Native Environment Research Articles

Integration of bioprinting advances and biomechanical strategies for in vitro lung modelling.

The recent occurrence of the Covid-19 pandemic and frequent wildfires have worsened pulmonary diseases and raised the urgent need for investigating host-pathogen interactions and advancing drug and vaccine therapies. Historically, research and experimental studies have relied on two-dimensional cell culture dishes and/or animal models, which suffer from physiological differences from the human lung. More recently, there has been investigation into the use of lung-on-a-chip models and organoids, while the use of bioprinting technologies has also emerged to fabricate three-dimensional constructs or lung models with enhanced physiological relevance. Concurrently, achievements have also been made to develop biomimetic strategies for simulating the in vivo biomechanical conditions induced by lung breathing, though challenges remain with incorporating these strategies with bioprinted models. Bioprinted models combined with advanced biomimetic strategies would represent a promising approach to advance disease discovery and therapeutic development. As inspired, this article briefly reviews the recent progress of both bioprinted in vitro lung models and biomechanical strategies, with a focus on native lung tissue microstructure and biomechanical properties, bioprinted constructs, and biomimetic strategies to mimic the native environment. This article also urges that the integration of bioprinting advances and biomimetic strategies would be essential to achieve synergistic effects for in vitro lung modelling. Key issues and challenges are also identified and discussed along with recommendations for future research.

Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling.

The discovery of Toll-like receptors (TLRs) represented a significant breakthrough that paved the way for the study of host-pathogen interactions in innate immunity. However, there are still major gaps in understanding TLR function, especially regarding the early dynamics of downstream TLR pathways. Here, we present a label-free optical biosensor-based assay as a method for detecting TLR activation in a native and label-free environment and defining the dynamics of TLR pathway activation. This technology is sufficiently sensitive to detect TLR signaling and readily discriminates between different TLR signaling pathways. We define pharmacological modulators of cell surface and endosomal TLRs and downstream signaling molecules and uncover TLR signaling signatures, including potential biased receptor signaling. These findings highlight that optical biosensor assays complement traditional assays that use a single endpoint and have the potential to facilitate the future design of selective drugs targeting TLRs and their downstream effector cascades.

Oviductal extracellular matrix hydrogels enhance in vitro culture of rabbit embryos and reduce deficiencies during assisted reproductive technologies.

In vitro embryo culture often falls short of mimicking the physiological dynamism occurring in the reproductive tract, prompting developmental plasticity in mammalian embryos with consequential genotypic and phenotypic deviations. Recent research highlights the potential of biological derivatives in in vitro culture to mitigate these effects, being the extracellular matrix (ECM) one of the most important components in retaining structural and biological signals derived from the native source tissue. Current bioengineering techniques could provide ECM-based biomaterials mimicking the native environment and offering optimal embryonic development. Rabbit oviducts (n = 24) were decellularized and solubilized to create tissue-specific ECM (OviECM) hydrogels. Following physicochemical characterization, these hydrogels were applied as coatings for the in vitro culture of two-cell embryos over 48h, along with embryos cultured under In vitro control conditions (n = 218/group), which were subsequently transferred to recipient females. A subset of embryos was recovered on day 6 for transcriptomic analysis (n = 75-80/group), while the remaining embryos were used to assess implantation and birth rates. Rabbit weights were monitored over 20 weeks post-delivery, with blood tests conducted at weeks 8 and 20. Bayesian inference methods were used for statistical analysis. Differences were considered relevant if P ≥ 0.8 (80%). No differences in embryo development and morphology were detected between the OviECM coating and In vitro control conditions. However, embryos cultured on these coatings exhibited upregulation of pathways involved in antigen presentation and immune system activation, as well as, increased cellular response to external stimulus and intracellular protein transport. The implantation and live birth rates were significantly higher in the coating group than in the In vitro control group (30.8% vs. 26.1% and 21.2% vs. 18.1%, respectively). During the first 20 weeks of life, the animals from the coating group showed higher weights than the In vitro control group P0 > 0.8. The animals of both experimental groups showed normal blood parameters. Implementation of OviECM coatings allows for improving in vitro conditions and decreases postnatal phenotypic deviations after assisted reproductive technology (ART). This study could initiate a new embryo culture techniques era to guarantee that ART is utilized in the most efficient and safest possible practice.

