Clinical pharmacist intervention to ensure safe stimulant prescribing practices at a Veterans Affairs facility.

Introduction: The Psychotropic Drug Safety Initiative (PDSI) is a national Veterans Affairs program that recommends obtaining cardiovascular vital signs semiannually and urine toxicology screening annually for veterans prescribed stimulants. The PDSI also recommends a risk review of concurrent central nervous system (CNS) depressants to ensure the benefits of coadministration with stimulants outweigh the risks. This project's purpose was to evaluate the occurrence of coprescriptions for CNS depressants and stimulants and encourage compliance with the PDSI recommendations to increase safe and appropriate management of veterans prescribed the combination. This study aimed to evaluate the occurrence of coprescriptions for CNS depressants and stimulants, evaluate compliance with stimulant monitoring recommendations, and measure the proportion of pharmacist recommendations implemented by the prescriber. Methods: This quality improvement project identified veterans with an outpatient prescription for a stimulant and any coprescription(s) for benzodiazepines, sedative-hypnotics, and/or opioids. A pharmacy intervention note was generated to request a risk review, provide recommendations for de-escalation, and notify the stimulant prescriber of overdue monitoring parameters. Impact was measured 60 days after intervention. Descriptive statistics and a McNemar test were used to compare preintervention and postintervention data. Results: From the 61 patients included, there were 67 unique prescriptions for benzodiazepines (49.3%), sedative-hypnotics (34.3%), and opioids (16.4%) in combination with a stimulant. Pharmacist intervention resulted in de-escalation of coprescribing for 9 patients (16.1%) and was associated with statistically significant improvement in compliance to stimulant monitoring recommendations. Discussion: Clinical pharmacists can assist in ensuring safe and appropriate monitoring and management of veterans prescribed stimulants.

Antibody-directed evolution reveals a mechanism for enhanced neutralization at the HIV-1 fusion peptide site.

The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display. Successive rounds of directed evolution by iterative selection of antibodies for binding to resistant HIV-1 strains establish a variant, VRC34.01_mm28, as a best-in-class antibody with 10-fold enhanced potency compared to the template antibody and ~80% breadth on a cross-clade 208-strain neutralization panel. Structural analyses demonstrate that the improved paratope expands the FP binding groove to accommodate diverse FP sequences of different lengths while also recognizing the HIV-1 Env backbone. These data reveal critical antibody features for enhanced neutralization breadth and potency against the FP site of vulnerability and accelerate clinical development of broad HIV-1 FP-targeting vaccines and therapeutics.

Hippocampal Mechanisms Support Cortisol-Induced Memory Enhancements.

Stress can powerfully influence episodic memory, often enhancing memory encoding for emotionally salient information. These stress-induced memory enhancements stand at odds with demonstrations that stress and the stress-related hormone cortisol can negatively affect the hippocampus, a brain region important for episodic memory encoding. To resolve this apparent conflict and determine whether and how the hippocampus supports memory encoding under cortisol, we combined behavioral assays of associative memory, high-resolution fMRI, and pharmacological manipulation of cortisol in a within-participant, double-blinded procedure (in both sexes). Behaviorally, hydrocortisone promoted the encoding of subjectively arousing, positive associative memories. Neurally, hydrocortisone led to enhanced functional connectivity between hippocampal subregions, which predicted subsequent memory enhancements for emotional associations. Cortisol also modified the relationship between hippocampal representations and associative memory: whereas hippocampal signatures of distinctiveness predicted memory under placebo, relative integration predicted memory under cortisol. Together, these data provide novel evidence that the human hippocampus contains the necessary machinery to support emotional associative memory enhancements under cortisol.SIGNIFICANCE STATEMENT Our daily lives are filled with stressful events, which powerfully shape the way we form episodic memories. For example, stress and stress-related hormones can enhance our memory for emotional events. However, the mechanisms underlying these memory benefits are unclear. In the current study, we combined functional neuroimaging, behavioral tests of memory, and double-blind, placebo-controlled hydrocortisone administration to uncover the effects of the stress-related hormone cortisol on the function of the human hippocampus, a brain region important for episodic memory. We identified novel ways in which cortisol can enhance hippocampal function to promote emotional memories, highlighting the adaptive role of cortisol in shaping memory formation.

