A specific fluorescence resonance energy quenching-based biosensor for measuring thrombin activity in whole blood.

BACKGROUND: At sites of vessel injury, thrombin acts as the central mediator of coagulation by catalyzing fibrin clot formation and platelet activation. Thrombin generation is most frequently measured in plasma samples using small-molecule substrates; however, these have low specificity for thrombin and limited utility in whole blood. Plasma assays are limited because they ignore the hemostatic contributions of blood cells and require anticoagulation and the addition of supraphysiological concentrations of calcium. OBJECTIVES: To overcome these limitations, we designed and characterized a fluorescence resonance energy quenching-based thrombin sensor (FTS) protein. METHODS: The fluorescence resonance energy quenching pair of mAmetrine and tTomato, separated by a thrombin recognition sequence, was developed. The protein was expressed using Escherichia coli, and purity was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cleavage of FTS was monitored by fluorescence using excitation at 406 nm and emission at 526 nm and 581 nm. RESULTS: Compared with small-molecule substrates, the FTS demonstrated high specificity for thrombin; it is not cleaved by thrombin or inhibited by α2-macroglobulin and interacts with thrombin's anion-binding exosite I. The FTS can effectively measure thrombin generation in plasma and in finger-prick whole blood, which allows it to be developed into a point-of-care test of thrombin generation. The FTS does not inhibit standard thrombin-generation assays. Lastly, FTS-based thrombin generation in nonanticoagulated finger-prick blood was delayed but enhanced compared with that in citrated plasma. CONCLUSION: The FTS will broaden our understanding of thrombin generation in ways that are not attainable with current methods.

Self-Propulsion by Directed Explosive Emulsification.

An active droplet system, programmed to repeatedly move autonomously at a specific velocity in a well-defined direction, is demonstrated. Coulombic energy is stored in oversaturated interfacial assemblies of charged nanoparticle-surfactants by an applied DC electric field and can be released on demand. Spontaneous emulsification is suppressed by an increase in the stiffness of the oversaturated assemblies. Rapidly removing the field releases the stored energy in an explosive event that propels the droplet, where thousands of charged microdroplets are ballistically ejected from the surface of the parent droplet. The ejection is made directional by a symmetry breaking of the interfacial assembly, and the combined interaction force of the microdroplet plume on one side of the droplet propels the droplet distances tens of times its size, making the droplet active. The propulsion is autonomous, repeatable, and agnostic to the chemical composition of the nanoparticles. The symmetry-breaking in the nanoparticle assembly controls the microdroplet velocity and direction of propulsion. This mechanism of droplet propulsion will advance soft micro-robotics, establishes a new type of active matter, and introduces new vehicles for compartmentalized delivery.

Training, Deployment Preparation, and Behavioral Health of New York National Guard Personnel Deployed to Assist with COVID-19 Decedent Work.

INTRODUCTION: A small body of research conducted mostly among civilians has shown that adequate training and preparation can prevent or reduce the development of behavioral health problems in first responders. Several civilian studies have shown that social support is protective against behavioral health problems. However, very few studies have examined the impact of these factors on the behavioral health of military first responders. Military first responders, who serve in the aftermath of natural disasters and disease outbreaks such as the coronavirus disease 2019 (COVID-19) pandemic, are often members of the National Guard (NG). The purpose of this study was to examine the impact of mortuary affairs training/handling human remains, role preparation, equipment preparation, and unit social support provided to families on the behavioral health of New York (NY) NG personnel deployed to assist the NY Office of Chief Medical Examiner with handling the remains of COVID-19 decedents. MATERIALS AND METHODS: We invited 410 NYNG personnel who deployed for the Office of Chief Medical Examiner mission to complete an anonymous online questionnaire 3 to 6 months post-mission. Of the 158 participants, we used the data of the 141 participants who provided consent. Standard behavioral health measures (depression, anxiety, post-traumatic stress disorder, alcohol misuse, and insomnia) as well as study-specific items designed to understand the unique dynamics of this deployment were included. Hierarchical logistic regression analysis was used to examine the relationships between mortuary training, role preparation, equipment preparation, and unit support with behavioral health. RESULTS: Close to two-thirds of the sample reported that they had not been trained in mortuary affairs/handling human remains before the mission. We also found that that lower levels of role preparation and unit support provided to the service members' families increased the odds of meeting criteria for one or more behavioral health problems, but that training in mortuary affairs and equipment preparation was unrelated to behavioral health. CONCLUSIONS: Our research points to the importance of emotionally and cognitively preparing service members for the specific dynamics of a deployment and the roles that that they are expected to play. Furthermore, it suggests that supporting the families of NG personnel during domestic missions can benefit the behavioral health of the NG personnel. Additional research is needed to corroborate these findings, particularly the impact of unit support provided to family members on service members' behavioral health.

