Specificity of Monoterpene Interactions with Insect Octopamine and Tyramine Receptors: Insights from in Silico Sequence and Structure Comparison.

Octopamine and tyramine receptors (OARs/TARs) are interesting targets for new insecticide development due to their unique roles in insects' physiological and cellular response and their specificity to invertebrates. Monoterpene compounds that bear resemblance to the natural ligands have been shown to bind to the OARs/TARs but elicit varied responses in different insect species. Using in silico methods, we attempt to investigate the molecular basis of monoterpene interactions and their specificity in different OARs and TARs of damaging or beneficial insects. Sequence and structure comparison revealed that the OARs/TARs studied generally have more similarities in terms of structure rather than sequence identity. Together with clustering and network analyses, we also revealed that the role of IL3 might be crucial in the identification of OAR and TAR and their unique function. Among the 35 monoterpenes subjected to ensemble docking, carvacrol had the most negative average binding energies with the target insect OARs and TARs. The differences in the key interacting residues of carvacrol with insect OARs and TARs could be the origin of variation in the responses of insect species to this monoterpene. Results suggest that carvacrol may be a potential natural-product-based insecticide, targeting multiple insect pests while being nonharmful to honeybees and Asian swallowtail butterflies. This work could provide insights into the development of effective species-specific natural-product-based insecticides that are more environmentally friendly than conventional insecticides.

Preclinical development of ZED8, an 89Zr immuno-PET reagent for monitoring tumor CD8 status in patients undergoing cancer immunotherapy.

BACKGROUND: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]. METHODS: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8+ tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data. RESULTS: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (KD) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound 89Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89Zr immuno-PET reagents. CONCLUSION: 89Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.

Automated segmentation of lungs and lung tumors in mouse micro-CT scans.

Here, we have developed an automated image processing algorithm for segmenting lungs and individual lung tumors in in vivo micro-computed tomography (micro-CT) scans of mouse models of non-small cell lung cancer and lung fibrosis. Over 3000 scans acquired across multiple studies were used to train/validate a 3D U-net lung segmentation model and a Support Vector Machine (SVM) classifier to segment individual lung tumors. The U-net lung segmentation algorithm can be used to estimate changes in soft tissue volume within lungs (primarily tumors and blood vessels), whereas the trained SVM is able to discriminate between tumors and blood vessels and identify individual tumors. The trained segmentation algorithms (1) significantly reduce time required for lung and tumor segmentation, (2) reduce bias and error associated with manual image segmentation, and (3) facilitate identification of individual lung tumors and objective assessment of changes in lung and individual tumor volumes under different experimental conditions.

Effect of molecular size on interstitial pharmacokinetics and tissue catabolism of antibodies.

Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for biologic therapies and expanding the need to understand how various structural aspects affect their distribution properties. To assess the effect of antibody size on systemic pharmacokinetics (PK) and tissue distribution with or without neonatal Fc receptor (FcRn) binding, we evaluated a series of non-mouse-binding anti-glycoprotein D monoclonal antibody formats, including IgG [~150 kDa], one-armed IgG [~100 kDa], IgG-HAHQ (attenuated FcRn binding) [~150 kDa], F(ab')2 [~100 kDa], and F(ab) [~50 kDa]. Tissue-specific concentration-time profiles were corrected for blood content based on vascular volumes and normalized based on interstitial volumes to allow estimation of interstitial concentrations and interstitial:serum concentration ratios. Blood correction demonstrated that the contribution of circulating antibody on total uptake was greatest at early time points and for highly vascularized tissues. Tissue interstitial PK largely mirrored serum exposure profiles. Similar interstitial:serum ratios were obtained for the two FcRn-binding molecules, IgG and one-armed IgG, which reached pseudo-steady-state kinetics in most tissues. For non-FcRn-binding molecules, interstitial:serum ratios changed over time, suggesting that these molecules did not reach steady-state kinetics during the study. Furthermore, concentration-time profiles of both intact and catabolized molecule were measured by a dual tracer approach, enabling quantification of tissue catabolism and demonstrating that catabolism levels were highest for IgG-HAHQ. Overall, these data sets provide insight into factors affecting preclinical distribution and may be useful in estimating interstitial concentrations and/or catabolism in human tissues.

