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Plasmids mediate gene exchange across taxonomic barriers through conjugation, shaping bacterial evolution for billions of years. While plasmid mobility can be harnessed for genetic engineering and drug-delivery applications, rapid plasmid-mediated spread of resistance genes has rendered most clinical antibiotics useless. To solve this urgent and growing problem, we must understand how plasmids spread across bacterial communities. Here, we applied machine-learning models to identify features that are important for extending the plasmid host range. We assembled an up-to-date dataset of more than thirty thousand bacterial plasmids, separated them into 1125 clusters, and assigned each cluster a distribution possibility score, taking into account the host distribution of each taxonomic rank and the sampling bias of the existing sequencing data. Using this score and an optimized plasmid feature pool, we built a model stack consisting of DecisionTreeRegressor, EvoTreeRegressor, and LGBMRegressor as base models and LinearRegressor as a meta-learner. Our mathematical modeling revealed that sequence brevity is the most important determinant for plasmid spread, followed by P-loop NTPases, mobility factors, and β-lactamases. Ours and other recent results suggest that small plasmids may broaden their range by evading host defenses and using alternative modes of transfer instead of autonomous conjugation. [ABSTRACT FROM AUTHOR]

There is scarce information concerning the role of sporadic clones in the dissemination of antimicrobial resistance genes (ARGs) within the nosocomial niche. We confirmed that the clinical Escherichia coli M19736 ST615 strain, one of the first isolates of Latin America that harbors a plasmid with an mcr-1 gene, could receive crucial ARG by transformation and conjugation using as donors critical plasmids that harbor blaCTX-M-15, blaKPC-2, blaNDM-5, blaNDM-1, or aadB genes. Escherichia coli M19736 acquired blaCTX-M-15, blaKPC-2, blaNDM-5, blaNDM-1, and aadB genes, being only blaNDM-1 maintained at 100% on the 10th day of subculture. In addition, when the evolved MDR-E. coli M19736 acquired sequentially blaCTX-M-15 and blaNDM-1 genes, the maintenance pattern of the plasmids changed. In addition, when the evolved XDR-E. coli M19736 acquired in an ulterior step the paadB plasmid, a different pattern of the plasmid’s maintenance was found. Interestingly, the evolved E. coli M19736 strains disseminated simultaneously the acquired conjugative plasmids in different combinations though selection was ceftazidime in all cases. Finally, we isolated and characterized the extracellular vesicles (EVs) from the native and evolved XDR-E. coli M19736 strains. Interestingly, EVs from the evolved XDR-E. coli M19736 harbored blaCTX-M-15 though the pDCAG1-CTX-M-15 was previously lost as shown by WGS and experiments, suggesting that EV could be a relevant reservoir of ARG for susceptible bacteria. These results evidenced the genetic plasticity of a sporadic clone of E. coli such as ST615 that could play a relevant transitional link in the clinical dynamics and evolution to multidrug/extensively/pandrug-resistant phenotypes of superbugs within the nosocomial niche by acting simultaneously as a vector and reservoir of multiple ARGs which later could be disseminated.

Abstract As emerging alternatives to natural enzymes, nanoscale materials featuring enzyme‐like catalytic behaviors (nanozymes) exhibit some attractive merits including robust activity, low cost, and easy‐to‐regulate performance. These merits have enabled them to be intensively used in the biomedical field in recent years. To remedy the lack of catalytic selectivity in most nanozymes, deoxyribonucleic acid (DNA) chains with specific recognition functions are utilized to integrate with nanozymes to produce various nanozyme–DNA combinations via adsorption/desorption. In the formed combinations, the DNA component provides the molecular/ionic recognition role, and the nanozyme part offers response with catalytically amplified signals, enabling them to detect analytes and biomarkers selectively and sensitively. To highlight this interesting topic, here we made a critical review of the interactions between nanozymes and DNA and their applications in biosensing and disease diagnosis. First, strategies for the conjugation of DNA chains onto nanozyme surface were introduced briefly. Then, the interactions between DNA and nanozymes were summarized in detail, where flexible modulations of nanozyme activity by DNA adsorption/desorption as well as various factors were analyzed, and potential impacts caused by nanozymes on the recognition characteristics of DNA chains were pointed out. After that, typical applications of DNA‐mediated nanozyme modulation in toxic ion sensing, health risk factor monitoring, and biomedical diagnosis were introduced. In the end, prospects of the combination of nanozymes and DNA chains were presented, and future challenges of the emerging field were also discussed, to attract more interest and effort to advance this promising area.

The presence of several interconnected electrochemical processes occurring on the surface of an electrode, strictly speaking, does not allow the use of the principle of independent reactions. Often, partial reactions of a complex multi-stage electrochemical process are coupled both through common intermediates and through the competitive adsorption of electroactive species. The presence of conjugation leads either to a change in the potential at which the corresponding electrochemical process becomes possible or to a change in the rate of partial processes. The latter is called kinetic coupling. This does not allow the simple calculation of the rate of each partial reaction as the difference between the current density of the target and background processes. The method of kinetic diagrams can be used to establish the kinetic patterns of such processes. This study shows that this method is applicable not only for the analysis of coupled electrochemical processes of various types, but can also be used in obtaining partial currents of the stages of a separate complex electrode reaction occurring in a background solution. As an example, options for the kinetic modelling of the total voltammogram of the anodic process on an Au electrode in an aqueous alkaline medium in the mode of linear potential change are considered. The stationary degrees of covering of the gold surface with various surface-active forms of oxygen are calculated depending on the electrode potential. It was established that the change in concentration of ОН- ions mainly affects the region of their adsorption potentials. A detailed analysis of stationary partial anodic processes in the Au|OH-,H2O system was carried out and the shape of the general stationary voltammogram was determined by calculation. The latter is in qualitative agreement with the experimental polarization dependence. It was shown that the type of calculated polarization dependence is determined by the degree of reversibility of individual stages and the rate of their occurrence. The performed analysis is necessary not only for the detailed scheme of the background anodic reaction on gold in an alkaline solution, but also for the ubsequent kinetic description of the electrooxidation process of organic substances on a gold electrode

