Bacterial Cells Research Articles

High-yield production of recombinant human myelin oligodendrocyte glycoprotein in SHuffle bacteria without a refolding step

Experimental autoimmune encephalomyelitis (EAE) is a model for central nervous system (CNS) autoimmune demyelinating diseases such as multiple sclerosis (MS) and MOG antibody-associated disease (MOGAD). Immunization with the extracellular domain of recombinant human MOG (rhMOG), which contains pathogenic antibody and T cell epitopes, induces B cell-dependent EAE for studies in mice. However, these studies have been hampered by rhMOG availability due to its insolubility when overexpressed in bacterial cells, and the requirement for inefficient denaturation and refolding. Here, we describe a new protocol for the high-yield production of soluble rhMOG in SHuffle cells, a commercially available E. coli strain engineered to facilitate disulfide bond formation in the cytoplasm. SHuffle cells can produce a soluble fraction of rhMOG yielding >100 mg/L. Analytical size exclusion chromatography multi-angle light scattering (SEC-MALS) and differential scanning fluorimetry of purified rhMOG reveals a homogeneous monomer with a high melting temperature, indicative of a well-folded protein. An in vitro proliferation assay establishes that purified rhMOG can be processed and recognized by T cells expressing a T cell receptor (TCR) specific for the immunodominant MOG35–55 peptide epitope. Lastly, immunization of wild-type, but not B cell deficient, mice with rhMOG resulted in robust induction of EAE, indicating a B cell-dependent induction. Our SHuffle cell method greatly simplifies rhMOG production by combining the high yield and speed of bacterial cell expression with enhanced disulfide bond formation and folding, which will enable further investigation of B cell-dependent EAE and expand human research of MOG in CNS demyelinating diseases.

Biochemistry, Mechanistic Intricacies, and Therapeutic Potential of Antimicrobial Peptides: An Alternative to Traditional Antibiotics.

The emergence of drug-resistant strains of pathogens becomes a major obstacle to treating human diseases. Antibiotics and antivirals are in the application for a long time but now these drugs are not much effective anymore against disease-causing drugresistant microbes and gradually it is becoming a serious complication worldwide. The development of new antibiotics cannot be a stable solution to treat drug-resistant strains due to their evolving nature and escaping antibiotics. At this stage, antimicrobial peptides (AMPs) may provide us with novel therapeutic leads against drug-resistant pathogens. Structurally, antimicrobial peptides are mostly α-helical peptide molecules with amphiphilic properties that carry the positive charge (cationic) and belong to host defense peptides. These positively charged AMPs can interact with negatively charged bacterial cell membranes and may cause the alteration in electrochemical potential on bacterial cell membranes and consequently lead to the death of microbial cells. In the present study, we will elaborate on the implication of AMPs in the treatment of various diseases along with their specific structural and functional properties. This review will provide information which assists in the development of new synthetic peptide analogues to natural AMPs. These analogues will eliminate the limitations of natural AMPs like toxicity and severe hemolytic activities.

6-Paradol exhibits antimicrobial, anti-quorum sensing and anti-virulence capacities on gram-negative bacteria: In vitro and in vivo studies

Grains of paradise, which are native to Africa, contain 6-paradol, an antibacterial compound found in plants of the Zingiberaceae family. In this study, the antimicrobial and anti-virulence properties of 6-paradol were evaluated against significant Gram-negative bacteria. The assessment encompassed the impact of 6-paradol on the bacterial cell membrane, efflux mechanisms, bacterial motility, biofilm formation, and extracellular enzyme production. The findings demonstrated promising anti-quorum sensing activity of 6-paradol. Moreover, the outcomes unveiled that the antimicrobial activity of 6-paradol is primarily stems from its substantial influence on the bacterial cell membrane and efflux mechanisms. Furthermore, 6-paradol exhibited potential anti-virulence activities by effectively reducing the generation of biofilm and virulent enzymes, impeding bacterial motility, and inhibiting in vivo bacterial pathogenesis. These anti-virulence effects were attributed to the compound's interference with quorum sensing systems and the downregulation of genes associated with these systems. Additionally, when combined with antibiotics, 6-paradol demonstrated synergistic effects. In conclusion, 6-paradol possesses noteworthy anti-virulence activities and can be employed as an auxiliary alongside antibiotics for treating aggressive Gram-negative infections. This highlights its potential as a valuable addition to therapeutic strategies.