The impact of in utero cannabis exposure on fetal growth.

BACKGROUND: The goal of this study was to examine if in utero cannabis exposure predicted reduced birth size and if these effects were evident in specific growth parameters as early as the second trimester. METHODS: Eligible women had an initial prenatal visit between January 1, 2010, and March 31, 2020, completed an anatomy ultrasound between 18-24 weeks' gestation, and had no self-reported alcohol, tobacco, or other biochemically verified drug use. The two primary study groups were cannabis users (n = 109) identified through self-report and urine toxicology screens, and a randomly selected control group of non-substance users (n = 171). Medical records were manually reviewed for background and medical information, anatomy ultrasound results, and birth size parameters. RESULTS: After controlling for significant confounders, regression results indicated significant (p < .05) deficits in birth weight associated with cannabis exposure, with a trend for increasing weight effects beginning in the second trimester. A significant decrease in head circumference was evident as early as the second trimester, with differences remaining significant until birth. Significant overall length and specific bone length deficits were not predicted by cannabis exposure, at birth or earlier in gestation, after control for confounding. CONCLUSIONS: Cannabis exposure predicted growth deficits at birth, with some effects evident as early as the second trimester. The biggest and earliest effects were seen for cranial size, with an adjusted deficit of more than 14 percentile points by birth. Overall weight was not impacted until at or near delivery.

Hippocampal mechanisms support cortisol-induced memory enhancements.

Stress can powerfully influence episodic memory, often enhancing memory encoding for emotionally salient information. These stress-induced memory enhancements stand at odds with demonstrations that stress and the stress-related hormone cortisol can negatively affect the hippocampus, a brain region important for episodic memory encoding. To resolve this apparent conflict and determine whether and how the hippocampus supports memory encoding under cortisol, we combined behavioral assays of associative memory, high-resolution functional magnetic resonance imaging (fMRI), and pharmacological manipulation of cortisol in a within-participant, double-blinded procedure. Hydrocortisone led to enhanced functional connectivity between hippocampal subregions, which predicted subsequent memory enhancements for emotional information. Cortisol also modified the relationship between hippocampal representations and memory: whereas hippocampal signatures of distinctiveness predicted memory under placebo, relative integration predicted memory under cortisol. Together, these data provide novel evidence that the human hippocampus contains the necessary machinery to support emotional memory enhancements under stress.

Nontarget analysis and fluorine atom balances of transformation products from UV/sulfite degradation of perfluoroalkyl contaminants.

Per- and polyfluoroalkyl substances (PFAS) are a class of thousands of highly fluorinated, anthropogenic compounds that are used in a wide variety of consumer applications. Due to their widespread use and high persistence, PFAS are ubiquitous in drinking water, which is of concern due to the threats these compounds pose to human health. Reduction via the hydrated electron is a promising technology for PFAS remediation and has been well-studied. However, since previous work rarely reports fluorine atom balances and often relies on suspect screening, some transformation products are likely unaccounted for. Therefore, we performed non-target analysis using high-resolution mass spectrometry on solutions of perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate (PFBS), perfluorooctanoate (PFOA), and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate (GenX) that had been treated with UV/sulfite to produce hydrated electrons. We determined fluorine atom balances for all compounds studied, finding high fluorine atom balances for PFOS and PFBS. PFOA and GenX had lower overall fluorine atom balances, likely due to the production of volatile or very polar transformation products that were not measured by our methods. Transformation products identified by our analysis were consistent with literature, with a few exceptions. Namely, shorter-chain perfluorosulfonates (PFSA) and their H/F substituted counterparts were also detected from PFOS. This is an unexpected result based on literature, as no documented pathway exists for the formation of shorter-chain PFSA during UV/sulfite treatment. Furthermore, the nontarget approach we employed allowed for identification of novel, unsaturated products from the hydrated electron treatment of perfluorooctanesulfonate (PFOS) that warrant further investigation.

