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Talanta Jun 2024A novel "double chemical bonding" electrochemical peptide biosensor 2FcP-GA-GDY(Fe)@NMIL-B was developed for highly selective, ultrasensitive, and ultrastable...
A novel "double chemical bonding" electrochemical peptide biosensor 2FcP-GA-GDY(Fe)@NMIL-B was developed for highly selective, ultrasensitive, and ultrastable identification of prostate-specific antigen (PSA). The C-Fe-O chemical bond linking Fe-Graphdiyne (Fe-GDY) with NH-MIL88B(Fe) (NMIL88B) as the first chemical bonding of electrode carrier Fe-GDY@NH-MIL88B(Fe) (GDY(Fe)@NMIL) significantly accelerates electron transport. With glutaraldehyde (GA) as a crosslinking agent, the Schiff-base -NC- formed by GDY(Fe)@NMIL nanocomposites links the two Fc molecules labeled peptides (2FcP) as the second chemical bonding, facilitating high-density attachment of peptides to the electrode carrier in a firm manner. When the PSA analyte is introduced to identify and cleave the specific peptide, the release of ferrocene from its head leads to a decrease in the electrical signal, enabling sensitive detection. The prepared sensing platform exhibits exceptional analytical performance for PSA with an extended linear response range from 10 fg mL to 50 ng mL. Additionally, the detection limit has been significantly reduced to an ultra-low level of only 0.94 fg mL, surpassing those reported in most literature by several orders of magnitude. Moreover, the 2FcP-GA-GDY(Fe)@NMIL-B sensor has excellent selectivity and stability while also showcasing great potential for practical application of PSA detection in human serum using the standard addition method.
PubMed: 38941809
DOI: 10.1016/j.talanta.2024.126459 -
Journal of Magnetic Resonance (San... Jun 2024Hyperpolarized water in dissolution dynamic nuclear polarization (dDNP) experiments has emerged as a promising method for enhancing nuclear magnetic resonance (NMR)...
Hyperpolarized water in dissolution dynamic nuclear polarization (dDNP) experiments has emerged as a promising method for enhancing nuclear magnetic resonance (NMR) signals, particularly in studies of proteins and peptides. Herein, we focus on the application of "proton exchange-doubly relayed" nuclear Overhauser effects (NOE) from hyperpolarized water to achieve positive signal enhancement of methyl groups in the side chain of an alanine-glycine peptide. In particular, we show a cascade hyperpolarization transfer. Initial proton exchange between solvent and amide introduces hyperpolarization into the peptide. Subsequently, intermolecular NOE relays the hyperpolarization first to Ala-H and then in a second step to the Ala-CH moiety. Both NOEs have negative signs. Hence, the twice-relayed NOE pathway leads to a positive signal enhancement of the methyl group with respect to the thermal equilibrium magnetization. This effect might indicate a way towards hyperpolarized water-based signal enhancement for methyl groups, which are often used for NMR studies of large proteins in solution.
PubMed: 38941676
DOI: 10.1016/j.jmr.2024.107727 -
ACS Sensors Jun 2024Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents...
Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become the standard of care in cancer surgeries. One of the key parameters to optimize in contrast agents is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a -(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker, as well as the positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased overall signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.
PubMed: 38941307
DOI: 10.1021/acssensors.4c00912 -
Journal of Comparative Physiology. A,... Jun 2024Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes,...
Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.
PubMed: 38940930
DOI: 10.1007/s00359-024-01706-5 -
Autophagy Jun 2024A multitude of cellular responses to intrinsic and extrinsic signals converge on macroautophagy/autophagy, a conserved catabolic process that degrades cytoplasmic...
A multitude of cellular responses to intrinsic and extrinsic signals converge on macroautophagy/autophagy, a conserved catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, particularly during starvation or stress. In addition to protein degradation, autophagy is deeply interconnected with unconventional protein secretion and polarized sorting at multiple levels within eukaryotic cells. Secretory autophagy (SA) has been recognized as a novel mechanism in which autophagosomes fuse with the plasma membrane and actively participate in the secretion of a series of cytosolic proteins, ranging from tissue remodeling factors to inflammatory molecules of the IL1 family. SA is partially controlled by the glucocorticoid-responsive, HSP90 co-chaperone FKBP5 and members of the SNARE proteins, SEC22B, SNAP23, SNAP29, STX3 and STX4. SA deregulation is implicated in several inflammatory pathologies, including cancer, cell death and degeneration. However, the key molecular mechanisms governing SA and its regulation remain elusive, as does its role in neuroinflammation and neurodegeneration. To further characterize SA and pinpoint its involvement in neuroinflammatory processes, we studied SA-relevant protein interaction networks in mouse brain, microglia and human postmortem brain tissue from control subjects and Alzheimer disease cases. We demonstrate that SA regulates neuroinflammation-mediated neurodegeneration via SKA2 and FKBP5 signaling.
