Protein Ions Research Articles

Toward Selective Transport of Monovalent Metal Ions with High Permeability Based on Crown Ether-Encapsulated Metal-Organic Framework Sub-Nanochannels.

Achieving selective transport of monovalent metal ions with high precision and permeability analogues to biological protein ion channels has long been explored for fundamental research and various applications, such as ion sieving, mineral extraction, and energy harvesting and conversion. However, it still remains a significant challenge to construct artificial nanofluidic devices to realize the trade-off effects between selective ion transportation and high ion permeability. In this work, we report a bioinspired functional micropipet with in situ growth of crown ether-encapsulated metal-organic frameworks (MOFs) inside the tip and realize selective transport of monovalent metal ions. The functional ion-selective micropipet with sub-nanochannels was constructed by the interfacial growth method with the formation of composite MOFs consisting of ZIF-8 and 15-crown-5. The resulting micropipet device exhibited obvious monovalent ion selectivity and high flux of Li+ due to the synergistic effects of size sieving in subnanoconfined space and specific coordination of 15-crown-5 toward Na+. The selectivity of Li+/Na+, Li+/K+, Li+/Ca2+, and Li+/Mg2+ with 15-crown-5@ZIF-8-functionalized micropipet reached 3.9, 5.2, 105.8, and 122.4, respectively, which had an obvious enhancement compared to that with ZIF-8. Notably, the ion flux of Li+ can reach up to 93.8 ± 3.6 mol h-1·m-2 that is much higher than previously reported values. Furthermore, the functional micropipet with 15-crown-5@ZIF-8 sub-nanochannels exhibited stable Li+ selectivity under various conditions, such as different ion concentrations, pH values, and mixed ion solutions. This work not only provides new opportunities for the development of MOF-based nanofluidic devices for selective ion transport but also facilitates the promising practical applications in lithium extraction from salt-like brines, sewage treatment, and other related aspects.

Exploring the fragmentation efficiency of proteins analyzed by MALDI-TOF-TOF tandem mass spectrometry using computational and statistical analyses.

Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) is a rapid technique for identifying intact proteins from unfractionated mixtures by top-down proteomic analysis. MS/MS allows isolation of specific intact protein ions prior to fragmentation, allowing fragment ion attribution to a specific precursor ion. However, the fragmentation efficiency of mature, intact protein ions by MS/MS post-source decay (PSD) varies widely, and the biochemical and structural factors of the protein that contribute to it are poorly understood. With the advent of protein structure prediction algorithms such as Alphafold2, we have wider access to protein structures for which no crystal structure exists. In this work, we use a statistical approach to explore the properties of bacterial proteins that can affect their gas phase dissociation via PSD. We extract various protein properties from Alphafold2 predictions and analyze their effect on fragmentation efficiency. Our results show that the fragmentation efficiency from cleavage of the polypeptide backbone on the C-terminal side of glutamic acid (E) and asparagine (N) residues were nearly equal. In addition, we found that the rearrangement and cleavage on the C-terminal side of aspartic acid (D) residues that result from the aspartic acid effect (AAE) were higher than for E- and N-residues. From residue interaction network analysis, we identified several local centrality measures and discussed their implications regarding the AAE. We also confirmed the selective cleavage of the backbone at D-proline bonds in proteins and further extend it to N-proline bonds. Finally, we note an enhancement of the AAE mechanism when the residue on the C-terminal side of D-, E- and N-residues is glycine. To the best of our knowledge, this is the first report of this phenomenon. Our study demonstrates the value of using statistical analyses of protein sequences and their predicted structures to better understand the fragmentation of the intact protein ions in the gas phase.

Bioactive metabolites from endophytes of African club moss- Selaginella kraussiana (Kunze) A. Braun exhibit broad-spectrum pharmaceutical utilities

