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Lenacapavir disrupts HIV-1 core integrity while stabilizing the capsid lattice

Lenacapavir (GS-6207; LEN) is a potent HIV-1 capsid inhibitor approved for treating multidrug-resistant infection. LEN binds to a hydrophobic pocket between neighboring capsid (CA) proteins in hexamers and stabilizes the capsid lattice, but its effect on HIV-1 capsids is not fully understood. Here, we labeled HIV-1 capsids with green fluorescent protein fused to CA (GFP-CA) or a fluid-phase GFP content marker (cmGFP) to assess LEN’s impact on HIV-1 capsids. HIV-1 cores labeled with GFP-CA, but not cmGFP, could be immunostained with an anti-GFP antibody and were less sensitive to the capsid-binding host restriction factor MX2, demonstrating that GFP-CA is incorporated into the capsid lattice and is a marker for capsid lattice stability, whereas cmGFP is an indicator of core integrity. LEN treatment of isolated HIV-1 cores resulted in a dose-dependent loss of cmGFP signal while preserving the GFP-CA signal, indicating that LEN disrupts core integrity but stabilizes the capsid lattice. In contrast, capsid inhibitor PF-3450074 (PF74) induced loss of core integrity and the capsid lattice. Electron microscopy of LEN- or PF74-treated viral cores revealed frequent breakage at the narrow end of the capsid and other morphological changes. Our results suggest that LEN treatment does not prevent nuclear envelope docking but inhibits nuclear import of cores with or without loss of core integrity. In contrast, PF74 treatment blocks nuclear import by inhibiting the nuclear envelope docking of viral cores, highlighting their different mechanisms of nuclear import inhibition.

Lung B cells in ectopic germinal centers undergo affinity maturation

The lungs are constantly exposed to the external environment and a myriad of antigenic challenges within the air. Chronic exposure to allergens and other airborne antigens can result in the formation of lymphocyte aggregates in the lung, which can harbor ectopic germinal centers (GCs). After allergen exposure, GCs that form in the lung are much smaller and less densely packed with B cells than lymph node GCs. Despite this, ectopic lung GCs support somatic hypermutation and affinity-based maturation as in lymph node GCs, and export memory B cells (MBCs) directly into the lung tissue. This demonstrates that the lung can locally diversify B cell responses and supports the generation of tissue MBC populations in situ.

Reconfigurable homochiral colloidal clusters assembled under orthogonally applied electric and magnetic fields

Chiral structures assembled from colloids are of great interest for applications in metamaterials and micromachines. However, similar to their molecular counterparts, these assemblies often result in racemic mixtures. Achieving homochirality by breaking the symmetry remains a significant challenge. Here, we report an approach to obtain single-handed clusters from colloidal dimers using orthogonal electric and magnetic fields. Applying an alternating-current electric field perpendicular to the substrate generates a mixture of chiral clusters with both handedness. However, symmetry is broken by superimposing a planar rotating magnetic field, favoring one chirality over the other. The cluster’s chirality can be precisely controlled in situ by adjusting the magnetic field’s direction and strength, as well as the electric field frequency. Remarkably, this method also induces uniform chirality in initially achiral clusters when exposed solely to the electric field. Both experimental and numerical analyses reveal that the stability of specific handedness depends on the competition between forces and torques generated by the magnetic field, electric field, and electrohydrodynamic flow. Furthermore, we propose a strategy for producing colloidal clusters with uniform sizes and single-handedness through dynamic tuning of the electric and magnetic fields. This work not only demonstrates the potential of integrating external fields but also provides a viable way to create reconfigurable chiral colloidal structures.