Evaluation of iodide chemical ionization mass spectrometry for gas and aerosol-phase per- and polyfluoroalkyl substances (PFAS) analysis.

Per- and polyfluoroalkyl substances (PFAS) are a class of ultra-persistent anthropogenic contaminants. PFAS are ubiquitous in environmental and built systems, but very few online methods exist for their characterization in atmospheric gases and aerosols. Iodide time-of-flight chemical ionization mass spectrometry (iodide-ToF-CIMS) is a promising technology for online characterization of PFAS in the atmosphere. Previous work using iodide-ToF-CIMS was successful in measuring gas-phase perfluoroalkyl carboxylic acids and fluorotelomer alcohols, but those are just two of the myriad classes of PFAS that are atmospherically relevant. Therefore, our first objective was to test other sample introduction methods coupled to iodide-TOF-CIMS to evaluate its ability to measure a wider suite of PFAS in both gas and aerosol phases. Using a variety of sample introduction techniques, we successfully measured gas-phase fluorotelomer alcohols (FTOHs), gas and aerosol-phase perfluoroalkyl carboxylic acids (PFCAs), and aerosol-phase perfluoroalkyl sulfonic acids and polyfluoroalkyl phosphoric acid diesters (PFSAs and diPAPs). We also determined iodide-ToF-CIMS response factors for these compounds by introducing known quantities using a Filter Inlet for Gases and AEROsols (FIGAERO). These response factors ranged from 400 to 6 × 104 ions per nanogram, demonstrating low limits of detection. Furthermore, PFAS are a poorly understood diverse class of molecules that exhibit unusual and often unexpected physicochemical properties due to their highly fluorinated nature. Since detection of PFAS with iodide-ToF-CIMS relies on the analyte molecule to either undergo proton transfer or adduct formation with iodide, understanding PFAS behavior during chemical ionization gives rise to a more fundamental understanding of these compounds. Through voltage scanning experiments and DFT calculations, we found that PFCAs and FTOHs readily form iodide adducts, while PFSAs and diPAPs preferentially undergo proton transfer to iodide. Generally, binding energy increased with increasing linear chain length, and PFCAs had stronger binding than FTOHs. Overall, our results suggest that iodide-ToF-CIMS can be used to measure even nonvolatile PFAS such as PFSAs and diPAPs in the aerosol phase in a semi-continuous online fashion.

Highly protective antimalarial antibodies via precision library generation and yeast display screening.

The monoclonal antibody CIS43 targets the Plasmodium falciparum circumsporozoite protein (PfCSP) and prevents malaria infection in humans for up to 9 mo following a single intravenous administration. To enhance the potency and clinical utility of CIS43, we used iterative site-saturation mutagenesis and DNA shuffling to screen precise gene-variant yeast display libraries for improved PfCSP antigen recognition. We identified several mutations that improved recognition, predominately in framework regions, and combined these to produce a panel of antibody variants. The most improved antibody, CIS43_Var10, had three mutations and showed approximately sixfold enhanced protective potency in vivo compared to CIS43. Co-crystal and cryo-electron microscopy structures of CIS43_Var10 with the peptide epitope or with PfCSP, respectively, revealed functional roles for each of these mutations. The unbiased site-directed mutagenesis and screening pipeline described here represent a powerful approach to enhance protective potency and to enable broader clinical use of antimalarial antibodies.

KIT Mutations Correlate with Higher Galectin Levels and Brain Metastasis in Breast and Non-Small Cell Lung Cancer.

To investigate a potential role for galectins as biomarkers that enable diagnosis or prognostication of breast or non-small cell lung cancer, the serum levels of galectins -1, -3, -7, -8, and -9 of cancer patients determined by ELISA assays were compared to the mutation status of 50 known cancer-critical genes, which were determined using multiplex PCR in tumors of the same patients. Mutations in the KIT proto-oncogene, which codes for the c-Kit protein, a receptor tyrosine kinase, correlated with higher levels of galectins -1, -3, -8, and -9 in breast cancer patients and galectin-1 in non-small cell lung cancer patients. Mutations in the KIT gene were more likely found in brain metastases from both of these primary cancers. The most common KIT mutation in our panel was p.M541L, a missense mutation in the transmembrane domain of the c-Kit protein. These results demonstrate an association between KIT oncogenic signaling and elevated serum galectins in patients with metastatic disease. Changes in protein trafficking and the glycocalyx composition of cancer cells may explain the observed alterations in galectin expression. This study can be useful for the targeted selection of receptor tyrosine kinase and galectin inhibitor anti-cancer treatments.

