Crystal Formation in Inflammation.

The formation and accumulation of crystalline material in tissues is a hallmark of many metabolic and inflammatory conditions. The discovery that the phase transition of physiologically soluble substances to their crystalline forms can be detected by the immune system and activate innate immune pathways has revolutionized our understanding of how crystals cause inflammation. It is now appreciated that crystals are part of the pathogenesis of numerous diseases, including gout, silicosis, asbestosis, and atherosclerosis. In this review we discuss current knowledge of the complex mechanisms of crystal formation in diseased tissues and their interplay with the nutrients, metabolites, and immune cells that account for crystal-induced inflammation.

Counting protein molecules for single-cell proteomics.

Technologies for counting protein molecules are enabling single-cell proteomics at increasing depth and scale. New advances in single-molecule methods by Brinkerhoff and colleagues promise to further increase the sensitivity of protein analysis and motivate questions about scaling up the counting of the human proteome.

Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells.

Transforming the vast knowledge from genetics, biochemistry, and structural biology into detailed molecular descriptions of biological processes inside cells remains a major challenge-one in sore need of better imaging technologies. For example, transcription involves the complex interplay between RNA polymerase II (Pol II), regulatory factors (RFs), and chromatin, but visualizing these dynamic molecular transactions in their native intracellular milieu remains elusive. Here, we zoom into single tagged genes using nanoscopy techniques, including an active target-locking, ultra-sensitive system that enables single-molecule detection in addressable sub-diffraction volumes, within crowded intracellular environments. We image, track, and quantify Pol II with single-molecule resolution, unveiling its dynamics during the transcription cycle. Further probing multiple functionally linked events-RF-chromatin interactions, Pol II dynamics, and nascent transcription kinetics-reveals detailed operational parameters of gene-regulatory mechanisms hitherto-unseen in vivo. Our approach sets the stage for single-molecule studies of complex molecular processes in live cells.

Reductionist Approach in Peptide-Based Nanotechnology.

The formation of ordered nanostructures by molecular self-assembly of proteins and peptides represents one of the principal directions in nanotechnology. Indeed, polyamides provide superior features as materials with diverse physical properties. A reductionist approach allowed the identification of extremely short peptide sequences, as short as dipeptides, which could form well-ordered amyloid-like ß-sheet-rich assemblies comparable to supramolecular structures made of much larger proteins. Some of the peptide assemblies show remarkable mechanical, optical, and electrical characteristics. Another direction of reductionism utilized a natural noncoded amino acid, α-aminoisobutryic acid, to form short superhelical assemblies. The use of this exceptional helix inducer motif allowed the fabrication of single heptad repeats used in various biointerfaces, including their use as surfactants and DNA-binding agents. Two additional directions of the reductionist approach include the use of peptide nucleic acids (PNAs) and coassembly techniques. The diversified accomplishments of the reductionist approach, as well as the exciting future advances it bears, are discussed.

Biological Insight from Super-Resolution Microscopy: What We Can Learn from Localization-Based Images.

Super-resolution optical imaging based on the switching and localization of individual fluorescent molecules [photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), etc.] has evolved remarkably over the last decade. Originally driven by pushing technological limits, it has become a tool of biological discovery. The initial demand for impressive pictures showing well-studied biological structures has been replaced by a need for quantitative, reliable data providing dependable evidence for specific unresolved biological hypotheses. In this review, we highlight applications that showcase this development, identify the features that led to their success, and discuss remaining challenges and difficulties. In this context, we consider the complex topic of defining resolution for this imaging modality and address some of the more common analytical methods used with this data.

Optical Tweezers: A Force to Be Reckoned With.

The 2018 Nobel Prize in Physics has been awarded jointly to Arthur Ashkin for the discovery and development of optical tweezers and their applications to biological systems and to Gérard Mourou and Donna Strickland for the invention of laser chirped pulse amplification. Here we focus on Arthur Ashkin and how his revolutionary work opened a window into the world of molecular mechanics and spurred the rise of single-molecule biophysics.

Nucleic Acid-Based Nanodevices in Biological Imaging.

The nanoscale engineering of nucleic acids has led to exciting molecular technologies for high-end biological imaging. The predictable base pairing, high programmability, and superior new chemical and biological methods used to access nucleic acids with diverse lengths and in high purity, coupled with computational tools for their design, have allowed the creation of a stunning diversity of nucleic acid-based nanodevices. Given their biological origin, such synthetic devices have a tremendous capacity to interface with the biological world, and this capacity lies at the heart of several nucleic acid-based technologies that are finding applications in biological systems. We discuss these diverse applications and emphasize the advantage, in terms of physicochemical properties, that the nucleic acid scaffold brings to these contexts. As our ability to engineer this versatile scaffold increases, its applications in structural, cellular, and organismal biology are clearly poised to massively expand.