The cingulate cortex facilitates auditory perception under challenging listening conditions

We often exert greater cognitive resources (i.e., listening effort) to understand speech under challenging acoustic conditions. This mechanism can be overwhelmed in those with hearing loss, resulting in cognitive fatigue in adults and potentially impeding language acquisition in children. However, the neural mechanisms that support listening effort are uncertain. Evidence from human studies suggests that the cingulate cortex is engaged under difficult listening conditions and may exert top–down modulation of the auditory cortex (AC). Here, we asked whether the gerbil cingulate cortex (Cg) sends anatomical projections to the AC that facilitate perceptual performance. To model challenging listening conditions, we used a sound discrimination task in which stimulus parameters were presented in either “Easy” or “Hard” blocks (i.e., long or short stimulus duration, respectively). Gerbils achieved statistically identical psychometric performance in Easy and Hard blocks. Anatomical tracing experiments revealed a strong, descending projection from layer 2/3 of the Cg1 subregion of the cingulate cortex to superficial and deep layers of the primary and dorsal AC. To determine whether Cg improves task performance under challenging conditions, we bilaterally infused muscimol to inactivate Cg1 and found that psychometric thresholds were degraded for only Hard blocks. To test whether the Cg-to-AC projection facilitates task performance, we chemogenetically inactivated these inputs and found that performance was only degraded during Hard blocks. Taken together, the results reveal a descending cortical pathway that facilitates perceptual performance during challenging listening conditions.

No compelling evidence of sex differences in brain maturation during COVID-19 lockdowns when the sexes are compared statistically

<i>LMX1B</i> missense-perturbation of regulatory element footprints disrupts serotonergic forebrain axon arborization

Pathogenic coding mutations are prevalent in human neuronal transcription factors (TFs) but how they disrupt development is poorly understood. Lmx1b is a master transcriptional regulator of postmitotic Pet1 neurons that give rise to mature serotonin (5-HT) neurons; over two hundred pathogenic heterozygous mutations have been discovered in human LMX1B, yet their impact on brain development has not been investigated. Here, we developed mouse models with different LMX1B DNA-binding missense mutations. Missense heterozygosity broadly altered Pet1 neuron transcriptomes, but expression changes converged on axon and synapse genes. Missense heterozygosity effected highly specific deficits in the postnatal maturation of forebrain serotonin axon arbors, primarily in the hippocampus and motor cortex, which was associated with spatial memory defects. Digital genomic footprinting (DGF) revealed that missense heterozygosity caused complete loss of Lmx1b motif protection and chromatin accessibility at sites enriched for a distal active enhancer/active promoter histone signature and homeodomain binding motifs; at other bound Lmx1b motifs, varying levels of losses, gains, or no change in motif binding and accessibility were found. The spectrum of footprint changes was strongly associated with synapse and axon genes. Further, Lmx1b missense heterozygosity caused wide disruption of Lmx1b-dependent GRNs comprising diverse TFs expressed in Pet1 neurons. These findings reveal an unanticipated continuum of Lmx1b missense-forced perturbations on Pet1 neuron regulatory element TF binding and accessibility. Our work illustrates DGF’s utility for gaining unique insight into how expressed TF missense mutations interfere with developing neuronal GRNs.

Quirks of track preservation and formation are more likely than pre-avian flight and ultrafast dinosaurs

Reactivation of memory-associated neurons induces downstream suppression of competing neuronal populations

Inducing apparent memory recall by tagging and optogenetically reactivating cells in the hippocampus was demonstrated over a decade ago. However, the hippocampal dynamics resulting from this reactivation remain largely unknown. While calcium imaging is commonly used as a measure of neuronal activity, GCaMP, the most common calcium indicator, cannot be used with optogenetic neuronal reactivation because both require blue light excitation. To resolve this overlap, we demonstrate optogenetic reactivation with a red-shifted opsin, ChrimsonR. We then conduct dual-color calcium imaging in CA1 during memory reactivation in DG. In addition to measuring population dynamics in CA1, CA1 cells tagged during the original experience were identified. In the fear-conditioned animals (FC+), nontagged cells in CA1 decreased their firing rate during stimulation, while tagged cells maintained their activity level. In the FC+ animals, as the behavioral effect of stimulation decreased across days, so did the changes in neural activity during stimulation. Our results both demonstrate the technical feasibility of calcium imaging during optogenetic reactivation of memory-associated neurons and advance our understanding of the dynamics underlying this reactivation.