PubMed: 38934263
DOI: 10.1080/15548627.2024.2373675 -
Chemistry (Weinheim An Der Bergstrasse,... Jun 2024Bioreduction of spin labels and polarizing agents (generally stable radicals) has been an obstacle limiting the in-cell applications of pulsed electron paramagnetic...
Bioreduction of spin labels and polarizing agents (generally stable radicals) has been an obstacle limiting the in-cell applications of pulsed electron paramagnetic resonance (EPR) spectroscopy and dynamic nuclear polarization (DNP). In this work, we have demonstrated that two semiquinone methide radicals (OXQM• and CTQM•) can be easily produced from the trityl-based quinone methides (OXQM and CTQM) via reduction by various reducing agents including biothiols and ascorbate under anaerobic conditions. Both radicals have relatively low pKa's and exhibit EPR single line signals at physiological pH. Moreover, the bioreduction of OXQM in three cell lysates enables quantitative generation of OXQM• which was most likely mediated by flavoenzymes. Importantly, the resulting OXQM• exhibited extremely high stability in the E.coli lysate under anaerobic conditions with 76- and 14.3-fold slower decay kinetics as compared to the trityl OX063 and a gem-diethyl pyrrolidine nitroxide. Intracellular delivery of OXQM into HeLa cells was also achieved by covalent conjugation with a cell-permeable peptide as evidenced by the stable intracellular EPR signal from the OXQM• moiety. Owing to extremely high resistance of OXQM• towards bioreduction, OXQM and its derivatives show great application potential in in-cell EPR and in-cell DNP studies for various cells which can endure short-term anoxic treatments.
PubMed: 38932665
DOI: 10.1002/chem.202400985 -
Vaccines Jun 2024The Bursa of Fabricius, an avian unique humoral immune organ, is instrumental to B cell development. Bursal-derived peptide BP9 fosters B-cell development and formation....
The Bursa of Fabricius, an avian unique humoral immune organ, is instrumental to B cell development. Bursal-derived peptide BP9 fosters B-cell development and formation. Yet, the exact mechanism wherein BP9 impacts B cell differentiation and antigenic presentation remains undefined. In this paper, B cell activation and differentiation in the spleen cells from mice immunized with the AIV vaccine and BP9 were detected following flow cytometry (FCM) analysis. Furthermore, the molecular mechanism of BP9 in B cell differentiation in vivo was investigated with RNA sequencing technology. To verify the potential functional mechanism of BP9 in the antigenic presentation process, the transcriptome molecular basis of chicken macrophages stimulated by BP9 was measured via high-throughput sequencing technology. The results proved that when given in experimental dosages, BP9 notably accelerated total B cells, and enhanced B-cell differentiation and plasma cell production. The gene expression profiles of B cells from mice immunized with 0.01 mg/mL BP9 and AIV vaccine disclosed that 0.01 mg/mL BP9 initiated the enrichment of several biological functions and significantly stimulated key B-cell pathways in immunized mice. Crucially, a total of 4093 differentially expressed genes were identified in B cells with BP9 stimulation, including 943 upregulated genes and 3150 downregulated genes. Additionally, BP9 induced various cytokine productions in the chicken macrophage HD11 cells and activated 9 upregulated and 20 downregulated differential miRNAs, which were involved in various signal and biological processes. Furthermore, BP9 stimulated the activation of multiple transcription factors in HD11 cells, which was related to antigen presentation processes. In summary, these results suggested that BP9 might promote B cell differentiation and induce antigen presentation, which might provide the valuable insights into the mechanism of B cell differentiation upon bursal-derived immunomodulating peptide stimulation and provide a solid experimental groundwork for enhancing vaccine-induced immunity.
PubMed: 38932336
DOI: 10.3390/vaccines12060607 -
Pharmaceutics May 2024Glucagon-like peptide-1 (GLP-1) is a multifunctional incretin hormone with various physiological effects beyond its well-characterized effect of stimulating... (Review)
Review
Glucagon-like peptide-1 (GLP-1) is a multifunctional incretin hormone with various physiological effects beyond its well-characterized effect of stimulating glucose-dependent insulin secretion in the pancreas. An emerging role for GLP-1 and its receptor, GLP-1R, in brain neuroprotection and in the suppression of inflammation, has been documented in recent years. GLP-1R is a G protein-coupled receptor (GPCR) that couples to Gs proteins that stimulate the production of the second messenger cyclic 3',5'-adenosine monophosphate (cAMP). cAMP, acting through its two main effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), exerts several anti-inflammatory (and some pro-inflammatory) effects in cells, depending on the cell type. The present review discusses the cAMP-dependent molecular signaling pathways elicited by the GLP-1R in cardiomyocytes, cardiac fibroblasts, central neurons, and even in adrenal chromaffin cells, with a particular focus on those that lead to anti-inflammatory effects by the GLP-1R. Fully elucidating the role cAMP plays in GLP-1R's anti-inflammatory properties can lead to new and more precise targets for drug development and/or provide the foundation for novel therapeutic combinations of the GLP-1R agonist medications currently on the market with other classes of drugs for additive anti-inflammatory effect.