Improving gender-affirming care in genetic counseling: Using educational tools that amplify transgender and/or gender non-binary community voices.

Transgender and/or gender non-binary (TGNB) individuals face significant health care disparities, including deficiencies in provider knowledge. To address this knowledge gap for genetic counselors, we developed, implemented, and analyzed an educational intervention on gender-affirming genetic counseling (GC) and care for TGNB patients. In partnership with the TGNB community, we designed a 5-module (length = 146 min ± 94 min) genetic counseling-targeted online learning program focused on gender-affirming care (Amplify). Content included elements of gender-affirming care, core components of gender-inclusive GC sessions, and cancer risk assessment/management. Video testimonials featuring TGNB individuals complemented learning within each module. Educational outcomes measured included comfort working with TGNB patients (n = 2 multiple choice questions (MCQs)), impact of education on knowledge (n = 25 MCQs), and clinical self-efficacy based on the Accreditation Council for Genetic Counseling competencies (n = 35 skills). Participants (n = 40), recruited through state and national GC organizations, completed all modules, and pre- and post-education/self-efficacy assessments. Pre-Amplify, 65% (n = 26/40) of participants endorsed feeling 'somewhat comfortable' working with TGNB patients. The average knowledge score was 77.6% (SD = 11.2%) with the lowest scores related to the gender affirmation process. After Amplify, overall knowledge improvement was statistically significant with an average 16.9% (p < 0.001) increase in score. Pre-Amplify, the average self-efficacy score was 78.4% (SD = 15.8%) with lowest scores seen in statements surrounding information gathering of family and medical histories. Post-Amplify, overall self-efficacy improvement was statistically significant with an average 13.8% (p < 0.001) increase in score. Linear regression did not identify an impact of practice specialty on participants' knowledge gains or self-efficacy. This study shows online modules are an effective form of gender-affirming care education for GCs. This intervention can positively improve the care practicing genetic counselors provide to patients and inform future decision-making about the development of gender-affirming care education for genetic counselors.

Remembering Election Night 2016: Subjective but not objective metrics of autobiographical memory vary with political affiliation, affective valence, and surprise.

Flashbulb memories represent a unique phenomenon linking research on cognition with research on emotion, yet most studies on this phenomenon have characterized collective events that are negative and unexpected in nature. In contrast, the 2016 American election of Donald Trump was a public, culturally shared event, eliciting extreme emotional responses that were positive for some individuals but negative for others, as well as varying levels of surprise. We longitudinally evaluated autobiographical memories for Election Night 2016 in a large online sample of Clinton supporters, Trump supporters, and third-party/nonvoters over a 12-month period, in terms of both objective memory metrics (information quantity and memory consistency) and subjective memory metrics (including memory confidence, metacognition, and sensory experience). Emotional responses to the election outcome varied widely, with Clinton supporters reporting highly negative responses, Trump supporters reporting highly positive responses, and third-party/nonvoters reporting mildly negative responses. Emotional intensity was enhanced in surprised versus nonsurprised individuals. Relative to third-party/nonvoters, Clinton and Trump supporters reported greater memory vividness, event importance, and sensory experience. Additionally, limited valence effects on subjective memory were observed (including higher memory confidence in Trump supporters and higher memory rehearsal in Clinton supporters). These differences in subjective experience were observed despite similar levels of information quantity and consistency as a function of valence. This characterization of memories for surprising positive events suggests they share many of the paradoxical qualities of memories for negative events often discussed as "flashbulb memories" but also points to potential differences in memory phenomenology for personal versus collectively experienced events. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Clinical tools and counseling considerations for breast cancer risk assessment and evaluation for hereditary cancer risk.