Design of a halogen bond catalyzed DNA endonuclease

In this study, we expand the repertoire of biological catalysts by showing that a halogen bond (X-bond) can functionally replace the magnesium (Mg 2+ ) cofactor in mouse endonuclease G ( m EndoG). We mutated the metal coordinating glutamate E136 in m EndoG to a meta- halotyrosine ( m X Y, X = chlorine or iodine) to form a m X Y- m EndoG construct that is both acid and base catalyzed. Under basic conditions, the enzyme is inactivated by the metal chelator ethylene diamine tetraacetic acid (EDTA), indicating that the halogen substituent facilitates deprotonation of the tyrosyl hydroxyl group, allowing recruitment of Mg 2+ to restore the metal-dependent catalytic center. At low pHs, we observe that the m X Y- m EndoG is resistant to EDTA inactivation and that the iodinated constructed is significantly more active than the chlorinated analogue. These results implicate a hydrogen bond (H-bond) enhanced X-bond as the catalyst in the m X Y- m EndoG, with asparagine N103 serving as the H-bond donor that communicates the protonation state of histidine H104 to the halogen. This model is supported by mutation studies and electrostatic potential (ESP) calculations on models for the protonated and unprotonated m X Y···N103···H104 system compared to the Mg 2+ coordination complex of the wild type. Thus, we have designed and engineered an enzyme that utilizes an unnatural catalyst in its active site—a catalytic X-bonding enzyme, or cX- Zyme—by controverting what constitutes a metal catalyst in biochemistry.

Prolonged signaling of backbone-modified glucagon‐like peptide‐ <b>1</b> analogues with diverse receptor trafficking

Signal duration and subcellular location are emerging as important facets of G protein–coupled receptor (GPCR) function. The glucagon-like peptide-1 receptor (GLP-1R), a clinically relevant class B1 GPCR, stimulates production of the second messenger cyclic adenosine monophosphate (cAMP) upon activation by the native hormone, GLP-1. cAMP production continues after the hormone–receptor complex has been internalized via endocytosis. Here, we report GLP-1 analogues that induce prolonged signaling relative to GLP-1. A single β-amino acid substitution at position 18, with the residue derived from ( S , S )- trans -2-aminocyclopentanecarboxylic acid (ACPC), enhances signaling duration with retention of receptor endocytosis. Pairing ACPC at position 18 with a second substitution, α-aminoisobutyric acid (Aib) at position 16, abrogates endocytosis, but prolonged signaling is maintained. Prolonged signaling is sensitive to the structure of the β residue at position 18. Cryoelectron microscopy structures of two GLP-1 analogues bound to the GLP-1R:Gs complex suggest substantial alterations to bound peptide structure and dynamics compared to the GLP-1:GLP-1R:Gs complex. These structural findings strengthen an emerging view that agonist dynamics in the receptor-bound state influence signaling profiles. Our results advance understanding of the structural underpinnings of receptor activation and introduce tools for exploring the impact of spatiotemporal signaling profiles following GLP-1R activation.

