Viral Capsid Trafficking along Treadmilling Tubulin Filaments in Bacteria.

Cargo trafficking along microtubules is exploited by eukaryotic viruses, but no such examples have been reported in bacteria. Several large Pseudomonas phages assemble a dynamic, tubulin-based (PhuZ) spindle that centers replicating phage DNA sequestered within a nucleus-like structure. Here, we show that capsids assemble on the membrane and then move rapidly along PhuZ filaments toward the phage nucleus for DNA packaging. The spindle rotates the phage nucleus, distributing capsids around its surface. PhuZ filaments treadmill toward the nucleus at a constant rate similar to the rate of capsid movement and the linear velocity of nucleus rotation. Capsids become trapped along mutant static PhuZ filaments that are defective in GTP hydrolysis. Our results suggest a transport and distribution mechanism in which capsids attached to the sides of filaments are trafficked to the nucleus by PhuZ polymerization at the poles, demonstrating that the phage cytoskeleton evolved cargo-trafficking capabilities in bacteria.

A phage tubulin assembles dynamic filaments by an atypical mechanism to center viral DNA within the host cell.

Tubulins are essential for the reproduction of many eukaryotic viruses, but historically, bacteriophage were assumed not to require a cytoskeleton. Here, we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with an unusual, extended C terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in-vitro-formed filaments, indicating how interactions between the C terminus and the following monomer drive polymerization. PhuZ forms a filamentous array that is required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. Thus, we show that PhuZ assembles a spindle-like array that functions analogously to the microtubule-based spindles of eukaryotes.

Expression optimization, purification, and biophysical characterization of a GluN2D-containing NMDA receptor.

N-methyl-d-aspartate (NMDA) receptors are hetero-tetrameric ion channels typically consisting of two GluN1 and two GluN2 subunits. A GluN2D subunit containing NMDA receptor dysfunction has been implicated in several neurological diseases, including schizophrenia; however, the lack of a purified GluN2D containing NMDA receptor has been a hurdle for structural and biophysical studies. Here, we present expression and purification strategies to generate human GluN2D containing NMDA receptor, confirm its hetero-tetrameric form using fluorescence size exclusion chromatography (FSEC) and evaluated its suitability for structural studies. The purification methodology outlined here will help in the development of GluN2D specific channel modulators and enable structure activity relationship (SAR) studies.

Correction: Striatopallidal neurons control avoidance behavior in exploratory tasks.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Striatopallidal neurons control avoidance behavior in exploratory tasks.

The dorsal striatum has been linked to decision-making under conflict, but the mechanism by which striatal neurons contribute to approach-avoidance conflicts remains unclear. We hypothesized that striatopallidal dopamine D2 receptor (D2R)-expressing neurons promote avoidance, and tested this hypothesis in two exploratory approach-avoidance conflict paradigms in mice: the elevated zero maze and open field. Genetic elimination of D2Rs on striatopallidal neurons (iMSNs), but not other neural populations, increased avoidance of the open areas in both tasks, in a manner that was dissociable from global changes in movement. Population calcium activity of dorsomedial iMSNs was disrupted in mice lacking D2Rs on iMSNs, suggesting that disrupted output of iMSNs contributes to heightened avoidance behavior. Consistently, artificial disruption of iMSN output with optogenetic stimulation heightened avoidance of open areas of these tasks, while inhibition of iMSN output reduced avoidance. We conclude that dorsomedial striatal iMSNs control approach-avoidance conflicts in exploratory tasks, and highlight this neural population as a potential target for reducing avoidance in anxiety disorders.

Ultra-Sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. METHODS: We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 patients with COVID-19. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. RESULTS: SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95% CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral-antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within 1 day) was statistically significant. CONCLUSIONS: The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.

Dissociating effects of error size, training duration, and amount of adaptation on the ability to retain motor memories.

Extensive computational and neurobiological work has focused on how the training schedule, i.e., the duration and rate at which an environmental disturbance is presented, shapes the formation of motor memories. If long-lasting benefits are to be derived from motor training, however, retention of the performance improvements gained during practice is essential. Thus a better understanding of mechanisms that promote retention could lead to the design of more effective training procedures. The few studies that have investigated how retention depends on the training schedule have suggested that the gradual exposure of a perturbation leads to improved retention of motor memory compared with an abrupt exposure. However, several of these previous studies showed small effects, and although some controlled the training duration and others the level of learning, none have controlled both. In the present study we disambiguated both of these effects from exposure rate by systematically varying the duration of training, type of trained dynamics, and exposure rate for these dynamics in human force-field adaptation. After controlling for both training duration and the amount of learning, we found essentially identical retention when comparing gradual and abrupt training for two different types of force-field dynamics. By contrast, we found that retention was markedly higher for long-duration compared with short-duration training for both types of dynamics. These results demonstrate that the duration of training has a far greater effect on the retention of motor memory than the exposure rate during training. We show that a multirate learning model provides a computational mechanism for these findings.NEW & NOTEWORTHY Previous studies have suggested that a gradual, incremental introduction of a novel environment is helpful for improving retention. However, we used experimental and computational approaches to demonstrate that previously reported improvements in retention associated with gradual introductions fail to persist when other factors, including the duration of training and the degree of initial learning, are accounted for.

