Since the adoption rate of e-grocery skyrocketed in the wake of the Covid-19 pandemic due to the influx of first-time e-grocery shoppers, grocery shopping behavior has been evolving and the travel effects of e-grocery are largely unknown. Thus, this study sought to examine the relationship between consumers’ grocery shopping behavior online and in-store, and the influencing factors (i.e., socio-demographic characteristics, household attributes, and personal attitudes). To achieve this, information relating to online and in-store grocery purchase frequencies, personal and household characteristics, and attitudes of more than 2,000 Florida residents were collected through an online survey. Using a bi-directional structural equation modeling (SEM) approach, our results show that online grocery shopping exhibited no significant effect on in-store grocery shopping frequency (i.e., neutrality), but in-store grocery shopping reduced the frequency of online grocery shopping (i.e., substitution). Also, a positive attitude toward some positive aspects of online shopping, preference for alternative travel modes, and tech savviness were associated with more frequent online grocery shopping, while cost consciousness and the joy of shopping encouraged more in-store shopping. Several socio-demographic and household attributes were also found to have direct and indirect effects mediated via attitudes on the shopping frequencies. Overall, this study provides insights into the demand and travel effects of e-grocery and highlights the need for retailers and transport planners to collaborate in order to mitigate the potential travel effects of e-grocery.

Research on grocery shopping channel preferences has been growing in the wake of the Covid-19 pandemic. However, few studies have utilized the discrete choice experiment (DCE) to elicit choices in hypothetical scenarios. Moreover, attitudinal factors, which may better explain preference heterogeneity, are rarely considered. Given that the evolution of shopping behavior in the context of the Covid-19 pandemic has huge implications for transportation planning and modeling, this study aims to examine consumers’ grocery shopping channel preferences through a DCE that was constructed with three grocery shopping channels (home delivery, curbside pickup, and in-store) and five time–cost attributes (product price, shopping time, delivery time, delivery cost, and travel time). 8,603 responses were elicited from 1,229 Florida residents between February and April 2021. Information on various aspects of respondents’ shopping attitudes as well as their socio-demographic and household attributes, grocery shopping activities, and distance to the grocery store were also collected. Using mixed logit modeling for analyses, results indicate that individuals with low education, in low- to middle-income earning households, with three or more household vehicles, and having full access to a vehicle tended to prefer in-store shopping. Also, perceived security risk, pro-alternative mobility options, pro-local store shopping, and shorter distances to grocery stores predisposed individuals toward in-store shopping. Alternatively, females, young and middle-aged individuals, workers, and individuals in large households tended to prefer home delivery and curbside pickup. Technology savviness, pro-environment, pro-online shopping, and shopping enjoyment were also drivers of home delivery and curbside pickup purchases, while cost and time consciousness did not show significant effects. Overall, the findings in this study have implications for retailers, transportation planners, and policymakers.

HIV-1 envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant [KD]) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD and whether B cells discriminate among proteins of similar affinities that bind with different kinetic rates. Here, we use a panel of Env proteins and Ramos B cell lines expressing immunoglobulin M (IgM) B cell receptors (BCRs) with specificity for CD4-binding-site broadly neutralizing antibodies to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD but on sensing of association rate and a threshold antigen-BCR half-life.

The adoption rate of e-grocery has skyrocketed in the wake of the Covid-19 pandemic due to the influx of first-time e-grocery shoppers due to Covid-19 safety concerns. However, how much of this trend will persist in the long term is largely unknown. To better estimate the demand for e-grocery and food deliveries, this study sought to examine consumers’ shopping frequencies for groceries and prepared food via both online and in-store channels, and identify the influencing factors such as socioeconomic and demographic characteristics, household attributes, and personal attitudes. To achieve this, information relating to the online and in-store shopping behavior, personal and household characteristics, and attitudes of more than 2,000 Florida residents were collected through an online survey. Using a structural equation modeling approach, our results show that online grocery shopping exhibits no significant effect on in-store grocery shopping frequency, in-store grocery shopping reduced the frequency for online grocery shopping. For prepared food, however, a complementarity effect was found in relation to the impact of online shopping on in-store shopping. Also, various personal and household attributes were found to have direct and indirect effects mediated via attitudes on shopping behavior. This study provides insights to better estimate the demand for e-grocery considering consumer behavior and preferences, which helps service providers and policy makers to better design and manage e-grocery services and address the transportation implications.

