Antibiotics have always been considered as one of the most relevant discoveries of the twentieth century. Unfortunately, the dawn of the antibiotic era has sadly corresponded to the rise of the phenomenon of antimicrobial resistance (AMR), which is a natural process whereby microbes evolve in such a way to withstand the action of drugs. In this context, the identification of new potential antimicrobial targets and/or the identification of new chemical entities as antimicrobial drugs are in great demand. To date, among the many possible approaches used to deal with antibiotic resistance is the use of antibiotic adjuvants that hit bacterial non-essential targets. In this review, the author focuses on the discovery of antibiotic adjuvants and on new tools to study and reduce the prevalence of resistant bacterial infections. Antibiotics have always been considered as one of the most relevant discoveries of the twentieth century and their widespread use has revolutionized healthcare since their introduction . The dawOver the last 30 years, antibacterial drug discovery has faced a discovery gap, and no new classes of antibacterials were introduced in the market until 2000, when linezolid, belonging to the class of oxazolidinones, was approved ,5.Unfortunately, the dawn of the antibiotic era has sadly corresponded to the rise of the phenomenon of antimicrobial resistance. It is widely accepted that, while the use of antimicrobial agents has led to the control and even eradication of infectious diseases, their misuse and/or overuse has led to the development of resistant strains. Based on his early observations, in 1954, Fleming had predicted that the inconsiderate use of this discovery could lead to the selection and propagation of selected mutants of bacteria resistant to antibiotics. Indeed, alarming signals of developing resistance were observed after only a few years of the golden age of antimicrobials  Figure .Antimicrobial resistance (AMR) is a natural phenomenon whereby bacteria evolve in such a way to withstand the action of drugs, making them apparently ineffective. The pressure that antimicrobials put on the pathogens is responsible for the selection of resistant strains . AlthougEnzymatic inactivation: An existing bacterial enzyme is modified to interact with an antibiotic in order to make them inactive towards bacteria. It is due to the transfer of the antibiotic resistance gene carried on plasmids. The most significant examples are beta-lactamase enzymes, which hydrolyze beta-lactams .P. aeruginosa and Acinetobacter spp.Drug extrusion by efflux pumps: These proteins, which are able to extrude a wide variety of compounds (including antibiotics) out of the cell, are overexpressed by the bacteria to extrude the antibiotic. This is an important mechanism of resistance in Decreased uptake by changes in the outer membrane permeability or by presence of porins: These variations interfere with the entrance of antibiotics.Modification of the drug target: These changes impede the binding of the antibiotic and limit its potency.There are four mechanisms through which bacteria become resistance to antibiotics  Figure :EnzymatiEnterecoccus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter. These bacteria are resistant to carbapenemes, that are considered as last resort antibiotics. Mycobacterium tuberculosis, the causative agent of tuberculosis, that has been added to this list, is considered as a top global health priority. They might cause serious and deadly infections such as bloodstream infections and pneumonia infections.An extensive list of AMR bacteria was recently published by the World Health Organization (WHO) on the fact sheet of 27 February 2017. Pathogens are classified as critical, high, and medium, and this classification is based on mortality, level of resistance, and treatability. The situation is highly critical in infections caused by the Gram-negative ESKAPE: These bacteria are resistant to many antibiotic drugs of the current pipeline, including carbapenems and third generation cephalosporins—the most efficacious antibiotics for treating multi-drug-resistant bacteria. That resistance is associated with high mortality due to the absence of new antimicrobials in the pipeline. This priority list of pathogens is to be considered as an indicator for the prioritization of incentives and funding for Research and Development (R&D) aimed at discovering new effective antibacterial drugs .Presently, multi-drug-resistant (MDR) bacteria cause about 25,000 deaths in Europe each year  and haveAs the protection of antibiotic therapy is diminishing, it is important to preserve its efficacy and to reinforce our current arsenal of antibacterial drugs. In addition, an improvement in antibiotic compliance  is imporThe first objective is hard to reach without significant government financing, and in the last few years, we have experienced a cut in investment by pharmaceutical companies for the discovery of new antibiotic drugs . The