Thrombin-activatable fibrinolysis inhibitor and sex modulate thrombus stability and pulmonary embolism burden in a murine model.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) levels are positively correlated with the risk of thrombosis. The mechanism of how TAFI affects venous thromboembolism (VTE) remains uncertain. In addition, the role of sex on the risk of VTE has also been studied. However, their association also remains unclear. OBJECTIVES: To investigate how TAFI and/or sex affect venous thrombus stability and consequent pulmonary embolism (PE). METHODS: Ferric chloride-induced thrombi were formed within the femoral veins of male and female wild-type (WT) or TAFI-knockout (Cpb2-/-) mice. Thrombi were imaged over 2 hours using intravital videomicroscopy to quantify embolization and thrombus size over time. Lungs were examined by immunohistochemistry to quantify (a) emboli and (b) fibrin composition of these emboli. RESULTS: Embolization events in female mice were higher than in males (7.9-fold in WT and 3.1-fold in Cpb2-/- mice). Although the maximal thrombus sizes were not different across groups, Cpb2-/- mice had thrombi that were, on average, 24% smaller at the end of the 2-hour experiment than WT mice. Loss of TAFI led to a 4.0- and 2.8-fold increase in PE burden in males and females, respectively, while sex had no influence. Pulmonary emboli in Cpb2-/- mice had higher fibrin composition compared with WT mice. CONCLUSION: Female mice had less stable venous thrombi than male mice, suggesting a higher risk of PE in females with deep vein thrombosis. Mice lacking TAFI had more thrombus degradation and higher PE burden than WT mice. These results confirm the role of TAFI in venous thrombosis.

Mixed Nanosphere Assemblies at a Liquid-Liquid Interface.

The in-plane packing of gold (Au), polystyrene (PS), and silica (SiO2) spherical nanoparticle (NP) mixtures at a water-oil interface is investigated in situ by UV-vis reflection spectroscopy. All NPs are functionalized with carboxylic acid such that they strongly interact with amine-functionalized ligands dissolved in an immiscible oil phase at the fluid interface. This interaction markedly increases the binding energy of these nanoparticle surfactants (NPSs). The separation distance between the Au NPSs and Au surface coverage are measured by the maximum plasmonic wavelength (λmax) and integrated intensities as the assemblies saturate for different concentrations of non-plasmonic (PS/SiO2) NPs. As the PS/SiO2 content increases, the time to reach intimate Au NP contact also increases, resulting from their hindered mobility. λmax changes within the first few minutes of adsorption due to weak attractive inter-NP forces. Additionally, a sharper peak in the reflection spectrum at NP saturation reveals tighter Au NP packing for assemblies with intermediate non-plasmonic NP content. Grazing incidence small angle X-ray scattering (GISAXS) and scanning electron microscopy (SEM) measurements confirm a decrease in Au NP domain size for mixtures with larger non-plasmonic NP content. The results demonstrate a simple means to probe interfacial phase separation behavior using in situ spectroscopy as interfacial structures densify into jammed, phase-separated NP films.

Validation of the time to attain maximal clot amplitude after reaching maximal clot formation velocity parameter as a measure of fibrinolysis using rotational thromboelastometry and its application in the assessment of fibrinolytic resistance in septic patients: a prospective observational study: communication from the ISTH SSC Subcommittee on Fibrinolysis.

