HIV-1 neutralizing antibodies in SHIV-infected macaques recapitulate structurally divergent modes of human V2 apex recognition with a single D gene.

Broadly neutralizing antibodies targeting the V2 apex of the HIV-1 envelope trimer are among the most common specificities elicited in HIV-1-infected humans and simian-human immunodeficiency virus (SHIV)-infected macaques. To gain insight into the prevalent induction of these antibodies, we isolated and characterized 11 V2 apex-directed neutralizing antibody lineages from SHIV-infected rhesus macaques. Remarkably, all SHIV-induced V2 apex lineages were derived from reading frame two of the rhesus DH3-15*01 gene. Cryo-EM structures of envelope trimers in complex with antibodies from nine rhesus lineages revealed modes of recognition that mimicked three canonical human V2 apex-recognition modes. Notably, amino acids encoded by DH3-15*01 played divergent structural roles, inserting into a hole at the trimer apex, H-bonding to an exposed strand, or forming part of a loop scaffold. Overall, we identify a DH3-15*01-signature for rhesus V2 apex broadly neutralizing antibodies and show that highly selected genetic elements can play multiple roles in antigen recognition. Highlights: Isolated 11 V2 apex-targeted HIV-neutralizing lineages from 10 SHIV-infected Indian-origin rhesus macaquesCryo-EM structures of Fab-Env complexes for nine rhesus lineages reveal modes of recognition that mimic three modes of human V2 apex antibody recognitionAll SHIV-elicited V2 apex lineages, including two others previously published, derive from the same DH3-15*01 gene utilizing reading frame twoThe DH3-15*01 gene in reading frame two provides a necessary, but not sufficient, signature for V2 apex-directed broadly neutralizing antibodiesStructural roles played by DH3-15*01-encoded amino acids differed substantially in different lineages, even for those with the same recognition modePropose that the anionic, aromatic, and extended character of DH3-15*01 in reading frame two provides a selective advantage for V2 apex recognition compared to B cells derived from other D genes in the naïve rhesus repertoireDemonstrate that highly selected genetic elements can play multiple roles in antigen recognition, providing a structural means to enhance recognition diversity.

Mosaic quadrivalent influenza vaccine single nanoparticle characterization.

Recent work by our laboratory and others indicates that co-display of multiple antigens on protein-based nanoparticles may be key to induce cross-reactive antibodies that provide broad protection against disease. To reach the ultimate goal of a universal vaccine for seasonal influenza, a mosaic influenza nanoparticle vaccine (FluMos-v1) was developed for clinical trial (NCT04896086). FluMos-v1 is unique in that it is designed to co-display four recently circulating haemagglutinin (HA) strains; however, current vaccine analysis techniques are limited to nanoparticle population analysis, thus, are unable to determine the valency of an individual nanoparticle. For the first time, we demonstrate by total internal reflection fluorescence microscopy and supportive physical-chemical methods that the co-display of four antigens is indeed achieved in single nanoparticles. Additionally, we have determined percentages of multivalent (mosaic) nanoparticles with four, three, or two HA proteins. The integrated imaging and physicochemical methods we have developed for single nanoparticle multivalency will serve to further understand immunogenicity data from our current FluMos-v1 clinical trial.

Implementing Programs to Initiate Buprenorphine for Opioid Use Disorder Treatment in High-Need, Low-Resource Emergency Departments: A Nonrandomized Controlled Trial.

STUDY OBJECTIVE: We hypothesized that implementation facilitation would enable us to rapidly and effectively implement emergency department (ED)-initiated buprenorphine programs in rural and urban settings with high-need, limited resources and dissimilar staffing structures. METHODS: This multicenter implementation study employed implementation facilitation using a participatory action research approach to develop, introduce, and refine site-specific clinical protocols for ED-initiated buprenorphine and referral in 3 EDs not previously initiating buprenorphine. We assessed feasibility, acceptability, and effectiveness by triangulating mixed-methods formative evaluation data (focus groups/interviews and pre/post surveys involving staff, patients, and stakeholders), patients' medical records, and 30-day outcomes from a purposive sample of 40 buprenorphine-receiving patient-participants who met research eligibility criteria (English-speaking, medically stable, locator information, nonprisoners). We estimated the primary implementation outcome (proportion receiving ED-initiated buprenorphine among candidates) and the main secondary outcome (30-day treatment engagement) using Bayesian methods. RESULTS: Within 3 months of initiating the implementation facilitation activities, each site implemented buprenorphine programs. During the 6-month programmatic evaluation, there were 134 ED-buprenorphine candidates among 2,522 encounters involving opioid use. A total of 52 (41.6%) practitioners initiated buprenorphine administration to 112 (85.1%; 95% confidence interval [CI] 79.7% to 90.4%) unique patients. Among 40 enrolled patient-participants, 49.0% (35.6% to 62.5%) were engaged in addiction treatment 30 days later (confirmed); 26 (68.4%) reported attending one or more treatment visits; there was a 4-fold decrease in self-reported overdose events (odds ratio [OR] 4.03; 95% CI 1.27 to 12.75). The ED clinician readiness increased by a median of 5.02 (95% CI: 3.56 to 6.47) from 1.92/10 to 6.95/10 (n(pre)=80, n(post)=83). CONCLUSIONS: The implementation facilitation enabled us to effectively implement ED-based buprenorphine programs across heterogeneous ED settings rapidly, which was associated with promising implementation and exploratory patient-level outcomes.