Inducible cell lines producing replication-defective human immunodeficiency virus particles containing envelope glycoproteins stabilized in a pretriggered conformation.

During the process by which human immunodeficiency virus (HIV-1) enters cells, the envelope glycoprotein (Env) trimer on the virion surface engages host cell receptors. Binding to the receptor CD4 induces Env to undergo transitions from a pretriggered, "closed" (State-1) conformation to more "open" (State 2/3) conformations. Most broadly neutralizing antibodies (bNAbs), which are difficult to elicit, recognize the pretriggered (State-1) conformation. More open Env conformations are recognized by poorly neutralizing antibodies (pNAbs), which are readily elicited during natural infection and vaccination with current Env immunogens. Env heterogeneity likely contributes to HIV-1 persistence by skewing antibody responses away from the pretriggered conformation. The conformationally flexible gp160 Env precursor on the infected cell or virion surface potentially presents multiple pNAb epitopes to the host immune system. Although proteolytic cleavage to produce the functional, mature Env trimer [(gp120/gp41)3] stabilizes State-1, many primary HIV-1 Envs spontaneously sample more open conformations. Here, we establish inducible cell lines that produce replication-defective HIV-1 particles with Env trimers stabilized in a pretriggered conformation. The mature Env is enriched on virus-like particles (VLPs). Using complementary approaches, we estimate an average of 25-50 Env trimers on each VLP. The stabilizing changes in Env limit the natural conformational heterogeneity of the VLP Env trimers, allowing recognition by bNAbs but not pNAbs. These defective VLPs provide a more homogeneous source of pretriggered Env trimers in a native membrane environment. Thus, these VLPs may facilitate the characterization of this functionally important Env conformation and its interaction with the immune system.IMPORTANCEA major impediment to the development of an effective HIV/AIDS vaccine is the inefficiency with which human immunodeficiency virus (HIV-1) envelope glycoproteins elicit antibodies that neutralize multiple virus strains. Neutralizing antibodies recognize a particular shape of the envelope glycoproteins that resides on the viral membrane before the virus engages the host cell. Here, we report the creation of stable cell lines that inducibly produce non-infectious HIV-like particles. The normally flexible envelope glycoprotein spikes on these virus-like particles have been stabilized in a conformation that is recognized by broadly neutralizing antibodies. These virus-like particles allow the study of the envelope glycoprotein conformation, its modification by sugars, and its ability to elicit desired neutralizing antibodies.

In Situ and Label-Free Quantification of Membrane Protein–Ligand Interactions Using Optical Imaging Techniques: A Review

Membrane proteins are crucial for various cellular processes and are key targets in pharmacological research. Their interactions with ligands are essential for elucidating cellular mechanisms and advancing drug development. To study these interactions without altering their functional properties in native environments, several advanced optical imaging methods have been developed for in situ and label-free quantification. This review focuses on recent optical imaging techniques such as surface plasmon resonance imaging (SPRi), surface plasmon resonance microscopy (SPRM), edge tracking approaches, and surface light scattering microscopy (SLSM). We explore the operational principles, recent advancements, and the scope of application of these methods. Additionally, we address the current challenges and explore the future potential of these innovative optical imaging strategies in deepening our understanding of biomolecular interactions and facilitating the discovery of new therapeutic agents.