Imaging Reveals Importance of Shape and Flexibility for Glomerular Filtration of Biologics.

Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for cancer immunotherapeutic drug discovery while also revealing limitations in knowledge of structure-activity relationships. The current understanding of renal filtration originates largely from data reported for dextrans, IgG, albumin, and selected globular proteins. For a one-armed IgG-based T-cell imaging agent, we observed higher renal signal than typically observed for bivalent IgGs, prompting us to explore the factors governing renal filtration of biologics. We constructed a small representative library of IgG-like formats with varied shapes and hinge flexibilities falling broadly into two categories: branched molecules including bivalent IgG and (scFv)2Fc, and nonbranched molecules including one-armed IgG, one-armed IgG with stacked Fab, and one-armed IgG with a rigid IgA2 hinge. Transmission electron microscopy revealed Y-shaped structures for the branched molecules and pseudo-linear structures for the nonbranched molecules. Single-photon emission CT imaging, autoradiography, and tissue harvest studies demonstrated higher renal uptake and catabolism for nonbranched molecules relative to branched molecules. Among the nonbranched molecules, the one-armed IgG with rigid IgA2 hinge molecule demonstrated higher kidney uptake and decreased systemic exposure relative to molecules with a more flexible hinge. Our results show that differences in shape and hinge flexibility drive the increased glomerular filtration of one-armed relative to bivalent antibodies and highlight the practical advantages of using imaging to assess renal filtration properties. These findings are particularly relevant for T-cell-dependent bispecific molecules, many of which have nonstandard antibody structures.

Valency of HER2 Targeting Antibodies Influences Tumor Cell Internalization and Penetration.

T-cell-dependent bispecific antibodies (TDB) have been a major advancement in the treatment of cancer, allowing for improved targeting and efficacy for large molecule therapeutics. TDBs are comprised of one arm targeting a surface antigen on a cancer cell and another targeting an engaging surface antigen on a cytotoxic T cell. To impart this function, the antibody must be in a bispecific format as opposed to the more conventional bivalent format. Through in vitro and in vivo studies, we sought to determine the impact of changing antibody valency on solid tumor distribution and catabolism. A bivalent anti-HER2 antibody exhibited higher catabolism than its full-length monovalent binding counterpart in vivo by both invasive tissue harvesting and noninvasive single photon emission computed tomography/X-ray computed tomography imaging despite similar systemic exposures for the two molecules. To determine what molecular factors drove in vivo distribution and uptake, we developed a mechanistic model for binding and catabolism of monovalent and bivalent HER2 antibodies in KPL4 cells. This model suggests that observed differences in cellular uptake of monovalent and bivalent antibodies are caused by the change in apparent affinity conferred by avidity as well as differences in internalization and degradation rates of receptor bound antibodies. To our knowledge, this is the first study to directly compare the targeting abilities of monovalent and bivalent full-length antibodies. These findings may inform diverse antibody therapeutic modalities, including T-cell-redirecting therapies and drug delivery strategies relying upon receptor internalization.

Mechanistic Modeling of Soluble Aß Dynamics and Target Engagement in the Brain by Anti-Aß mAbs in Alzheimer's Disease.

BACKGROUND: Anti-amyloid-ß (Aß) monoclonal antibodies (mAbs) are currently in development for treating Alzheimer's disease. OBJECTIVES: To address the complexity of Aß target engagement profiles, improve the understanding of crenezumab Pharmacokinetics (PK) and Aß Pharmacodynamics (PD) in the brain, and facilitate comparison of anti-Aß therapies with different binding characteristics. METHODS: A mechanistic mathematical model was developed describing the distribution, elimination, and binding kinetics of anti-Aß mAbs and Aß (monomeric and oligomeric forms of Aß1-40 and Aß1-42) in the brain, Cerebrospinal Fluid (CSF), and plasma. Physiologically meaningful values were assigned to the model parameters based on the previous data, with remaining parameters fitted to clinical measurements of Aß concentrations in CSF and plasma, and PK/PD data of patients undergoing anti-Aß therapy. Aß target engagement profiles were simulated using a Monte Carlo approach to explore the impact of biological uncertainty in the model parameters. RESULTS: Model-based estimates of in vivo affinity of the antibody to monomeric Aß were qualitatively consistent with the previous data. Simulations of Aß target engagement profiles captured observed mean and variance of clinical PK/PD data. CONCLUSION: This model is useful for comparing target engagement profiles of different anti-Aß therapies and demonstrates that 60 mg/kg crenezumab yields a significant increase in Aß engagement compared with lower doses of solanezumab, supporting the selection of 60 mg/kg crenezumab for phase 3 studies. The model also provides evidence that the delivery of sufficient quantities of mAb to brain interstitial fluid is a limiting step with respect to the magnitude of soluble Aß oligomer neutralization.