The TRPV3 channel is a mediator of zinc influx and homeostasis in murine oocytes

Zinc (Zn 2+ ) homeostasis is essential for gametogenesis and reproduction, and its deficiency causes infertility. Oocytes contain higher Zn 2+ levels than somatic cells, and Zn 2+ concentrations in oocytes are far higher than those of other transition metals and increase even more during maturation in preparation for fertilization. Remarkably, it is unknown what transporter(s) or channel(s) mediate Zn 2+ influx in oocytes and whether they are expressed uniformly throughout folliculogenesis. Here, we showed that the functional expression of a member of the t ransient r eceptor p otential family, vanilloid 3, TRPV3, closely follows the dynamics of intracellular Zn 2+ during oocyte maturation, raising the prospect that these events may be functionally linked. Using microfluorometry, we monitored in oocytes of Trpv3 null females the expected rise in Zn 2+ concentrations during maturation. Surprisingly, Zn 2+ levels did not climb, and the overall FluoZin3 signal in Trpv3 null eggs was lower than in control eggs. Electrophysiological recordings showed a large TRPV3 current induced by the agonist 2-APB in WT eggs supplemented with extracellular Zn 2+ that was absent in Trpv3 null eggs; TRPV3 showed a clear preference for Zn 2+ over Ca 2+ . Trpv3 null eggs displayed features associated with Zn 2+ deficient conditions, such as lower IP 3 R1 function, abnormal cortical granule distribution, and disturbed cytoskeletal organization with distinct actin nucleation disorders. Notably, Trpv3 null eggs demonstrated undisturbed Zn 2+ sparks. Our results suggest that TRPV3 is a pivotal member of the Zn 2+ toolkit, mediating Zn 2+ intake during maturation. They also indicate that distinct transporters or channels mediate Zn 2+ influx throughout folliculogenesis.

Following phospholipid transfer through the OmpF ₃ –MlaA–MlaC lipid shuttle with native mass spectrometry

The maintenance of lipid asymmetry (Mla) system in gram-negative bacteria transfers phospholipids between the outer and inner membrane to maintain the outer membrane asymmetry. Misplaced phospholipids are extracted from the outer leaflet of the outer membrane by MlaA, transferred to the periplasmic lipid transporter MlaC, and shuttled to the inner membrane. We set out to investigate the lipid transfer between MlaA and MlaC using native mass spectrometry, with the aim of determining the lipid preferences of MlaC and whether MlaA preselected lipids for MlaC. First, we characterized the lipids that copurified with overexpressed MlaC, phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), and following delipidation noted a headgroup-independent enrichment of cyclopropane lipids. Under native expression conditions, we found that PG is three-fold enriched on MlaC compared to its abundance in the membrane. Next, we isolated and characterized OmpF 3 –MlaA complexes and demonstrated their ability to enhance loading of delipidated MlaC with bacterial and nonbacterial phospholipids. We then captured the intact ternary lipid shuttle (OmpF 3 –MlaA–MlaC) and demonstrated that PG dissociates this transient complex, releasing lipid-bound MlaC. Together our results point to a high population of endogenous PG on periplasmic MlaC, which likely arises from disassembly of the lipid shuttle to maintain lipid asymmetry for cell viability.

Signaling networks in cancer stromal senescent cells establish malignant microenvironment

The tumor microenvironment (TME) encompasses various cell types, blood and lymphatic vessels, and noncellular constituents like extracellular matrix (ECM) and cytokines. These intricate interactions between cellular and noncellular components contribute to the development of a malignant TME, such as immunosuppressive, desmoplastic, angiogenic conditions, and the formation of a niche for cancer stem cells, but there is limited understanding of the specific subtypes of stromal cells involved in this process. Here, we utilized p16-Cre ERT2 -tdTomato mouse models to investigate the signaling networks established by senescent cancer stromal cells, contributing to the development of a malignant TME. In pancreatic ductal adenocarcinoma (PDAC) allograft models, these senescent cells were found to promote cancer fibrosis, enhance angiogenesis, and suppress cancer immune surveillance. Notably, the selective elimination of senescent cancer stromal cells improves the malignant TME, subsequently reducing tumor progression in PDAC. This highlights the antitumor efficacy of senolytic treatment alone and its synergistic effect when combined with conventional chemotherapy. Taken together, our findings suggest that the signaling crosstalk among senescent cancer stromal cells plays a key role in the progression of PDAC and may be a promising therapeutic target.

