Charge-solvated versus protonated salt forms of cyclodepsipeptide toxins in electrospray: Dissociation of alkali-cationized forms enables straightforward sequencing of cereulide.

Bacillus cereus is responsible for foodborne outbreaks worldwide. Among the produced toxins, cereulide induces nausea and vomiting after 30 min to 6 h following the consumption of contaminated foods. Cereulide, a cyclodepsipeptide, is an ionophore selective to K+ in solution. In electrospray, the selectivity is reduced as [M + Li]+; [M + Na]+ and [M + NH4]+ can also be detected without adding corresponding salts. Two forms are possible for alkali-cationized ions: charge-solvated (CS) that exclusively dissociates by releasing a bare alkali ion and protonated salt (PS), yielding alkali product ions by covalent bond cleavages (CBC) promoted by mobile proton. Based on a modified peptide cleavage nomenclature, the PS product ion series (b, a, [b + H2O] and [b + CnH2nO] [n = 4, 5]) are produced by Na+/Li+/K+-cationized cereulide species that specifically open at ester linkages followed by proton mobilization promoting competitive ester CBC as evidenced under resonant collision activation. What is more, unlike the sodiated or lithiated cereulide, which regenerates little or no alkali cation, the potassiated forms lead to an abundant K+ regeneration. This occurs by splitting of (i) the potassiated CS forms with an appearance threshold close to that of the PS first fragment ion generation and (ii) eight to four potassiated residue product ions from the PS forms. Since from Na+/Li+-cationized cereulide, (i) the negligible Na+/Li+ regeneration results in a higher sensibility than that of potassiated forms that abundantly releasing K+, and (ii) a better sequence recovering, the use of Na+ (or Li+) should be more pertinent to sequence isocereulides and other cyclodepsipeptides.

Phytic Acid-Promoted Deposition of Gold Nanoparticles with Grafted Cationic Polymer Brushes for the Construction of Synergistic Contact-Killing and Photothermal Bactericidal Coatings.

Medical implants are constantly facing the risk of bacterial infections, especially infections caused by multidrug resistant bacteria. To mitigate this problem, gold nanoparticles with alkyl bromide moieties (Au NPs-Br) on the surfaces were prepared. Xenon light irradiation triggered the plasmon effect of Au NPs-Br to induce free radical graft polymerization of 2-(dimethylamino)ethyl methacrylate (DMAEMA), leading to the formation of poly(DMAEMA) brush-grafted Au NPs (Au NPs-g-PDM). The Au NPs-g-PDM nanocomposites were conjugated with phytic acid (PA) via electrostatic interaction and van der Waals interaction. The as-formed aggregates were deposited on the titanium (Ti) substrates to form the PA/Au NPs-g-PDM (PAP) hybrid coatings through surface adherence of PA and the gravitational effect. Synergistic bactericidal effects of contact-killing caused by the cationic PDM brushes, and local heating generated by the Au NPs under near-infrared irradiation, conferred strong antibacterial effects on the PAP-deposited Ti (Ti-PAP) substrates. The synergistic bactericidal effects reduced the threshold temperature required for the photothermal sterilization, which in turn minimized the secondary damage to the implant site. The Ti-PAP substrates exhibited 97.34% and 99.97% antibacterial and antiadhesive efficacy, respectively, against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), compared to the control under in vitro antimicrobial assays. Furthermore, the as-constructed Ti-PAP surface exhibited a 99.42% reduction in the inoculated S. aureus under in vivo assays. In addition, the PAP coatings exhibited good biocompatibility in the hemolysis and cytotoxicity assays as well as in the subcutaneous implantation of rats.

Intermolecular hydrogen bonding behavior of amino acid radical cations.

Amino acid and peptide radicals are of broad interest due to their roles in biochemical oxidative damage, pathogenesis and protein radical catalysis, among others. Using density functional theory (DFT) calculations at the ωB97X-D/def2-QZVPPD//ωB97X-D/def2-TZVPP level of theory, we systematically investigated the hydrogen bonding between water and fourteen α-amino acids (Ala, Asn, Cys, Gln, Gly, His, Met, Phe, Pro, Sel, Ser, Thr, Trp, and Tyr) in both neutral and radical cation forms. For all amino acids surveyed, stronger hydrogen-bonding interactions with water were observed upon single-electron oxidation, with the greatest increases in hydrogen-bonding strength occurring in Gly, Ala and His. We demonstrate that the side chain has a significant impact on the most favorable hydrogen-bonding modes experienced by amino acid radical cations. Our computations also explored the fragmentation of amino acid radical cations through the loss of a COOH radical facilitated by hydrogen bonding. The most favorable pathways provided stabilization of the resulting cationic fragments through hydrogen bonding, resulting in more favorable thermodynamics for the fragmentation process. These results indicate that non-covalent interactions with the environment have a profound impact on the structure and chemical fate of oxidized amino acids.

