Co-, N-doped carbon dot nanozymes based on an untriggered ROS generation approach for anti-biofilm activities and in vivo anti-bacterial treatment.

Bacterial infections originating from food, water, and soil are widely recognized as significant global public health concerns. Biofilms are implicated in approximately two-thirds of bacterial infections. In recent times, nanomaterials have emerged as potential agents for combating biofilms and bacteria, with many of them being activated by light and H2O2 to generate reactive oxygen species (ROS). However, this energy-consuming and extrinsic substrate pattern poses many challenges for practical application. Consequently, there is a pressing need to develop methods for the untriggered generation of ROS to effectively address biofilm and bacterial infections. In this study, we investigated the oxidase-like activity of the Co,N-doped carbon dot (CoNCD) nanozyme, which facilitated the oxidation of ambient O2 to generate 1O2 in the absence of light and H2O2 supplementation; this resulted in effective biofilm cleavage and enhanced bactericidal effects. CoNCDs could become a potential candidate for wound healing and treatment of acute peritonitis in vivo, which can be primarily attributed to the spontaneous production of ROS. This study presents a convenient ROS generator that does not necessitate any specific triggering conditions. The nanozyme properties of CoNCDs exhibit significant promise as a potential remedy for diseases, specifically as an anti-biofilm and anti-bacterial agent.

A novel self-assembled nucleobase-nanofiber platform of CDN to activate the STING pathway for synergistic cancer immunotherapy.

2', 3'-cGAMP (CDN) as cGAS-STING pathway agonist is extensively used in tumor treatment. However, due to its negatively charged nature (containing two phosphate groups) and high hydrophilicity, CDN faces challenges in crossing cell membranes, resulting in reduced efficiency of its use. Additionally, CDN is susceptible to inactivation through phosphodiesterase hydrolysis. Therefore, the development of a new drug delivery system for CDN is necessary to prevent hydrolysis and enhance targeted accumulation in tumors, as well as improve cellular uptake for STING activation. In this study, we have developed peptide-polymer nanofibers (PEG-Q11) that incorporate thymine (T) and arginine (R) residues to facilitate complexation with CDN through the principles of Watson-Crick base pairing with thymine and favorable electrostatic interactions and bidentate hydrogen bonding with arginine side chains. The entrapment efficiency (EE) of PEG-Q11T3R4@CDN was found to be 51% higher than that of PEG-Q11@CDN. Due to its favorable biocompatibility, PEG-Q11T3R4@CDN was employed for immunotherapy in mouse CT26 tumors. In local tumor treatment, the administration of PEG-Q11T3R4@CDN at a low dose and through a single injection exhibited inhibitory effects. Furthermore, the local injection of PEG-Q11T3R4@CDN resulted in systemic therapeutic responses, effectively suppressing tumor metastasis by activating CD8 + T cells to target distant tumors. This research not only underscores the potential of PEG-Q11T3R4@CDN as an efficient therapeutic agent but also highlights its ability to achieve long-lasting systemic therapeutic outcomes following local treatment. Consequently, PEG-Q11T3R4@CDN represents a promising strategy for immunization.

Synthetic K+ Channels Constructed by Rebuilding the Core Modules of Natural K+ Channels in an Artificial System.

Different types of natural K+ channels share similar core modules and cation permeability characteristics. In this study, we have developed novel artificial K+ channels by rebuilding the core modules of natural K+ channels in artificial systems. All the channels displayed high selectivity for K+ over Na+ and exhibited a selectivity sequence of K+ ≈Rb+ during the transport process, which is highly consistent with the cation permeability characteristics of natural K+ channels. More importantly, these artificial channels could be efficiently inserted into cell membranes and mediate the transmembrane transport of K+ , disrupting the cellular K+ homeostasis and eventually triggering the apoptosis of cells. These findings demonstrate that, by rebuilding the core modules of natural K+ channels in artificial systems, the structures, transport behaviors, and physiological functions of natural K+ channels can be mimicked in synthetic channels.

Fluoro-functionalized ionic covalent organic frameworks (F-iCOFs) for highly selective enrichment and sensitive determination of perfluorinated sulfonates by MALDI-MS.

