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The HIV-1 capsid has emerged as a tractable target for antiretroviral therapy. Lenacapavir, developed by Gilead Sciences, is the first capsid-targeting drug approved for medical use. Here we investigate the effect of Lenacapavir on HIV capsid stability and uncoating. We employ a single particle approach that simultaneously measures capsid content release and lattice persistence. We demonstrate that Lenacapavir's potent antiviral activity is predominantly due to lethal hyperstabilisation of the capsid lattice and resultant loss of compartmentalisation. This study highlights that disrupting capsid metastability is a powerful strategy for the development of novel antivirals.

Teaming permits cognitively complex work to be rapidly executed by multiple entities. As artificial agents (AAs) participate in increasingly complex cognitive work, they hold the promise of moving beyond tools to becoming effective members of human–agent teams. Coordination has been identified as the critical process that enables effective teams and is required to achieve the vision of tightly coupled teams of humans and AAs. This paper characterizes coordination on the axes of types, content, and cost. This characterization is grounded in the human and AA literature and is evaluated to extract design implications for human–agent teams. These design implications are the mechanisms, moderators, and models employed within human–agent teams, which illuminate potential AA design improvements to support coordination.

The HIV capsid is a multifunctional protein capsule that mediates the delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here, we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labeled capsid-binding analytes ("prey" molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using the fluorescent capsid as the bait further allows the quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for the discovery and characterization of molecules used by the HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays, utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms.

The human immunodeficiency virus (HIV) capsid is a cone-shaped capsule formed from the viral capsid protein (CA), which is arranged into a lattice of hexamers and pentamers. The capsid comprises multiple binding interfaces for the recruitment of host proteins and macromolecules used by the virus to establish infection. Here, we coassembled CA proteins engineered for pentamer cross-linking and fluorescence labeling, into spherical particles. The CA spheres, which resemble the pentamer-rich structure of the end caps of the native HIV capsid, were immobilized onto surfaces as biorecognition elements for fluorescence microscopy-based quantification of host protein binding. The capsid-binding host protein cyclophilin A (CypA) is bound to CA spheres with the same affinity as CA tubes but at a higher CypA/CA stoichiometry, suggesting that the level of recruitment of CypA to the HIV capsid is dependent on curvature.

Autonomous unmanned aerial systems (UAS) could supplement and eventually subsume a substantial portion of the mission set currently executed by remote pilots, making UAS more robust, responsive, and numerous than can be achieved by teleoperation alone. Unfortunately, the development of robust autonomous systems is difficult, costly, and time-consuming. Furthermore, the resulting systems often make little reuse of proven software components and offer limited adaptability for new tasks. This work presents a development platform for UAS which promotes behavioral flexibility. The platform incorporates the unified behavior framework (a modular, extensible autonomy framework), the robotic operating system (a robotic software framework), and PX4 (an open-source flight controller). Simulation of UBF agents identify a combination of reactive robotic control strategies effective for small-scale navigation tasks by a UAS in the presence of obstacles. Finally, flight tests provide a partial validation of the simulated results. The development platform presented in this work offers robust and responsive behavioral flexibility for UAS agents in simulation and reality using a methodology originally proven on ground robots.

Nature microbiology 7(11), 1762 - 1776 (2022). doi:10.1038/s41564-022-01247-0, Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation., Published by Nature Publishing Group, London

Knowledge transfer among research disciplines can lead to substantial research progress. At first glance, astronaut health and rare diseases may be seen as having little common ground for such an exchange. However, deleterious health conditions linked to human space exploration may well be considered as a narrow sub-category of rare diseases. Here, we compare and contrast research and healthcare in the contexts of rare diseases and space health and identify common barriers and avenues of improvement. The prevalent genetic basis of most rare disorders contrasts sharply with the occupational considerations required to sustain human health in space. Nevertheless small sample sizes and large knowledge gaps in natural history are examples of the parallel challenges for research and clinical care in the context of both rare diseases and space health. The two areas also face the simultaneous challenges of evidence scarcity and the pressure to deliver therapeutic solutions, mandating expeditious translation of research knowledge into clinical care. Sharing best practices between these fields, including increasing participant involvement in all stages of research and ethical sharing of standardized data, has the potential to contribute to humankind’s efforts to explore ever further into space while caring for people on Earth in a more inclusive fashion.

