Differential Proteomic Analysis of Low-Dose Chronic Paralytic Shellfish Poisoning.

Shellfish poisoning is a common food poisoning. To comprehensively characterize proteome changes in the whole brain due to shellfish poisoning, Tandem mass tag (TMT)-based differential proteomic analysis was performed with a low-dose chronic shellfish poisoning model in mice. A total of 6798 proteins were confidently identified, among which 123 proteins showed significant changes (fold changes of >1.2 or <0.83, p < 0.05). In positive regulation of synaptic transmission, proteins assigned to a presynaptic membrane (e.g., Grik2) and synaptic transmission (e.g., Fmr1) changed. In addition, altered proteins in nervous system development were observed, suggesting that mice suffered nerve damage due to the nervous system being activated. Ion transport in model mice was demonstrated by a decrease in key enzymes (e.g., Kcnj11) in voltage-gated ion channel activity and solute carrier family (e.g., Slc38a3). Meanwhile, alterations in transferase activity proteins were observed. In conclusion, these modifications observed in brain proteins between the model and control mice provide valuable insights into understanding the functional mechanisms underlying shellfish poisoning.

A protein standard absolute quantification strategy for enhanced absolute quantification of ricin in complex matrices using in vitro synthesized mutant holoprotein as internal standard by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Ricin is a highly toxic protein toxin that poses a potential bioterrorism threat due to its potency and widespread availability. However, the accurate quantification of ricin through absolute mass spectrometry (MS) using a protein standard absolute quantification (PSAQ) strategy is not widely practiced. This limitation primarily arises from the presence of interchain disulfide bonds, which hinder the production of full-length isotope-labeled ricin as an internal standard (IS) in vitro. In this study, we have developed a novel approach for the absolute quantification of ricin in complex matrices using recombinant single-chain and full-length mutant ricin as the protein IS, instead of isotope-labeled ricin, in conjunction with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The amino acid sequence of the ricin mutant internal standard (RMIS) was designed by introducing site mutations in specific amino acids of trypsin/Glu-C enzymatic digestion marker peptides of ricin. To simplify protein expression, the A-chain and B-chain of RMIS were directly linked to replace the original interchain disulfide bonds. The RMISs were synthesized using an Escherichia coli expression system. An appropriate RMIS was selected as the protein IS based on consistent digestion efficiency, UHPLC-MS/MS behavior, antibody recognition function, lectin activity, and proper depurination activity with intact ricin. The RMIS was utilized to simultaneously quantify A- and B-chain marker peptides of ricin through UHPLC-MS/MS. This method was thoroughly validated using a milk matrix. By employing internal protein standards, this quantitative strategy overcomes the challenges posed by variations in extraction recoveries, matrix effects, and digestion efficiency encountered when working with different matrices. Consequently, calibration curves generated from milk matrix-spiked samples were utilized to accurately and precisely quantify ricin in river water and plasma samples. Moreover, the established method successfully detected intact ricin in samples obtained from the sixth Organization for the Prohibition of Chemical Weapons (OPCW) exercise on biotoxin analysis. This study presents a novel PSAQ strategy that enables the accurate quantification of ricin in complex matrices.

Study on the Analgesic Activity of Peptide from Conus achates.

BACKGROUND: As a peptide originally discovered from Conus achates by mass spectrometry and cDNA sequencing, Ac6.4 contains 25 amino acid residues and three disulfide bridges. Our previous study found that this peptide possesses 80% similarity to MVIIA by BLAST and that MVIIA is a potent and selective blocker of N-type voltage-sensitive calcium channels in neurons. OBJECTIVE: To recognize the target protein and analgesic activity of Ac6.4 from Conus achates. METHODS: In the present study, we synthesized Ac6.4, expressed the Trx-Ac6.4 fusion protein, tested Ac6.4 for its inhibitory activity against Cav2.2 in CHO cells and investigated Ac6.4 and Trx-Ac6.4 for their analgesic activities in mice. RESULTS: Data revealed that Ac6.4 had strong inhibitory activity against Cav2.2 (IC50 = 43.6 nM). After intracranial administration of Ac6.4 (5, 10, 20 µg/kg) and Trx-Ac6.4 (20, 40, 80 µg/kg), significant analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent. CONCLUSION: This study expands our knowledge of the peptide Ac6.4 and provides new possibilities for developing Cav2.2 inhibitors and analgesic drugs.

Precise sequencing of single protected-DNA fragment molecules for profiling of protein distribution and assembly on DNA.

