Proteins are responsible for the occurrence and treatment of many diseases, and therefore protein sequencing will revolutionize proteomics and clinical diagnostics. Biological nanopore approach has ...proved successful for single‐molecule DNA sequencing, which resolves the identities of 4 natural deoxyribonucleotides based on the current blockages and duration times of their translocations across the nanopore confinement. However, open challenges still remain for biological nanopores to sequentially identify each amino acid (AA) of single proteins due to the inherent complexity of 20 proteinogenic AAs in charges, volumes, hydrophobicity and structures. Herein, we focus on recent exciting advances in biological nanopores for single‐molecule protein sequencing (SMPS) from native protein unfolding, control of peptide translocation, AA identification to applications in disease detection.
Nanopore electrochemistry offers a bright prospect for single‐molecule protein sequencing by measuring specific interactions between amino acids based on their natural structure and chemistry continuity and diversity. This Minireview focusses on recent advances in biological nanopores from protein unfolding, peptide translocation, amino acid identification to diagnostic application.
Inspired by the biological processes of molecular recognition and transportation across membranes, nanopore techniques have evolved in recent decades as ultrasensitive analytical tools for individual ...molecules. In particular, nanopore-based single-molecule DNA/RNA sequencing has advanced genomic and transcriptomic research due to the portability, lower costs and long reads of these methods. Nanopore applications, however, extend far beyond nucleic acid sequencing. In this Review, we present an overview of the broad applications of nanopores in molecular sensing and sequencing, chemical catalysis and biophysical characterization. We highlight the prospects of applying nanopores for single-protein analysis and sequencing, single-molecule covalent chemistry, clinical sensing applications for single-molecule liquid biopsy, and the use of synthetic biomimetic nanopores as experimental models for natural systems. We suggest that nanopore technologies will continue to be explored to address a number of scientific challenges as control over pore design improves.
Abstract
Background
The triglyceride-glucose index (TyG index) has been regarded as a reliable alternative marker of insulin resistance and an independent predictor of cardiovascular outcomes. ...Whether the TyG index predicts adverse cardiovascular events in patients with diabetes and acute coronary syndrome (ACS) remains uncertain. The aim of this study was to investigate the prognostic value of the TyG index in patients with diabetes and ACS.
Methods
A total of 2531 consecutive patients with diabetes who underwent coronary angiography for ACS were enrolled in this study. Patients were divided into tertiles according to their TyG index. The primary outcomes included the occurrence of major adverse cardiovascular events (MACEs), defined as all-cause death, non-fatal myocardial infarction and non-fatal stroke. The TyG index was calculated as the ln (fasting triglyceride level mg/dL × fasting glucose level mg/dL/2).
Results
The incidence of MACE increased with TyG index tertiles at a 3-year follow-up. The Kaplan–Meier curves showed significant differences in event-free survival rates among TyG index tertiles (P = 0.005). Multivariate Cox hazards regression analysis revealed that the TyG index was an independent predictor of MACE (95% CI 1.201–1.746; P < 0.001). The optimal TyG index cut-off for predicting MACE was 9.323 (sensitivity 46.0%; specificity 63.6%; area under the curve 0.560; P = 0.001). Furthermore, adding the TyG index to the prognostic model for MACE improved the C-statistic value (P = 0.010), the integrated discrimination improvement value (P = 0.001) and the net reclassification improvement value (P = 0.019).
Conclusions
The TyG index predicts future MACE in patients with diabetes and ACS independently of known cardiovascular risk factors, suggesting that the TyG index may be a useful marker for risk stratification and prognosis in patients with diabetes and ACS.