Sexual reproduction of Zygnematophyceae by conjugation is a less investigated topic due to the difficulties of the induction of this process and zygospore ripening under laboratory conditions. For this study, we collected field sampled zygospores of Spirogyra mirabilis and three additional Spirogyra strains in Austria and Greece. Serial block-face scanning electron microscopy was performed on high pressure frozen and freeze substituted zygospores and 3D reconstructions were generated, allowing a comprehensive insight into the process of zygospore maturation, involving storage compound and organelle rearrangements. Chloroplasts are drastically changed, while young stages contain both parental chloroplasts, the male chloroplasts are aborted and reorganised as ‘secondary vacuoles’ which initially contain plastoglobules and remnants of thylakoid membranes. The originally large pyrenoids and the volume of starch granules is significantly reduced during maturation (young: 8 ± 5 µm³, mature: 0.2 ± 0.2 µm³). In contrast, lipid droplets (LDs) increase significantly in number upon zygospore maturation, while simultaneously getting smaller (young: 21 ± 18 µm³, mature: 0.1 ± 0.2 and 0.5 ± 0.9 µm³). Only in S. mirabilis the LD volume increases (34 ± 29 µm³), occupying ~50% of the zygospore volume. Mature zygospores contain barite crystals as confirmed by Raman spectroscopy with a size of 0.02 - 0.05 µm³. The initially thin zygospore cell wall (~0.5 µm endospore, ~0.8 µm exospore) increases in thickness and develops a distinct, electron dense mesospore, which has a reticulate appearance (~1.4 µm) in Spirogyra sp. from Greece. The exo- and endospore show cellulose microfibrils in a helicoidal pattern. In the denser endospore, pitch angles of the microfibril layers were calculated: ~18 ± 3° in S. mirabilis, ~20 ± 3° in Spirogyra sp. from Austria and ~38 ± 8° in Spirogyra sp. from Greece. Overall this study gives new insights into Spirogyra sp. zygospore development, crucial for survival during dry periods and dispersal of this genus.

This study investigated the grafting chlorogenic acid (CA) onto myosin, utilizing various techniques including conventional method, ultrasound, microwave, and combination of ultrasound and microwave (UM). The grafting efficiency was as follows: conventional method

Emerging pollutants such as microplastics in water environments readily accumulate microorganisms on their surfaces, forming biofilms and concentrating antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Consequently, microplastic biofilms have attracted the attention of researchers. Horizontal gene transfer (HGT) of ARGs is one of the primary ways that bacteria acquire antibiotic resistance. Most studies focus on the effects of nanomaterials on suspended bacteria, but microplastic biofilms as hotspots for horizontal gene transfer also warrant significant investigation. This study primarily explored and compared the effects of nano-titanium dioxide on the conjugation transfer frequency of ARGs in suspended bacteria and microplastic biofilms. Nano-titanium dioxide could promote ARG conjugation in both suspended bacteria and microplastic biofilms, with a greater effect on the former. The mechanism involved nano-titanium dioxide promoting the production of reactive oxygen species (ROS) in suspended and biofilm bacteria, increasing the synthesis of outer membrane proteins, enhancing the cell membrane permeability, and elevating the expression levels of conjugation-related genes, thereby facilitating the conjugation transfer of ARGs. Biofilm bacteria, being heavily encased and protected by extracellular polymeric substances (EPS), exhibit greater resistance to external environmental pressure, resulting in the weaker impact of nano-titanium dioxide on biofilm bacteria compared to suspended bacteria. This study reveals the risk of ARG conjugation transfer within microplastic biofilms induced by nanomaterials, providing valuable insights into the risks of microplastic and antibiotic resistance dissemination in water environments.

Public health faces daily challenges due to increasing reports of pathogenic microorganisms with new antimicrobial resistance. Klebsiella michiganensis, an emerging pathogen, poses difficulty in its identification using conventional techniques. This study presents the first documented case of NDM-1-producing K. michiganensis in Brazil, identified as the new ST418. Initially, the isolate from a tracheal secretion was misidentified as K. oxytoca. However, accurate identification was achieved through ANI analyses. Whole-genome sequencing was conducted to characterize the genetic context of the resistance genes, to identify virulence factors, and to construct a phylogenetic tree. The blaNDM-1 gene was found to be harbored on an IncFIB plasmid approximately 112 kb in length, which was transferable in conjugation assays. The detection of carbapenem resistance genes in this species highlights the importance of public health vigilance, as it may serve as a reservoir and disseminator of significant resistance genes.

Glycoconjugation is a well-established technology for vaccine development: linkage of the polysaccharide (PS) antigen to an appropriate carrier protein overcomes the limitations of PS T-independent antigens, making them effective in infants and providing immunological memory. Glycoconjugate vaccines have been successful in reducing the burden of different diseases globally. However, many pathogens still require a vaccine, and many of them display a variety of glycans on their surface that have been proposed as key antigens for the development of high-valency glycoconjugate vaccines. CDAP chemistry represents a generic conjugation strategy that is easily applied to PS with different structures. This chemistry utilizes common groups to a large range of PS and proteins, e.g., hydroxyl groups on the PS and amino groups on the protein. Here, new fast analytical tools to study CDAP reaction have been developed, and reaction conditions for PS activation and conjugation have been extensively investigated. Mathematical models have been built to identify reaction conditions to generate conjugates with wanted characteristics and successfully applied to a large number of bacterial PSs from different pathogens, e.g., Klebsiella pneumoniae, Salmonella Paratyphi A, Salmonella Enteritidis, Salmonella Typhimurium, Shighella sonnei and Shigella flexneri. Furthermore, using Salmonella Paratyphi A O-antigen and CRM197 as models, a design of experiment approach has been used to study the impact of conjugation conditions and conjugate features on immunogenicity in rabbits. The approach used can be rapidly extended to other PSs and accelerate the development of high-valency glycoconjugate vaccines.

Photocatalysis technology is an economical and effective new energy technology which depends on the conversion and storage of light energy through an energy transfer process or charge transfer process. Recently, organic semiconductor photocatalytic materials with the advantages of controllable structure, broad spectral response, designability, and flexibility have received wide attention. In particular, the organic polymeric materials containing poly-perylene diimides (PDI) show significant promise in the realm of photocatalysis due to their impressive catalytic capabilities and wide spectral reactivity. However, a poor charge separation and transportation (CST) process undermines their photocatalytic efficiency in most polymer photocatalysts, as well as in PDI photocatalysts. In this context, we propose a new strategy through regulating the monomer symmetry to construct highly efficient PDI photocatalysts. As proof-of-concept, a series of new PDI-based organic supramolecular photocatalytic materials with full visible spectral response from the perspectives of both the π-π conjugated structure and the symmetry of chain structure are successfully synthesized. Meanwhile, the structural compositions, morphology features, electrical properties, and photocatalytic performances of those obtained PDI photocatalysts were systematically studied. The results shown that the as-prepared PDI-1,5NDA exhibits 1.6-fold and 3.7-fold higher levels of photosynthesis of H2O2 activity than those of PDI-1,4NDA and PDI-PDA, respectively, which could be ascribe to its lower symmetry and large π-conjugate systems greatly enhances the separation of charge carriers.