Multiple DNA-interacting protein molecules are often dynamically distributed and/or assembled along a DNA molecule to adapt to their intricate functions temporally. However, analytical technology for measuring such binding behaviours is still missing. Here, we demonstrate the unique capacity of a supernuclease for a highly efficient cutting of the unprotected-DNA segments and with complete preservation of the protein-occluded DNA segments at near single-nucleotide resolution. By exploring this high-resolution cutting, an unprecedented assay that allows a precise sequencing of single protected-DNA fragment molecules (SPDFMS) was developed. As relevant applications, relevant information was gained on the respective distribution/assembly patterns and coordinated displacement of single-stranded DNA-binding protein and recombinase RecA, two model proteins, on DNA. Benefiting from this assay, we also for the first time provide direct measurement of the length of single RecA nucleofilaments, showing the predominant stoichiometry of 5-7 RecA monomers per RecA nucleofilament under physiologically relevant conditions. This innovative assay appears as a promising analytical tool for studying diverse protein-DNA interactions implicated in DNA replication, transcription, recombination, repair, and gene editing.

One-pot intramolecular cyclization of 5-hydroxymethylcytosine for sequencing DNA hydroxymethylation at single-base resolution.

Here we establish a one-pot reaction to directly convert the DNA base 5-hydroxymethylcytosine (5hmC) to an intramolecular cyclization nucleobase, which loses both protons of the exocyclic N4-amino group and thus is recognized as thymine (T) by DNA polymerase. Based on this 5hmC-specific reaction, a prospective bisulfite-free strategy for 5hmC sequencing is proposed. This is also the first example to show modified DNA labeling in non-water solvent-dominant media for DNA sequencing.

Formic Acid of ppm Enhances LC-MS/MS Detection of UV Irradiation-Induced DNA Dimeric Photoproducts.

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are genotoxic DNA lesions and mainly generated on thymine-thymine (T-T) dinucleotides upon UV irradiation. Regarding the sensitivity, specificity, and accuracy of analytical methods, it is of first choice to develop a reliable assay for simultaneous detection of these DNA lesions using liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, the dilemma is the low detection sensitivity of the phosphate-containing dimeric photoproducts even using most favorable negative-ion mode for LC-MS/MS analysis. Unexpectedly, we observed that the detection sensitivity of T-T CPD and 6-4PP could be significantly improved using formic acid/acetic acid (∼ppm) as an additive of the mobile phase for reversed-phase LC-MS/MS analysis. This is the first demonstration of the enhancement of LC-MS/MS signals by formic acid/acetic acid in negative-ion mode. Of note, these acidic agents are often used for positive-ion mode in LC-MS assays. Benefited from the developed method, we could quantify both T-T CPD and 6-4PP in mouse embryonic stem cells upon UVC irradiation at low dosage. This sensitive method is applicable to the screening and identification of genes involved in formation, signaling, and repair of UV lesion.

A template-assisted strategy to synthesize a dilute CoNi alloy incorporated into ultramicroporous carbon for high performance supercapacitor application.

Carbon is widely studied as an electrical double-layer capacitor (EDLC) electrode material due to its high specific surface area and good electronic conductivity. However, the low capacitance and energy density limit its further commercial applications. Herein, we report a facile and novel synthesis of dilute CoNi alloy nanoparticles embedded into ultramicroporous carbon (CoNi/UMCs) via a template-assisted strategy using SBA-15 as a template. The CoNi alloy serves as not only the electron collector to decrease the conductivity during tests, but also creates CoNi oxide/oxhydroxide on the alloy surface in an alkali solution, promoting redox reactions in pseudocapacitors, enhancing the performance of supercapacitors. Interestingly, the final morphology of the composite is not transferred from SBA-15 and the ultramicropores absolutely come from sucrose itself. However, the presence of SBA-15 can definitely enlarge the surface area of the CoNi/UMCs. After carefully tailoring the loading of CoNi, we find that 0.95% CoNi/UMCs exhibit a high surface area of 613 m2 g-1 with regular ultramicropores of 0.57 nm. Due to the synergistic effect of porous carbon and CoNi alloy, the unique 0.95% CoNi/UMCs exhibit a high specific capacitance of up to 268 F g-1 at 0.25 A g-1 in 6 M KOH aqueous solution and a high capacitance retention ratio of 97.8% after 10 000 cycles.

Simultaneous Modulation of Composition and Oxygen Vacancies on Hierarchical ZnCo2 O4 /Co3 O4 /NC-CNT Mesoporous Dodecahedron for Enhanced Oxygen Evolution Reaction.