PHLPP2 is a pseudophosphatase that lost activity in the metazoan ancestor

The phosphoinositide 3-kinase (PI3K) pathway is a major regulator of cell and organismal growth. Consequently, hyperactivation of PI3K and its downstream effector kinase, Akt, is observed in many human cancers. Pleckstrin homology domain leucine-rich repeat-containing protein phosphatases (PHLPP), two paralogous members of the metal-dependent protein phosphatase family, have been reported as negative regulators of Akt signaling and, therefore, tumor suppressors. However, the stoichiometry and identity of the bound metal ion(s), mechanism of action, and enzymatic specificity of these proteins are not known. Seeking to fill these gaps in our understanding of PHLPP biology, we unexpectedly found that PHLPP2 has no catalytic activity in vitro. Instead, we found that PHLPP2 is a pseudophosphatase with a single zinc ion bound in its catalytic center. Furthermore, we found that cancer genomics data do not support the proposed role of PHLPP1 or PHLPP2 as tumor suppressors. Phylogenetic analyses revealed an ancestral phosphatase that arose more than 1,000 Mya, but that lost activity at the base of the metazoan lineage. Surface conservation indicates that while PHLPP2 has lost catalytic activity, it may have retained substrate binding. Finally, using phylogenomics, we identify coevolving genes consistent with a scaffolding role for PHLPP2 on membranes. In summary, our results provide a molecular explanation for the inconclusive results that have hampered research on PHLPP and argue for a focus on the noncatalytic roles of PHLPP1 and PHLPP2.

Childhood muscle growth: Reference curves for lower leg muscle volumes and their clinical application in cerebral palsy

Skeletal muscles grow substantially during childhood. However, quantitative information about the size of typically developing children’s muscles is sparse. Here, the objective was to construct muscle-specific reference curves for lower leg muscle volumes in children aged 5 to 15 y. Volumes of 10 lower leg muscles were measured from magnetic resonance images of 208 typically developing children and 78 ambulant children with cerebral palsy. Deep learning was used to automatically segment the images. Reference curves for typical childhood muscle volumes were constructed with quantile regression. The median total leg muscle volume of a 15-y-old child is nearly five times that of a 5-y-old child. Between the ages of 5 and 15, boys typically have larger muscles than girls, both in absolute terms (medians are greater by 5 to 20%) and per unit of body weight (1 to 13%). Muscle volumes vary widely between children of a particular age: the range of volumes for the central 80% of the distribution (i.e., between the 10th and 90th centiles) is more than 40% of the median volume. Reference curves for individual muscle volumes have a similar shape to reference curves for total lower leg muscle volume. Confidence bands about the centile curves were wide, especially at the youngest and oldest ages. Nonetheless, the reference curves can be used with confidence to identify small-for-age muscles (centile &lt; 10). We show that 56% of children with cerebral palsy in our cohort had total lower leg muscle volumes that were small-for-age and that 80% had at least one lower leg muscle that was small-for-age.

Validating new limits for human thermoregulation

Recent projections suggest that large geographical areas will soon experience heat and humidity exceeding limits for human thermoregulation. The survivability limits modeled in that research were based on laboratory studies suggesting that humans cannot effectively thermoregulate in wet bulb temperatures (T wb ) above 26 to 31 °C, values considerably lower than the widely publicized theoretical threshold of 35 °C. The newly proposed empirical limits were derived from the T wb corresponding to the core temperature inflection point in participants exposed to stepped increases in air temperature or relative humidity in a climate-controlled chamber. Despite the increasing use of these thermal-step protocols, their validity has not been established. We used a humidity-step protocol to estimate the T wb threshold for core temperature inflection in 12 volunteers. To determine whether this threshold truly demarcates the T wb above which thermoregulation is impossible, each participant was subsequently exposed to T wb above (~33.7 °C, T above ) and below (~30.9 °C, T below ) their respective inflection point (~32.3 °C, T wb ) for up to 9 h (in random order). Core temperature rose continuously in T above . It was projected that core temperatures associated with heat stroke (40.2 °C) would occur within 10 h. While T below was also uncompensable, the core temperature rate of rise was considerably lower than in T above such that it would take &gt;24 h to reach 40.2 °C. Our study supports thermal-step protocols as an effective technique for evaluating survivability limits for heat exposure and provides a direct assessment of the limits of human thermoregulation.

