The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response.

The induction of type I interferons by the bacterial secondary messengers cyclic di-GMP (c-di-GMP) or cyclic di-AMP (c-di-AMP) is dependent on a signaling axis that involves the adaptor STING, the kinase TBK1 and the transcription factor IRF3. Here we identified the heliase DDX41 as a pattern-recognition receptor (PRR) that sensed both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacted with c-di-GMP. Knockdown of DDX41 via short hairpin RNA in mouse or human cells inhibited the induction of genes encoding molecules involved in the innate immune response and resulted in defective activation of STING, TBK1 and IRF3 in response to c-di-GMP or c-di-AMP. Our results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by DDX41, which forms a complex with STING to signal to TBK1-IRF3 and activate the interferon response.

Enhancing Extracellular Vesicle Analysis by Integration of Large-Volume Sample Stacking in Capillary Electrophoresis with Asymmetrical Flow Field-Flow Fractionation.

Extracellular vesicles (EVs) play important roles in cell-cell communication and pathological development. Cargo profiling for the EVs present in clinical specimens can provide valuable insights into their functions and help discover effective EV-based markers for diagnostic and therapeutic purposes. However, the highly abundant and complex matrix components pose significant challenges for specific identification of low-abundance EV cargos. Herein, we combine asymmetrical flow field-flow fractionation (AF4) with large-volume sample stacking and capillary electrophoresis (LVSS/CE), to attain EVs with high purity for downstream protein profiling. This hyphenated system first separates the EVs from the contamination of smaller serum proteins by AF4, and second resolves the EVs from the coeluted, nonvesicular matrix components by CE following LVSS. The optimal LVSS condition permits the injection of 10-fold more EVs into CE compared to the nonstacking condition without compromising separation resolution. Collection and downstream analysis of the highly pure EVs after CE separation were demonstrated in the present work. The high EV purity yields a much-improved labeling efficiency when detected by fluorescent antibodies compared to those collected from the one-dimension separation of AF4, and permits the identification of more EV-specific cargos by LC-MS/MS compared to those isolated by ultracentrifugation (UC), the exoEasy Maxi Kit, and AF4. Our results strongly support that AF4-LVSS/CE can improve EV isolation and cargo analysis, opening up new opportunities for understanding EV functions and their applications in the biomedical fields.

Combining Excellent Selectivity with Broad Target Scope: Biosensing with Arrayed Deep Cavitand Hosts.

Simple macrocyclic water-soluble hosts such as cucurbiturils, cyclophanes, and calixarenes have long been used for biosensing via indicator displacement assays. Using multiple hosts and dyes in an arrayed format allows pattern recognition-based "chemical nose" sensing, which confers exquisite selectivity, even rivaling the abilities of biological recognition tools such as antibodies. However, a challenge in indicator displacement-based biosensing with macrocyclic hosts is that selectivity and scope are often inversely correlated: strong selectivity for a specific target can limit wide application, and broad scope sensing can suffer from a lack of selectivity between similar targets. This problem can be addressed by using water-soluble, self-folding deep cavitands as hosts. These flexible bowl-shaped receptors can be easily functionalized with different motifs at the upper and lower rim, and the large cavities can bind many different fluorescent dyes, causing either fluorescence enhancement or quenching upon binding.Cavity-based affinity is strongest for NMe3+ groups such as trimethyl-lysine, and we have exploited this for the site-selective recognition of post-translational lysine methylations in oligopeptides. The host recognizes the NMe3+ group, and by applying differently functionalized hosts in an arrayed format, discrimination between identical modifications at different positions on the oligopeptide is possible. Multiple recognition elements can be exploited for selectivity, including a defined, yet "breathable" cavity, and variable upper rim functions oriented toward the target.While the performance of the host/guest sensing system is impressive for lysine methylations, the most important advance is the use of multiple different sensing mechanisms that can target a broad range of different biorelevant species. The amphiphilic deep cavitands can both bind fluorescent dyes and interact with charged biomolecules. These non-cavity-based interactions, when paired with additives such as heavy metal ions, modulate fluorescence response in an indirect manner, and these different mechanisms allow selective recognition of serine phosphorylation, lysine acetylation, and arginine citrullination. Other targets include heavy metals, drugs of abuse, and protein isoforms. Furthermore, the hosts can be applied in supramolecular tandem assays of enzyme function: the broad scope allows analysis of such different enzymes as chromatin writers/erasers, kinases, and phosphatases, all from a single host scaffold. Finally, the indirect sensing concept allows application in sensing different oligonucleotide secondary structures, including G-quadruplexes, hairpins, triplexes, and i-motifs. Discrimination between DNA strands with highly similar structures such as G-quadruplex strands with bulges and vacancies can be achieved. Instead of relying on a single highly specific fluorescent probe, the synthetic hosts tune the fluorophore-DNA interaction, introducing multiple recognition equilibria that modulate the fluorescence signal. By applying machine learning algorithms, a classification model can be established that can accurately predict the folding state of unknown sequences. Overall, the unique recognition profile of self-folded deep cavitands provides a powerful, yet simple sensing platform, one that can be easily tuned for a wide scope of biorelevant targets, in complex biological media, without sacrificing selectivity in the recognition.