Site-Specific Fluorescent Labeling of Hemagglutinin-Specific Antigen Binding Fragment through Amine Chemistry Revealed by Mass Spectrometry.

To capture the structure of assembled hemagglutinin (HA) nanoparticles at single-particle resolution, HA-specific antigen binding fragments (Fabs) were labeled by fluorescent (FLR) dyes as probes to highlight the HA trimers displayed on the assembled tetravalent HA nanoparticles for a qualitative localization microscopic study. The FLR dyes were conjugated to the Fabs through N-hydroxysuccinimide (NHS) ester mediated amine coupling chemistry. The labeling profile, including labeling ratio, distribution, and site-specific labeling occupancy, can affect the imaging results and introduce inconsistency. To evaluate the labeling profile so as to evaluate the labeling efficiency, a combination of intact mass measurement by MALDI-MS and peptide mapping through LC-MS/MS was implemented. At the intact molecular level, the labeling ratio and distribution were determined. Through peptide mapping, the labeled residues were identified and the corresponding site-specific labeling occupancy was measured. A systematic comparative investigation of four different FLR-labeled 1H01-Fabs (generated from H1 strain HA specific mAb 1H01) allowed accurate profiling of the labeling pattern. The data indicate that the labeling was site-specific and semiquantitative. This warrants the consistency of single-particle fluorescent imaging experiments and allows a further imaging characterization of the single nanoparticles.

Development and validation of a mass spectrometry based analytical method to quantify the ratios in hemagglutinin trimers in quadrivalent influenza nanoparticle vaccine - FluMos-v1.

A quadrivalent influenza nanoparticle vaccine (FluMos-v1) offers long-lasting protection against multiple influenza virus strains and is composed of four strains of hemagglutinin trimer (HAT) assembled around a pentamer core. Here we report an LC-MS/MS analytical development and validation method to measure the percentage of each HAT component in FluMos-v1.

Large-Scale Interlaboratory DI-FT-ICR MS Comparability Study Employing Various Systems.

Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics. Extended comparisons employing different FT-ICR MS instruments with qualitative direct infusion analysis are scarce since the majority of detected compounds cannot be quantified. The extent to which observations can be reproduced by different laboratories remains unknown. We set up a preliminary study which encompassed a set of 17 laboratories around the globe, diverse in instrumental characteristics and applications, to analyze the same sets of extracts from commercially available standard human blood plasma and Standard Reference Material (SRM) for blood plasma (SRM1950), which were delivered at different dilutions or spiked with different concentrations of pesticides. The aim of this study was to assess the extent to which the outputs of differently tuned FT-ICR mass spectrometers, with different technical specifications, are comparable for setting the frames of a future DI-FT-ICR MS ring trial. We concluded that a cluster of five laboratories, with diverse instrumental characteristics, showed comparable and representative performance across all experiments, setting a reference to be used in a future ring trial on blood plasma.

A multispecific antibody prevents immune escape and confers pan-SARS-CoV-2 neutralization.

Despite effective countermeasures, SARS-CoV-2 persists worldwide due to its ability to diversify and evade human immunity1. This evasion stems from amino-acid substitutions, particularly in the receptor-binding domain of the spike, that confer resistance to vaccines and antibodies 2-16. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different receptor binding domain (RBD) sites17,18 into multispecific antibodies. Here, we describe multispecific antibodies, including a trispecific that prevented virus escape >3000-fold more potently than the most effective clinical antibody or mixtures of the parental antibodies. Despite being generated before the evolution of Omicron, this trispecific antibody potently neutralized all previous variants of concern and major Omicron variants, including the most recent BA.4/BA.5 strains at nanomolar concentrations. Negative stain electron microscopy revealed that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated inter-spike binding. An optimized trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2 and BA.5, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. Such multispecific antibodies decrease the likelihood of SARS-CoV-2 escape, simplify treatment, and maximize coverage, providing a strategy for universal antibody therapies that could help eliminate pandemic spread for this and other pathogens.