Silver nanoparticles in medicine and technology: Synthesis, functionality and future prospects

Nanomaterials, particularly nanoparticles (NPs), have emerged as forefront materials of the 21st century due to their unique properties and potential applications. Silver nanoparticles (AgNPs) are among the most attractive inorganic nanomaterials, widely used due to their significant antibacterial properties, broad-spectrum activity, and potential applications across various fields, including health, food storage, textiles, and environmental solutions. AgNPs exhibit a large surface-area-to-volume ratio, enabling enhanced interaction with bacterial cells, making them effective in medical and industrial applications. Despite concerns over their toxicity, AgNP-based products have been approved by multiple regulatory bodies. Nanoparticle synthesis methods are generally divided into physical, chemical, and biological approaches. Although physical and chemical methods are efficient, they involve toxic chemicals and energy-intensive processes. Biosynthesis, particularly plant-based, offers an eco-friendly alternative, reducing the environmental and health risks associated with chemical agents. The review also explores the therapeutic potential of AgNPs in drug delivery systems, especially in topical formulations like nanoemulsions, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs). These nanocarriers enhance drug stability, skin penetration, and controlled release, making them ideal for dermal and transdermal delivery. Moreover, the use of AgNPs in dentistry is highlighted for their bactericidal properties, which help prevent infections and implant failure, with the potential for enhancing therapeutic outcomes without common drawbacks like tooth staining. Overall, AgNPs exhibit promising applications in biomedical, pharmaceutical, and dental fields, and their novel functionalities continue to advance the development of nanotechnology-driven solutions.

Use of stable isotope combined with intact cell lipidomic by routine MALDI mass spectrometry analysis for rapid drug susceptibility assay in mycobacteria.

Rapid, accurate, and easy-to-perform diagnostic assays are required to address the current need for the diagnosis of resistant pathogens. That is particularly the case for mycobacteria, such as the human pathogen Mycobacterium tuberculosis, which requires up to 2weeks for the determination of the drug susceptibility profile using the conventional broth microdilution method. To address this challenge, we investigated the incorporation of deuterium, the stable isotope of hydrogen, into lipids as a read out of the drug susceptibility profile. Deuterium is incorporated into newly synthesized proteins or lipids in place of hydrogen as bacterial cells grow, increasing the mass of the macromolecules, which can then be observed via matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). As proof-of-concept, we used the non-pathogenic Mycobacterium smegmatis mc2155 strain, which is susceptible to the aminoglycoside antibiotic kanamycin, and M.smegmatis mc2155 containing the empty vector pVV16, which is kanamycin-resistant. Bacteria were incubated in a culture medium containing 50% of deuterium oxide (D2O) and either 1 or 2 times the minimal inhibitory concentration (MIC50) of kanamycin. Lipids were then analyzed using the MBT lipid Xtract matrix combined with routine MALDI mass spectrometry in the positive ion mode to evaluate the changes in the lipid profile. Using this approach, we were able to distinguish susceptible from resistant bacteria in less than 5h, a process that would take 72 h using the conventional broth microdilution method. We therefore propose a solution for the rapid determination of drug susceptibility profiles using a phenotypic assay combining D2O stable isotope labelling and lipid analysis by routine MALDI mass spectrometry.

The ability in managing reactive oxygen species affects Escherichia coli persistence to ampicillin after nutrient shifts.

This research delves into the intriguing realm of bacterial persistence and its profound implications for biofilms, infections, and antibiotic efficacy. The study focuses on Escherichia coli and how the switch from different carbon sources to fatty acids influences the formation of persister-resilient bacterial cells resistant to antibiotics. The findings reveal a striking variation in survival rates, with a significant number of cells surviving ampicillin treatment after transitioning from glucose to oleic acid. The key revelation is the role of reactive oxygen species (ROS) in cell killing, particularly after switching from gluconeogenic carbons. The timing of ROS bursts aligns with the rapid killing phase, highlighting the critical impact of oxidative stress regulation on persistence frequency. This research provides valuable insights into bacterial persistence mechanisms, offering potential avenues for targeted therapeutic interventions to combat bacterial resistance effectively.

