Scalable Processes for Culturing Meat Using Edible Scaffolds.

There is increasing consumer demand for alternative animal protein products that are delicious and sustainably produced to address concerns about the impacts of mass-produced meat on human and planetary health. Cultured meat has the potential to provide a source of nutritious dietary protein that both is palatable and has reduced environmental impact. However, strategies to support the production of cultured meats at the scale required for food consumption will be critical. In this review, we discuss the current challenges and opportunities of using edible scaffolds for scaling up the production of cultured meat. We provide an overview of different types of edible scaffolds, scaffold fabrication techniques, and common scaffold materials. Finally, we highlight potential advantages of using edible scaffolds to advance cultured meat production by accelerating cell growth and differentiation, providing structure to build complex 3D tissues, and enhancing the nutritional and sensory properties of cultured meat. Expected final online publication date for the Annual Review of Food Science and Technology, Volume 15 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Pre-existing humoral immunity and complement pathway contribute to immunogenicity of adeno-associated virus (AAV) vector in human blood.

AAV gene transfer is a promising treatment for many patients with life-threatening genetic diseases. However, host immune response to the vector poses a significant challenge for the durability and safety of AAV-mediated gene therapy. Here, we characterize the innate immune response to AAV in human whole blood. We identified neutrophils, monocyte-related dendritic cells, and monocytes as the most prevalent cell subsets able to internalize AAV particles, while conventional dendritic cells were the most activated in terms of the CD86 co-stimulatory molecule upregulation. Although low titers (≤1:10) of AAV neutralizing antibodies (NAb) in blood did not have profound effects on the innate immune response to AAV, higher NAb titers (≥1:100) significantly increased pro-inflammatory cytokine/chemokine secretion, vector uptake by antigen presenting cells (APCs) and complement activation. Interestingly, both full and empty viral particles were equally potent in inducing complement activation and cytokine secretion. By using a compstatin-based C3 and C3b inhibitor, APL-9, we demonstrated that complement pathway inhibition lowered CD86 levels on APCs, AAV uptake, and cytokine/chemokine secretion in response to AAV. Together these results suggest that the pre-existing humoral immunity to AAV may contribute to trigger adverse immune responses observed in AAV-based gene therapy, and that blockade of complement pathway may warrant further investigation as a potential strategy for decreasing immunogenicity of AAV-based therapeutics.

The impact of physical, biochemical, and electrical signaling on Schwann cell plasticity.

Peripheral nervous system (PNS) injuries are an ongoing health care concern. While autografts and allografts are regarded as the current clinical standard for traumatic injury, there are inherent limitations that suggest alternative remedies should be considered for therapeutic purposes. In recent years, nerve guidance conduits (NGCs) have become increasingly popular as surgical repair devices, with a multitude of various natural and synthetic biomaterials offering potential to enhance the design of conduits or supplant existing technologies entirely. From a cellular perspective, it has become increasingly evident that Schwann cells (SCs), the primary glia of the PNS, are a predominant factor mediating nerve regeneration. Thus, the development of severe nerve trauma therapies requires a deep understanding of how SCs interact with their environment, and how SC microenvironmental cues may be engineered to enhance regeneration. Here we review the most recent advancements in biomaterials development and cell stimulation strategies, with a specific focus on how the microenvironment influences the behavior of SCs and can potentially lead to functional repair. We focus on microenvironmental cues that modulate SC morphology, proliferation, migration, and differentiation to alternative phenotypes. Promotion of regenerative phenotypic responses in SCs and other non-neuronal cells that can augment the regenerative capacity of multiple biomaterials is considered along with innovations and technologies for traumatic injury.

Mechanical stimulation of a bioactive, functionalized PVDF-TrFE scaffold provides electrical signaling for nerve repair applications.

Traumatic nerve injuries have limited success in achieving full functional recovery, with current clinical solutions often including implementation of nerve grafts or the use of nerve conduits to guide damaged axons across injury gaps. In search of alternative, and complimentary solutions, piezoelectric biomaterials demonstrate immense potential for tissue engineering applications. Piezoelectric poly(vinylidene fluoride-triflouroethylene) (PVFD-TrFE) scaffolds can be harnessed to non-invasively stimulate and direct function of key peripheral nervous system (PNS) cells in regeneration strategies. In this study, electrospun PVDF-TrFE was characterized, fabricated into a 3D scaffold, and finally rendered bioactive with the incorporation of a cell-secreted, decellularized extracellular matrix (dECM). PVDF-TrFE scaffolds were characterized extensively for piezoelectric capacity, mechanical properties, and cell-material interactions with fibroblasts and Schwann cells. Through functionalization of PVDF-TrFE scaffolds with a native, cell-assembled dECM, the ability to promote cell adhesion and enhanced viability was also demonstrated. Additionally, incorporation of bioactive functionalization improved the assembly of key regenerative ECM proteins and regenerative growth factors. PVDF-TrFE scaffolds were then fabricated into a conduit design that retained key physical, chemical, and piezoelectric properties necessary for PNS repair. This work shows great promise for multi-cue, electrospun biomaterials for regeneration of the PNS in traumatic injury.