BACKGROUND: In sepsis, fibrinolysis resistance correlates with worse outcomes. Practically, rotational thromboelastometry (ROTEM) is used to report residual clot amplitude relative to maximum amplitude at specified times after clot formation clot lysis indices (CLIs). However, healthy individuals can exhibit similar CLIs, thus making it challenging to solely diagnose the low fibrinolytic state. Furthermore, CLI does not include the kinetics of clot formation, which can affect overall fibrinolysis. Therefore, a more nuanced analysis, such as time to attain maximal clot amplitude after reaching maximal clot formation velocity (t-AUCi), is needed to better identify fibrinolysis resistance in sepsis. OBJECTIVES: To evaluate the correlation between the degree of fibrinolytic activation and t-AUCi in healthy or septic individuals. METHODS: Whole blood (n = 60) from septic or healthy donors was analyzed using tissue factor-activated (EXTEM) and nonactivated (NATEM) ROTEM assays. Lysis was initiated with tissue-type plasminogen activator, and CLI and t-AUCi were calculated. Standard coagulation tests and plasma fibrinolysis markers (D-dimer, plasmin-α2-antiplasmin complex, plasminogen activator inhibitor type 1, and plasminogen) were also measured. RESULTS: t-AUCi values decreased with increasing fibrinolytic activity and correlated positively with CLI for different degrees of clot lysis both in EXTEM and NATEM. t-AUCi cutoff value of 1962.0 seconds in EXTEM predicted low fibrinolytic activity with 81.8% sensitivity and 83.7% specificity. In addition, t-AUCi is not influenced by clot retraction. CONCLUSION: Whole-blood point-of-care ROTEM analyses with t-AUCi offers a more rapid and parametric evaluation of fibrinolytic potential compared with CLI, which can be used for a more rapid and accurate diagnosis of fibrinolysis resistance in sepsis.

US Soldiers' Individual and Unit-level Factors Associated with Perceptions of Disinformation in the Military Context.

INTRODUCTION: Although the US Government considers threats of misinformation, disinformation, and mal-information to rise to the level of terrorism, little is known about service members' experiences with disinformation in the military context. We examined soldiers' perceptions of disinformation impact on the Army and their units. We also investigated associations between disinformation perceptions and soldiers' sociodemographic characteristics, reported use of fact-checking, and perceptions of unit cohesion and readiness. METHODS: Active-duty soldiers (N = 19,465) across two large installations in the Southwest US completed an anonymous online survey. RESULTS: Sixty-six percent of soldiers agreed that disinformation has a negative impact on the Army. Thirty-three percent of soldiers perceived disinformation as a problem in their unit. Females were more likely to agree that disinformation has a negative impact on the Army and is a problem in their unit. Higher military rank was associated with lower odds of agreeing that disinformation is a problem in units. Most soldiers were confident about their ability to recognize disinformation (62%) and reported using fact-checking resources (53%), and these factors were most often endorsed by soldiers who agreed that disinformation is a problem for the Army and their unit. Soldiers' perceptions of unit cohesion and readiness were negatively associated with the perception that disinformation is a problem in their unit. CONCLUSION: While the majority of soldiers viewed disinformation as a problem across the Army, fewer perceived it as problematic within their units. Higher levels of reported fact-checking were most evident among those who perceived disinformation as a problem, suggesting that enhancing awareness of the problem of disinformation alone could help mitigate its deleterious impact. Perceptions of disinformation problems within units were associated with soldiers' perceptions of lower unit cohesion and readiness, highlighting misinformation, disinformation, and mal-information's impact on force readiness. Limitations and future directions are discussed.

Evidence for Enhanced Tracer Diffusion in Densely Packed Interfacial Assemblies of Hairy Nanoparticles.

Nearly monodisperse nanoparticle (NP) spheres attached to a nonvolatile ionic liquid surface were tracked by in situ scanning electron microscopy to obtain the tracer diffusion coefficient Dtr as a function of the areal fraction ϕ. The in situ technique resolved both tracer (gold) and background (silica) particles for ∼1-2 min, highlighting their mechanisms of diffusion, which were strongly dependent on ϕ. Structure and dynamics at low and moderate ϕ paralleled those reported for larger colloidal spheres, showing an increase in order and a decrease in Dtr by over 4 orders of magnitude. However, ligand interactions were more important near jamming, leading to different caging and jamming dynamics for smaller NPs. The normalized Dtr at ultrahigh ϕ depended on particle diameter and ligand molecular weight. Increasing the PEG molecular weight by a factor of 4 increased Dtr by 2 orders of magnitude at ultrahigh ϕ, indicating stronger ligand lubrication for smaller particles.

4D intravital imaging studies identify platelets as the predominant cellular procoagulant surface in a mouse model of hemostasis.

