Engineering RNA export for measurement and manipulation of living cells.

A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins.

Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types. Using different glycoproteins in eVLPs alters their cellular tropism. Single injections of eVLPs into mice support therapeutic levels of base editing in multiple tissues, reducing serum Pcsk9 levels 78% following 63% liver editing, and partially restoring visual function in a mouse model of genetic blindness. In vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and nonviral delivery.

Identification of a therapeutic interfering particle-A single-dose SARS-CoV-2 antiviral intervention with a high barrier to resistance.

Viral-deletion mutants that conditionally replicate and inhibit the wild-type virus (i.e., defective interfering particles, DIPs) have long been proposed as single-administration interventions with high genetic barriers to resistance. However, theories predict that robust, therapeutic DIPs (i.e., therapeutic interfering particles, TIPs) must conditionally spread between cells with R0 >1. Here, we report engineering of TIPs that conditionally replicate with SARS-CoV-2, exhibit R0 >1, and inhibit viral replication 10- to 100-fold. Inhibition occurs via competition for viral replication machinery, and a single administration of TIP RNA inhibits SARS-CoV-2 sustainably in continuous cultures. Strikingly, TIPs maintain efficacy against neutralization-resistant variants (e.g., B.1.351). In hamsters, both prophylactic and therapeutic intranasal administration of lipid-nanoparticle TIPs durably suppressed SARS-CoV-2 by 100-fold in the lungs, reduced pro-inflammatory cytokine expression, and prevented severe pulmonary edema. These data provide proof of concept for a class of single-administration antivirals that may circumvent current requirements to continually update medical countermeasures against new variants.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Emerging and re-emerging themes in co-transcriptional pre-mRNA splicing.

Proper gene expression requires the collaborative effort of multiple macromolecular machines to produce functional messenger RNA. As RNA polymerase II (RNA Pol II) transcribes DNA, the nascent pre-messenger RNA is heavily modified by other complexes such as 5' capping enzymes, the spliceosome, the cleavage, and polyadenylation machinery as well as RNA-modifying/editing enzymes. Recent evidence has demonstrated that pre-mRNA splicing and 3' end cleavage can occur on similar timescales as transcription and significantly cross-regulate. In this review, we discuss recent advances in co-transcriptional processing and how it contributes to gene regulation. We highlight how emerging areas-including coordinated splicing events, physical interactions between the RNA synthesis and modifying machinery, rapid and delayed splicing, and nuclear organization-impact mRNA isoforms. Coordination among RNA-processing choices yields radically different mRNA and protein products, foreshadowing the likely regulatory importance of co-transcriptional RNA folding and co-transcriptional modifications that have yet to be characterized in detail.

Nuclear mRNPs are compact particles packaged with a network of proteins promoting RNA-RNA interactions.

Messenger RNAs (mRNAs) are at the center of the central dogma of molecular biology. In eukaryotic cells, these long ribonucleic acid polymers do not exist as naked transcripts; rather, they associate with mRNA-binding proteins to form messenger ribonucleoprotein (mRNP) complexes. Recently, global proteomic and transcriptomic studies have provided comprehensive inventories of mRNP components. However, knowledge of the molecular features of distinct mRNP populations has remained elusive. We purified endogenous nuclear mRNPs from Saccharomyces cerevisiae by harnessing the mRNP biogenesis factors THO and Sub2 in biochemical procedures optimized to preserve the integrity of these transient ribonucleoprotein assemblies. We found that these mRNPs are compact particles that contain multiple copies of Yra1, an essential protein with RNA-annealing properties. To investigate their molecular and architectural organization, we used a combination of proteomics, RNA sequencing, cryo-electron microscopy, cross-linking mass spectrometry, structural models, and biochemical assays. Our findings indicate that yeast nuclear mRNPs are packaged around an intricate network of interconnected proteins capable of promoting RNA-RNA interactions via their positively charged intrinsically disordered regions. The evolutionary conservation of the major mRNA-packaging factor (yeast Yra1 and Aly/REF in metazoans) points toward a general paradigm governing nuclear mRNP packaging.

Localization-delocalization transition for light particles in turbulence.