To develop vaccines for certain key global pathogens such as HIV, it is crucial to elicit both neutralizing and non-neutralizing Fc-mediated effector antibody functions. Clinical evidence indicates that non-neutralizing antibody functions including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) contribute to protection against several pathogens. In this study, we demonstrated that conjugation of HIV Envelope (Env) antigen gp120 to a self-assembling nanofiber material named Q11 induced antibodies with higher breadth and functionality when compared to soluble gp120. Immunization with Q11-conjugated gp120 vaccine (gp120-Q11) demonstrated higher tier 1 neutralization, ADCP, and ADCC as compared to soluble gp120. Moreover, Q11 conjugation altered the Fc N-glycosylation profile of antigen-specific antibodies, leading to a phenotype associated with increased ADCC in animals immunized with gp120-Q11. Thus, this nanomaterial vaccine strategy can enhance non-neutralizing antibody functions possibly through modulation of immunoglobulin G Fc N-glycosylation.

Abstract The rise in e-commerce adoption and decline in in-store shopping at the peak of the COVID-19 pandemic sparked optimism among researchers and transport planners on the potential of e-commerce in reducing shopping trips. However, fluctuations in consumers’ attitudes and shopping habits across the COVID-19 timeline add to the mix and necessitate an investigation into the travel effects of consumers’ evolving shopping behavior. In this regard, this study examines the interactions between the frequency of online shopping and shopping trips and the mediating effects of shoppers’ attitudes in early 2021. Over 2,000 Florida residents were surveyed to explore how various socio-economic, demographic and household characteristics, and attitudes influenced their shopping behavior for four types of non-grocery experience goods, namely clothing, shoes, watches, and jewelry (CSWJ); beauty and health products (BH); toys, kids, and baby supplies (TKB); home, garden, and tools (HGT). Structural equation models were developed to analyze their shopping patterns for each of the products, and results showed that the interactions between online and in-store purchase frequencies for CSWJ and TKB exhibited complementarity effects in both directions. For the other product types, online shopping had positive effects on in-store shopping frequency, but no effects were found in the reverse direction. Results also showed that personal attitudes played important roles in individuals’ shopping decisions, and there were significant mediating effects for a number of personal and household attributes. Tech savviness and the preferences toward alternative modes and online shopping benefits had direct positive influences on online shopping frequency, while the joy of shopping (i.e., shopping for leisure) and privacy and security concerns tended to increase in-store shopping frequency. Concerns regarding unattended delivery had negative impacts on online shopping frequency, while preferences toward the benefits of online shopping showed negative impacts on in-store shopping frequency. Based on these findings, the traffic implications of the expected higher delivery demand and passenger travel are discussed.

The Membrane Proximal External Region (MPER) of HIV Envelope represents a key target for vaccine development due to high neutralization breadth and potency of MPER-specific broadly neutralizing antibodies (bnAbs). However, neutralizing antibody responses to MPER epitopes are restricted by tolerance control and the MPER epitope is absent from many HIV immunogens under clinical investigation. Using computational design and yeast display, a candidate germline-targeting (GT5) immunogen was developed that bound strongly to the inferred human unmutated common ancestor (UCA) of the distal MPER bnAb DH511, as well as to several human DH511-like potential precursor antibodies. We studied the immunogenicity of a multimeric nanoparticle of the GT5 immunogen in a knock-in mouse line expressing human DH511.UCA B cell receptors (BCRs). Naïve DH511.UCA knock-in mice exhibited a reduction in overall B cell numbers, and DH511.UCA-bearing B cells expressed low levels of surface IgM and IgD, suggesting that DH511.UCA expression is subject to immune tolerance control. Nonetheless, following immunization with GT5 nanoparticles mixed with a saponin/monophosphoryl lipid A adjuvant, knock-in mice mounted robust anti-GT5 humoral responses, including anti-GT5 IgG in serum and GT5-specific germinal center B cells and T follicular helper cells in lymphoid tissues. Sequencing analysis of IgG+ GT5-specific B cells revealed improbable mutations in knock-in immunoglobulin genes of DH511.UCA. These studies will guide further optimization of immunogens with potential to select for development of bnAbs against MPER epitopes. Supported by a grant from NIH (P01-AI138211)

HIV-1 Envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant, KD) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD, and whether B cells discriminate between proteins of similar affinities but that bind with different kinetic rates. Here we used a panel of Env proteins and Ramos B cell lines expressing IgM BCRs with specificity for CD4 binding-site broadly neutralizing (bnAb) or a precursor antibody to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD, but on sensing of association rate and a threshold antigen-BCR half-life.

