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Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody–envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires. The immunogen RC1 facilitates recognition of the V3-glycan patch on the envelope of HIV-1 and elicits specific serological responses in mice and macaques, making it a possible priming immunogen for sequential vaccination strategies in humans. [ABSTRACT FROM AUTHOR]

Many shipping companies are unwilling to share their raw data because of data privacy concerns. However, certain problems in the maritime industry become much more solvable or manageable if data are shared—for instance, the problem of reducing ship fuel consumption and thus emissions. In this study, we develop a two-stage method based on federated learning (FL) and optimization techniques to predict ship fuel consumption and optimize ship sailing speed. Because FL only requires parameters rather than raw data to be shared during model training, it can achieve both information sharing and data privacy protection. Our experiments show that FL develops a more accurate ship fuel consumption prediction model in the first stage and thus helps obtain the optimal ship sailing speed setting in the second stage. The proposed two-stage method can reduce ship fuel consumption by 2.5%–7.5% compared to models using the initial individual data. Moreover, our proposed FL framework protects the data privacy of shipping companies while facilitating the sharing of information among shipping companies. • A two-stage model for predicting ship fuel consumption and optimizing sailing speed. • Reducing ship fuel consumption and thus emissions. • A federated learning (FL) mechanism to protect data privacy. • Analysis of the integrity of alliance parties in the FL mechanism. • Numerical experiments showing the effectiveness of the proposed method. [ABSTRACT FROM AUTHOR]

The transportation of construction and demolition waste (CDW) is an essential problem in waste management. The CDW generated in large construction projects is typically transported by designated trucks. However, the disposal of scattered CDW produced by individuals or small projects is typically not planned, leading to its illegal dumping. Hong Kong aims to recycle scattered CDW by an appointment-recycling mechanism that involves a three-layered transportation network consisting of construction sites, recycling locations, and disposal facilities. In this study, we develop a two-stage Stackelberg game model to minimize the social costs of this three-layered transportation network. In the first stage, we consider the optimal decisions regarding the recycling locations, assigned trucks, and transport routes from the perspective of the government. In the second stage, the delivery routes for small projects are identified based on the government decisions. We transform the proposed model into a single-level integer program (IP) and conduct numerical experiments to demonstrate its efficiency and effectiveness. To the best of our knowledge, this is the first study to focus on the collection of scattered CDW. Thus, our study contributes to the literature on CDW transportation. Moreover, this study helps address a practical problem, i.e., the illegal dumping of scattered CDW, which is a common concern in many cities. In particular, even small amounts of CDW must be appropriately treated because it may contain hazardous materials. [ABSTRACT FROM AUTHOR]

Meta-learning aims to use the knowledge from previous tasks to facilitate the learning of novel tasks. Many meta-learning models elaborately design various task-shared inductive bias, and learn it from a large number of tasks, so the generalization capability of the learned inductive bias depends on the diversity of the training tasks. A common assumption in meta-learning is that the training tasks and the test tasks come from the same or similar task distributions. However, this is usually not strictly satisfied in practice, so meta-learning models need to cope with various novel in-domain or cross-domain tasks. To this end, we propose to use task augmentation to increase the diversity of training tasks, thereby improving the generalization capability of meta-learning models. Concretely, we consider the worst-case problem around the base task distribution, and derive the adversarial task augmentation method which can generate inductive bias-adaptive 'challenging' tasks. Our method can be used as a simple plug-and-play module for various meta-learning models, and improve their generalization capability. We conduct extensive experiments under in-domain and cross-domain few-shot learning and unsupervised few-shot learning settings, and evaluate our method on different types of data (images and text). Experimental results show that our method can effectively improve the generalization capability of various meta-learning models under different settings. [ABSTRACT FROM AUTHOR]

The construction industry has been severely affected by the COVID-19 pandemic and the associated restrictions on person-to-person contacts issued by the government. A construction site usually has a high number of workers working at the same time; therefore, the question of how to ensure their safety during the pandemic—that is, how to protect them from getting infected—has become an urgent problem. In this study, we propose a bi-objective integer programming model to establish the optimal schedule plan under COVID-19 regulations. We develop a solution method and conduct numerical experiments to solve and validate our model. The optimal schedule plan can avoid contacts between workers of different groups while minimizing the total costs of complying with government policy. Our proposed model can be applied in practice to help project managers establish a reasonable and cost-effective schedule plan. This study contributes to reducing the operating costs of contractors and protecting the health of construction workers. • Designing work–rest–lunch schedule for construction sites in the COVID-19 pandemic. • Assigning rest and lunch areas to workers in different groups. • A bi-objective integer program to minimize cost and avoid contacts between workers. • The validity of the proposed model and solution method is demonstrated. [ABSTRACT FROM AUTHOR]

