Biowelding 3D-Printed Biodigital Brick of Seashell-Based Biocomposite by Pleurotus ostreatus Mycelium.

Mycelium biocomposites are eco-friendly, cheap, easy to produce, and have competitive mechanical properties. However, their integration in the built environment as durable and long-lasting materials is not solved yet. Similarly, biocomposites from recycled food waste such as seashells have been gaining increasing interest recently, thanks to their sustainable impact and richness in calcium carbonate and chitin. The current study tests the mycelium binding effect to bioweld a seashell biocomposite 3D-printed brick. The novelty of this study is the combination of mycelium and a non-agro-based substrate, which is seashells. As well as testing the binding capacity of mycelium in welding the lattice curvilinear form of the V3 linear Brick model (V3-LBM). Thus, the V3-LBM is 3D printed in three separate profiles, each composed of five layers of 1 mm/layer thickness, using seashell biocomposite by paste extrusion and testing it for biowelding with Pleurotus ostreatus mycelium to offer a sustainable, ecofriendly, biomineralized brick. The biowelding process investigated the penetration and binding capacity of the mycelium between every two 3D-printed profiles. A cellulose-based culture medium was used to catalyse the mycelium growth. The mycelium biowelding capacity was investigated by SEM microscopy and EDX chemical analysis of three samples from the side corner (S), middle (M), and lateral (L) zones of the biowelded brick. The results revealed that the best biowelding effect was recorded at the corner and lateral zones of the brick. The SEM images exhibited the penetration and the bridging effect achieved by the dense mycelium. The EDX revealed the high concentrations of carbon, oxygen, and calcium at all the analyzed points on the SEM images from all three samples. An inverted relationship between carbon and oxygen as well as sodium and potassium concentrations were also detected, implying the active metabolic interaction between the fungal hyphae and the seashell-based biocomposite. Finally, the results of the SEM-EDX analysis were applied to design favorable tessellation and staking methods for the V3-LBM from the seashell-mycelium composite to deliver enhanced biowelding effect along the Z axis and the XY axis with <1 mm tessellation and staking tolerance.

Lipid nanoparticle delivery limits antisense oligonucleotide activity and cellular distribution in the brain after intracerebroventricular injection.

Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery in vivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs.

The New Standard Is Biodigital: Durable and Elastic 3D-Printed Biodigital Clay Bricks.

In a previously published study, the authors explained the formal design efficiency of the 3D-printed biodigital clay bricks 3DPBDCB: a project that aimed to change the conventional methods of clay brick design and mass production. This was achieved by employing the behavioural algorithms of reaction-diffusion and the shortest path that were extracted from the exact material physical properties and hydrophilic behaviours of clay and controlled material deposition 3D printing to create sustainable clay bricks. Sustainability in their use in the built environment and their production processes, with full potential sustainability aspects such as passive cooling, thermal and acoustical insulation, and bio receptivity. The current work studies the mechanical properties of the 3D-printed biodigital clay bricks as elastic and durable clay bricks whose properties depend mainly on their geometrical composition and form. Each of the three families of the 3D-printed biodigital clay bricks (V1, V2, V3) includes the linear model of a double line of 0.5 cm thickness and a bulk model of 55% density were tested for compression and elasticity and compared to models of standard industrial clay bricks. The results revealed that the best elasticity pre-cracking was achieved by the V2 linear model, followed by the V3 linear model, which also achieved the highest post-cracking elasticity-enduring until 150 N pre-cracking and 200 N post-cracking, which makes the V3 linear model eligible for potential application in earthquake-resistant buildings. While the same model V3-linear achieved the second-best compressive strength enduring until 170 N. The best compressive strength was recorded by the V1 linear and bulk model enduring up to 240 N without collapsing, exceeding the strength and resistance of the industrial clay bricks with both models, where the bulk and the perforated collapsed at 200 N and 140 N, respectively. Thus, the mass production and integration of the V1 bulk and linear model and the V3 linear model are recommended for the construction industry and the architectural built environment for their multi-aspect sustainability and enhanced mechanical properties.

Allosteric inhibition of HTRA1 activity by a conformational lock mechanism to treat age-related macular degeneration.

