand separation distance and multivalent lectin binding at the bilayer interface. The binding results using the O-linked glucosyl conjugate were consistent with a simple model in which binding kinetics are governed by the density of noninteracting glucose ligands, whereas the N-linked glycoconjugate exhibited binding kinetics consistent with interacting or clustering conjugates. From the noninteracting ligand model, an effective binding site separation of the sugar sites for concanavalin A of 3.6-4.3 nm was determined and a critical ligand density above which binding kinetics are zeroth order with respect to the amount of glycoconjugate present at the bilayer was observed. We also report cryo-transmission electron microscopy (cryo-TEM) images of conjugated vesicles showing morphological changes (multilayering) upon aggregation of unilamellar vesicles with concanavalin A.