Spatially resolved isotope tracing reveals tissue metabolic activity.

Isotope tracing has helped to determine the metabolic activities of organs. Methods to probe metabolic heterogeneity within organs are less developed. We couple stable-isotope-labeled nutrient infusion to matrix-assisted laser desorption ionization imaging mass spectrometry (iso-imaging) to quantitate metabolic activity in mammalian tissues in a spatially resolved manner. In the kidney, we visualize gluconeogenic flux and glycolytic flux in the cortex and medulla, respectively. Tricarboxylic acid cycle substrate usage differs across kidney regions; glutamine and citrate are used preferentially in the cortex and fatty acids are used in the medulla. In the brain, we observe spatial gradations in carbon inputs to the tricarboxylic acid cycle and glutamate under a ketogenic diet. In a carbohydrate-rich diet, glucose predominates throughout but in a ketogenic diet, 3-hydroxybutyrate contributes most strongly in the hippocampus and least in the midbrain. Brain nitrogen sources also vary spatially; branched-chain amino acids contribute most in the midbrain, whereas ammonia contributes in the thalamus. Thus, iso-imaging can reveal the spatial organization of metabolic activity.

Reef Cover, a coral reef classification for global habitat mapping from remote sensing.

Coral reef management and conservation stand to benefit from improved high-resolution global mapping. Yet classifications underpinning large-scale reef mapping to date are typically poorly defined, not shared or region-specific, limiting end-users' ability to interpret outputs. Here we present Reef Cover, a coral reef geomorphic zone classification, developed to support both producers and end-users of global-scale coral reef habitat maps, in a transparent and version-based framework. Scalable classes were created by focusing on attributes that can be observed remotely, but whose membership rules also reflect deep knowledge of reef form and functioning. Bridging the divide between earth observation data and geo-ecological knowledge of reefs, Reef Cover maximises the trade-off between applicability at global scales, and relevance and accuracy at local scales. Two case studies demonstrate application of the Reef Cover classification scheme and its scientific and conservation benefits: 1) detailed mapping of the Cairns Management Region of the Great Barrier Reef to support management and 2) mapping of the Caroline and Mariana Island chains in the Pacific for conservation purposes.

Cholesterol-binding translocator protein TSPO regulates steatosis and bile acid synthesis in nonalcoholic fatty liver disease.

Translocator protein (TSPO, 18 kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and protein kinase RNA-like ER kinase/ATF4/CCAAT-enhancer-binding protein homologous protein pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. A TSPO ligand 19-Atriol blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD.

Implementation facilitation to introduce and support emergency department-initiated buprenorphine for opioid use disorder in high need, low resource settings: protocol for multi-site implementation-feasibility study.

BACKGROUND: For many reasons, the emergency department (ED) is a critical venue to initiate OUD interventions. The prevailing culture of the ED has been that substance use disorders are non-emergent conditions better addressed outside the ED where resources are less constrained. This study, its rapid funding mechanism, and accelerated timeline originated out of the urgent need to learn whether ED-initiated buprenorphine (BUP) with referral for treatment of OUD is generalizable, as well as to develop strategies to facilitate its adoption across a variety of ED settings and under real-world conditions. It both complements and uses methods adapted from Project ED Health (CTN-0069), a Hybrid Type 3 implementation-effectiveness study of using Implementation Facilitation (IF) to integrate ED-initiated BUP and referral programs. METHODS: ED-CONNECT (CTN 0079) was a three-site implementation study exploring the feasibility, acceptability, and impact of introducing ED-initiated BUP in rural and urban settings with high-need, limited resources, and different staffing structures. We used a multi-faceted approach to develop, introduce and iteratively refine site-specific ED clinical protocols and implementation plans for opioid use disorder (OUD) screening, ED-initiated BUP, and referral for treatment. We employed a participatory action research approach and use mixed methods incorporating data derived from abstraction of medical records and administrative data, assessments of recruited ED patient-participants, and both qualitative and quantitative inquiry involving staff from the ED and community, patients, and other stakeholders. DISCUSSION: This study was designed to provide the necessary, time-sensitive understanding of how to identify OUD and initiate treatment with BUP in the EDs previously not providing ED-initiated BUP, in communities in which this intervention is most needed: high need, low resource settings. TRIAL REGISTRATION: The study was prospectively registered on ClinicalTrials.gov (NCT03544112) on June 01, 2018: https://clinicaltrials.gov/ct2/show/NCT03544112 .