Abstract B cell activation is dependent on the productive engagement of an antigen (Ag) with the B cell receptor (BCR). How complex protein Ags that bind with different affinities and kinetic rates affect the strength of B cell activation is not fully understood. We used a panel of HIV-1 Env proteins of varying affinities and each expressed in either monomeric or multimeric forms to investigate the role of binding rates on B cell activation and Ag-induced BCR down-modulation. Ramos cells expressing the CD4 binding-site CH31 IgM BCRs were functional for Ag-specific activation and gave Ca-flux responses that was not dependent on the overall affinity or dissociation rates (off-rates) but on the association rates (on-rates) of Ag binding. Monomeric Ags did not induce flux responses and required multimerization, and the strength of activation was dependent on the on-rate of the tetrameric Ags. Comparison of half-life of the tetrameric Ags indicated a requirement of a half-life threshold for both activation and BCR down-modulation. In contrast, trimeric Ags that bound with faster on-rates (ka>104 M−1s−1) did not require higher-order multimerization (6-mer or 20-mer) for Ca-flux responses. These results provide support to a kinetic model in which B cell activation is dependent on the rates of receptor occupancy, and an above threshold half-life of the Ag-BCR complex.

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible “up” or receptor-inaccessible “down” conformations. Here, we report that the commonly used stabilized S ectodomain construct “2P” is sensitive to cold temperature, and that this cold sensitivity is resolved in a “down” state stabilized spike. Our results will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.

A major challenge in developing an effective vaccine against HIV-1 is the genetic diversity of its viral envelope. Because of the broad range of sequences exhibited by HIV-1 strains, protective antibodies must be able to bind and neutralize a widely mutated viral envelope protein. No vaccine has yet been designed which induces broadly neutralizing or protective immune responses against HIV in humans. Nanomaterial-based vaccines have shown the ability to generate antibody and cellular immune responses of increased breadth and neutralization potency. Thus, we have developed supramolecular nanofiber-based immunogens bearing the HIV gp120 envelope glycoprotein. These immunogens generated antibody responses that had increased magnitude and binding breadth compared to soluble gp120. By varying gp120 density on nanofibers, we determined that increased antigen valency was associated with increased antibody magnitude and germinal center responses. This study presents a proof-of-concept for a nanofiber vaccine platform generating broad, high binding antibody responses against the HIV-1 envelope glycoprotein.

Murine double minute 2 (MDM2), a negative regulator of the p53 tumor suppressor protein, is overexpressed in several human cancers. Herein we investigate the feasibility of developing 18F-labeled compounds based on the small molecule inhibitor SP-141 for imaging tumor MDM2 expression levels with positron emission tomography (PET). Three nonradioactive fluorinated SP-141 analogues, 1–3, were synthesized, and their binding to the MDM2 protein was analyzed by surface plasmon resonance (SPR). One of these, a fluoroethoxy analogue, was labeled with fluorine-18 (18F) using 18F-fluorethyl bromide to provide [18F]1 and evaluated in vitro and in vivo. SPR analysis confirmed the binding of the fluorinated analogues to MDM2 at 1.25–20 µM concentrations. Cell uptake studies revealed high uptake (67.5–71.4 %/mg protein) and specificity of [18F]1 in MCF7 and HepG2 cells. The uptake of [18F]1 in these cells could be modulated using 100 µM SP-141, potentially reflecting changes in MDM2 expression because of p53 activation by SP-141. [18F]1 exhibited stable uptake and retention in HepG2 tumor xenografts (~3 %ID/g) in vivo, but poor clearance from blood and other normal tissues, yielding low tumor-to-background ratios (< 2) at 2 h post injection. Our results suggest that [18F]1 has suboptimal characteristics for in vivo evaluation as a PET tracer for MDM2, but warrant radiolabeling and assessment of the other fluorinated analogues synthesized in this work, 2 and 3, and potentially other molecular scaffolds for developing MDM2 targeted radiotracers.

