Exploring single-molecule interactions: heparin and FGF-1 proteins through solid-state nanopores.

Detection and characterization of protein-protein interactions are essential for many cellular processes, such as cell growth, tissue repair, drug delivery, and other physiological functions. In our research, we have utilized emerging solid-state nanopore sensing technology, which is highly sensitive to better understand heparin and fibroblast growth factor 1 (FGF-1) protein interactions at a single-molecule level without any modifications. Understanding the structure and behavior of heparin-FGF-1 complexes at the single-molecule level is very important. An abnormality in their formation can lead to life-threatening conditions like tumor growth, fibrosis, and neurological disorders. Using a controlled dielectric breakdown pore fabrication approach, we have characterized individual heparin and FGF-1 (one of the 22 known FGFs in humans) proteins through the fabrication of 17 ± 1 nm nanopores. Compared to heparin, the positively charged heparin-binding domains of some FGF-1 proteins translocationally react with the pore walls, giving rise to a distinguishable second peak with higher current blockade. Additionally, we have confirmed that the dynamic FGF-1 is stabilized upon binding with heparin-FGF-1 at the single-molecule level. The larger current blockades from the complexes relative to individual heparin and the FGF-1 recorded during the translocation ensure the binding of heparin-FGF-1 proteins, forming binding complexes with higher excluded volumes. Taken together, we demonstrate that solid-state nanopores can be employed to investigate the properties of individual proteins and their complex interactions, potentially paving the way for innovative medical therapies and advancements.

An Importance-Performance Analysis of Korean Middle School Students' Health Management Awareness during the Post-COVID-19 Era Depending on Sex.

This study aimed to conduct importance-performance analyses (IPAs) based on Korean middle school students' health management awareness during the post-coronavirus disease 2019 (COVID-19) era. Data were collected from 867 Korean middle school students (13-15 years old) via online and offline surveys between May and June 2023. Frequency analysis, reliability analysis, IPA based on the entire student group, and IPA depending on sex were carried out with the collected data, which revealed the following. First, regardless of sex, the IPA results indicated that four factors of mental health were located in the third quadrant, with one factor of the same variable in the fourth quadrant. The three factors of disease management were located in the third quadrant. Regarding physical activity, two factors were located in the first quadrant, one in the second quadrant, and one in the third quadrant. Regarding sleep management, two factors were located in the second quadrant, one in the third quadrant, and one in the first quadrant. Regarding eating management, two factors were located in the third quadrant and one in the fourth quadrant. Regarding the social distancing variable, all four factors were located in the third quadrant. Regarding hygiene management, two factors were located in the first quadrant, one in the third quadrant, and one in the fourth quadrant. Furthermore, the IPA results indicated sex differences in regular sports and vigorous movement activities associated with physical activity. Additionally, a sex difference was observed in regular diet associated with eating management. This study proposed possible measures for encouraging middle school students to recognize the importance of health and increase their health-related performance during the COVID-19 endemic phase.

A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion.

Fluorescence resonance energy transfer (FRET) reporters are commonly used in the final stages of nucleic acid amplification tests to indicate the presence of nucleic acid targets, where fluorescence is restored by nucleases that cleave the FRET reporters. However, the need for dual labelling and purification during manufacturing contributes to the high cost of FRET reporters. Here we demonstrate a low-cost silver nanocluster reporter that does not rely on FRET as the on/off switching mechanism, but rather on a cluster transformation process that leads to fluorescence color change upon nuclease digestion. Notably, a 90 nm red shift in emission is observed upon reporter cleavage, a result unattainable by a simple donor-quencher FRET reporter. Electrospray ionization-mass spectrometry results suggest that the stoichiometric change of the silver nanoclusters from Ag13 (in the intact DNA host) to Ag10 (in the fragments) is probably responsible for the emission colour change observed after reporter digestion. Our results demonstrate that DNA-templated silver nanocluster probes can be versatile reporters for detecting nuclease activities and provide insights into the interactions between nucleases and metallo-DNA nanomaterials.

Cloning and Expression Analysis of Bioluminescence Genes in Omphalotus guepiniiformis Reveal Stress-Dependent Regulation of Bioluminescence.

