Comparison of Free-Breathing 3D Phase-Resolved Functional Lung (PREFUL) MRI With Dynamic 19 F Ventilation MRI in Patients With Obstructive Lung Disease and Healthy Volunteers.

BACKGROUND: Non-contrast-enhanced 1 H magnetic resonance imaging (MRI) with full lung coverage shows promise for assessment of regional lung ventilation but a comparison with direct ventilation measurement using 19 F MRI is lacking. PURPOSE: To compare ventilation parameters calculated using 3D phase-resolved functional lung (PREFUL) MRI with 19 F MRI. STUDY TYPE: Prospective. POPULATION: Fifteen patients with asthma, 14 patients with chronic obstructive lung disease, and 13 healthy volunteers. FIELD STRENGTH/SEQUENCE: A 3D gradient-echo pulse sequence with golden-angle increment and stack-of-stars encoding at 1.5 T. ASSESSMENT: All participants underwent 3D PREFUL MRI and 19 F MRI. For 3D PREFUL, static regional ventilation (RVent) and dynamic flow-volume cross-correlation metric (FVL-CM) were calculated. For both parameters, ventilation defect percentage (VDP) values and ventilation defect (VD) maps (including a combination of both parameters [VDPCombined ]) were determined. For 19 F MRI, images from eight consecutive breaths under volume-controlled inhalation of perfluoropropane were acquired. Time-to-fill (TTF) and wash-in (WI) parameters were extracted. For all 19 F parameters, a VD map was generated and the corresponding VDP values were calculated. STATISTICAL TESTS: For all parameters, the relationship between the two techniques was assessed using a Spearman correlation (r). Differences between VDP values were compared using Bland-Altman analysis. For regional comparison of VD maps, spatial overlap and Sørensen-Dice coefficients were computed. RESULTS: 3D PREFUL VDP values were significantly correlated to VDP measures by 19 F (r range: 0.59-0.70). For VDPRVent , no significant bias was observed with VDP of the third and fourth breath (bias range = -6.8:7.7%, P range = 0.25:0.30). For VDPFVL-CM , no significant bias was found with VDP values of fourth-eighth breaths (bias range = -2.0:12.5%, P range = 0.12:0.75). The overall spatial overlap of all VD maps increased with each breath, ranging from 61% to 81%, stabilizing at the fourth breath. DATA CONCLUSION: 3D PREFUL MRI parameters showed moderate to strong correlation with 19 F MRI. Depending on the 3D PREFUL VD map, the best regional agreement was found to 19 F VD maps of third-fifth breath. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY: Stage 2.

Feasibility, Repeatability, and Correlation to Lung Function of Phase-Resolved Functional Lung (PREFUL) MRI-derived Pulmonary Artery Pulse Wave Velocity Measurements.

BACKGROUND: Pulse wave velocity (PWV) in the pulmonary arteries (PA) is a marker of vascular stiffening. Currently, only phase-contrast (PC) MRI-based options exist to measure PA-PWV. PURPOSE: To test feasibility, repeatability, and correlation to clinical data of Phase-Resolved Functional Lung (PREFUL) MRI-based calculation of PA-PWV. STUDY TYPE: Retrospective. SUBJECTS: 79 (26 female) healthy subjects (age range 19-78), 58 (24 female) patients with chronic obstructive pulmonary disease (COPD, age range 40-77), 60 (33 female) patients with suspected pulmonary hypertension (PH, age range 28-85). SEQUENCE: 2D spoiled gradient echo, 1.5T. ASSESSMENT: PA-PWV was measured from PREFUL-derived cardiac cycles based on the determination of temporal and spatial distance between lung vasculature voxels using a simplified (sPWV) method and a more comprehensive (cPWV) method including more elaborate distance calculation. For 135 individuals, PC MRI-based PWV (PWV-QA) was measured. STATISTICAL TESTS: Intraclass-correlation-coefficient (ICC) and coefficient of variation (CoV) were used to test repeatability. Nonparametric tests were used to compare cohorts. Correlation of sPWV/cPWV, PWV-QA, forced expiratory volume in 1 sec (FEV1 ) %predicted, residual volume (RV) %predicted, age, and right heart catheterization (RHC) data were tested. Significance level α = 0.05 was used. RESULTS: sPWV and cPWV showed no significant differences between repeated measurements (P-range 0.10-0.92). CoV was generally lower than 15%. COPD and PH patients had significantly higher sPWV and cPWV than healthy subjects. Significant correlation was found between sPWV or cPWV and FEV1 %pred. (R = -0.36 and R = -0.44), but not with RHC (P-range -0.11 - 0.91) or age (P-range 0.23-0.89). Correlation to RV%pred. was significant for cPWV (R = 0.42) but not for sPWV (R = 0.34, P = 0.055). For all cohorts, sPWV and cPWV were significantly correlated with PWV-QA (R = -0.41 and R = 0.48). DATA CONCLUSION: PREFUL-derived PWV is feasible and repeatable. PWV is increased in COPD and PH patients and correlates to airway obstruction and hyperinflation. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY: Stage 2.