Capturing real-time electron transfer, enzyme activity, molecular dynamics, and biochemical messengers in living cells is essential for understanding the signaling pathways and cellular ...communications. However, there is no generalizable method for characterizing a broad range of redox-active species in a single living cell at the resolution of cellular compartments. Although nanoelectrodes have been applied in the intracellular detection of redox-active species, the fabrication of nanoelectrodes to maximize the signal-to-noise ratio of the probe remains challenging because of the stringent requirements of 3D fabrication. Here, we report an asymmetric nanopore electrode-based amplification mechanism for the real-time monitoring of NADH in a living cell. We used a two-step 3D fabrication process to develop a modified asymmetric nanopore electrode with a diameter down to 90 nm, which allowed for the detection of redox metabolism in living cells. Taking advantage of the asymmetric geometry, the above 90% potential drop at the two terminals of the nanopore electrode converts the faradaic current response into an easily distinguishable bubble-induced transient ionic current pattern. Therefore, the current signal was amplified by at least 3 orders of magnitude, which was dynamically linked to the presence of trace redox-active species. Compared to traditional wire electrodes, this wireless asymmetric nanopore electrode exhibits a high signal-to-noise ratio by increasing the current resolution from nanoamperes to picoamperes. The asymmetric nanopore electrode achieves the highly sensitive and selective probing of NADH concentrations as low as 1 pM. Moreover, it enables the real-time nanopore monitoring of the respiration chain (i.e., NADH) in a living cell and the evaluation of the effects of anticancer drugs in an MCF-7 cell. We believe that this integrated wireless asymmetric nanopore electrode provides promising building blocks for the future imaging of electron transfer dynamics in live cells.
Anxiety is common in patients suffering from chronic pain. Here, we report anxiety-like behaviors in mouse models of chronic pain and reveal that nNOS-expressing neurons in ventromedial prefrontal ...cortex (vmPFC) are essential for pain-induced anxiety but not algesia, using optogenetic and chemogenetic strategies. Additionally, we determined that excitatory projections from the posterior subregion of paraventricular thalamic nucleus (pPVT) provide a neuronal input that drives the activation of vmPFC nNOS-expressing neurons in our chronic pain models. Our results suggest that the pain signal becomes an anxiety signal after activation of vmPFC nNOS-expressing neurons, which causes subsequent release of nitric oxide (NO). Finally, we show that the downstream molecular mechanisms of NO likely involve enhanced glutamate transmission in vmPFC CaMKIIα-expressing neurons through S-nitrosylation-induced AMPAR trafficking. Overall, our data suggest that pPVT excitatory neurons drive chronic pain-induced anxiety through activation of vmPFC nNOS-expressing neurons, resulting in NO-mediated AMPAR trafficking in vmPFC pyramidal neurons.
Protein nanopores offer an inexpensive, label-free method of analysing single oligonucleotides. The sensitivity of the approach is largely determined by the characteristics of the pore-forming ...protein employed, and typically relies on nanopores that have been chemically modified or incorporate molecular motors. Effective, high-resolution discrimination of oligonucleotides using wild-type biological nanopores remains difficult to achieve. Here, we show that a wild-type aerolysin nanopore can resolve individual short oligonucleotides that are 2 to 10 bases long. The sensing capabilities are attributed to the geometry of aerolysin and the electrostatic interactions between the nanopore and the oligonucleotides. We also show that the wild-type aerolysin nanopores can distinguish individual oligonucleotides from mixtures and can monitor the stepwise cleavage of oligonucleotides by exonuclease I.
Posttranslational modifications (PTMs) affect protein function/dysfunction, playing important roles in the occurrence and development of tauopathies including Alzheimer's disease. PTM detection is ...significant and still challenging due to the requirements of high sensitivity to identify the subtle structural differences between modifications. Herein, in terms of the unique geometry of the aerolysin (AeL) nanopore, we elaborately engineered a T232K AeL nanopore to detect the acetylation and phosphorylation of Tau segment (Pep). By replacing neutral threonine (T) with positively charged lysine (K) at the 232 sites, the T232K and K238 rings of this engineered T232K AeL nanopore corporately work together to enhance electrostatic trapping of the acetylated and phosphorylated Tau peptides. Translocation speed of the monophosphorylated Pep‐P was decelerated by up to 46 folds compared to the wild‐type (WT) AeL nanopore. The prolonged residences within the T232K AeL nanopore enabled to simultaneously identify the monoacetylated Pep‐Ac, monophosphorylated Pep‐P, di‐modified Pep‐P‐Ac and non‐modified Pep. The tremendous potential is demonstrated for PTM sensing by manipulating non‐covalent interactions between nanopores and single analytes.