Effect of Modulating FcRn Binding on Direct and Pretargeted Tumor Uptake of Full-length Antibodies.

Full-length antibodies lack ideal pharmacokinetic properties for rapid targeted imaging, prompting the pursuit of smaller peptides and fragments. Nevertheless, studying the disposition properties of antibody-based imaging agents can provide critical insight into the pharmacology of their therapeutic counterparts, particularly for those coupled with potent payloads. Here, we evaluate modulation of binding to the neonatal Fc receptor (FcRn) as a protein engineering-based pharmacologic strategy to minimize the overall blood pool background with directly labeled antibodies and undesirable systemic click reaction of radiolabeled tetrazine with circulating pretargeted trans-cyclooctene (TCO)-modified antibodies. Noninvasive SPECT imaging of mice bearing HER2-expressing xenografts was performed both directly (111In-labeled antibody) and indirectly (pretargeted TCO-modified antibody followed by 111In-labeled tetrazine). Pharmacokinetic modulation of antibodies was achieved by two distinct methods: Fc engineering to reduce binding affinity to FcRn, and delayed administration of an antibody that competes with binding to FcRn. Tumor imaging with directly labeled antibodies was feasible in the absence of FcRn binding, rapidly attaining high tumor-to-blood ratios, but accompanied by moderate liver and spleen uptake. Pretargeted imaging of tumors with non-FcRn-binding antibody was also feasible, but systemic click reaction still occurred, albeit at lower levels than with parental antibody. Our findings demonstrate that FcRn binding impairment of full-length IgG antibodies moderately lowers tumor accumulation of radioactivity, and shifts background activity from blood pool to liver and spleen. Furthermore, reduction of FcRn binding did not eliminate systemic click reaction, but yielded greater improvements in tumor-to-blood ratio when imaging with directly labeled antibodies than with pretargeting.

Biodistribution and efficacy of an anti-TENB2 antibody-drug conjugate in a patient-derived model of prostate cancer.

TENB2, a transmembrane proteoglycan protein, is a promising target for antibody drug conjugate (ADC) therapy due to overexpression in human prostate tumors and rapid internalization. We previously characterized how predosing with parental anti-TENB2 monoclonal antibody (mAb) at 1 mg/kg in a patient-derived LuCap77 explant model with high (3+) TENB2 expression could (i) block target-mediated intestinal uptake of tracer (& 0.1 mg/kg) levels of radiolabeled anti-TENB2-monomethyl auristatin E ADC while preserving tumor uptake, and (ii) maintain efficacy relative to ADC alone. Here, we systematically revisit this strategy to evaluate the effects of predosing on tumor uptake and efficacy in LuCap96.1, a low TENB2-expressing (1+) patient-derived model that is more responsive to ADC therapy than LuCap77. Importantly, rather than using tracer (& 0.1 mg/kg) levels, radiolabeled ADC tumor uptake was assessed at 1 mg/kg - one of the doses evaluated in the tumor growth inhibition study - in an effort to bridge tissue distribution (PK) with efficacy (PD). Predosing with mAb up to 1 mg/kg had no effect on efficacy. These findings warrant further investigations to determine whether predosing prior to ADC therapy might improve therapeutic index by preventing ADC disposition and possible toxicological liabilities in antigen-expressing healthy tissues.

VCAM-1 Density and Tumor Perfusion Predict T-cell Infiltration and Treatment Response in Preclinical Models.

