Multiplex Detection of Foodborne Pathogens using 3D Nanostructure Swab and Deep Learning-Based Classification of Raman Spectra.

Proactive management of foodborne illness requires routine surveillance of foodborne pathogens, which requires developing simple, rapid, and sensitive detection methods. Here, a strategy is presented that enables the detection of multiple foodborne bacteria using a 3D nanostructure swab and deep learning-based Raman signal classification. The nanostructure swab efficiently captures foodborne pathogens, and the portable Raman instrument directly collects the Raman signals of captured bacteria. a deep learning algorithm has been demonstrated, 1D convolutional neural network with binary labeling, achieves superior performance in classifying individual bacterial species. This methodology has been extended to mixed bacterial populations, maintaining accuracy close to 100%. In addition, the gradient-weighted class activation mapping method is used to provide an investigation of the Raman bands for foodborne pathogens. For practical application, blind tests are conducted on contaminated kitchen utensils and foods. The proposed technique is validated by the successful detection of bacterial species from the contaminated surfaces. The use of a 3D nanostructure swab, portable Raman device, and deep learning-based classification provides a powerful tool for rapid identification (≈5 min) of foodborne bacterial species. The detection strategy shows significant potential for reliable food safety monitoring, making a meaningful contribution to public health and the food industry.

High order assembly of multiple protein cages with homogeneous sizes and shapes via limited cage surface engineering.

Protein cages are attractive building blocks to build high order materials such as 3D cage lattices, which offer accurately ordered bio-templates. However, controlling the size or valency of these cage-to-cage assemblies is extremely difficult due to highly multivalent and symmetric cage structures. Here, various high order cage assemblies with homogeneous sizes and geometries are constructed by developing an anisotropic ferritin cage with limitedly exposed binding modules, leucine zipper. The anisotropic ferritin is produced as expressed in cells without the need of complex in vitro cage fabrication by careful subunit manipulation. Ferritin cages with limitedly exposed zippers are assembled around a core ferritin with fully exposed opposing zippers, generating homogeneous high order structures, whereas two fully exposed ferritins are assembled into heterogeneous cage aggregates. Diverse fully exposed core cages are prepared by varying the zipper-ferritin fusion geometries and even by using larger cage structures. With these core cages and the anisotropic ferritin, a range of high order cage assemblies with diverse ferritin valencies (3 to over 12) and sizes (over 40 nm) are created. Cell surface binding and internalization of cage structures are greatly varied by assembly sizes, where high order ferritins are clearly more effective than monomeric ferritin.

Elution-free DNA detection using CRISPR/Cas9-mediated light-up aptamer transcription: Toward all-in-one DNA purification and detection tube.

Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications.

Lipid coated protein condensates as stable protocells with selective uptake abilities for biomolecules.

To create cell-like synthetic systems, spatial confinement that is stable against environmental changes and selective uptake of diverse biomolecules into these compartments are key initial conditions. However, fabrication of protocells with these two features has been extremely difficult. Here, we used fully protein-based liquid condensates and a lipid coating on these condensates to construct highly stable protocells with an uptake ability for outside biomolecules. Condensates with an extremely high density of 6His-tagged proteins were coated with Ni(ii)-NTA(nitrilotriacetic acid)-modified lipids. High condensate rigidity and specific 6His-Ni-NTA interactions enabled the formation of lipid-protein protocells, which are stable even after centrifugations. In addition, immobile lipid coatings on condensates were permeable to outside biomolecules. When binding modules were fused into condensate-forming proteins, the resulting functionalized condensate-protocells could strongly and selectively uptake various outside proteins through specific protein interactions.

Protein Cages Engineered for Interaction-Driven Selective Encapsulation of Biomolecules.

Hollow protein cages have become attractive drug delivery vehicles with high biocompatibility and precise functional/structural manipulability. However, difficulties in effective cargo loading inside the cages have been limiting further development of protein cage-based drug carriers. Here, we developed a specific interaction-driven encapsulation and cellular delivery strategy for various biomolecules by engineering a porous protein cage. The computationally designed hyperstable mi3 protein cage was circularly permuted to fuse the cancer targeting RGD tripeptide to the cage surface and SpyTag (ST), which forms a covalent bond with SpyCatcher (SC), to the cage inner cavity. SC-fused proteins with different sizes and charges could be stably and actively encapsulated in the engineered nanocage via the ST/SC reaction. Cargo protein encapsulation inside the cage was directly confirmed by cryo-electron microscopy (EM) structure determination. In addition, SC-fused monomeric avidin was added to the nanocage to encapsulate various biotinylated (nonprotein) cargos such as oligonucleotides and the anticancer drug doxorubicin. All cargo molecules loaded onto the engineered mi3 were effectively delivered to cells. This work introduces a highly versatile cargo loading/delivery strategy, where loading/delivery interactions, cargo molecules, and cell targeting moieties can be further varied for optimal cellular drug delivery.