Overcoming the blood–brain barrier (BBB) remains a significant hurdle in effective drug delivery to the brain. While the BBB serves as a crucial protective barrier, it poses challenges in delivering therapeutic agents to their intended targets within the brain parenchyma. To enhance drug delivery for the treatment of neurological diseases, several delivery technologies to circumvent the BBB have been developed in the last few years. Among them, nanoparticles (NPs) are one of the most versatile and promising tools. Here, we summarize the characteristics of NPs that facilitate BBB penetration, including their size, shape, chemical composition, surface charge, and importantly, their conjugation with various biological or synthetic molecules such as glucose, transferrin, insulin, polyethylene glycol, peptides, and aptamers. Additionally, we discuss the coating of NPs with surfactants. A comprehensive overview of the common in vitro and in vivo models of the BBB for NP penetration studies is also provided. The discussion extends to discussing BBB impairment under pathological conditions and leveraging BBB alterations under pathological conditions to enhance drug delivery. Emphasizing the need for future studies to uncover the inherent therapeutic properties of NPs, the review advocates for their role beyond delivery systems and calls for efforts translating NPs to the clinic as therapeutics. Overall, NPs stand out as a highly promising therapeutic strategy for precise BBB targeting and drug delivery in neurological disorders.

Astringency of phenolic-rich foods is a key tactile perception responsible for acceptability/rejection of plant extracts as ingredients in formulations. Covalent conjugation of phenolic extracts with plant proteins might be a promising strategy to control astringency, but suffers from a lack of mechanistic understanding from the lubrication point of view. To shed light on this, this ex vivo study evaluated the effect of conjugation of a phenolic grape seed extract (GSE) with legume protein (lupin, LP) on tribological and surface adsorption performance of GSE in the absence and presence of human saliva (ex vivo). Tribological results confirmed GSE had an inferior lubrication capacity as compared to LP. The lubrication performance of LP-GSE dispersions was comparable to their corresponding LP dispersion (p > 0.05) when covalently conjugated with LP (LP-GSE) with increasing LP:GSE ratio up to 1:0.04 w/w and at a specific degree of conjugation (DC: 2%). Tribological and surface adsorption measurements confirmed the tendency of GSE to interact with human saliva (ex vivo, n = 17 subjects), impairing the lubricity of salivary films. The covalent bonding of LP to GSE hindered GSE's interaction with human saliva, implying the potential influence of covalent conjugation on attenuating astringency. LP appeared to compete with human saliva for surface adsorption and governed the lubrication behaviour in LP-GSE dispersions. Findings from this study provide valuable knowledge to guide the rational design of sustainable, functional foods using conjugation of phenolics with plant proteins to incorporate larger proportions of health-promoting phenolics while controlling astringency, which needs validation by sensory trials.

Plasmids pNP40 and pUC11B encode two prevalent yet divergent conjugation systems, which have been characterized in detail recently. Here, we report the elucidation of the putative adhesins of the pNP40 and pUC11B conjugation systems, encoded by traAd and trsAd, respectively. Despite their significant sequence divergence, TraAd and TrsAd represent the most conserved component between the pNP40- and the pUC11B-encoded conjugation systems and share similar peptidoglycan-hydrolase domains. Protein structure prediction using AlphaFold2 highlighted the structural similarities between their predicted domains, as well as the potential homo-dimeric state of both proteins. Expression of the putative surface adhesins resulted in a cell clumping phenotype not only among cells expressing these surface adhesins but also between adhesin-expressing and non-producing cells. Furthermore, mutant derivatives of plasmids pNP40 or pUC11B carrying a mutation in traAd or trsAd, respectively, were shown to act as efficient donors provided the corresponding recipient expresses either traAd or trsAd, thus demonstrating in trans reciprocal complementarity of these proteins in conjugation systems.

ABSTRACT Plasmid-dependent phages infect bacteria carrying conjugative plasmids by recognizing the plasmid-encoded pilus. Despite the high abundance of conjugative plasmids in diverse environments, plasmid-dependent phages have not been widely studied. Since conjugative plasmids often carry antimicrobial resistance genes (ARGs), interfering with conjugation could reduce the spread of ARGs and avoid the appearance of multiresistant superbugs. Our aim was to isolate and characterize plasmid-dependent phages able to infect bacteria carrying diverse conjugative plasmids belonging to the most common plasmid families among Gram-negative pathogens. We isolated two lytic phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncN plasmid pKM101. Both phages, named Lu221 and Hi226, are novel dsDNA viruses within the class Caudoviricetes with genomes of approximately 76 kb. They showed broad host range infecting Escherichia coli, S. enterica, Kluyvera sp., and Enterobacter sp. carrying conjugative plasmids. They recognize plasmid-encoded receptors from 12 out of 15 tested plasmids, all of them carrying resistance determinants. Phages Lu221 and Hi226 could have the potential to help combat the antimicrobial resistance crisis by reducing ARGs present in conjugative plasmids. IMPORTANCE This work was undertaken because plasmid-dependent phages can reduce the prevalence of conjugative plasmids and can be leveraged to prevent the acquisition and dissemination of ARGs by bacteria. The two novel phages described in this study, Lu221 and Hi226, can infect Escherichia coli, Salmonella enterica, Kluyvera sp. and Enterobacter sp. carrying conjugative plasmids. This was verified with plasmids carrying resistance determinants and belonging to the most common plasmid families among Gram-negative pathogens. Therefore, the newly isolated phages could have the potential to help control the spread of ARGs and thus help combat the antimicrobial resistance crisis.

ABSTRACTNeisseria gonorrhoeae causes sexually transmitted infection (STI) gonorrhoea and is on the WHO critical list due to increasing antimicrobial resistance (AMR). The bacterium can carry a conjugative plasmid, pConj, which prevents the use of tetracycline or doxycycline for treating gonorrhea, and is responsible for spread of a β-lactamase plasmid, pbla; over 90% of gonococcal isolates also harbor a small cryptic plasmid, pCryp. We systematically analyzed the presence of these plasmids in other Neisseria spp., including Neisseria meningitidis, which causes sepsis/meningitis. pConj is the most frequently found plasmid in the meningococcus and is in many clonal complexes. The plasmid is associated with meningococcal carriage rather than disease, indicating that pConj imposes fitness costs during systemic disease. Phylogenetic analyses reveal that pConj is genetically diverse in N. meningitidis, indicating that it shares a long evolutionary history with the meningococcus and that the plasmid has been transferred at least twice from N. meningitidis to N. gonorrhoeae. Following the first transfer, gonococcal isolates carrying the plasmid underwent clonal expansion and disseminated pConj to other gonococcal lineages. The second introduction was associated with an altered conjugation machinery which reduces conjugation efficiency. Therefore, in contrast to chromosomal resistance which has evolved through introduction of genes from commensals, gonococcal plasmid-mediated resistance has arisen through transfer from another pathogen, N. meningitidis. Further antibiotic pressure from the use of doxycycline for post-exposure prophylaxis against STIs is likely to promote plasmid-mediated AMR in both N. gonorrhoeae and N. meningitidis.IMPORTANCEHorizontal gene transfer (HGT) is a major influence in driving the spread of antimicrobial resistance (AMR) in many bacteria. A conjugative plasmid which is widespread in Neisseria gonorrhoeae, pConj, prevented the use of tetracycline/doxycycline for treating gonococcal infection. Here, we show that pConj evolved in the related pathogen, Neisseria meningitidis, and has been acquired by the gonococcus from the meningococcus on multiple occasions. Following its initial acquisition, pConj spread to different gonococcal lineages; changes in the plasmid’s conjugation machinery associated with another transfer event limit spread in the gonococcal populations. Our findings have important implications for the use of doxycycline to prevent bacterial sexually transmitted disease which is likely to exacerbate the spread of AMR through HGT in pathogenic bacteria.