Cationic Lipid Pairs Enhance Liver-to-Lung Tropism of Lipid Nanoparticles for In Vivo mRNA Delivery.

Much of current clinical interest has focused on mRNA therapeutics for the treatment of lung-associated diseases, such as infections, genetic disorders, and cancers. However, the safe and efficient delivery of mRNA therapeutics to the lungs, especially to different pulmonary cell types, is still a formidable challenge. In this paper, we proposed a cationic lipid pair (CLP) strategy, which utilized the liver-targeted ionizable lipid and its derived quaternary ammonium lipid as the CLP to improve liver-to-lung tropism of four-component lipid nanoparticles (LNPs) for in vivo mRNA delivery. Interestingly, the structure-activity investigation identified that using liver-targeted ionizable lipids with higher mRNA delivery performance and their derived lipid counterparts is the optimal CLP design for improving lung-targeted mRNA delivery. The CLP strategy was also verified to be universal and suitable for clinically available ionizable lipids such as SM-102 and ALC-0315 to develop lung-targeted LNP delivery systems. Moreover, we demonstrated that CLP-based LNPs were safe and exhibited potent mRNA transfection in pulmonary endothelial and epithelial cells. As a result, we provided a powerful CLP strategy for shifting the mRNA delivery preference of LNPs from the liver to the lungs, exhibiting great potential for broadening the application scenario of mRNA-based therapy.

Combination of PEGylation and Cationization on Phospholipid-Coated Cyclosporine Nanosuspensions for Enhanced Ocular Drug Delivery.

Strong precorneal clearance mechanisms including reflex blink, constant tear drainage, and rapid mucus turnover constitute great challenges for eye drops for effective drug delivery to the ocular epithelium. In this study, cyclosporine A (CsA) for the treatment of dry eye disease (DED) was selected as the model drug. Two strategies, PEGylation for mucus penetration and cationization for potent cellular uptake, were combined to construct a novel CsA nanosuspension (NS@lipid-PEG/CKC) by coating nanoscale drug particles with a mixture of lipids, DSPE-PEG2000, and a cationic surfactant, cetalkonium chloride (CKC). NS@lipid-PEG/CKC with the mean size ∼173 nm and positive zeta potential ∼+40 mV showed promoted mucus penetration, good cytocompatibility, more cellular uptake, and prolonged precorneal retention without obvious ocular irritation. More importantly, NS@lipid-PEG/CKC recovered tear production and goblet cell density more efficiently than the commercial cationic nanoemulsion on a dry eye disease rat model. All results indicated that a combination of PEGylation and cationization might provide a promising strategy to coordinate mucus penetration and cellular uptake for enhanced drug delivery to the ocular epithelium for nanomedicine-based eye drops.

Gene Transfection Efficiency Improvement with Lipid Conjugated Cationic Carbon Dots.

An ideal vehicle with a high transfection efficiency is crucial for gene delivery. In this study, a type of cationic carbon dot (CCD) known as APCDs were first prepared with arginine (Arg) and pentaethylenehexamine (PEHA) as precursors and conjugated with oleic acid (OA) for gene delivery. By tuning the mass ratio of APCDs to OA, APCDs-OA conjugates, namely, APCDs-0.5OA, APCDs-1.0OA, and APCDs-1.5OA were synthesized. All three amphiphilic APCDs-OA conjugates show high affinity to DNA through electrostatic interactions. APCDs-0.5OA exhibit strong binding with small interfering RNA (siRNA). After being internalized by Human Embryonic Kidney (HEK 293) and osteosarcoma (U2OS) cells, they could distribute in both the cytoplasm and the nucleus. With APCDs-OA conjugates as gene delivery vehicles, plasmid DNA (pDNA) that encodes the gene for the green fluorescence protein (GFP) can be successfully delivered in both HEK 293 and U2OS cells. The GFP expression levels mediated by APCDs-0.5OA and APCDs-1.0OA are ten times greater than that of PEI in HEK 293 cells. Furthermore, APCDs-0.5OA show prominent siRNA transfection efficiency, which is proven by the significantly downregulated expression of FANCA and FANCD2 proteins upon delivery of FANCA siRNA and FANCD2 siRNA into U2OS cells. In conclusion, our work demonstrates that conjugation of CCDs with a lipid structure such as OA significantly improves the gene transfection efficiency, providing a new idea about the designation of nonviral carriers in gene delivery systems.