The search for potent natural compounds from plant and microbial sources is increasing logarithmically to combat various types of health issues of which multidrug-resistant microbes and problems related to oxidative stress are the prime ones. Novel secondary metabolites from endophytes have always been potent alternatives to contemporary antimicrobials and antioxidants. The present study shows that two potent endophytic fungal isolates Ascochyta medicaginicola SK16 and Phomopsis sp. SK5 synthesises non-proteinaceous, thermostable secondary metabolites with broad-spectrum antimicrobial, antioxidative, and cytotoxic activity. Bioassay-guided fractionation revealed that the Ethyl acetate (EA) extract of the cell-free culture extract (CFCE) of SK5 and SK16 constitutes ten and eleven secondary metabolites of which 2,4, -di tert butyl phenol, aziridine- 1,2-amino ethyl, actinobolin, bactobolin, azulene, menthyl acetate, phenol, p-[2-(methylamino)ethyl], and imidazole components were significant. SK16 exhibited strong antimicrobial activity against seventeen pathogens including Methicillin-resistant Staphylococcus aureus (MRSA), and C. albicans with a minimum microbicidal concentration of 100- 400 µg mL−1. These metabolites disrupt the microbial cell membranes causing lethal leakage of biomacromolecules- nucleic acid, protein, and potassium ions. Enzymes involved in the EMP pathway, TCA cycle, and gluconeogenesis were also blocked. Both the biofilm formation and transition of Candida cells from the yeast-to-hyphal form were inhibited. SK5 metabolites are potent radical scavengers with an IC50 of 34.25 ± 0.87 – 195.21 ± 5.73 µg mL−1 against DPPH, ABTS, FRAP, and H2O2 free radical generators and significantly increased antioxidant parameters in treated peritoneal macrophage cells of male Wistar rats. Also, SK5 metabolites were cytotoxic against breast (T47D), pancreatic (MIA Paca-2), lung (A549) and colon (HCT-116) cancer cells. The present findings suggest that metabolites from endophytes of African club moss hold potent pharmaceutical utilities.

Genome-scale annotation of protein binding sites via language model and geometric deep learning.

Revealing protein binding sites with other molecules, such as nucleic acids, peptides, or small ligands, sheds light on disease mechanism elucidation and novel drug design. With the explosive growth of proteins in sequence databases, how to accurately and efficiently identify these binding sites from sequences becomes essential. However, current methods mostly rely on expensive multiple sequence alignments or experimental protein structures, limiting their genome-scale applications. Besides, these methods haven't fully explored the geometry of the protein structures. Here, we propose GPSite, a multi-task network for simultaneously predicting binding residues of DNA, RNA, peptide, protein, ATP, HEM, and metal ions on proteins. GPSite was trained on informative sequence embeddings and predicted structures from protein language models, while comprehensively extracting residual and relational geometric contexts in an end-to-end manner. Experiments demonstrate that GPSite substantially surpasses state-of-the-art sequence-based and structure-based approaches on various benchmark datasets, even when the structures are not well-predicted. The low computational cost of GPSite enables rapid genome-scale binding residue annotations for over 568,000 sequences, providing opportunities to unveil unexplored associations of binding sites with molecular functions, biological processes, and genetic variants. The GPSite webserver and annotation database can be freely accessed at https://bio-web1.nscc-gz.cn/app/GPSite.

Genome-scale annotation of protein binding sites via language model and geometric deep learning

Revealing protein binding sites with other molecules, such as nucleic acids, peptides, or small ligands, sheds light on disease mechanism elucidation and novel drug design. With the explosive growth of proteins in sequence databases, how to accurately and efficiently identify these binding sites from sequences becomes essential. However, current methods mostly rely on expensive multiple sequence alignments or experimental protein structures, limiting their genome-scale applications. Besides, these methods haven’t fully explored the geometry of the protein structures. Here, we propose GPSite, a multi-task network for simultaneously predicting binding residues of DNA, RNA, peptide, protein, ATP, HEM, and metal ions on proteins. GPSite was trained on informative sequence embeddings and predicted structures from protein language models, while comprehensively extracting residual and relational geometric contexts in an end-to-end manner. Experiments demonstrate that GPSite substantially surpasses state-of-the-art sequence-based and structure-based approaches on various benchmark datasets, even when the structures are not well-predicted. The low computational cost of GPSite enables rapid genome-scale binding residue annotations for over 568,000 sequences, providing opportunities to unveil unexplored associations of binding sites with molecular functions, biological processes, and genetic variants. The GPSite webserver and annotation database can be freely accessed at https://bio-web1.nscc-gz.cn/app/GPSite.

Major changes in domain arrangements are associated with the evolution of termites.

