Physiologic dentin regeneration: its past, present, and future perspectives.

Regenerative dentistry has rapidly progressed since the advancement of stem cell biology and material science. However, more emphasis has been placed on the success of tissue formation than on how well the newly generated tissue retains the original structure and function. Once dentin is lost, tertiary dentinogenesis can be induced by new odontoblastic differentiation or re-activation of existing odontoblasts. The characteristic morphology of odontoblasts generates the tubular nature of dentin, which is a reservoir of fluid, ions, and a number of growth factors, and protects the inner pulp tissue. Therefore, understanding the dynamic but delicate process of new dentin formation by odontoblasts, or odontoblast-like cells, following dentinal defects is crucial. In this regard, various efforts have been conducted to identify novel molecules and materials that can promote the regeneration of dentin with strength and longevity. In this review, we focus on recent progress in dentin regeneration research with biological molecules identified, and discuss its potential in future clinical applications.

Combined effect of electrical energy and graphene oxide on Enterococcus faecalis biofilms.

This study aimed to investigate the effects of electrical energy and its synergistic activity with graphene oxide (GO) in Enterococcus faecalis (E. faecalis) biofilms. The viability of E. faecalis biofilms was analyzed by colony-forming units, crystal violet staining, and confocal laser scanning microscopy. The morphologies of the biofilms and the bacterial organelles were observed by scanning electron microscopy and transmission emission microscopy (TEM), respectively. Application of electrical energy combined with 0.2% sodium hypochlorite (NaOCl) on E. faecalis in biofilms significantly decreased the bacterial viability and biofilm biomass compared to the 0.2% NaOCl-only-treated group. Furthermore, additional application of GO showed similar antibacterial effects to 0.5% NaOCl. Notably, TEM observation revealed that the bacteria treated with electric energy and GO showed damaged cell membranes. The results suggest that combination of electrical energy and GO enhances antibacterial activity of NaOCl and has the potential to be applied to root canal irrigation protocols.

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.

Mechanical properties and sustainable bacterial resistance effect of strontium-modified phosphate-based glass microfiller in dental composite resins.

Dental composite resins are widely used in dental restorations. However, their clinical application is limited by the occurrence of secondary caries. Strontium-modified phosphate-based glass (Sr-PBG) is a material known to have a sustainable bacterial resistance effect. The mechanical properties (in particular, flexural strength, modulus of elasticity, and hardness) of dental materials determine their function. Therefore, this study aimed to investigate the mechanical and ion-releasing properties as well as the sustainable bacterial resistance effect of bioactive resin composites containing Sr-PBG. The data were analyzed by ANOVA and Tuckey's tests (p < 0.05). We incorporated a Sr-PBG microfiller at 3, 6, and 9 wt.% concentrations into a commercially available composite resin and investigated the mechanical properties (flexural strength, elastic modulus, and micro hardness), ion release characteristics, and color of the resultant resins. In addition, we examined the antibacterial effects of the composite resins against Streptococcus mutans (S. mutans). The mechanical properties of the Sr-PBG groups differed only slightly from those of the control group (p > 0.05). However, the optical density at 600 nm of S. mutans incubated on the experimental group was significantly lower compared to that observed with the control (p < 0.05) both before and after thermocycling between 5 and 55 â„ƒ for 850 cycles (dwell time: 45 s). Therefore, strontium-modified resin materials exhibited a sustainable bacterial resistance effect in vitro while maintaining some of the mechanical properties of ordinary acrylic resins.

A novel orthodontic adhesive containing zinc-doped phosphate-based glass for preventing white spot lesions.