Capturing the mechanics of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis enables selective uptake of molecules into cells in response to changing cellular needs. It occurs through assembly of coat components around the plasma membrane that determine vesicle contents and facilitate membrane bending to form a clathrin-coated transport vesicle. In this review we discuss recent cryo-electron microscopy structures that have captured a series of events in the life cycle of a clathrin-coated vesicle. Both single particle analysis and tomography approaches have revealed details of the clathrin lattice structure itself, how AP2 may interface with clathrin within a coated vesicle and the importance of PIP2 binding for assembly of the yeast adaptors Sla2 and Ent1 on the membrane. Within cells, cryo-electron tomography of clathrin in flat lattices and high-speed AFM studies provided new insights into how clathrin morphology can adapt during CCV formation. Thus, key mechanical processes driving clathrin-mediated endocytosis have been captured through multiple techniques working in partnership.

UAV rapidly-deployable stage sensor with electro-permanent magnet docking mechanism for flood monitoring in undersampled watersheds.

The availability of historical flood data is vital in recognizing weather-related trends and outlining necessary precautions for at-risk communities. Flood frequency, magnitude, endurance, and volume are traditionally recorded using established streamgages; however, the material and installation costs allow only a few streamgages in a region, which yield a narrow data selection. In particular, stage, the vertical water height in a water body, is an important parameter in determining flood trends. This work investigates a low-cost, compact, rapidly-deployable alternative to traditional stage sensors that will allow for denser sampling within a watershed and a more detailed record of flood events. The package uses a HC-SR04 ultrasonic sensor to measure stage, onboard memory for recording flood events, and an electropermanet magnet (EPM) to enable Unmanned Aerial Vehicle (UAV) deployments. Optional modules for solar panels and wireless communication can also be added to extend package longevity or allow wireless control of the EPM. The stage sensor package was found to have a range of 0.02 to 4 m with a 6.9 mm accuracy and capable of a 6.4 day long deployment. With the total cost of production at 271.37 USD, it is a cheaper and more flexible alternative to traditional stage sensors that will enable dense sensor networks and rapid response to flooding events.

Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the ß2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of ß2 hinge-appendage (ß2HA). We find that the ß2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, ß2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of ß2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that ß2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.

Clathrin: the molecular shape shifter.

Clathrin is best known for its contribution to clathrin-mediated endocytosis yet it also participates to a diverse range of cellular functions. Key to this is clathrin's ability to assemble into polyhedral lattices that include curved football or basket shapes, flat lattices or even tubular structures. In this review, we discuss clathrin structure and coated vesicle formation, how clathrin is utilised within different cellular processes including synaptic vesicle recycling, hormone desensitisation, spermiogenesis, cell migration and mitosis, and how clathrin's remarkable 'shapeshifting' ability to form diverse lattice structures might contribute to its multiple cellular functions.

Inhibitory Molecules PD-1, CD73 and CD39 Are Expressed by CD8+ T Cells in a Tissue-Dependent Manner and Can Inhibit T Cell Responses to Stimulation.

The salivary gland is an important tissue for persistence and transmission of multiple viruses. Previous work showed that salivary gland tissue-resident CD8+ T cells elicited by viruses were poorly functional ex vivo. Using a model of persistent murine cytomegalovirus (MCMV) infection, we now show that CD8+ T cells in the salivary gland and other non-lymphoid tissues of mice express multiple molecules associated with T cell exhaustion including PD-1, CD73 and CD39. Strikingly however, these molecules were expressed independently of virus or antigen. Rather, PD-1-expressing T cells remained PD-1+ after migration into tissues regardless of infection, while CD73 was activated on CD8+ T cells by TGF-ß signaling. Blockade of PD-L1, but not CD73, improved cytokine production by salivary gland T cells ex vivo and increased the expression of granzyme B after stimulation within the salivary gland. Nevertheless, salivary-gland localized CD8+ T cells could kill PD-L1-expressing targets in vivo, albeit with modest efficiency, and this was not improved by PD-L1 blockade. Moreover, the impact of PD-L1 blockade on granzyme B expression waned with time. In contrast, the function of kidney-localized T cells was improved by CD73 blockade, but was unaffected by PD-L1 blockade. These data show that tissue localization per se is associated with expression of inhibitory molecules that can impact T cell function, but that the functional impact of this expression is context- and tissue-dependent.