Chronotopes of exile and loss in Philip O'Sullivan Beare's Zoilomastix (c. 1626)☆

AbstractThis essay explores the relationship between an early modern exile and his native environment, as depicted in Philip O'Sullivan Beare's unfinished natural history Zoilomastix. Writing by turns in Latin, Spanish and Gaelic from the safety of the Habsburg court, O'Sullivan Beare marshalled Ciceronian rhetoric and Plinian wonder to argue for the conservation of interwoven natural and cultural environments. One of the earliest examples of national historiographies written from exile by erudite Irish figures, Zoilomastix has most often been read in terms of its political theology. Though understandable, such readings obscure O'Sullivan Beare's idiosyncratic treatments of self, state and nature. Additionally, such readings only partially consider the author's attempts to engage with the debates on ethnicity, natural law and resource capture which directed the actions of the English state. Adopting the dialogic framework of Mikhail Bakhtin's chronotope, this essay therefore explores the extent to which the genre of natural history was utilized to inform and influence contested narratives. It argues that O'Sullivan Beare's autobiographical account, and its relevance to the forthcoming ‘loss’ of his Gaelic world, has much to tell us in a time of ecological crisis.

Selective Methods Promote Protein Solid-State NMR.

Solid-state nuclear magnetic resonance (ssNMR) is indispensable for studying the structures, dynamics, and interactions of insoluble proteins in native or native-like environments. While ssNMR includes numerous nonselective techniques for general analysis, it also provides various selective methods that allow for the extraction of precise details about proteins. This perspective highlights three key aspects of selective methods: selective signals of protein segments, selective recoupling, and site-specific insights into proteins. These methods leverage protein topology, labeling strategies, and the tailored manipulation of spin interactions through radio frequency (RF) pulses, significantly promoting the field of protein ssNMR. With ongoing advancements in higher magnetic fields and faster magic angle spinning (MAS), there remains an ongoing need to enhance the selectivity and efficiency of selective ssNMR methods, facilitating deeper atomic-level insights into complex biological systems.

What Is the Protonation State of Proteins in Crystals? Insights from Constant pH Molecular Dynamics Simulations.

X-ray crystallography is an important technique to determine the positions of atoms in a protein crystal. However, because the native environment in which proteins function, is not a crystal, but a solution, it is not a priori clear if the crystal structure represents the functional form of the protein. Because the protein structure and function often depend critically on the pH, the question arises whether proton affinities are affected by crystallization. X-ray diffraction usually does not reveal protons, which makes it difficult to experimentally measure pKa shifts in crystals. Here, we investigate whether this challenge can be addressed by performing in silico titration with constant pH molecular dynamics (MD) simulations. We compare the computed pKa values of proteins between solution and crystal environment and analyze these differences in the context of molecular interactions. For the proteins considered in this work, pKa shifts were mostly found for residues at the crystal interfaces, where the environment is more apolar in the crystal than in water. Although convergence was an obstacle, our simulations suggest that in principle it is possible to apply constant pH MD to protein crystals routinely and assess the effect of crystallization on protein function more systematically than with standard MD simulations. We also highlight technical challenges that need to be addressed to make MD simulations of crystals more reliable.

Clonal dynamics after allogeneic haematopoietic cell transplantation.

Allogeneic haematopoietic cell transplantation (HCT) replaces the stem cells responsible for blood production with those from a donor1,2. Here, to quantify dynamics of long-term stem cell engraftment, we sequenced genomes from 2,824 single-cell-derived haematopoietic colonies of ten donor-recipient pairs taken 9-31 years after HLA-matched sibling HCT3. With younger donors (18-47 years at transplant), 5,000-30,000 stem cells had engrafted and were still contributing to haematopoiesis at the time of sampling; estimates were tenfold lower with older donors (50-66 years). Engrafted cells made multilineage contributions to myeloid, B lymphoid and T lymphoid populations, although individual clones often showed biases towards one or other mature cell type. Recipients had lower clonal diversity than matched donors, equivalent to around 10-15 years of additional ageing, arising from up to 25-fold greater expansion of stem cell clones. A transplant-related population bottleneck could not explain these differences; instead, phylogenetic trees evinced two distinct modes of HCT-specific selection. In pruning selection, cell divisions underpinning recipient-enriched clonal expansions had occurred in the donor, preceding transplant-their selective advantage derived from preferential mobilization, collection, survival ex vivo or initial homing. In growth selection, cell divisions underpinning clonal expansion occurred in the recipient's marrow after engraftment, most pronounced in clones with multiple driver mutations. Uprooting stem cells from their native environment and transplanting them to foreign soil exaggerates selective pressures, distorting and accelerating the loss of clonal diversity compared to the unperturbed haematopoiesis of donors.

