Potently neutralizing and protective human antibodies against SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health1 and the medical countermeasures available so far are limited2,3. Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-24. Here we analyse a large panel of human monoclonal antibodies that target the spike (S) glycoprotein5, and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (SRBD) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the SRBD, as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In two mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130 or a combination of both of these antibodies protected mice from weight loss and reduced the viral burden and levels of inflammation in the lungs. In addition, passive transfer of either of two of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents.

Proximity-dependent mapping of the HCMV US28 interactome identifies RhoGEF signaling as a requirement for efficient viral reactivation.

Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.

The Δ40p53 isoform inhibits p53-dependent eRNA transcription and enables regulation by signal-specific transcription factors during p53 activation.

The naturally occurring Δ40p53 isoform heterotetramerizes with wild-type p53 (WTp53) to regulate development, aging, and stress responses. How Δ40p53 alters WTp53 function remains enigmatic because their co-expression causes tetramer heterogeneity. We circumvented this issue with a well-tested strategy that expressed Δ40p53:WTp53 as a single transcript, ensuring a 2:2 tetramer stoichiometry. Human MCF10A cell lines expressing Δ40p53:WTp53, WTp53, or WTp53:WTp53 (as controls) from the native TP53 locus were examined with transcriptomics (precision nuclear run-on sequencing [PRO-seq] and RNA sequencing [RNA-seq]), metabolomics, and other methods. Δ40p53:WTp53 was transcriptionally active, and, although phenotypically similar to WTp53 under normal conditions, it failed to induce growth arrest upon Nutlin-induced p53 activation. This occurred via Δ40p53:WTp53-dependent inhibition of enhancer RNA (eRNA) transcription and subsequent failure to induce mRNA biogenesis, despite similar genomic occupancy to WTp53. A different stimulus (5-fluorouracil [5FU]) also showed Δ40p53:WTp53-specific changes in mRNA induction; however, other transcription factors (TFs; e.g., E2F2) could then drive the response, yielding similar outcomes vs. WTp53. Our results establish that Δ40p53 tempers WTp53 function to enable compensatory responses by other stimulus-specific TFs. Such modulation of WTp53 activity may be an essential physiological function for Δ40p53. Moreover, Δ40p53:WTp53 functional distinctions uncovered herein suggest an eRNA requirement for mRNA biogenesis and that human p53 evolved as a tetramer to support eRNA transcription.

Identifying patterns of neurocognitive dysfunction through direct comparison of children with leukemia, central nervous system tumors, and sickle cell disease.

PURPOSE: To quantify and compare the magnitude and type of neurocognitive dysfunction in at-risk children with central nervous system (CNS) tumors, acute lymphoblastic leukemia (ALL), and sickle cell disease (SCD) using a common instrument and metric to directly compare these groups with each other. METHODS: Fifty-three participants between the ages of 7 and 12 years (n = 27 ALL, n = 11 CNS tumor, n = 15 SCD) were enrolled and assessed using the NIH Toolbox Cognition Battery (NIHTCB). Participants with ALL or CNS tumor were 0-18 months posttherapy, while participants with SCD possessed the SS or Sß0 genotype, took hydroxyurea, and had no known history of stroke. RESULTS: Independent sample t-tests showed that participants with ALL and CNS tumor experienced greatest deficits in processing speed (ALL d = -0.96; CNS tumor d = -1.2) and inhibitory control and attention (ALL d = -0.53; CNS tumor d = -0.97) when compared with NIHTCB normative data. Participants with SCD experienced deficits in cognitive flexibility only (d = -0.53). Episodic memory was relatively spared in all groups (d = -0.03 to -0.32). There were no significant differences in function when groups were compared directly with each other by analysis of variance. CONCLUSIONS: Use of a common metric to quantify the magnitude and type of neurocognitive dysfunction across at-risk groups of participants by disease shows that participants perform below age-expected norms in multiple domains and experience dysfunction differently than one another. This approach highlights patterns of dysfunction that can inform disease- and domain-specific interventions.

Tracheal Narrowing and Its Impact on Anesthesia Care in Patients With Morquio A (Mucopolysaccharidosis Type IVA): An Observational Study.