Detection of Serratia marcescens and Mierococcus lysodeikticus Bacterial Cells Using Cerium Oxide/Transition Metal Dichalcogenides Heterostructure‐Based Surface Plasmon Resonance Sensor

This article explores a novel surface plasmon resonance sensor for detecting the Serratia marcescens and Mierococcus lysodeikticus bacteria cells employing the cerium oxide (CeO2) and transition metal dichalcogenides heterostructure. The proposed sensor is designed based on the Kretschmann configuration, where silver (Ag) is deposited over a prism's surface to excite the surface plasmons. The transfer matrix method and angular interrogation technique are exploited for analyzing the sensor's performance at a wavelength of 633 nm. Firstly, the optimization of the prism and metal film is analyzed by selecting the minimum reflectance, better sensitivity, and quality factor. Secondly, the influence of the proposed structure in the sensor is executed by the performances of different structures, which are made with defined layers. Thirdly, different sensing parameters are analyzed for the considered bacterial cells, resulting in the maximum attained parameters being a sensitivity of 369.40° RIU−1, QF of 151.35 RIU−1, and detection accuracy of 7.57. Finally, the comparative study also shows the noteworthy enhancement using the proposed sensor compared with existing work. Therefore, the proposed sensor can be used as a carrier for detecting the bacterial cells and establishing a new platform for biomolecular and biomedical applications.

MOF-Enhanced Phototherapeutic Wound Dressings Against Drug-Resistant Bacteria.

Phototherapy is a low-risk alternative to traditional antibiotics against drug-resistant bacterial infections. However, optimizing phototherapy agents, refining treatment conditions, and addressing misuse of agents, remain a formidable challenge. This study introduces a novel concept leveraging the unique customizability of metal-organic frameworks (MOFs) to house size-matched dye molecules in "single rooms". The mesoporous iron(III) carboxylate nanoMOF, MIL-100(Fe), and the hydrophobic heptamethine cyanine photothermal dye (Cy7), IR775, are selected as model systems. Their combination is predicted to minimize dye-dye interactions, leading to exceptional photostability and efficient light-to-heat conversion. Furthermore, MIL-100(Fe) preserves the antimicrobial nature of hydrophobic IR775, enabling it to disrupt bacterial cell envelopes. Through electrospinning, MIL-100(Fe)@IR775 nanoparticles are shaped into a gelatin-based film dressing for the treatment of skin wounds infected by Methicillin-resistant Staphylococcus aureus (MRSA). Activation of the dressing requires only a portable near-infrared light-emitting diode (NIR LED) and induces both low-dose photodynamic therapy (LPDT) and mild-temperature photothermal therapy (MPTT). Combined with the antimicrobial properties of IR775 and ferroptosis-like lipid peroxidation induced by MIL-100(Fe), the photoactive dressing eradicates MRSA and the healing is as quick as the uninfected wounds. This safe, cost-effective, and multifunctional therapeutic wound dressing offers a promising solution to overcome the current bottleneck in phototherapy.

Engineered Outer Membrane Vesicles as Nanosized Immune Cell Engagers for Enhanced Solid Tumor Immunotherapy.

Although tumor immunotherapy has achieved significant success in recent years, tackling solid tumors remains a formidable challenge. Here, we present an approach that utilizes outer membrane vesicles (OMVs) from bacterial cells as scaffolds to engage immune cells in solid tumor immunotherapy. Two types of nanobodies targeting CD47/SIRPα and PD-1/PD-L1 pathways were simultaneously conjugated onto the surfaces of the OMVs in divalent and trivalent forms using orthogonal SpyCatcher-SpyTag and SnoopCatcher-SnoopTag chemistry. This resulted in the generation of an OMV-based nanosized immune cell engager (OMV-NICE) with dual-targeting abilities. In vitro assays confirmed the retention of the function of the two nanobodies on the OMV-NICE, as evidenced by the synergistically enhanced macrophage phagocytosis and T cell cytotoxicity against tumor cells. In vivo studies using a B16-F10 melanoma mouse model also revealed the superior antitumor activity of OMV-NICE compared to those of unconjugated nanobodies and OMVs alone. Subsequent mechanistic investigations further supported the enhanced recruitment of macrophages and T cells to the tumor region by OMV-NICE. Overall, this work expands the current repertoire of immune cell engagers, and the developed OMV-NICE platform holds great promise for broad applications, particularly in solid tumor immunotherapy.