Small bubbles in fluids rise to the surface due to Archimede's force. Remarkably, in turbulent flows this process is severely hindered by the presence of vortex filaments, which act as moving potential wells, dynamically trapping light particles and bubbles. Quantifying the statistical weights and roles of vortex filaments in turbulence is, however, still an outstanding experimental and computational challenge due to their small scale, fast chaotic motion, and transient nature. Here we show that, under the influence of a modulated oscillatory forcing, the collective bubble behavior switches from a dynamically localized to a delocalized state. Additionally, we find that by varying the forcing frequency and amplitude, a remarkable resonant phenomenon between light particles and small-scale vortex filaments emerges, likening particle behavior to a forced damped oscillator. We discuss how these externally actuated bubbles can be used as a type of microscopic probe to investigate the space-time statistical properties of the smallest turbulence scales, allowing to quantitatively measure physical characteristics of vortex filaments. We develop a superposition model that is in excellent agreement with the simulation data of the particle dynamics which reveals the fraction of localized/delocalized particles as well as characteristics of the potential landscape induced by vortices in turbulence. Our approach paves the way for innovative ways to accurately measure turbulent properties and to the possibility to control light particles and bubble motions in turbulence with potential applications to oceanography, medical imaging, drug/gene delivery, chemical reactions, wastewater treatment, and industrial mixing.

Elimination of virus-like particles reduces protein aggregation and extends replicative lifespan in Saccharomyces cerevisiae.

A major consequence of aging and stress, in yeast to humans, is an increased accumulation of protein aggregates at distinct sites within the cells. Using genetic screens, immunoelectron microscopy, and three-dimensional modeling in our efforts to elucidate the importance of aggregate annexation, we found that most aggregates in yeast accumulate near the surface of mitochondria. Further, we show that virus-like particles (VLPs), which are part of the retrotransposition cycle of Ty elements, are markedly enriched in these sites of protein aggregation. RNA interference-mediated silencing of Ty expression perturbed aggregate sequestration to mitochondria, reduced overall protein aggregation, mitigated toxicity of a Huntington's disease model, and expanded the replicative lifespan of yeast in a partially Hsp104-dependent manner. The results are in line with recent data demonstrating that VLPs might act as aging factors in mammals, including humans, and extend these findings by linking VLPs to a toxic accumulation of protein aggregates and raising the possibility that they might negatively influence neurological disease progression.

Automotive braking is a source of highly charged aerosol particles.

Although the last several decades have seen a dramatic reduction in emissions from vehicular exhaust, nonexhaust emissions (e.g., brake and tire wear) represent an increasingly significant class of traffic-related particulate pollution. Aerosol particles emitted from the wear of automotive brake pads contribute roughly half of the particle mass attributed to nonexhaust sources, while their relative contribution to urban air pollution overall will almost certainly grow coinciding with vehicle fleet electrification and the transition to alternative fuels. To better understand the implications of this growing prominence, a more thorough understanding of the physicochemical properties of brake wear particles (BWPs) is needed. Here, we investigate the electrical properties of BWPs as emitted from ceramic and semi-metallic brake pads. We show that up to 80% of BWPs emitted are electrically charged and that this fraction is strongly dependent on the specific brake pad material used. A dependence of the number of charges per particle on charge polarity and particle size is also demonstrated. We find that brake wear produces both positive and negative charged particles that can hold in excess of 30 elementary charges and show evidence that more negative charges are produced than positive. Our results will provide insights into the currently limited understanding of BWPs and their charging mechanisms, which potentially have significant implications on their atmospheric lifetimes and thus their relevance to climate and air quality. In addition, our study will inform future efforts to remove BWP emissions before entering the atmosphere by taking advantage of their electric charge.

Air Pollution-Related Neurotoxicity Across the Life Span.

Air pollution is a complex mixture of gases and particulate matter, with adsorbed organic and inorganic contaminants, to which exposure is lifelong. Epidemiological studies increasingly associate air pollution with multiple neurodevelopmental disorders and neurodegenerative diseases, findings supported by experimental animal models. This breadth of neurotoxicity across these central nervous system diseases and disorders likely reflects shared vulnerability of their inflammatory and oxidative stress-based mechanisms and a corresponding ability to produce brain metal dyshomeo-stasis. Future research to define the responsible contaminants of air pollution underlying this neurotoxicity is critical to understanding mechanisms of these diseases and disorders and protecting public health.