Interactions between innate antiviral factors at mucosal surfaces and HIV-1 virions contribute to the natural inefficiency of HIV-1 transmission and are a platform to inform the development of vaccine and nonvaccine strategies to block mucosal HIV-1 transmission. Tenascin-C (TNC) is a large, hexameric extracellular matrix glycoprotein identified in breast milk and genital fluids that broadly neutralizes HIV-1 via interaction with the HIV-1 Envelope (Env) variable 3 (V3) loop. In this report, we characterize the specific determinants of the interaction between TNC and the HIV-1 Env. We observed that TNC binding and neutralization of HIV-1 is dependent on the TNC fibrinogen-like globe (fbg) and fibronectin-type III (fn) domains, oligomerization, and its newly-mapped glycan structure. Moreover, we observed that TNC-mediated neutralization is also dependent on Env V3 residues 321/322 and 326/327, which surround the IGDIR motif of the V3 loop, as well the N332 glycan, which is critical to the broadly neutralizing activity of glycan-dependent V3-specific antibodies such as PGT128. Our results demonstrate a striking parallel between innate and adaptive immune mechanisms of broad HIV neutralization and provide further insight into the host protein-virus interactions responsible for the natural inefficiency of mucosal HIV-1 transmission.

Engineering better bnAbs A highly effective HIV vaccine has been the goal of vaccinologists for nearly 35 years. A successful vaccine would need to induce broadly neutralizing antibodies (bnAbs) that are capable of neutralizing multiple HIV strains (see the Perspective by Agazio and Torres). Steichen et al. report a strategy in which the first vaccine shot can lead to immune responses that generate desired bnAbs. By combining knowledge of human antibody repertoires and structure to guide design, they validated candidate immunogens through functional preclinical testing. Saunders et al. designed immunogens with differences in binding strength for bnAb precursors, which enabled selection of rare mutations after immunization. The immunogens promoted bnAb precursor maturation in humanized mice and macaques. Science , this issue p. eaax4380 , p. eaay7199 ; see also p. 1197

Somatic mutations within antibody variable and framework regions (FWR) can alter thermostability and structural flexibility, but their impact on functional potency is unclear. Here we study thermostability and use molecular dynamics (MD) simulations to assess the role of FWR mutations during maturation of HIV-1 broadly neutralizing antibodies (bnAbs). The tested bnAbs show lower thermostability than their unmutated ancestor antibodies. FWR mutations in the Fab elbow region are frequently observed in HIV-1 bnAbs and MD simulations show that such FWR mutations alter interdomain flexibility in two HIV-1 bnAbs. In a CD4-binding site lineage, reversion mutations result in a loss of neutralization potency in an early intermediate and affinity-matured bnAb against autologous and heterologous Tier-2 viruses, respectively. Elbow region reversion mutations in a glycan-V3 bnAb modestly reduces potency against an autologous virus isolate. Thus, selection of mutations in the Fab elbow region impacts interdomain conformational flexibility and paratope plasticity during bnAb development.

Viral glycoproteins are a primary target for host antibody responses. However, glycans on viral glycoproteins can hinder antibody recognition since they are self glycans derived from the host biosynthesis pathway. During natural HIV-1 infection, neutralizing antibodies are made against glycans on HIV-1 envelope glycoprotein (Env). However, such antibodies are rarely elicited with vaccination. Previously, the vaccine-induced, macaque antibody DH501 was isolated and shown to bind to high mannose glycans on HIV-1 Env. Understanding how DH501 underwent affinity maturation to recognize glycans could inform vaccine induction of HIV-1 glycan antibodies. Here, we show that DH501 Env glycan reactivity is mediated by both germline-encoded residues that contact glycans, and somatic mutations that increase antibody paratope flexibility. Only somatic mutations in the heavy chain were required for glycan reactivity. The paratope conformation was fragile as single mutations within the immunoglobulin fold or complementarity determining regions were sufficient for eliminating antibody function. Taken together, the initial germline VHDJH rearrangement generated contact residues capable of binding glycans, and somatic mutations were required to form a flexible paratope with a cavity conducive to HIV-1 envelope glycan binding. The requirement for the presence of most somatic mutations across the heavy chain variable region provides one explanation for the difficulty in inducing anti-Env glycan antibodies with HIV-1 Env vaccination.