Domains as functional protein units and their rearrangements along the phylogeny can shed light on the functional changes of proteomes associated with the evolution of complex traits like eusociality. This complex trait is associated with sterile soldiers and workers, and long-lived, highly fecund reproductives. Unlike in Hymenoptera (ants, bees, and wasps), the evolution of eusociality within Blattodea, where termites evolved from within cockroaches, was accompanied by a reduction in proteome size, raising the question of whether functional novelty was achieved with existing rather than novel proteins. To address this, we investigated the role of domain rearrangements during the evolution of termite eusociality. Analysing domain rearrangements in the proteomes of three solitary cockroaches and five eusocial termites, we inferred more than 5,000 rearrangements over the phylogeny of Blattodea. The 90 novel domain arrangements that emerged at the origin of termites were enriched for several functions related to longevity, such as protein homeostasis, DNA repair, mitochondrial activity, and nutrient sensing. Many domain rearrangements were related to changes in developmental pathways, important for the emergence of novel castes. Along with the elaboration of social complexity, including permanently sterile workers and larger, foraging colonies, we found 110 further domain arrangements with functions related to protein glycosylation and ion transport. We found an enrichment of caste-biased expression and splicing within rearranged genes, highlighting their importance for the evolution of castes. Furthermore, we found increased levels of DNA methylation among rearranged compared to non-rearranged genes suggesting fundamental differences in their regulation. Our findings indicate the importance of domain rearrangements in the generation of functional novelty necessary for termite eusociality to evolve.

Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange.

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.

Knock-out of dipeptidase CN2 in human proximal tubular cells disrupts dipeptide and amino acid homeostasis and para- and transcellular solute transport.

Although of potential biomedical relevance, dipeptide metabolism has hardly been studied. We found the dipeptidase carnosinase-2 (CN2) to be abundant in human proximal tubules, which regulate water and solute homeostasis. We therefore hypothesized, that CN2 has a key metabolic role, impacting proximal tubular transport function. A knockout of the CN2 gene (CNDP2-KO) was generated in human proximal tubule cells and characterized by metabolomics, RNA-seq analysis, paracellular permeability analysis and ion transport. CNDP2-KO in human proximal tubule cells resulted in the accumulation of cellular dipeptides, reduction of amino acids and imbalance of related metabolic pathways, and of energy supply. RNA-seq analyses indicated altered protein metabolism and ion transport. Detailed functional studies demonstrated lower CNDP2-KO cell viability and proliferation, and altered ion and macromolecule transport via trans- and paracellular pathways. Regulatory and transport protein abundance was disturbed, either as a consequence of the metabolic imbalance or the resulting functional disequilibrium. CN2 function has a major impact on intracellular amino acid and dipeptide metabolism and is essential for key metabolic and regulatory functions of proximal tubular cells. These findings deserve invivo analysis of the relevance of CN2 for nephron function and regulation of body homeostasis.

Formation and Application of Polymer Spherulite-like Patterns of Halloysite Nanotubes by Evaporation-Induced Self-Assembly.

Halloysite nanotubes (HNTs) are one-dimensional clay nanomaterials featuring distinct tubular structures and unique surface charges. HNTs can readily form ordered assembly structures under specific conditions, which shows significant potential applications in optical and biological fields. In this study, sodium hexametaphosphate (SHMP) was employed as a stabilizer to prepare polymer spherulite-like patterns via the evaporation-induced self-assembly (EISA) technique. The incorporation of SHMP enhanced the repulsion force among the nanotubes and the surface potential, which facilitated the orderly deposition of HNTs. The influence of HNT concentration, SHMP concentration, drying temperature, and substrate on the polymer spherulites-like pattern has been investigated in detail. The optimal conditions were 10 wt % HNT dispersion, 0.6 wt % SHMP concentration, 30 °C as drying temperature, and glass substrates. In addition, by changing the droplet volume and shape of the three-phase contact line, patterns of different sizes and shapes can be achieved. Bovine serum albumin or metal salt compounds were incorporated into the dispersion of SHMP-modified HNTs, which altered the charge and the self-assembled patterns with different area ratios. Thus, this technology can be utilized for the analysis and comparison of protein and metal ion concentration accurately. This study creates the correlation between the structural parameters and the preparation process involved in creating polymer spherulite-like patterns of modified HNTs and offers fresh insights into potential applications for the self-assembly of HNT droplets in the realms of anticounterfeiting and solution concentration analysis.

Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry.

The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra. By using the charge-detection method called individual ion mass spectrometry, we demonstrate how complex mixtures of phosphoproteoforms and their fragment ions can be reproducibly handled to provide a "bird's eye" view of signaling activity through mapping proteoform landscapes in a pathway. Using this approach, the overall stoichiometry and distribution of 0-4 phosphorylations on MEK1 was determined in a cellular model of drug-resistant metastatic melanoma. This approach can be generalized to other multiply modified proteoforms, for which PTM combinations are key to their function and drug action.