PubMed: 38931817
DOI: 10.3390/pharmaceutics16060693 -
Nutrients Jun 2024Corn peptide (CP) is a short, naturally occurring, and physiologically active peptide generated from corn-protease-catalyzed hydrolysis. CP plays a role in preventing...
Corn peptide (CP) is a short, naturally occurring, and physiologically active peptide generated from corn-protease-catalyzed hydrolysis. CP plays a role in preventing obesity-related disorders, but its impact on reducing inflammation is unknown. Hence, this study examined the possible protective effects of corn peptide powder (CPP) against the harmful effects of lipopolysaccharide (LPS), with a particular emphasis on reducing oxidative damage and inflammation in adipocytes. Hence, mature 3T3-L1 adipocytes underwent exposure to 10 ng/mL LPS, with or without CPP (10 and 20 μg/mL). LPS stimulation increased reactive oxygen species and superoxide anion generation. However, this effect was reduced in a dose-dependent manner by pretreatment with CPP. CPP treatment elevated the mRNA expressions of the antioxidant enzymes manganese superoxide dismutase (mnSOD) and glutathione peroxidase 1 (Gpx1) while reducing the mRNA expressions of the cytosolic reactive oxygen species indicators p40 and p67 (NADPH oxidase 2). In addition, CPP inhibited the monocyte chemoattractant protein-1, tumor necrosis factor-alpha, Toll-like receptor 4, and nuclear factor kappa B mRNA expressions induced by LPS. These findings demonstrate that CPP may ameliorate adipocyte dysfunction by suppressing oxidative damage and inflammatory responses through a new mechanism known as Toll-like receptor 4/nuclear factor kappa B-mediated signaling.
Topics: Animals; Mice; 3T3-L1 Cells; Adipocytes; Lipopolysaccharides; Zea mays; Reactive Oxygen Species; Inflammation; Toll-Like Receptor 4; Oxidative Stress; Superoxide Dismutase; Powders; Peptides; Glutathione Peroxidase; NF-kappa B; Antioxidants; Glutathione Peroxidase GPX1; Signal Transduction; Chemokine CCL2; Tumor Necrosis Factor-alpha; Anti-Inflammatory Agents
PubMed: 38931278
DOI: 10.3390/nu16121924 -
International Journal of Molecular... Jun 2024The SLC35 (Solute Carrier 35) family members acting as nucleotide sugar transporters are typically localized in the endoplasmic reticulum or Golgi apparatus. It is,...
The SLC35 (Solute Carrier 35) family members acting as nucleotide sugar transporters are typically localized in the endoplasmic reticulum or Golgi apparatus. It is, therefore, intriguing that some reports document the presence of orphan transporters SLC35F1 and SLC35F6 within the endosomal and lysosomal system. Here, we compared the subcellular distribution of these proteins and found that they are concentrated in separate compartments; i.e., recycling endosomes for SLC35F1 and lysosomes for SLC35F6. Swapping the C-terminal tail of these proteins resulted in a switch of localization, with SLC35F1 being trafficked to lysosomes while SLC35F6 remained in endosomes. This suggested the presence of specific sorting signals in these C-terminal regions. Using site-directed mutagenesis, fluorescence microscopy, and cell surface biotinylation assays, we found that the EQERLL signal located in the cytoplasmic tail of human SLC35F6 is involved in its lysosomal sorting (as previously shown for this conserved sequence in mouse SLC35F6), and that SLC35F1 localization in the recycling pathway depends on two YXXΦ-type signals: a YKQF sequence facilitates its internalization from the plasma membrane, while a YTSL motif prevents its transport to lysosomes, likely by promoting SLC35F1 recycling to the cell surface. Taken together, these results support that some SLC35 members may function at different levels of the endosomal and lysosomal system.
Topics: Lysosomes; Endosomes; Humans; Protein Transport; Animals; Nucleotide Transport Proteins; HeLa Cells; Mice; Golgi Apparatus; Amino Acid Sequence; Protein Sorting Signals; HEK293 Cells; Cell Membrane
PubMed: 38928424
DOI: 10.3390/ijms25126718