Bioluminescence is a type of chemiluminescence that arises from a luciferase-catalyzed oxidation reaction of luciferin. Molecular biology and comparative genomics have recently elucidated the genes involved in fungal bioluminescence and the evolutionary history of their clusters. However, most studies on fungal bioluminescence have been limited to observing the changes in light intensity under various conditions. To understand the molecular basis of bioluminescent responses in Omphalotus guepiniiformis under different environmental conditions, we cloned and sequenced the genes of hispidin synthase, hispidin-3-hydroxylase, and luciferase enzymes, which are pivotal in the fungal bioluminescence pathway. Each gene showed high sequence similarity to that of other luminous fungal species. Furthermore, we investigated their transcriptional changes in response to abiotic stresses. Wound stress enhanced the bioluminescence intensity by increasing the expression of bioluminescence pathway genes, while temperature stress suppressed the bioluminescence intensity via the non-transcriptional pathway. Our data suggested that O. guepiniiformis regulates bioluminescence to respond differentially to specific environmental stresses. To our knowledge, this is the first study on fungal bioluminescence at the gene expression level. Further studies are required to address the biological and ecological meaning of different bioluminescence responses in changing environments, and O. quepiniiformis could be a potential model species.

Real-time monitoring of Ti(IV) metal ion binding of transferrin using a solid-state nanopore.

Transferrin, a central player in iron transport, has been recognized not only for its role in binding iron but also for its interaction with other metals, including titanium. This study employs solid-state nanopores to investigate the binding of titanium ions [Ti(IV)] to transferrin in a single-molecule and label-free manner. We demonstrate the novel application of solid-state nanopores for single-molecule discrimination between apo-transferrin (metal-free) and Ti(IV)-transferrin. Despite their similar sizes, Ti(IV)-transferrin exhibits a reduced current drop, attributed to differences in translocation times and filter characteristics. Single-molecule analysis reveals Ti(IV)-transferrin's enhanced stability and faster translocations due to its distinct conformational flexibility compared to apo-transferrin. Furthermore, our study showcases solid-state nanopores as real-time monitors of biochemical reactions, tracking the gradual conversion of apo-transferrin to Ti(IV)-transferrin upon the addition of titanium citrate. This work offers insights into Ti(IV) binding to transferrin, promising applications for single-molecule analysis and expanding our comprehension of metal-protein interactions at the molecular level.

Navigation and Control of Motion Modes with Soft Microrobots at Low Reynolds Numbers.

This study investigates the motion characteristics of soft alginate microrobots in complex fluidic environments utilizing wireless magnetic fields for actuation. The aim is to explore the diverse motion modes that arise due to shear forces in viscoelastic fluids by employing snowman-shaped microrobots. Polyacrylamide (PAA), a water-soluble polymer, is used to create a dynamic environment with non-Newtonian fluid properties. Microrobots are fabricated via an extrusion-based microcentrifugal droplet method, successfully demonstrating the feasibility of both wiggling and tumbling motions. Specifically, the wiggling motion primarily results from the interplay between the viscoelastic fluid environment and the microrobots' non-uniform magnetization. Furthermore, it is discovered that the viscoelasticity properties of the fluid influence the motion behavior of the microrobots, leading to non-uniform behavior in complex environments for microrobot swarms. Through velocity analysis, valuable insights into the relationship between applied magnetic fields and motion characteristics are obtained, facilitating a more realistic understanding of surface locomotion for targeted drug delivery purposes while accounting for swarm dynamics and non-uniform behavior.

Over One Million DNA and Protein Events Through Ultra-Stable Chemically-Tuned Solid-State Nanopores.

