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At least 505 records · Page 28

Exploring the Structural, Biochemical, and Functional Diversity of Glycoside Hydrolase Family 12 from Penicillium subrubescens

Glycoside hydrolases (GHs) play an essential role in plant biomass degradation and modification for the sustainable production of biochemicals. The filamentous Ascomycete fungus Penicillium subrubescens contains a higher number of GH12 candidates compared to related species. Therefore, we aimed to compare P. subrubescens GH12s for their ability and substrate specificity for plant cell wall polysaccharide degradation and species’ potential as a source of novel enzymes for plant biomass valorization. Our re-evaluated phylogenetic analysis of fungal GH12 members showed that the P. subrubescens GH12s were located in different (new) clades. Biochemical characterization marked PsEglA as an endoglucanase and four other P. subrubescens GH12s (i.e., PsXegA–D) as xyloglucanases. Interestingly, structural features of PsXegD and PsXegE were more comparable to those of Basidiomycete GH12 xyloglucanases with a unique open substrate-binding cleft. PsUegA displayed dual xyloglucanase and endoglucanase activity and also showed distinct structural features. Comparative transcriptome analysis supported the functional diversity of P. subrubescens GH12s in plant biomass degradation. The gene encoding PsUegA was expressed under diverse conditions, suggesting a scouting role for this enzyme.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Towards sustainable production and utilization of plant-biomass-based nanomaterials: a review and analysis of recent developments

Abstract Plant-biomass-based nanomaterials have attracted great interest recently for their potential to replace petroleum-sourced polymeric materials for sustained economic development. However, challenges associated with sustainable production of lignocellulosic nanoscale polymeric materials (NPMs) need to be addressed. Producing materials from lignocellulosic biomass is a value-added proposition compared with fuel-centric approach. This report focuses on recent progress made in understanding NPMs—specifically lignin nanoparticles (LNPs) and cellulosic nanomaterials (CNMs)—and their sustainable production. Special attention is focused on understanding key issues in nano-level deconstruction of cell walls and utilization of key properties of the resultant NPMs to allow flexibility in production to promote sustainability. Specifically, suitable processes for producing LNPs and their potential for scaled-up production, along with the resultant LNP properties and prospective applications, are discussed. In the case of CNMs, terminologies such as cellulose nanocrystals (CNCs) and cellulose nanofibrils (CNFs) used in the literature are examined. The term cellulose nano-whiskers (CNWs) is used here to describe a class of CNMs that has a morphology similar to CNCs but without specifying its crystallinity, because most applications of CNCs do not need its crystalline characteristic. Additionally, progress in enzymatic processing and drying of NPMs is also summarized. Finally, the report provides some perspective of future research that is likely to result in commercialization of plant-based NPMs.

09 BIOMASS FUELS↗

Carbohydrate-aromatic interface and molecular architecture of lignocellulose

Abstract Plant cell walls constitute the majority of lignocellulosic biomass and serve as a renewable resource of biomaterials and biofuel. Extensive interactions between polysaccharides and the aromatic polymer lignin make lignocellulose recalcitrant to enzymatic hydrolysis, but this polymer network remains poorly understood. Here we interrogate the nanoscale assembly of lignocellulosic components in plant stems using solid-state nuclear magnetic resonance and dynamic nuclear polarization approaches. We show that the extent of glycan-aromatic association increases sequentially across grasses, hardwoods, and softwoods. Lignin principally packs with the xylan in a non-flat conformation via non-covalent interactions and partially binds the junction of flat-ribbon xylan and cellulose surface as a secondary site. All molecules are homogeneously mixed in softwoods; this unique feature enables water retention even around the hydrophobic aromatics. These findings unveil the principles of polymer interactions underlying the heterogeneous architecture of lignocellulose, which may guide the rational design of more digestible plants and more efficient biomass-conversion pathways.

59 BASIC BIOLOGICAL SCIENCES↗

Mixed-Linkage Glucan Is the Main Carbohydrate Source and Starch Is an Alternative Source during Brachypodium Grain Germination

Seeds of the model grass Brachypodium distachyon are unusual because they contain very little starch and high levels of mixed-linkage glucan (MLG) accumulated in thick cell walls. It was suggested that MLG might supplement starch as a storage carbohydrate and may be mobilised during germination. In this work, we observed massive degradation of MLG during germination in both endosperm and nucellar epidermis. The enzymes responsible for the MLG degradation were identified in germinated grains and characterized using heterologous expression. By using mutants targeting MLG biosynthesis genes, we showed that the expression level of genes coding for MLG and starch-degrading enzymes was modified in the germinated grains of knocked-out cslf6 mutants depleted in MLG but with higher starch content. Our results suggest a substrate-dependent regulation of the storage sugars during germination. These overall results demonstrated the function of MLG as the main carbohydrate source during germination of Brachypodium grain. More astonishingly, cslf6 Brachypodium mutants are able to adapt their metabolism to the lack of MLG by modifying the energy source for germination and the expression of genes dedicated for its use.