Electrochemical water splitting is a promising way for the sustainable production of hydrogen, but the efficiency of the overall water-splitting reaction largely depends on the oxygen evolution reaction (OER) because of its sluggish kinetics. Herein, a series of hierarchical ZnCo2 O4 /Co3 O4 /NC-CNT (NC-CNT=nitrogen-rich carbon nanotube) mesoporous dodecahedrons grafted to carbon nanotubes have been synthesized from ZnCo bimetallic zeolitic imidazolate frameworks (ZnCo-ZIFs) through sequential pyrolysis in nitrogen and mild oxidation in air. The simultaneous modulation of oxygen vacancies, composition, and hierarchical mesoporous architecture remarkably enhanced their electronic conduction and the amount and reactivity of accessible actives; thus boosting their intrinsic activity in the OER. The optimal ZnCo2 O4 /Co3 O4 /NC-CNT-700 sample exhibited a large current density of 50 mA cm-2 at a potential of 1.65 V, a small Tafel slope of 88.5 mV dec-1 , and superior stability in alkaline media. This work should provide a facile strategy for the rational design of advanced OER catalysts by simultaneous engineering of oxygen vacancies and composition.

Strongly Coupled FeNi Alloys/NiFe2O4@Carbonitride Layers-Assembled Microboxes for Enhanced Oxygen Evolution Reaction.

Hydrogen produced from electrocatalytic water splitting is a promising route due to the sustainable powers derived from the solar and wind energy. However, the sluggish kinetics at the anode for water splitting makes the highly effective and inexpensive electrocatalysts desirable in oxygen evolution reaction (OER) by structure and composition modulations. Metal-organic frameworks (MOFs) have been intensively used as the templates/precursors to synthesize complex hollow structures for various energy-related applications. Herein, an effective and facile template-engaged strategy originated from bimetal MOFs is developed to construct hollow microcubes assembled by interconnected nanopolyhedron, consisting of intimately dominant FeNi alloys coupled with a small NiFe2O4 oxide, which was confined within carbonitride outer shell (denoted as FeNi/NiFe2O4@NC) via one-step annealing treatment. The optimized FeNi/NiFe2O4@NC exhibits excellent electrocatalytic performances toward OER in alkaline media, showing 10 mA·cm-2 at η = 316 mV, lower Tafel slope (60 mV·dec-1), and excellent durability without decay after 5000 CV cycles, which also surpasses the IrO2 catalyst and most of non-noble catalysts in the OER, demonstrating a great perspective. The superior OER performance is ascribed to the hollow interior for fast mass transport, in situ formed strong coupling between FeNi alloys and NiFe2O4 for electron transfer, and the protection of carbonitride layers for long stability.

Metallic Cobalt Encapsulated in Bamboo-Like and Nitrogen-Rich Carbonitride Nanotubes for Hydrogen Evolution Reaction.

Despite being technically possible, the hydrogen production by means of electrocatalytic water splitting is still practically unreachable mainly because of the lack of inexpensive and high active catalysts. Herein, a novel and facile approach by melamine polymerization, exfoliation and Co(2+)-assisted thermal annealing is developed to fabricate Co nanoparticles embedded in bamboo-like and nitrogen-rich carbonitride nanotubes (Co@NCN). The electronic interaction between the embedded Co nanoparticles and N-rich carbonitride nanotubes could strongly promote the HER performance. The optimized Co@NCN-800 exhibits outstanding HER activity with an onset potential of -89 mV (vs RHE), a large exchange current density of 62.2 µA cm(-2), and small Tafel slope of 82 mV dec(-1), as well as excellent stability (5000 cycles) in acid media, demonstrating the potential for the replacement of Pt-based catalysts. Control experiments reveal that the superior performance should be ascribed to the synergistic effects between embedded Co nanoparticles and N-rich carbonitride nanotubes, which originate from the high pyridinic N content, fast charge transfer rate from Co particles to electrodes via electronic coupling, and porous and bamboo-like carbonitride nanotubes for more active sites in HER.

Co-Doped MoS2 Nanosheets with the Dominant CoMoS Phase Coated on Carbon as an Excellent Electrocatalyst for Hydrogen Evolution.

Highly active and low-cost catalysts for hydrogen evolution reaction (HER) are crucial for the development of efficient water splitting. Molybdenum disulfide (MoS2) nanosheets possess unique physical and chemical properties, which make them promising candidates for HER. Herein, we reported a facile, effective, and scalable strategy by a deposition-precipitation method to fabricate metal-doped (Fe, Co, Ni) molybdenum sulfide with a few layers on carbon black as noble metal-free electrocatalysts for HER. The CoMoS phase after thermal annealing in Co-doped MoS2 plays a crucial role for the enhanced HER. The optimized Co-doped MoS2 catalyst shows superior HER performance with a high exchange current density of 0.03 mA·cm(-2), low onset potential of 90 mV, and small Tafel slope of 50 mV·dec(-1), which also exhibits excellent stability of 10000 cycles with negligible loss of the cathodic current. The superior HER activity originates from the synergistically structural and electronic modulations between MoS2 and Co ions, abundant defects in the active edge sites, as well as the good balance between active sites and electronic conductivity. Thanks to their ease of synthesis, low cost, and high activity, the Co-doped MoS2 catalysts appear to be promising HER catalysts for electrochemical water splitting.