Cholesterol sulfate-mediated ion-pairing facilitates the self-nanoassembly of hydrophilic cationic mitoxantrone.

HYPOTHESIS: Hydrophilic cationic drugs such as mitoxantrone hydrochloride (MTO) pose a significant delivery challenge to the development of nanodrug systems. Herein, we report the use of a hydrophobic ion-pairing strategy to enhance the nano-assembly of MTO. EXPERIMENTS: We employed biocompatible sodium cholesteryl sulfate (SCS) as a modification module to form stable ion pairs with MTO, which balanced the intermolecular forces and facilitated nano-assembly. PEGylated MTO-SCS nanoassemblies (pMS NAs) were prepared via nanoprecipitation. We systematically evaluated the effect of the ratio of the drug module (MTO) to the modification module (SCS) on the nanoassemblies. FINDINGS: The increased lipophilicity of MTO-SCS ion pair could significantly improve the encapsulation efficiency (∼97 %) and cellular uptake efficiency of MTO. The pMS NAs showed prolonged blood circulation, maintained the same level of tumor antiproliferative activity, and exhibited reduced toxicity compared with the free MTO solution. It is noteworthy that the stability, cellular uptake, cytotoxicity, and in vivo pharmacokinetic behavior of the pMS NAs increased in proportion to the molar ratio of SCS to MTO. This study presents a self-assembly strategy mediated by ion pairing to overcome the challenges commonly associated with the poor assembly ability of hydrophilic cationic drugs.

Structural Elucidation of Ubiquitin via Gas-Phase Ion/Ion Cross-Linking Reactions Using Sodium-Cationized Reagents Coupled with Infrared Multiphoton Dissociation.

Accurate structural determination of proteins is critical to understanding their biological functions and the impact of structural disruption on disease progression. Gas-phase cross-linking mass spectrometry (XL-MS) via ion/ion reactions between multiply charged protein cations and singly charged cross-linker anions has previously been developed to obtain low-resolution structural information on proteins. This method significantly shortens experimental time relative to conventional solution-phase XL-MS but has several technical limitations: (1) the singly deprotonated N-hydroxysulfosuccinimide (sulfo-NHS)-based cross-linker anions are restricted to attachment at neutral amine groups of basic amino acid residues and (2) analyzing terminal cross-linked fragment ions is insufficient to unambiguously localize sites of linker attachment. Herein, we demonstrate enhanced structural information for alcohol-denatured A-state ubiquitin obtained from an alternative gas-phase XL-MS approach. Briefly, singly sodiated ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) cross-linker anions enable covalent cross-linking at both ammonium and amine groups. Additionally, covalently modified internal fragment ions, along with terminal b-/y-type counterparts, improve the determination of linker attachment sites. Molecular dynamics simulations validate experimentally obtained gas-phase conformations of denatured ubiquitin. This method has identified four cross-linking sites across 8+ ubiquitin, including two new sites in the N-terminal region of the protein that were originally inaccessible in prior gas-phase XL approaches. The two N-terminal cross-linking sites suggest that the N-terminal half of ubiquitin is more compact in gas-phase conformations. By comparison, the two C-terminal linker sites indicate the signature transformation of this region of the protein from a native to a denatured conformation. Overall, the results suggest that the solution-phase secondary structures of the A-state ubiquitin are conserved in the gas phase. This method also provides sufficient sensitivity to differentiate between two gas-phase conformers of the same charge state with subtle structural variations.

Enhancing selective ammonium transport in membrane electrochemical systems.