The trimeric serine protease HTRA1 is a genetic risk factor associated with geographic atrophy (GA), a currently untreatable form of age-related macular degeneration. Here, we describe the allosteric inhibition mechanism of HTRA1 by a clinical Fab fragment, currently being evaluated for GA treatment. Using cryo-EM, X-ray crystallography and biochemical assays we identify the exposed LoopA of HTRA1 as the sole Fab epitope, which is approximately 30 Å away from the active site. The cryo-EM structure of the HTRA1:Fab complex in combination with molecular dynamics simulations revealed that Fab binding to LoopA locks HTRA1 in a non-competent conformational state, incapable of supporting catalysis. Moreover, grafting the HTRA1-LoopA epitope onto HTRA2 and HTRA3 transferred the allosteric inhibition mechanism. This suggests a conserved conformational lock mechanism across the HTRA family and a critical role of LoopA for catalysis, which was supported by the reduced activity of HTRA1-3 upon LoopA deletion or perturbation. This study reveals the long-range inhibition mechanism of the clinical Fab and identifies an essential function of the exposed LoopA for activity of HTRA family proteases.

A Systematic Approach for Liposome and Lipodisk Preclinical Formulation Development by Microfluidic Technology.

Lipid nanoparticles have transformed the drug delivery field enhancing the therapeutic drug performance of small molecules and biologics with several approved drug products. However, in industry, these more complex drug delivery systems such as liposomes require more material and time to develop. Here, we report a liposome and lipodisk decision tree with model compounds of diverse physicochemical properties to understand how to resourcefully optimize encapsulation efficiency (EE) for these lipid-based drug delivery systems. We have identified trends with physicochemical properties such as Log P, where higher Log P compounds such as curcumin were able to efficiently load into the lipid bilayer resulting in high EE with altering the drug/lipid (D/L) ratio. Moderate Log P compounds such as cyclosporine A and dexamethasone had significantly higher encapsulation in lipodisks, which contain higher amounts of PEG lipid compared to liposomes. The EE of negative Log P compounds, like acyclovir, remained low regardless of altering the D/L ratio and PEG concentrations. In this study, microfluidic techniques were employed to fabricate liposomes and lipodisks formulations allowing for a reproducible strategy for formulation development. Both liposome and lipodisk of curcumin demonstrated enhanced in vivo performance compared with a conventional formulation in the rat pharmacokinetic study. This combination of approaches with multiple model compounds and lipid-based drug delivery systems provides a systematic guidance to effective strategies to generate higher EE with minimal drug waste and expedite the process for preclinical development when applied to industry compounds.

3D-Printed Biodigital Clay Bricks.

Construction materials and techniques have witnessed major advancements due to the application of digital tools in the design and fabrication processes, leading to a wide array of possibilities, especially in additive digital manufacturing tools and 3D printing techniques, scales, and materials. However, possibilities carry responsibilities with them and raise the question of the sustainability of 3D printing applications in the built environment in terms of material consumption and construction processes: how should one use digital design and 3D printing to achieve minimum material use, minimum production processes, and optimized application in the built environment? In this work, we propose an optimized formal design of "Biodigital Barcelona Clay Bricks" to achieve sustainability in the use of materials. These were achieved by using a bottom-up methodology of biolearning to extract the formal grammar of the bricks that is suitable for their various applications in the built environment as building units, thereby realizing the concept of formal physiology, as well as employing the concept of fractality or pixilation by using 3D printing to create the bricks as building units on an architectural scale. This enables the adoption of this method as an alternative construction procedure instead of conventional clay brick and full-scale 3D printing of architecture on a wider and more democratic scale, avoiding the high costs of 3D printing machines and lengthy processes of the one-step, 3D-printed, full-scale architecture, while also guaranteeing minimum material consumption and maximum forma-function coherency. The "Biodigital Barcelona Clay Bricks" were developed using Rhinoceros 3D and Grasshopper 3D + Plugins (Anemone and Kangaroo) and were 3D printed in clay.

Impact of surfactant selection and incorporation on in situ nanoparticle formation from amorphous solid dispersions.