• Co-occurrence among diseases is crucial to knowledge discovery in medicine. • Existing researches are mainly based on clinical experience rather than data driven. • The distance in the disease embedding space reflects the co-occurrence relationship. • Existing medical concept embedding models focus on prediction tasks. • The first work to use disease embedding to study co-occurrence relationships. The co-occurrence relationship among diseases facilitates the knowledge discovery in the medical field. However, due to limited data, previous researches are mainly based on clinician experience and simple statistics which make it difficult to discover deep associations among diseases. Treating the diagnoses in an electronic medical record (EMR) as interrelated random variables, we use Markov random fields to model the co-occurrence relationship among diseases and propose Di2Vec to learn distributed representations of diseases. The diseases having high co-occurrence frequency will be very close to each other in the embedding space. Considering the hierarchical structure in each diagnosis code, we introduce the subword embedding and explore its impact on the quality of embeddings, where the embedding of each diagnosis is expressed as the sum of its subword embedding. Qualitative and Quantitative experiments show that our Di2Vec can make the embeddings of diseases with high co-occurrence frequency close to each other, and can also outperform Skip-gram and CBOW when use these embeddings as the feature representations for medical expense prediction. Using subword embedding will make the disease embeddings to have better clustering property, but to a certain extent, it loss the co-occurrence information contained in the disease embeddings. [ABSTRACT FROM AUTHOR]

Magnetic mesoporous silicon was used as the core and P(MAA-co-NIPAM) polymer as the shell. Among them, MAA plays the role of pH response and NIPAM plays the role of temperature response. A core-shell stimulation responsive drug carrier Fe 3 O 4 /mSiO 2 /P(MAA-co-NIPAM) was constructed. The structure, magnetic properties, drug adsorption and drug release behavior of Fe 3 O 4 /mSiO 2 /P(MAA-co-NIPAM) nanoparticles were systematically studied. results show that Fe 3 O 4 /mSiO 2 /P(MAA-co-NIPAM) nanoparticles have obvious core-shell structure, and its saturation magnetization value was 9.77 emu/g. Dox was used as the drug model to investigate the drug loading and release ability of the samples. The results showed that the carrier had dual stimulation response to pH and temperature, and the release rate was higher at pH 6 and 40 ℃. The drug loading rate and entrapment efficiency were 11.43% and 76.21%, respectively. It has a potential application. [Display omitted] [ABSTRACT FROM AUTHOR]

A fault protection method is proposed for flexible direct-current (DC) power systems based on the cosine similarity of currents in current-limiting reactors. The typical characteristics of external and internal faults in a flexible DC power system were analyzed. The principles of cosine similarity and the fault current characteristics of current-limiting reactors were used to distinguish between internal and external faults. The ratio of the average positive and negative voltages of the current-limiting reactor was then used to distinguish the fault types and fault line. Finally, a simulation model of a circular flexible DC power system was constructed using MATLAB/Simulink software (2018b). The simulation results show that the proposed protection scheme can quickly and accurately identify the location and type of faults, and has a strong ability to withstand fault resistance and is less affected by distributed capacitance, noise, and communication delay. [ABSTRACT FROM AUTHOR]