Recovering ammonia nitrogen from wastewater is a sustainable strategy that simultaneously addresses both nitrogen removal and fertilizer production. Membrane electrochemical system (MES), which utilizes electrochemical redox reactions to transport ammonium ions through cation exchange membranes, has been considered as an effective technology for ammonia recovery from wastewater. In this study, we develop a mathematical model to systematically investigate the impact of co-existing ions on the transport of ammonium (NH4+) ions in MES. Our analysis elucidates the importance of pH values on both the NH4+ transport and inert ion (Na+) transport. We further comprehensively assess the system performance by varying the concentration of Na+ in the system. We find that while the inert cation in the initial anode compartment competes with NH4+ transport, NH4+ dominates the cation transport in most cases. The transport number of Na+ surpasses NH4+ only if the fraction of Na+ to total cation is extremely high (>88.5%). Importantly, introducing Na+ ions into the cathode compartment significantly enhances the ammonia transport due to the Donnan dialysis. The analysis of selective ion transport provides valuable insights into optimizing both selectivity and efficiency in ammonia recovery from wastewater.

Influence of clay minerals on pH and major cation concentrations in acid-leached sands: Column experiments and reactive-transport modeling.

A series of laboratory experiments are conducted to simulate the acidification and subsequent recovery of a sand aquifer exploited by in situ recovery (ISR) mining. A sulfuric acid solution (pH 2) is first injected into a column packed with sand from the Zoovch Ovoo uranium roll front deposit (Mongolia). Solutions representative of local groundwater or enriched in cations (Na+, Mg2+) are then circulated through the column to simulate the inflow of aquifer water. pH and major ion concentrations (Na+, Cl-, SO42-, Ca2+, Mg2+, K+) measured at the column outlet reproduce the overall evolution of porewater chemistry observed in the field. The presence of minor quantities of swelling clay minerals (≈6 wt% smectite) is shown to exert an important influence on the behavior of inorganic cations, particularly H+, via ion-exchange reactions. Numerical models that consider ion-exchange on smectite as the sole solid-solution interaction are able to reproduce variations in pH and cation concentrations in the column experiments. This highlights the importance of clay minerals in controlling H+ mobility and demonstrates that sand from the studied aquifer can be described to a first order as an ion-exchanger. The present study confirms the key role of clay minerals in controlling water chemistry in acidic environments through ion-exchange processes. In a context of managing the long-term environmental footprint of industrial and mining activities (ISR, acid mine drainage…), this work will bring insights for modeling choices and identification of key parameters to help operators to define their production and/or remediation strategies.

Membraneless channels sieve cations in ammonia-oxidizing marine archaea.

Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle1,2. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine environment at the cell surface and their subsequent channelling to the cell membrane of N. maritimus. Here we elucidate the structure of the molecular machinery responsible for this process, comprising the surface layer (S-layer), using electron cryotomography and subtomogram averaging from cells. We supplemented our in situ structure of the ammonium-binding S-layer array with a single-particle electron cryomicroscopy structure, revealing detailed features of this immunoglobulin-rich and glycan-decorated S-layer. Biochemical analyses showed strong ammonium binding by the cell surface, which was lost after S-layer disassembly. Sensitive bioinformatic analyses identified similar S-layers in many ammonia-oxidizing archaea, with conserved sequence and structural characteristics. Moreover, molecular simulations and structure determination of ammonium-enriched specimens enabled us to examine the cation-binding properties of the S-layer, revealing how it concentrates ammonium ions on its cell-facing side, effectively acting as a multichannel sieve on the cell membrane. This in situ structural study illuminates the biogeochemically essential process of ammonium binding and channelling, common to many marine microorganisms that are fundamental to the nitrogen cycle.

Strong cation-exchange combined with mass spectrometry reveals the glycoform heterogeneity of sialylated glycoproteins.

Sialylation is an important modification of proteins, related to protein life and bioactivity. However, the evaluation of sialylation is only based on the average molecular composition by peptide mapping and glycan profiling because sialylated proteins are usually too heterogeneous to obtain good quality mass spectra by conventional intact mass analysis methods. In this study, a simple strong cation exchange-mass spectroscopy (SCX-MS) method was developed for intact mass analysis of sialylated glycoproteins. The developed SCX-MS method provided good separation for sialylated glycoproteins and had an inherent characteristic of native MS. Thus, the intact mass analysis of highly heterogeneous glycoprotein, which cannot be obtained by reversed-phase liquid chromatography (RPLC)-MS and size exclusion chromatography (SEC)-MS methods, can be well analyzed using the current SCX-MS method. First, the method was developed and optimized using the etanercept monomer. Conditions including MS parameters, flow rate, and gradient were investigated. Then, the developed method was used to analyze a new recombinant vaccine, protein 1. Similar to the etanercept monomer, the intact molecular information of protein 1, which cannot be obtained by RPLC-MS and SEC-MS, can be achieved using SCX-MS. Combined with information obtained on peptide mapping and glycan profiles obtained by LC-MS, the new vaccine was well characterized. Finally, the SCX-MS method was used to quickly evaluate the batch-to-batch reproducibility of protein 1. It was much faster than peptide mapping and glycan profiling methods and can provide information complementary to these strategies. It should be useful for many applications where speed and comprehensive characterization are required, such as recombinant sialylated vaccines and fusion proteins.

