Data_Sheet_1_Effect of Heat Treatment on the Property, Structure, and Aggregation of Skim Milk Proteins.PDF

To study the mechanism of heat-induced protein aggregates, skim milk was heated at 55, 65, 75, 85, and 95°C for 30 s. Then, the sulfhydryl content, surface hydrophobicity, and secondary structure of heat-treated skim milk were studied. Treating skim milk at different temperatures induced a decrease in sulfhydryl content (75.9% at 95°C) and an increase in surface hydrophobicity (44% at 95°C) with a disrupted secondary structure containing random coil, β-sheet, and β-turn of skim milk proteins. The change in these properties facilitated aggregate formation through disulfide bonds and hydrophobicity interaction. Microstructural observation also showed a higher degree of aggregation when skim milk was heated at 85 and 95°C. The result of two-dimensional polyacrylamide gel electrophoresis demonstrated that the aggregates consisted of a high proportion of κ-casein, β-lactoglobulin, and other whey proteins.</p

Extending the Family of UNCG-like Tetraloop Motifs: NMR Structure of a CACG Tetraloop from Coxsackievirus B3^†

Stable RNA tetraloop motifs are found frequently in biologically active RNAs. These motifs carry out a wide variety of functions in RNA folding, in RNA−RNA and RNA−protein interactions. A great deal of knowledge about the structures and functions of tetraloop motifs has accumulated largely due to intensive theoretical, biochemical, and biophysical studies on three most frequently occurring families of tetraloop sequences, namely, the cUNCGg, the cGNRAg, and the gCUUGc sequences. Our knowledge surely is not exhaustive, and efforts are still being made to gain a better understanding. Here we report the NMR structure of a uCACGg tetraloop that occurs naturally within the cloverleaf RNA structure of the 5‘-UTR of coxsackievirus B3. This tetraloop is the major determinant for interaction between the cloverleaf RNA and viral 3C protease, which is an essential part of a ribonucleoprotein complex that plays a critical role in the regulation of viral translation and replication. Our structure shows that the CACG tetraloop is closed by a wobble U·G base pair. The structure of the CACG tetraloop is stabilized by extensive base stacking and hydrogen bonding interactions strikingly similar to those previously reported for the cUUCGg tetraloop. Identification of these hallmark structural features strongly supports the existence of an extended YNCG tetraloop family. The U·G base pair closing the stem and the A residue in the loop introduce some small structural and themodynamic distinctions from the canonical cUUCGg tetraloop that may be important for recognition by the viral 3C protease

Innovative Integration of Phase-Change Microcapsules with Metal–Organic Frameworks into an Intelligent Biosensing System for Enhancing Dopamine Detection

This work focuses on an interdisciplinary issue in energy management and biosensing techniques. Aiming at enhancing the biosensing detection of dopamine at high ambient temperatures, we developed an innovative integration of phase-change microcapsules with a metal–organic framework (MOF) based on zeolitic imidazolate framework-8 to develop an intelligent electrochemical biosensing system with a thermal self-regulation function. We first fabricated a type of electroactive microcapsules containing a MOF-anchored polypyrrole/SiO2 double-layered shell and a phase-change material (PCM) core. The resultant microcapsules not only exhibit a regular spherical morphology with a layer-by-layer core–shell microstructure but also display an effective temperature-regulation capability to enhance enzymatic bioactivity under phase-change enthalpies of around 124.0 J·g–1 along with good thermal impact resistance and excellent thermal cycling stability for long-term use in thermal energy management. These electroactive microcapsules were then used to modify a working electrode together with laccase as a biocatalyst to construct a thermal self-regulatory biosensor. With a high sensitivity of 3.541 μA·L·μmol–1·cm–2 and a low detection limit of 0.0069 μmol·L–1 at 50 °C, this biosensor exhibits much better determination effectiveness toward dopamine at higher temperatures than conventional biosensors thanks to in situ thermal management derived from its PCM core in the electroactive microcapsules. This study offers a promising approach for development of intelligent thermal self-regulatory biosensors with an enhanced detection capability to identify various chemicals accurately in a wide range of applicable temperatures