Precise temperature regulation is a crucial guarantee for many microfluidic analyses. This study presents a linear cooling method of microfluidic chips based on a single TEC, which can achieve synchronous observation of samples. A numerical model was developed and experimentally verified to analyze the temperature responses under different driving current mechanisms of TEC. Based on simulation and experimental analysis, this study proposes to achieve linear cooling based on TEC driven by polynomial function current mechanism. The implementation process is clarified, and the iterative method to obtain the polynomial function current mechanism is provided and elaborated. Using a commercial TEC, the linear cooling of the microfluidic chip from 25.2 °C to −19.7 °C was successfully achieved through both simulation and experiment. The linear cooling rates ranging from 24 °C/min to 41 °C/min with linearity higher than 0.998 were obtained. Moreover, the influencing factors of linear cooling were discussed. It is found that the temperature of the TEC hot side has a significant impact on the linear cooling of the sample cell, while the impact of nitrogen gas temperature is almost negligible. Results also indicate that both the minimum and maximum cooling rates increase as TE-element length increases and TE-element height decreases. [Display omitted] • This work proposes a linear cooling method of microfluidic chip based on TEC. • Linear cooling of microfluidic chip was achieved through simulation and experiment. • Temperature responses of microfluidic chip cooled by TEC were analyzed. • Different driving current mechanisms of TEC were discussed. • Influencing factors and optimization of the linear cooling were discussed. [ABSTRACT FROM AUTHOR]

Governments and contractors have different goals in handling construction and demolition waste (CDW), which makes it challenging to design CDW recycling networks. This paper develops a Stackelberg game model that governments can use to decide the optimal locations and types of CDW disposal facilities by considering the interests of both the government and contractors. The proposed model is proven to be efficient and practical through a series of real-world case studies. The findings elucidate and manage the different goals of governments and contractors and can help government design an optimal CDW recycling network in practice. Future research could tackle the problem of designing a multiperiod recycling network for managing CDW. • This study designs an optimal construction waste disposal network by considering into account different goals of governments and contractors. • A two-stage Stackelberg game model is proposed to trade off interests between governments and contractors. • The two-stage Stackelberg game model is transformed to a single-level mixed-integer linear programming. • Real-world case studies demonstrate the validity of the proposed model. • The findings provide insights for government to design an optimal construction waste recycling network in practice. [ABSTRACT FROM AUTHOR]

Prefabrication is a sustainable construction method that improves productivity and reduces environmental footprints. Prefabricated panel is one typical type of components widely used in practice. When designing panel stacking plans for transportation, practitioners should consider numerous practical factors, e.g., panel stability and reshufflings. However, in current practice, stacking plans are made on the basis of planners' intuition and experience. A scientific method that can yield satisfactory panel stacking plans is thus required. Motivated by this real need, this paper aims to address a practical problem for practitioners: how to stack prefabricated panels transported by A-frame trailers with high stability and little reshuffling. This paper extends the literature by innovatively formulating a bi-objective mixed-integer programming model that incorporates all the aforementioned factors and objectives. A non-dominated sorting genetic algorithm is designed to approximate the Pareto optimal solutions. We conduct extensive numerical experiments to validate the effectiveness, efficiency, and applicability of our proposed model and algorithm. The developed model and algorithm are capable of automatically performing calculations and effectively generating vertical stacking plans of high quality for planners in real-world settings within seconds. Future research may be devoted to the optimization of stacking plans for other types of prefabricated components or the horizontal stacking plans for components transported by flat-bed trailers. [ABSTRACT FROM AUTHOR]

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-G s complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD. [Display omitted] • RAMP2 binds to the glucagon receptor and acts as a negative allosteric modulator • RAMP2 promotes extracellular receptor dynamics resulting in an inactive cytosolic face • Cryo-EM shows an unproductive complex formation with a dynamic extracellular domain • Dynamic allostery is an endogenous GPCR regulatory mechanism Receptor activity-modifying protein 2 functions as a negative allosteric modulator of GCGR by enhancing extracellular receptor dynamics that stabilizes the inactive state of the intracellular surface. [ABSTRACT FROM AUTHOR]

Good water quality is critical to public health and aquatic ecological security of global reservoirs. In stratified reservoirs, increasing near-term management demands foster extremely high monitoring and forecasting needs. In this study, a management assistant for stratified reservoirs (MASR) was developed, including a wave-driven monitoring platform and interpretation platform for multiple reservoir water quality variables. The wave-driven platform can adapt to the characteristics of water level changes and transmit the monitoring data through a mobile network to the reservoir manager, which are processed by the interpretation platform in real time for near-term management. After several months of application, MASR monitored 1237 groups of valid profile water quality data with an acceptable error, which showed a strong capacity for capturing the water quality dynamics in a stratified reservoir. The effective identification of thermal stratification structures and anoxic zones can help managers to design withdrawal schemes for reservoirs. Moreover, the prediction of algae state based on the back propagation (BP) neural network provided the basis for making operation plans to proactively control algae blooms. Our study provides an economical and convenient solution for stratified reservoirs to address near-term management issues. [Display omitted] • MASR was developed to observe multiple water quality variables of stratified reservoirs profile. • MASR could identify thermal stratification and anoxic zone, and predict the algae blooms in real time. • Near-term withdrawal schemes and operation plans can be design based on the monitoring and analysis results from MASR. [ABSTRACT FROM AUTHOR]