Abstract Nanoparticles (NPs) are promising therapeutic delivery agents, with the number and manner of presentation of cell‐binding ligands on the NP affecting the eventual fate of the therapeutic. Whenever NPs are conjugated with biomolecules, a heterogeneous population of decorated NPs will be produced and these subpopulations of particle‐ligand structures need to be characterized for a reliable interpretation of NP‐based data. An optical microscopy method is reported to quantitatively evaluate the conjugation on a single‐particle basis in samples consisting of gold NPs (GNPs) decorated with holo‐transferrin fluorescently labeled with Alexa647 (Tf). Widefield fluorescence and extinction microscopy are employed on NP‐ligand constructs, alongside a correlative analysis that spatially co‐localizes diffraction‐limited sources of fluorescence with the optical extinction by individual GNPs. A photobleaching step analysis estimates the number of fluorophores contributing to the detected emission rate. The method quantifies the number of fluorescent biomolecules attached per GNP, the numbers of unconjugated GNPs and unbound Tf present within the mixed population, and the size and intraparticle clustering propensity of conjugated GNPs. A high variability is found in the number of Tf ligands per GNP within the GNP population, when analyzed at the single‐particle level, unraveling a non‐trivial statistical distribution not accessible in ensemble‐averaged approaches.

Infectious diseases resulting from various strains of pathogenic bacteria are among the leading causes of death and morbidity worldwide. The currently-available antibacterial medications developed from natural or synthetic sources are the cornerstone of bacterial infection intervention, but somehow the mounting spread and expansion of multidrug-resistant harmful bacteria has become a major concern for medical-providing communities and pharmaceutical-releasing companies. The pervasiveness of coumarin-based products in nature and the simplicity of their lab chemical synthesis, in addition to the ability of the coumarin backbone to communicate, may interact with the bacterial DNA gyrase at the B subunit and hinder DNA secondary structure by abolishing ATPase activity; thus, coumarin-based products and their conjugates may have the potential to act as antibacterial agents. It is conceivable that conjugation of the coumarin chemical nucleus with other heterocyclic-based antibacterial scaffolds may present opportunities for the creation of novel antibacterial drugs. This presentation may be based on the fact that several coumarin-heterocycle conjugates, including clorobiocin, coumermycin A1, and novobiocin, have already been used in medical methods for the treatment of various infectious bacterial diseases. This study explores, during the period of the last 5 years, the relationships between the structure and activity of coumarin-heterocycle conjugates with potential for antibacterial action. This investigation also explored the potential for developing these conjugates into antibacterial medicines.

ABSTRACTNon-caloric artificial sweeteners have been widely permitted as table sugar substitutes with high intensities of sweetness. They can pass through the intestinal tract without significant metabolization and frequently encounter the gut microbiome, which is composed of diverse bacterial species and is a pool of antibiotic resistance genes (ARGs). However, little is known about whether these sweeteners could accelerate the spread of ARGs in the gut microbiome. Here, we established an in vitro conjugation model by using Escherichia coli that carries chromosome-inserted Tn7 lacIq-pLpp-mCherry and plasmid-encoded gfpmut3b gene as the donor and murine fecal bacteria as the recipient. We found that four commonly used artificial sweeteners (saccharin, sucralose, aspartame, and acesulfame potassium) can increase reactive oxygen species (ROS) production and promote plasmid-mediated conjugative transfer to the gut microbiome. Cell sorting and 16S rRNA gene amplicon sequencing analysis of fecal samples reveal that the tested sweeteners can promote the broad-host-range plasmid permissiveness to both Gram-negative and Gram-positive gut bacteria. The increased plasmid permissiveness was also validated with a human pathogen Klebsiella pneumoniae. Collectively, our study demonstrates that non-caloric artificial sweeteners can induce oxidative stress and boost the plasmid-mediated conjugative transfer of ARGs among the gut microbiota and a human pathogen. Considering the soaring consumption of these sweeteners and the abundance of mobile ARGs in the human gut, our results highlight the necessity of performing a thorough risk assessment of antibiotic resistance associated with the usage of artificial sweeteners as food additives.

Introduction: Type IV secretion systems (T4SSs) are integral parts of the conjugation process in enteric bacteria. These secretion systems are encoded within the transfer (tra) regions of plasmids, including those that harbor antimicrobial resistance (AMR) genes. The conjugal transfer of resistance plasmids can lead to the dissemination of AMR among bacterial populations.Methods: To facilitate the analyses of the conjugation-associated genes, transfer related genes associated with key groups of AMR plasmids were identified, extracted from GenBank and used to generate a plasmid transfer gene dataset that is part of the Virulence and Plasmid Transfer Factor Database at FDA, serving as the foundation for computational tools for the comparison of the conjugal transfer genes. To assess the genetic feature of the transfer gene database, genes/proteins of the same name (e.g., traI/TraI) or predicted function (VirD4 ATPase homologs) were compared across the different plasmid types to assess sequence diversity. Two analyses tools, the Plasmid Transfer Factor Profile Assessment and Plasmid Transfer Factor Comparison tools, were developed to evaluate the transfer genes located on plasmids and to facilitate the comparison of plasmids from multiple sequence files. To assess the database and associated tools, plasmid, and whole genome sequencing (WGS) data were extracted from GenBank and previous WGS experiments in our lab and assessed using the analysis tools.Results: Overall, the plasmid transfer database and associated tools proved to be very useful for evaluating the different plasmid types, their association with T4SSs, and increased our understanding how conjugative plasmids contribute to the dissemination of AMR genes.