Walking bumblebees see faster.

The behavioural state of animals has profound effects on neuronal information processing. Locomotion changes the response properties of visual interneurons in the insect brain, but it is still unknown if it also alters the response properties of photoreceptors. Photoreceptor responses become faster at higher temperatures. It has therefore been suggested that thermoregulation in insects could improve temporal resolution in vision, but direct evidence for this idea has so far been missing. Here, we compared electroretinograms from the compound eyes of tethered bumblebees that were either sitting or walking on an air-supported ball. We found that the visual processing speed strongly increased when the bumblebees were walking. By monitoring the eye temperature during recording, we saw that the increase in response speed was in synchrony with a rise in eye temperature. By artificially heating the head, we show that the walking-induced temperature increase of the visual system is sufficient to explain the rise in processing speed. We also show that walking accelerates the visual system to the equivalent of a 14-fold increase in light intensity. We conclude that the walking-induced rise in temperature accelerates the processing of visual information-an ideal strategy to process the increased information flow during locomotion.

Intratumoral delivery of TransCon™ TLR7/8 Agonist promotes sustained anti-tumor activity and local immune cell activation while minimizing systemic cytokine induction.

BACKGROUND: Intratumoral (IT) delivery of toll-like receptor (TLR) agonists has shown encouraging anti-tumor benefit in preclinical and early clinical studies. However, IT delivery of TLR agonists may lead to rapid effusion from the tumor microenvironment (TME), potentially limiting the duration of local inflammation and increasing the risk of systemic adverse events. METHODS: To address these limitations, TransCon™ TLR7/8 Agonist-an investigational sustained-release prodrug of resiquimod that uses a TransCon linker and hydrogel technology to achieve sustained and predictable IT release of resiquimod-was developed. TransCon TLR7/8 Agonist was characterized for resiquimod release in vitro and in vivo, in mice and rats, and was assessed for anti-tumor efficacy and pharmacodynamic activity in mice. RESULTS: Following a single IT dose, TransCon TLR7/8 Agonist mediated potent tumor growth inhibition which was associated with sustained resiquimod release over several weeks with minimal induction of systemic cytokines. TransCon TLR7/8 Agonist monotherapy promoted activation of antigen-presenting cells in the TME and tumor-draining lymph nodes, with evidence of activation and expansion of CD8+ T cells in the tumor-draining lymph node and TME. Combination of TransCon TLR7/8 Agonist with systemic immunotherapy further promoted anti-tumor activity in TransCon TLR7/8 Agonist-treated tumors. In a bilateral tumor setting, combination of TransCon TLR7/8 Agonist with systemic IL-2 potentiated tumor growth inhibition in both injected and non-injected tumors and conferred protection against tumor rechallenge following complete regressions. CONCLUSIONS: Our findings show that a single dose of TransCon TLR7/8 Agonist can mediate sustained local release of resiquimod in the TME and promote potent anti-tumor effects as monotherapy and in combination with systemic immunotherapy, supporting TransCon TLR7/8 Agonist as a novel intratumoral TLR agonist for cancer therapy. A clinical trial to evaluate the safety and efficacy of TransCon TLR7/8 Agonist, as monotherapy and in combination with pembrolizumab, in cancer patients is currently ongoing (transcendIT-101; NCT04799054).

Inflammatory response of monocytes to ambient particles varies by highway proximity.