Identifying cognitively normal (CN) older adults who will convert to cognitive impairment (CI) due to Alzheimer's disease is crucial for early intervention. Clinical and neuroimaging measures were acquired from 301 CN adults who converted to CI within 15 years of baseline, and 294 who did not. Regional volumes and brain age measures were extracted from T 1 -weighted magnetic resonance images. Linear discriminant analysis compared non-converters' characteristics against those of short-, mid-, and long-term converters. Conversion was associated with clinical measures such as hearing impairment and self-reported memory decline. Converters' brain volumes were smaller than non-converters' across 48 frontal, temporal, and subcortical structures. Brain age measures of 12 structures were correlated with shorter times to conversion. Conversion prediction accuracy increased from 81.5% to 90.5% as time to conversion decreased. Proximity to CI conversion is foreshadowed by anatomic features of brain aging that enhance the accuracy of predicting conversion., (© 2024. The Author(s).)

The mu opioid receptor (μOR) is a target for clinically used analgesics. However, adverse effects, such as respiratory depression and physical dependence, necessitate the development of alternative treatments. Recently we reported a novel strategy to design functionally selective opioids by targeting the sodium binding allosteric site in μOR with a supraspinally active analgesic named C6guano . Presently, to improve systemic activity of this ligand, we used structure-based design, identifying a new ligand named RO76 where the flexible alkyl linker and polar guanidine guano group is swapped with a benzyl alcohol, and the sodium site is targeted indirectly through waters. A cryoEM structure of RO76 bound to the μOR-G i complex confirmed that RO76 interacts with the sodium site residues through a water molecule, unlike C6guano which engages the sodium site directly. Signaling assays coupled with APEX based proximity labeling show binding in the sodium pocket modulates receptor efficacy and trafficking. In mice, RO76 was systemically active in tail withdrawal assays and showed reduced liabilities compared to those of morphine. In summary, we show that targeting water molecules in the sodium binding pocket may be an avenue to modulate signaling properties of opioids, and which may potentially be extended to other G-protein coupled receptors where this site is conserved., Competing Interests: The authors declare the following competing financial interest(s): S.M. is a cofounder of Sparian Biosciences. R.O.D. holds equity in Septerna, Inc. Other authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The µ-opioid receptor (µOR) is a well-established target for analgesia 1 , yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl 2 , a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to 'steer' hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1's activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints., (© 2024. The Author(s).)

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain 1 . Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the G protein-coupling 7-transmembrane domain 2 . In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and G protein binding., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI. Reduction of HRG by PDI enhances the procoagulant and anticoagulant activities of HRG by neutralization of endothelial heparan sulfate (HS) and inhibition of factor XII (FXIIa) activity, respectively. Murine HRG deficiency (Hrg -/- ) leads to delayed onset but enhanced formation of thrombus compared to WT. However, in the combined FXII deficiency (F12 -/- ) and HRG deficiency (by siRNA or Hrg -/- ), there is further thrombosis reduction compared to F12 -/- alone, confirming HRG's procoagulant activity independent of FXIIa. Mutation of target disulfides of PDI leads to a gain-of-function mutant of HRG that promotes its activities during coagulation. Thus, PDI-HRG pathway fine-tunes thrombosis by promoting its rapid initiation via neutralization of HS and preventing excessive propagation via inhibition of FXIIa., (© 2024. The Author(s).)

Background: Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMβ2) on neutrophils remains elusive., Methods: Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy., Results: ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury., Conclusions: Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy., Competing Interests: Disclosures None.