Ciliated protists are highly differentiated unicellular eukaryotes that possess special sexual processes (conjugation and autogamy) that rely on their unique nuclear dimorphism, i.e., the presence of both a germline micronucleus (MIC) and a somatic macronucleus (MAC) in a single cell. The sexual processes show a high diversity in different ciliates. To better understand the differentiation and evolution of sexual processes in closely related species, we investigated the nuclear events during conjugation in Paramecium jenningsi strain GZ, a poorly studied close relative of the well-known P. aurelia complex. The main results include: (1) the conjugation process takes about 48 h, including three prezygotic divisions (meiosis I, II, and mitosis) and three postzygotic divisions; (2) the MICs are dominated by the "parachute" phase at the prophase of meiosis I; (3) after meiosis II, a variable number of nuclei undergo the third prezygotic division, and the two products near the paroral cone become the genetically identical migratory and stationary pronuclei; (4) the synkaryon divides three times to form the MIC and the MAC anlagen, but only one nuclear product of the first postzygotic division completes the next two divisions; and (5) an extra cell division is required to complete the last step of conjugation, during which two MIC anlagen undergo mitotic division while two MAC anlagen are distributed between the daughter cells without division. The comparison of the nuclear events during conjugation in representatives of the class Oligohymenophorea reveals that usually there are three prezygotic divisions and a variable number of postzygotic divisions. However, the number of selected and differentiated nuclei after each division differs among species. This study provided a new model ciliate for further investigations of nuclear selection and differentiation as well as nuclear morphology during meiosis. [ABSTRACT FROM AUTHOR]

Biofilms as a form of adaptation are beneficial for bacterial survival and may be hot spots for horizontal gene transfer, including conjugation. The aim of this research was to characterize the biofilm biomass, viable cell ratios and conjugative transfer of the pOX38 plasmid, an F-plasmid derivative, from the Escherichia coli N4i pOX38 strain (donor) into a uropathogenic E. coli DL82 strain (recipient) within dual-species biofilms with one of the following opportunistic pathogenic bacteria: Klebsiella pneumoniae, Enterococcus faecalis or Pseudomonas aeruginosa. Dual-species biofilms of E. coli with K. pneumoniae or P. aeruginosa but not E. faecalis were more massive and possessed more exopolysaccharide matrix compared to single-species biofilms of donor and recipient cells. Correlation between biofilm biomass and exopolysaccharide matrix was rs = 0.888 in dual-species biofilms. In dual-species biofilm with E. faecalis the proportion of E. coli was the highest, while in the biofilm with P. aeruginosa and K. pneumoniae, the E. coli was less abundant. The conjugative frequencies of plasmid transfer in dual-species biofilms of E. coli with E. faecalis and P. aeruginosa were reduced. A decrease in conjugative frequency was also observed when cell-free supernatants (CFSs) of E. faecalis and P. aeruginosa were added to the E. coli conjugation mixture. Further, the activity of the autoinducer AI-2 in the CFSs of the E. coli conjugation mixture was reduced when bacteria or CFSs of E. faecalis and P. aeruginosa were added to the E. coli conjugation mixture. Hence, the intercellular and interspecies interactions in dual-species biofilms depend on the partners involved. [ABSTRACT FROM AUTHOR]

L-Asparaginase exists as an effective chemotherapeutic drug for various cancer cell types. However, the L-asparaginase enzyme in its free state is unstable and has reduced half-life period with numerous side effects. Here, as a green synthesis approach, we have tried a nanocarrier to reduce its side effects and thereby the environmental pollution and toxicity. We used leaf extract of Mesosphaerum suaveolens (L.) Kuntze, commonly called Pignut, Wild spikenard or Horsehound, to produce a Mg-ZnO nanocomposite, which has antitumor activity by arresting the cell cycle of tumour cells. To the synthesized nanocomposite, extracted L-asparaginase enzyme from Aspergillus terreus was impregnated. The presence of ZnO helps to preserve the biological activity of immobilized L-asparaginase along with MgO making the nanocarrier biocompatible and biodegradable. This helps in increasing the stability and anticancer property of the L-asparaginase. The enzyme-impregnated nanobiocomposite was characterized using UV-Vis spectroscopy which ensured the existence of the Asp-Mg-ZnO in the nanobiocomposite. The presence of functional group and the crystallinity nature of the nanobiocomposite were studied using FT-IR and XRD, respectively. The morphological structure and elemental composition of the nanobiocomposite were determined using the SEM-EDAX. The anticancer activity of the synthesized Asp-Mg-ZnO nanobiocomposite was determined using the MTT assay, which helped to determine the viability and proliferation capacity of the cancer cells. [ABSTRACT FROM AUTHOR]

Neomycin is the first-choice antibiotic for the treatment of porcine enteritis caused by enterotoxigenic Escherichia coli. Resistance to this aminoglycoside is on the rise after the increased use of neomycin due to the ban on zinc oxide. We ident ified the neomycin resistance determinants and plasmid contents in a historical collection of 128 neomycin-resistant clinical E. coli isolates from Danish pig farms. All isolates were characterized by whole-genome sequencing and antimicrobial susceptibility testing, followed by conjugation experiments and long-read sequencing of eight selected representative strains. We detected 35 sequence types (STs) with ST100 being the most prevalent lineage (38.3%). Neomycin resistance was associated with two resistance genes, namely aph(3')-Ia and aph(3')-Ib, which were identified in 93% and 7% of the isolates, respectively. The aph(3')-Ia was found on different large conjugative plasmids belonging to IncI1a, which was present in 67.2% of the strains, on IncHI1, IncHI2, and IncN, as well as on a multicopy ColRNAI plasmid. All these plasmids except ColRNAI carried genes encoding resistance to other antimicrobials or heavy metals, highlighting the risk of co-selection. The aph(3')-Ib gene occurred on a 19 kb chimeric, mobilizable plasmid that contained elements tracing back its origin to distantly related genera. While aph(3')-Ia was flanked by either Tn903 or Tn4352 derivatives, no clear association was observed between aph(3')-Ib and mobile genetic elements. In conclusion, the spread of neomycin resistance in porcine clinical E. coli is driven by two resistance determinants located on distinct plasmid scaffolds circulating within a highly diverse population dominated by ST100. [ABSTRACT FROM AUTHOR]

This paper studies conjugation-invariant norms on SL 2 (R) for R a ring of S-algebraic integers with infinitely many units in a number field. We will show that barring some differences in the proofs the properties of these norms strongly resemble related properties of conjugation-invariant norms on arithmetic Chevalley groups of higher rank. Specifically, we prove two properties: First, we show that the diameter of each word norm on SL 2 (R) defined by finitely many conjugacy classes has an upper bound linear in the number of conjugacy-classes. Second, we show that all non-discrete conjugation-invariant norms on SL 2 (R) have a profinite norm-completion. Both of these properties of conjugation-invariant norms are already known for higher rank arithmetic Chevalley groups. [ABSTRACT FROM AUTHOR]