Production of a fungal aspartic protease via solid‐state fermentation using a rotating drum bioreactor

AbstractBACKGROUNDSolid‐state fermentation (SSF) has gained attention recently as a promising approach for producing a range of high‐value industrial products. SSF has several benefits, including a high product yield, a lesser chance of contamination, a resemblance to some fungi and bacteria's native environment, a lower energy need, and cost‐effectiveness. Additionally, the enzyme extraction from concentrated solutions facilitates the recovery procedure. Mucor racemosus, a filamentous fungus, has the potential to produce a milk‐clotting enzyme (aspartic protease) cultivated on various combinations of the solid media to investigate the effect of media on the production and activity of the milk‐clotting enzyme.RESULTSThe fermentation was scaled up by utilizing Terrafors‐IS Infors HT in‐situ sterilizable rotating solid‐state bioreactor to cultivate the fungus. The effect of agitation (rotation on the day of inoculation and day 1), aeration (air flow rates of 1 L and 2 L/min), and moisture content (60% and 90%), during SSF were investigated. The profiles of various operational parameters (that is, temperature, pressure, flow rate, exit O2, and exit CO2) were monitored using the provided software, and data were collected at 10‐min intervals during the whole course of fermentation. The results suggest that aeration has a remarkable effect on the quantity of biomass produced, but it doesn't assure high enzyme production. The highest milk clotting activity of ~570 U/mL was recorded with a moisture content of 60% and a flow rate of 1 L/min.CONCLUSIONA high yield of ~570 U/mL of the enzyme with desired characteristics was obtained with minimum moisture availability at low temperatures. The results narrated in this study validate the SSF process for producing aspartic protease from wheat bran using a rotating solid‐state bioreactor to be used as a potential milk coagulant in the cheese industry. © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

On the Properties of Styrene-Maleic Acid Copolymer-Lipid Nanoparticles: A Solution NMR Perspective.

The production of functionally active membrane proteins (MPs) in an adequate membrane environment is a key step in structural biology. Polymer-lipid particles based on styrene and maleic acid (SMA) represent a promising type of membrane mimic, as they can extract properly folded MPs directly from their native lipid environment. However, the original SMA polymer is sensitive to acidic pH levels, which has led to the development of several modifications: SMA-EA, SMA-QA, and others. Here, we introduce a novel SMA derivative with a negatively charged taurine moiety, SMA-tau, and investigate the formation and characteristics of lipid-SMA-EA and lipid-SMA-tau membrane-mimicking particles. Our findings demonstrate that both polymers can form nanodiscs with a patch of lipid bilayer that can undergo phase transitions at temperatures close to those of the lipid bilayer membranes. Finally, we discuss the potential applications of these SMAs for NMR spectroscopy.

SPOT: spatial proteomics through on-site tissue-protein-labeling

BackgroundSpatial proteomics seeks to understand the spatial organization of proteins in tissues or at different subcellular localization in their native environment. However, capturing the spatial organization of proteins is challenging. Here, we present an innovative approach termed Spatial Proteomics through On-site Tissue-protein-labeling (SPOT), which combines the direct labeling of tissue proteins in situ on a slide and quantitative mass spectrometry for the profiling of spatially-resolved proteomics.Materials and MethodsEfficacy of direct TMT labeling was investigated using seven types of sagittal mouse brain slides, including frozen tissues without staining, formalin-fixed paraffin-embedded (FFPE) tissues without staining, deparaffinized FFPE tissues, deparaffinized and decrosslinked FFPE tissues, and tissues with hematoxylin & eosin (H&E) staining, hematoxylin (H) staining, eosin (E) staining. The ability of SPOT to profile proteomes at a spatial resolution was further evaluated on a horizontal mouse brain slide with direct TMT labeling at eight different mouse brain regions. Finally, SPOT was applied to human prostate cancer tissues as well as a tissue microarray (TMA), where TMT tags were meticulously applied to confined regions based on the pathological annotations. After on-site direct tissue-protein-labeling, tissues were scraped off the slides and subject to standard TMT-based quantitative proteomics analysis.ResultsTissue proteins on different types of mouse brain slides could be directly labeled with TMT tags. Moreover, the versatility of our direct-labeling approach extended to discerning specific mouse brain regions based on quantitative outcomes. The SPOT was further applied on both frozen tissues on slides and FFPE tissues on TMAs from prostate cancer tissues, where a distinct proteomic profile was observed among the regions with different Gleason scores.ConclusionsSPOT is a robust and versatile technique that allows comprehensive profiling of spatially-resolved proteomics across diverse types of tissue slides to advance our understanding of intricate molecular landscapes.