The 24-h savings of adaptation to novel movement dynamics initially reflects the recall of previous performance.

Humans rapidly adapt reaching movements in response to perturbations (e.g., manipulations of movement dynamics or visual feedback). Following a break, when reexposed to the same perturbation, subjects demonstrate savings, a faster learning rate compared with the time course of initial training. Although this has been well studied, there are open questions on the extent early savings reflects the rapid recall of previous performance. To address this question, we examined how the properties of initial training (duration and final adaptive state) influence initial single-trial adaptation to force-field perturbations when training sessions were separated by 24 h. There were two main groups that were distinct based on the presence or absence of a washout period at the end of day 1 (with washout vs. without washout). We also varied the training duration on day 1 (15, 30, 90, or 160 training trials), resulting in 8 subgroups of subjects. We show that single-trial adaptation on day 2 scaled with training duration, even for similar asymptotic levels of learning on day 1 of training. Interestingly, the temporal force profile following the first perturbation on day 2 matched that at the end of day 1 for the longest training duration group that did not complete the washout. This correspondence persisted but was significantly lower for shorter training durations and the washout subject groups. Collectively, the results suggest that the adaptation observed very early in reexposure results from the rapid recall of the previously learned motor recalibration but is highly dependent on the initial training duration and final adaptive state.NEW & NOTEWORTHY The extent initial readaptation reflects the recall of previous motor performance is largely unknown. We examined early single-trial force-field adaptation on the second day of training and distinguished initial retention from recall. We found that the single-trial adaptation following the 24-h break matched that at the end of the first day, but this recall was modified by the training duration and final level of learning on the first day of training.

The decay of motor adaptation to novel movement dynamics reveals an asymmetry in the stability of motion state-dependent learning.

Motor adaptation paradigms provide a quantitative method to study short-term modification of motor commands. Despite the growing understanding of the role motion states (e.g., velocity) play in this form of motor learning, there is little information on the relative stability of memories based on these movement characteristics, especially in comparison to the initial adaptation. Here, we trained subjects to make reaching movements perturbed by force patterns dependent upon either limb position or velocity. Following training, subjects were exposed to a series of error-clamp trials to measure the temporal characteristics of the feedforward motor output during the decay of learning. The compensatory force patterns were largely based on the perturbation kinematic (e.g., velocity), but also showed a small contribution from the other motion kinematic (e.g., position). However, the velocity contribution in response to the position-based perturbation decayed at a slower rate than the position contribution to velocity-based training, suggesting a difference in stability. Next, we modified a previous model of motor adaptation to reflect this difference and simulated the behavior for different learning goals. We were interested in the stability of learning when the perturbations were based on different combinations of limb position or velocity that subsequently resulted in biased amounts of motion-based learning. We trained additional subjects on these combined motion-state perturbations and confirmed the predictions of the model. Specifically, we show that (1) there is a significant separation between the observed gain-space trajectories for the learning and decay of adaptation and (2) for combined motion-state perturbations, the gain associated to changes in limb position decayed at a faster rate than the velocity-dependent gain, even when the position-dependent gain at the end of training was significantly greater. Collectively, these results suggest that the state-dependent adaptation associated with movement velocity is relatively more stable than that based on position.

ROBucket: A low cost operant chamber based on the Arduino microcontroller.

The operant conditioning chamber is a cornerstone of animal behavioral research. Operant boxes are used to assess learning and motivational behavior in animals, particularly for food and drug reinforcers. However, commercial operant chambers cost several thousands of dollars. We have constructed the Rodent Operant Bucket (ROBucket), an inexpensive and easily assembled open-source operant chamber based on the Arduino microcontroller platform, which can be used to train mice to respond for sucrose solution or other liquid reinforcers. The apparatus contains two nose pokes, a drinking well, and a solenoid-controlled liquid delivery system. ROBucket can run fixed ratio and progressive ratio training schedules, and can be programmed to run more complicated behavioral paradigms. Additional features such as motion sensing and video tracking can be added to the operant chamber through the array of widely available Arduino-compatible sensors. The design files and programming code are open source and available online for others to use.

OmpA and OmpC are critical host factors for bacteriophage Sf6 entry in Shigella.