Effects of Nano-Electrospray Ionization Emitter Position on Unintentional In-Source Activation of Peptide and Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) typically introduces protein ions into the gas phase through nano-electrospray ionization (nESI). Many nESI setups have mobile stages for tuning the ion signal and extent of co-solute and salt adduction. However, tuning the position of the emitter capillary in nESI can have unintended downstream consequences for collision-induced unfolding or collision-induced dissociation (CIU/D) experiments. Here, we show that relatively small variations in the nESI emitter position can shift the midpoint (commonly called the "CID50" or "CIU50") potential of CID breakdown curves and CIU transitions by as much as 8 V on commercial instruments. A spatial "map" of the shift in CID50 for the loss of heme from holomyoglobin onto the emitter position on a Waters Synapt G2-Si mass spectrometer shows that emitter positions closer to the instrument inlet can result in significantly greater in-source activation, whereas different effects are found on an Agilent 6545XT instrument for the ions studied. A similar effect is observed for CID of the singly protonated leucine enkephalin peptide and Shiga toxin 1 subunit B homopentamer on the Waters Synapt G2-Si instrument. In-source activation effects on a Waters Synapt G2-Si are also investigated by examining the RMSD between CIU fingerprints acquired at different emitter positions and the shifts in CIU50 for structural transitions of bovine serum albumin and NIST monoclonal antibody.

A58 UTILIZATION OF HYPOXIA-TOLERANT ORGANISMS AS A MODEL IN THE STUDY OF ISCHEMIC-REPERFUSION INJURY IN TRANSPLANT HEPATOLOGY

Abstract Background Ischemic-reperfusion injury (IRI) is a barrier to successful liver transplantation. This complex process is initiated by an episode of hypoxia and decreased adenosine triphosphate (ATP) production. Mammalian hepatocytes are susceptible to prolonged hypoxia and may experience irreversible damage with ATP depletion. Yet, facultative anaerobes have developed physiological hepatoprotective strategies to tolerate hypoxic stress. The painted turtle (Chrysemys picta belli) and the common goldfish (Carassius auratus) are able to survive under severe hypoxia for weeks to months. Aims Introducing hypoxia-tolerant organisms as a suitable animal model for studying IRI in transplant hepatology. Methods A comprehensive search of PubMed, OVID, CINAHL, and Cochrane databases up to May 2023 was conducted to identify all studies reporting experimental evidence and hepatoprotective pathways in hypoxia-tolerant organisms (HTOs). Each article was qualitatively assessed. The primary focus was on cell death, intracellular ion gradient, mitochondrial function, and reactive oxygen species (ROS) with hypoxia and IRI. Results HTOs have increased glycogen storage with a greater ATP yield from anaerobic metabolism (glycolysis). Approximately 15-30% of liver mass in HTOs is composed of glycogen which is massive compared to 5-6% in mammals. Turtle isolated hepatocytes can tolerate 10 hours of anoxia by reducing cellular metabolic demand by 90% through decreased protein synthesis and ion channel activity. Goldfish hepatocytes can tolerate 6 hours of anoxia via similar strategies. In mammals, mitochondria undergo depolarization, Ca2+ efflux, cellular swelling, and apoptosis with anoxia. Yet, a depolarized mitochondrial membrane potential is regulated to provide cellular protection in HTOs. The ability to preserve mitochondrial function and electrochemical gradients with oxygen lack is an important contributor to cellular survival. Even in mammalian hepatocytes, if the mitochondrial integrity is maintained, cells are better able to tolerate ischemic insults. Finally, formation of radicals with reperfusion leads to hepatocyte apoptosis and necrosis in the mammalian model. Meanwhile, regulated ROS levels in turtles and goldfish cells, prevent cell death and IRI. Conclusions IRI is of significant interest in transplant hepatology. Although extremely valuable, the traditional mammalian models are vulnerable to hypoxia. Meanwhile, HTOs have undergone years of adaptation with thousands of genes dedicated to hypoxia tolerance. Utilizing these organisms can provide a broader understanding of IRI and avoid irreversible tissue damage in transplant hepatology. Funding Agencies CIHR

Informatic challenges and advances in illuminating the druggable proteome

The understudied members of the druggable proteomes offer promising prospects for drug discovery efforts. While large-scale initiatives have generated valuable functional information on understudied members of the druggable gene families, translating this information into actionable knowledge for drug discovery requires specialized informatics tools and resources. Here, we review the unique informatics challenges and advances in annotating understudied members of the druggable proteome. We demonstrate the application of statistical evolutionary inference tools, knowledge graph mining approaches, and protein language models in illuminating understudied protein kinases, pseudokinases, and ion channels.