Extracellular vesicles and particles impact the systemic landscape of cancer.

Intercellular cross talk between cancer cells and stromal and immune cells is essential for tumor progression and metastasis. Extracellular vesicles and particles (EVPs) are a heterogeneous class of secreted messengers that carry bioactive molecules and that have been shown to be crucial for this cell-cell communication. Here, we highlight the multifaceted roles of EVPs in cancer. Functionally, transfer of EVP cargo between cells influences tumor cell growth and invasion, alters immune cell composition and function, and contributes to stromal cell activation. These EVP-mediated changes impact local tumor progression, foster cultivation of pre-metastatic niches at distant organ-specific sites, and mediate systemic effects of cancer. Furthermore, we discuss how exploiting the highly selective enrichment of molecules within EVPs has profound implications for advancing diagnostic and prognostic biomarker development and for improving therapy delivery in cancer patients. Altogether, these investigations into the role of EVPs in cancer have led to discoveries that hold great promise for improving cancer patient care and outcome.

X-ray 3D imaging-based microunderstanding of granular mixtures: Stiffness enhancement by adding small fractions of soft particles.

This research focuses on performing ultrasound propagation measurements and micro-X-ray computed tomography (µXRCT) imaging on prestressed granular packings prepared with biphasic mixtures of monodisperse glass and rubber particles at different compositions/fractions. Ultrasound experiments employing piezoelectric transducers, mounted in an oedometric cell (complementing earlier triaxial cell experiments), are used to excite and detect longitudinal ultrasound waves through randomly prepared mixtures of monodisperse stiff/soft particles. While the fraction of the soft particles is increasing linearly from zero, the effective macroscopic stiffness of the granular packings transits nonlinearly and nonmonotonically toward the soft limit, remarkably via an interesting stiffer regime for small rubber fractions between 0.1 ≲ ν ≲ 0.2. The contact network of dense packings, as accessed from µXRCT, plays a key role in understanding this phenomenon, considering the structure of the network, the chain length, the grain contacts, and the particle coordination. While the maximum stiffness is due to surprisingly shortened chains, the sudden drop in elastic stiffness of the mixture packings, at ν ≈ 0.4, is associated with chains of particles that include both glass and rubber particles (soft chains); for ν ≲ 0.3, the dominant chains include only glass particles (hard chains). At the drop, ν ≈ 0.4, the coordination number of glass and rubber networks is approximately four and three, respectively, i.e., neither of the networks are jammed, and the chains need to include particles from another species to propagate information.

Lagrangian large eddy simulations via physics-informed machine learning.

High-Reynolds number homogeneous isotropic turbulence (HIT) is fully described within the Navier-Stokes (NS) equations, which are notoriously difficult to solve numerically. Engineers, interested primarily in describing turbulence at a reduced range of resolved scales, have designed heuristics, known as large eddy simulation (LES). LES is described in terms of the temporally evolving Eulerian velocity field defined over a spatial grid with the mean-spacing correspondent to the resolved scale. This classic Eulerian LES depends on assumptions about effects of subgrid scales on the resolved scales. Here, we take an alternative approach and design LES heuristics stated in terms of Lagrangian particles moving with the flow. Our Lagrangian LES, thus L-LES, is described by equations generalizing the weakly compressible smoothed particle hydrodynamics formulation with extended parametric and functional freedom, which is then resolved via Machine Learning training on Lagrangian data from direct numerical simulations of the NS equations. The L-LES model includes physics-informed parameterization and functional form, by combining physics-based parameters and physics-inspired Neural Networks to describe the evolution of turbulence within the resolved range of scales. The subgrid-scale contributions are modeled separately with physical constraints to account for the effects from unresolved scales. We build the resulting model under the differentiable programming framework to facilitate efficient training. We experiment with loss functions of different types, including physics-informed ones accounting for statistics of Lagrangian particles. We show that our L-LES model is capable of reproducing Eulerian and unique Lagrangian turbulence structures and statistics over a range of turbulent Mach numbers.