59 BASIC BIOLOGICAL SCIENCES↗

Chemo-Enzymatic Synthesis of Long-Chain Oligosaccharides for Studying Xylan-Modifying Enzymes

Plant research is hampered in several aspects by a lack of pure oligosaccharide samples that closely represent structural features of cell wall glycans. An alternative to purely chemical synthesis to access these oligosaccharides is chemo-enzymatic synthesis using glycosynthases. These enzymes enable the ligation of oligosaccharide donors, when activated for example as α-glycosyl fluorides, with suitable acceptor oligosaccharides. Herein, the synthesis of xylan oligosaccharides up to dodecasaccharides is reported, with glycosynthase-mediated coupling reactions as key steps. The xylo-oligosaccharide donors were protected at the non-reducing end with a 4-O-tetrahydropyranyl (THP) group to prevent polymerization. Installation of an unnatural 3-O-methylether substituent at the reducing end xylose of the oligosaccharides ensured good water solubility. Biochemical assays demonstrated enzymatic activity for the xylan acetyltransferase XOAT1 from Arabidopsis thaliana, xylan arabinofuranosyl-transferase XAT3 enzymes from rice and switchgrass, and the xylan glucuronosyltransferase GUX3 from Arabidopsis thaliana. In case of the glucuronosyltransferase GUX3, MALDI-MS/MS analysis of the reaction product suggested that a single glucuronosyl substituent was installed primarily at the central xylose residues of the dodecasaccharide acceptor, demonstrating the value of long-chain acceptors for assaying biosynthetic glycosyltransferases.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

SPRUCE Wood Anatomy of Picea mariana and Larix laricina in SPRUCE Experimental Plots, Marcell Experimental Forest, Minnesota, July 2023

Branch samples were collected in July 2023 to measure wood anatomical traits on two dominant conifer species, Picea mariana and Larix laricina, in a bog forest at the SPRUCE (Spruce and Peatland Responses Under Changing Environments) experiment in northern Minnesota. Anatomical measurements were made on the annual rings of those branches with dates ranging from 2011-2023. Wood anatomical measurements include annual tracheid diameter, tracheid density, cell wall thickness, thickness-to-span-ratio, and conduit lumen fraction in both earlywood and latewood. Wood anatomical samples were prepared using a portable sliding microtome (G.S.L.-1 lightweight microtome, WSL) and a light microscope (Leica DM2500). This dataset spans 2011–2023, with full branch-level coverage for 2020–2023, whereas some thinner branches formed only in recent years and therefore do not contain rings from earlier years. By providing annual, treatment-specific anatomical measurements, this dataset can help quantify structural acclimation to global change, clarify links among phenology, wood formation, and hydraulic traits, and improve predictions of forest growth response under future climates. This dataset contains one data file in comma separate (.csv) format. Additional metadata are provided: one data dictionary and a file-level metadata file in comma separate (.csv) format and a user guide in PDF (*.pdf) format.

annual tracheid diameter↗

Engineering the green algae Chlamydomonas incerta for recombinant protein production

Chlamydomonas incerta , a genetically close relative of the model green alga Chlamydomonas reinhardtii , shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii , this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta ’s capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii , C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii . For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii , as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta ’s potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.

cell membranes↗

Genetic Modification of KNAT7 Transcription Factor Expression Enhances Saccharification and Reduces Recalcitrance of Woody Biomass in Poplars

The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 ( PtKNAT7 ) overexpression ( PtKNAT7 -OE) and antisense suppression ( PtKNAT7 -AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium -mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 ( AtKNAT7-OE ) in transgenic poplars. In all these constructs, the expression of KNAT7 transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three KNAT7 knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of KNAT7 expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.