Tunable Single-Cell Extraction for Molecular Analyses.

Because of cellular heterogeneity, the analysis of endogenous molecules from single cells is of significant interest and has major implications. While micromanipulation or cell sorting followed by cell lysis is already used for subsequent molecular examinations, approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Here, we demonstrate the quantitative extraction from single cells with spatiotemporal control using fluidic force microscopy. We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances. This approach has uncovered the ability of cells to withstand extraction of up to several picoliters and opens opportunities to study cellular dynamics and cell-cell communication under physiological conditions at the single-cell level.

Immunoengineering: how nanotechnology can enhance cancer immunotherapy.

Although cancer immunotherapy can lead to durable outcomes, the percentage of patients who respond to this disruptive approach remains modest to date. Encouragingly, nanotechnology can enhance the efficacy of immunostimulatory small molecules and biologics by altering their co-localization, biodistribution, and release kinetics.

Are nanopore technologies ready for the proteomic challenge primetime?

Novel techniques for single-protein molecule sequencing are rapidly becoming the focus of contemporary biomedical research. Here, Brinkerhoff et al. (2021) report a significant progress in nanopore-based rereading of DNA-peptide conjugates.

POINT technology illuminates the processing of polymerase-associated intact nascent transcripts.

Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly because of technical limitations in purifying authentic nascent transcripts. We present a new approach to characterize nascent RNA, called polymerase intact nascent transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5' ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano), and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5' P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that co-transcriptional splicing either occurs immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.

Near-complete depolymerization of polyesters with nano-dispersed enzymes.

Successfully interfacing enzymes and biomachinery with polymers affords on-demand modification and/or programmable degradation during the manufacture, utilization and disposal of plastics, but requires controlled biocatalysis in solid matrices with macromolecular substrates1-7. Embedding enzyme microparticles speeds up polyester degradation, but compromises host properties and unintentionally accelerates the formation of microplastics with partial polymer degradation6,8,9. Here we show that by nanoscopically dispersing enzymes with deep active sites, semi-crystalline polyesters can be degraded primarily via chain-end-mediated processive depolymerization with programmable latency and material integrity, akin to polyadenylation-induced messenger RNA decay10. It is also feasible to achieve processivity with enzymes that have surface-exposed active sites by engineering enzyme-protectant-polymer complexes. Poly(caprolactone) and poly(lactic acid) containing less than 2 weight per cent enzymes are depolymerized in days, with up to 98 per cent polymer-to-small-molecule conversion in standard soil composts and household tap water, completely eliminating current needs to separate and landfill their products in compost facilities. Furthermore, oxidases embedded in polyolefins retain their activities. However, hydrocarbon polymers do not closely associate with enzymes, as their polyester counterparts do, and the reactive radicals that are generated cannot chemically modify the macromolecular host. This study provides molecular guidance towards enzyme-polymer pairing and the selection of enzyme protectants to modulate substrate selectivity and optimize biocatalytic pathways. The results also highlight the need for in-depth research in solid-state enzymology, especially in multi-step enzymatic cascades, to tackle chemically dormant substrates without creating secondary environmental contamination and/or biosafety concerns.

Phase Separation Comes of Age: From Phenomenology to Single Molecules.

In this issue of Molecular Cell, Pitchiaya et al. (2019) use high-resolution single-molecule microscopy to dissect the localization of different types of RNAs with processing bodies (PBs) in cells, revealing novel insights about their dynamic recruitment to PBs.

A nanofluidic spiking synapse.

Currently, the nanofluidic synapse can only perform basic neuromorphic pulse patterns. One immediate problem that needs to be addressed to further its capability of brain-like computing is the realization of a nanofluidic spiking device. Here, we report the use of a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate membrane to achieve bionic ionic current-induced spiking. In addition to the simulation of various electrical pulse patterns, our synapse could produce transmembrane ionic current-induced spiking, which is highly analogous to biological action potentials with similar phases and excitability. Moreover, the spiking properties could be modulated by ions and neurochemicals. We expect that this work could contribute to biomimetic spiking computing in solution.

Multimodal neuro-nanotechnology: Challenging the existing paradigm in glioblastoma therapy.