Cancer immunotherapies have demonstrated durable responses in a range of different cancers. However, only a subset of patients responds to these therapies. We set out to test if non-invasive imaging of tumor perfusion and vascular inflammation may be able to explain differences in T-cell infiltration in pre-clinical tumor models, relevant for treatment outcomes. Tumor perfusion and vascular cell adhesion molecule (VCAM-1) density were quantified using magnetic resonance imaging (MRI) and correlated with infiltration of adoptively transferred and endogenous T-cells. MRI biomarkers were evaluated for their ability to detect tumor rejection 3 days after T-cell transfer. Baseline levels of these markers were used to assess their ability to predict PD-L1 treatment response. We found correlations between MRI-derived VCAM-1 density and infiltration of endogenous or adoptively transferred T-cells in some preclinical tumor models. Blocking T-cell binding to endothelial cell adhesion molecules (VCAM-1/ICAM) prevented T-cell mediated tumor rejection. Tumor rejection could be detected 3 days after adoptive T-cell transfer prior to tumor volume changes by monitoring the extracellular extravascular volume fraction. Imaging tumor perfusion and VCAM-1 density before treatment initiation was able to predict the response of MC38 tumors to PD-L1 blockade. These results indicate that MRI based assessment of tumor perfusion and VCAM-1 density can inform about the permissibility of the tumor vasculature for T-cell infiltration which may explain some of the observed variance in treatment response for cancer immunotherapies.

Deficient Functioning of Frontostriatal Circuits During the Resolution of Cognitive Conflict in Cannabis-Using Youth.

OBJECTIVE: Disturbances in self-regulatory control are involved in the initiation and maintenance of addiction, including cannabis use disorder. In adults, long-term cannabis use is associated with disturbances in frontostriatal circuits during tasks that require the engagement of self-regulatory control, including the resolution of cognitive conflict. Understudied are the behavioral and neural correlates of these processes earlier in the course of cannabis use disentangled from effects of long-term use. The present study investigated the functioning of frontostriatal circuits during the resolution of cognitive conflict in cannabis-using youth. METHOD: Functional magnetic resonance imaging data were acquired from 28 cannabis-using youth and 32 age-matched healthy participants during the performance of a Simon task. General linear modeling was used to compare patterns of brain activation during correct responses to conflict stimuli across groups. Psychophysiologic interaction analyses were used to examine conflict-related frontostriatal connectivity across groups. Associations of frontostriatal activation and connectivity with cannabis use measures were explored. RESULTS: Decreased conflict-related activity was detected in cannabis-using versus healthy control youth in frontostriatal regions, including the ventromedial prefrontal cortex, striatum, pallidum, and thalamus. Frontostriatal connectivity did not differ across groups, but negative connectivity between the ventromedial prefrontal cortex and striatum was detected in the 2 groups. CONCLUSION: These findings are consistent with previous reports of cannabis-associated disturbances in frontostriatal circuits in adults and point to the specific influence of cannabis on neurodevelopmental changes in youth. Future studies should examine whether frontostriatal functioning is a reliable marker of cannabis use disorder severity and a potential target for circuit-based interventions.

Tissue Physiology of Cynomolgus Monkeys: Cross-Species Comparison and Implications for Translational Pharmacology.

We previously performed a comparative assessment of tissue-level vascular physiological parameters in mice and rats, two of the most commonly utilized species in translational drug development. The present work extends this effort to non-human primates by measuring tissue- and organ-level vascular volumes (Vv), interstitial volumes (Vi), and blood flow rates (Q) in cynomolgus monkeys. These measurements were accomplished by red blood cell labeling, extracellular marker infusion, and rubidium chloride bolus distribution, respectively, the same methods used in previous rodent measurements. In addition, whole-body blood volumes (BV) were determined across species. The results demonstrate that Vv, Vi, and Q, measured using our methods scale approximately by body weight across mouse, rat, and monkey in the tissues considered here, where allometric analysis allowed extrapolation to human parameters. Significant differences were observed between the values determined in this study and those reported in the literature, including Vv in muscle, brain, and skin and Q in muscle, adipose, heart, thymus, and spleen. The impact of these differences for selected tissues was evaluated via sensitivity analysis using a physiologically based pharmacokinetic model. The blood-brain barrier in monkeys was shown to be more impervious to an infused radioactive tracer, indium-111-pentetate, than in mice or rats. The body weight-normalized total BV measured in monkey agreed well with previously measured value in rats but was lower than that in mice. These findings have important implications for the common practice of scaling physiological parameters from rodents to primates in translational pharmacology.