Influence of DIBMA Polymer Length on Lipid Nanodisc Formation and Membrane Protein Extraction.

Polymer-based lipid nanoparticles like styrene-maleic acid lipid particles have revolutionized the study of membrane proteins. More recently, alternative polymers such as poly(diisobutylene-alt-maleic acid) (DIBMA) have been used in this field. DIBMA is commonly synthesized via conventional radical copolymerization. In order to study the influence of its chain length on lipid nanodisc formation and membrane protein extraction, we synthesized DIBMA with molar masses varying from 1.2-12 kDa via RAFT-mediated polymerization. For molar masses in the range of 3-7 kDa, the rate of lipid nanodisc formation was the highest and similar to those of poly(styrene-co-maleic acid) (SMA) and commercially available DIBMA. ZipA solubilization efficiency was significantly higher than for commercially available DIBMA and similar to SMA (circa 75%). Furthermore, RAFT-made DIBMA with a molar mass of 1.2-3.9 kDa showed a much cleaner separation on SDS-PAGE, without the smearing that is typically seen for SMA and commercially available DIBMA.

Development of a Piezoelectric PVDF-TrFE Fibrous Scaffold to Guide Cell Adhesion, Proliferation, and Alignment.

Severe peripheral nervous system injuries currently hold limited therapeutic solutions. Existing clinical techniques such as autografts, allografts, and newer nerve guidance conduits have shown variable outcomes in functional recovery, adverse immune responses, and in some cases low or minimal availability. This can be attributed in part to the lack of chemical, physical, and electrical cues directing both nerve guidance and regeneration. To address this pressing clinical issue, electrospun nanofibers and microfibers composed of piezoelectric polyvinylidene flouride-triflouroethylene (PVDF-TrFE) have been introduced as an alternative template for tissue engineered biomaterials, specifically as it pertains to their relevance in soft tissue and nerve repair. Here, biocompatible scaffolds of PVDF-TrFE are fabricated and their ability to generate an electrical response to mechanical deformations and produce a suitable regenerative microenvironment is examined. It is determined that 20% (w/v) PVDF-TrFE in (6:4) dimethyl formamide (DMF):acetone solvent maintains a desirable piezoelectric coefficient and the proper physical and electrical characteristics for tissue regeneration. Further, it is concluded that scaffolds of varying thickness promoted the adhesion and alignment of Schwann cells and fibroblasts. This work offers a prelude to further advancements in nanofibrous technology and a promising outlook for alternative, autologous remedies to peripheral nerve damage.

The MiDAC histone deacetylase complex is essential for embryonic development and has a unique multivalent structure.

MiDAC is one of seven distinct, large multi-protein complexes that recruit class I histone deacetylases to the genome to regulate gene expression. Despite implications of involvement in cell cycle regulation and in several cancers, surprisingly little is known about the function or structure of MiDAC. Here we show that MiDAC is important for chromosome alignment during mitosis in cancer cell lines. Mice lacking the MiDAC proteins, DNTTIP1 or MIDEAS, die with identical phenotypes during late embryogenesis due to perturbations in gene expression that result in heart malformation and haematopoietic failure. This suggests that MiDAC has an essential and unique function that cannot be compensated by other HDAC complexes. Consistent with this, the cryoEM structure of MiDAC reveals a unique and distinctive mode of assembly. Four copies of HDAC1 are positioned at the periphery with outward-facing active sites suggesting that the complex may target multiple nucleosomes implying a processive deacetylase function.

Bacteriophage K1F targets Escherichia coli K1 in cerebral endothelial cells and influences the barrier function.