Despite being essential for successful infection, the molecular cues involved in host recognition and genome transfer of viruses are not completely understood. Bacterial outer membrane proteins A and C co-purify in lipid vesicles with bacteriophage Sf6, implicating both outer membrane proteins as potential host receptors. We determined that outer membrane proteins A and C mediate Sf6 infection by dramatically increasing its rate and efficiency. We performed a combination of in vivo studies with three omp null mutants of Shigella flexneri, including classic phage plaque assays and time-lapse fluorescence microscopy to monitor genome ejection at the single virion level. Cryo-electron tomography of phage 'infecting' outer membrane vesicles shows the tail needle contacting and indenting the outer membrane. Lastly, in vitro ejection studies reveal that lipopolysaccharide and outer membrane proteins are both required for Sf6 genome release. We conclude that Sf6 phage entry utilizes either outer membrane proteins A or C, with outer membrane protein A being the preferred receptor.

Identification of the bacteriophage nucleus protein interaction network.

In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against host immune factors. By segregating the genome from the host cytoplasm, however, the 'phage nucleus' introduces the need to specifically translocate messenger RNA and proteins through the nuclear shell and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear-shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggest that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging and may also participate in messenger RNA and/or protein translocation.

Identification of the bacteriophage nucleus protein interaction network.

In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against DNA-targeting immune factors. By segregating the genome from the host cytoplasm, however, the "phage nucleus" introduces the need to specifically transport mRNA and proteins through the nuclear shell, and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggests that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging, and may also participate in mRNA and/or protein transport.

The indirect pathway of the basal ganglia promotes transient punishment but not motor suppression.

Optogenetic stimulation of Adora2a receptor-expressing spiny projection neurons (A2A-SPNs) in the striatum drives locomotor suppression and transient punishment, results attributed to activation of the indirect pathway. The sole long-range projection target of A2A-SPNs is the external globus pallidus (GPe). Unexpectedly, we found that inhibition of the GPe drove transient punishment but not suppression of movement. Within the striatum, A2A-SPNs inhibit other SPNs through a short-range inhibitory collateral network, and we found that optogenetic stimuli that drove motor suppression shared a common mechanism of recruiting this inhibitory collateral network. Our results suggest that the indirect pathway plays a more prominent role in transient punishment than in motor control and challenges the assumption that activity of A2A-SPNs is synonymous with indirect pathway activity.

The variable conversion of neutralizing anti-SARS-CoV-2 single-chain antibodies to IgG provides insight into RBD epitope accessibility.

Monoclonal antibody (mAb) therapies have rapidly become a powerful class of therapeutics with applications covering a diverse range of clinical indications. Though most widely used for the treatment of cancer, mAbs are also playing an increasing role in the defense of viral infections, most recently with palivizumab for prevention and treatment of severe RSV infections in neonatal and pediatric populations. In addition, during the COVID-19 pandemic, mAbs provided a bridge to the rollout of vaccines; however, their continued role as a therapeutic option for those at greatest risk of severe disease has become limited due to the emergence of neutralization resistant Omicron variants. Although there are many techniques for the identification of mAbs, including single B cell cloning and immunization of genetically engineered mice, the low cost, rapid throughput and technological simplicity of antibody phage display has led to its widespread adoption in mAb discovery efforts. Here we used our 27-billion-member naïve single-chain antibody (scFv) phage library to identify a panel of neutralizing anti-SARS-CoV-2 scFvs targeting diverse epitopes on the receptor binding domain (RBD). Although typically a routine process, we found that upon conversion to IgG, a number of our most potent clones failed to maintain their neutralization potency. Kinetic measurements confirmed similar affinity to the RBD; however, mechanistic studies provide evidence that the loss of neutralization is a result of structural limitations likely arising from initial choice of panning antigen. Thus this work highlights a risk of scFv-phage panning to mAb conversion and the importance of initial antigen selection.

Recurrent pattern completion drives the neocortical representation of sensory inference.

When sensory information is incomplete or ambiguous, the brain relies on prior expectations to infer perceptual objects. Despite the centrality of this process to perception, the neural mechanism of sensory inference is not known. Illusory contours (ICs) are key tools to study sensory inference because they contain edges or objects that are implied only by their spatial context. Using cellular resolution, mesoscale two-photon calcium imaging and multi-Neuropixels recordings in the mouse visual cortex, we identified a sparse subset of neurons in the primary visual cortex (V1) and higher visual areas that respond emergently to ICs. We found that these highly selective 'IC-encoders' mediate the neural representation of IC inference. Strikingly, selective activation of these neurons using two-photon holographic optogenetics was sufficient to recreate IC representation in the rest of the V1 network, in the absence of any visual stimulus. This outlines a model in which primary sensory cortex facilitates sensory inference by selectively strengthening input patterns that match prior expectations through local, recurrent circuitry. Our data thus suggest a clear computational purpose for recurrence in the generation of holistic percepts under sensory ambiguity. More generally, selective reinforcement of top-down predictions by pattern-completing recurrent circuits in lower sensory cortices may constitute a key step in sensory inference.