Exploiting the advantages of cationic copolymers and AgBr nanoparticles to optimize the antibacterial activity of chitosan.

Recently, the chitosan (CS)-based composites have attracted increasing attention for controlling and preventing the spread of pathogenic microorganisms. Herein, an amphiphilic copolymer containing epoxy and quaternary ammonium groups (PBGDBr) was synthesized via three common acrylate monomers. The epoxy groups of this copolymer were then crosslinked with the amino groups of CS to synthesize a natural/synthetic (PBGDBr-C) composite to increase the water solubility of CS under alkaline conditions and enhance its antibacterial activity based on chemical contact-type modes. Moreover, silver bromide nanoparticles (AgBr NPs)-decorated PBGDBr-C (AgBr@PBGDBr-C) composite was prepared, which aimed to endow the final AgBr@PBGDBr-C composite with a photodynamic antibacterial mode relying on the formation of Ag/AgBr nanostructures catalyzed by visible light on AgBr NPs. The results showed that the final composite possessed satisfactory bactericidal effects at concentrations higher than 64 and 128 µg/mL against Escherichia coli and Staphylococcus aureus, respectively. Additionally, The L929 cells treated with the final composite retained high cell viability (>80 %) at a concentration of 128 µg/mL, indicating its low toxicity to L929 cells. Overall, our synthetic strategy exploits a multi-modal system that enables chemical-photodynamic synergies to treat infections caused by pathogenic bacteria while delaying the development of bacterial resistance.

Carrageenan/calcium alginate composite hydrogel filtration membranes for efficient cationic dye separation.

The development of biopolymer-based filtration systems for water remediation applications is an extremely fascinating area of research. In this paper, we developed a biopolymer-based filtration system using sodium alginate (NaAlg) and carrageenan (Car) for the removal of the toxic cationic dye, methylene blue (MB). The membrane's properties were assessed using FTIR, TGA, UTM, FESEM, EDS, XRD, and water uptake, revealing commendable thermomechanical stability (5.79 MPa), good hydrophilicity, and compatibility. The experimental results further revealed that lambda Car/calcium alginate (λ-Car/CaAlg) exhibited superior dye rejection (100%) and flux (11.67 L m-2 h-1) compared to kappa Car/CaAlg (κ-Car/CaAlg) (99.22% and 11.19 L m-2 h-1) and plain alginate (CaAlg) (99.63% and 9.79 L m-2 h-1). The high MB rejection rate was attributed to the sieving mechanism and electrostatic interaction. A rejection rate of 100% was achieved at an initial MB concentration of 10 mg/L, pressure of 0.1 MPa, pH of 7, and temperature of 25°C. Furthermore, the hydrogel membranes demonstrated excellent recyclability over nine cycles, indicating their potential for water treatment applications.

Strategies for Improved pDNA Loading and Protection Using Cationic and Neutral LNPs with Industrial Scalability Potential Using Microfluidic Technology.

Purpose: In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive optimizations are needed for each NMed type and genetic material. This article strives to improve LNPs for pDNA loading, protection, and delivery, while minimizing toxicity. Methods: The microfluidic technique was optimized to form cationic or neutral LNPs to load pDNA. Classical "post-formulation" DNA addition vs "pre" addition in the aqueous phase were compared. All formulations were characterized (size, homogeneity, zeta potential, morphology, weight yield, and stability), then tested for loading efficiency, nuclease protection, toxicity, and cell uptake. Results: Optimized LNPs formulated with DPPC: Chol:DOTAP 1:1:0.1 molar ratio and 10 µg of DOPE-Rhod, had a size of 160 nm and good homogeneity. The chemico-physical characteristics of cationic LNPs worsened when adding 15 µg/mL of pDNA with the "post" method, while maintaining their characteristics up to 100 µg/mL of pDNA with the "pre" addition remaining stable for 30 days. Interestingly, neutral LNPs formulated with the same method loaded up to 50% of the DNA. Both particles could protect the DNA from nucleases even after one month of storage, and low cell toxicity was found up to 40 µg/mL LNPs. Cell uptake occurred within 2 hours for both formulations with the DNA intact in the cytoplasm, outside of the lysosomes. Conclusion: In this study, the upcoming microfluidic technique was applied to two strategies to generate pDNA-LNPs. Cationic LNPs could load 10x the amount of DNA as the classical approach, while neutral LNPs, which also loaded and protected DNA, showed lower toxicity and good DNA protection. This is a big step forward at minimizing doses and toxicity of LNP-based gene therapy.