Biology and host specificity of the stem-boring weevil Gasteroclisus tricostalis, a candidate agent for the biological control of fireweed (Senecio madagascariensis) in Australia

ABSTRACT Senecio madagascariensis Poiret (Asteraceae) is a target for biological control in Australia, due to substantial impacts on agriculture and native environments. Studies in the plant’s native range in South Africa prioritised the stem-boring weevil Gasteroclisus tricostalis (Thunberg) (Curculionidae) as a candidate agent. In this study, we assessed the biology and host specificity of G. tricostalis during laboratory trials conducted in both South Africa and Australia. No-choice trials in South Africa revealed that, despite oviposition on 15 non-target Senecio species (seven Australian and eight South African), there was significantly lower oviposition relative to S. madagascariensis, with no survival to pupation or adulthood on any non-target species. In contrast, no-choice tests in quarantine in Australia, recorded oviposition on 12 non-target Australian native Senecio species, with oviposition on two species not significantly different to that on S. madagascariensis. Furthermore in Australia, larvae developed to adulthood on five non-target species, in numbers that were not significantly different to S. madagascariensis. These discrepancies can mostly be explained by the lower phylogenetic distance from S. madagascariensis to the susceptible non-target species tested in Australia and, likely, optimal plant growth conditions in quarantine in two cases. Although no-choice tests are renowned for their conservative nature, preliminary paired-choice trials in both South Africa and Australia failed to demonstrate clear oviposition preferences for S. madagascariensis. These results suggest that, given the high diversity of the genus Senecio in Australia, the weevil is likely unsuitable for release and further trials were suspended.

Interplay between native plant performance and environment shapes resistance to aquatic plant invasion

Abstract In the limited number of studies on biotic resistance in freshwater ecosystems, experimental studies have supported that native plants play a role in resisting exotic plant invasion, but field observations have often reported positive relationships between exotic and native plant richness. To determine the reasons for the lack of evidence supporting biotic resistance in field observations, we surveyed the richness, biomass and coverage of exotic and native aquatic plants in 2801 quadrats at 287 sites in various freshwater ecosystems of China and examined the impacts of scale and environmental factors on the relationships between native and exotic plant richness and performance. At both the site and quadrat scales, we observed a positive correlation between the richness of exotic and native plants. However, at the quadrat scale, high native plant richness was associated with a reduction in the biomass of exotic emergent and floating plants as well as the biomass of exotic submerged plants in low‐nutrient water bodies. An increase in native plant performance, a metric that integrates the biomass and coverage of native plants, was associated with a decrease in both the richness and biomass of exotic plants at both scales. Furthermore, with increasing native plant performance at the quadrat scale, the richness of exotic plants declined more strongly in low‐nutrient water bodies than in high‐nutrient water bodies, and the decline in the biomass of exotic emergent and floating plants was more severe in shallow water than in deep water. Synthesis. Our results indicate that native plant performance is more important than richness for resistance to invasion in freshwater ecosystems, and the strength of biotic resistance is related to environmental factors. This study highlights the importance of environmental variables and multiple native community features and invasibility metrics in field observational studies of invasion.

Tunable Blended Collagen I/II and Collagen I/III Hydrogels as Tissue Mimics.