Overall survival in AML patients remains poor. Selection of the right treatment for the right patient will contribute to better response rates and survival. Biological data associated with response and survival may inform development of novel treatment strategies. In this thesis, we generated data to optimize prediction of survival and treatment response in AML patients. We reviewed commonly used culture techniques for AML samples, that are at the basis of targeted therapy development and response-prediction experiments. We identified several pitfalls that should be taken into account when planning and interpreting data from ex vivo drug response experiments. With the described recommendations, we aim to maximize the reproducibility and meaningfulness of ex vivo experiments with primary patient AML cells. Although novel treatment strategies are of paramount importance to improve clinical outcomes in AML patients and the development of novel agents has surged, chemotherapy is likely to remain the basis of curative treatment for the coming years. To better understand response mechanisms towards currently used chemotherapeutic regimens, we investigated molecular processes that determine their cytotoxic effects. Our data suggest that distinct gene expression profiles are associated with ex vivo drug response, and may confer a priori drug resistance in leukemic cells. Gene function and expression may be affected by genetic mutations, or may be altered by other (epigenetic) mechanisms, independent of mutations. These genetic aberrations often have prognostic implications for AML patients. The frequency of TP53 mutations and their prognostic impact in pediatric AML were unclear, as well as the impact of differential expression of p53 pathway genes. Therefore, we analyzed TP53 alterations in bone marrow samples of 229 children with de novo AML. Our results indicate that TP53 alterations in children are rare, but that alterations in TP53 – and differential expression of its pathway genes – are associated with poor survival in pediatric AML. Mutations that frequently occur in AML, are internal tandem duplications of FLT3 (FLT3-ITD). Although an established adverse prognostic marker in adult AML, its precise role in pediatric AML was unclear, as well as the preferred measurement level (DNA or RNA/cDNA). Our results show that in pediatric AML, a high FLT3-ITD allelic ratio (FLT3-ITD-AR) is predictive for poor overall survival. Importantly, only RNA/cDNA-based FLT3-ITD-AR measurements – and not DNA measurements – have prognostic value in pediatric AML patients. Also in adults, we showed that the use of RNA/cDNA enables better detection of minor FLT3-ITD clones than DNA. This primarily impacts treatment selection and not prognostication as the additional identified FLT3-ITD clones only represent minor clones with low allelic ratios. However, more AML patients would be eligible for treatment with FLT3-TKIs using a cDNA-based approach. Next, profiled primary AML samples at the phosphoproteomic level to characterize FLT3-ITD AML and identify phosphorylation profiles associated with FLT3-TKI response. We demonstrated that ERK1/2 activity is associated with impaired response towards gilteritinib, and that modulation of the RAS-RAF-MEK-ERK axis may be a promising approach to improve responses and reduce resistance towards FLT3-TKIs. In future research, these analyses should ideally be performed directly on clinical specimens from patients treated with midostaurin and gilteritinib to provide more direct mechanisms of clinical response towards FLT3-TKIs. Curative pharmacological options remain limited for AML and come at a high cost of severe side effects, and thus we are still awaiting novel treatments that improve duration and quality of life of all AML patients. We anticipate that in the future, refined patient selection based on integral prediction models for response and survival, combined with novel targeted agents and/or combination strategies will maximize clinical benefit of currently available therapies, as well as of novel therapies in development.

Human immunodeficiency virus (HIV) is the most extensively researched human pathogen. Despite this massive scientific endeavour, several fundamental viral processes remain enigmatic. One such critical process is uncoating—the event that releases the viral genome from the proteinaceous shell of the capsid during infection. While this process is conceptually simple, the molecular underpinnings, timing, regulation, and cellular location of uncoating remain contentious. This review describes the hurdles that have limited our understanding in this area and presents recently deployed in vitro and in cellulo techniques that have been developed expressly with the aim of directly visualising capsid uncoating at the single-particle level and understanding the mechanics behind this essential aspect of HIV infection.

Many firms generate revenue by operating systems or fleets, such as welding robots, rental cars, aircraft, etc. The contribution of service parts to the availability of the system or fleet is well ...

Trim-Away is a recently developed technology that exploits off-the-shelf antibodies and the RING E3 ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically activated upon target engagement, either during its normal immune function or when repurposed for targeted protein degradation, is unknown. Here we show that a mechanism of target-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce virus neutralization or drive Trim-Away. We harness this mechanism for selective degradation of disease-causing huntingtin protein containing long polyglutamine tracts and expand the Trim-Away toolbox with highly active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has implications for targeted protein degradation technologies.