Single-cell Sequencing of Circulating Human Plasmablasts during Staphylococcus aureus Bacteremia.

Staphylococcus aureus is the major cause of healthcare-associated infections, including life-threatening conditions as bacteremia, endocarditis, and implant-associated infections. Despite adequate antibiotic treatment, the mortality of S. aureus bacteremia remains high. This calls for different strategies to treat this infection. In past years, sequencing of Ab repertoires from individuals previously exposed to a pathogen emerged as a successful method to discover novel therapeutic monoclonal Abs and understand circulating B cell diversity during infection. In this paper, we collected peripheral blood from 17 S. aureus bacteremia patients to study circulating plasmablast responses. Using single-cell transcriptome gene expression combined with sequencing of variable heavy and light Ig genes, we retrieved sequences from >400 plasmablasts revealing a high diversity with >300 unique variable heavy and light sequences. More than 200 variable sequences were synthesized to produce recombinant IgGs that were analyzed for binding to S. aureus whole bacterial cells. This revealed four novel monoclonal Abs that could specifically bind to the surface of S. aureus in the absence of Ig-binding surface SpA. Interestingly, three of four mAbs showed cross-reactivity with Staphylococcus epidermidis. Target identification revealed that the S. aureus-specific mAb BC153 targets wall teichoic acid, whereas cross-reactive mAbs BC019, BC020, and BC021 target lipoteichoic acid. All mAbs could induce Fc-dependent phagocytosis of staphylococci by human neutrophils. Altogether, we characterize the active B cell responses to S. aureus in infected patients and identify four functional mAbs against the S. aureus surface, of which three cross-react with S. epidermidis.

High-Density Fermentation of Lactobacillus plantarum P6: Enhancing Cell Viability via Sodium Alginate Enrichment

Lactobacillus plantarum exhibits a wide range of beneficial physiological functions, including maintaining intestinal microbiota balance, reducing serum cholesterol, and promoting digestive health. According to the specific nutrient requirements of Lactobacillus plantarum P6, we investigated the effects of various carbon sources, nitrogen sources, trace elements, growth-promoting substances, as well as the initial pH and inoculum size on the growth of Lactobacillus plantarum P6 under fermentation conditions. The optimal growth conditions for Lactobacillus plantarum P6 were identified to facilitate high-density fermentation in small-scale fermenter production, achieving a cell concentration of 1.03 × 1011 CFU/mL. This resulted in a 2.5-fold increase in bacterial wet weight, and fermentation time was reduced to 12 h when utilizing a specific medium enriched with 0.2% sodium alginate. It is hypothesized that sodium alginate forms a protective film around the bacterial cells, promoting cell aggregation and enhancing self-coalescence, potentially triggering a bacterial community effect. These results provide a basis for the industrial-scale high-density cultivation of Lactobacillus plantarum, offering potential for enhanced biotechnological applications.

Effect of Surface-Immobilized States of Antimicrobial Peptides on Their Ability to Disrupt Bacterial Cell Membrane Structure

Antimicrobial peptide (AMP) surfaces are widely used to inhibit biofilm formation and bacterial infection. However, endpoint-immobilized AMPs on surfaces are totally different from free-state AMPs due to the constraints of the surface. In this work, the interactions between AMPs and bacterial cell membranes were analyzed through coarse-grained molecular dynamics and all-atom molecular dynamics simulations. This AMP disrupted membrane structure by altering the thickness and curvature of the membrane. Furthermore, the effect of surface-immobilized states of AMPs on their ability to disrupt membrane structure was revealed. The immobilized AMPs in the freeze-N system could bind to the membrane and disrupt the membrane structure through electrostatic forces between positively charged N-terminal amino acid residues and the negatively charged membrane, while the immobilized AMPs in the freeze-C system were repelled. The results will aid in the rational design of new AMP surfaces with enhanced efficacy and stability.