Interplay between platelets, coagulation/fibrinolytic factors, and endothelial cells (ECs) is necessary for effective hemostatic plug formation. This study describes a novel four-dimensional (4D) imaging platform to visualize and quantify hemostatic plug components with high spatiotemporal resolution. Fibrin accumulation following laser-induced endothelial ablation was observed at the EC-platelet plug interface, controlled by the antagonistic balance between fibrin generation and breakdown. Phosphatidylserine (PS) was first detected in close physical proximity to the fibrin ring, followed by exposure across the endothelium. Impaired PS exposure in cyclophilinD -/- mice resulted in a significant reduction in fibrin accumulation. Adoptive transfer and inhibitor studies demonstrated a key role for platelets, but not ECs, in fibrin generation during hemostatic plug formation. Inhibition of fibrinolysis with tranexamic acid (TXA) led to increased fibrin accumulation in WT mice, but not in cyclophilinD -/- mice or WT mice treated with antiplatelet drugs. These studies implicate platelets as the functionally dominant procoagulant surface during hemostatic plug formation. In addition, they suggest that impaired fibrin formation due to reduced platelet procoagulant activity is not reversed by TXA treatment.

Relaxing Wrinkles in Jammed Interfacial Assemblies.

Dynamic covalent bonding has emerged as a mean by which stresses in a network can be relaxed. Here, the strength of the bonding of ligands to nanoparticles at the interface between two immiscible liquids affect the same results in jammed assemblies of nanoparticle surfactants. Beyond a critical degree of overcrowding induced by the compression of jammed interfacial assemblies, the bonding of ligands to nanoparticles (NPs) can be broken, resulting in a desorption of the NPs from the interface. This reduces the areal density of nanoparticle surfactants at the interface, allowing the assemblies to relax, not to a fluid state but rather another jammed state. The relaxation of the wrinkles caused by the compression reflects the tendency of these assemblies to eliminate areas of high curvature, favoring a more planar geometry. This enabled the generation of giant vesicular and multivesicular structures from these assemblies.

A collaborative approach to improving representation in viral genomic surveillance.

The lack of routine viral genomic surveillance delayed the initial detection of SARS-CoV-2, allowing the virus to spread unfettered at the outset of the U.S. epidemic. Over subsequent months, poor surveillance enabled variants to emerge unnoticed. Against this backdrop, long-standing social and racial inequities have contributed to a greater burden of cases and deaths among minority groups. To begin to address these problems, we developed a new variant surveillance model geared toward building 'next generation' genome sequencing capacity at universities in or near rural areas and engaging the participation of their local communities. The resulting genomic surveillance network has generated more than 1,000 SARS-CoV-2 genomes to date, including the first confirmed case in northeast Louisiana of Omicron, and the first and sixth confirmed cases in Georgia of the emergent BA.2.75 and BQ.1.1 variants, respectively. In agreement with other studies, significantly higher viral gene copy numbers were observed in Delta variant samples compared to those from Omicron BA.1 variant infections, and lower copy numbers were seen in asymptomatic infections relative to symptomatic ones. Collectively, the results and outcomes from our collaborative work demonstrate that establishing genomic surveillance capacity at smaller academic institutions in rural areas and fostering relationships between academic teams and local health clinics represent a robust pathway to improve pandemic readiness.

SHock-INduced Endotheliopathy (SHINE): A mechanistic justification for viscoelastography-guided resuscitation of traumatic and non-traumatic shock.

Irrespective of the reason for hypoperfusion, hypocoagulable and/or hyperfibrinolytic hemostatic aberrancies afflict up to one-quarter of critically ill patients in shock. Intensivists and traumatologists have embraced the concept of SHock-INduced Endotheliopathy (SHINE) as a foundational derangement in progressive shock wherein sympatho-adrenal activation may cause systemic endothelial injury. The pro-thrombotic endothelium lends to micro-thrombosis, enacting a cycle of worsening perfusion and increasing catecholamines, endothelial injury, de-endothelialization, and multiple organ failure. The hypocoagulable/hyperfibrinolytic hemostatic phenotype is thought to be driven by endothelial release of anti-thrombogenic mediators to the bloodstream and perivascular sympathetic nerve release of tissue plasminogen activator directly into the microvasculature. In the shock state, this hemostatic phenotype may be a counterbalancing, yet maladaptive, attempt to restore blood flow against a systemically pro-thrombotic endothelium and increased blood viscosity. We therefore review endothelial physiology with emphasis on glycocalyx function, unique biomarkers, and coagulofibrinolytic mediators, setting the stage for understanding the pathophysiology and hemostatic phenotypes of SHINE in various etiologies of shock. We propose that the hyperfibrinolytic phenotype is exemplified in progressive shock whether related to trauma-induced coagulopathy, sepsis-induced coagulopathy, or post-cardiac arrest syndrome-associated coagulopathy. Regardless of the initial insult, SHINE appears to be a catecholamine-driven entity which early in the disease course may manifest as hyper- or hypocoagulopathic and hyper- or hypofibrinolytic hemostatic imbalance. Moreover, these hemostatic derangements may rapidly evolve along the thrombohemorrhagic spectrum depending on the etiology, timing, and methods of resuscitation. Given the intricate hemochemical makeup and changes during these shock states, macroscopic whole blood tests of coagulative kinetics and clot strength serve as clinically useful and simple means for hemostasis phenotyping. We suggest that viscoelastic hemostatic assays such as thromboelastography (TEG) and rotational thromboelastometry (ROTEM) are currently the most applicable clinical tools for assaying global hemostatic function-including fibrinolysis-to enable dynamic resuscitation with blood products and hemostatic adjuncts for those patients with thrombotic and/or hemorrhagic complications in shock states.