Epidemiological studies have demonstrated associations of chronic respiratory disease with near-roadway pollutant exposure, effects that were independent of those of regional air pollutants. However, there has been limited study of the potential mechanisms for near-roadway effects. Therefore, we examined the in vitro effect of respirable particulate matter (PM) collected adjacent to a major Los Angeles freeway and at an urban background location. PM was collected on filters during two consecutive 15-day periods. Oxidative stress and inflammatory response (intracellular reactive oxygen species [ROS], IL-1ß, IL-6, IL-8, and TNF-α) to PM aqueous extract was assessed in THP-1 cells, a model for evaluating monocyte/macrophage lineage cell responses. The near-roadway PM induced statistically significantly higher levels of IL-6, IL-8, and TNF-α (P < 0.01) and a near significant increase in IL-1ß (P = 0.06) but did not induce ROS activity (P = 0.17). The contrast between urban background and near-roadway PM-induced inflammatory cytokines was similar in magnitude to that corresponding to temporal differences between the two collection periods. PM-induced proinflammatory protein expression was attenuated by antioxidant pretreatment, and PM stimulation enhanced the activity of protein kinases, including extracellular signal-regulated kinase and c-Jun N-terminal kinase. Pretreatment of THP-1 cells with kinase inhibitors reduced PM-induced proinflammatory mediator expression. The proinflammatory response was also reduced by pretreatment with polymyxin B, suggesting a role for endotoxin. However, the patterns of PM-induced protein kinase response and the attenuation of inflammatory responses by antioxidant or polymyxin B pretreatment did not vary between near-roadway and urban background locations. We conclude that near-roadway PM produced greater inflammatory response than urban background PM, a finding consistent with emerging epidemiologic findings, but these differences were not explained by PM endotoxin content or by MAPK pathways. Nevertheless, THP-1 cells may be a model for the development of biologically relevant metrics of long-term spatial variation in exposure for study of chronic disease.

Heterotrimeric G-protein signaling is critical to pathogenic processes in Entamoeba histolytica.

Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGßγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.

Artificially-generated consolidations and balanced augmentation increase performance of U-net for lung parenchyma segmentation on MR images.

PURPOSE: To improve automated lung segmentation on 2D lung MR images using balanced augmentation and artificially-generated consolidations for training of a convolutional neural network (CNN). MATERIALS AND METHODS: From 233 healthy volunteers and 100 patients, 1891 coronal MR images were acquired. Of these, 1666 images without consolidations were used to build a binary semantic CNN for lung segmentation and 225 images (187 without consolidations, 38 with consolidations) were used for testing. To increase CNN performance of segmenting lung parenchyma with consolidations, balanced augmentation was performed and artificially-generated consolidations were added to all training images. The proposed CNN (CNNBal/Cons) was compared to two other CNNs: CNNUnbal/NoCons-without balanced augmentation and artificially-generated consolidations and CNNBal/NoCons-with balanced augmentation but without artificially-generated consolidations. Segmentation results were assessed using Sørensen-Dice coefficient (SDC) and Hausdorff distance coefficient. RESULTS: Regarding the 187 MR test images without consolidations, the mean SDC of CNNUnbal/NoCons (92.1 ± 6% (mean ± standard deviation)) was significantly lower compared to CNNBal/NoCons (94.0 ± 5.3%, P = 0.0013) and CNNBal/Cons (94.3 ± 4.1%, P = 0.0001). No significant difference was found between SDC of CNNBal/Cons and CNNBal/NoCons (P = 0.54). For the 38 MR test images with consolidations, SDC of CNNUnbal/NoCons (89.0 ± 7.1%) was not significantly different compared to CNNBal/NoCons (90.2 ± 9.4%, P = 0.53). SDC of CNNBal/Cons (94.3 ± 3.7%) was significantly higher compared to CNNBal/NoCons (P = 0.0146) and CNNUnbal/NoCons (P = 0.001). CONCLUSIONS: Expanding training datasets via balanced augmentation and artificially-generated consolidations improved the accuracy of CNNBal/Cons, especially in datasets with parenchymal consolidations. This is an important step towards a robust automated postprocessing of lung MRI datasets in clinical routine.

Structural determinants of affinity enhancement between GoLoco motifs and G-protein alpha subunit mutants.