Antimicrobial activity of ceftibuten/polymyxin B combination against polymyxin/carbapenem-resistant Klebsiella pneumoniae.

To evaluate the synergistic effect of a ceftibuten and polymyxin B combination and to determine its capacity to overcome polymyxin B resistance in polymyxin/carbapenem-resistant (PC-R) Klebsiella pneumoniae. To investigate the combination's antibacterial efficacy, antimicrobial susceptibility tests using broth microdilution methods, chequerboard assays and time-kill testing were performed. Antibiofilm activity was also assessed. The treatment's effect on the bacterial cell membrane was examined by quantifying intracellular protein leakage and conducting scanning electron microscopy. Haemocompatibility tests were conducted to evaluate toxicity. Additionally, an infection model was established using Swiss mice to assess in vivo antimicrobial activity. The ceftibuten/polymyxin B combination demonstrated synergistic effects against several PC-R strains of K. pneumoniae, as determined by the FIC index (FICI) values, which ranged from 0.15 to 0.37. This combination was efficacious, exhibiting bactericidal activity at twice the MIC. Ceftibuten/polymyxin B also demonstrated antibiofilm activity. Additionally, ceftibuten/polymyxin B neither damaged the bacterial membrane nor exhibited haemolytic activity. Based on these findings, the in vivo therapeutic potential was investigated and it was found that ceftibuten/polymyxin B significantly decreased the bacterial load in the peritoneal lavage fluid of mice, revealing its effectiveness in treating infections caused by PC-R K. pneumoniae. The ceftibuten/polymyxin B combination exhibited synergistic effects in vitro and in vivo, and thus might be a promising therapeutic alternative for treating PC-R K. pneumoniae infections. As the combination was efficacious in preclinical models, researchers may further investigate its potential in clinical studies.

CD36 deficiency protects lipopolysaccharide-induced sepsis via inhibiting CerS6-mediated endoplasmic reticulum stress

The type 2 scavenger receptor CD36 functions not only as a long chain fatty acid transporter, but also as a pro-inflammatory mediator. Ceramide is the simple N-acylated form of sphingosine and exerts distinct biological activity depending on its acyl chain length. Six ceramide synthases (CerS) in mammals determine the chain length of ceramide species, and CerS6 mainly produces C16-ceramide. Endotoxin-induced septic shock shows high mortality, but the pathophysiologic role of sphingolipids involved in this process has been hardly investigated. This paper aims to highlight the different role of CerS isoforms in endotoxin-induced inflammatory responses and the regulatory role of CD36 in CerS6 protein degradation with an emphasis as the potential therapeutic candidates in humans. Lipopolysaccharide (LPS), the endotoxin of the Gram-negative bacterial cell wall, was treated to induce endotoxin-induced inflammation both in vitro and in vivo. CerS6-derived C16-ceramide propagated LPS-induced inflammatory responses activating various intracellular signaling pathways, such as mitogen-activated protein kinase and nuclear factor-κB, resulting in the formation of inflammasome complex and pro-inflammatory cytokines. Mechanistically, CerS6-derived C16-ceramide augmented inflammatory responses via endoplasmic reticulum stress, and CerS6 protein stability was regulated by CD36. Finally, CerS6 protein expression and LPS-induced lethality were strikingly reduced in CD36 knockout mice. Collectively, our findings show that CerS6-derived C16-ceramide plays a pivotal role in endotoxin-induced inflammation and suggest CerS6 and its regulator CD36 as possible targets for therapy under life-threatening inflammation such as septic shock.