Assays to quantify fibrinolysis: strengths and limitations. Communication from the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee on fibrinolysis.

Fibrinolysis is a series of enzymatic reactions that degrade insoluble fibrin. Plasminogen activators convert the zymogen plasminogen to the active serine protease plasmin, which cleaves and solubilizes crosslinked fibrin clots into fibrin degradation products. The quantity and quality of fibrinolytic enzymes, their respective inhibitors, and clot structure determine overall fibrinolysis. The quantity of protein can be measured by antigen-based assays, and both quantity and quality can be assessed using functional assays. Furthermore, variations of commonly used assays have been reported, which are tailored to address the role(s) of specific fibrinolytic factors and cellular elements (eg, platelets, neutrophils, and red blood cells). Although the concentration and/or activity of a protein can be quantified, how these individual components contribute to the overall fibrinolysis outcome can be challenging to determine. This difficulty is due to temporal changes within and around the thrombi during the clot breakdown, particularly the fibrin matrix structure, and composition. Furthermore, terms such as "fibrinolytic activity/potential," "plasminogen activation," and "plasmin activity" are often used interchangeably despite having different definitions. The purpose of this review is to 1) summarize the assays measuring fibrinolysis activity and potential, 2) facilitate the interpretation of data generated by these assays, and 3) summarize the strengths and limitations of these assays.

Comparison of three dosing intervals for the primary vaccination of the SARS-CoV-2 mRNA Vaccine (BNT162b2) on magnitude, neutralization capacity and durability of the humoral immune response in health care workers: A prospective cohort study.

OBJECTIVES: The dosing interval of a primary vaccination series can significantly impact on vaccine immunogenicity and efficacy. The current study compared 3 dosing intervals for the primary vaccination series of the BNT162b2 mRNA COVID-19 vaccine, on humoral immune response and durability against SARS-CoV-2 ancestral and Beta variants up to 9 months post immunization. METHODS: Three groups of age- and sex-matched healthcare workers (HCW) who received 2 primary doses of BNT162b2 separated by 35-days, 35-42 days or >42-days were enrolled. Vaccine induced antibody titers at 3 weeks, 3 and 6-9 months post-second dose were assessed. RESULTS: There were 309 age- and sex-matched HCW (mean age 43 [sd 13], 58% females) enrolled. Anti-SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibody titers showed significant waning in levels beyond 35 days post first dose. The second dose induced a significant rise in antibody titers, which peaked at 3 weeks and then declined at variable rates across groups. The magnitude, consistency and durability of response was greater for anti-Spike than anti-RBD antibodies; and for IgG than IgA or IgM. Compared to the shorter schedules, a longer interval of >42 days offered the highest binding and neutralizing antibody titers against SARS-CoV-2 ancestral and Beta (B1.351) variants beyond 3 months post-vaccination. CONCLUSIONS: This is the first comprehensive study to compare 3 dosing intervals for the primary vaccination of BNT162b2 mRNA COVID-19 vaccine implemented in the real world. These findings suggest that delaying the second dose beyond 42 days can potentiate and prolong the humoral response against ancestral and Beta variants of SARS-CoV-2 up to 9 months post-vaccination.

Cross-immunity against SARS-COV-2 variants of concern in naturally infected critically ill COVID-19 patients.