reaTaken together, it is crucial to look for new and more potent anti-infective drugs and to develop antibiotics with different scaffolds and with new mechanisms of action. Large efforts are being made, particularly in academia, to investigate the potential of an exploited essential process in bacteria and to develop new molecules that target them, as well as to study the biochemical basis of these targets. In addition, promising success has been achieved with those approaches that are focused on targeting non-essential pathways in order to minimize the impact of resistance to antibiotics by the use of an antibiotic “adjuvant” in combination with an antibiotic .It has already been proven that synergy and drug combinations are a winning strategy in fighting MDR bacteria and might help in protecting the already existing drugs through the use of antibiotic adjuvants. The most known and successful example is the combination of Amoxicillin and clavulanic acid. Amoxicillin is a potent beta-lactam that is inactivated by beta-lactamases, and clavulanic acid is an inhibitor of beta-lactamases endowed with a weak antibacterial activity. This association, of an antibiotic molecule together with an antibiotic adjuvant, has led to Augmentin, which was the best-selling drug in 2001. Antibiotic adjuvants are molecules with weak or absent antimicrobial activity but that are able to enhance the activity of antibiotics, minimizing or blocking resistance. Antibiotic adjuvants can also suppress intrinsic resistance and this has led to an expansion of the activity spectrum of antibiotics. As a matter of fact, in the literature, there are examples reporting the use of Gram-positive selective antibiotics to treat infections caused by Gram-negative. This is also a good strategy with the antibiotics for which the toxicity is a problem, such as colistin. In this case, antibiotic adjuvants that increase bacterial susceptibility to antibiotics, would allow them to be efficacious at lower doses, thereby mitigating potential side effects [To date, three main types of antibiotic adjuvants have been developed to block the aforementioned mechanisms of antibiotic resistance: (a) beta-lactamase inhibitors, (b) efflux pumps inhibitors, and (c) outer membrane permeabilizers. In In addition, to enhance the antimicrobial action of drugs, new potential inhibitors are designed to target pathogenicity, in other words, the capacity of the bacterium to cause an infection . This apIn this review, the author provides an overview of the antibiotic adjuvant strategies.Efflux pumps (EPs) are among the most well-known examples of bacterial mechanisms that confer cross-resistance to different antibiotics . This tyMFS, ABC, SMR, and MATE families are expressed in both Gram-positive and Gram-negative bacteria, while the RND one is characteristic of the Gram-negative bacteria . The ABCPseudomonas aeruginosa, in which the knocking-down of the mexB gene produces mutants which are more susceptible to different classes of antibiotic  [Apparent poor antibiotics permeability: in some bacteria, it can be related to the expression of efflux pumps, which confers a resistance . The beslactams) .Cross-resistance to unrelated antibiotic classes: Cross-resistance comprises evolutionary events of the adaptation of antibiotics, or any other antimicrobial drug, which decreases the organism’s sensitivity to multiple drugs. This can be due, generally, to a high exposure to a given antibiotic.E. coli strain that expresses both beta-lactamases and efflux pumps, and which is also insensitive to beta-lactams [Wide spectrum resistance can be observed in bacteria in which active efflux functions synergistically with other mechanisms of resistance, for example, in the -lactams . Thus, i-lactams .Mutations can be favored in bacteria overexpressing efflux pumps. Indeed, in that condition, antibiotic targets become exposed to subinhibitory concentrations and can mutate to inhibit the effect of antibiotics , eventuaIt is widely believed that they usually confer a moderate level of resistance and the consequences of antibiotic active efflux can be summarized as follows:E. coli [The active efflux of antibiotics was described for the first time 30 years ago. At that time, the presence of plasmid-encoded proteins able to extrude tetracycline and confer resistance to this antibiotic in E. coli  was studNowadays, efflux pumps can be considered as potential antibacterial targets, due to their role in antibiotic resistance, and the development of inhibitors could improve the therapeutic arsenal against resistant pathogens. In the context of antibiotic combination therapy, efflux pumps are different from other mechanisms of resistance (such as beta-lactamases) that work on a specific family of antibiotics. Indeed, a single efflux pump can extrude a wide range of different families of antibiotics and, for this reason, their inhibition will increase the bacterial susceptibility and their combination could work with several