BACKGROUND: Recently, tracheal narrowing has been recognized as a significant comorbid condition in patients with Morquio A, also known as mucopolysaccharidosis IVA. We studied a large cohort of patients with Morquio A to describe the extent of their tracheal narrowing and its relationship to airway management during anesthesia care. METHODS: This is an observational study, collecting data retrospectively, of a cohort of patients with Morquio A. Ninety-two patients with Morquio A syndrome were enrolled, among whom 44 patients had their airway evaluated by computed tomography angiography and had undergone an anesthetic within a year of the evaluation. Our hypothesis was that the tracheal narrowing as evaluated by computed tomography angiography increases with age in patients with Morquio A. The primary aim of the study was to examine the degree of tracheal narrowing in patients with Morquio A and describe the difficulties encountered during airway management, thus increasing awareness of both the tracheal narrowing and airway management difficulties in this patient population. In addition, the degree of tracheal narrowing was evaluated for its association with age or spirometry parameters using Spearman's rank correlation. Analysis of variance followed by the Bonferroni test was used to further examine the age-based differences in tracheal narrowing for the 3 age groups: 1 to 10 years, 11 to 20 years, and >21 years. RESULTS: Patient age showed a positive correlation with tracheal narrowing ( rs= 0.415; 95% confidence interval [95% CI], 0.138-0.691; P = .005) with older patients having greater narrowing of the trachea. Among spirometry parameters, FEF25%-75% showed an inverse correlation with tracheal narrowing as follows: FEF25%-75% versus tracheal narrowing: ( rs = -0.467; 95% CI, -0.877 to -0.057; P = .007). During anesthetic care, significant airway management difficulties were encountered, including cancelation of surgical procedures, awake intubation using flexible bronchoscope, and failed video laryngoscopy attempts. CONCLUSIONS: Clinically significant tracheal narrowing was present in patients with Morquio A, and the degree of such narrowing likely contributed to the difficulty with airway management during their anesthetic care. Tracheal narrowing worsens with age, but the progression appears to slow down after 20 years of age. In addition to tracheal narrowing, spirometry values of FEF25%-75% may be helpful in the overall evaluation of the airway in patients with Morquio A.

ClonoMatch: a tool for identifying homologous immunoglobulin and T-cell receptor sequences in large databases.

SUMMARY: B-cell receptor (BCR) and T-cell receptor (TCR) repertoires are generated through somatic DNA rearrangements and are responsible for the molecular basis of antigen recognition in the immune system. Next-generation sequencing (NGS) of DNA and the falling cost of sequencing due to continued development of these technologies have made sequencing assays an affordable way to characterize the repertoire of adaptive immune receptors (sometimes termed the 'immunome'). Many new workflows have been developed to take advantage of NGS and have placed the resulting immunome datasets in the public domain. The scale of these NGS datasets has made it challenging to search through the Complementarity-determining region 3 (CDR3), which is responsible for imparting specific antibody-antigen interactions. Thus, there is an increasing demand for sequence analysis tools capable of searching through CDR3s from immunome data collections containing millions of sequences. To address this need, we created a software package called ClonoMatch that facilitates rapid searches in bulk immunome data for BCR or TCR sequences based on their CDR3 sequence or V3J clonotype. AVAILABILITY AND IMPLEMENTATION: Documentation, software support and the codebase are all available at https://github.com/crowelab/clonomatch. This software is distributed under the GPL v3 license.

F-actin homeostasis through transcriptional regulation and proteasome-mediated proteolysis.

Many organisms possess multiple and often divergent actins whose regulation and roles are not understood in detail. For example, Chlamydomonas reinhardtii has both a conventional actin (IDA5) and a highly divergent one (NAP1); only IDA5 is expressed in normal proliferating cells. We showed previously that the drug latrunculin B (LatB) causes loss of filamentous (F-) IDA5 and strong up-regulation of NAP1, which then provides essential actin function(s) by forming LatB-resistant F-NAP1. RNA-sequencing analyses now show that this up-regulation of NAP1 reflects a broad transcriptional response, much of which depends on three proteins (LAT1, LAT2, and LAT3) identified previously as essential for NAP1 transcription. Many of the LAT-regulated genes contain a putative cis-acting regulatory site, the "LRE motif." The LatB transcriptional program appears to be activated by loss of F-IDA5 and deactivated by formation of F-NAP1, thus forming an F-actin-dependent negative-feedback loop. Multiple genes encoding proteins of the ubiquitin-proteasome system are among those induced by LatB, resulting in rapid degradation of IDA5 (but not NAP1). Our results suggest that IDA5 degradation is functionally important because nonpolymerizable LatB-bound IDA5 interferes with the formation of F-NAP1. The genes for the actin-interacting proteins cofilin and profilin are also induced. Cofilin induction may further the clearance of IDA5 by promoting the scission of F-IDA5, whereas profilin appears to function in protecting monomeric IDA5 from degradation. This multifaceted regulatory system allows rapid and quantitative turnover of F-actin in response to cytoskeletal perturbations and probably also maintains F-actin homeostasis under normal growth conditions.