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to mechanical stress. This paper details an innovative adaptation of the fluorescence-coupled micropipette aspiration (fMPA) assay. The fMPA assay introduces the capability to administer precise mechanical forces while concurrently monitoring the live-cell mechanotransduction processes mediated by ion channels. The sophisticated setup incorporates a precision-engineered borosilicate glass micropipette connected to a finely regulated water reservoir and pneumatic aspiration system, facilitating controlled pressure application with increments as refined as ± 1 mmHg. A significant enhancement is the integration of epi-fluorescence imaging, allowing for the simultaneous observation and quantification of cell morphological changes and intracellular calcium fluxes during aspiration. The fMPA assay, through its synergistic combination of epi-fluorescence imaging with micropipette aspiration, sets a new standard for the study of cell mechanosensing within mechanically challenging environments. This multifaceted approach is adaptable to various experimental setups, providing critical insights into the single-cell mechanosensing mechanisms.

Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP). Biologic inhibitors have not yet been reported and could have substantial therapeutic advantages over current small molecules because they can be recombinantly engineered into multifunctional formats and immunotherapies. Here we used phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1 and discovered a VH domain that allosterically inhibited the hydrolysis of cGAMP and adenosine triphosphate (ATP). We solved a 3.2 Å-resolution cryo-electron microscopy structure for the VH inhibitor complexed with ENPP1 that confirmed its new allosteric binding pose. Finally, we engineered the VH domain into multispecific formats and immunotherapies, including a bispecific fusion with an anti-PD-L1 checkpoint inhibitor that showed potent cellular activity., (© 2023. The Author(s).)

von Willebrand factor (VWF) is the protective carrier of procoagulant factor VIII (FVIII) in the shear forces of the circulation, prolonging its half-life and delivering it to the developing thrombus. Using force spectroscopy, VWF-FVIII complex formation is characterized by catch-bond behavior in which force first decelerates then accelerates bond dissociation. Patients with mutations in VWF at the FVIII binding site phenocopies hemophilia A and the most common mutations are of cysteine residues involving multiple disulfide bonds. From differential cysteine alkylation and mass spectrometry experiments, 13 VWF disulfide bonds at the FVIII binding site were found to exist in formed and unformed states, and binding of FVIII results in partial formation of 12 of the VWF bonds. Force spectroscopy studies indicate that the VWF-FVIII bond stiffens in response to force and this feature of the interaction is ablated when VWF disulfide bonds are prevented from forming, resulting in slip-only bond behavior. Exposure of VWF to pathological fluid shear forces ex vivo and in vivo causes partial cleavage of all 13 disulfide bonds, further supporting their malleable nature. These findings demonstrate that FVIII binding to VWF involves dynamic changes in the covalent states of several VWF disulfides that are required for productive interaction in physiological shear forces., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Nosema ceranae is an intracellular parasite invading the midgut of honeybees, which causes serious nosemosis implicated in honeybee colony losses worldwide. The core gut microbiota is involved in protecting against parasitism, and the genetically engineering of the native gut symbionts provides a novel and efficient way to fight pathogens. Here, using laboratory-generated bees mono-associated with gut members, we find that Snodgrassella alvi inhibit microsporidia proliferation, potentially via the stimulation of host oxidant-mediated immune response. Accordingly, N. ceranae employs the thioredoxin and glutathione systems to defend against oxidative stress and maintain a balanced redox equilibrium, which is essential for the infection process. We knock down the gene expression using nanoparticle-mediated RNA interference, which targets the γ-glutamyl-cysteine synthetase and thioredoxin reductase genes of microsporidia. It significantly reduces the spore load, confirming the importance of the antioxidant mechanism for the intracellular invasion of the N. ceranae parasite. Finally, we genetically modify the symbiotic S. alvi to deliver dsRNA corresponding to the genes involved in the redox system of the microsporidia. The engineered S. alvi induces RNA interference and represses parasite gene expression, thereby inhibits the parasitism significantly. Specifically, N. ceranae is most suppressed by the recombinant strain corresponding to the glutathione synthetase or by a mixture of bacteria expressing variable dsRNA. Our findings extend our previous understanding of the protection of gut symbionts against N. ceranae and provide a symbiont-mediated RNAi system for inhibiting microsporidia infection in honeybees., (© 2023. The Author(s).)