Functional differentiation and genetic diversity of rice cation exchanger (CAX) genes and their potential use in rice improvement.

Cation exchanger (CAX) genes play an important role in plant growth/development and response to biotic and abiotic stresses. Here, we tried to obtain important information on the functionalities and phenotypic effects of CAX gene family by systematic analyses of their expression patterns, genetic diversity (gene CDS haplotypes, structural variations, gene presence/absence variations) in 3010 rice genomes and nine parents of 496 Huanghuazhan introgression lines, the frequency shifts of the predominant gcHaps at these loci to artificial selection during modern breeding, and their association with tolerances to several abiotic stresses. Significant amounts of variation also exist in the cis-regulatory elements (CREs) of the OsCAX gene promoters in 50 high-quality rice genomes. The functional differentiation of OsCAX gene family were reflected primarily by their tissue and development specific expression patterns and in varied responses to different treatments, by unique sets of CREs in their promoters and their associations with specific agronomic traits/abiotic stress tolerances. Our results indicated that OsCAX1a and OsCAX2 as general signal transporters were in many processes of rice growth/development and responses to diverse environments, but they might be of less value in rice improvement. OsCAX1b, OsCAX1c, OsCAX3 and OsCAX4 was expected to be of potential value in rice improvement because of their associations with specific traits, responsiveness to specific abiotic stresses or phytohormones, and relatively high gcHap and CRE diversity. Our strategy was demonstrated to be highly efficient to obtain important genetic information on genes/alleles of specific gene family and can be used to systematically characterize the other rice gene families.

Golgi-associated retrograde protein (GARP) complex-dependent endosomes to trans Golgi network retrograde trafficking is controlled by Rab4b.

BACKGROUND: The trafficking of cargoes from endosomes to the trans-Golgi network requires numerous sequential and coordinated steps. Cargoes are sorted into endosomal-derived carriers that are transported, tethered, and fused to the trans-Golgi network. The tethering step requires several complexes, including the Golgi-associated retrograde protein complex, whose localization at the trans-Golgi network is determined by the activity of small GTPases of the Arl and Rab family. However, how the Golgi-associated retrograde protein complex recognizes the endosome-derived carriers that will fuse with the trans-Golgi network is still unknown. METHODS: We studied the retrograde trafficking to the trans-Golgi network by using fluorescent cargoes in cells overexpressing Rab4b or after Rab4b knocked-down by small interfering RNA in combination with the downregulation of subunits of the Golgi-associated retrograde protein complex. We used immunofluorescence and image processing (Super Resolution Radial Fluctuation and 3D reconstruction) as well as biochemical approaches to characterize the consequences of these interventions on cargo carriers trafficking. RESULTS: We reported that the VPS52 subunit of the Golgi-associated retrograde protein complex is an effector of Rab4b. We found that overexpression of wild type or active Rab4b increased early endosomal to trans-Golgi network retrograde trafficking of the cation-independent mannose-6-phosphate receptor in a Golgi-associated retrograde protein complex-dependent manner. Conversely, overexpression of an inactive Rab4b or Rab4b knockdown attenuated this trafficking. In the absence of Rab4b, the internalized cation-independent mannose 6 phosphate receptor did not have access to VPS52-labeled structures that look like endosomal subdomains and/or endosome-derived carriers, and whose subcellular distribution is Rab4b-independent. Consequently, the cation-independent mannose-6-phosphate receptor was blocked in early endosomes and no longer had access to the trans-Golgi network. CONCLUSION: Our results support that Rab4b, by controlling the sorting of the cation-independent mannose-6-phosphate receptor towards VPS52 microdomains, confers a directional specificity for cargo carriers en route to the trans-Golgi network. Given the importance of the endocytic recycling in cell homeostasis, disruption of the Rab4b/Golgi-associated retrograde protein complex-dependent step could have serious consequences in pathologies.