antimicrobials.There are several ways for inhibiting efflux pumps: (i) interfering with efflux gene expression, (ii) adding functional groups to the drug substrate to hamper recognition, (iii) interfering with the assembly of channel proteins, (iv) developing small-molecules as substrate analogues able to block the efflux pump activity, or (v) able to disjoin the energy transfer mechanism of the pump, or (vi) able to obstruct the channel ,36.Therefore, it is possible to corroborate that inhibition of efflux might lead to a variety of positive effects: (i) increasing the activity of the antibacterial drugs subject to efflux, (ii) keeping the concentration of the drug at the therapeutic dose, and (iii) shortening the duration of treatment by reducing multi-drug tolerance ,38.The most widely exploited strategy is the development of efflux pump inhibitors (EPIs), which are intended for combination therapy with specific antibiotics.EPIs are small molecules that are able to bind efflux pumps and block their extrusion activity. EPIs, usually, do not have intrinsic antibacterial activity. For this reason, these compounds are further tested for synergy with different concentrations of antibiotics against a single concentration of inhibitor in bacterial strains containing efflux pumps. Inhibitors showing an eight-fold or more synergistic reduction in MIC are further evaluated by using the fractional inhibitory concentration (FIC) method .One of the inconveniences in targeting these pumps is linked to the variety of physiological functions they are involved in, which can cause related toxicities when they are blocked, especially for the EPIs derived for a drug repurposing approach. Indeed, the main issue in such combination therapy is related to the need to use EPIs at high doses, underlying potential on- and off-targets side effects. In this context, research is focused on finding compounds that selectively inhibit pumps working only in prokaryotic cells . Given tSimilar effects were described with the phenothiazines, calcium channel antagonists, selective inhibitors of serotonin reuptake, or proton pump inhibitors. Derivatives lacking the pharmacological activity of the cognate compound are now developed, as described for inhibitors of serotonin reuptake or of omeprazole. To date, the only documented inhibitor is currently MP-601 2,  administacrB gene [Among the many strategies by which it is possible to inhibit efflux pumps’ activity, there are compounds able to compete with the antibiotics for their extrusion. The EPI lead compound is a dipeptide amide, named phenylalanine-arginine-β-naphthylamide PaβN, 3, , which icrB gene .K. pneumoniae and E. aerogenes [Quinolines have shown inhibition of antibiotic efflux in isolates of the multi-drug-resistant bacteria. Indeed, it was shown that a number of quinoline derivatives are capable of potentiating activity of antibiotics through the inactivation of the AcrAB-ToIC (RND family) efflux transporters . Studieserogenes .N-heterocyclic compounds, in particular the arylpiperazine derivatives  derivatives 14–17, , and theThirteen small molecules were rationally synthesized and their activities were compared with TZ. Such newly synthetized compounds resulted as less toxic toward human macrophages, while preserving high synergistic activity when administrated in combination with the first-line  drugs currently used for the treatment of tuberculosis .The production of enzymes able to deactivate antibiotics is one of the resistance mechanisms used by bacteria to withstand the effect of antibiotic drugs .β-lactam antibiotics, the first natural antibacterial compounds successfully developed, and subsequently modified, still represent a very important class of antibiotics thanks to their antibacterial activity and selectivity. However, their introduction to the market has been rapidly followed by the spreading of resistant strains on a global scale. In Gram-positive bacteria, hydrolysis of the first commercial penicillin by β-lactamases was the first reported resistance mechanism to β-lactams.The mechanism of action through which these antibiotics work involves the inactivation of transpeptidases that are required for the last step of bacterial cell wall biosynthesis. Bacterial strains produce β-lactamases, which destroy the β-lactam ring, leading to a loss of efficacy of antibiotics containing this functional group.The β-lactam ring is the key element responsible for antibiotic activity because of its electrophilicity, through which it irreversibly acylates the penicillin-binding proteins (PBPs). PBPs are responsible for the synthesis of peptidoglycan that is responsible for the structural integrity of the bacterial cell wall. To preserve the cell wall, the β-lactamases synthesized by bacteria are able to hydrolyze β-lactams-based antibiotics through the generation of an inactive open cycle, and the degree and the extent of hydrolysis depends on the type (and quantity) of beta-lactamases produced by the