Memristive Response and Capacitive Spiking in Aqueous Ion Transport through Two-Dimensional Nanopore Arrays.

In living organisms, information is processed in interconnected symphonies of ionic currents spiking through protein ion channels. As a result of dynamic switching of their conductive states, ion channels exhibit a variety of current-voltage nonlinearities and memory effects. Fueled by the promise of computing architectures entirely different from von Neumann, recent attempts to identify and harness similar phenomena in artificial nanofluidic environments focused on demonstrating analogue circuit elements with memory. Here we explore aqueous ionic transport through two-dimensional (2D) membranes featuring arrays of ion-trapping crown-ether-like pores. We demonstrate that for aqueous salts featuring ions with different ion-pore binding affinities, memristive effects emerge through coupling between the time-delayed state of the system and its transport properties. We also demonstrate a nanopore array that behaves as a capacitor with a strain-tunable built-in barrier, yielding behaviors ranging from current spiking to an ohmic response. By focusing on the illustrative underlying mechanisms, we demonstrate that realistically observable memory effects may be achieved in nanofluidic systems featuring crown-porous 2D membranes.

Inductive and Capacitive Hysteresis of Current-Voltage Curves: Unified Structural Dynamics in Solar Energy Devices, Memristors, Ionic Transistors, and Bioelectronics

Hysteresis observed in the current-voltage curves of both electronic and ionic devices is a phenomenon where the curve's shape is altered on the basis of the measurement speed. This effect is driven by internal processes that introduce a time delay in the response to an external stimulus, leading to measurements being dependent on the history of the past disturbances. This hysteresis effect has posed challenges, particularly in solution-processed photovoltaic devices such as halide perovskite solar cells, where it significantly complicates the evaluation of performance quality. In other devices, such as memristors and organic electrochemical transistors for neuromorphic applications, hysteresis is an inherent aspect of their functionality, facilitating transitions between different conductivity states. Natural and artificial ionically conducting channels also exhibit pronounced hysteresis, a crucial component for generating action potentials in neurons. In this study, we aim to categorize various forms of hysteresis by identifying shared elements among diverse physical, chemical, and biological conducting systems. Our method involves examining hysteresis from multiple angles, using simplified models that capture essential response types. We analyze system behavior using techniques such as linear sweep voltammetry and impedance spectroscopy and transient currents resulting from small voltage steps. Our investigation reveals two primary hysteresis types based on how current responds to rapid sweep rates: capacitive hysteresis and inductive hysteresis. These terms correspond to the dominant component in the equivalent circuit, determining the transient time response. Remarkably, these concepts provide insights into vastly different systems, spanning solar cells, capacitors, transistors, electrofluidic nanopores, and protein ion channels. The consistency in electrical responses across the different cases enables the identification of the primary cause of hysteresis. We also elucidate the frequency dependence of hysteresis and the stepwise responses of solar cells, illustrating how fundamental relaxations contribute to the overall surplus or deficit of current during extensive voltage sweeps that define the current-voltage curve. Published by the American Physical Society 2024

Fabrication, characterization, and application of pea protein isolate-polyphenol-iron complexes with antioxidant and antibacterial activity

Plant proteins are becoming increasingly popular as functional ingredients in foods because of their health, sustainability, and environmental benefits. The functional performance of plant proteins can often be improved by combining them with other food ingredients. For instance, they can be combined with polyphenols and metal ions to form surface active protein-polyphenol-iron complexes with biological activity. In this study, ternary complexes of pea protein isolate (PPI), epigallocatechin-3-gallate (EGCG) and iron (Fe3+) was prepared using a simple molecular self-assembly method. The formation mechanism, characteristics, and functional properties of these complexes were then characterized. Our results showed that PPI, EGCG, and Fe3+ could spontaneously combine to form nano-sheet networks held together by hydrogen bonds, phenolic hydroxyl coordination bonds, and electrostatic interactions. The addition of Fe3+ increased the fraction of disordered secondary structures, reduced the thermal denaturation temperature, reduced the surface hydrophobicity, increased the particle size, and altered the surface potential of the proteins in a dose dependent manner. Optimizing the EGCG-to-Fe3+ ratio in the complexes (1:2 w/w) led to the formation of protein composites with improved surface, antioxidant, and antibacterial activities. This study provided valuable insights into the interaction mechanisms of proteins, polyphenols, and metal ions in ternary complexes. These complexes may be used as multifunctional ingredients in plant-based foods because of their beneficial antioxidant, antimicrobial, nutritional, and emulsification properties.