Stability, long lifetime, resilience against clogging, low noise, and low cost are five critical cornerstones of solid-state nanopore technology. Here, a fabrication protocol is described wherein >1 million events are obtained from a single solid-state nanopore with both DNA and protein at the highest available lowpass filter (LPF, 100 kHz) of the Axopatch 200B-the highest event count mentioned in literature. Moreover, a total of ≈8.1 million events are reported in this work encompassing the two analyte classes. With the 100 kHz LPF, the temporally attenuated population is negligible while with the more ubiquitous 10 kHz, ≈91% of the events are attenuated. With DNA experiments, the pores are operational for hours (typically >7 h) while the average pore growth is merely ≈0.16 ± 0.1 nm h-1 . The current noise is exceptionally stable with traces typically showing <10 pA h-1 increase in noise. Furthermore, a real-time method to clean and revive pores clogged with analyte with the added benefit of minimal pore growth during cleaning (< 5% of the original diameter) is showcased. The enormity of the data collected herein presents a significant advancement to solid-state pore performance and will be useful for future ventures such as machine learning where large amounts of pristine data are a prerequisite.

Kynurenine pathway can be a potential biomarker of fatigue in primary Sjögren's syndrome.

OBJECTIVES: Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease with low quality of life caused by various constitutional symptoms and glandular dysfunction. Although fatigue is one of the most frequent symptoms in pSS, its aetiology or biomarkers are poorly elucidated. We investigated potential relationship between severity of fatigue and the kynurenine pathway in pSS. METHODS: Clinical data and blood samples of 81 patients were obtained from a prospective cohort for pSS and compared with age- and sex-matched healthy controls (HC). Severity of fatigue was defined according to the fatigue domain scores in the ESSPRI. Potential biomarkers related to the kynurenine pathway were determined using ELISA. RESULTS: Of the total, 44 patients were defined as the "severe fatigue (ESSPRI fatigue ≥ 5)" group, whereas 37 as the "less fatigue (ESSPRI fatigue < 5)". Serum tryptophan levels in the severe fatigue group were significantly lower while those of kynurenine were higher. Serum interferon gamma, IDO1, and quinolinic acid levels were mostly higher in the less fatigue group. Kynurenine/tryptophan ratios were distinctly higher in the severe fatigue group than both HC and the less fatigue group (p < 0.001). This ratio showed a strong degree of positive correlation (r = 0.624, p < 0.001) with severity of fatigue in pSS while the other markers showed fair degrees of correlation. CONCLUSIONS: Serum markers related to the kynurenine pathway, especially the kynurenine/tryptophan ratio, may be associated with severity of fatigue in pSS. These results can provide guidance for further investigations on fatigue in pSS.

Use of a solid-state nanopore for profiling the transferrin receptor protein and distinguishing between transferrin receptor and its ligand protein.

A nanopore device is capable of providing single-molecule level information of an analyte as they translocate through the sensing aperture-a nanometer-sized through-hole-under the influence of an applied electric field. In this study, a silicon nitride (Six Ny )-based nanopore was used to characterize the human serum transferrin receptor protein (TfR) under various applied voltages. The presence of dimeric forms of TfR was found to decrease exponentially as the applied electric field increased. Further analysis of monomeric TfR also revealed that its unfolding behaviors were positively dependent on the applied voltage. Furthermore, a comparison between the data of monomeric TfR and its ligand protein, human serum transferrin (hSTf), showed that these two protein populations, despite their nearly identical molecular weights, could be distinguished from each other by means of a solid-state nanopore (SSN). Lastly, the excluded volumes of TfR were experimentally determined at each voltage and were found to be within error of their theoretical values. The results herein demonstrate the successful application of an SSN for accurately classifying monomeric and dimeric molecules while the two populations coexist in a heterogeneous mixture.

Spontaneous symmetry breaking propulsion of chemically coated magnetic microparticles.

Chemically coated micro/nanoparticles are often used in medicine to enhance drug delivery and increase drug up-take into specific areas of the body. Using a recently discovered spontaneous symmetry breaking propulsion mechanism, we demonstrate that chemically coated microparticles can swim through mucus solution under precise navigation and that certain functionalizations can dynamically change propulsion behavior. For this investigation biotin, Bitotin-PEG3-amine, and biotin chitosan were chemically functionalized onto the surfaces of magnetic microparticles using an avidin-biotin complex. These chemicals were chosen because they are used prolifically in drug delivery applications, with PEG and chitosan having well known mucoadhesive effects. Coated microparticles were then suspended in mucus synthesized from porcine stomach mucins and propelled using rotating magnetic fields. The relationship between different chemical coatings, microparticle velocity, and controllability were thoroughly explored and discussed. Results indicate that the biotinylated surface coatings altered the propulsion behavior of microparticles, with performance differences interlinked to both magnetic field properties and localized mucus properties. Precisely controlled drug carrying microparticles are envisioned to help supplant traditional drug delivery methods and enhance existing medical techniques utilizing micro/nanoparticles.