Multivalent binding is an efficient means to enhance the affinity and specificity of chemical probes targeting multidomain proteins in order to study their function and role in disease. While the theory of multivalent binding is straightforward, physical and structural characterization of bivalent binding encounters multiple technical difficulties. We present a case study where a combination of experimental techniques and computational simulations was used to comprehensively characterize the binding and structure affinity relationships for a series of Bromosporine-based bivalent bromodomain ligands with a bivalent protein, Transcription Initiation Factor TFIID subunit 1 (TAF1). Experimental techniques Isothermal Titration Calorimetry, X-ray Crystallography, Circular Dichroism, Size Exclusion Chromatography-Multi-Angle Light Scattering, and Surface Plasmon Resonance were used to determine structures, binding affinities, and kinetics of monovalent ligands and bivalent ligands with varying linker lengths. The experimental data for monomeric ligands were fed into explicit computational simulations, in which both ligand and protein species were present in a broad range of concentrations, and in up to a 100 s time regime, to match experimental conditions. These simulations provided accurate estimates for apparent affinities (in good agreement with experimental data), individual dissociation microconstants and other microscopic details for each type of protein ligand complex. We conclude that the expected efficiency of bivalent ligands in a cellular context is difficult to estimate by a single technique in vitro, due to higher order associations favored at the concentrations used, and other complicating processes. Rather, a combination of structural, biophysical, and computational approaches should be utilized to estimate and characterize multivalent interactions.

Early recruitment of non-classical monocytes and their macrophage derivatives is associated with augmented tissue repair and improved integration of biomaterial constructs. A promising therapeutic approach to recruit these subpopulations is by elevating local concentrations of chemoattractants such as fractalkine (FKN, CX3CL1). However, delivering recombinant or purified proteins is not ideal due to their short half-lives, suboptimal efficacy, immunogenic potential, batch variabilities, and cost. Here we report an approach to enrich endogenous FKN, obviating the need for delivery of exogenous proteins. In this study, modified FKN-binding-aptamers are integrated with poly(ethylene glycol) diacrylate to form aptamer-functionalized hydrogels ("aptagels") that localize, dramatically enrich and passively release FKN in vitro for at least one week. Implantation in a mouse model of excisional skin injury demonstrates that aptagels enrich endogenous FKN and stimulate significant local increases in Ly6CloCX3CR1hi non-classical monocytes and CD206+ M2-like macrophages. The results demonstrate that orchestrators of inflammation can be manipulated without delivery of foreign proteins or cells and FKN-aptamer functionalized biomaterials may be a promising approach to recruit anti-inflammatory subpopulations to sites of injury. Aptagels are readily synthesized, highly customizable and could combine different aptamers to treat complex diseases in which regulation or enrichment of multiple proteins may be therapeutic.

During replication in yeast, the three B family DNA replicases frequently incorporate ribonucleotides (rNMPs) into DNA, and their presence in the nuclear genome can affect genome stability. This prompted us to examine ribonucleotide incorporation by the fourth B family member, Pol , the enzyme responsible for the majority of damage-induced mutagenesis in eukaryotes. We first show that Pol inserts rNMPs into DNA and can extend primer termini containing 3′-ribonucleotides. We then measure rNMP incorporation by Pol in the presence of its cofactors, RPA, RFC and PCNA and at normal cellular dNTP and rNTP concentrations that exist under unstressed conditions. Under these conditions, Pol stably incorporates one rNMP for every 200–300 dNMPs incorporated, a frequency that is slightly higher than for the high ranslesion synthesis fidelity replicative DNA polymerases. Under damage-induced conditions wherein cellular dNTP concentrations are elevated 5-fold, Pol only incorporates one rNMP per 1300 dNMPs. Functional interaction of Pol with the mutasome assembly factor Rev1 gives comparable rNMP incorporation frequencies. These results suggest that ribonucleotide incorporation into DNA during Pol -mediated mutagenesis in vivo may be rare. © 2014 Elsevier B.V. All rights reserved.