Low-Dose Exposure of WS2 Nanosheets Induces Differential Apoptosis in Lung Epithelial Cells.

Escalating the production and application of tungsten disulfide (WS2) nanosheets inevitably increases environmental human exposure and warrants the necessity of studies to elucidate their biological impacts. Herein, we assessed the toxicity of WS2 nanosheets and focused on the impacts of low doses (≤10 µg/mL) on normal (BEAS-2B) and tumorigenic (A549) lung epithelial cells. The low doses, which approximate real-world exposures, were found to induce cell apoptosis, while doses ≥ 50 µg/mL cause necrosis. Focused studies on low-dose exposure to WS2 nanosheets revealed more details of the impacts on both cell lines, including reduction of cell metabolic activity, induction of lipid peroxidation in cell membranes, and uncoupling of mitochondrial oxidative phosphorylation that led to the loss of ATP production. These phenomena, along with the expression situations of a few key proteins involved in apoptosis, point toward the occurrence of mitochondria-dependent apoptotic signaling in exposed cells. Substantial differences in responses to WS2 exposure between normal and tumorigenic lung epithelial cells were noticed as well. Specifically, BEAS-2B cells experienced more adverse effects and took up more nanosheets than A549 cells. Our results highlight the importance of dose and cell model selection in the assessment of nanotoxicity. By using doses consistent with real-world exposures and comparing normal and diseased cells, we can gain knowledge to guide the development of safety precautions for mitigating the adverse impacts of nanomaterial exposure on human health.

Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles.

Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples.

Applications of Synthetic Receptors in Bioanalysis and Drug Transport.

Synthetic receptors are powerful tools for molecular recognition. They can bind to guests with high selectivity and affinity, and their structures are tunable and diversified. These features, plus the relatively low cost and high simplicity in synthesis and modification, support the feasibility of array-based molecular analysis with synthetic receptors for improved selectivity in the recognition of a wide range of targets. More attractively, host-guest interaction is reversible and guest displacement allows biocompatible and gentle release of the host-bound molecules, simplifying the stimulation designs needed to control analyte sensing, enrichment, and transportation. Here, we highlight a few recent advancements in using synthetic receptors for molecular analysis and manipulation, with the focus on macrocyclic receptors and their applications in displacement sensing, separation, imaging, and drug transport.

Biological Impacts of Reduced Graphene Oxide Affected by Protein Corona Formation.