Piezotronic Effect-Assisted Photoelectrochemical Exosomal MicroRNA Monitoring Based on an Electron Donor Self-Supplying Strategy

Exosomal microRNAs (miRNAs) as newly emerging reliable and noninvasive biomarkers have demonstrated a significant function in early cancer diagnosis. Photoelectrochemical (PEC) biosensing has attracted unprecedented attention in exosomal miRNA monitoring due to its inherent advantages of both electrochemical and optical techniques; however, the severe charge carrier recombination greatly restricts the PEC assay performance. Herein, a high-sensitive PEC strategy assisted by the piezoelectric effect is designed based on Bi2WO6/Cu2S heterojunctions and implemented for the monitoring of exosomal miRNAs. The introduction of the piezoelectric effect enables promoted electron–hole transfer and separation, thereby improving the analytical sensitivity. In addition, a target reprogramming metal–organic framework-capped CaO2 (MOF@CaO2) hybrids is prepared, in which MOF@CaO2 being responsive to exosomal miRNAs induces exposure of the capped CaO2 to H2O and then triggers self-supplying of H2O2, which effectively suppresses the electron–hole recombination, giving rise to an amplified photocurrent and a decrease in the cost of the reaction. Benefiting from the coupled sensitization strategy, the as-fabricated PEC strategy exhibits high sensitivity, specificity, low cost, and ease of use for real-time analysis of exosomal miRNAs within the effectiveness linear range of 0.1 fM–1 μM. The present work demonstrates promising external field coupling-enhanced PEC bioassay and offers innovative thoughts for applying this strategy in other fields

NMR Structures of Loop B RNAs from the Stem−Loop IV Domain of the <i>Enterovirus </i>Internal Ribosome Entry Site: A Single C to U Substitution Drastically Changes the Shape and Flexibility of RNA^†^,^‡

The 5‘-untranslated region of positive-strand RNA viruses harbors many cis-acting RNA structural elements that are important for various viral processes such as replication, translation, and packaging of new virions. Among these is loop B RNA of the stem−loop IV domain within the internal ribosomal entry site (IRES) of enteroviruses, including Poliovirus type 1 (PV1). Studies on PV1 have shown that specific recognition of loop B by the first KH (hnRNP K homology) domain of cellular poly(rC)-binding protein 2 (PCBP2) is essential for efficient translation of the viral mRNA. Here we report the NMR solution structures of two representative sequence variants of enteroviral loop B RNA. The two RNA variants differ at only one position (C vs U) within a six-nucleotide asymmetric internal loop sequence that is the binding site for the PCBP2 KH1 domain. Surprisingly, the two RNAs are drastically different in the overall shape and local dynamics of the bulge region. The RNA with the 5‘-AUCCCU bulge sequence adopts an overall L shape. Its bulge nucleotides, especially the last four, are highly flexible and not very well defined by NMR. The RNA with the 5‘-AUUCCU bulge sequence adopts an overall U shape, and its bulge sequence exhibits only limited flexibility. A detailed analysis of the two RNA structures and their dynamic properties, as well as available sequence data and known KH domain−RNA complex structures, not only provides insights into how loop B RNA might be recognized by the PCBP2 KH1 domain but also suggests a possible correlation between structural flexibility and pre-existing structural features for protein recognition

Solution Structure of a Consensus Stem-Loop D RNA Domain that Plays Important Roles in Regulating Translation and Replication in Enteroviruses and Rhinoviruses^†^,^‡