OBJECTIVES: This study aimed at demonstrating the remineralization effect of the enamel around the brackets to aid reduction in white spot lesions (WSLs) with use of zinc-doped phosphate-based glass (Zn-PBG) containing orthodontic adhesives. METHODS: Zn-PBG powder was synthesized, and particle morphology, size, and density were evaluated. Orthodontic adhesives with increasing loading percentage of Zn-PBG powder were prepared: ZnPG3 (3 wt.%), ZnPG6 (6 wt.%), and ZnPG9 (9 wt.%). Brackets were bonded on the etched enamel surface and stored in distilled water (DW) for 1 h. Following, Shear bond strength (SBS) along with adhesive remnant index were analyzed. The release of calcium (Ca), phosphorus (P), and zinc (Zn) from adhesive specimens in DW was evaluated after 7, 15 and 30 days of immersion. The remineralization effect was confirmed by microhardness and surface morphology analysis with scanning electron microscopy. RESULTS: The SBS value was observed between 20 and 22 MPa on enamel surface. The concentration of Ca, P and Zn released in DW increased with loading percentage of Zn-PBG. The microhardness increased in the experimental groups after immersion in artificial saliva for 7 days. Apatite-like crystal formation was observed after 30 days in the ZnPG 9 group. CONCLUSIONS: The orthodontic adhesive containing Zn-PBG with an optimal SBS performance has an enamel remineralization effect, and therefore can aid in prevention of WSLs. CLINICAL SIGNIFICANCE: The orthodontic adhesive containing Zn-PBG is clinically advantageous as it can promote remineralization and resist the formation of WSLs that may occur during orthodontic therapy.

Analysis of Weather Factors on Aircraft Cancellation using a Multilayer Complex Network.

Airlines provide one of the most popular and important transportation services for passengers. While the importance of the airline industry is rising, flight cancellations are also increasing due to abnormal weather factors, such as rainfall and wind speed. Although previous studies on cancellations due to weather factors considered both aircraft and weather factors concurrently, the complex network studies only treated the aircraft factor with a single-layer network. Therefore, the aim of this study was to apply a multilayer complex network (MCN) method that incorporated three different factors, namely, aircraft, rainfall, and wind speed, to investigate aircraft cancellations at 14 airports in the Republic of Korea. The results showed that rainfall had a greater impact on aircraft cancellations compared with wind speed. To find out the most important node in the cancellation, we applied centrality analysis based on information entropy. According to the centrality analysis, Jeju Airport was identified as the most influential node since it has a high demand for aircraft. Also, we showed that characteristics and factors of aircraft cancellation should be appropriately defined by links in the MCN. Furthermore, we verified the applicability of the MCN method in the fields of aviation and meteorology. It is expected that the suggested methodology in this study can help to understand aircraft cancellation due to weather factors.

Soluble prefusion-closed HIV-envelope trimers with glycan-covered bases.

Soluble HIV-1-envelope (Env) trimers elicit immune responses that target their solvent-exposed protein bases, the result of removing these trimers from their native membrane-bound context. To assess whether glycosylation could limit these base responses, we introduced sequons encoding potential N-linked glycosylation sites (PNGSs) into base-proximal regions. Expression and antigenic analyses indicated trimers bearing six-introduced PNGSs to have reduced base recognition. Cryo-EM analysis revealed trimers with introduced PNGSs to be prone to disassembly and introduced PNGS to be disordered. Protein-base and glycan-base trimers induced reciprocally symmetric ELISA responses, in which only a small fraction of the antibody response to glycan-base trimers recognized protein-base trimers and vice versa. EM polyclonal epitope mapping revealed glycan-base trimers -even those that were stable biochemically- to elicit antibodies that recognized disassembled trimers. Introduced glycans can thus mask the protein base but their introduction may yield neo-epitopes that dominate the immune response.

Effect of strontium substitution on functional activity of phosphate-based glass.

Phosphate-based glass (PBG) is a bioactive agent, composed of a glass network with phosphate as the primary component and can be substituted with various therapeutic ions for functional enhancement. Strontium (Sr) has been shown to stimulate osteogenic activity and inhibit pro-inflammatory responses. Despite this potential, there are limited studies that focus on the proportion of Sr substituted and its impact on the functional activity of resulting Sr-substituted PBG (PSr). In this study, focusing on the cellular biological response we synthesized and investigated the functional activity of PSr by characterizing its properties and comparing the effect of Sr substitution on cellular bioactivity. Moreover, we benchmarked the optimal composition against 45S5 bioactive glass (BG). Our results showed that PSr groups exhibited a glass structure and phosphate network like that of PBG. The release of Sr and P was most stable for PSr6, which showed favorable cell viability. Furthermore, PSr6 elicited excellent early osteogenic marker expression and inhibition of pro-inflammatory cytokine expression, which was significant compared to BG. In addition, compared to BG, PSr6 had markedly higher expression of osteopontin in immunocytochemistry, higher ALP expression in osteogenic media, and denser alizarin red staining in vitro. We also observed a comparable in vivo regenerative response in a 4-week rabbit calvaria defect model. Therefore, based on the results of this study, PSr6 could be identified as the functionally optimized composition with the potential to be applied as a valuable bioactive component of existing biomaterials used for bone regeneration.