Integrating multimodal neuro- and nanotechnology-enabled precision immunotherapies with extant systemic immunotherapies may finally provide a significant breakthrough for combatting glioblastoma (GBM). The potency of this approach lies in its ability to train the immune system to efficiently identify and eradicate cancer cells, thereby creating anti-tumor immune memory while minimizing multi-mechanistic immune suppression. A critical aspect of these therapies is the controlled, spatiotemporal delivery of structurally defined nanotherapeutics into the GBM tumor microenvironment (TME). Architectures such as spherical nucleic acids or poly(beta-amino ester)/dendrimer-based nanoparticles have shown promising results in preclinical models due to their multivalency and abilities to activate antigen-presenting cells and prime antigen-specific T cells. These nanostructures also permit systematic variation to optimize their distribution, TME accumulation, cellular uptake, and overall immunostimulatory effects. Delving deeper into the relationships between nanotherapeutic structures and their performance will accelerate nano-drug development and pave the way for the rapid clinical translation of advanced nanomedicines. In addition, the efficacy of nanotechnology-based immunotherapies may be enhanced when integrated with emerging precision surgical techniques, such as laser interstitial thermal therapy, and when combined with systemic immunotherapies, particularly inhibitors of immune-mediated checkpoints and immunosuppressive adenosine signaling. In this perspective, we highlight the potential of emerging treatment modalities, combining advances in biomedical engineering and neurotechnology development with existing immunotherapies to overcome treatment resistance and transform the management of GBM. We conclude with a call to action for researchers to leverage these technologies and accelerate their translation into the clinic.

The Promise and Potential of Metal-Organic Frameworks and Covalent Organic Frameworks in Vaccine Nanotechnology.

The immune system's complexity and ongoing evolutionary struggle against deleterious pathogens underscore the value of vaccination technologies, which have been bolstering human immunity for over two centuries. Despite noteworthy advancements over these 200 years, three areas remain recalcitrant to improvement owing to the environmental instability of the biomolecules used in vaccines─the challenges of formulating them into controlled release systems, their need for constant refrigeration to avoid loss of efficacy, and the requirement that they be delivered via needle owing to gastrointestinal incompatibility. Nanotechnology, particularly metal-organic frameworks (MOFs) and covalent organic frameworks (COFs), has emerged as a promising avenue for confronting these challenges, presenting a new frontier in vaccine development. Although these materials have been widely explored in the context of drug delivery, imaging, and cancer immunotherapy, their role in immunology and vaccine-related applications is a recent yet rapidly developing field. This review seeks to elucidate the prospective use of MOFs and COFs for biomaterial stabilization, eliminating the necessity for cold chains, enhancing antigen potency as adjuvants, and potentializing needle-free delivery of vaccines. It provides an expansive and critical viewpoint on this rapidly evolving field of research and emphasizes the vital contribution of chemists in driving further advancements.

Ultrasound-Based Micro-/Nanosystems for Biomedical Applications.

Due to the intrinsic non-invasive nature, cost-effectiveness, high safety, and real-time capabilities, besides diagnostic imaging, ultrasound as a typical mechanical wave has been extensively developed as a physical tool for versatile biomedical applications. Especially, the prosperity of nanotechnology and nanomedicine invigorates the landscape of ultrasound-based medicine. The unprecedented surge in research enthusiasm and dedicated efforts have led to a mass of multifunctional micro-/nanosystems being applied in ultrasound biomedicine, facilitating precise diagnosis, effective treatment, and personalized theranostics. The effective deployment of versatile ultrasound-based micro-/nanosystems in biomedical applications is rooted in a profound understanding of the relationship among composition, structure, property, bioactivity, application, and performance. In this comprehensive review, we elaborate on the general principles regarding the design, synthesis, functionalization, and optimization of ultrasound-based micro-/nanosystems for abundant biomedical applications. In particular, recent advancements in ultrasound-based micro-/nanosystems for diagnostic imaging are meticulously summarized. Furthermore, we systematically elucidate state-of-the-art studies concerning recent progress in ultrasound-based micro-/nanosystems for therapeutic applications targeting various pathological abnormalities including cancer, bacterial infection, brain diseases, cardiovascular diseases, and metabolic diseases. Finally, we conclude and provide an outlook on this research field with an in-depth discussion of the challenges faced and future developments for further extensive clinical translation and application.

High-speed AFM reveals the dynamics of single biomolecules at the nanometer scale.

Atomic force microscopy allows visualization of biomolecules with nanometer resolution under physiological conditions. Recent advances have improved the time resolution of the technique from minutes to tens of milliseconds, meaning that it is now possible to watch single biomolecules in action in real time. Here, we review this development.

DNAforge: a design tool for nucleic acid wireframe nanostructures.

DNAforge is an online tool that provides a unified, user-friendly interface to several recent design methods for DNA and RNA wireframe nanostructures, with the possibility of integrating additional methods into the same framework. Currently, DNAforge supports three design methods for DNA nanostructures and two for RNA nanostructures. The tool enables the design, visualisation and sequence generation for highly complex wireframe nanostructures with a simple fully automated process. DNAforge is freely accessible at https://dnaforge.org/.