Applications of reduced graphene oxide (rGO) in many different areas have been gradually increasing owing to its unique physicochemical characteristics, demanding more understanding of their biological impacts. Herein, we assessed the toxicological effects of rGO in mammary epithelial cells. Because the as-synthesized rGO was dissolved in sodium cholate to maintain a stable aqueous dispersion, we hypothesize that changing the cholate concentration in the dispersion may alter the surface property of rGO and subsequently affect its cellular toxicity. Thus, four types of rGO were prepared and compared: rGO dispersed in 4 and 2 mg/mL sodium cholate, labeled as rGO and concentrated-rGO (c-rGO), respectively, and rGO and c-rGO coated with a protein corona through 1 h incubation in culture media, correspondingly named pro-rGO and pro-c-rGO. Notably, c-rGO and pro-c-rGO exhibited higher toxicity than rGO and pro-rGO and also caused higher reactive oxygen species production, more lipid membrane peroxidation, and more significant disruption of mitochondrial-based ATP synthesis. In all toxicological assessments, pro-c-rGO induced more severe adverse impacts than c-rGO. Further examination of the material surface, protein adsorption, and cellular uptake showed that the surface of c-rGO was coated with a lower content of surfactant and adsorbed more proteins, which may result in the higher cellular uptake observed with pro-c-rGO than pro-rGO. Several proteins involved in cellular redox mediation were also more enriched in pro-c-rGO. These results support the strong correlation between dispersant coating and corona formation and their subsequent cellular impacts. Future studies in this direction could reveal a deeper understanding of the correlation and the specific cellular pathways involved and help gain knowledge on how the toxicity of rGO could be modulated through surface modification, guiding the sustainable applications of rGO.

Incineration-Generated Polyethylene Micro-Nanoplastics Increase Triglyceride Lipolysis and Absorption in an In Vitro Small Intestinal Epithelium Model.

Despite mounting evidence of micro-nanoplastics (MNPs) in food and drinking water, little is known of the potential health risks of ingested MNPs, and nothing is known of their potential impact on nutrient digestion and absorption. We assessed the effects of environmentally relevant secondary MNPs generated by incineration of polyethylene (PE-I), on digestion and absorption of fat in a high fat food model using a 3-phase in vitro simulated digestion coupled with a tri-culture small intestinal epithelium model. The presence of 400 µg/mL PE-I increased fat digestion by 33% and increased fat absorption by 147 and 145% 1 and 2 h after exposure. Analysis of the PE-I lipid corona during digestion revealed predominantly triacylglycerols with enrichment of fatty acids in the small intestinal phase. Protein corona analysis showed enrichment of triacylglycerol lipase and depletion of ß-casein in the small intestinal phase. These findings suggest digestion of triacylglycerol by lipase on the surface of lipid-coated MNPs as a potential mechanism. Further studies are needed to investigate the mechanisms underlying the greater observed increase in fat absorption, to verify these results in an animal model, and to determine the MNP properties governing their effects on lipid digestion and absorption.

Recent (2018-2020) development in capillary electrophoresis.

Development of new capillary electrophoresis (CE) methodology and instrumentation, as well as application of CE to solve new problems, remains an active research area because of the attractive features of CE compared to other separation techniques. In this review, we focus on the representative works about sample preconcentration, separation media or capillary coating development, detector construction, and multidimensional separation in CE, which are judiciously selected from the papers published in 2018-2020.

Machine Learning Aids Classification and Discrimination of Noncanonical DNA Folding Motifs by an Arrayed Host:Guest Sensing System.

An arrayed host:guest fluorescence sensor system can discriminate among and classify multiple different noncanonical DNA structures by exploiting selective molecular recognition. The sensor is highly selective and can discriminate between folds as similar as native G-quadruplexes and those with bulges or vacancies. The host and guest can form heteroternary complexes with DNA strands, with the host acting as mediator between the DNA and dye, modulating the emission. By applying machine learning algorithms to the sensing data, prediction of the folding state of unknown DNA strands is possible with high fidelity.

A DNA aptamer for binding and inhibition of DNA methyltransferase 1.