Chemical modification of the enzymes with biospecific macromolecules is used in various fields of biotechnology to impart new functions or improve their properties and is a fast and convenient way to get the final products. The preparation of highly active, stable, and functionally active conjugates of the thermostable luciferase through the NH2-groups or free SH-groups of the enzyme with target molecules of different molecular weight (albumin, avidin from chicken eggs, antibodies, and progesterone) is described. The obtained conjugates were successfully tested as a reporter in bioluminescent immunoassay for the detection of the molecules and pathogens. Thus, the luc-albumin (Luc-Alb) and luc-insulin (Luc-Ins) conjugates were used in competitive ELISA for the detection of an analyte (albumin or insulin) in the samples. Luc-progesterone (Luc-Pg) was used in the rapid homogeneous immunoassay of progesterone by the BRET technique with the detection limit of 0.5 ng/ml. Luciferase conjugates with avidin (Luc-Avi) and secondary and primary antibodies (Luc-RAM and Luc-Sal) were used for enzyme immunoassay detection of Salmonella paratyphi A cells with the cell detection limit of 5 × 104 CFU/ml. To reduce the detection limit of Salmonella cells, we developed a pseudo-homogeneous bioluminescent enzyme immunoassay of cells using a new matrix for the analyte capture—polystyrene microparticles coated with Pluronic F108, covalently labeled with Sal antibodies. This allowed to achieve efficient trapping of cells from solution, significantly reduced nonspecific sorption and decreased the cell detection limit to 2.7 × 103 CFU/ml without prior concentration of the sample. The methodology that was developed in this study can be applied for the development of novel bioanalytical systems based on firefly luciferases. [ABSTRACT FROM AUTHOR]

Cross-interface spread and aggregation of antibiotic resistance genes (ARGs) at human-animal-environment has become a major problem in public health and environmental ecosystems, ARGs and mobile genetic elements (MGEs) carrying ARGs have been recognized as emerging environmental contaminants. Heavy metal-mediated horizontal transfer of ARGs increases the potential risk of ARG enrichment in indigenous microorganisms. Currently, the study of horizontal transfer of ARGs mediated by heavy metals and other emerging contaminants has become a hot research topic in the field of environment and its related disciplines. The review outlines the effects of heavy metals as co-selectants on the conjugation of ARGs in the environment, focusing on the mechanisms associated with the increased conjugation of ARGs induced by heavy metals from a molecular biological perspective. The heavy metal-mediated conjugation of ARGs was the main reason for the increased abundance of ARGs, and the heavy metal-induced changes in conjugation frequency were mainly related to their concentrations, which is consistent with the hormesis. Related regulatory mechanisms include regulation of reactive oxygen species (ROS) production and initiation of SOS responses, enhancement of cell membrane permeability, and effects on conjugation transfer-related gene expression and ATP synthesis. Elucidation of the mechanisms that enhance the horizontal transfer of ARGs in contaminated environments with heavy metals (or compound heavy metals) and antibiotics under different environmental conditions is a key direction for future research. It can provide some reference for the study of heavy metal-mediated environmental behavior of ARGs. [ABSTRACT FROM AUTHOR]

Sixteen conjugates of dihydroartemisinin and zerumbone with 2-mercapto-1,3,4-oxadiazoles were synthesized and structurally elucidated by ID NMR, 2D NMR, and HRMS spectra. The cytotoxic screening results showed that all the conjugates of dihydroartemisinin with 2-mercapto-1,3,4-oxadiazoles (19a-h) exhibited cytotoxic activity against two human cancer cell lines, HepG2 and LU-I, with the IC50 values ranging from 2.22 to 40.69 µM. Among dihydroartemisinin conjugates, conjugate 19b displayed the strongest activity against both HepG2 and LU-I cell lines, with the IC50 values of 3.49 and 2.22 µM, respectively. The zerumbone conjugates (20a-h) expressed their cytotoxic activity stronger than that of 19a-h series, with IC50 values ranging from 1.54 to 2.00 µM. In addition, all 16 compounds exhibited an impressively inhibitory effect against EGFR tyrosine kinase with binding affinities ranging from -8.61 to -10.2 kcal/mol, higher than that of the erlotinib drug (-7.50 kcal/mol), a co-crystallized inhibitor of EGFR receptor. Both conjugates ( 19a and 20a) containing the 2-hydroxyphenyl-2mercapto-1,3,4-oxadiazole moiety had the best binding energies (-10.2 and -9.494 kcal/mol, respectively) on the EGFR tyrosine kinase. Furthermore, the potential interactions and binding patterns between the compounds and the relevant amino acid residues revealed the most significant contribution to amplifying their efficacy against this protein, according to docking studies, which identified both hydrogen bonds and hydrophobic contacts at the active site of the EGFR protein. [ABSTRACT FROM AUTHOR]

Nisin is a naturally occurring bioactive small peptide produced by Lactococcus lactis subsp. lactis and belongs to the Type A (I) lantibiotics. Due to its potent antimicrobial activity, it has been broadly employed to preserve various food materials as well as to combat a variety of microbial pathogens. The present review discusses the antimicrobial properties of nisin and different types of their derivatives employed to treat microbial pathogens with a detailed underlying mechanism of action. Several alternative strategies such as combination, conjugation, and nanoformulations have been discussed in order to address several issues such as rapid degradation, instability, and reduced activity due to the various environmental factors that arise in the applications of nisin. Furthermore, the evolutionary relationship of many nisin genes from different nisin-producing bacterial species has been investigated. A detailed description of the natural and bioengineered nisin variants, as well as the underlying action mechanisms, has also been provided. The chemistry used to apply nisin in conjugation with natural or synthetic compounds as a synergetic mode of antimicrobial action has also been thoroughly discussed. The current review will be useful in learning about recent and past research that has been performed on nisin and its derivatives as antimicrobial agents. [ABSTRACT FROM AUTHOR]

In recent decades, there has been a rapid increase in the prevalence of multidrug‐resistant pathogens, posing a challenge to modern antibiotic‐based medicine. This has highlighted the need for novel treatments that can specifically affect the target microorganism without disturbing other co‐inhabiting species, thus preventing the development of dysbiosis in treated patients. Moreover, there is a pressing demand for tools to effectively manipulate complex microbial populations. One of the approaches suggested to address both issues was to use conjugation as a tool to modify the microbiome by either editing the genome of specific bacterial species and/or the removal of certain taxonomic groups. Conjugation involves the transfer of DNA from one bacterium to another, which opens up the possibility of introducing, modifying or deleting specific genes in the recipient. In response to this proposal, there has been a significant increase in the number of studies using this method for gene delivery in bacterial populations. This MicroReview aims to provide a detailed overview on the use of conjugation for microbiome engineering, and at the same time, to initiate a discussion on the potential, limitations and possible future directions of this approach. [ABSTRACT FROM AUTHOR]