Valence-controlled protein conjugation on nanoparticles via re-arrangeable multivalent interactions of tandem repeat protein chains.

Precise control of the number of conjugated proteins on a nanoparticle surface has long been a highly challenging task. Here, we developed a one-pot, purification-free strategy for valency-controlled conjugation of tandem repeat protein chains on gold nanoparticles. Protein chains were designed to contain multiple, regularly spaced binding modules, which can multivalently interact with coating molecules on nanoparticle surfaces. We discovered that a slow increase of this interaction strength facilitates full participation of repeated binding modules on a protein chain for surface binding (as well as dynamic rearrangement) on a single nanoparticle, which resulted in stable protein chain wrapping around nanoparticles. By varying the protein chain length, a defined number of protein chains were conjugated on gold nanoparticles with difference sizes. Various high-order nanoparticle structures were accurately assembled with these valence-controlled protein-particle conjugates. The present strategy offers a highly dynamic but controlled protein coating approach on solid surfaces of diverse nanostructures. In addition, this work also provides a valuable clue to understand dynamic binding processes of multivalent repeat proteins.

Bactericidal Effect of Cecropin A Fused Endolysin on Drug-Resistant Gram-Negative Pathogens.

The rapid spread of superbugs leads to the escalation of infectious diseases, which threatens public health. Endolysins derived from bacteriophages are spotlighted as promising alternative antibiotics against multi-drug resistant bacteria. In this study, we isolated and characterized the novel Salmonella typhimurium phage PBST08. Bioinformatics analysis of the PBST08 genome revealed putative endolysin ST01 with a lysozyme-like domain. Since the lytic activity of the purified ST01 was minor, probably owing to the outer membrane, which blocks accessibility to peptidoglycan, antimicrobial peptide cecropin A (CecA) was fused to the N-terminus of ST01 to disrupt the outer membrane. The resulting CecA::ST01 has been shown to have increased bactericidal activity against gram-negative pathogens including Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Enterobacter cloacae and the most affected target was A. baumannii. In the presence of 0.25 µM CecA::ST01, A. baumannii ATCC 17978 strain was completely killed and CCARM 12026 strain was wiped out by 0.5 µM CecA::ST01, which is a clinical isolate of A. baumannii and resistant to multiple drugs including carbapenem. Moreover, the larvae of Galleria mellonella could be rescued up to 58% or 49% by the administration of CecA::ST01 upon infection by A. baumannii 17978 or CCARM 12026 strain. Finally, the antibacterial activity of CecA::ST01 was verified using 31 strains of five gram-negative pathogens by evaluation of minimal inhibitory concentration. Thus, the results indicate that a fusion of antimicrobial peptide to endolysin can enhance antibacterial activity and the spectrum of endolysin where multi-drug resistant gram-negative pathogens can be efficiently controlled.

Ligation-free isothermal nucleic acid amplification.

In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.

Determinants for intrinsically disordered protein recruitment into phase-separated protein condensates.

Multivalent interactions between amino acid residues of intrinsically disordered proteins (IDPs) drive phase separation of these proteins into liquid condensates, forming various membrane-less organelles in cells. These interactions between often biased residues of IDPs are also likely involved in selective recruitment of many other IDPs into condensates. However, determining factors for this IDP recruitment into protein condensates are not understood yet. Here, we quantitatively examined recruitment tendencies of various IDPs with different sequence compositions into IDP-clustered condensates both in vitro as well as in cells. Condensate-forming IDP scaffolds, recruited IDP clients, and phase separation conditions were carefully varied to find key factors for selective IDP partitioning in protein condensates. Regardless of scaffold sequences, charged residues in client IDPs assured potent IDP recruitment, likely via strong electrostatic interactions, where positive residues could further enhance recruitment, possibly with cation-pi interactions. Notably, poly-ethylene glycol, a widely used crowding reagent for in vitro phase separation, abnormally increased IDP recruitment, indicating the need for careful use of crowding conditions. Tyrosines of IDP clients also strongly participated in recruitment both in vitro and in cells. Lastly, we measured recruitment degrees by more conventional interactions between folded proteins instead of disordered proteins. Surprisingly, recruitment forces by an even moderate protein interaction (K d ∼ 5 µM) were substantially stronger than those by natural IDP-IDP interactions. The present data offer valuable information on how cells might organize protein partitioning on various protein condensates.