Fabrication and application of a continuously regenerated cationic impurity removal device for ion chromatography.

A continuously regenerated cationic impurity removal device (CR-CRD) has been fabricated and applied for ion chromatography (IC). The removal of cationic impurities is realized by electrodialytically replacing the cationic impurities with hydronium ions. The device is configured in a sandwich structure and the central eluent channel is respectively isolated from both electrodes by stacked cation exchange membranes and a bipolar membrane (BPM) plus stacked anion exchange membranes. The eluent channel is packed with cation exchange resins in hydronium form and their continuous regeneration can be achieved by electrodialysis. A desirable feature of the device is gas-free, and no degasser is required. It showed sufficient ability to remove cationic impurities, as indicated by > 99.9 % removal of 10 mL of 1 mM LiOH solution injected (∼10 µmol) or continuous removal of 1 mM LiOH solution at the flow rate of 1 mL/min (1 µmol/min). A useful application was for sample pretreatment in nuclear power industry, by eliminating strong matrix interference of the sample containing LiOH (1 mM) and boric acid (2000 mg/L) with trace anion analysis.

Targeted mutagenesis of negatively charged amino acids outlining the substrate translocation path within the human organic cation transporter 3.

Recently published cryo-EM structures of human organic cation transporters of the SLC22 family revealed seven, sequentially arranged glutamic and aspartic acid residues, which may be relevant for interactions with positively charged substrates. We analyzed the functional consequences of removing those negative charges by creating D155N, E232Q, D382N, E390Q, E451Q, E459Q, and D478N mutants of OCT3. E232Q, E459Q, and D478N resulted in a lack of localization in the outer cell membrane and no relevant uptake activity. However, D155N and E451Q showed a substrate-specific loss of transport activity, whereas E390Q had no remaining activity despite correct membrane localization. In contrast, D382N showed almost wild-type-like uptake. D155 is located at the entrance to the substrate binding pocket and could, therefore be involved in guiding cationic substrates towards the inside of the binding pocket. For E390, we confirm its critical function for transporter function as it was recently shown for the corresponding position in OCT1. Interestingly, E451 seems to be located at the bottom of the binding pocket in the outward-open confirmation of the transporter. Substrate-specific loss of transport activity of the E451Q variant suggests an essential role in the transport cycle of specific substances as part of an opportunistic binding site. In general, our study highlights the impact of the cryo-EM structures in guiding mutagenesis studies to understand the molecular level of transporter-ligand interactions, and it also confirms the importance of testing multiple substrates in mutagenesis studies of polyspecific OCTs.

Synergistic optical sensing: Selective colorimetric analysis of copper in environmental and biological samples.

A groundbreaking optical sensing membrane has been engineered for the accurate assessment of copper ions. The pliable poly(vinyl chloride) membrane is formulated through the integration of sodium tetraphenylborate (Na-TPB), 4-(2-hydroxy-4-nitro azobenzene)-2-methyl-quinoline (HNAMQ), and tri-n-octyl phosphine oxide (TOPO), in conjunction with o-nitrophenyl octyl ether (o-NPOE). The sensor membrane undergoes a thorough investigation of its composition to optimize performance, revealing that HNAMQ serves a dual role as both an ionophore and a chromoionophore. Simultaneously, TOPO contributes to enhancing the complexation of HNAMQ with copper ions. Demonstrating a linear range for Cu2+ ions spanning from 5.0 × 10-9 to 7.5 × 10-6 M, the proposed sensor membrane showcases detection and quantification limits of 1.5 × 10-9 and 5.0 × 10-9 M, respectively. Rigorous assessments of potential interferences from other cations and anions revealed no observable disruptions in the detection of Cu2+. With no discernible HNAMQ leaching, the membrane demonstrates rapid response times and excellent durability. The sensor exhibits remarkable selectivity for Cu2+ ions and can be regenerated through exposure to 0.05 M EDTA. Successful application of the sensor in determining the presence of Cu2+ in biological (blood, liver and meat), soil, food (coffee, black tea, sour cherry juice, black currant, and milk powder) and environmental water samples underscores its efficacy.