Endocannabinoids (eCBs) are endogenous ligands of the cannabinoid receptor 1 (CB1), a G protein-coupled receptor that regulates a number of therapeutically relevant physiological responses. Hence, understanding the structural and functional consequences of eCB-CB1 interactions has important implications for designing effective drugs targeting this receptor. To characterize the molecular details of eCB interaction with CB1, we utilized AMG315, an analog of the eCB anandamide to determine the structure of the AMG315-bound CB1 signaling complex. Compared to previous structures, the ligand binding pocket shows some differences. Using docking, molecular dynamics simulations, and signaling assays we investigated the functional consequences of ligand interactions with the "toggle switch" residues F200 3.36 and W356 6.48 . Further, we show that ligand-TM2 interactions drive changes to residues on the intracellular side of TM2 and are a determinant of efficacy in activating G protein. These intracellular TM2 rearrangements are unique to CB1 and are exploited by a CB1-specific allosteric modulator., (© 2023. The Author(s).)

Drugs targeting the μ-opioid receptor (μOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two μOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and β-arrestin recruitment. Cryo-EM structures of μOR-Gi1 complex with MP (2.5 Å) and LFT (3.2 Å) revealed that the two ligands engage distinct subpockets, and molecular dynamics simulations showed additional differences in the binding site that promote distinct active-state conformations on the intracellular side of the receptor where G proteins and β-arrestins bind. These observations highlight how drugs engaging different parts of the μOR orthosteric pocket can lead to distinct signaling outcomes., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and β-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance β-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs., (© 2023. The Author(s).)

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose 1 . Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site 2 found in µOR 3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp 2.50 residue in the Na + site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at G i subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest G z efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for G i , G o and G z subtypes and arrestins, thus modulating their in vivo pharmacology., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The μ-opioid receptor (μOR) is the major target for opioid analgesics. Activation of μOR initiates signaling through G protein pathways as well as through β-arrestin recruitment. μOR agonists that are biased towards G protein signaling pathways demonstrate diminished side effects. PZM21, discovered by computational docking, is a G protein biased μOR agonist. Here we report the cryoEM structure of PZM21 bound μOR in complex with G i protein. Structure-based evolution led to multiple PZM21 analogs with more pronounced G i protein bias and increased lipophilicity to improve CNS penetration. Among them, FH210 shows extremely low potency and efficacy for arrestin recruitment. We further determined the cryoEM structure of FH210 bound to μOR in complex with G i protein and confirmed its expected binding pose. The structural and pharmacological studies reveal a potential mechanism to reduce β-arrestin recruitment by the μOR, and hold promise for developing next-generation analgesics with fewer adverse effects., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca 2+ ) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca 2+ permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca 2+ influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (- 2.94 to 0 mmH 2 O) and a pneumatic high-speed pressure clamp (- 25 to 0 mmHg). To visualize Ca 2+ influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca 2+ levels immediately after aspirating individual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic clamp showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic clamp as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner., (© 2022. The Author(s).)

Mechanobiology is an emerging field at the interface of biology and mechanics, investigating the roles of mechanical forces within biomolecules, organelles, cells, and tissues. As a highlight, the recent advances of micropipette-based aspiration assays and dynamic force spectroscopies such as biomembrane force probe (BFP) provide unprecedented mechanobiological insights with excellent live-cell compatibility. In their classic applications, these assays measure force-dependent ligand-receptor-binding kinetics, protein conformational changes, and cellular mechanical properties such as cortical tension and stiffness. In recent years, when combined with advanced microscopies in high spatial and temporal resolutions, these biomechanical nanotools enable characterization of receptor-mediated cell mechanosensing and subsequent organelle behaviors at single-cellular and molecular level. In this review, we summarize the latest developments of these assays for live-cell mechanobiology studies. We also provide perspectives on their future upgrades with multimodal integration and high-throughput capability., (© 2022. The Author(s).)