An easily prepared fluoro-functionalized ionic covalent organic framework (F-iCOF) has been implemented into MALDI-TOF MS, enabling the highly selective enrichment and sensitive determination of perfluorinated sulfonate (PFS) contaminants in a rapid and convenient manner. The good thermal stability and excellent optical absorption properties of F-iCOF makes it a brilliant matrix with no background noise. Moreover, benefitting from the large surface area, appropriate pore size, good water dispersibility, and abundant fluorine atom and cationic characteristic of F-iCOF, it exhibited superior adsorption capacity and enrichment selectivity towards PFSs. Good signal responses for PFSs were obtained in the presence of various interfering compounds such as BSA, HA, or even more than 100-fold excess of glutamic acid and similar in structure sodium alkyl sulfonates, highlighting the specific selectivity of F-iCOF. Calibration curves for potassium perflurobutane sulfonate (PFBSK) in tap water and whole blood were established with good linear correlation in the range 1-500 pg mL-1. The limits of detection and quantification for PFBSK were as low as 0.04 pg mL-1 and 0.05 pg mL-1, respectively, which are comparable or better than the existing methods for the determination of PFSs.

High-throughput profiling volatiles in edible oils by cooling assisted solid-phase microextraction technique for sensitive discrimination of edible oils adulteration.

In this study, a cooling assisted solid-phase microextraction technique (CA-SPME) was proposed and used for identifying volatile and semi-volatile compounds in edible oil innovatively coupled to gas chromatography-mass spectrometry. Compared with regular SPME technique, CA-SPME presented significantly higher extraction efficiencies for analytes in edible oil due to its synergistic effect of heating and cooling. After optimization of the extraction conditions including heating temperature, cooling temperature, extraction time, and added amount of edible oil, thirty-eight, thirty-six, twenty-nine, and thirty-three kinds of compounds in peanut oil, olive oil, canola oil, and soybean oil were successfully identified, respectively, using DVB/CAR/PDMS coating with extraction time of 30 min and edible oil amounts of 20 µL. Principal component analysis, partial least squares discriminant analysis, and hierarchical clustering analysis (HCA) were performed to evaluate the potential of proposed method in discriminating edible oils adulteration (peanut oil adulterated with canola oil, peanut oil adulterated with soybean oil, olive oil adulterated with canola oil) subsequently. Results demonstrated that the method was useful in successful discrimination of pure and adulterated edible oils with adulteration percentages ranging from 0.5 to 10%. Furthermore, volatiles contributing to classifications between pure and adulterated edible oils were also illustrated based on variable importance for the projection analysis and distributions of volatiles in HCA heatmaps. The proposed method provided a novel strategy for sensitive detection of edible oil adulteration without any other sample pretreatment.

Fullerenol as a water-soluble MALDI-MS matrix for rapid analysis of small molecules and efficient quantification of saccharin sodium in foods.

Due to the strong background interferences in the low-mass region and poor reproducibility of conventional organic matrices, it is of great importance to develop a novel matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to qualitatively and quantitatively analyze small molecules. In this work, water-soluble fullerenol C60(OH)24-26 was selected as a MALDI matrix for the analysis of low-molecular-weight compounds in consideration of optical absorption property, water solubility and stability. Compared with the traditional matrices, fullerenol demonstrated lower background interference and stronger peak intensity. In addition, the hydrophilic fullerenol could avoid the heterogeneous crystallization in sample preparation, increase the reproducibility and sensitivity of MALDI-MS, and ameliorate quantitative analysis of small molecules. With saccharin as model analyte, quantitative analysis was carried out using fullerenol as matrix. The results demonstrated satisfying reproducibility and good tolerance to salt. The limit-of-detection of the quantitative analysis was as low as 4 pmol, and the linear range is 1-100 µg mL-1 with R2 greater than 0.99. The analytical results also showed excellent precision and accuracy, low matrix effect and good recovery rate. Fullerenol as a potential matrix was further validated in the quantification of saccharin sodium in different real food samples, such as nuts and drinks. This work not only confirms the potential of fullerenol for the quantitative analysis in food field, but also provides a new technique for rapid analysis of small molecules.

Effect of charge status on the ion transport and antimicrobial activity of synthetic channels.

A class of unimolecular channels formed by pillararene-gramicidin hybrid molecules are presented. The charge status of the peptide domain in these channels has a significant impact on their ion transport and antimicrobial activity. These channels exhibited different membrane-association abilities between microbial cells and mammalian cells. One of the channels displayed a higher antimicrobial activity towards S. aureus (IC50 = 0.55 µM) and negligible hemolytic toxicity, showing potential to serve as a systemic antibiotic.

Three-dimensional pompon-like Au/ZnO porous microspheres as solid phase microextraction coating for determination of volatile fatty acids from foot odor.