Critically ill patients infected with SARS-CoV-2 display adaptive immunity, but it is unknown if they develop cross-reactivity to variants of concern (VOCs). We profiled cross-immunity against SARS-CoV-2 VOCs in naturally infected, non-vaccinated, critically ill COVID-19 patients. Wave-1 patients (wild-type infection) were similar in demographics to Wave-3 patients (wild-type/alpha infection), but Wave-3 patients had higher illness severity. Wave-1 patients developed increasing neutralizing antibodies to all variants, as did patients during Wave-3. Wave-3 patients, when compared to Wave-1, developed more robust antibody responses, particularly for wild-type, alpha, beta and delta variants. Within Wave-3, neutralizing antibodies were significantly less to beta and gamma VOCs, as compared to wild-type, alpha and delta. Patients previously diagnosed with cancer or chronic obstructive pulmonary disease had significantly fewer neutralizing antibodies. Naturally infected ICU patients developed adaptive responses to all VOCs, with greater responses in those patients more likely to be infected with the alpha variant, versus wild-type.

A collaborative approach to improve representation in viral genomic surveillance.

The lack of routine viral genomic surveillance delayed the initial detection of SARS-CoV-2, allowing the virus to spread unfettered at the outset of the U.S. epidemic. Over subsequent months, poor surveillance enabled variants to emerge unnoticed. Against this backdrop, long-standing social and racial inequities have contributed to a greater burden of cases and deaths among minority groups. To begin to address these problems, we developed a new variant surveillance model geared toward building microbial genome sequencing capacity at universities in or near rural areas and engaging the participation of their local communities. The resulting genomic surveillance network has generated more than 1,000 SARS-CoV-2 genomes to date, including the first confirmed case in northeast Louisiana of Omicron, and the first and sixth confirmed cases in Georgia of the emergent BA.2.75 and BQ.1.1 variants, respectively. In agreement with other studies, significantly higher viral gene copy numbers were observed in Delta variant samples compared to those from Omicron BA.1 variant infections, and lower copy numbers were seen in asymptomatic infections relative to symptomatic ones. Collectively, the results and outcomes from our collaborative work demonstrate that establishing genomic surveillance capacity at smaller academic institutions in rural areas and fostering relationships between academic teams and local health clinics represent a robust pathway to improve pandemic readiness. Author summary: Genomic surveillance involves decoding a pathogen’s genetic code to track its spread and evolution. During the pandemic, genomic surveillance programs around the world provided valuable data to scientists, doctors, and public health officials. Knowing the complete SARS-CoV-2 genome has helped detect the emergence of new variants, including ones that are more transmissible or cause more severe disease, and has supported the development of diagnostics, vaccines, and therapeutics. The impact of genomic surveillance on public health depends on representative sampling that accurately reflects the diversity and distribution of populations, as well as rapid turnaround time from sampling to data sharing. After a slow start, SARS-CoV-2 genomic surveillance in the United States grew exponentially. Despite this, many rural regions and ethnic minorities remain poorly represented, leaving significant gaps in the data that informs public health responses. To address this problem, we formed a network of universities and clinics in Louisiana, Georgia, and Mississippi with the goal of increasing SARS-CoV-2 sequencing volume, representation, and equity. Our results demonstrate the advantages of rapidly sequencing pathogens in the same communities where the cases occur and present a model that leverages existing academic and clinical infrastructure for a powerful decentralized genomic surveillance system.

Sustained Oncogenic Signaling in the Cytostatic State Enables Targeting of Nonproliferating Persistent Cancer Cells.