The HIV capsid self-assembles a protective conical shell that simultaneously prevents host sensing whilst permitting the import of nucleotides to drive DNA synthesis. This is accomplished through the construction of dynamic, highly charged pores at the centre of each capsid multimer. The clustering of charges required for dNTP import is strongly destabilising and it is proposed that HIV uses the metabolite IP6 to coordinate the pore during assembly. Here we have investigated the role of inositol phosphates in coordinating a ring of positively charged lysine residues (K25) that forms at the base of the capsid pore. We show that whilst IP5, which can functionally replace IP6, engages an arginine ring (R18) at the top of the pore, the lysine ring simultaneously binds a second IP5 molecule. Dose dependent removal of K25 from the pore severely inhibits HIV infection and concomitantly prevents DNA synthesis. Cryo-tomography reveals that K25A virions have a severe assembly defect that inhibits the formation of mature capsid cones. Monitoring both the kinetics and morphology of capsids assembled in vitro reveals that while mutation K25A can still form tubes, the ability of IP6 to drive assembly of capsid cones has been lost. Finally, in single molecule TIRF microscopy experiments, capsid lattices in permeabilised K25 mutant virions are rapidly lost and cannot be stabilised by IP6. These results suggest that the coordination of IP6 by a second charged ring in mature hexamers drives the assembly of conical capsids capable of reverse transcription and infection., Author summary HIV protects its RNA genome while copying it into DNA by carrying out reverse transcription inside its capsid. This is accomplished by importing nucleotides through highly charged pores at the centre of each capsid multimer. These pores contain two rings of positively charged residues–R18 and K25 –but assembling capsids with these features is challenging because they are intrinsically destabilising. Here we show that the metabolite IP6 coordinates both residues within the pore to drive the assembly of stable capsids capable of nucleotide import. R18 or K25 mutants lose infectivity and the ability to synthesise DNA but have differing assembly phenotypes. Mutant K25A is unable to undergo efficient capsid assembly, while replacing K25 with a neutral polar residue partially restores assembly but not infectivity. We propose that IP6-driven assembly is conserved by HIV not because it is the only way to build a capsid, but because it allows the construction of a capsid with a charged pore that can import nucleotides.

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.

The implementation and testing of autonomous and cooperative unmanned systems is challenging due to the inherent design complexity, infinite test spaces, and lack of autonomy specific measures. Simulation provides a low cost alternative to flight tests, allowing researchers to rapidly iterate on the design before fielding. To expedite this process, an Autonomous System Reference Architecture allows researchers to utilize existing software modules to rapidly develop algorithms for autonomous systems and test them in included simulation environments. In this paper, we implement ASRA on a cooperative wide area search scenario as a test bed to study ASRA’s utility for rapid prototyping and evaluation of autonomous and cooperative systems. Through a face centered cubic design of experiments, selected autonomy metrics are studied to provide a response surface model to characterize the system and provide a tool to optimize mission control parameters and maximize mission performance.

Across many industries, systems are exceeding their intended design lives, whether they are ships, bridges or military aircraft. As a result failure rates can increase and unanticipated wear or failure conditions can arise. Health monitoring research and application has the potential to more safely lengthen the service life of a range of systems through utilization of sensor data and knowledge of failure mechanisms to predict component life remaining. A further benefit of health monitoring when combined across an entire platform is system health management. System health management is an enabler of condition based maintenance, which allows repair or replacement based on material condition, not a set time. Replacement of components based on condition can enable cost savings through fewer parts being used and the associated maintenance costs. The goal of this research is to show the management of system health can provide savings in maintenance and logistics cost while increasing vehicle availability through the approach of condition based maintenance.This work examines the impact of prediction accuracy uncertainty in remaining useful life prognostics for a squadron of 12 aircraft. The uncertainty in this research is introduced in the system through an uncertainty factor applied to the useful life prediction. An ARENA discrete event simulation is utilized to explore the effect of prediction error on availability, reliability, and maintenance and logistics processes. Aircraft are processed through preflight, flight, and post-flight operations, as well as maintenance and logistics activities. A baseline case with traditional time driven maintenance is performed for comparison to the condition based maintenance approach of this research.This research does not consider cost or decision making processes, instead focusing on utilization parameters of both aircraft and manpower. The occurrence and impact of false alarms on system performance is examined. The results show the potential availability, reliability, and maintenance benefits of a health monitoring system and explore the diagnostic uncertainty.

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SUMMARYTrim-Away is a powerful new technology that exploits off-the-shelf antibodies and the E3 RING ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically-activated upon substrate engagement during either its normal immune function or when re-purposed for targeted protein degradation is unknown. Here we show that a mechanism of substrate-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce an antiviral response or drive Trim-Away. We harness this mechanism to expand the Trim-Away toolbox with highly-active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has important implications for targeted protein degradation technologies.