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging.

Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder 'click' reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by 125I) and subsequent accumulation of an 111In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of 111In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using 111In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.

A Preclinical Population Pharmacokinetic Model for Anti-CD20/CD3 T-Cell-Dependent Bispecific Antibodies.

CD20 is a cell-surface receptor expressed by healthy and neoplastic B cells and is a well-established target for biologics used to treat B-cell malignancies. Pharmacokinetic (PK) and pharmacodynamic (PD) data for the anti-CD20/CD3 T-cell-dependent bispecific antibody BTCT4465A were collected in transgenic mouse and nonhuman primate (NHP) studies. Pronounced nonlinearity in drug elimination was observed in the murine studies, and time-varying, nonlinear PK was observed in NHPs, where three empirical drug elimination terms were identified using a mixed-effects modeling approach: i) a constant nonsaturable linear clearance term (7 mL/day/kg); ii) a rapidly decaying time-varying, linear clearance term (t½  = 1.6 h); and iii) a slowly decaying time-varying, nonlinear clearance term (t½  = 4.8 days). The two time-varying drug elimination terms approximately track with time scales of B-cell depletion and T-cell migration/expansion within the central blood compartment. The mixed-effects NHP model was scaled to human and prospective clinical simulations were generated.

Validation of a noninvasive monitor to continuously trend individual responses to hypovolemia.

BACKGROUND: Humans are able to compensate for significant blood loss with little change in traditional vital signs, limiting early detection and intervention. We hypothesized that the Compensatory Reserve Index (CRI), a new hemodynamic parameter that trends changes in intravascular volume relative to the individual patient's response to hypovolemia, would accurately trend each subject's progression from normovolemia to decompensation (systolic blood pressure < 80) and back to normovolemia in humans. METHODS: Men and women, ages 19 years to 36 years, underwent stepwise (~333 mL aliquot) removal and replacement of 20% blood volume (men, 15 mL/kg; women, 13 mL/kg) via a large bore intravenous (i.v.) line. During each experiment, subjects were monitored with four CipherOx CRI Tablets. Withdrawn blood was reinfused at the end of each experiment. RESULTS: Forty-two subjects (24 men; 18 women) were enrolled in the study, of which 32 completed the protocol. Seven subjects became symptomatic and collapsed (systolic blood pressure < 80), six never achieving maximum blood loss; each was rescued with a saline infusion followed by reinfusion of their stored blood. The mean CRI at baseline for all 42 subjects was 0.9 ± 0.04. The mean CRI for the 32 subjects while asymptomatic at maximum blood loss was 0.611 ± 0.028. For the asymptomatic subjects, the average blood loss volume was 1018 mL ± 286 mL. In comparison, the mean CRI at maximum blood loss for the seven subjects who collapsed was 0.15 ± 0.007 and their average blood loss volume was 860 ± 183 mL. Mean CRI after reinfusion of blood was 0.89 ± 0.02. In addition symptomatic subjects demonstrated three times larger average decrease in CRI per liter of blood removed, 0.85 versus 0.28 for asymptomatic subjects. CONCLUSION: CRI trends change in intravascular volume relative to an individual's response to hypovolemia and is sensitive to the differing risks associated with individuals' differing tolerance to volume loss. LEVEL OF EVIDENCE: Prognostic study, level II.

Reward-Based Spatial Learning in Teens With Bulimia Nervosa.

OBJECTIVE: To assess the functioning of mesolimbic and fronto-striatal areas involved in reward-based spatial learning in teenaged girls with bulimia nervosa (BN) that might be involved in the development and maintenance of maladaptive behaviors characteristic of the disorder. METHOD: We compared functional magnetic resonance imaging blood oxygen level-dependent response in 27 adolescent girls with BN to that of 27 healthy, age-matched control participants during a reward-based learning task that required learning to use extra-maze cues to navigate a virtual 8-arm radial maze to find hidden rewards. We compared groups in their patterns of brain activation associated with reward-based spatial learning versus a control condition in which rewards were unexpected because they were allotted pseudo-randomly to experimentally prevent learning. RESULTS: Both groups learned to navigate the maze to find hidden rewards, but group differences in brain activity associated with maze navigation and reward processing were detected in the fronto-striatal regions and right anterior hippocampus. Unlike healthy adolescents, those with BN did not engage the right inferior frontal gyrus during maze navigation, activated the right anterior hippocampus during the receipt of unexpected rewards (control condition), and deactivated the left superior frontal gyrus and right anterior hippocampus during expected reward receipt (learning condition). These patterns of hippocampal activation in the control condition were significantly associated with the frequency of binge-eating episodes. CONCLUSION: Adolescents with BN displayed abnormal functioning of the anterior hippocampus and fronto-striatal regions during reward-based spatial learning. These findings suggest that an imbalance in control and reward circuits may arise early in the course of BN. Clinical trial registration information-An fMRI Study of Self-Regulation in Adolescents With Bulimia Nervosa; https://clinicaltrials.gov/; NCT00345943.