In this study, facile fabrication of three-dimensional (3D) pompon-like gold/zinc oxide (Au/ZnO) porous microspheres by hydrothermal procedure was investigated. These microspheres were utilized as solid phase microextraction (SPME) coating for determination of volatile fatty acids (VFAs) from foot odor coupling with gas chromatography-mass spectrometry (GC-MS). SEM and TEM characterizations showed that as-prepared material was composed of 1D porous nanowires and presented a uniform coating on stainless-steel wire. The extraction of VFAs including propanoic acid, butyric acid, isobutanoic acid, isovaleric acid, hexanoic acid, and heptylic acid was carried out by headspace model after sampling from human foot using cotton wool strips. Following optimization of extraction parameters including extraction temperature and time and desorption temperature and time, the as-prepared SPME coating presented better extraction efficiency than commercial DVB/CAR/PDMS fiber towards all the VFAs due to its excellent properties. Under the optimized conditions, the method exhibited good linearity (0.5-200 ng) with regression coefficients (R2) ranging from 0.9836 to 0.9981 for all the analytes. The limits of detection ranged from 0.017 to 0.098 ng. Single fiber repeatability varied from 6.5% to 11.2% and the fiber-to-fiber reproducibility ranged from 8.6% to 12.3%. The proposed method was successfully applied for extraction and determination of VFAs from foot odor after sampling using cotton wool strips.

Distinct Dynamic and Conformational Features of Human STING in Response to 2'3'-cGAMP and c-di-GMP.

The human stimulator of interferon genes protein (hSTING) can bind cyclic dinucleotides (CDNs) to activate the production of type I interferons and inflammatory cytokines. These CDNs can be either bacterial secondary messengers, 3'3'-CDNs, or endogenous 2'3'-cGAMP. cGAMP, with a unique 2'-5' bond, is the most potent activator of hSTING among all CDNs. However, current understanding of the molecular principles underlying the unique ability of 2'3'-cGAMP to potently activate hSTINGs other than 3'3'-CDNs remains incomplete. In this work, molecular dynamics simulations were used to provide an atomistic picture of the binding of 2'3'-cGAMP and one 3'3'-CDN (c-di-GMP) to hSTING. The results suggest that hSTING binds more strongly to 2'3'-cGAMP than to c-di-GMP, which prefers to bind with a more open and flexible state of hSTING. Finally, a potential "dock-lock-anchor" mechanism is proposed for the activation of hSTING upon the binding of a potent ligand. It is believed that deep insights into understanding the binding of hSTING with 3'3'-CDNs and the endogenous 2'3'-cGAMP would help to establish the principles underlying powerful 2'3'-cGAMP signaling and the nature of hSTING activation, as well as related drug design.

Artificial K+ Channels Formed by Pillararene-Cyclodextrin Hybrid Molecules: Tuning Cation Selectivity and Generating Membrane Potential.

A class of artificial K+ channels formed by pillararene-cyclodextrin hybrid molecules have been designed and synthesized. These channels efficiently inserted into lipid bilayers and displayed high selectivity for K+ over Na+ in fluorescence and electrophysiological experiments. The cation transport selectivity of the artificial channels is tunable by varying the length of the linkers between pillararene and cyclodexrin. The shortest channel showed specific transmembrane transport preference for K+ over all alkali metal ions (selective sequence: K+ > Cs+ > Rb+ > Na+ > Li+ ), and is rarely observed for artificial K+ channels. The high selectivity of this artificial channel for K+ over Na+ ensures specific transmembrane translocation of K+ , and generated stable membrane potential across lipid bilayers.

A unimolecular channel formed by dual helical peptide modified pillar[5]arene: correlating transmembrane transport properties with antimicrobial activity and haemolytic toxicity.

Five unimolecular channels with different lengths are presented. The varying length of these channels has significant impact on their transmembrane transport properties, which are directly correlated with their antimicrobial activity and inversely correlated with their haemolytic toxicity. By further structural optimization, these new channels could reach high antimicrobial activity and very low haemolytic toxicity, with the potential to serve as systemic antibiotics.

Covalent Attachment of Daptomycin to Ti6Al4V Alloy Surfaces by a Thioether Linkage to Inhibit Colonization by Staphylococcus aureus.

Infections are a devastating complication of titanium alloy orthopedic implants. Current therapies include antibiotic-impregnated bone cement and antibiotic-containing coatings. Daptomycin (DAP) (1) is a novel peptide antibiotic that penetrates the cell membranes of Gram-positive bacteria. Few DAP-resistant strains have appeared so far. We hypothesized that when DAP covalently bonded via a flexible, hydrophilic spacer it could prevent bacterial colonization of titanium alloy surfaces. We designed and synthesized a series of DAP conjugates for bonding to the surface of Ti6Al4V foils through tetra(ethylene glycol) spacers via thioether linkages. The stability and antimicrobial activity of the attached conjugates were evaluated using Staphylococcus aureus ATCC 25923. Colonization of the Ti6Al4V foils was inhibited by 72% at 8 h and 54% at 24 h. The strategy described in this report provides a new, more facile way to prepare bactericidal Ti6Al4V implants.