Collagen (Col) is commonly used as a natural biomaterial for biomedical applications. Although Col I is the most prevalent col type employed, many collagen types work together in vivo to confer function and biological activity. Thus, blending collagen types can better recapitulate many native environments. This work investigates how hydrogel properties can be tuned through blending collagen types (col I/II and col I/III) and by varying polymerization temperatures. Col I/II results in poorly developed fibril networks, which softened the gels, especially at lower polymerization temperatures. Conversely, col I/III hydrogels exhibit well-connected fibril networks with localized areas of fine fibrils and result in stiffer hydrogels. A decreased molecular mass recovery rate is observed in blended hydrogels. The altered fibril morphologies, mechanical properties, and biological signals of the blended gels can be leveraged to alter cell responses and can be used as models for different tissue types (e.g., healthy vs fibrotic tissue). Furthermore, the biomimetic hydrogel properties are a tool that can be used to modulate the transport of drugs, nutrients, and wastes in tissue engineering applications.

Modulation of heteromeric glycine receptor function through high concentration clustering.

Ion channels are targeted by many drugs for treating neurological, musculoskeletal, renal and other diseases. These drugs bind to and alter the function of individual channels to achieve desired therapeutic effects. However, many ion channels function in high concentration clusters in their native environment. It is unclear if and how clustering modulates ion channel function. Human heteromeric glycine receptors (GlyRs) are the major inhibitory neurotransmitter receptors in the spinal cord and are active targets for developing chronic pain medications. We show that the α2β heteromeric GlyR assembles with the master postsynaptic scaffolding gephyrin (GPHN) into micron-sized clustered at the plasma membrane after heterologous expression. The inhibitory trans- synaptic adhesion protein neuroligin-2 (NL2) further increases both the cluster sizes and GlyR concentration. The apparent glycine affinity increases monotonically as a function of GlyR concentration but not with cluster size. We also show that ligand re-binding to adjacent GlyRs alters kinetics but not chemical equilibrium. A positively charged N- terminus sequence of the GlyR β subunit was further identified essential for glycine affinity modulation through clustering. Taken together, we propose a mechanism where clustering enhances local electrostatic potential, which in turn concentrates ions and ligands, modulating the function of GlyR. This mechanism is likely universal across ion channel clusters found ubiquitously in biology and provides new perspectives in possible pharmaceutical development.

Illuminating cellular architecture and dynamics with fluorescence polarization microscopy.

Ever since Robert Hooke's 17th century discovery of the cell using a humble compound microscope, light-matter interactions have continuously redefined our understanding of cell biology. Fluorescence microscopy has been particularly transformative and remains an indispensable tool for many cell biologists. The subcellular localization of biomolecules is now routinely visualized simply by manipulating the wavelength of light. Fluorescence polarization microscopy (FPM) extends these capabilities by exploiting another optical property - polarization - allowing researchers to measure not only the location of molecules, but also their organization or alignment within larger cellular structures. With only minor modifications to an existing fluorescence microscope, FPM can reveal the nanoscale architecture, orientational dynamics, conformational changes and interactions of fluorescently labeled molecules in their native cellular environments. Importantly, FPM excels at imaging systems that are challenging to study through traditional structural approaches, such as membranes, membrane proteins, cytoskeletal networks and large macromolecular complexes. In this Review, we discuss key discoveries enabled by FPM, compare and contrast the most common optical setups for FPM, and provide a theoretical and practical framework for researchers to apply this technique to their own research questions.