A novel group of conjugative plasmids of Pseudomonas is characterized. The prototype plasmid pPPUT-Tik1-1 (153,663 bp), isolated from a permafrost strain of P. putida Tik1, carries a defective mercury transposon, Tn501, and a streptomycin resistance transposon, Tn5393. Ten plasmids and 34 contigs with backbone regions closely related to pPPUT-Tik1-1 have been found in GenBank. Two of these plasmids from clinical strains of P. putida and P. fulva are almost identical to the ancient plasmid. A characteristic feature of this group of plasmids is the presence of two genes encoding the initiators of replication (repA1 and repA2). None of these genes have high similarity with plasmid replication genes belonging to known incompatibility groups. It has been demonstrated that while pPPUT-Tik1-1-like plasmids have homologous backbone regions, they significantly differ by the molecular structure and the predicted functions of their accessory regions. Some of the pPPUT-Tik1-1-related plasmids carry determinants of antibiotic resistance and/or heavy metal salts. Some plasmids are characterized by the ability to degrade xenobiotics. Plasmids related to pPPUT-Tik1-1 are characterized by a narrow host range and are found in various species of the Pseudomonas genus. Interestingly, we also found shorter plasmid variants containing the same replication module, but lacking conjugation genes and containing other structural changes that strongly distinguish them from plasmids related to pPPUT-Tik1-1, indicating that the structure of the replication module cannot be used as the sole criterion for classifying plasmids. Overall, the results suggest that the plasmids of the novel group can be spread using conjugation in environmental and clinical strains of Pseudomonas and may play diverse adaptive functions due to the presence of various accessory regions. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Metallacyclopentadienes play a vital role in transition‐metal mediated and catalyzed cycloaddition reactions of alkynes. Though versatile reactions of metallacyclopentadienes with alkynes have been disclosed, [2+1] cycloadditions of metallacyclopentadienes and alkynes have never been discovered. In present work, we report the formal [2+1] cycloadditions of a metallacyclopentadiene unit with a broad scope of commercial alkynes, providing a facile strategy to construct tetracyclic conjugated compounds. The deuterated experiment indicates a metal vinylidene intermediate has been involved in [2+1] cycloaddition. Moreover, the electrophilic substitution reaction of the tetracyclic conjugated compound with the aid of density functional theory (DFT) calculated Fukui functions is investigated. These tetracyclic conjugated compounds exhibit broad absorption spectra in the whole visible region which could be employed as potential photovoltaic materials. [ABSTRACT FROM AUTHOR]

The first case of successful suppression of the coordination of a lithium atom with a dialkylamino group by the effective conjugation of the latter with the aromatic core has been discovered. This effect controls regioselectivity of the bromine−lithium exchange in 4,6,7,9‐tetrabromo‐1,3‐dimethyl‐2,3‐dihydro‐1H‐perimidine, which leads to products with the most effective conjugation. As a result, the product of this quadruple exchange demonstrates no tendency of the coordination of the NMe groups to neighboring lithium atoms despite the absence of steric restrictions. Experimental results are explained by means of quantum chemical calculations: geometry optimization, natural bond analysis and scans using the modredundant scheme. [ABSTRACT FROM AUTHOR]

Introduction: Many Gram-negative plant- and animal-pathogenic bacteria employ type IV secretion (T4S) systems to transport proteins or DNA/protein complexes into eukaryotic or bacterial target cells. T4S systems have been divided into minimized and expanded T4S systems and resemble the VirB/ VirD4 T4S system from the plant pathogen Agrobacterium tumefaciens and the Icm/Dot T4S system from the human pathogen Legionella pneumophila, respectively. The only known plant pathogen with both types of T4S systems is Xanthomonas euvesicatoria which is the causal agent of bacterial spot disease on pepper and tomato plants. Results and discussion: In the present study, we show that virB/virD4 and icm/dot T4S genes are expressed and encode components of oligomeric complexes corresponding to known assemblies of VirB/VirD4 and Icm/Dot proteins. Both T4S systems are dispensable for the interaction of X. euvesicatoria with its host plants and do not seem to confer contact-dependent lysis of other bacteria, which was previously shown for the chromosomally encoded VirB/VirD4 T4S system from Xanthomonas axonopodis pv. citri. The corresponding chromosomal T4S gene cluster from X. euvesicatoria is incomplete, however, the second plasmid-localized vir gene cluster encodes a functional VirB/VirD4 T4S system which contributes to plasmid transfer. In agreement with this finding, we identified the predicted relaxase TraI as substrate of the T4S systems from X. euvesicatoria. TraI and additional candidate T4S substrates with homology to T4S effectors from X. axonopodis pv. citri interact with the T4S coupling protein VirD4. Interestingly, however, the predicted C-terminal VirD4-binding sites are not sufficient for T4S, suggesting the contribution of additional yet unknown mechanisms to the targeting of T4S substrates from X. euvesicatoria to both VirB/VirD4 and Icm/Dot T4S systems. [ABSTRACT FROM AUTHOR]

A novel uricase producing marine bacterium Priestia flexa alkaAU was isolated and identified. The 16S rDNA and the uricase coding gene were sequenced, analyzed and submitted to GenBank. The uricase from Priestia flexa alkaAU (PFU) was purified, determined to be 58.87 kDa, and conjugated with carboxymethyl chitosan (CMCS) by ionic gelation. CMCS conjugation had no effect on the optimum pH of PFU but decreased the optimum temperature by 10 °C. CMCS conjugation increased the specific activity of PFU by 53% at the human body temperature (37 °C) and small intestine's pH (pH 6.8). Uricase thermostabilizing ability of CMCS was significant in the range of 37–80 °C but not at lower temperatures. For improvement of the pH stability of PFU, CMCS was more effective at pHs 3–5 than pHs 6–11. CMCS increased the half-life of PFU against artificial intestinal fluid by 1.5 folds, which demonstrated the potential capability of CMCS–PFU for oral administration. [ABSTRACT FROM AUTHOR]