Spray dried amorphous solid dispersions (ASDs) stand as one of the most effective formulation strategies to address issues of low aqueous solubility when developing new chemical entities.An emerging research topic focusing on the formation of amorphous nanoparticles or nanodroplets from ASD formulations has attracted attention recently. These ASD nanoparticlescan be highly beneficial and able to further increase oral bioavailability. The incorporation of surfactants in ASD formulations has been shown to facilitate the formation of these nanoparticles. Therefore, understanding the mechanism of surfactant-promoted nanoparticle formation becomes critical for the rational design of ASD formulations. This work demonstrated the importance of inclusion of the surfactant within the ASD composition for nanoparticle formation. In contrast, when a surfactant is added externally (e.g., by inclusion in the dosing vehicle), only a limited degree of nanoparticle formation was observed even at the optimized surfactant-to-drug ratios. A variety of different surfactants were also assessed for understanding their impact on ASD nanoparticle formation. The spray drying systems containing nonionic surfactants, Tween 80 and Vitamin E TPGS, produced higher amounts of in situ ASD nanoparticles when compared to an anionic surfactant, sodium lauryl sulfate (SLS). The ASD nanoparticles produced by the Genentech developmental compound, GDC-0334, were highly stable and retained their original particle size and amorphous feature for at least 18 h under biorelevant conditions. The high degree of nanoparticle formation from spray dried GDC-0334 containing Tween 80 combined with the superior physical stability of the nanoparticles also translated to enhanced in vivo performance in a rat pharmacokinetics study.

Imaging Reveals Importance of Shape and Flexibility for Glomerular Filtration of Biologics.

Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for cancer immunotherapeutic drug discovery while also revealing limitations in knowledge of structure-activity relationships. The current understanding of renal filtration originates largely from data reported for dextrans, IgG, albumin, and selected globular proteins. For a one-armed IgG-based T-cell imaging agent, we observed higher renal signal than typically observed for bivalent IgGs, prompting us to explore the factors governing renal filtration of biologics. We constructed a small representative library of IgG-like formats with varied shapes and hinge flexibilities falling broadly into two categories: branched molecules including bivalent IgG and (scFv)2Fc, and nonbranched molecules including one-armed IgG, one-armed IgG with stacked Fab, and one-armed IgG with a rigid IgA2 hinge. Transmission electron microscopy revealed Y-shaped structures for the branched molecules and pseudo-linear structures for the nonbranched molecules. Single-photon emission CT imaging, autoradiography, and tissue harvest studies demonstrated higher renal uptake and catabolism for nonbranched molecules relative to branched molecules. Among the nonbranched molecules, the one-armed IgG with rigid IgA2 hinge molecule demonstrated higher kidney uptake and decreased systemic exposure relative to molecules with a more flexible hinge. Our results show that differences in shape and hinge flexibility drive the increased glomerular filtration of one-armed relative to bivalent antibodies and highlight the practical advantages of using imaging to assess renal filtration properties. These findings are particularly relevant for T-cell-dependent bispecific molecules, many of which have nonstandard antibody structures.

Discovery of a dual pathway aggregation mechanism for a therapeutic constrained peptide.

Constrained peptides (CPs) have emerged as attractive candidates for drug discovery and development. To fully unlock the therapeutic potential of CPs, it is crucial to understand their physical stability and minimize the formation of aggregates that could induce immune responses. Although amyloid like aggregates have been researched extensively, few studies have focused on aggregates from other peptide scaffolds (e.g., CPs). In this work, a streamlined approach to effectively profile the nature and formation pathway of CP aggregates was demonstrated. Aggregates of various sizes were detected and shown to be amorphous. Though no major changes were found in peptide structure upon aggregation, these aggregates appeared to have mixed natures, consisting of primarily non-covalent aggregates with a low level of covalent species. This co-existence phenomenon was also supported by two kinetic pathways observed in time- and temperature-dependent aggregation studies. Furthermore, a stability study with 8 additional peptide variants exhibited good correlation between aggregation propensity and peptide hydrophobicity. Therefore, a dual aggregation pathway was proposed, with the non-covalent aggregates driven by hydrophobic interactions, whereas the covalent ones formed through disulfide scrambling. Overall, the workflow presented here provides a powerful strategy for comprehensive characterization of peptide aggregates and understanding their mechanisms of formation.