Methamphetamine-Induced Bowel Ischemia in a 50-Year-Old Male.

Methamphetamine intoxication is a known risk factor for nonocclusive mesenteric ischemia (NOMI). We describe a case of a 50-year-old male with a history of polysubstance abuse who presented to the Emergency Department with severe abdominal pain and coffee-ground emesis. Computed tomographic (CT) imaging demonstrated portal venous gas and diffuse colonic wall thickening concerning for ischemic colitis. The patient underwent exploratory laparotomy with resection of the ascending colon as well as a necrotic section of the jejunum. Further embolic workup was negative with a subjective history of amphetamine use prior to presentation. NOMI has a high fatality rate, and we recommend providers include drug-induced bowel infarction on their differential when presented with findings of ischemic bowel of unclear etiology.

Subcellular organization of viral particles during maturation of nucleus-forming jumbo phage.

Many eukaryotic viruses assemble mature particles within distinct subcellular compartments, but bacteriophages are generally assumed to assemble randomly throughout the host cell cytoplasm. Here, we show that viral particles of Pseudomonas nucleus-forming jumbo phage PhiPA3 assemble into a unique structure inside cells we term phage bouquets. We show that after capsids complete DNA packaging at the surface of the phage nucleus, tails assemble and attach to capsids, and these particles accumulate over time in a spherical pattern, with tails oriented inward and the heads outward to form bouquets at specific subcellular locations. Bouquets localize at the same fixed distance from the phage nucleus even when it is mispositioned, suggesting an active mechanism for positioning. These results mark the discovery of a pathway for organizing mature viral particles inside bacteria and demonstrate that nucleus-forming jumbo phages, like most eukaryotic viruses, are highly spatially organized during all stages of their lytic cycle.

IgG-like bispecific antibodies with potent and synergistic neutralization against circulating SARS-CoV-2 variants of concern.

Monoclonal antibodies are a promising approach to treat COVID-19, however the emergence of SARS-CoV-2 variants has challenged the efficacy and future of these therapies. Antibody cocktails are being employed to mitigate these challenges, but neutralization escape remains a major challenge and alternative strategies are needed. Here we present two anti-SARS-CoV-2 spike binding antibodies, one Class 1 and one Class 4, selected from our non-immune human single-chain variable fragment (scFv) phage library, that are engineered into four, fully-human IgG-like bispecific antibodies (BsAb). Prophylaxis of hACE2 mice and post-infection treatment of golden hamsters demonstrates the efficacy of the monospecific antibodies against the original Wuhan strain, while promising in vitro results with the BsAbs demonstrate enhanced binding and distinct synergistic effects on neutralizing activity against circulating variants of concern. In particular, one BsAb engineered in a tandem scFv-Fc configuration shows synergistic neutralization activity against several variants of concern including B.1.617.2. This work provides evidence that synergistic neutralization can be achieved using a BsAb scaffold, and serves as a foundation for the future development of broadly reactive BsAbs against emerging variants of concern.

Distinct Kinematic Adjustments over Multiple Timescales Accompany Locomotor Skill Development in Mice.

Robust locomotion is critical to many species' survival, yet the mechanisms by which efficient locomotion is learned and maintained are poorly understood. In mice, a common paradigm for assaying locomotor learning is the rotarod task, in which mice learn to maintain balance atop of an accelerating rod. However, the standard metric for learning in this task is improvements in latency to fall, which gives little insight into the rich kinematic adjustments that accompany locomotor learning. In this study, we developed a rotarod-like task called the RotaWheel in which changes in paw kinematics are tracked using high-speed cameras as mice learn to stay atop an accelerating wheel. Using this device, we found that learning was accompanied by stereotyped progressions of paw kinematics that correlated with early, intermediate, and late stages of performance. Within the first day, mice sharpened their interlimb coordination using a timed pause in the forward swing of their forepaws. Over the next several days, mice reduced their stride length and took shorter, quicker steps. By the second week of training, mice began to use a more variable locomotor strategy, where consecutive overshoots or undershoots in strides were selected across paws to drive forward and backward exploration of the wheel. Collectively, our results suggest that mouse locomotor learning occurs through multiple mechanisms evolving over separate time courses and involving distinct corrective actions. These data provide insights into the kinematic strategies that accompany locomotor learning and establish an experimental platform for studying locomotor skill learning in mice.