Anthropogenic and biogenic CO2 fluxes in the Boston urban region.

With the pending withdrawal of the United States from the Paris Climate Accord, cities are now leading US actions toward reducing greenhouse gas emissions. Implementing effective mitigation strategies requires the ability to measure and track emissions over time and at various scales. We report CO2 emissions in the Boston, MA, urban region from September 2013 to December 2014 based on atmospheric observations in an inverse model framework. Continuous atmospheric measurements of CO2 from five sites in and around Boston were combined with a high-resolution bottom-up CO2 emission inventory and a Lagrangian particle dispersion model to determine regional emissions. Our model-measurement framework incorporates emissions estimates from submodels for both anthropogenic and biological CO2 fluxes, and development of a CO2 concentration curtain at the boundary of the study region based on a combination of tower measurements and modeled vertical concentration gradients. We demonstrate that an emission inventory with high spatial and temporal resolution and the inclusion of urban biological fluxes are both essential to accurately modeling annual CO2 fluxes using surface measurement networks. We calculated annual average emissions in the Boston region of 0.92 kg C·m-2·y-1 (95% confidence interval: 0.79 to 1.06), which is 14% higher than the Anthropogenic Carbon Emissions System inventory. Based on the capability of the model-measurement approach demonstrated here, our framework should be able to detect changes in CO2 emissions of greater than 18%, providing stakeholders with critical information to assess mitigation efforts in Boston and surrounding areas.

Early postnatal exposure to a cafeteria diet interferes with recency and spatial memory, but not open field habituation in adolescent rats.

The cafeteria diet (CD), an experimental diet that mimics the obesogenic Western diet, can impair memory in adult rats. However, the suckling period is also particularly susceptible to diet-induced behavioural modification. Here, following exposure to CD feeding during lactation, 24- to 26-day-old offspring were tested to determine maternal dietary effects on either open field habituation, object location (OL) learning or on recency learning. Whereas no impact on habituation learning could be demonstrated, both OL and recency memory were impaired. In controls (C), OL memory was shown both after a 5 min (p < .05) or 60 min (p < .001) inter-trial interval (ITI). After the 60 min ITI, the difference between C and CD was significant (p < .05). Learning did not occur in the CD group at any time point and was not observed after the 24hr ITI in in either group. Whereas control rats demonstrated intact recency memory (p < .00001), no learning occurred in the CD group. Both groups differed significantly in their exploration ratios (p < .01). This study suggests a detrimental effect of exposure to an unhealthy Western diet during lactation, on cognitive functions in adolescent rats. These results could have implications for human cognition in the context of obesity epidemic.

PyIR: a scalable wrapper for processing billions of immunoglobulin and T cell receptor sequences using IgBLAST.

BACKGROUND: Recent advances in DNA sequencing technologies have enabled significant leaps in capacity to generate large volumes of DNA sequence data, which has spurred a rapid growth in the use of bioinformatics as a means of interrogating antibody variable gene repertoires. Common tools used for annotation of antibody sequences are often limited in functionality, modularity and usability. RESULTS: We have developed PyIR, a Python wrapper and library for IgBLAST, which offers a minimal setup CLI and API, FASTQ support, file chunking for large sequence files, JSON and Python dictionary output, and built-in sequence filtering. CONCLUSIONS: PyIR offers improved processing speed over multithreaded IgBLAST (version 1.14) when spawning more than 16 processes on a single computer system. Its customizable filtering and data encapsulation allow it to be adapted to a wide range of computing environments. The API allows for IgBLAST to be used in customized bioinformatics workflows.

Using Lidar Technology To Assess Urban Air Pollution and Improve Estimates of Greenhouse Gas Emissions in Boston.