Evaluation of Biomechanical Stability of Teeth Tissue According to Crown Materials: A Three-Dimensional Finite Element Analysis.

The biomechanical effects of dental tissue according to various dental crown materials were investigated using finite element analysis. Bone, prepared tooth, root canal, and periodontal ligament were modeled based on computed tomography. Depending on the characteristics of the crown material, it was classified into zirconia, hybrid ceramic, gold alloy, and acrylic resin. A loading force of 200 N was applied in the vertical direction to the occlusal surface of the crown, and analysis was performed under the condition that all interfaces were tied. The results demonstrate that the highest von Mises stress was shown in the prepared tooth of the acrylic resin model, which is a temporary prosthesis, and the pulpal pressure was also the highest. Additionally, among the final prosthesis, the highest stress was shown in the hybrid ceramic model prepared teeth. The properties of restoration materials can be a factor influencing the tooth structure. Thus, in order to make a correct decision when selecting a material for restorative treatment, it is necessary to understand, analyze, and evaluate the properties of these restoration materials.

HIV-1 neutralizing antibodies elicited in humans by a prefusion-stabilized envelope trimer form a reproducible class targeting fusion peptide.

Elicitation of antibodies that neutralize the tier-2 neutralization-resistant isolates that typify HIV-1 transmission has been a long-sought goal. Success with prefusion-stabilized envelope trimers eliciting autologous neutralizing antibodies has been reported in multiple vaccine-test species, though not in humans. To investigate elicitation of HIV-1 neutralizing antibodies in humans, here, we analyze B cells from a phase I clinical trial of the "DS-SOSIP"-stabilized envelope trimer from strain BG505, identifying two antibodies, N751-2C06.01 and N751-2C09.01 (named for donor-lineage.clone), that neutralize the autologous tier-2 strain, BG505. Though derived from distinct lineages, these antibodies form a reproducible antibody class that targets the HIV-1 fusion peptide. Both antibodies are highly strain specific, which we attribute to their partial recognition of a BG505-specific glycan hole and to their binding requirements for a few BG505-specific residues. Prefusion-stabilized envelope trimers can thus elicit autologous tier-2 neutralizing antibodies in humans, with initially identified neutralizing antibodies recognizing the fusion-peptide site of vulnerability.

The effect of 6% hydroxyethyl starch 130/0.4 preloading on the blood glucose levels in diabetic patients undergoing orthopedic surgery with spinal anesthesia: a randomized pilot study.

BACKGROUND: Perioperative hyperglycemia can occur in surgical patients and may increase postoperative morbidity and mortality, especially in patients with diabetes. Therefore, we conducted the present study to evaluate whether the administration of 6% hydroxyethyl starch (HES)-130/0.4 increases blood glucose levels in patients with diabetes. METHODS: Forty patients undergoing lower limb surgery under spinal anesthesia were randomly allocated into two groups according to the fluids administered 20 min before spinal anesthesia (Group L, lactated Ringer's solution; Group H, 6% HES-130/0.4). Patient characteristics, intraoperative variables, blood glucose levels, mean blood pressure (MBP), and heart rate (HR) were recorded at five time-points (0, 20, 60, 120, and 240 min). RESULTS: A total of 39 patients were analyzed (Group L, n = 20; Group H, n = 19). The amount of intraoperative fluid was significantly higher in Group L than in Group H (718.2 ml vs. 530.0 ml, P = 0.010). There were no significant differences in the changes in blood glucose levels, HR, or MBP between the two groups (P = 0.737, P = 0.896, and P = 0.141, respectively). Serial changes in mean blood glucose levels from baseline also showed no significant differences between the groups (P = 0.764). CONCLUSIONS: There were no significant changes in blood glucose levels when lactated Ringer's solution or 6% HES-130 was used. When compared to the lactated Ringer's solution, no evidence that 6% HES-130/0.4 produces hyperglycemia in diabetic patients could be found. Further evaluation of larger populations is needed.