Stem-loop D from the cloverleaf RNA is a highly conserved domain within the 5‘-UTR of enteroviruses and rhinoviruses. Interaction between the stem-loop D RNA and the viral 3C or 3CD proteins constitutes an essential feature of a ribonucleoprotein complex that plays a critical role in regulating viral translation and replication. Here we report the solution NMR structure of a 38-nucleotide RNA with a sequence that encompasses the entire stem-loop D domain and corresponds to the consensus sequence found in enteroviruses and rhinoviruses. Sequence variants corresponding to Poliovirus type 1 and Coxsackievirus B3 have virtually the same structure, based on small differences in chemical shifts. A substantial number (136) of 1H−13C one-bond residual dipolar coupling (RDC) values were used in the structure determination in addition to conventional distance and torsion angle restraints. Inclusion of the RDC restraints was essential for achieving well-defined structures, both globally and locally. The structure of the consensus stem-loop D is an elongated A-type helical stem capped by a UACG tetraloop with a wobble UG closing base pair. Three consecutive pyrimidine base pairs (two UU and one CU pair) are present in the middle of the helical stem, creating distinctive local structural features such as a dramatically widened major groove. A dinucleotide bulge is located near the base of the stem. The bulge itself is flexible and not as well defined as the other parts of the molecule, but the flanking base pairs are intact. The peculiar spatial arrangement of the distinctive structural elements implies that they may work synergistically to achieve optimal binding affinity and specificity toward the viral 3C or 3CD proteins

Photoelectrochemical Detection of Exosomal miRNAs by Combining Target-Programmed Controllable Signal Quenching Engineering

MicroRNAs extracted from exosomes (exosomal miRNAs) have recently emerged as promising biomarkers for early prognosis and diagnosis. Thus, the development of an effective approach for exosomal miRNA monitoring has triggered extensive attention. Herein, a sensitive photoelectrochemical (PEC) biosensing platform is demonstrated for exosomal miRNA assay via the target miRNA-powered λ-exonuclease for the amplification strategy. The metal–organic framework (MOF)-decorated WO3 nanoflakes heterostructure is constructed and implemented as the photoelectrode. Also, a target exosomal miRNA-activatable programmed release nanocarrier was fabricated, which is responsible for signal control. Hemin that acted as the electron acceptor was prior entrapped into the programmed control release nanocarriers. Once the target exosomal miRNAs-21 was introduced, the as-prepared programmed release nanocarriers were initiated to trigger the release of hemin, which enabled the quenching of the photocurrent. Under the optimized conditions, the level of exosomal miRNAs-21 could be accurately tracked ranging from 1 fM to 0.1 μM with a low detection limit of 0.5 fM. The discoveries illustrate the possibility for the rapid and efficient diagnosis and prognosis prediction of diseases based on the detection of exosomal miRNAs-21 and would provide feasible approaches for the fabrication of an efficient platform for clinical applications

Paper-Supported Self-Powered System Based on a Glucose/O₂ Biofuel Cell for Visual MicroRNA-21 Sensing

The exploitation of self-powered devices that get rid of the power source restriction represents the development tendency of sensing systems. Herein, a paper-supported glucose/O2 biofuel cell (BFC)-based self-powered sensing platform for visual analysis was developed. The BFC device utilized gold nanoparticle-modified paper fibers as the electrode to wire glucose oxidase (GOx) and bilirubin oxidase for the fabrication of bioanodes and biocathodes. To implement an assay protocol, a target-responsive cargo release system based on mesoporous silica nanocarriers controlled by microRNA-21 (miRNA-21) was designed. During the BFC operation, undesired H2O2, the side product of glucose oxidation which would be deleterious for GOx, was generated, leading to inevitable degeneration of BFC performance. On the basis of the H2O2-mediated iodide oxidation reaction to form iodine that further modulated the starch chromogenic reaction, undesired H2O2 could be effectively removed, resulting in remarkably improved BFC performance as well as providing a means for visual signal readout. Thanks to the dual output signals (maximum power output density or length of blue bar), enhanced analysis reliability and sensitive detection of miRNA-21 over a range of 5 fM to 100 pM were achieved. Moreover, this study demonstrates a proof of concept in visualized BFC-based self-powered systems for sensing applications and provides a blueprint to advance future sensors and analysis devices powered by BFCs in a wide variety of in vitro applications