Many advanced therapeutics possess cytostatic properties that suppress cancer cell growth without directly inducing death. Treatment-induced cytostatic cancer cells can persist and constitute a reservoir from which recurrent growth and resistant clones can develop. Current management approaches primarily comprise maintenance and monitoring because strategies for targeting nonproliferating cancer cells have been elusive. Here, we used targeted therapy paradigms and engineered cytostatic states to explore therapeutic opportunities for depleting treatment-mediated cytostatic cancer cells. Sustained oncogenic AKT signaling was common, while nonessential, in treatment-mediated cytostatic cancer cells harboring PI3K-pathway mutations, which are associated with cancer recurrence. Engineering oncogenic signals in quiescent mammary organotypic models showed that sustained, aberrant activation of AKT sensitized cytostatic epithelial cells to proteasome inhibition. Mechanistically, sustained AKT signaling altered cytostatic state homeostasis and promoted an oxidative and proteotoxic environment, which imposed an increased proteasome dependency for maintaining cell viability. Under cytostatic conditions, inhibition of the proteasome selectively induced apoptosis in the population with aberrant AKT activation compared with normal cells. Therapeutically exploiting this AKT-driven proteasome vulnerability was effective in depleting treatment-mediated cytostatic cancer cells independent of breast cancer subtype, epithelial origin, and cytostatic agent. Moreover, transient targeting during cytostatic treatment conditions was sufficient to reduce recurrent tumor growth in spheroid and mouse models. This work identified an AKT-driven proteasome-vulnerability that enables depletion of persistent cytostatic cancer cells harboring PTEN-PI3K pathway mutations, revealing a viable strategy for targeting nonproliferating persistent cancer cell populations before drug resistance emerges. SIGNIFICANCE: This study finds that sustained oncogenic signaling in therapy-induced cytostatic cancer cells confers targetable vulnerabilities to deplete persistent cancer cell populations and reduce cancer recurrence.

Relaxation and Aging of Nanosphere Assemblies at a Water-Oil Interface.

The relaxation and aging of an assembly of spherical nanoparticles (NPs) at a water-oil interface are characterized in situ by grazing incidence X-ray photon correlation spectroscopy. The dynamics of the interfacial assembly is measured while the interface saturates with NPs. Weak attractions between NPs lead to gel-like structures in the assembly, where the in-plane ordering is inhibited by the broad size distribution of the NPs. Structural rearrangements on the length scale of the NP-NP center-to-center distances proceed by intermittent fluctuations instead of continuous cooperative motions. The coexistence of rapid and slow NP populations is confirmed, as commonly observed in soft glass-forming materials. Dynamics are increasingly slowed as the NPs initially segregate to the locally clustered interface. The structural relaxation of the NPs in these localized clusters is 5 orders of magnitude slower than that of free particles in the bulk. When the interface is nearly saturated, the time for relaxation increases suddenly due to the onset of local jamming, and the dynamics slow exponentially afterward until the system reaches collective jamming by cooperative rearrangements. This investigation provides insights into structural relaxations near the glass transition and the evolution of the structure and dynamics of the assemblies as they transition from an isotropic liquid to a dense disordered film.

Suicide Behavior Results From the U.S. Army's Suicide Prevention Leadership Tool Study: The Behavioral Health Readiness and Suicide Risk Reduction Review (R4).

INTRODUCTION: The U.S. Army developed a new tool called the Behavioral Health Readiness and Suicide Risk Reduction Review (R4) for suicide prevention. A 12-month evaluation study with the primary objective of testing the hypothesis (H1) that Army units receiving R4 would demonstrate improved outcomes in suicidal-behavior measures following the intervention, relative to control, was then conducted. The results of analyses to answer H1 are herein presented. MATERIALS AND METHODS: The R4 intervention (R4-tools/instructions/orientation) evaluation study, Institutional Review Board approved and conducted in May 2019-June 2020, drew samples from two U.S. Army divisions and employed a repeated measurement in pre-/post-quasi-experimental design, including a nonequivalent, but comparable, business-as-usual control. Intervention effectiveness was evaluated using self-report responses to suicide-related measures (Suicide Behaviors Questionnaire-Revised/total-suicide behaviors/ideations/plans/attempts/non-suicidal self-injuries) at 6-/12-month intervals. Analyses examined baseline to follow-up linked and cross-sectional cohorts, incidence/prevalence, and intervention higher-/lower-use R4 subanalyses. RESULTS: Both divisions demonstrated favorable in-study reductions in total-suicide burden, with relatively equivalent trends for total-suicide behaviors, total-suicide risk (Suicide Behaviors Questionnaire-Revised), suicidal ideations, and non-suicidal self-injuries. Although both demonstrated reductions in suicide plans, the control showed a more robust trend. Neither division demonstrated a significant reduction in suicide attempts, but subgroup analyses showed a significant reduction in pre-coronavirus disease 2019-attempt incidence among those with higher-use R4 relative to control. CONCLUSIONS: There is no evidence of harm associated with the R4 intervention. R4 effectiveness as a function of R4 itself requires confirmatory study. R4 is judged an improvement (no evidence of harm + weak evidence of effectiveness) over the status quo (no safety data or effectiveness studies) with regard to tool-based decision-making support for suicide prevention in the U.S. Army.