The Effect of Passive Heat Stress and Exercise-Induced Dehydration on the Compensatory Reserve During Simulated Hemorrhage.

Compensatory reserve represents the proportion of physiological responses engaged to compensate for reductions in central blood volume before the onset of decompensation. We hypothesized that compensatory reserve would be reduced by hyperthermia and exercise-induced dehydration, conditions often encountered on the battlefield. Twenty healthy males volunteered for two separate protocols during which they underwent lower-body negative pressure (LBNP) to hemodynamic decompensation (systolic blood pressure <80 mm Hg). During protocol #1, LBNP was performed following a passive increase in core temperature of ∼1.2°C (HT) or a normothermic time-control period (NT). During protocol #2, LBNP was performed following exercise during which: fluid losses were replaced (hydrated), fluid intake was restricted and exercise ended at the same increase in core temperature as hydrated (isothermic dehydrated), or fluid intake was restricted and exercise duration was the same as hydrated (time-match dehydrated). Compensatory reserve was estimated with the compensatory reserve index (CRI), a machine-learning algorithm that extracts features from continuous photoplethysmograph signals. Prior to LBNP, CRI was reduced by passive heating [NT: 0.87 (SD 0.09) vs. HT: 0.42 (SD 0.19) units, P <0.01] and exercise-induced dehydration [hydrated: 0.67 (SD 0.19) vs. isothermic dehydrated: 0.52 (SD 0.21) vs. time-match dehydrated: 0.47 (SD 0.25) units; P <0.01 vs. hydrated]. During subsequent LBNP, CRI decreased further and its rate of change was similar between conditions. CRI values at decompensation did not differ between conditions. These results suggest that passive heating and exercise-induced dehydration limit the body's physiological reserve to compensate for further reductions in central blood volume.

Weeding Out the Truth: Adolescents and Cannabis: Case and Discussion.

This clinical case conference discusses the case of an adolescent presenting with a marijuana use disorder. Information about a real patient is presented to expert clinicians, who respond to the information by sharing their reasoning and recommendations, followed by a summary of the clinical discussion.

Reactivity Controlling Factors for an Aromatic Carbon-Centered σ,σ,σ-Triradical: The 4,5,8-Tridehydroisoquinolinium Ion.

The chemical properties of the 4,5,8-tridehydroisoquinolinium ion (doublet ground state) and related mono- and biradicals were examined in the gas phase in a dual-cell Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer. The triradical abstracted three hydrogen atoms in a consecutive manner from tetrahydrofuran (THF) and cyclohexane molecules; this demonstrates the presence of three reactive radical sites in this molecule. The high (calculated) electron affinity (EA=6.06 eV) at the radical sites makes the triradical more reactive than two related monoradicals, the 5- and 8-dehydroisoquinolinium ions (EA=4.87 and 5.06 eV, respectively), the reactivity of which is controlled predominantly by polar effects. Calculated triradical stabilization energies predict that the most reactive radical site in the triradical is not position C4, as expected based on the high EA of this radical site, but instead position C5. The latter radical site actually destabilizes the 4,8-biradical moiety, which is singlet coupled. Indeed, experimental reactivity studies show that the radical site at C5 reacts first. This explains why the triradical is not more reactive than the 4-dehydroisoquinolinium ion because the C5 site is the intrinsically least reactive of the three radical sites due to its low EA. Although both EA and spin-spin coupling play major roles in controlling the overall reactivity of the triradical, spin-spin coupling determines the relative reactivity of the three radical sites.