Reconciling ecology and evolutionary game theory or “When not to think cooperation”

Evolutionary game theory (EGT)—overwhelmingly employed today for the study of cooperation in various systems, from microbes to cancer and from insect to human societies—started with the seminal 1973 paper by Maynard Smith and Price showing that limited animal conflict can be selected at the individual level. Owing to the explanatory potential of this paper and enabled by the powerful machinery of the soon-to-be-developed replicator dynamics, EGT took off at an accelerated pace and began to shape expectations across systems and scales. But, even as EGT has expanded its reach, and even as its mathematical foundations expanded with the development of adaptive dynamics and inclusion of stochastic processes, the replicator equation remains, half a century later, its most widely used equation. Owing to its early development and its staying power, the replicator dynamics has helped set both the baseline expectations and the terminology of the field. However, much like the original 1973 paper, replicator dynamics rests on the assumption that individual differences in reproduction are determined only by the payoff from the game (i.e., in isolation, all individuals, regardless of their strategy, have identical intrinsic growth rates). Here, we argue that this assumption limits the scope of replicator dynamics to such an extent as to warrant not just a more deliberative application process, but also a reconsideration of the broad predictions and terminology that it has generated. Simultaneously, we reestablish a dialog with ecology that can be mutually fruitful, e.g., by providing an explanation for how diverse ecological communities can assemble evolutionarily.

Removing masculine defaults in the hiring process

Onboard recordings reveal how bats maneuver under severe acoustic interference

Echolocating bats rely on active acoustic sensing to perceive their environment. When multiple bats fly together, echolocating simultaneously, the calls emitted by nearby conspecifics could interfere with and mask the echoes necessary for orientation. Nowhere is this impairment of sensing more dramatic than when thousands of bats emerge from a cave at the same time. Here, we tracked the movement of tens of greater mouse-tailed bats flying within a group of thousands. By mounting miniature microphones onboard some of the bats, we monitored the acoustic scene from the point of view of an individual bat within the echolocating collective. We found that bats experienced a very high level of conspecific acoustic masking when emerging from their cave, which dropped within seconds as the bats spread out in space. A comprehensive sensorimotor model, based on the unique data that we collected, revealed how bats content with this severe echo masking almost without collisions. Our results demonstrate that even under severe masking, bats are hardly impaired sensorially, and we suggest how they are able to maneuver smoothly and avoid collisions, even at high densities, without applying a jamming avoidance response.

Microparticle impact–induced bond strength in metals peaks with velocity

Supersonic impact of metallic microparticles onto metallic substrates generates extreme interfacial deformation and high contact pressures, enabling solid-state metallic bonding. Although higher impact velocities are generally believed to improve bond quality and mechanical properties in materials formed by supersonic impact deposition, here we report a peak in bond strength for single microparticle impact bonding, followed by a decline at higher impact velocities. Our in situ micromechanical measurements of interfacial strength for Al microparticles bonded to Al substrates reveal a three-fold increase from the critical bonding velocity (800 m/s) to a peak strength around 1,060 m/s. Interestingly, further increase in impact velocity results in a rapid decline in local interfacial strength. The decline continues up to the highest velocity studied, 1,337 m/s, which is well below the threshold required to induce melting or erosion. We show that a mechanistic transition from material strengthening to intensified elastic recovery is responsible for the peak strength in impact-induced bonding, with evidence linking the intensified elastic recovery to adiabatic softening at high impact velocities. Beyond 1,000 m/s for Al, interfacial damage induced by the intensified elastic recovery offsets the strength gain from higher impact velocities, resulting in a net decline in interfacial strength. This mechanistic understanding shall offer insights into the optimal design of processes that rely on impact bonding.