Simulation of the planetary boundary layer (PBL) is key for forecasting air quality and estimating greenhouse gas (GHG) emissions in cities. Here we conducted the first long-term and continuous study of PBL heights (PBLHs) in Boston, MA, using a compact lidar instrument. We developed an image recognition algorithm to estimate PBLHs from the lidar measurements and evaluated simulations of the PBL from seven numerical weather prediction (NWP) model versions, which showed different systematic errors and variability in simulating the PBLHs (discrepancies from -2.5 to 4.0 km). The NWP model with the best overall agreement for the fully developed PBL had R2 = 0.72 and a bias of only 0.128 km. However, this model predicted a notable number of anomalously high carbon dioxide concentrations at ground stations, because it occasionally significantly underestimated the PBLH. We also developed a novel method that combines lidar data with footprints from a Lagrangian particle dispersion model to identify long-range transport of air pollution in the nocturnal residual layer. Our framework was powerful in evaluating the performance of models used to estimate air pollution and GHG emissions in cities, which is critical to track progress on emission reduction targets and guide effective policies.

Impact of Pre-exposure History and Host Genetics on Antibody Avidity Following Norovirus Vaccination.

Background: Development of high avidity, broadly neutralizing antibodies (Abs) is a priority after vaccination against rapidly evolving, widely disseminated viruses like human norovirus. After vaccination with a multivalent GI.1 and GII.4c norovirus virus-like particle (VLP) vaccine candidate adjuvanted with alum and monophosphoryl lipid A (MPL), blockade Ab titers peaked early, with no increase in titer following a second vaccine dose. Methods: Blockade Ab relative avidity was evaluated by measuring the slope of blockade Ab neutralization curves. Results: Blockade Ab avidity to the GI.1 vaccine component peaked at day 35 (7 days after dose 2). Avidities to heterotypic genogroup I VLPs were not sustained at day 35 after vaccination or GI.1 infection, as measured from archived sera. Only secretor-positive participants maintained high avidity blockade Ab to GI.1 at day 180. Avidity to the GII.4c vaccine component peaked at day 7, remained elevated through day 180, and was not secretor dependent. Avidity to an immunologically novel GII.4 strain VLP correlated with preexisting Ab titer to an ancestral strain Epitope A. Conclusions: Host genetics and pre-exposure history shape norovirus vaccine Ab responses, including blockade Ab avidity. Avidity of potentially neutralizing Ab may be an important metric for evaluating vaccine responses to highly penetrant viruses with cross-reactive serotypes.

The Effects of a Sports Nutrition Education Intervention on Nutritional Status, Sport Nutrition Knowledge, Body Composition, and Performance during Off Season Training in NCAA Division I Baseball Players.

This study investigated the effects of a sport nutrition education intervention (SNEI) on dietary intake, knowledge, body composition, and performance in NCAA Division I baseball players. Resistance trained NCAA Division I baseball players (82.4 ± 8.2 kg; 1.83 ± 0.06 m; 13.7 ± 5 % body fat) participated in the study during 12 weeks of off-season training. Fifteen players volunteered for SNEI while 15 players matched for position served as controls (C) for body composition and performance. The nutrition intervention group (NI) received a 90 min SNEI encompassing energy intake (Kcal), carbohydrate (CHO), protein (PRO), fat, food sources, and hydration. Sport nutrition knowledge questionnaires were administered to NI pre and post. Nutritional status was determined by three-day dietary logs administered to NI pre and post. Body composition and performance (5-10-5 shuttle test, vertical jump, broad jump, 1 RM squat) were measured pre and post for C and NI. Knowledge increased in NI. Pro and fat, but not CHO intake increased in NI. FM decreased pre to post in NI (11.5 ± 4.8 vs. 10.5 ± 5.4 kg) but not C (11.3 ± 4.7 vs. 11.9 ± 4.5 kg). FFM increased pre to post with no differences between groups. The 5-10-5 shuttle times decreased significantly more in NI (4.58 ± 0.15 vs. 4.43 ± 0.13 sec) compared to C (4.56 ± 0.18 vs. 4.50 ± 0.16 sec). Jump and squat performance increased pre to post with no differences between groups. Our findings indicate that an off season SNEI is effective at improving sport nutrition knowledge and some, but not all, nutrient intakes and performance measures in Division I baseball players.