Aims: Vibrio parahaemolyticus is an important foodborne pathogen worldwide, which can cause gastroenteritis. This study aimed to investigate the effect of quorum sensing system LuxS/AI-2-related gene luxS on the biological characteristics and antimicrobial resistance of V. parahaemolyticus Vp2015094 from shellfish, which carried a multi-antimicrobial-resistant plasmid. Methods and Results: The critical gene luxS related to the synthesis of AI-2 in V. parahaemolyticus Vp2015094 was knocked out by homologous recombination with suicide plasmid. The effect of luxS on the biological characteristics of V. parahaemolyticus was determined by comparing the growth, AI-2 activity, motility, biofilm formation ability, and antibiotic resistance between the wildtype strain and the luxS deletion mutant. Compared with wildtype strain, the production of AI-2, the motility and biofilm formation ability, antimicrobial resistance, and conjugation frequency of luxS deletion mutant strain were decreased. The transcriptome sequencing showed that the transcriptional levels of many genes related to motility, biofilm formation, antimicrobial resistance, and conjugation were significantly downregulated after luxS deletion. Conclusions: Quorum sensing system LuxS/AI-2-related gene luxS in V. parahaemolyticus Vp2015094 played an important role in growth characteristics, biofilm formation, antimicrobial resistance, and resistance genes’ transfer. [ABSTRACT FROM AUTHOR]

This study reports on the successful conjugation of SARS-CoV-2 S1 spike protein fragments with gold nanoparticles (AuNPs) that were synthesised with Ultrasonic Spray Pyrolysis (USP). This method enables the continuous synthesis of AuNPs with a high degree of purity, round shapes, and the formation of a surface that allows various modifications. The conjugation mechanism of USP synthesized AuNPs with SARS-CoV-2 S1 spike protein fragments was investigated. A gel electrophoresis experiment confirmed the successful conjugation of AuNPs with SARS-CoV-2 S1 fragments indirectly. X-ray Photoelectron Spectroscopy (XPS) analysis confirmed the presence of characteristic O1s and N1s peaks, which indicated that specific binding between AuNPs and SARS-CoV-2 S1 spike protein fragments takes place via a peptide bond formed with the citrate stabiliser. This bond is coordinated to the AuNP's surface and the N-terminals of the protein, with the conjugate displaying the expected response within a prototype LFIA test. This study will help in better understanding the behaviour of AuNPs synthesised with USP and their potential use as sensors in colorimetric or electrochemical sensors and LFIA tests. [ABSTRACT FROM AUTHOR]

Rapid and reliable point-of-care (POC) diagnostic tests can have a significant impact on global health. One of the most common approaches for developing POC systems is the use of target-specific biomolecules. However, the conjugation of biomolecules can result in decreased activity, which may compromise the analytical performance and accuracy of the developed systems. To overcome this challenge, we present a polymer-based cross-linking protocol for controlled and directed conjugation of biological molecules. Our protocol utilizes a bifunctional thiol-polyethylene glycol (PEG)-hydrazide polymer to enable site-directed conjugation of IgG antibodies to the surface of screen-printed metal electrodes. The metal surface of the electrodes is first modified with thiolated PEG molecules, leaving the hydrazide groups available to react with the aldehyde group in the Fc fragments of the oxidized IgG antibodies. Using anti-Klebsiella pneumoniae carbapenemase-2 (KPC-2) antibody as a model antibody used for antimicrobial resistance (AMR) testing, our results demonstrate a ~10-fold increase in antibody coupling compared with the standard N-hydroxysuccinimide (NHS)-based conjugation chemistry and effective capture (>94%) of the target KPC-2 enzyme antigen on the surface of modified electrodes. This straightforward and easy-to-perform strategy of site-directed antibody conjugation can be engineered for coupling other protein- and non-protein-based biological molecules commonly used in POC testing and development, thus enhancing the potential for improved diagnostic accuracy and performance. [ABSTRACT FROM AUTHOR]

In this article, we introduce a new class of conjugations in the scalar-valued Hardy space H C 2 (D) and provide a characterization of a complex symmetric Toeplitz operator T ϕ with respect to these newly introduced conjugations in various cases. Moreover, we obtain a characterization of a complex symmetric block Toeplitz operator T Φ on the vector-valued Hardy space H C 2 2 (D) with respect to certain conjugations introduced in [Câmara MC, Kliś-Garlicka K, Ptak M. Complex symmetric completions of partial operator matrices. Linear and Multilinear Algebra. 2019; DOI: 10.1080/03081087.2019.1631246], [Kang D, Ko E, Lee JE. Remarks on complex symmetric Toeplitz operators. Linear Multilinear Algebra. 2020; DOI: 10.1080/03081087.2020.1842847], [Ko E, Lee JE. Remark on complex symmetric operator matrices. Linear Multilinear Algebra. 2019;67(6):1198–1216]. [ABSTRACT FROM AUTHOR]

Context: Chronic inflammation is a risk factor for diabetes, but it can also be a complication of diabetes, leading to severe diabetes and causing many other clinical manifestations. Inflammation is a major emerging complication in both type I and type II diabetes, which causes increasing interest in targeting inflammation to improve and control diabetes. Diabetes with insulin resistance and impaired glucose utilization in humans and their underlying mechanism is not fully understood. But a growing understanding of the intricacy of the insulin signaling cascade in diabetic inflammatory cells reveals potential target genes and their proteins responsible for severe insulin resistance. With this baseline concept, the current project explores the binding affinities of the hyaluronic acid anti-diabetic compounds conjugates to such target proteins in diabetic inflammatory cells and their molecular geometries. A range of 48 anti-diabetic compounds was screened against aldose reductase binding pocket 3 protein target through in silico molecular docking, and results revealed that three compounds viz, metformin (CID:4091), phenformin (CID:8249), sitagliptin (CID:4,369,359), possess significant binding affinity out of 48 chosen drugs. Further, these three anti-diabetic compounds were conjugated with hyaluronic acid (HA), and their binding affinity and their molecular geometrics towards aldose reductase enzyme were screened compared with the free form of the drug. The molecular geometries of three shortlisted drugs (metformin, phenformin, sitagliptin) and their HA conjugates were also explored through density functional theory studies, and it proves their good molecular geometry towards pocket 3 of aldose reductase target. Further, MD simulation trajectories affirm that HA conjugates possess good binding affinity and simulation trajectories with protein target aldose reductase than a free form of the drug. Our current study unravels the new mechanism of drug targeting for diabetes through HA conjugation for inflammatory diabetes. HA conjugates act as novel drug candidates for treating inflammatory diabetes; however, it needs further human clinical trials. Methods: For ligand structure, PubChem, ACD chem sketch, and online structure file generator platform are utilized for ligand preparation. Target protein aldose reductase obtained from protein database (PDB). For molecular docking analysis, AutoDock Vina (Version 4) was utilized. pKCSM online server used to predict ADMET properties of the above three shortlisted drugs from the docking study. Using mol-inspiration software (version 2011.06), three shortlisted compounds' bioactivity scores were predicted. DFT analysis for three shortlisted anti-diabetic drugs and their hyaluronic acid conjugates were calculated using a functional B3LYP set of Gaussian 09 software. Molecular dynamics simulation calculations for six chosen protein–ligand complexes were done through YASARA dynamics software and AMBER14 force field. [ABSTRACT FROM AUTHOR]