Artificial protein assemblies with well-defined supramolecular protein nanostructures.

Nature uses a wide range of well-defined biomolecular assemblies in diverse cellular processes, where proteins are major building blocks for these supramolecular assemblies. Inspired by their natural counterparts, artificial protein-based assemblies have attracted strong interest as new bio-nanostructures, and strategies to construct ordered protein assemblies have been rapidly expanding. In this review, we provide an overview of very recent studies in the field of artificial protein assemblies, with the particular aim of introducing major assembly methods and unique features of these assemblies. Computational de novo designs were used to build various assemblies with artificial protein building blocks, which are unrelated to natural proteins. Small chemical ligands and metal ions have also been extensively used for strong and bio-orthogonal protein linking. Here, in addition to protein assemblies with well-defined sizes, protein oligomeric and array structures with rather undefined sizes (but with definite repeat protein assembly units) also will be discussed in the context of well-defined protein nanostructures. Lastly, we will introduce multiple examples showing how protein assemblies can be effectively used in various fields such as therapeutics and vaccine development. We believe that structures and functions of artificial protein assemblies will be continuously evolved, particularly according to specific application goals.

Experience of a Neuro-Emergency Expert in the Emergency Department during One Year of the COVID-19 Pandemic.

We aimed to evaluate the overall clinical characteristics of patients treated by a neuro-emergency expert dedicated to the emergency department (ED) as an attending neurologist during the COVID-19 pandemic. We included adult patients who visited the ED between 1 January and 31 December 2020 and were treated by a neuro-emergency expert. We retrospectively obtained and analyzed the data on patients' clinical characteristics and outcome. The neuro-emergency expert treated 1155 patients (mean age, 62.9 years). The proportion of aged 18-40 years was the lowest, and the most common modes of arrival were public ambulance (50.6%) and walk-in (42.3%). CT and MRI examinations were performed in 94.4 and 33.1% of cases, respectively. The most frequent complaints were dizziness (31.8%), motor weakness (24.2%), and altered mental status (15.8%). The ED diagnoses were acute ischemic stroke (19.8%), benign paroxysmal positional vertigo (14.2%), vestibular neuritis (9.9%), and seizure (8.8%). The mean length of stay in the ED was 207 min. Of the patients, 55.0% were admitted to the hospital, and 41.8% were discharged for outpatient follow-up. Despite the longer stay and the complexity and difficulty of neurological diseases during the COVID-19 pandemic, the accurate diagnosis and treatment provided by a neuro-emergency expert can be presented as a good model in the ED.

Quantification of tire wear particles in road dust from industrial and residential areas in Seoul, Korea.

In this study, we examined tire and road wear microparticles (TRWMPs) in road dust along the Seoul metropolitan area, from industrial and residential areas. The road dust samples were collected via vacuum sweep methods and then filtered to obtain particles with diameters less than 75 µm. To quantify the TRWMPs in road dust, we used the raw materials of tire components, natural rubber (NR), and styrene-butadiene rubber (SBR), as standard materials. We evaluated the usability of the pyrolyzer-gas chromatography/mass spectrometry py-GC/MS method introduced in ISO/TS 20593 by confirming the decomposition temperatures of the NR and SBR by thermogravimetric (TG) and evolved gas analysis (EGA)-MS. The average of TRWMPs in industrial and residential area road dust were 22,581 and 9818 µg/g, respectively, indicating that the industrial area has 2.5 times higher TRWMPs concentration. Further, the NR, the main component of truck bus radial, to SBR, the main component of passenger car radial, ratio was slightly higher in the industrial area than in the residential area. This presumably means that the high traffic volume, including heavy duty vehicles in industrial areas, affected the higher concentration of TRWMPs. This study reveals the growing evidence of the importance of TRWMPs in road dust and how TRWMPs quantity can impact the air quality of the Seoul metropolitan area.

Development of PM10 and PM2.5 cyclones for small sampling ports at stationary sources: Numerical and experimental study.