Bacteria-inspired magnetically actuated rod-like soft robot in viscous fluids.

This paper seeks to design, develop, and explore the locomotive dynamics and morphological adaptability of a bacteria-inspired rod-like soft robot propelled in highly viscous Newtonian fluids. The soft robots were fabricated as tapered, hollow rod-like soft scaffolds by applying a robust and economic molding technique to a polyacrylamide-based hydrogel polymer. Cylindrical micro-magnets were embedded in both ends of the soft scaffolds, which allowed bending (deformation) and actuation under a uniform rotating magnetic field. We demonstrated that the tapered rod-like soft robot in viscous Newtonian fluids could perform two types of propulsion; boundary rolling was displayed when the soft robot was located near a boundary, and swimming was displayed far away from the boundary. In addition, we performed numerical simulations to understand the swimming propulsion along the rotating axis and the way in which this propulsion is affected by the soft robot's design, rotation frequency, and fluid viscosity. Our results suggest that a simple geometrical asymmetry enables the rod-like soft robot to perform propulsion in the low Reynolds number (Re≪ 1) regime; these promising results provide essential insights into the improvements that must be made to integrate the soft robots into minimally invasivein vivoapplications.

Rolling Motion of a Soft Microsnowman under Rotating Magnetic Field.

This paper demonstrates a manipulation of snowman-shaped soft microrobots under a uniform rotating magnetic field. Each microsnowman robot consists of two biocompatible alginate microspheres with embedded magnetic nanoparticles. The soft microsnowmen were fabricated using a microfluidic device by following a centrifuge-based microfluidic droplet method. Under a uniform rotating magnetic field, the microsnowmen were rolled on the substrate surface, and the velocity response for increasing magnetic field frequencies was analyzed. Then, a microsnowman was rolled to follow different paths, which demonstrated directional controllability of the microrobot. Moreover, swarms of microsnowmen and single alginate microrobots were manipulated under the rotating magnetic field, and their velocity responses were analyzed for comparison.

Diagnosis of Central Sensitization and Its Effects on Postoperative Outcomes following Total Knee Arthroplasty: A Systematic Review and Meta-Analysis.

Central sensitization (CS) has been extensively researched as a cause of persistent pain after total knee arthroplasty (TKA). This systematic review study sought to investigate the diagnosis of CS in patients who underwent TKA for knee osteoarthritis (OA) and the effect of CS on clinical outcomes after TKA. Three comprehensive databases, including MEDLINE, EMBASE, and the Cochrane Library, were searched for studies that evaluated the outcomes of TKA in knee OA patients with CS. Data extraction, risk of bias assessment, and (where appropriate) meta-analysis were performed. The standardized mean difference (SMD) with a 95% confidence interval was used to assess the different scales of pain. A total of eight studies were selected, including two retrospective studies and five prospective observational studies. One study used additional randomized controlled trial data. Five studies were finally included in the meta-analysis. All studies had a minimum follow-up period of 3 months. The Central Sensitization Inventory (CSI), whole-body pain diagram, and quantitative sensory testing (QST) were used for measuring CS. The pooled analysis showed that patients with CS had more severe postoperative pain after TKA (SMD, 0.65; 95% CI, 0.40−0.90; p < 0.01) with moderate heterogeneity (I2 = 60%). In patients who underwent TKA with knee OA, CSI is most often used for the diagnosis of CS, and the QST and whole-body pain diagram are also used. CS is closely associated with more severe and persistent pain after TKA.

Heme oxygenase-1 gene delivery for altering high mobility group box-1 protein in pancreatic islet.