59 BASIC BIOLOGICAL SCIENCES↗

Exploring Plant Cis –Regulatory Elements at Single–Cell Resolution: Overcoming Biological and Computational Challenges to Advance Plant Research

Cis-regulatory elements (CREs) are important sequences for gene expression and for plant biological processes such as development, evolution, domestication, and stress response. However, studying CREs in plant genomes has been challenging. The totipotent nature of plant cells, coupled with inability to maintain plant cell types in culture and the inherent technical challenges posed by the cell wall have limited our understanding of how plant cell types acquire and maintain their identities and respond to the environment via CRE usage. Furthermore, advances in single cell epigenomics have revolutionized the field identifying cell-type-specific CREs. These new technologies have the potential to significantly advance our understanding of plant CRE biology, and shed light on how the regulatory genome gives rise to diverse plant phenomena. However, there are significant biological and computational challenges associated with analyzing single cell epigenomic datasets. In this review, we discuss the historical and foundational underpinnings of plant single-cell research, challenges and common pitfalls in analysis of plant single-cell epigenomic data, and highlight biological challenges unique to plants. Additionally, we discuss how the application of single-cell epigenomic data in various contexts stands to transform our understanding of the importance of CREs in plant genomes.

59 BASIC BIOLOGICAL SCIENCES↗

Tip-growing cells of the moss Ceratodon purpureus Are gravitropic in high-density media

Gravity sensing in plants and algae is hypothesized to rely upon either the mass of the entire cell or that of sedimenting organelles (statoliths). Protonemata of the moss Ceratodon purpureus show upward gravitropism and contain amyloplasts that sediment. If moss sensing were whole-cell based, then media denser than the cell should prevent gravitropism or reverse its direction. Cells that were inverted or reoriented to the horizontal displayed distinct negative gravitropism in solutions of iodixanol with densities of 1.052 to 1.320 as well as in bovine serum albumin solutions with densities of 1.037 to 1.184 g cm(-3). Studies using tagged molecules of different sizes and calculations of diffusion times suggest that both types of media penetrate through the apical cell wall. Estimates of the density of the apical cell range from 1.004 to 1.085. Because protonemata grow upward when the cells have a density that is lower than the surrounding medium, gravitropic sensing probably utilizes an intracellular mass in moss protonemata. These data provide additional support for the idea that sedimenting amyloplasts function as statoliths in gravitropism.

NASA Discipline Plant Biology↗

Gene Expression Measurement Module (GEMM) for space application: Design and validation

In order to facilitate studies on the impact of the space environment on biological systems, we have developed a prototype of GEMM (Gene Expression Measurement Module) - an automated, miniaturized, integrated fluidic system for in-situ measurements of gene expression in microbial samples. The GEMM instrument is capable of (1) lysing bacterial cell walls, (2) extracting and purifying RNA released from cells, (3) hybridizing the RNA to probes attached to a microarray and (4) providing electrochemical readout, all in a microfluidics cartridge. To function on small, uncrewed spacecraft, the conventional, laboratory protocols for both sample preparation and hybridization required significant modifications. Biological validation of the instrument was carried out on Synechococcus elongatus, a photosynthetic cyanobacterium known for its metabolic diversity and resilience to adverse conditions. It was demonstrated that GEMM yielded reliable, reproducible gene expression profiles. GEMM is the only high throughput instrument that can be deployed in near future on space platforms other than the ISS to advance biological research in space. It can also prove useful for numerous terrestrial applications in the field.

Kianoosh Peyvan↗

Cell-Free Gene Expression: Methods and Applications

Cell-free gene expression (CFE) systems empower synthetic biologists to build biological molecules and processes outside of living intact cells. The foundational principle is that precise, complex biomolecular transformations can be conducted in purified enzyme or crude cell lysate systems. This concept circumvents mechanisms that have evolved to facilitate species survival, bypasses limitations on molecular transport across the cell wall, and provides a significant departure from traditional, cell-based processes that rely on microscopic cellular “reactors.” In addition, cell-free systems are inherently distributable through freeze-drying, which allows simple distribution before rehydration at the point-of-use. Furthermore, as cell-free systems are nonliving, they provide built-in safeguards for biocontainment without the constraints attendant on genetically modified organisms. These features have led to a significant increase in the development and use of CFE systems over the past two decades. Here, we discuss recent advances in CFE systems and highlight how they are transforming efforts to build cells, control genetic networks, and manufacture biobased products.

59 BASIC BIOLOGICAL SCIENCES↗

Plant Single-Cell Solutions for Energy and the Environment (Second Workshop Report)