DNA methyltransferases (DNMTs) are enzymes responsible for establishing and maintaining DNA methylation in cells. DNMT inhibition is actively pursued in cancer treatment, dominantly through the formation of irreversible covalent complexes between small molecular compounds and DNMTs that suffers from low efficacy and high cytotoxicity, as well as no selectivity towards different DNMTs. Herein, we discover aptamers against the maintenance DNA methyltransferase, DNMT1, by coupling Asymmetrical Flow Field-Flow Fractionation (AF4) with Systematic Evolution of Ligands by EXponential enrichment (SELEX). One of the identified aptamers, Apt. #9, contains a stem-loop structure, and can displace the hemi-methylated DNA duplex, the native substrate of DNMT1, off the protein on sub-micromolar scale, leading for effective enzymatic inhibition. Apt. #9 shows no inhibition nor binding activity towards two de novo DNMTs, DNMT3A and DNMT3B. Intriguingly, it can enter cancer cells with over-expression of DNMT1, colocalize with DNMT1 inside the nuclei, and inhibit the activity of DNMT1 in cells. This study opens the possibility of exploring the aptameric DNMT inhibitors being a new cancer therapeutic approach, by modulating DNMT activity selectively through reversible interaction. The aptamers could also be valuable tools for study of the functions of DNMTs and the related epigenetic mechanisms.

Asymmetrical Flow Field Flow Fractionation Coupled to Nanoparticle Tracking Analysis for Rapid Online Characterization of Nanomaterials.

Increasing applications of nanomaterials in consumer goods, industrial products, medical practices, etc., calls for the development of tools for rapid separation, quantification, and sizing of nanoparticles to ensure their safe and sustainable employment. While many techniques are available for characterization of pure, homogeneous nanomaterial preparations, particle sizing and counting remains difficult for heterogeneous mixtures that resulted from imperfect synthesis conditions, aggregation from product instability, or degradation during storage. Herein, nanoparticle tracking analysis (NTA) was coupled to asymmetrical flow field flow fraction (AF4) using a splitter manifold to enable online particle separation and counting. The high pressure and flow rate in AF4 were reduced to the levels compatible with NTA by the proper flow splitting design, and a syringe pump was employed to withdraw fluid through the exit port of the NTA and maintain consistent flow rates entering NTA for proper particle sizing. Successful AF4-NTA coupling was demonstrated by analyzing a mixture of polystyrene particles with the average diameters of ∼50, 100, and 200 nm. Good correlation was observed between the amount of each type of particle injected to and measured by the hyphenated system. The particle concentrations acquired using online and offline coupling of AF4-NTA also agreed well with each other. The nonspherical nanoparticles like gold nanorods and hexagonal boron nitride nanosheets were also analyzed to demonstrate the versatile applicability of this system. Our work has proved that AF4-NTA can achieve accurate online particle counting on different populations of the nanomaterials in a mixture, which cannot be done by either AF4 or NTA alone. It will be a valuable tool for rapid characterization of heterogeneous nanomaterial solutions without purification to fulfill the regulation requirement on the nanomaterial-containing products.

Physical and chemical template-blocking strategies in the exponential amplification reaction of circulating microRNAs.

The detection of circulating miRNA through isothermal amplification wields many attractive advantages over traditional methods, such as reverse transcription RT-qPCR. However, it is challenging to control the background signal produced in the absence of target, which severely hampers applications of such methods for detecting low abundance targets in complex biological samples. In the present work, we employed both the cobalt oxyhydroxide (CoOOH) nanoflakes and the chemical modification of hexanediol to block non-specific template elongation in exponential amplification reaction (EXPAR). Adsorption by the CoOOH nanoflakes and the hexanediol modification at the 3' end effectively prevented no-target polymerization on the template itself and thus greatly improved the performance of EXPAR, detecting as low as 10 aM of several miRNA targets, including miR-16, miR-21, and miR-122, with the fluorescent DNA staining dye of SYBR Gold™. Little to no cross-reactivity was observed from the interfering strands present in 10-fold excess. Besides contributing to background reduction, the CoOOH nanoflakes strongly adsorbed nucleic acids and isolated them from a complex sample matrix, thus permitting successful detection of the target miRNA in the serum. We expect that simple but sensitive template-blocking EXPAR could be a valuable tool to help with the discovery and validation of miRNA markers in biospecimens. Graphical abstract.