Membrane protein structures provide atomic level insight into essential biochemical processes and facilitate protein structure-based drug design. However, the inherent instability of these bio-macromolecules outside lipid bilayers hampers their structural and functional study. Detergent micelles can be used to solubilize and stabilize these membrane-inserted proteins in aqueous solution, thereby enabling their downstream characterizations. Membrane proteins encapsulated in detergent micelles tend to denature and aggregate over time, highlighting the need for development of new amphiphiles effective for protein solubility and stability. In this work, we present newly-designed maltoside detergents containing a pendant chain attached to a glycerol-decorated tris(hydroxymethyl)methane (THM) core, designated GTMs. One set of the GTMs has a hydrophobic pendant (ethyl chain; E-GTMs), and the other set has a hydrophilic pendant (methoxyethoxylmethyl chain; M-GTMs) placed in the hydrophobic-hydrophilic interfaces. The two sets of GTMs displayed profoundly different behaviors in terms of detergent self-assembly and protein stabilization efficacy. These behaviors mainly arise from the polarity difference between two pendants (ethyl and methoxyethoxylmethyl chains) that results in a large variation in detergent conformation between these sets of GTMs in aqueous media. The resulting high hydrophobic density in the detergent micelle interior is likely responsible for enhanced efficacy of the M-GTMs for protein stabilization compared to the E-GTMs and a gold standard detergent DDM. A representative GTM, M-GTM-O12, was more effective for protein stability than some recently developed detergents including LMNG. This is the first case study investigating the effect of pendant polarity on detergent geometry correlated with detergent efficacy for protein stabilization. STATEMENT OF SIGNIFICANCE: This study introduces new amphiphiles for use as biochemical tools in membrane protein studies. We identified a few hydrophilic pendant-bearing amphiphiles such as M-GTM-O11 and M-GTM-O12 that show remarkable efficacy for membrane protein solubilization and stabilization compared to a gold standard DDM, the hydrophobic counterparts (E-GTMs) and a significantly optimized detergent LMNG. In addition, detergent results obtained in the current study reveals the effect of detergent pendant polarity on protein solubility and stability. Thus, the current study represents both significant chemical and conceptual advance. The detergent tools and design principle introduced here advance protein science and facilitate structure-based drug design and development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Distantly related species entering similar biological niches often adapt by evolving similar morphological and physiological characters. How much genomic molecular convergence (particularly of highly constrained coding sequence) contributes to convergent phenotypic evolution, such as echolocation in bats and whales, is a long-standing fundamental question. Like others, we find that convergent amino acid substitutions are not more abundant in echolocating mammals compared to their outgroups. However, we also ask a more informative question about the genomic distribution of convergent substitutions by devising a test to determine which, if any, of more than 4,000 tissue-affecting gene sets is most statistically enriched with convergent substitutions. We find that the gene set most overrepresented ( q -value = 2.2e-3) with convergent substitutions in echolocators, affecting 18 genes, regulates development of the cochlear ganglion, a structure with empirically supported relevance to echolocation. Conversely, when comparing to nonecholocating outgroups, no significant gene set enrichment exists. For aquatic and high-altitude mammals, our analysis highlights 15 and 16 genes from the gene sets most affected by molecular convergence which regulate skin and lung physiology, respectively. Importantly, our test requires that the most convergence-enriched set cannot also be enriched for divergent substitutions, such as in the pattern produced by inactivated vision genes in subterranean mammals. Showing a clear role for adaptive protein-coding molecular convergence, we discover nearly 2,600 convergent positions, highlight 77 of them in 3 organs, and provide code to investigate other clades across the tree of life., Competing Interests: The authors declare no competing interest.

HIV-1 Env, a trimer of gp120-gp41 heterodimers, mediates membrane fusion after binding host receptor CD4. Receptor binding displaces V1V2 loops from Env's apex, allowing coreceptor binding and opening Env to enable gp41-mediated fusion. We present 3.54 Å and 4.06 Å cryoelectron microscopy structures of partially open soluble native-like Env trimers (SOSIPs) bound to CD4. One structure, a complex with a coreceptor-mimicking antibody that binds both CD4 and gp120, stabilizes the displaced V1V2 and reveals its structure. Comparing partially and fully open Envs with closed Envs shows that gp41 rearrangements are independent of the CD4-induced rearrangements that result in V1V2 displacement and formation of a 4-stranded bridging sheet. These findings suggest ordered conformational changes before coreceptor binding: (1) gp120 opening inducing side-chain rearrangements and a compact gp41 central helix conformation, and (2) 4-stranded bridging-sheet formation and V1V2 displacement. These analyses illuminate potential receptor-induced Env changes and inform design of therapeutics disrupting viral entry., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)