Small particles with large impact: Insights into the unresolved roles of innate immunity in extracellular vesicle-mediated cardiovascular calcification.

Extracellular vesicles (EVs) are critical in the initiation and progression of cardiovascular calcification, and immune cell infiltration and inflammation have a central role in this process. EVs egress from various cardiovascular cell types, which when acquiring specific properties, become calcifying. These calcifying EVs form nidi for microcalcification, which can progress to the macrocalcification lesions that are visualized clinically. We make the distinction between inflammatory-driven and mineral dysregulation-driven calcification, which both share EVs as a central initiator. In inflammation-mediated calcification, inflammation precedes microcalcification and results from EV release from macrophages. Local cellular crosstalk mediated by EVs as well as circulating EVs and other inflammatory nanoparticles, such as calciprotein particles and lipoproteins, are also critical in the progression of cardiovascular calcification. It is imperative that future work links the already established and to be discovered roles of inflammation and innate immunity in cardiovascular calcification to these key signaling and functional roles of these nanoparticles. It remains an understudied area with high potential to unravel mechanistic roles and has important implications in drug target research.

The expanding family of neutrophil-derived extracellular vesicles.

Neutrophils are immune cells involved in several inflammatory and homeostatic processes. Their capacity to release cargo can be classified based on whether the cargo is released on its own, or in conjunction with plasma membrane structures. Examples of plasma membrane-free secretion modes are degranulation, neutrophil extracellular trap (NET) release, and cytokine release through inflammasome formation. The most studied membrane-covered neutrophil-derived structures are exosomes and ectosomes that are collectively called extracellular vesicles (EV). Apoptotic vesicles are another recognized EV subtype. Over the last decade, additional membrane-covered neutrophil-derived structures were characterized: migratory cytoplasts, migrasomes, and elongated neutrophil-derived structures (ENDS). All these structures are smaller than the neutrophils, cannot reproduce themselves, and thus meet the latest consensus definition of EVs. In this review, we focus on the less well-studied neutrophil EVs: apoptotic vesicles, cytoplasts, migrasomes, and ENDS.

Virus-like particles as robust tools for functional assessment: Deciphering the pathogenicity of ABCA4 genetic variants of uncertain significance.

The retina-specific ABCA transporter, ABCA4, is essential for vision, and its genetic variants are associated with a wide range of inherited retinal degenerative diseases, leading to blindness. Of the 1630 identified missense variants in ABCA4, ∼50% are of unknown pathogenicity (variants of unknown significance, VUS). This genetic uncertainty presents three main challenges: (i) inability to predict disease-causing variants in relatives of inherited retinal degenerative disease patients with multiple ABCA4 mutations; (ii) limitations in developing variant-specific treatments; and (iii) difficulty in using these variants for future disease prediction, affecting patients' life-planning and clinical trial participation. To unravel the clinical significance of ABCA4 genetic variants at the level of protein function, we have developed a virus-like particle-based system that expresses the ABCA4 protein and its variants. We validated the efficacy of this system in the enzymatic characterization (ATPase activity) of VLPs harboring ABCA4 and two variants of established pathogenicity: p.N965S and p.C1488R. Our results were consistent with previous reports and clinical phenotypes. We also applied this platform to characterize the VUS p.Y1779F and observed a functional impairment, suggesting a potential pathogenic impact. This approach offers an efficient, high-throughput method for ABCA4 VUS characterization. Our research points to the significant promise of the VLP-based system in the functional analysis of membrane proteins, offering important perspectives on the disease-causing potential of genetic variants and shedding light on genetic conditions involving such proteins.

Illuminating bunyavirus entry into host cells with fluorescence.

Bunyavirales constitute the largest order of enveloped RNA viruses, many members of which cause severe diseases in humans and domestic animals. In recent decades, innovative fluorescence-based methods have paved the way to visualize and track single fluorescent bunyaviral particles in fixed and live cells. This technological breakthrough has enabled imaging of the early stages of infection and the quantification of every step in the bunyavirus cell entry process. Here, we describe the latest procedures for rendering bunyaviral particles fluorescent and discuss the advantages and disadvantages of each approach in light of the most recent advances in fluorescence detection and monitoring of bunyavirus entry. In this mini-review, we also illustrate how fluorescent viral particles are a powerful tool for deciphering the cellular entry process of bunyaviruses, the vast majority of which have not yet been analyzed.