Plants are important sources of energy and materials, and they collectively represent a critical component of Earth’s ecosystem. With increasing environmental stresses due to climate change and intensive agricultural practices, the need for resilient plants is greater than ever before. To secure plant resources for bioenergy, biomaterials, food, and ecosystem adaptation, a deeper understanding of the fundamental biology of plants at a cellular level is urgently needed. Plants contain a multitude of specialized cell types that compose tissues and organs. Pathogens often target specific cell types within plants, and the response of one cell to a particular stimulus is likely to be distinct from its neighbor because of underlying molecular and contextual differences. Understanding how these responses are distributed among cells, the main goal of single-cell approaches, will substantially enhance our ability to use targeted engineering for improving plant productivity and resilience. Furthermore, single-cell approaches are necessary to understand the interactions between plants and other ecosystem members such as fungi, bacteria, and archaea. Unlocking these gene-response mechanisms at a cellular level can improve our ability to adapt plants to environmental stresses, increasing their utility as feedstocks for biomaterials and bioenergy. Recent advances in high-throughput sequencing, mass spectrometry, microfluidics and miniaturization, artificial intelligence and machine learning, and bioinformatics have greatly improved our ability to detect and understand processes at a cellular level. In mammalian systems, single-cell transcriptomics has already led to many advances, such as newly identified cell types and cell-targeted treatment of diseases, and mass spectrometry-based single-cell proteomics has recently been demonstrated as a promising emerging technology. However, plant single-cell omics has lagged behind mammalian approaches due to the high cost of the technologies relative to available resources and to the innate biological features of plants, including the complexity of the cell wall and polyploidy. To better understand how single-cell methods could enable plant science, Lawrence Berkeley National Laboratory (Berkeley Lab) hosted a workshop on April 29, 2021, that brought together a diverse group of leaders in plant and/or single-cell biology. Attendees represented federal research programs and domestic and international academic institutions. During the workshop, three presenters described the current state of research in both experimental and computational approaches. While the focus of the workshop was on factors preventing plant biology researchers from fully adopting single-cell methodologies, workshop participants agreed that most barriers could be overcome with focused, strategic investment and coordinated efforts among institutions leading to significant scientific discoveries that would be difficult to obtain using more conventional technologies.

59 BASIC BIOLOGICAL SCIENCES↗

Distribution of calmodulin in corn seedlings - Immunocytochemical localization in coleoptiles and root apices

Immunofluorescence techniques have been used to study the distribution of calmodulin in several tissues in etiolated corn (Zea mays, var. Bear Hybrid) seedlings. Uniform staining was seen in the background cytoplasm of most cell types. Cell walls and vacuoles were not stained. In coleoptile mesophyll cells the nucleoplasm of most nuclei was stained as was the stroma of most amyloplasts. The lumen border of mature tracheary elements in coleoptiles also stained. In the rootcap the most intensely stained regions were the cytoplasms of columella cells and of the outermost cells enmeshed in the layer of secreted slime. Nuclei in the rootcap cells did not stain distinctly, but those in all cell types of the root meristem did. Also in the root meristem, the cytoplasm of metaxylem elements stained brightly. These results are compared and contrasted with previous data on the localization of calmodulin in pea root apices and epicotyls and discussed in relation to current hypotheses on mechanisms of gravitropism.

Dauwalder, M.↗

Elevated levels of N-lauroylethanolamine, an endogenous constituent of desiccated seeds, disrupt normal root development in Arabidopsis thaliana seedlings

N-Acylethanolamines (NAEs) are prevalent in desiccated seeds of various plant species, and their levels decline substantially during seed imbibition and germination. Here, seeds of Arabidopsis thaliana (L.) Heynh. were germinated in, and seedlings maintained on, micromolar concentrations of N-lauroylethanolamine (NAE 12:0). NAE 12:0 inhibited root elongation, increased radial swelling of root tips, and reduced root hair numbers in a highly selective and concentration-dependent manner. These effects were reversible when seedlings were transferred to NAE-free medium. Older seedlings (14 days old) acclimated to exogenous NAE by increased formation of lateral roots, and generally, these lateral roots did not exhibit the severe symptoms observed in primary roots. Cells of NAE-treated primary roots were swollen and irregular in shape, and in many cases showed evidence, at the light- and electron-microscope levels, of improper cell wall formation. Microtubule arrangement was disrupted in severely distorted cells close to the root tip, and endoplasmic reticulum (ER)-localized green fluorescent protein (mGFP5-ER) was more abundant, aggregated and distributed differently in NAE-treated root cells, suggesting disruption of proper cell division, endomembrane organization and vesicle trafficking. These results suggest that NAE 12:0 likely influences normal cell expansion in roots by interfering with intracellular membrane trafficking to and/or from the cell surface. The rapid metabolism of NAEs during seed imbibition/germination may be a mechanism to remove this endogenous class of lipid mediators to allow for synchronized membrane reorganization associated with cell expansion.