The genome of SARS-CoV-2 (SARS2) encodes for two viral proteases (NSP3/ papain-like protease and NSP5/ 3C-like protease or major protease) that are responsible for cleaving viral polyproteins for successful replication. NSP3 and NSP5 of SARS-CoV (SARS1) are known interferon antagonists. Here, we examined whether the protease function of SARS2 NSP3 and NSP5 target proteins involved in the host innate immune response. We designed a fluorescent based cleavage assay to rapidly screen the protease activity of NSP3 and NSP5 on a library of 71 human innate immune proteins (HIIPs), covering most pathways involved in human innate immunity. By expressing each of these HIIPs with a genetically encoded fluorophore in a cell-free system and titrating in the recombinant protease domain of NSP3 or NSP5, we could readily detect cleavage of cognate HIIPs on SDS-page gels. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type- I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of IL-6 and inflammatory response observed in COVID-19 patients. Surprisingly, both NLRP12 and TAB1 have each two distinct cleavage sites. We demonstrate that in mice, the second cleavage site of NLRP12 is absent. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for in-depth studies into the pathophysiology of COVID-19 and should facilitate the search or development of more effective animal models for severe COVID-19. Finally, we discovered that one particular species of bats, David’s Myotis, possesses the five cleavage sites found in humans for NLRP12, TAB1 and IRF3. These bats are endemic from the Hubei province in China and we discuss its potential role as reservoir for the evolution of SARS1 and SASR2.

The type one interferon induced restriction factor Myxovirus resistance B (MxB) restricts HIV-1 nuclear entry evidenced by inhibition of 2-LTR but not linear forms of viral DNA. The HIV-1 capsid is the key determinant of MxB sensitivity and cofactor binding defective HIV-1 capsid mutants P90A (defective for cyclophilin A and Nup358 recruitment) and N74D (defective for CPSF6 recruitment) have reduced dependency on nuclear transport associated cofactors, altered integration targeting preferences and are not restricted by MxB expression. This has suggested that nuclear import mechanism may determine MxB sensitivity. Here we have use genetics to separate HIV-1 nuclear import cofactor dependence from MxB sensitivity. We provide evidence that MxB sensitivity depends on HIV-1 capsid conformation, rather than cofactor recruitment. We show that depleting CPSF6 to change nuclear import pathway does not impact MxB sensitivity, but mutants that recapitulate the effect of Cyclophilin A binding on capsid conformation and dynamics strongly impact MxB sensitivity. We demonstrate that HIV-1 primary isolates have different MxB sensitivities due to cytotoxic T lymphocyte (CTL) selected differences in Gag sequence but similar cofactor dependencies. Overall our work demonstrates a complex relationship between cyclophilin dependence and MxB sensitivity likely driven by CTL escape. We propose that cyclophilin binding provides conformational flexibility to HIV-1 capsid facilitating simultaneous evasion of capsid-targeting restriction factors including TRIM5 as well as MxB.

Levels of Automation (LOA) provide a method for describing authority granted to automated system elements to make individual decisions. However, these levels are technology-centric and provide little insight into overall system operation. The current research discusses an alternate classification scheme, referred to as the Level of Human Control Abstraction (LHCA). LHCA is an operator-centric framework that classifies a system&rsquo, s state based on the required operator inputs. The framework consists of five levels, each requiring less granularity of human control: Direct, Augmented, Parametric, Goal-Oriented, and Mission-Capable. An analysis was conducted of several existing systems. This analysis illustrates the presence of each of these levels of control, and many existing systems support system states which facilitate multiple LHCAs. It is suggested that as the granularity of human control is reduced, the level of required human attention and required cognitive resources decreases. Thus, it is suggested that designing systems that permit the user to select among LHCAs during system control may facilitate human-machine teaming and improve the flexibility of the system.

Purpose From on-board automotive diagnostics to real-time aircraft state of health, the implementation of health monitoring and management systems are an increasing trend. Further, reductions in operating budgets are forcing many companies and militaries to consider new operating and support environments. Combined with longer service lives for aircraft and other systems, maintenance and operations processes must be reconsidered. The majority of research efforts focus on health monitoring techniques and technologies, leaving others to determine the maintenance and logistics impact on the systems. The paper aims to discuss these issues. Design/methodology/approach This research analyzes the impact of a health monitoring system on a squadron of aircraft. Flight, maintenance and logistics operations are stochastically modeled to determine the impact of program decisions on supply metrics. An arena discrete event simulation is utilized to conduct this research on 20 components on each of the 12 aircraft modeled. Costs and availability are recorded for comparison across three sparing scenarios to include economic order quantity (EOQ) for baseline and health monitoring cases and a just-in-time (JIT) health monitoring set of simulations. Findings Data are presented for EOQ and JIT supply methods. A comparison of health monitoring enabled supply to current methods shows cost savings and availability gains. The different methodologies are compared and discussed as a trade-space for programmatic decisions. Originality/value This work demonstrates the ability of health monitoring systems and condition based maintenance to affect supply ordering decisions. The development of trade-spaces within operating environments is demonstrated along with the ability to conduct cost benefit analyses.