Expression of DUX4 in zebrafish development recapitulates facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy characterized by an asymmetric progressive weakness and wasting of the facial, shoulder and upper arm muscles, frequently accompanied by hearing loss and retinal vasculopathy. FSHD is an autosomal dominant disease linked to chromosome 4q35, but the causative gene remains controversial. DUX4 is a leading candidate gene as causative of FSHD. However, DUX4 expression is extremely low in FSHD muscle, and there is no DUX4 animal model that mirrors the pathology in human FSHD. Here, we show that the misexpression of very low levels of human DUX4 in zebrafish development recapitulates the phenotypes seen in human FSHD patients. Microinjection of small amounts of human full-length DUX4 (DUX4-fl) mRNA into fertilized zebrafish eggs caused asymmetric abnormalities such as less pigmentation of the eyes, altered morphology of ears, developmental abnormality of fin muscle, disorganization of facial musculature and/or degeneration of trunk muscle later in development. Moreover, DUX4-fl expression caused aberrant localization of myogenic cells marked with α-actin promoter-driven enhanced green fluorescent protein outside somite boundary, especially in head region. These abnormalities were rescued by coinjection of the short form of DUX4 (DUX4-s). Our results suggest that the misexpression of DUX4-fl, even at extremely low level, can recapitulate the phenotype observed in FSHD patients in a vertebrate model. These results strongly support the current hypothesis for a role of DUX4 in FSHD pathogenesis. We also propose that DUX4 expression during development is important for the pathogenesis of FSHD.

Particle conformation regulates antibody access to a conserved GII.4 norovirus blockade epitope.

UNLABELLED: GII.4 noroviruses (NoVs) are the primary cause of epidemic viral acute gastroenteritis. One primary obstacle to successful NoV vaccination is the extensive degree of antigenic diversity among strains. The major capsid protein of GII.4 strains is evolving rapidly, resulting in the emergence of new strains with altered blockade epitopes. In addition to characterizing these evolving blockade epitopes, we have identified monoclonal antibodies (MAbs) that recognize a blockade epitope conserved across time-ordered GII.4 strains. Uniquely, the blockade potencies of MAbs that recognize the conserved GII.4 blockade epitope were temperature sensitive, suggesting that particle conformation may regulate functional access to conserved blockade non-surface-exposed epitopes. To map conformation-regulating motifs, we used bioinformatics tools to predict conserved motifs within the protruding domain of the capsid and designed mutant VLPs to test the impacts of substitutions in these motifs on antibody cross-GII.4 blockade. Charge substitutions at residues 310, 316, 484, and 493 impacted the blockade potential of cross-GII.4 blockade MAbs with minimal impact on the blockade of MAbs targeting other, separately evolving blockade epitopes. Specifically, residue 310 modulated antibody blockade temperature sensitivity in the tested strains. These data suggest access to the conserved GII.4 blockade antibody epitope is regulated by particle conformation, temperature, and amino acid residues positioned outside the antibody binding site. The regulating motif is under limited selective pressure by the host immune response and may provide a robust target for broadly reactive NoV therapeutics and protective vaccines. IMPORTANCE: In this study, we explored the factors that govern norovirus (NoV) cross-strain antibody blockade. We found that access to the conserved GII.4 blockade epitope is regulated by temperature and distal residues outside the antibody binding site. These data are most consistent with a model of NoV particle conformation plasticity that regulates antibody binding to a distally conserved blockade epitope. Further, antibody "locking" of the particle into an epitope-accessible conformation prevents ligand binding, providing a potential target for broadly effective drugs. These observations open lines of inquiry into the mechanisms of human NoV entry and uncoating, fundamental biological questions that are currently unanswerable for these noncultivatable pathogens.

A transcription factor (TF) inference method that broadly measures TF activity and identifies mechanistically distinct TF networks.

TF profiler is a method of inferring transcription factor regulatory activity, i.e. when a TF is present and actively regulating transcription, directly directly from nascent sequencing assays such as PRO-seq and GRO-seq. Transcription factors orchestrate transcription and play a critical role in cellular maintenance, identity and response to external stimuli. While ChIP assays have measured DNA localization, they fall short of identifying when and where transcription factors are actively regulating transcription. Our method, on the other hand, uses RNA polymerase activity to infer TF activity across hundreds of data sets and transcription factors. Based on these classifications we identify three distinct classes of transcription factors: ubiquitous factors that play roles in cellular homeostasis, driving basal gene programs across tissues and cell types, tissue specific factors that act almost exclusively at enhancers and are themselves regulated at transcription, and stimulus responsive TFs which are regulated post-transcriptionally but act predominantly at enhancers. TF profiler is broadly applicable, providing regulatory insights on any PRO-seq sample for any transcription factor with a known binding motif.

Cellular zinc status alters chromatin accessibility and binding of p53 to DNA.