Transperons: RNA operons as effectors of coordinated gene expression in eukaryotes.

Coordinated gene expression allows spatiotemporal control of cellular processes and is achieved by the cotranscription/translation of functionally related genes/proteins. Prokaryotes evolved polycistronic messages (operons) to confer expression from a single promoter to efficiently cotranslate proteins functioning on the same pathway. Yet, despite having far greater diversity (e.g., gene number, distribution, modes of expression), eukaryotic cells employ individual promoters and monocistronic messages. Although gene expression is modular, it does not account for how eukaryotes achieve coordinated localized translation. The RNA operon theory states that mRNAs derived from different chromosomes assemble into ribonucleoprotein particles (RNPs) that act as functional operons to generate protein cohorts upon cotranslation. Work in yeast has now validated this theory and shown that intergenic associations and noncanonical histone functions create pathway-specific RNA operons (transperons) that regulate cell physiology. Herein the involvement of chromatin organization in transperon formation and programmed gene coexpression is discussed.

Rational design of polymer-based particulate vaccine adjuvants.

Vaccination is considered one of the major milestones in modern medicine, facilitating the control and eradication of life-threatening infectious diseases. Vaccine adjuvants are a key component of many vaccines, serving to steer antigen-specific immune responses and increase their magnitude. Despite major advances in the field of adjuvant research over recent decades, our understanding of their mechanism of action remains incomplete. This hinders our capacity to further improve these adjuvant technologies, so addressing how adjuvants induce and control the induction of innate and adaptive immunity is a priority. Investigating how adjuvant physicochemical properties, such as size and charge, exert immunomodulatory effects can provide valuable insights and serve as the foundation for the rational design of vaccine adjuvants. Most clinically applied adjuvants are particulate in nature and polymeric particulate adjuvants present advantages due to stability, biocompatibility profiles, and flexibility in terms of formulation. These properties can impact on antigen release kinetics and biodistribution, cellular uptake and targeting, and drainage to the lymphatics, consequently dictating the induction of innate, cellular, and humoral adaptive immunity. A current focus is to apply rational design principles to the development of adjuvants capable of eliciting robust cellular immune responses including CD8+ cytotoxic T-cell and Th1-biased CD4+ T-cell responses, which are required for vaccines against intracellular pathogens and cancer. This review highlights recent advances in our understanding of how particulate adjuvants, especially polymer-based particulates, modulate immune responses and how this can be used as a guide for improved adjuvant design.

Palmitoylation of SARS-CoV-2 Envelope protein is central to virus particle formation.

The Envelope (E) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an integral structural protein in the virus particles. However, its role in the assembly of virions and the underlying molecular mechanisms are yet to be elucidated, including whether the function of E protein is regulated by post-translational modifications. In the present study, we report that SARS-CoV-2 E protein is palmitoylated at C40, C43, and C44 by palmitoyltransferases zDHHC3, 6, 12, 15, and 20. Mutating these three cysteines to serines (C40/43/44S) reduced the stability of E protein, decreased the interaction of E with structural proteins Spike, Membrane, and Nucleocapsid, and thereby inhibited the production of virus-like particles (VLPs) and VLP-mediated luciferase transcriptional delivery. Specifically, the C40/43/44S mutation of E protein reduced the density of VLPs. Collectively, these results demonstrate that palmitoylation of E protein is vital for its function in the assembly of SARS-CoV-2 particles.IMPORTANCEIn this study, we systematically examined the biochemistry of palmitoylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) E protein and demonstrated that palmitoylation of SARS-CoV-2 E protein is required for virus-like particle (VLP) production and maintaining normal particle density. These results suggest that palmitoylated E protein is central for proper morphogenesis of SARS-CoV-2 VLPs in densities required for viral infectivity. This study presents a significant advancement in the understanding of how palmitoylation of viral proteins is vital for assembling SARS-CoV-2 particles and supports that palmitoyl acyltransferases can be potential therapeutic targets for the development of SARS-CoV-2 inhibitors.