Breast cancer is a complex and heterogeneous disease. Unfortunately, it is the most common malignancy diagnosed in women in the USA, with 281,550 new cases of invasive breast cancer and 49,290 new cases of noninvasive breast cancer are diagnosed per year. In England, it is currently estimated that approximately 1 in 7 (14%) women will be diagnosed with breast cancer in their lifetime. In the UK in 2017, 54,700 women and 390 men were diagnosed with breast cancer. The risk of breast cancer is influenced by many factors, including but not limited to age, family history, reproductive history, hormonal exposure, proliferative breast lesions, physical activity, alcohol use, tobacco use, breast density, and environmental exposures. Breast cancer risk assessment is a critical part of public health. By identifying women at high risk for breast cancer, personalized recommendations can be deployed with regards to modes of screening, the age to initiate breast screening, and the frequency for completing such screens. In addition, breast cancer risk assessment can assist in determining a woman's eligibility for interventions to reduce risk, either through the use of chemoprevention medications or through surgical means with risk-reducing bilateral mastectomy. This chapter summarizes breast cancer risk assessment models and discusses interventions to reduce breast cancer risk to aid in reducing morbidity and mortality from breast cancer.

Antibody screening at reduced pH enables preferential selection of potently neutralizing antibodies targeting SARS-CoV-2.

Antiviral monoclonal antibody (mAb) discovery enables the development of antibody-based antiviral therapeutics. Traditional antiviral mAb discovery relies on affinity between antibody and a viral antigen to discover potent neutralizing antibodies, but these approaches are inefficient because many high affinity mAbs have no neutralizing activity. We sought to determine whether screening for anti-SARS-CoV-2 mAbs at reduced pH could provide more efficient neutralizing antibody discovery. We mined the antibody response of a convalescent COVID-19 patient at both physiological pH (7.4) and reduced pH (4.5), revealing that SARS-CoV-2 neutralizing antibodies were preferentially enriched in pH 4.5 yeast display sorts. Structural analysis revealed that a potent new antibody called LP5 targets the SARS-CoV-2 N-terminal domain supersite via a unique binding recognition mode. Our data combine with evidence from prior studies to support antibody screening at pH 4.5 to accelerate antiviral neutralizing antibody discovery.

Increased Circulating Levels of Galectin Proteins in Patients with Breast, Colon, and Lung Cancer.

Galectins are proteins with high-affinity ß-galactoside-binding sites that function in a variety of signaling pathways through interactions with glycoproteins. The known contributions of galectins-1, -3, -7, -8, and -9 to angiogenesis, metastasis, cell division, and evasion of immune destruction led us to investigate the circulating levels of these galectins in cancer patients. This study compares galectin concentrations by enzyme-linked immunosorbent assay (ELISA) from each stage of breast, lung, and colon cancer. Galectins-1 and -7, which share a prototype structure, were found to have statistically significant increases in breast and lung cancer. Of the tandem-repeat galectins, galectin-8 showed no statistically significant change in these cancer types, but galectin-9 was increased in colon and lung cancer. Galectin-3 is the only chimera-type galectin and was increased in all stages of breast, colon, and lung cancer. In conclusion, there were significant differences in the galectin levels in patients with these cancers compared with healthy controls, and galectin levels did not significantly change from stage to stage. These findings suggest that further research on the roles of galectins early in disease pathogenesis may lead to novel indications for galectin inhibitors.

Evaluation of BCL6 and SIRT1 as Non-Invasive Diagnostic Markers of Endometriosis.