A novel regulator CdsR negatively regulates cell motility in Bacillus thuringiensis

Cell motility increases the fitness of bacterial cells. Previous research focused on the transcriptional regulator CdsR, which represses cellular autolysis and promotes spore formation in Bacillus thuringiensis. However, the targets of CdsR are mostly unknown. Here, we reported a new function of CdsR in regulating cell motility. Mutation of cdsR results in increase of cell mobility, and a number of related genes were upregulated compared to wild type HD73. Thus, we investigated the transcription of the fla/che gene cluster, which involves in cell mobility and comprises eight operons/genes, including motAB1, cheY-yrhK, lamB-cheR, yaaR-fliG2, cheV-mogR, hag1, hag2, and yjbJ-flgG. Additionally, the motAB2 operon was discovered, which consists of homologs genes motA2 and motB2 that are like motA1 and motB1. Through promoter-lacZ fusion assays and EMSA experiments, it was discovered that CdsR directly regulates the motAB1, cheY-yrhK, lamB-cheR, yaaR-fliG2, cheV-mogR, hag1, hag2, yjbJ-flgG, and motAB2 operons by binding to their promoter regions. Importantly, it was confirmed that CdsR is a metalloregulator and the binding to promoter can be inhibited by Cu (II) ions. This research enhances our understanding of the regulation of cell mobility in B. thuringiensis.

The role of prokaryotic argonautes in resistance to type II topoisomerases poison ciprofloxacin.

Argonaute proteins are programmable nucleases found in all domains of life. Eukaryotic argonautes (eAgos) participate in genetic regulation, antiviral response, and transposon silencing during RNA interference. Prokaryotic argonautes (pAgos) are much more diverse than eAgos and have been implicated in defense against invading genetic elements. Recently, it was shown that pAgos protect bacterial cells from a topoisomerase poison ciprofloxacin, raising a possibility that they may play a role in DNA replication and/or repair. Here, we discuss possible models of pAgo-mediated ciprofloxacin resistance. We propose that pAgos could (i) participate in chromosome decatenation as a backup to topoisomerases; (ii) participate in the processing of DNA repair intermediates formed after topoisomerase poisoning, or (iii) induce SOS response that generally affects DNA repair and antibiotic resistance. These hypotheses should guide future investigations of the involvement of pAgos in the emergence of resistance to ciprofloxacin and, possibly, other antibiotics.

Comparative evaluation of surface roughness and bacterial adhesion on two bioactive cements: an in-vitro study

BackgroundDental restorative materials are recognized as artificial niches that facilitate the adherence and accumulation of oral microorganisms. To mitigate oral diseases and extend the lifespan of restorations, it is advantageous to use dental materials that exhibit low susceptibility to bacterial adhesion.ObjectiveTo evaluate and compare bacterial adhesion on two bioactive restorative materials, a glass hybrid restorative, and an alkasite with a nanohybrid resin composite as a positive control. The secondary objectives were to compare the surface roughness (SR) of the materials and determine the correlation between the bacterial adhesion and the SR.Materials and methodsThe samples consisted of 33 polished discs of each material: Group A: Tetric® N-Ceram (nanohybrid resin composite), Group B: Equia Forte™ HT Fil (glass hybrid restorative) and Group C: Cention N® (alkasite). Streptococcus mutans cultures were inoculated and after 24-hours of incubation, bacterial adhesion was measured by measuring optical density (OD) and number of colony forming units (CFUs). After 96-hours incubation, the bacterial cell count was determined using scanning electron microscopy (SEM). SR was assessed using surface profilometer.ResultsAlkasite had significantly lower OD and CFUs (p < 0.001 and p = 0.015 respectively). According to the SEM analysis, the glass hybrid restorative had lower mean bacterial cell count with no significant difference between the groups. The nanohybrid composite had the smoothest surface that was significantly lower than the alkasite and glass hybrid restorative (p = 0.002). None of the groups demonstrated a correlation between bacterial adhesion and SR.ConclusionAlkasite impedes bacterial adhesion better than the glass hybrid restorative and nanohybrid composite, while smoother surfaces are achieved with the nanohybrid composite.