High Mass Analysis with a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: From Inorganic Salt Clusters to Antibody Conjugates and Beyond.

Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high m/z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high m/z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1k monoclonal antibody (mAb; 148.2 kDa); an IgG1-mertansine drug conjugate (148.5 kDa, drug-to-antibody ratio; DAR 2.26); an IgG1-siRNA conjugate (159.1 kDa; ribonucleic acid to antibody ratio; RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR MS that impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole-ToF (Q-ToF) and Orbitrap instrumentation. This report serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m/z.

Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

Understanding the structural complexity of dissolved organic matter: isomeric diversity.

In the present work, the advantages of ESI-TIMS-FT-ICR MS to address the isomeric content of dissolved organic matter are studied. While the MS spectra allowed the observation of a high number of peaks (e.g., PAN-L: 5004 and PAN-S: 4660), over 4× features were observed in the IMS-MS domain (e.g., PAN-L: 22 015 and PAN-S: 20 954). Assuming a total general formula of CxHyN0-3O0-19S0-1, 3066 and 2830 chemical assignments were made in a single infusion experiment for PAN-L and PAN-S, respectively. Most of the identified chemical compounds (∼80%) corresponded to highly conjugated oxygen compounds (O1-O20). ESI-TIMS-FT-ICR MS provided a lower estimate of the number of structural and conformational isomers (e.g., an average of 6-10 isomers per chemical formula were observed). Moreover, ESI-q-FT-ICR MS/MS at the level of nominal mass (i.e., 1 Da isolation) allowed for further estimation of the number of isomers based on unique fragmentation patterns and core fragments; the later suggested that multiple structural isomers could have very closely related CCS. These studies demonstrate the need for ultrahigh resolution TIMS mobility scan functions (e.g., R = 200-500) in addition to tandem MS/MS isolation strategies.

Using Tumor Explants for Imaging Mass Spectrometry Visualization of Unlabeled Peptides and Small Molecules.

Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry has emerged as a powerful, label-free technique to visualize penetration of small molecules in vivo and in vitro, including in 3D cell culture spheroids; however, some spheroids do not grow sufficiently large to provide enough area for imaging mass spectrometry. Here, we describe an ex vivo method for visualizing unlabeled peptides and small molecules in tumor explants, which can be divided into pieces of desired size, thus circumventing the size limitations of many spheroids. As proof-of-concept, a small molecule drug (4-hydroxytamoxifen), as well as a peptide drug (cyclosporin A) and peptide chemical probe, can be visualized after in vitro incubation with tumor explants so that this technique may provide a solution to robing cell penetration by unlabeled peptides.

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer.

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.

Towards the Analysis of High Molecular Weight Proteins and Protein complexes using TIMS-MS.

In the present work, we demonstrate the potential and versatility of TIMS for the analysis of proteins, DNA-protein complexes and protein-protein complexes in their native and denatured states. In addition, we show that accurate CCS measurement are possible and in good agreement with previously reported CCS values using other IMS analyzers (<5% difference). The main challenges for the analysis of high mass proteins and protein complexes in the mobility and m/z domain are described. That is, the analysis of high molecular weight systems in their native state may require the use of higher electric fields or a compromise in the TIMS mobility resolution by reducing the bath gas velocity in order to effectively trap at lower electric fields. This is the first report of CCS measurements of high molecular weight biomolecules and biomolecular complexes (~ 150 kDa) using TIMS-MS.

Gated Trapped Ion Mobility Spectrometry Coupled to Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Analysis of molecules by ion mobility spectrometry coupled with mass spectrometry (IMS-MS) provides chemical information on the three dimensional structure and mass of the molecules. The coupling of ion mobility to trapping mass spectrometers has historically been challenging due to the large differences in analysis time between the two devices. In this paper we present a modification of the trapped ion mobility (TIMS) analysis scheme termed "Gated TIMS" that allows efficient coupling to a Fourier Transform Ion Cyclotron Resonance (FT-ICR) analyzer. Analyses of standard compounds and the influence of source conditions on the TIMS distributions produced by ion mobility spectra of labile ubiquitin protein ions are presented. Ion mobility resolving powers up to 100 are observed. Measured collisional cross sections of ubiquitin ions are in excellent qualitative and quantitative agreement to previous measurements. Gated TIMS FT-ICR produces results comparable to those acquired using TIMS/time-of-flight MS instrument platforms as well as numerous drift tube IMS-MS studies published in the literature.