Nature-inspired protein mineralization strategies for nanoparticle construction: advancing effective cancer therapy.

Recently, nanotechnology has shown great potential in the field of cancer therapy due to its ability to improve the stability and solubility and reduce side effects of drugs. The biomimetic mineralization strategy based on natural proteins and metal ions provides an innovative approach for the synthesis of nanoparticles. This strategy utilizes the unique properties of natural proteins and the mineralization ability of metal ions to combine nanoparticles through biomimetic mineralization processes, achieving the effective treatment of tumors. The precise control of the mineralization process between proteins and metal ions makes it possible to obtain nanoparticles with the ideal size, shape, and surface characteristics, thereby enhancing their stability and targeting ability in vivo. Herein, initially, we analyze the role of protein molecules in biomineralization and comprehensively review the functions, properties, and applications of various common proteins and metal particles. Subsequently, we systematically review and summarize the application directions of nanoparticles synthesized based on protein biomineralization in tumor treatment. Specifically, we discuss their use as efficient drug delivery carriers and role in mediating monotherapy and synergistic therapy using multiple modes. Also, we specifically review the application of nanomedicine constructed through biomimetic mineralization strategies using natural proteins and metal ions in improving the efficiency of tumor immunotherapy.

In Vitro Patch-Clamp.

The patch-clamp technique is one of the most useful tools to analyze the function of electrically active cells such as neurons. This technique allows for the analysis of proteins (ion channels and receptors), cells (neurons), and synapses that are the building blocks of neuronal networks. Cortical development involves coordinated changes in functional measures at each of these levels of analysis that reflect both cellular and circuit maturation. This chapter explains the technical and theoretical basis of patch-clamp methodology and introduces several examples of how this technique can be applied in the context of cortical development.

Application of a novel numerical simulation to biochemical reaction systems.

Recent advancements in omics and single-cell analysis highlight the necessity of numerical methods for managing the complexity of biological data. This paper introduces a simulation program for biochemical reaction systems based on the natural number simulation (NNS) method. This novel approach ensures the equitable treatment of all molecular entities, such as DNA, proteins, H2O, and hydrogen ions (H+), in biological systems. Central to NNS is its use of stoichiometric formulas, simplifying the modeling process and facilitating efficient and accurate simulations of diverse biochemical reactions. The advantage of this method is its ability to manage all molecules uniformly, ensuring a balanced representation in simulations. Detailed in Python, NNS is adept at simulating various reactions, ranging from water ionization to Michaelis-Menten kinetics and complex gene-based systems, making it an effective tool for scientific and engineering research.

Effects of Amino Acid Additives on Protein Stability during Electrothermal Supercharging in ESI-MS.

The surprising formation of highly charged protein ions from aqueous ammonium bicarbonate solution is a fascinating phenomenon referred to as electrothermal supercharging (ETS). Although the precise mechanism involved is not clearly understood, previous studies predominantly suggest that ETS is due to native protein destabilization in the presence of bicarbonate anion inside the electrospray ionization droplets under high temperatures and spray voltages. To evaluate existing hypotheses surrounding the underlying mechanism of ETS, the effects of several additives on protein charging under ETS conditions were investigated. The changes in the protein charge state distributions were compared by measuring the ratios between the intensities of highest intensity charge states of native and unfolded protein envelopes and shifts in the lowest and highest observed charge states. This study demonstrated that source temperature plays a more important role in ETS compared to spray voltage, especially when using a nebulized microelectrospray ionization source. Moreover, the effect of amino acids on ETS were generally in good agreement with the extensive literature available on the stabilization or destabilization of proteins by these additives in bulk solution. Among the natural amino acids, protein supercharging was significantly reduced by proline and glycine; however, imidazole provided the highest degree of noncovalent complex stabilization against ETS, outperforming the amino acids. Overall, our study shows that the simple addition of stabilizing reagents such as proline and imidazole can reduce the extent of apparent protein unfolding and supercharging in ammonium bicarbonate solution and provide evidence against the roles of charge depletion and thermal unfolding during ETS.