Zn2+ is an essential metal required by approximately 850 human transcription factors. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Using ATAC-seq to profile regions of accessible chromatin coupled with transcription factor enrichment analysis, we examined how increases and decreases in the labile zinc pool affect chromatin accessibility and transcription factor enrichment. We found 685 transcription factor motifs were differentially enriched, corresponding to 507 unique transcription factors. The pattern of perturbation and the types of transcription factors were notably different at promoters versus intergenic regions, with zinc-finger transcription factors strongly enriched in intergenic regions in elevated Zn2+ To test whether ATAC-seq and transcription factor enrichment analysis predictions correlate with changes in transcription factor binding, we used ChIP-qPCR to profile six p53 binding sites. We found that for five of the six targets, p53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc alter chromatin accessibility and transcription factor binding to DNA.

Benchmarking AlphaFold2 on peptide structure prediction.

Recent advancements in computational tools have allowed protein structure prediction with high accuracy. Computational prediction methods have been used for modeling many soluble and membrane proteins, but the performance of these methods in modeling peptide structures has not yet been systematically investigated. We benchmarked the accuracy of AlphaFold2 in predicting 588 peptide structures between 10 and 40 amino acids using experimentally determined NMR structures as reference. Our results showed AlphaFold2 predicts α-helical, ß-hairpin, and disulfide-rich peptides with high accuracy. AlphaFold2 performed at least as well if not better than alternative methods developed specifically for peptide structure prediction. AlphaFold2 showed several shortcomings in predicting Φ/Ψ angles, disulfide bond patterns, and the lowest RMSD structures failed to correlate with lowest pLDDT ranked structures. In summary, computation can be a powerful tool to predict peptide structures, but additional steps may be necessary to analyze and validate the results.

A NitroPure Nitrocellulose Membrane-Based Grapevine Virus Sampling Kit: Development and Deployment to Survey Japanese Vineyards and Nurseries.

We developed a NitroPure Nitrocellulose (NPN) membrane-based method for sampling and storing grapevine sap for grapevine virus detection. We devised an efficient nucleic acid extraction method for the NPN membrane, resulting in 100% amplification success for grapevine leafroll-associated virus 2 (GLRaV2) and 3 (GLRaV3), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A, grapevine virus B, and grapevine red blotch virus (GRBV). This method also allowed the storage of recoverable nucleic acid for 18 months at room temperature. We created a sampling kit to survey GLRaV2, GLRaV3, and GRBV in Japanese vineyards. We tested the kits in the field in 2018 and then conducted mail-in surveys in 2020-2021. The results showed a substantial prevalence of GLRaV3, with 48.5% of 132 sampled vines being positive. On the other hand, only 3% of samples tested positive for GLRaV2 and none for GRBV.

TSWIFT: Tower Spectrometer on Wheels for Investigating Frequent Timeseries for high-throughput phenotyping of vegetation physiology.

BACKGROUND: Remote sensing instruments enable high-throughput phenotyping of plant traits and stress resilience across scale. Spatial (handheld devices, towers, drones, airborne, and satellites) and temporal (continuous or intermittent) tradeoffs can enable or constrain plant science applications. Here, we describe the technical details of TSWIFT (Tower Spectrometer on Wheels for Investigating Frequent Timeseries), a mobile tower-based hyperspectral remote sensing system for continuous monitoring of spectral reflectance across visible-near infrared regions with the capacity to resolve solar-induced fluorescence (SIF). RESULTS: We demonstrate potential applications for monitoring short-term (diurnal) and long-term (seasonal) variation of vegetation for high-throughput phenotyping applications. We deployed TSWIFT in a field experiment of 300 common bean genotypes in two treatments: control (irrigated) and drought (terminal drought). We evaluated the normalized difference vegetation index (NDVI), photochemical reflectance index (PRI), and SIF, as well as the coefficient of variation (CV) across the visible-near infrared spectral range (400 to 900 nm). NDVI tracked structural variation early in the growing season, following initial plant growth and development. PRI and SIF were more dynamic, exhibiting variation diurnally and seasonally, enabling quantification of genotypic variation in physiological response to drought conditions. Beyond vegetation indices, CV of hyperspectral reflectance showed the most variability across genotypes, treatment, and time in the visible and red-edge spectral regions. CONCLUSIONS: TSWIFT enables continuous and automated monitoring of hyperspectral reflectance for assessing variation in plant structure and function at high spatial and temporal resolutions for high-throughput phenotyping. Mobile, tower-based systems like this can provide short- and long-term datasets to assess genotypic and/or management responses to the environment, and ultimately enable the spectral prediction of resource-use efficiency, stress resilience, productivity and yield.