One-pot formation of hydrazide macrocycles with modified cavities: an example of pH-sensitive unimolecular cation channels.

A series of pH-sensitive, cation-selective hydrazide macrocyclic channels have been synthesized. The macrocyclic channels bear multiple carboxyls in the inner cavity, which have a significant impact on their membrane-incorporation ability and NH4+ transport activity. Moreover, the K+/Cl- selectivities of the macrocyclic channels can be tuned by the pH value of the electrolyte.

Functionalized hydrazide macrocycle ion channels showing pH-sensitive ion selectivities.

Transmembrane channels formed by functionalized hydrazide macrocycles are reported. The different pH values of buffer solutions have a significant effect on the K+/Cl- selectivity of the macrocycles. This unique transport behavior is mainly induced by the different distributions of charges in the tubular channels under various pH values.

Evaluation of a PACAP Peptide Analogue Labeled with (68)Ga Using Two Different Chelating Agents.

OBJECTIVE: The authors have conjugated chelating agents (DOTA and NODAGA) with a peptide (pituitary adenylate cyclase-activating peptide [PACAP] analogue) that has a high affinity for VPAC1 receptors expressed on cancer cells. To determine a suitable chelating agent for labeling with (68)Ga, they have compared the labeling kinetics and stability of these peptide conjugates. METHODS: For labeling, (68)GaCl3 was eluted in 0.1 M HCl from a [(68)Ge-(68)Ga] generator. The influences of peptide concentration, pH, and temperature on the radiolabeling efficiency were studied. The stability was evaluated in saline, human serum, DTPA, transferrin, and metallic ions (FeCl3, CaCl2, and ZnCl2). Cell binding assay was performed using human breast cancer cells (T47D). Tissue biodistribution was studied in normal athymic nude mice. RESULTS: Optimal radiolabeling (>95.0%) of the DOTA-peptide conjugates required a higher (50°C-90°C) temperature and 10 minutes of incubation at pH 2-5. The NODAGA-peptide conjugate needed incubation only at 25°C for 10 minutes. Both radiocomplexes were stable in saline, serum, as well as against transchelation and transmetallation. Cell binding at 37°C for 15 minutes of incubation with (68)Ga-NODAGA-peptide was 34.0% compared to 24.5% for (68)Ga-DOTA-peptide. Tissue biodistribution at 1 hour postinjection of both (68)Ga-labeled peptide conjugates showed clearance through the kidneys. CONCLUSIONS: NODAGA-peptide showed more convenient radiolabeling features than that of DOTA-peptide.

Ecosystem Functions Connecting Contributions from Ecosystem Services to Human Wellbeing in a Mangrove System in Northern Taiwan.

The present study examined a mangrove ecosystem in northern Taiwan to determine how the various components of ecosystem function, ecosystem services and human wellbeing are connected. The overall contributions of mangrove services to specific components of human wellbeing were also assessed. A network was developed and evaluated by an expert panel consisting of hydrologists, ecologists, and experts in the field of culture, landscape or architecture. The results showed that supporting habitats was the most important function to human wellbeing, while water quality, habitable climate, air quality, recreational opportunities, and knowledge systems were services that were strongly linked to human welfare. Security of continuous supply of services appeared to be the key to a comfortable life. From a bottom-up and top-down perspective, knowledge systems (a service) were most supported by ecosystem functions, while the security of continuous supply of services (wellbeing) had affected the most services. In addition, the overall benefits of mangrove services to human prosperity concentrated on mental health, security of continuous supply of services, and physical health.

Fluorescence detection of KRAS2 mRNA hybridization in lung cancer cells with PNA-peptides containing an internal thiazole orange.