Air pollution caused by particulate matter (PM) has become a serious issue, and significant research has focused on managing large stationary emission sources, i.e., the primary sources of PM. Currently, the U.S. Environmental Protection Agency (EPA) Method 201A and ISO 23210 are predominantly employed to measure the PM emissions at large stationary sources. Method 201A is designated as a standard test method in Korea, but it is difficult to measure PM10 and PM2.5 simultaneously owing to the size of the full-set cyclone. In large stationary emission sources, the use of a serial connection of PM10 and PM2.5 cyclones is unsuitable for measurements at conventional sampling ports featuring diameters of approximately 100 mm. Therefore, in this study, PM10 and PM2.5 cyclones were developed to replace the cyclones currently used in Method 201A. The developed cyclones featured a cutoff diameter, which was confirmed by numerical and experimental analyses that were close to Method 201A. Moreover, there was an increase in the stiffness of collection efficiency. The hook adaptor, which is a key accessory used in Method 201A, was found to be applicable to the newly developed cyclones. This alternative method will help reduce the measurement time by simultaneously measuring TSP, PM10, and PM2.5 and eliminates the costs of installing or refurbishing additional sampling ports at existing large stationary sources.

Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions.

Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated via the classical concept of liquid-liquid phase separation, biomolecular behaviors such as protein interactions inside these liquid compartments have been largely unexplored. Here we report quantitative measurements of changes in protein interactions for the proteins recruited into membrane-less compartments (termed client proteins) in living cells. Under a wide range of phase separation conditions, protein interaction signals were vastly increased only inside compartments, indicating greatly enhanced proximity between recruited client proteins. By employing an in vitro phase separation model, we discovered that the operational proximity of clients (measured from client-client interactions) could be over 16 times higher than the expected proximity from actual client concentrations inside compartments. We propose that two aspects should be considered when explaining client proximity enhancement by phase separation compartmentalization: (1) clients are selectively recruited into compartments, leading to concentration enrichment, and more importantly, (2) recruited clients are further localized around compartment-forming scaffold protein networks, which results in even higher client proximity.

Behavior control of membrane-less protein liquid condensates with metal ion-induced phase separation.

Phase separation of specific biomolecules into liquid droplet-like condensates is a key mechanism to form membrane-less organelles, which spatio-temporally organize diverse biochemical processes in cells. To investigate the working principles of these biomolecular condensates as dynamic reaction centers, precise control of diverse condensate properties is essential. Here, we design a strategy for metal ion-induced clustering of minimal protein modules to produce liquid protein condensates, the properties of which can be widely varied by simple manipulation of the protein clustering systems. The droplet forming-minimal module contains only a single receptor protein and a binding ligand peptide with a hexahistidine tag for divalent metal ion-mediated clustering. A wide range of protein condensate properties such as droplet forming tendency, droplet morphology, inside protein diffusivity, protein recruitment, and droplet density can be varied by adjusting the nature of receptor/ligand pairs or used metal ions, metal/protein ratios, incubation time, binding motif variation on recruited proteins, and even spacing between receptor/ligand pairs and the hexahistidine tag. We also demonstrate metal-ion-induced protein phase separation in cells. The present phase separation strategy provides highly versatile protein condensates, which will greatly facilitate investigation of molecular and structural codes of droplet-forming proteins and the monitoring of biomolecular behaviors inside diverse protein condensates.

Multivalent-Interaction-Driven Assembly of Discrete, Flexible, and Asymmetric Supramolecular Protein Nano-Prisms.

Current approaches to design monodisperse protein assemblies require rigid, tight, and symmetric interactions between oligomeric protein units. Herein, we introduce a new multivalent-interaction-driven assembly strategy that allows flexible, spaced, and asymmetric assembly between protein oligomers. We discovered that two polygonal protein oligomers (ranging from triangle to hexagon) dominantly form a discrete and stable two-layered protein prism nanostructure via multivalent interactions between fused binding pairs. We demonstrated that protein nano-prisms with long flexible peptide linkers (over 80 amino acids) between protein oligomer layers could be discretely formed. Oligomers with different structures could also be monodispersely assembled into two-layered but asymmetric protein nano-prisms. Furthermore, producing higher-order architectures with multiple oligomer layers, for example, 3-layered nano-prisms or nanotubes, was also feasible.

A new air-washing method to clean fabric filters clogged with submicron fume particles: A pilot-scale study.