Monitoring the crosstalk between methylation and phosphorylation on histone peptides with host-assisted capillary electrophoresis.

Post-translational modifications (PTMs) greatly increase protein diversity and regulate their functions by changing the structures, properties, and molecular interactions of proteins. In peptide regions with high density of PTMs, PTMs can influence modification on residues in proximity or even at distal positions, adding another layer of regulation. Methods that can monitor the activities of PTM enzymes on peptides carrying multiple modifications are valuable tools for better understanding of PTM crosstalk. Herein, we developed a host-assisted capillary electrophoresis (CE) method to separate histone peptides with methylation and phosphorylation and applied it to monitor the crosstalk between serine phosphorylation and lysine methylation when they were added by Aurora B kinase and G9a lysine methyltransferase, respectively. A synthetic receptor molecule, 4-hexasulfonatocalix[6]arene (CX6), was included in the CE buffer to improve the resolution of the corresponding substrates and products. A linear polyacrylamide-coated capillary was employed to effectively reduce wall adsorption of the cationic histone peptides. The peptide substrates were labeled with fluorescein to enhance their detectability during CE separation. Our method successfully revealed that the activity of G9a methyltransferase was completely inhibited by the adjacent phosphorylation, while 25% reduction in the activity of Aurora B kinase was observed with the presence of dimethylation on the nearby residue. The PTM crosstalk was examined not only using a pure peptide substrate, but also in a competitive reaction environment, in which the modified and unmodified peptides were mixed and the enzyme actions on both peptides were monitored simultaneously. Our work demonstrates that host-assisted CE is an effective method for study of PTM crosstalk, which could offer the advantages of fast separation, high resolution, and low sample consumption. Graphical abstract.

Rapid Enrichment and Detection of Extracellular Vesicles Enabled by CuS-Enclosed Microgels.

Extracellular vesicles (EVs) are cell-derived membranous vesicles that exist in nearly all biological fluids, including blood and urine; and carry a great number of cargo molecules such as protein, nucleic acids, and lipid. They may play important roles in cell-cell communication and modulation of pathological processes, which, however, are not yet well understood, calling for highly sensitive, specific, and rapid methods for EV detection and quantification in biological samples. Here, we report the CuS-enclosed microgels that not only help enrich EVs carrying specific protein markers from complex biomatrices, but also produce strong chemiluminescence (CL) to realize sensitive detection of the target EVs. A detection limit of 104 EV particles/mL was achieved with these microgels by targeting EV proteins like CD63 and HER2, with a dynamic range up to 108 particles/mL. Direct detection of EVs in human serum and cell culture medium without tedious sample preparation was demonstrated, consuming much less sample compared to ELISA and Western Blot. We envision that our method will be valuable for quick quantification of EVs in biological samples, benefiting disease monitoring and functional study.

Mapping Molecular Structure of Protein Locating on Nanoparticles with Limited Proteolysis.

The molecular structure of a protein could be altered when it is attached to nanoparticles (NPs), affecting the performance of NPs present in biological systems. Limited proteolysis coupled with LC-MS/MS could reveal the changes in protein structure when it binds to a variety of entities, including macro-molecules and small drugs, but it has not yet been applied to study protein-NP interaction. Herein, adsorption of proteins, transferrin, and catalase on the polystyrene (PS) or iron oxide (IO) NPs was analyzed with this method. Both increased and decreased proteolytic efficiency in certain regions on the proteins were observed. Identification of the peptides affected by protein-NP interaction led to proper prediction of alterations to protein function as well as to colloidal stability of NPs. Overall, the present work has demonstrated the utility of limited proteolysis in helping to elucidate the potential biological outcomes of the protein-NP conjugate, obtaining knowledge to guide improvement of the rational design of the protein-conjugated NPs for biomedical applications and to understand the biological behaviors of the engineered NPs.