Seasonal changes in the viability and abundance of bacterial cells in the snowpack ecosystem of a High Arctic ice cap

ABSTRACT Microbes play an essential role in nutrient turnover within Arctic environments, and their contribution to biogeochemical cycles can depend on several factors, including but not limited to cell viability. In this study, we employed the SYBR-PI dual cell stain to epifluorescence microscopy to enumerate proportions of potentially viable and non-viable bacterial cell populations within a melting snowpack on an ice cap, Foxfonna in Svalbard. Non-viable cells dominated on Foxfonna (2.5 ± 0.36 × 107 cells m−2) during the June to early July period, when biological production was usually at its peak. Furthermore, non-viable cells also dominated the total cell abundance within superimposed ice (223 ± 242 cells mL−1) and glacial ice (695 ± 717 cells mL−1) beneath the snow. We propose that the rapid, early loss of cell viability was caused by a number of abiotic and biotic factors. Hence, necromass (dead cell residue) contributed to the export of organic matter to downstream ecosystems.

Antibacterial action of novel zeolite-based compositions depends upon doping with Ag(+) and Сu(2+) cations

Recently, there is a growing interest to exploration of sorption and catalytic properties of solid nanomaterials, in particular natural zeolites, as well as to study of their antimicrobial effects with the aim of potential using them as a principal component of disinfection and degassing remedies. The purpose of this work was to study the antimicrobial action of compositions based on the Transcarpathian clinoptilolite (CL) doped with Ag+ and Сu2+ cations or Ag microparticles (MPs). These compositions were subjected to mechanochemical modification in ethanol medium and with the addition of plant (Actinidia arguta) extract used as an antioxidant. Mechanochemical treatment (MChT) of all forms of CL MPs led to their grinding which caused better contact of CL with bacterial cells, while an increased content of larger pores improved their access to the active sites on the surface of the CL MPs. Treatment of CL samples with metallic silver used as a dopant with the help of the extract of Actinidia arguta plant did not increase the antibacterial activity regardless of treatment time. Treatment of AgNO3 with ethanol slightly increased the antibacterial action of the CL MPs towards Gram-positive bacteria and decreased it towards Gram-negative bacteria. The CL samples doped with copper and treated with ethanol and plant (Actinidia arguta) extract demonstrated comparable toxic action towards Bacillus subtilis regardless of grinding conditions. While such a treatment caused a significant decrease in the antibacterial activity towards Staphylococcus aureus and Pseudomonas aeruginosa strains, compared to the action of samples that were not treated with that plant extract. To address the potential biochemical mechanisms of the antibacterial action of the created zeolite-based compositions, their influence on generation of the reactive oxygen species (ROS) was studied using diphenylpicrylhydrazyl (DPH) fluorescent dye. Most versions of the CL composites demonstrated time-dependent antioxidant effect comparable with the effect of the ascorbic acid used as a positive control. Thus, the ROS generation is not the mechanism that is responsible for the antibacterial action of the created CL-based compositions. Probably, that action is explained by the peculiarities of interaction of doped CL microparticles with the surface of the bacterial cells. Keywords: antimicrobial action, clinoptilolite-based compositions, doping with Ag+ and Сu2+ cations, physicochemical treatment

A Clickase-Mediated Immunoassay Based on Nanopore and Bionic Signal Labels for Ultrasensitive, Portable, and On-Site Detection of Ricin.

It is of particular importance to develop an effective method that possesses several merits simultaneously of rapid, ultrasensitive, portable, and on-site detection potential for food safety detection. Herein, we propose a clickase-mediated immunoassay based on nanopore and bionic signal labels for the detection of ricin. The introduction of Cu/Cys clickase and nanopore simultaneously effectively addressed the inherent limitations of natural enzymes and colorimetric signal output, respectively. Using this method, bionic signal labels can be easily formed through DNA and Gram-positive bacterial cell wall terminal peptide fragments (labeled by alkynyl and azide, respectively) and vancomycin. Translocation of the D-P@vancomycin through the nanopore generated highly specific oscillation current traces. This method showed a great on-site detection potential and superior analytical performance owing to the combination of the specificity of antibodies, high CuAAC click reaction catalytic efficiency of clickase, ultrasensitivity of the nanopore, and high signal resolution of D-P@vancomycin. Moreover, the practical applicability of the established method was also verified, achieving a limit of detection (LOD) down to 200.9 ag/mL with a wide linear relationship under the optimized conditions. In conclusion, this method is promising for rapid, portable, ultrasensitive, and on-site food safety detection.