This study investigates a new air-washing cleaning system that directly injects compressed air on the filter surface for filter regeneration in a fabric filter (FF) dust collector. A pilot-scale FF is designed to test the new system and to compare it with the conventional pulse-jet cleaning system with regard to filter clogging by fume particles. A pleated filter with a filtration area of 2.4 m2 is installed in the FF and a thermal steel spraying gun is used to supply the fume particles. Pressure drop and particle emission concentration are monitored to examine the effect of the new system on filter regeneration and collection efficiency. The results show that the air-washing cleaning is effective for filter regeneration, as it allows the FF to operate stably for a long time, whereas the pulse-jet cleaning fails to achieve filter regeneration, resulting in a continuously increasing pressure drop. In addition, air-washing cleaning shows better performance on collection efficiency than the pulse-jet cleaning method, as it reduces the outlet particulate matter concentration to less than half that of the pulse-jet cleaning.

Determination of the emission rate for ultrafine and accumulation mode particles as a function of time during the pan-frying of fish.

Particulate matter (PM) from cooking is considered one of the most harmful indoor air pollutants causing numerous adverse health effects, and it is essential to comprehend the characteristics of the particles generated from cooking to prevent these problems. In this study, we investigated PM from the pan-frying of salmon using number concentration and developed emission rates as a function of time for ultrafine particles (UFPs < 100 nm) and accumulation mode particles (AMPs 0.1-1 µm). The newly defined emission rates vary significantly with time and are very different from the conventionally determined rates that do not consider the variation of particle concentration with time. The emission rate of UFPs decreased over time after a sharp rise, whereas that of AMPs continued to increase, resulting in a change in the proportions of UFPs and AMPs in the total PM from 93 to 7% to 72 and 28%, respectively. Particle-particle interactions such as coagulation and coalescence were observed between primary particles via high resolution transmission electron microscopy (HR-TEM), which is a plausible reason for the decreasing emission rate of UFPs with time. The emission rate as a function of time can serve as a tool to estimate PM from cooking, as well as to monitor the change trends through phenomena such as agglomeration.

Interplay between intrinsically disordered proteins inside membraneless protein liquid droplets.

Intrinsically disordered proteins (IDPs) in cells phase separate to form diverse membraneless organelles, which have condensed liquid droplet-like properties and often contain multiple IDPs. However, how potential interactions between different IDPs affect the dynamic behavior of these protein droplets is largely unknown. Here, we develop a rapid IDP clustering system to generate protein droplets with varied residue compositions and examine diverse interacting IDPs inside droplets. Three different IDP droplets actively recruited other diverse IDPs inside droplets with extremely varied enrichment (inside/outside) degrees (over 100-fold variation) under highly crowded conditions. The recruited IDPs were mostly mobile even inside highly immobile droplets. Among the five tested IDPs, the disordered region of Ddx4 helicase with its unique multiple charged residue blocks was noticeably influenced by droplet mobility. We also discovered that droplets of different IDPs could rapidly fuse to each other. Interestingly, some droplets were heterogeneously fused with segregated subcompartments, and this segregation was enhanced by droplet maturation and was more apparent for specific IDP pairs, in which the polar and charged residue compositions are highly different. The present study not only reports multiple peculiar behaviors of interacting IDP pairs inside droplets but also provides valuable information on generating membraneless organelle models with controllable droplet properties.

Monomeric Covalent-Avidin for Rapid and Covalent Labeling of Quantum Dots to Cell Surface Proteins.

With high brightness and photostability, quantum dots (QDs) are potent probes for long-term imaging of dynamic cell surface proteins, but practical methods to covalently label QDs to target proteins for stable imaging are largely lacking. Here, a small covalent-bond forming protein (Covalent-avidin)/peptide pair is introduced, which provides a recombinant protein-based rapid and covalent QD labeling strategy. Covalent-avidin is constructed by optimized fusion of circular permuted monomeric avidin to SpyCatcher, which forms an isopeptide bond with the SpyTag peptide. Covalent-avidin-conjugated QDs allow for strong and irreversible QD labeling to the biotinylated SpyTag-fused adrenergic receptor on live cells in 2 min. In addition, QDs with only a minimum number of conjugated Covalent-avidin show more stable receptor labeling than commercially available streptavidin-conjugated QDs, also with minimal unwanted clustering of labeled receptors. Monomeric Covalent-avidin will be a valuable protein linker for diverse other nanolabeling structures with beneficial properties such as covalent linkages and facile valency control.