Surface engineering of inorganic solid-state electrolytes via interlayers strategy for developing long-cycling quasi-all-solid-state lithium batteries.

Lithium metal batteries (LMBs) with inorganic solid-state electrolytes are considered promising secondary battery systems because of their higher energy content than their Li-ion counterpart. However, the LMB performance remains unsatisfactory for commercialization, primarily owing to the inability of the inorganic solid-state electrolytes to hinder lithium dendrite propagation. Here, using an Ag-coated Li6.4La3Zr1.7Ta0.3O12 (LLZTO) inorganic solid electrolyte in combination with a silver-carbon interlayer, we demonstrate the production of stable interfacially engineered lab-scale LMBs. Via experimental measurements and computational modelling, we prove that the interlayers strategy effectively regulates lithium stripping/plating and prevents dendrite penetration in the solid-state electrolyte pellet. By coupling the surface-engineered LLZTO with a lithium metal negative electrode, a high-voltage positive electrode with an ionic liquid-based liquid electrolyte solution in pouch cell configuration, we report 800 cycles at 1.6 mA/cm2 and 25 °C without applying external pressure. This cell enables an initial discharge capacity of about 3 mAh/cm2 and a discharge capacity retention of about 85%.

Poly (Methyl Methacrylate)-Containing Silver-Phosphate Glass Exhibits Potent Antimicrobial Activity without Deteriorating the Mechanical and Biological Properties of Dental Prostheses.

Poly (methyl methacrylate) (PMMA) is a commonly used denture material with poor antimicrobial effects. This study investigated the antimicrobial effects of PMMA-containing silver-phosphate glass. We fabricated a novel material comprising PMMA-containing silver-phosphate glass. Then, microhardness, flexural strength, and gloss unit were analyzed. Antimicrobial activity against Streptococcus mutans and Candida albicans was investigated. Colony-forming units were counted, and antimicrobial rates were measured. Biocompatibility tests were performed using a colorimetric MTT assay for evaluating cell metabolic activity. The microhardness, flexural strength, and gloss unit of the experimental groups (with silver-phosphate glass) were not significantly different from those of the control group (no silver-phosphate glass) (P > 0.05), which showed clinically valid values. With increasing proportions of silver-phosphate glass, the antimicrobial activity against the two microorganisms increased (P < 0.05). Furthermore, S. mutans showed more than 50% antimicrobial activity in 4%, 6%, and 8% experimental groups, C. albicans showed more than 50% antimicrobial activity in 6% and 8% groups, and a statistically significant difference in antimicrobial activity was observed compared to the control (P < 0.05). The cell viability of the experimental groups was not significantly different from that of the control group (P > 0.05). Both control and experimental groups showed approximately 100% cell viability. These results suggest that silver-phosphate glass is a promising antimicrobial material in dentistry.

RING-Type E3 Ubiquitin Ligases AtRDUF1 and AtRDUF2 Positively Regulate the Expression of PR1 Gene and Pattern-Triggered Immunity.

The importance of E3 ubiquitin ligases from different families for plant immune signaling has been confirmed. Plant RING-type E3 ubiquitin ligases are members of the E3 ligase superfamily and have been shown to play positive or negative roles during the regulation of various steps of plant immunity. Here, we present Arabidopsis RING-type E3 ubiquitin ligases AtRDUF1 and AtRDUF2 which act as positive regulators of flg22- and SA-mediated defense signaling. Expression of AtRDUF1 and AtRDUF2 is induced by pathogen-associated molecular patterns (PAMPs) and pathogens. The atrduf1 and atrduf2 mutants displayed weakened responses when triggered by PAMPs. Immune responses, including oxidative burst, mitogen-activated protein kinase (MAPK) activity, and transcriptional activation of marker genes, were attenuated in the atrduf1 and atrduf2 mutants. The suppressed activation of PTI responses also resulted in enhanced susceptibility to bacterial pathogens. Interestingly, atrduf1 and atrduf2 mutants showed defects in SA-mediated or pathogen-mediated PR1 expression; however, avirulent Pseudomonas syringae pv. tomato DC3000-induced cell death was unaffected. Our findings suggest that AtRDUF1 and AtRDUF2 are not just PTI-positive regulators but are also involved in SA-mediated PR1 gene expression, which is important for resistance to P. syringae.