GoLoco motif proteins bind to the inhibitory G(i) subclass of G-protein α subunits and slow the release of bound GDP; this interaction is considered critical to asymmetric cell division and neuro-epithelium and epithelial progenitor differentiation. To provide protein tools for interrogating the precise cellular role(s) of GoLoco motif/Gα(i) complexes, we have employed structure-based protein design strategies to predict gain-of-function mutations that increase GoLoco motif binding affinity. Here, we describe fluorescence polarization and isothermal titration calorimetry measurements showing three predicted Gα(i1) point mutations, E116L, Q147L, and E245L; each increases affinity for multiple GoLoco motifs. A component of this affinity enhancement results from a decreased rate of dissociation between the Gα mutants and GoLoco motifs. For Gα(i1)(Q147L), affinity enhancement was seen to be driven by favorable changes in binding enthalpy, despite reduced contributions from binding entropy. The crystal structure of Gα(i1)(Q147L) bound to the RGS14 GoLoco motif revealed disorder among three peptide residues surrounding a well defined Leu-147 side chain. Monte Carlo simulations of the peptide in this region showed a sampling of multiple backbone conformations in contrast to the wild-type complex. We conclude that mutation of Glu-147 to leucine creates a hydrophobic surface favorably buried upon GoLoco peptide binding, yet the hydrophobic Leu-147 also promotes flexibility among residues 511-513 of the RGS14 GoLoco peptide.

Structural determinants of RGS-RhoGEF signaling critical to Entamoeba histolytica pathogenesis.

G protein signaling pathways, as key components of physiologic responsiveness and timing, are frequent targets for pharmacologic intervention. Here, we identify an effector for heterotrimeric G protein α subunit (EhGα1) signaling from Entamoeba histolytica, the causative agent of amoebic colitis. EhGα1 interacts with this effector and guanosine triphosphatase-accelerating protein, EhRGS-RhoGEF, in a nucleotide state-selective fashion. Coexpression of EhRGS-RhoGEF with constitutively active EhGα1 and EhRacC leads to Rac-dependent spreading in Drosophila S2 cells. EhRGS-RhoGEF overexpression in E. histolytica trophozoites leads to reduced migration toward serum and lower cysteine protease activity, as well as reduced attachment to, and killing of, host cells. A 2.3 Å crystal structure of the full-length EhRGS-RhoGEF reveals a putative inhibitory helix engaging the Dbl homology domain Rho-binding surface and the pleckstrin homology domain. Mutational analysis of the EhGα1/EhRGS-RhoGEF interface confirms a canonical "regulator of G protein signaling" domain rather than a RhoGEF-RGS ("rgRGS") domain, suggesting a convergent evolution toward heterotrimeric and small G protein cross-talk.

A capture coupling method for the covalent immobilization of hexahistidine tagged proteins for surface plasmon resonance.

Surface plasmon resonance (SPR) is a robust method to detect and quantify macromolecular interactions; however, to measure binding interactions, one component must be immobilized on a sensor surface. This is typically achieved using covalent immobilization via free amines or thiols, or noncovalent immobilization using high-affinity interactions such as biotin/streptavidin or antibody/antigen. In this chapter we describe a robust method to covalently immobilize His(6) fusion proteins on the sensor surface for SPR analysis.

A homogeneous method to measure nucleotide exchange by α-subunits of heterotrimeric G-proteins using fluorescence polarization.

The mainstay of assessing guanosine diphosphate release by the α-subunit of a heterotrimeric G-protein is the [³5S]guanosine 5'-O-(3-thiotriphosphate) (GTPγS) radionucleotide-binding assay. This assay requires separation of protein-bound GTPγS from free GTPγS at multiple time points followed by quantification via liquid scintillation. The arduous nature of this assay makes it difficult to quickly characterize multiple mutants, determine the effects of individual variables (e.g., temperature and Mg(2+) concentration) on nucleotide exchange, or screen for small molecule modulators of Gα nucleotide binding/cycling properties. Here, we describe a robust, homogeneous, fluorescence polarization assay using a red-shifted fluorescent GTPγS probe that can rapidly determine the rate of GTPγS binding by Gα subunits.

A high throughput fluorescence polarization assay for inhibitors of the GoLoco motif/G-alpha interaction.

The GoLoco motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.