We previously developed reporter-peptide nucleic acid (PNA)-peptides for sequence-specific radioimaging and fluorescence imaging of particular mRNAs in cells and tumors. However, a direct test for PNA-peptide hybridization with RNA in the cytoplasm would be desirable. Thiazole orange (TO) dye at the 5' end of a hybridization agent shows a strong increase in fluorescence quantum yield when stacked upon a 5' terminal base pair, in solution and in cells. We hypothesized that hybridization agents with an internal TO could distinguish a single base mutation in RNA. Thus, we designed KRAS2 PNA-IGF1 tetrapeptide agents with an internal TO adjacent to the middle base of the 12th codon, a frequent site of cancer-initiating mutations. Our molecular dynamics calculations predicted a disordered bulge with weaker hybridization resulting from a single RNA mismatch. We observed that single-stranded PNA-IGF1 tetrapeptide agents with an internal TO showed low fluorescence, but fluorescence escalated 5-6-fold upon hybridization with KRAS2 RNA. Circular dichroism melting curves showed ∼10 °C higher Tm for fully complementary vs single base mismatch TO-PNA-peptide agent duplexes with KRAS2 RNA. Fluorescence measurements of treated human lung cancer cells similarly showed elevated cytoplasmic fluorescence intensity with fully complementary vs single base mismatch agents. Sequence-specific elevation of internal TO fluorescence is consistent with our hypothesis of detecting cytoplasmic PNA-peptide:RNA hybridization if a mutant agent encounters the corresponding mutant mRNA.

Determining efficacy of breast cancer therapy by PET imaging of HER2 mRNA.

INTRODUCTION: Monitoring the effectiveness of therapy early and accurately continues to be challenging. We hypothesize that determination of Human Epidermal Growth Factor Receptor 2 (HER2) mRNA in malignant breast cancer (BC) cells by positron emission tomography (PET) imaging, before and after treatment, would reflect therapeutic efficacy. METHOD: WT4340, a peptide nucleic acid (PNA) 12-mer complementary to HER2 mRNA was synthesized together with -CSKC, a cyclic peptide, which facilitated internalization of the PNA via IGFR expressed on BC cells, and DOTA that chelated Cu-64. Mice (n = 8) with BT474 ER+/HER2+ human BC received doxorubicin (DOX, 1.5mg/kg) i.p. once a week for six weeks. Mice (n = 8) without DOX served as controls. All mice were PET imaged with F-18-FDG and 48 h later with Cu-64-WT4340. PET imaging were performed before and 72 h after each treatment. Standardized uptake values (SUVs) were determined and percent change calculated. Animal body weight (BW) and tumor volume (TV) were measured. RESULTS: SUVs for Cu-64-WT4340 after DOX treatment declined by 54% ± 17% after the second dose, 41% ± 15% after the fourth dose, and 29% ± 7% after the sixth dose, compared with 42% ± 22%, 31% ± 18%, and 13% ± 9% (p<0.05) for F-18-FDG. In untreated mice, the corresponding percent SUVs for Cu-64-WT4340 were 145% ± 82%, 165% ± 39%, and 212% ± 105% of pretreatment SUV, compared with 108% ± 28%, 151% ± 8%, and 152% ± 35.5%, (p<0.08) for F-18-FDG. TV in mice after second dose was 114.15% ± 61.83%, compared with 144.7% ± 64.4% for control mice. BW of DOX-treated mice was 103.4% ± 7.6% of pretreatment, vs. 100.1% ± 4.3% for control mice. CONCLUSION: Therapeutic efficacy was apparent sooner by molecular PET imaging than by determination of reduction in TV.

Effects of hypoxanthine substitution in peptide nucleic acids targeting KRAS2 oncogenic mRNA molecules: theory and experiment.

Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multimutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick base pairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA:PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA:PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition.

Fluorescent peptide-PNA chimeras for imaging monoamine oxidase A mRNA in neuronal cells.

Monoamine oxidases (MAO) catalyze the oxidative deamination of many biogenic amines and are integral proteins found in the mitochondrial outer membrane. Changes in MAO-A levels are associated with depression, trait aggression, and addiction. Here we report the synthesis, characterization, and in vitro evaluation of novel fluorescent peptide-peptide nucleic acid (PNA) chimeras for MAOA mRNA imaging in live neuronal cells. The probes were designed to include MAOA-specific PNA dodecamers, separated by an N-terminal spacer to a µ-opioid receptor targeting peptide (DAMGO), with a spacer and a fluorophore on the C-terminus. The probe was successfully delivered into human SH-SY5Y neuroblastoma cells through µ-opioid receptor-mediated endocytosis. The K(d) by flow cytometry was 11.6 ± 0.8 nM. Uptake studies by fluorescence microscopy showed ∼5-fold higher signal in human SH-SY5Y neuroblastoma cells than in negative control CHO-K1 cells that lack µ-opioid receptors. Moreover, a peptide-mismatch control sequence showed no significant uptake in SH-SY5Y cells. Such mRNA imaging agents with near-infrared fluorophores might enable real time imaging and quantitation of neuronal mRNAs in live animal models.