Selective Array-Based Sensing of Anabolic Steroids in Aqueous Solution by Host-Guest Reporter Complexes.

Arrayed complexes of a water-soluble deep cavitand and two fluorescent indicators show selective sensing of anabolic-androgenic steroids in aqueous environments. By combining the host-guest complexes with small amounts of heavy metal ions, discrimination between steroids that vary in structure by only a single π bond is possible. The sensing occurs through a triggered aggregation mechanism, which can be mediated by both the presence of metal ions and the steroids. The use of both "turn-on" and "turn-off" fluorophores is essential for good discrimination. As low as 10 µm steroid can be detected, and the discrimination is selective in steroid samples spiked into human urine.

Analysis of circulating non-coding RNAs in a non-invasive and cost-effective manner.

Non-coding RNAs (ncRNAs) participate in regulation of gene expression, and are highly relevant to pathological development. They are found to be stably present in diverse body fluids, including those in the circulatory system, which can be sampled non-invasively for clinical tests. Thus, circulating ncRNAs have great potential to be disease biomarkers. However, tremendous efforts are desired to discover and utilize ncRNAs as biomarkers in clinical diagnosis, calling for technological advancement in analysis of circulating ncRNAs in biospecimens. Hence, this review summarizes the recent developments in this area, highlighting the works devoted to cancer diagnosis and prognosis. Three main directions are focused: 1) Extraction and purification of ncRNAs from body fluids; 2) Quantification of the purified circulating ncRNAs; and 3) Microfluidic platforms for integration of both steps to enable point-of-care diagnostics. These technologies have laid a solid foundation to move forward the applications of circulating ncRNAs in disease diagnosis and cure.

Selective Sensing of Phosphorylated Peptides and Monitoring Kinase and Phosphatase Activity with a Supramolecular Tandem Assay.

Simple tuning of a host:guest pair allows selective sensing of different peptide modifications, exploiting orthogonal recognition mechanisms. Excellent selectivity for either lysine trimethylations or alcohol phosphorylations is possible by simply varying the fluorophore guest. The phosphorylation sensor can be modulated by the presence of small (µM) concentrations of metal ions, allowing array-based sensing. Phosphorylation at serine, threonine, and tyrosine can be selectively sensed via discriminant analysis. The phosphopeptide sensing is effective in the presence of small-molecule phosphates such as ATP, which in turn enables the sensor to be employed in continuous optical assays of both serine kinase and tyrosine phosphatase activity. The activity of multiple different kinases can be monitored, and the sensor is capable of detecting the phosphorylation of peptides containing multiple different modifications, including lysine methylations and acetylation. A single deep cavitand can be used as a "one size fits all" sensor that can selectively detect multiple different modifications to oligopeptides, as well as monitoring the function of their post-translational modification writer and eraser enzymes in complex systems.

Separation of Methylated Histone Peptides via Host-Assisted Capillary Electrophoresis.

Lysine methylation in protein is one important epigenetic mechanism that regulates diverse biological processes but is challenging to study due to the large variability in methylation levels and sites. Here, we show that supramolecular hosts such as calixarenes and cucurbiturils can be applied in the background electrolyte (BGE) of capillary electrophoresis (CE) for highly effective separation of post-translationally methylated histone peptides. The molecular recognition event causes a shift in the electrophoretic mobility of the peptide, allowing affinity measurement for binding between the synthetic receptor and various methylated lysine species. Successful separation of the H3 peptides carrying different methylation levels at the K9 position can be achieved using CX4 and CX6 as the BGE additives in CE, enabling monitoring of the activity of the histone lysine demethylase JMJD2E. This reveals the power of combining high resolution CE with synthetic hosts for study of protein methylation, and the method should be capable of analyzing complex biological samples for better understanding of the functions of histone methylation.