Bacterial neonatal meningitis results in high mortality and morbidity rates for those affected. Although improvements in diagnosis and treatment have led to a decline in mortality rates, morbidity rates have remained relatively unchanged. Bacterial resistance to antibiotics in this clinical setting further underlines the need for developing other technologies, such as phage therapy. We exploited an in vitro phage therapy model for studying bacterial neonatal meningitis based on Escherichia coli (E. coli) EV36, bacteriophage (phage) K1F and human cerebral microvascular endothelial cells (hCMECs). We show that phage K1F is phagocytosed and degraded by constitutive- and PAMP-dependent LC3-assisted phagocytosis and does not induce expression of inflammatory cytokines TNFα, IL-6, IL-8 or IFNß. Additionally, we observed that phage K1F temporarily decreases the barrier resistance of hCMEC cultures, a property that influences the barrier permeability, which could facilitate the transition of immune cells across the endothelial vessel in vivo. Collectively, we demonstrate that phage K1F can infect intracellular E. coli EV36 within hCMECs without themselves eliciting an inflammatory or defensive response. This study illustrates the potential of phage therapy targeting infections such as bacterial neonatal meningitis and is an important step for the continued development of phage therapy targeting antibiotic-resistant bacterial infections generally.

Stochastic Expansions Maintain the Clonal Stability of CD8+ T Cell Populations Undergoing Memory Inflation Driven by Murine Cytomegalovirus.

CMV is an obligate and persistent intracellular pathogen that continually drives the production of highly differentiated virus-specific CD8+ T cells in an Ag-dependent manner, a phenomenon known as memory inflation. Extensive proliferation is required to generate and maintain inflationary CD8+ T cell populations, which are counterintuitively short-lived and typically exposed to limited amounts of Ag during the chronic phase of infection. An apparent discrepancy therefore exists between the magnitude of expansion and the requirement for ongoing immunogenic stimulation. To address this issue, we explored the clonal dynamics of memory inflation. First, we tracked congenically marked OT-I cell populations in recipient mice infected with murine CMV (MCMV) expressing the cognate Ag OVA. Irrespective of numerical dominance, stochastic expansions were observed in each population, such that dominant and subdominant OT-I cells were maintained at stable frequencies over time. Second, we characterized endogenous CD8+ T cell populations specific for two classic inflationary epitopes, M38 and IE3. Multiple clonotypes simultaneously underwent Ag-driven proliferation during latent infection with MCMV. In addition, the corresponding CD8+ T cell repertoires were stable over time and dominated by persistent clonotypes, many of which also occurred in more than one mouse. Collectively, these data suggest that stochastic encounters with Ag occur frequently enough to maintain oligoclonal populations of inflationary CD8+ T cells, despite intrinsic constraints on epitope display at individual sites of infection with MCMV.

Nano-encapsulated Escherichia coli Divisome Anchor ZipA, and in Complex with FtsZ.

The E. coli membrane protein ZipA, binds to the tubulin homologue FtsZ, in the early stage of cell division. We isolated ZipA in a Styrene Maleic Acid lipid particle (SMALP) preserving its position and integrity with native E. coli membrane lipids. Direct binding of ZipA to FtsZ is demonstrated, including FtsZ fibre bundles decorated with ZipA. Using Cryo-Electron Microscopy, small-angle X-ray and neutron scattering, we determine the encapsulated-ZipA structure in isolation, and in complex with FtsZ to a resolution of 1.6 nm. Three regions can be identified from the structure which correspond to, SMALP encapsulated membrane and ZipA transmembrane helix, a separate short compact tether, and ZipA globular head which binds FtsZ. The complex extends 12 nm from the membrane in a compact structure, supported by mesoscale modelling techniques, measuring the movement and stiffness of the regions within ZipA provides molecular scale analysis and visualisation of the early divisome.

Cryo-EM of multiple cage architectures reveals a universal mode of clathrin self-assembly.

Clathrin forms diverse lattice and cage structures that change size and shape rapidly in response to the needs of eukaryotic cells during clathrin-mediated endocytosis and intracellular trafficking. We present the cryo-EM structure and molecular model of assembled porcine clathrin, providing insights into interactions that stabilize key elements of the clathrin lattice, namely, between adjacent heavy chains, at the light chain-heavy chain interface and within the trimerization domain. Furthermore, we report cryo-EM maps for five different clathrin cage architectures. Fitting structural models to three of these maps shows that their assembly requires only a limited range of triskelion leg conformations, yet inherent flexibility is required to maintain contacts. Analysis of the protein-protein interfaces shows remarkable conservation of contact sites despite architectural variation. These data reveal a universal mode of clathrin assembly that allows variable cage architecture and adaptation of coated vesicle size and shape during clathrin-mediated vesicular trafficking or endocytosis.

SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis.

Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.