Microalgal oils, depending on their degree of unsaturation, can be utilized as either nutritional supplements or fuels; thus, a feedstock with genetically designed and tunable degree of unsaturation ...is desirable to maximize process efficiency and product versatility. Systematic profiling of ex vivo （in yeast）, in vitro, and in vivo activities of type-2 diacylglycerol acyltransferases in Nannochloropsis oceanica （NoDGAT2s or NoDGTTs）, via reverse genetics, revealed that NoDGAT2A prefers saturated fatty acids （SFAs）, NoDGAT2D prefers monounsaturated fatty acids （MUFAs）, and NoDGAT2C exhibits the strongest activity toward polyunsaturated fatty acids （PUFAs）. As NoDGAT2A, 2C, and 2D originated from the green alga, red alga, and eukaryotic host ancestral participants of secondary endosymbiosis, respectively, a mecha- nistic model of oleaginousness was unveiled, in which the indigenous and adopted NoDGAT2s formulated functional complementarity and specific transcript abundance ratio that underlie a rigid SFA：MUFA：PUFA hierarchy in triacylglycerol （TAG）. By rationally modulating the ratio of NoDGAT2A＇：2C~D transcripts, a bank of N. oceanica strains optimized for nutritional supplement or fuel production with a wide range of degree of unsaturation were created, in which proportion of SFAs, MUFAs, and PUFAs in TAG varied by 1.3-, 3.7-, and 11.2-fold, respectively. This established a novel strategy to simultaneously improve productivity and quality of oils from industrial microalgae.
Aggregation-induced emission (AIE) as a unique photophysical process has been intensively explored for their features in fields from optical sensing, bioimaging to optoelectronic devices. However, ...all AIE luminogens (AIEgens) hardly recover into the initial dispersed state after illuminating at the ultimate aggregated state, which limits AIEgens to achieve reversible sensing and reproducible devices. To real-time manipulate the emission of AIEgen, here we take the advantage of confined space in the quartz nanopore to achieve a nanopore-size-dependent restriction of AIEgens for reversible conversions of "on-to-off" and "off-to-on" emission. By electrochemically manipulating 26 fL AIEgen solution inside nanopore confinement, AIE illuminates while moves along nanopore from the constricted tip to inside cavity at a velocity of 1.4-2.2 μm s
, and vice versa. We further apply this dynamic manipulation for a target delivery of AIEgen into single cells, which opens up new possibility to design powerful and practical AIE applications.
Oxygen evolution reaction (OER) is a key half‐reaction in many electrochemical transformations, and efficient electrocatalysts are critical to improve its kinetics which is typically sluggish due to ...its multielectron‐transfer nature. Perovskite oxides are a popular category of OER catalysts, while their activity remains insufficient under the conventional adsorbate evolution reaction scheme where scaling relations limit activity enhancement. The lattice oxygen‐mediated mechanism (LOM) has been recently reported to overcome such scaling relations and boost the OER catalysis over several doped perovskite catalysts. However, direct evidence supporting the LOM participation is still very little because the doping strategy applied would introduce additional active sites that may mask the real reaction mechanism. Herein, a dopant‐free, cation deficiency manipulation strategy to tailor the bulk diffusion properties of perovskites without affecting their surface properties is reported, providing a perfect platform for studying the contribution of LOM to OER catalysis. Further optimizing the A‐site deficiency achieves a perovskite candidate with excellent intrinsic OER activity, which also demonstrates outstanding performance in rechargeable Zn–air batteries and water electrolyzers. These findings not only corroborate the key role of LOM in OER electrocatalysis, but also provide an effective way for the rational design of better catalyst materials for clean energy technologies.
A dopant‐free, cation‐deficiency manipulation strategy is reported to regulate the bulk oxygen‐ion diffusion properties of perovskites without altering their surface properties, offering a perfect platform to understand the role of lattice‐oxygen participation in oxygen evolution reaction electrocatalysis. Optimizing the cation deficiency level identifies a perovskite candidate with excellent catalytic activity applicable in Zn–air batteries and water electrolyzers.
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