Cryo-EM structures of prefusion SIV envelope trimer.

Simian immunodeficiency viruses (SIVs) are lentiviruses that naturally infect non-human primates of African origin and seeded cross-species transmissions of HIV-1 and HIV-2. Here we report prefusion stabilization and cryo-EM structures of soluble envelope (Env) trimers from rhesus macaque SIV (SIVmac) in complex with neutralizing antibodies. These structures provide residue-level definition for SIV-specific disulfide-bonded variable loops (V1 and V2), which we used to delineate variable-loop coverage of the Env trimer. The defined variable loops enabled us to investigate assembled Env-glycan shields throughout SIV, which we found to comprise both N- and O-linked glycans, the latter emanating from V1 inserts, which bound the O-link-specific lectin jacalin. We also investigated in situ SIVmac-Env trimers on virions, determining cryo-electron tomography structures at subnanometer resolutions for an antibody-bound complex and a ligand-free state. Collectively, these structures define the prefusion-closed structure of the SIV-Env trimer and delineate variable-loop and glycan-shielding mechanisms of immune evasion conserved throughout SIV evolution.

Zinc-modified phosphate-based glass micro-filler improves Candida albicans resistance of auto-polymerized acrylic resin without altering mechanical performance.

Colonization of auto-polymerized acrylic resin by pathogenic Candida albicans is a common problem for denture users. In this study, zinc-modified phosphate-based glass was introduced into an auto-polymerized acrylic resin at concentrations of 3, 5, and 7 wt.%. The mechanical or physical properties (flexural strength, elastic modulus, microhardness, and contact angle), surface morphology of the resultant materials, and the antimicrobial effect on C. albicans were investigated. There were no statistical differences in the mechanical properties between the control and the zinc-modified phosphate-based glass samples (p > 0.05); however, the number of C. albicans colony-forming units was significantly lower in the control group (p < 0.05). Scanning electron microscopy revealed that C. albicans tended not to adhere to the zinc-modified-phosphate-based glass samples. Thus, the zinc-modified materials retained the advantageous mechanical properties of unaltered acrylic resins, while simultaneously exhibiting a strong antimicrobial effect in vitro.

Jasmonate regulates plant resistance to Pectobacterium brasiliense by inducing indole glucosinolate biosynthesis.

Pectobacterium brasiliense (P. brasiliense) is a necrotrophic bacterium that causes the soft rot disease in Brassica rapa. However, the mechanisms underlying plant immune responses against necrotrophic bacterial pathogens with a broad host range are still not well understood. Using a flg22-triggered seedling growth inhibition (SGI) assay with 455 Brassica rapa inbred lines, we selected six B. rapa flagellin-insensitive lines (Brfin2-7) and three B. rapa flagellin-sensitive lines (Brfs1-3). Brfin lines showed compromised flg22-induced immune responses (oxidative burst, mitogen-activated protein kinase (MAPK) activation, and seedling growth inhibition) compared to the control line R-o-18; nevertheless, they were resistant to P. brasiliense. To explain this, we analyzed the phytohormone content and found that most Brfin lines had higher P. brasiliense-induced jasmonic acid (JA) than Brfs lines. Moreover, MeJA pretreatment enhanced the resistance of B. rapa to P. brasiliense. To explain the correlation between the resistance of Brfin lines to P. brasiliense and activated JA signaling, we analyzed pathogen-induced glucosinolate (GS) content in B. rapa. Notably, in Brfin7, the neoglucobrassicin (NGBS) content among indole glucosinolates (IGS) was significantly higher than that in Brfs2 following P. brasiliense inoculation, and genes involved in IGSs biosynthesis were also highly expressed. Furthermore, almost all Brfin lines with high JA levels and resistance to P. brasiliense had higher P. brasiliense-induced NGBS levels than Brfs lines. Thus, our results show that activated JA-mediated signaling attenuates flg22-triggered immunity but enhances resistance to P. brasiliense by inducing indole glucosinolate biosynthesis in Brassica rapa. This study provides novel insights into the role of JA-mediated defense against necrotrophic bacterial pathogens within a broad host range.