Non-NASA Center↗

Thermal Expansion of Polyurethane Foam

Closed cell foams are often used for thermal insulation. In the case of the Space Shuttle, the External Tank uses several thermal protection systems to maintain the temperature of the cryogenic fuels. A few of these systems are polyurethane, closed cell foams. In an attempt to better understand the foam behavior on the tank, we are in the process of developing and improving thermal-mechanical models for the foams. These models will start at the microstructural level and progress to the overall structural behavior of the foams on the tank. One of the key properties for model characterization and verification is thermal expansion. Since the foam is not a material, but a structure, the modeling of the expansion is complex. It is also exacerbated by the anisoptropy of the material. During the spraying and foaming process, the cells become elongated in the rise direction and this imparts different properties in the rise direction than in the transverse directions. Our approach is to treat the foam as a two part structure consisting of the polymeric cell structure and the gas inside the cells. The polymeric skeleton has a thermal expansion of its own which is derived from the basic polymer chemistry. However, a major contributor to the thermal expansion is the volume change associated with the gas inside of the closed cells. As this gas expands it exerts pressure on the cell walls and changes the shape and size of the cells. The amount that this occurs depends on the elastic and viscoplastic properties of the polymer skeleton. The more compliant the polymeric skeleton, the more influence the gas pressure has on the expansion. An additional influence on the expansion process is that the polymeric skeleton begins to breakdown at elevated temperatures and releases additional gas species into the cell interiors, adding to the gas pressure. The fact that this is such a complex process makes thermal expansion ideal for testing the models. This report focuses on the thermal expansion tests and the response of the microstructure. A novel optical method is described which is appropriate for measuring thermal expansion at high temperatures without influencing the thermal expansion measurement. Detailed microstructural investigations will also be described which show cell expansion as a function of temperature. Finally, a phenomenological model on thermal expansion will be described.

Lerch, Bradley A.↗

A systems genetic analysis identifies putative mechanisms and candidate genes regulating vessel traits in poplar wood

Wood is the water conducting tissue of tree stems. Like most angiosperm trees, poplar wood contains water-conducting vessel elements whose functional properties affect water transport and growth rates, as well as susceptibility to embolism and hydraulic failure during water stress and drought. Here we used a unique hybrid poplar pedigree carrying genomically characterized chromosomal insertions and deletions to undertake a systems genomics analysis of vessel traits. We assayed gene expression in wood forming tissues from clonal replicates of genotypes covering dosage quantitative trait loci with insertions and deletions, genotypes with extreme vessel trait phenotypes, and control genotypes. A gene co-expression analysis was used to assign genes to modules, which were then used in integrative analyses to identify modules associated with traits, to identify putative molecular and cellular processes associated with each module, and finally to identify candidate genes using multiple criteria including dosage responsiveness. These analyses identified known processes associated with vessel traits including stress response, abscisic acid and cell wall biosynthesis, and in addition identified previously unexplored processes including cell cycle and protein ubiquitination. We discuss our findings relative to component processes contributing to vessel trait variation including signaling, cell cycle, cell expansion, and cell differentiation.

59 BASIC BIOLOGICAL SCIENCES↗

Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells

Mycobacterium tuberculosis (Mtb) infection elicits both protein and lipid antigen-specific T cell responses. However, the incorporation of lipid antigens into subunit vaccine strategies and formulations has been underexplored, and the characteristics of vaccine-induced Mtb lipid-specific memory T cells have remained elusive. Mycolic acid (MA), a major lipid component of the Mtb cell wall, is presented by human CD1b molecules to unconventional T cell subsets. These MA-specific CD1b-restricted T cells have been detected in the blood and disease sites of Mtb-infected individuals, suggesting that MA is a promising lipid antigen for incorporation into multicomponent subunit vaccines. In this study, we utilized the enhanced stability of bicontinuous nanospheres (BCN) to efficiently encapsulate MA for in vivo delivery to MA-specific T cells, both alone and in combination with an immunodominant Mtb protein antigen (Ag85B). Pulmonary administration of MA-loaded BCN (MA-BCN) elicited MA-specific T cell responses in humanized CD1 transgenic mice. Simultaneous delivery of MA and Ag85B within BCN activated both MA- and Ag85B-specific T cells. Notably, pulmonary vaccination with MA-Ag85B-BCN resulted in the persistence of MA, but not Ag85B, within alveolar macrophages in the lung. Vaccination of MA-BCN through intravenous or subcutaneous route, or with attenuated Mtb likewise reproduced MA persistence. Moreover, MA-specific T cells in MA-BCN-vaccinated mice differentiated into a T follicular helper-like phenotype. Overall, the BCN platform allows for the dual encapsulation and in vivo activation of lipid and protein antigen-specific T cells and leads to persistent lipid depots that could offer long-lasting immune responses.

59 BASIC BIOLOGICAL SCIENCES↗