Spatial transcriptomics of human and murine Neurofibromatosis Type I fracture pseudarthroses reveal impaired BMP signaling

Abstract Introduction/Objective In Neurofibromatosis Type 1 (NF1), caused by mutations in the NF1 tumor-suppressor gene, children form fibrotic nonunions known as pseudarthroses following long bone fractures. Here, we integrate spatial transcriptomics of human and murine samples to investigate the pathogenesis underlying pseudoarthrosis development. Methods/Case Report We performed spatial transcriptomics on 10-day post-fracture calluses from control and periosteum-specific Nf1-deficient (Nf1Postn) mice as well as a human NF1 patient pseudarthrosis specimen. Differential gene expression and SpatialTime analyses were performed. BMP signaling was validated by immunohistochemical staining of tissues for phospho-SMAD1/5/8. Results (if a Case Study enter NA) Spatial transcriptomics detected eight cell clusters, including bone, cartilage, and muscle within mouse fracture calluses. In the control, clusters involved in fracture repair were enriched for genes associated with extracellular matrix and collagen formation (progenitor and cartilage clusters), cartilage and endochondral bone processes (ossifying perichondrium cluster), and bone remodeling (woven bone cluster). Expression of TGF-beta pathway genes was highest near the fracture plane, while BMP and Wnt pathways were activated further from the fracture. In the Nf1Postn fracture, healing was significantly delayed, lacking a robust callus and with a lower abundance of reparative skeletal cell clusters. While expression of TGF-beta pathway genes was similar to control, BMP pathway expression was minimal within the Nf1Postn fracture, which was further demonstrated by a lack of phospho-SMAD1/5/8 staining. In the human pseudarthrosis, while TGF-beta pathway genes were expressed near the fracture plane as expected, there was no enrichment of BMP pathway expression in the pseudarthrosis tissue and no significant phospho-SMAD1/5/8 staining. Conclusion Spatial transcriptomics allows for the simultaneous unbiased analysis of multiple signal transduction pathways within their native tissue environment. Our analyses 1) revealed the cellular diversity required for fracture healing, 2) demonstrated the spatially-restricted expression of key morphogenetic pathways within a fracture callus, and 3) provides in situ evidence for impaired BMP pathway activation associated with NF1 fracture pseudarthroses.

ExoSloNano: Multi-Modal Nanogold Tags for identification of Macromolecules in Live Cells & Cryo-Electron Tomograms.

In situ cryo-Electron Microscopy (cryo-EM) enables the direct interrogation of structure-function relationships by resolving macromolecular structures in their native cellular environment. Tremendous progress in sample preparation, imaging and data processing over the past decade has contributed to the identification and determination of large biomolecular complexes. However, the majority of proteins are of a size that still eludes identification in cellular cryo-EM data, and most proteins exist in low copy numbers. Therefore, novel tools are needed for cryo-EM to identify the vast majority of macromolecules across multiple size scales (from microns to nanometers). Here, we introduce and validate novel nanogold probes that enable the detection of specific proteins using cryo-ET (cryo-Electron Tomography) and resin-embedded correlated light and electron microscopy (CLEM). We demonstrate that these nanogold probes can be introduced into live cells, in a manner that preserves intact molecular networks and cell viability. We use this system to identify both cytoplasmic and nuclear proteins by room temperature EM, and resolve associated structures by cryo-ET. We further employ gold particles of different sizes to enable future multiplexed labeling and structural analysis. By providing high efficiency protein labeling in live cells and molecular specificity within cryo-ET tomograms, we establish a broadly enabling tool that significantly expands the proteome available to electron microscopy.

Direct Mapping of Polyclonal Epitopes in Serum by HDX-MS.

Elucidating the interactions that drive antigen recognition is central to understanding antibody-mediated protection and is vital for the rational design of immunogens. Often, structural knowledge of epitopes targeted by antibodies is derived from isolated studies of monoclonal antibodies, for which numerous structural techniques exist. In contrast, there are very few approaches capable of mapping the full scope of antigen surfaces targeted by polyclonal sera through the course of a natural antibody response. Here, we develop an approach using immobilized antigen coupled to hydrogen/deuterium exchange with mass spectrometry (HDX-MS) to probe epitope targeting in the context of the fully native serum environment. Using the well-characterized Staphylococcal enterotoxin B (SEB) as a model system, we show that complex combinations of epitopes can be detected and subtle differences across different antisera can be discerned. This work reveals new insight into how neutralizing antibodies and antisera target SEB and, more importantly, establishes a novel method for directly mapping the epitope landscape of polyclonal sera.