Pancreatic islet transplantation is a promising strategy for the treatment of type I diabetes. High-mobility group box-1 (HMGB1), highly expressed in islet cells, is a potent immune stimulator in immune rejection. Heme oxygenase-1 (HO1) gene therapy can modulate the release of HMGB1 by altering intracellular molecules for successful cell transplantation. After delivery of the heme oxygenase-1 (HO1) gene to islet cells using an adeno-associated viral vector (AAV), it was evaluated the changes in cytoplasmic Ca2+ ions and calcineurin activity as well as histone acetyltransferase (HAT) and Poly(ADP) ribose polymerase-1 (PARP-1). Inhibition of HMGB1 release was evaluated through altering these intracellular molecules. Then, after transplantation of HO1-transduced islets, the therapeutic effect of them was evaluated through measuring blood glucose level to diabetic mice and through immunohistochemical analysis. The transduced HO1 gene significantly inhibited HMGB1 release in islets that was under the cell damage by hypoxia exposure. It was confirmed that this result was initially due to the decrease in cytoplasmic Ca2+ ion concentration and calcineurin activity. In addition, the delivered HO1 gene simultaneously reduced the activity of HAT and PARP-1, which are involved in the translocation of HMGB1 from the nucleus to the cytoplasm. As a result, when the HO1 gene-transduced islets were transplanted into diabetic mice, the treatment efficiency of diabetes was effectively improved by increasing the survival rate of the islets. Collectively, these results suggest that HO1 gene transfer can be used for successful islet transplantation by altering the activity of intracellular signal molecules and reducing HMGB1 release.

Self-Induced Back-Action Actuated Nanopore Electrophoresis (SANE) Sensor for Label-Free Detection of Cancer Immunotherapy-Relevant Antibody-Ligand Interactions.

We fabricated a novel single molecule nanosensor by integrating a solid-state nanopore and a double nanohole nanoaperture. The nanosensor employs Self-Induced Back-Action (SIBA) for optical trapping and enables SIBA-Actuated Nanopore Electrophoresis (SANE) for concurrent acquisition of bimodal optical and electrical signatures of molecular interactions. This work describes how to fabricate and use the SANE sensor to quantify antibody-ligand interactions. We describe how to analyze the bimodal optical-electrical data to improve upon the discrimination of antibody and ligand versus bound complex compared to electrical measurements alone. Example results for specific interaction detection are described for T-cell receptor-like antibodies (TCRmAbs) engineered to target peptide-presenting Major Histocompatibility Complex (pMHC) ligands, representing a model of target ligands presented on the surface of cancer cells. We also describe how to analyze the bimodal optical-electrical data to discriminate between specific and non-specific interactions between antibodies and ligands. Example results for non-specific interactions are shown for cancer-irrelevant TCRmAbs targeting the same pMHCs, as a control. These example results demonstrate the utility of the SANE sensor as a potential screening tool for ligand targets in cancer immunotherapy, though we believe that its potential uses are much broader.

Investigating protein translocation in the presence of an electrolyte concentration gradient across a solid-state nanopore.

Electrolyte chemistry plays an important role in the transport properties of analytes through nanopores. Here, we report the translocation properties of the protein human serum transferrin (hSTf) in asymmetric LiCl salt concentrations with either positive (Ctrans /Ccis < 1) or negative chemical gradients (Ctrans /Ccis > 1). The cis side concentration was fixed at 4 M for positive chemical gradients and at 0.5 M LiCl for negative chemical gradients, while the trans side concentration varied between 0.5 to 4 M which resulted in six different configurations, respectively, for both positive and negative gradient types. For positive chemical gradient conditions, translocations were observed in all six configurations for at least one voltage polarity whereas with negative gradient conditions, dead concentrations where no events at either polarity were observed. The flux of Li+ and Cl- ions and their resultant cation or anion enrichment zones, as well as the interplay of electrophoretic and electroosmotic transport directions, would determine whether hSTf can traverse across the pore.

An Analysis of Health Perceptions and Performance in Elementary Students in Korea during the ongoing COVID-19 Pandemic.