Assessment of Zinc-Bound Phosphate-Based Glass-Coated Denture-Relining Material with Antifungal Efficacy for Inhibiting Denture Stomatitis.

This study investigated the surface properties, biocompatibility, and antifungal activity against Candida albicans of a denture-relining material coated with zinc-bound phosphate-based glass. First, zinc-bound phosphate-based glass was fabricated. A polymerized denture-relining disk was coated with zinc-bound phosphate-based glass (2%, 4%, and 6%). The surface properties of the control and experimental groups were measured, including the wettability, microhardness, color difference, and gloss. The biocompatibility was evaluated using the MTT assay according to ISO 10993-5. The antifungal activity was investigated by counting the number of colony-forming units of Candida albicans. The results were analyzed using a one-way ANOVA and Tukey's test (p = 0.05). The results of this study indicate that, despite the antimicrobial effect of zinc-bound phosphate-based glass, a coated denture-relining material does not degrade the surface properties and biocompatibility. Therefore, this novel material is considered promising for use as a dental material with antimicrobial properties that can potentially prevent denture stomatitis.

C3-Symmetric Aromatic Core of Griffithsin Is Essential for Potent Anti-HIV Activity.

Lectins, carbohydrate-binding proteins of nonimmune origin, bind to carbohydrates and glycan shields present on the surfaces of cells and viral spike proteins. Lectins thus hold great promise as therapeutic and diagnostic proteins, exemplified by their potent antiviral activities and the desire to engineer synthetic carbohydrate receptors based on lectin recognition principles. Here, we describe a new carbohydrate-binding architectural motif─namely, a C3-symmetric tyrosine-based aromatic core, present in the therapeutic lectin griffithsin (GRFT). By using structure-based amino acid substitutions, X-ray crystallography, molecular dynamics (MD) simulations, and HIV-1 neutralization assays, we show that this core is critical for potent (pM) antiviral activity and nanomolar binding to the glycan shield largely consisting of high mannose glycans. Crystal structures and MD simulations show that CH-π interactions stabilize the aromatic cluster to maintain the three pseudo-symmetric carbohydrate-binding sites, nonaromatic amino acid substitutions (Tyr to Ala) abrogate antiviral activity, and increasing the aromatic CH-π edge-to-centroid interface via a Tyr to Trp substitution yields a GRFT variant with improved potency and increased residence time of Man-9 observed in MD simulations. NMR titrations of a Tyr-to-Ala variant indicate that disruption of the aromatic prevents the intermolecular crosslinking between two equivalents of Man-9 and one carbohydrate-binding face observed in wild-type GRFT and known to be critical for picomolar potency of this lectin. This C3-symmetric aromatic core defines a new recognition motif for the design of carbohydrate receptors and suggests principles for engineering known lectins to have increased affinity and stability.

Antigenic analysis of the HIV-1 envelope trimer implies small differences between structural states 1 and 2.

The conformationally dynamic HIV-1 envelope trimer (Env) is the target of broadly neutralizing antibodies (bnAbs) that block viral entry. Single-molecule Förster resonance energy transfer (smFRET) has revealed that HIV-1 Env exists in at least three conformational states on the virion. Prior to complete host-receptor engagement (State 3), Env resides most prevalently in the smFRET-defined State 1, which is preferentially recognized by most bnAbs that are elicited by natural infection. smFRET has also revealed that soluble trimers containing prefusion-stabilizing disulfide and isoleucine-to-proline substitutions reside primarily in State 2, which is a required intermediate between States 1 and 3. While high-resolution Env structures have been determined for States 2 and 3, the structure of these trimers in State 1 is unknown. To provide insight into the State 1 structure, here we characterized antigenic differences between smFRET-defined states and then correlated these differences with known structural differences between States 2 and 3. We found that cell surface-expressed Env was enriched in each state using state-enriching antibody fragments or small-molecule virus entry inhibitors and then assessed binding to HIV-1 bnAbs preferentially binding different states. We observed small but consistent differences in binding between Env enriched in States 1 and 2, and a more than 10-fold difference in binding to Env enriched in these states versus Env enriched in State 3. We conclude that structural differences between HIV-1 Env States 1 and 3 are likely more than 10-fold greater than those between States 1 and 2, providing important insight into State 1.