(1) Background: Endometriosis is characterized by the presence of endometrial glands and stroma outside of the uterus and is often associated with severe pelvic pain and infertility. Our study explored the utilization of B-Cell Lymphoma 6 (BCL6) and Sirtuin 1 (SIRT1) as potential biomarkers in serum, plasma, urine, and cervical mucus for a non-invasive diagnostic test for endometriosis. BCL6 was chosen based on its previously reported elevated expression in endometrial biopsies, and SIRT1 is co-expressed and upregulated in the endometrium of women with endometriosis. (2) Methods: BCL6 and SIRT1 levels were measured using enzyme-linked immunoassay (ELISA) in samples from 20 women with endometriosis (ten with stages I/II and ten with stages III/IV) and ten women without endometriosis. (3) Results: Levels of SIRT1 in sera showed a statistically significant elevation in advanced stages III/IV compared to controls and stages I/II. No significant differences were found in other bodily fluids for SIRT1 or any bodily fluids tested for BCL6. (4) Conclusions: These results suggest some potential of SIRT1 expression within serum as a predictor of advanced asymptomatic stages of endometriosis. Using immunohistochemistry (IHC) staining and H-SCORE values for the elevated BCL6 (and potentially SIRT1) levels in endometrial biopsy samples seems to have higher diagnostic potential based on the previously published studies.

Paired heavy- and light-chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses.

Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2.

Atmospheric aging enhances the ice nucleation ability of biomass-burning aerosol.

Ice-nucleating particles (INPs) in biomass-burning aerosol (BBA) that affect cloud glaciation, microphysics, precipitation, and radiative forcing were recently found to be driven by the production of mineral phases. BBA experiences extensive chemical aging as the smoke plume dilutes, and we explored how this alters the ice activity of the smoke using simulated atmospheric aging of authentic BBA in a chamber reactor. Unexpectedly, atmospheric aging enhanced the ice activity for most types of fuels and aging schemes. The removal of organic carbon particle coatings that conceal the mineral-based ice-active sites by evaporation or oxidation then dissolution can increase the ice activity by greater than an order of magnitude. This represents a different framework for the evolution of INPs from biomass burning where BBA becomes more ice active as it dilutes and ages, making a larger contribution to the INP budget, resulting cloud microphysics, and climate forcing than is currently considered.

Paired heavy and light chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses.

Understanding protective mechanisms of antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We discovered a new antibody, 910-30, that targets the SARS-CoV-2 ACE2 receptor binding site as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. We performed sequence and structural analyses to explore how antibody features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer revealed its binding interactions and ability to disassemble spike. Despite heavy chain sequence similarity, biophysical analyses of IGHV3-53/3-66 antibodies highlighted the importance of native heavy:light pairings for ACE2 binding competition and for SARS-CoV-2 neutralization. We defined paired heavy:light sequence signatures and determined antibody precursor prevalence to be ~1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These data reveal key structural and functional neutralization features in the IGHV3-53/3-66 public antibody class to accelerate antibody-based medical interventions against SARS-CoV-2. HIGHLIGHTS: A molecular study of IGHV3-53/3-66 public antibody responses reveals critical heavy and light chain features for potent neutralizationCryo-EM analyses detail the structure of a novel public antibody class member, antibody 910-30, in complex with SARS-CoV-2 spike trimerCryo-EM data reveal that 910-30 can both bind assembled trimer and can disassemble the SARS-CoV-2 spikeSequence-structure-function signatures defined for IGHV3-53/3-66 class antibodies including both heavy and light chainsIGHV3-53/3-66 class precursors have a prevalence of 1:44,000 B cells in healthy human antibody repertoires.

Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.

Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.

Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.

Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes. Funding: Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID). Support for this work was also provided by COVID-19 Fast Grants, the Jack Ma Foundation, the Self Graduate Fellowship Program, and NIH grants DP5OD023118, R21AI143407, and R21AI144408. Some of this work was performed at the Columbia University Cryo-EM Center at the Zuckerman Institute, and some at the Simons Electron Microscopy Center (SEMC) and National Center for Cryo-EM Access and Training (NCCAT) located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310). Conflict of Interest: The authors declare that they have no conflict of interest. Ethical Approval: Peripheral blood mononuclear cells (PBMCs) for B cell sorting were obtained from a convalescent SARS-CoV-2 patient (collected 75 days post symptom onset under an IRB approved clinical trial protocol, VRC 200 - ClinicalTrials.gov Identifier: NCT00067054) and a healthy control donor from the NIH blood bank pre-SARS-CoV-2 pandemic.