During the coronavirus disease 2019 (COVID-19) pandemic, social distancing guidelines changed lifestyles, including increased sedentary time, physical inactivity, and disrupted sleep patterns among children. The purpose of the present study is to analyze the health awareness (mental health, disease, physical activity, sleep, eating habit, and hygiene health management) of elementary school students during the COVID-19 pandemic, and use the importance-performance analysis (IPA) technique to identify gender differences in health perceptions. We collected data on 1006 students, which was analyzed using frequency analysis, reliability testing, independent sample t-tests, and importance-performance analysis (IPA). A median importance value of 0.163 and a median performance value of 4.048 were selected as cross points to distribute the IPA matrix into four quadrants. The highest performance was given for wearing a mask and sanitary practice; the IPA matrix indicated that the sense of belonging, happiness, trust, and movement activity were located in quadrant I. Children's regular physical activity and level of physical activity were low, especially that of girls. Children's sleep management was poor. Their physical activity and sleep-related factors must be improved under the facilitation of the national government, public education institutions, and families.

Fine-tuning-based Transfer Learning for Characterization of Adeno-Associated Virus.

Accurate and precise identification of adeno-associated virus (AAV) vectors play an important role in dose-dependent gene therapy. Although solid-state nanopore techniques can potentially be used to characterize AAV vectors by capturing ionic current, the existing data analysis techniques fall short of identifying them from their ionic current profiles. Recently introduced machine learning methods such as deep convolutional neural network (CNN), developed for image identification tasks, can be applied for such classification. However, with smaller data set for the problem in hand, it is not possible to train a deep neural network from scratch for accurate classification of AAV vectors. To circumvent this, we applied a pre-trained deep CNN (GoogleNet) model to capture the basic features from ionic current signals and subsequently used fine-tuning-based transfer learning to classify AAV vectors. The proposed method is very generic as it requires minimal preprocessing and does not require any handcrafted features. Our results indicate that fine-tuning-based transfer learning can achieve an average classification accuracy between 90 and 99% in three realizations with a very small standard deviation. Results also indicate that the classification accuracy depends on the applied electric field (across nanopore) and the time frame used for data segmentation. We also found that the fine-tuning of the deep network outperforms feature extraction-based classification for the resistive pulse dataset. To expand the usefulness of the fine-tuning-based transfer learning, we have tested two other pre-trained deep networks (ResNet50 and InceptionV3) for the classification of AAVs. Overall, the fine-tuning-based transfer learning from pre-trained deep networks is very effective for classification, though deep networks such as ResNet50 and InceptionV3 take significantly longer training time than GoogleNet.

Propulsion of magnetically actuated achiral planar microswimmers in Newtonian and non-Newtonian fluids.

Magnetic achiral planar microswimmers can be massively fabricated at low cost and are envisioned to be useful for in vivo biomedical applications. To understand locomotion in representative in vivo environments, we investigated the swimming performance of achiral planar microswimmers in methylcellulose solutions. We observed that these microswimmers displayed very similar swimming characteristics in methylcellulose solutions as in water. Furthermore, this study indicated that the range of precession angles increased as the concentration of MC solution increased. Last, it was demonstrated that achiral planar microswimmers with similar precession angles exhibited nearly the same dimensionless speeds in different concentrations of the methylcellulose solutions. Upon understanding swimmer kinematics, more effective control over the achiral planar microswimmers can be achieved to perform multiple biomedical tasks in in vivo environments.

Flagellated Janus particles for multimodal actuation and transport.

Catalytic Janus particles rely on chemical decomposition to self-propel and have displayed enormous potential for targeted drug delivery and cellular penetration. Catalytic propulsion mechanisms are limiting, however, with fuel requirements and specialized fluid properties being necessary to achieve propulsion. We have improved the dynamic propulsion of catalytic Janus particles by functionalizing flagellar filaments to one of their hemispheres. Flagellated Janus particles, torqued by rotating magnetic fields, swim along their rotation axis using the explicit chirality and flexibility of flagella, mimicking flagellar rotation of live bacteria. Depending on the working fluid, flagellated Janus particles can propel using either catalytic or swimming propulsion. We demonstrate experimentally that flagellated Janus particles behave predictably under the two actuation modes and can precisely follow trajectories under closed-loop feedback control. Flagellated Janus particles were demonstrated to swim in both Newtonian and shear-thickening fluids. These are the first Janus particles developed that can be propelled interchangeably between catalytic and flagellar swimming propulsion, allowing two distinct propulsion mechanisms for future use within in vivo operations.