A TRPA1 inhibitor suppresses neurogenic inflammation and airway contraction for asthma treatment.

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.

Automated high-throughput preparation and characterization of oligonucleotide-loaded lipid nanoparticles.

Lipid nanoparticles (LNPs) are increasingly employed to improve delivery efficiency and therapeutic efficacy of nucleic acids. Various formulation parameters can affect the quality attributes of these nanoparticle formulations, but currently there is a lack of systemic screening approaches to address this challenge. Here, we developed an automated high-throughput screening (HTS) workflow for streamline preparation and analytical characterization of LNPs loaded with antisense oligonucleotides (ASOs) in a full 96-well plate within 3 hrs. ASO-loaded LNPs were formulated by an automated solvent-injection method using a robotic liquid handler, and assessed for particle size distribution, encapsulation efficiency, and stability with different formulation compositions and ASO loadings. Results indicated that the PEGylated lipid content significantly affected the particle size distribution, while the ionizable lipid / ASO charge ratio impacted the encapsulation efficiency of ASOs. Furthermore, results from our HTS approach correlated with those from the state-of-the-art scale-up method using a microfluidic formulator, therefore opening up a new avenue for robust formulation development and design of experiment methods, while reducing material usage by 10 folds, improving analytical outputs and accumulation of information by 100 folds.

Bivalent antibody pliers inhibit ß-tryptase by an allosteric mechanism dependent on the IgG hinge.

Human ß-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the ß-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits ß-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive ß-tryptase monomers, and may provide an alternative strategy for antibody engineering.

Targeted IgMs agonize ocular targets with extended vitreal exposure.

Treatment of ocular disease is hindered by the presence of the blood-retinal barrier, which restricts access of systemic drugs to the eye. Intravitreal injections bypass this barrier, delivering high concentrations of drug to the targeted tissue. However, the recommended dosing interval for approved biologics is typically 6-12 weeks, and frequent travel to the physician's office poses a substantial burden for elderly patients with poor vision. Real-world data suggest that many patients are under-treated. Here, we investigate IgMs as a novel platform for treating ocular disease. We show that IgMs are well-suited to ocular administration due to moderate viscosity, long ocular exposure, and rapid systemic clearance. The complement-dependent cytotoxicity of IgMs can be readily removed with a P436G mutation, reducing safety liabilities. Furthermore, dodecavalent binding of IgM hexamers can potently activate pathways implicated in the treatment of progressive blindness, including the Tie2 receptor tyrosine kinase signaling pathway for the treatment of diabetic macular edema, or the death receptor 4 tumor necrosis family receptor pathway for the treatment of wet age-related macular degeneration. Collectively, these data demonstrate the promise of IgMs as therapeutic agonists for treating progressive blindness.

Antigen physiochemical properties allosterically effect the IgG Fc-region and Fc neonatal receptor affinity.

The neonatal Fc receptor (FcRn) is a key membrane protein that plays an integral role in serum immunoglobulin (IgG) recycling, which extends the half-life of antibody. In addition, FcRn is known to traffic antigen-bound immunoglobulins (Ag-IgGs), and to interact with immune complexes to facilitate the antigen cross-presentation of peptides derived from the immune complexes in antigen-presenting cells (APCs). Studies on the IgG-FcRn molecular interactions have primarily focused on the Fc region, and only recently have shown the potential impact of the antigen-binding fragment physiochemical properties on FcRn binding. However, the effect of the antigen physiochemical properties on IgG structure as it relates to Ag-IgG-FcRn binding is not well understood. Here we used an IgG-peptide antigen complex as a model system to investigate the structural effects of the antigen's physiochemical properties on the IgG structure, and the subsequent effects of Ag-IgG-FcRn interactions. We used hydroxyl radical footprinting-mass spectrometry to investigate the structural impact on an IgG upon antigen binding, and observed that the physicochemical properties of the antigen differentially induce conformational changes in the IgG FcRn binding region. The extent of these structural changes directly correlates to the magnitude of the affinity differences between the Ag-IgG complexes and FcRn. Moreover, the antigen's physicochemical properties differentially induce structural differences within the Ag-IgG-FcRn ternary complex. We also provide electron microscopy data that shows corroborating Fab-FcRn interactions, and confirms the hypothesis of potential 2:1 FcRn:IgG binding stoichiometry. These data demonstrate antigen-induced Fc structural rearrangements affect both the affinity toward FcRn and the trimeric antigen-IgG-FcRn complex, providing novel molecular insights in the first steps toward understanding interactions of FcRn-containing large(r)-sized immune complex.

Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab.

Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers.

Employing Laccase-Producing Aspergillus sydowii NYKA 510 as a Cathodic Biocatalyst in Self-Sufficient Lighting Microbial Fuel Cell.

In the present work, we isolated and identified Aspergillus sydowii NYKA 510 as the most potent laccase producer. Its medium constituents were optimized to produce the highest possible amount of laccase, which was after 7 days at 31°C and pH 5.2. Banana peel and peptone excelled in inducing laccase production at concentrations of 15.1 and 2.60 g/l, respectively. Addition of copper sulfate elevated enzyme yield to 145%. The fungus was employed in a microbial fuel cell (MFC). The best performance was obtained at 2000 Ω achieving 0.76 V, 380 mAm-2, 160 mWm-2, and 0.4 W. A project to design a self-sufficient lighting unit was implemented by employing a system of 2 sets of 4 MFCs each, connected in series, for electricity generation. A scanning electron microscopy image of A. sydowii NYKA 510 was utilized in algorithmic form generation equations for the design. The mixed patterning and patterned customized mass approach were developed by the authors and chosen for application in the design.

Production, characterization, and in vivo half-life extension of polymeric IgA molecules in mice.

IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance that is thought to be mediated by glycan receptor scavenging of recombinantly produced IgA monomer bearing incompletely sialylated N-linked glycans. Here, we present a comprehensive biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA: removal of all N-linked glycosylation sites creating an aglycosylated polymeric IgA and engineering in FcRn binding with the generation of a polymeric IgG-IgA Fc fusion. While previous reports and the results presented in this study indicate that glycan-mediated clearance plays a major role for monomeric IgA, systemic clearance of polymeric IgA in mice is predominantly controlled by mechanisms other than glycan receptor clearance, such as pIgR-mediated transcytosis. The developed IgA platform now provides the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure.

Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin.

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.

Picomolar zinc binding modulates formation of Bcl10-nucleating assemblies of the caspase recruitment domain (CARD) of CARD9.

The caspase recruitment domain-containing protein 9 (CARD9)-B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse pathogens. This signaling pathway requires a critical caspase recruitment domain (CARD)-CARD interaction between CARD9 and Bcl10 that promotes downstream activation of factors, including NF-κB and the mitogen-activated protein kinase (MAPK) p38. Despite these insights, CARD9 remains structurally uncharacterized, and little mechanistic understanding of its regulation exists. We unexpectedly found here that the CARD in CARD9 binds to Zn2+ with picomolar affinity-a concentration comparable with the levels of readily accessible Zn2+ in the cytosol. NMR solution structures of the CARD9-CARD in the apo and Zn2+-bound states revealed that Zn2+ has little effect on the ground-state structure of the CARD; yet the stability of the domain increased considerably upon Zn2+ binding, with a concomitant reduction in conformational flexibility. Moreover, Zn2+ binding inhibited polymerization of the CARD9-CARD into helical assemblies. Here, we also present a 20-Å resolution negative-stain EM (NS-EM) structure of these filamentous assemblies and show that they adopt a similar helical symmetry as reported previously for filaments of the Bcl10 CARD. Using both bulk assays and direct NS-EM visualization, we further show that the CARD9-CARD assemblies can directly template and thereby nucleate Bcl10 polymerization, a capacity considered critical to propagation of the CARD9-Bcl10 signaling cascade. Our findings indicate that CARD9 is a potential target of Zn2+-mediated signaling that affects Bcl10 polymerization in innate immune responses.