microorganisms. Gram-negative release β-lactamases in the periplasmatic space to prevent the antibiotic from reaching its target in the cytoplasmatic membrane , while GNowadays, hundreds of β-lactamases have been discovered to be endowed with the same general mechanism of action. They differ from each other for the amino acid sequences and this has led to a different affinity for different substrates. In general, β-lactamases are classified in two different methods: One is Ambler classification and is based on a structural characterization, the other one is Bush and Jacoby classification and is based on a functional characterization ,57.Enterobacteriaceae, such as E. coli, K. pneumoniae, and P. mirabilis [The huge amount of β-lactams antibiotics used in therapy has promoted the synthesis of a particular class of β-lactamases, called extended-spectrum β-lactamases (ESBL), which are able to hydrolyze most β-lactam antibiotics, and these are characterized especially in irabilis ,59. Carbirabilis .In this context, due to the growing increase in newly discovered β-lactamases, there is an urgent need of new and effective β-lactamases inhibitors as antibiotic adjuvants in antibiotic therapy .Two strategies are pursued to overcome β-lactamase-mediated resistance to β-lactams: (i) The development of β-lactamase-stable antibiotics, for example cephalosporins and carbapenems, that are stable to hydrolysis by β-lactamases, and (ii) the development of selective β-lactamase inhibitors (BLIs) to be used in co-administration with a β-lactam antibiotic.The selection of the inhibitor that can be combined with a specific β-lactam antibiotic is a complex step that takes into consideration different requirements: a) The ability of the inhibitors to protect the antibiotic against enzymatic hydrolysis, b) the dose of inhibitor required to ensure this protection, and c) the viability and stability of the combination.Streptomyces clavuligerus secondary metabolite, accounted for an important step in the field of antibacterial discovery. This β-lactam is capable to inactivate most of the β-lactamases, showing good antibacterial activity. This led to the development of the first β-lactam-β-lactamase inhibitor combination, Augmentin (amoxycillin/clavulanic acid) [In this scenario, the discovery of clavulanic acid 18, , a Strepic acid) . This coP. aeruginosa [N. gonorrhoeae [Acinetobacter spp. and Burkholderia cepacian, with MIC values <8 and 10 µg/mL, respectively, but does not show activity against strains with multiple resistance mechanisms [Indeed, following the discovery of clavulanic acid, a medicinal chemistry campaign was started aiming at the synthesis of several penicillanic acid sulfones endowed with β-lactamase inhibitory activity. Among these, sulbactam and tazobactam  scaffold, arose. Avibactam is the first inhibitor of this class. It has a larger spectrum of activity compared to clavulanic acid. Avibactam 21,  has beenAnother new class of synthetic non β-lactam β-lactamase inhibitors are made up of boronic acids . Among tResistance to β-lactams continues to spread, especially in Gram-negative organisms ,73. NowaGenerally, the antibiotics used in therapy exert their antibacterial action, hitting the respective targets inside the cell. This requires the antibiotics to cross the bacterial membrane(s), to be able to reach their target(s). To withstand the permeation of antibiotics, Gram-negative are protected by the presence of a further level of defense, which is the outer membrane .Indeed, the Gram-negative’s outer membrane, which is mainly composed of polyanionic lipopolysaccharides and porins, limits the entrance of xenobiotic such as antibiotics. As a consequence, some antibacterials show reduced efficacy in treating Gram-negative infections because of the complex architecture of their wall.As a matter of fact, the complex architecture and composition of the membrane wall heavily affects the bacterium vulnerability toward the antibiotic. It is not surprising, therefore, that most of the occurring resistant strains usually adopt protein mutations, at the outer membrane level, in order to overcome the action of some antibacterial drugs.Hydrophobic compound (such as macrolides and rifampicin) cross the lipid bilayer through passive transport mechanisms.Hydrophilic molecules  diffuse through active transport mechanisms, taking advantage of their ability to interact with peculiar porins ,75.For penetration across the bacterial wall, antibiotics use two different strategies, depending on the chemical nature of the small-molecule:In this context, the bacterial outer membrane represents a potential target able to deal with bacterial resistance, and a better knowledge of its structure will surely increase our ability to develop new classes of antibiotics, endowed with a peculiar mechanism of actions .The use of antibiotic adjuvants to increase the membrane permeability propensity  proved to be a good strategy to improve the entrance of antibiotics. In general, permeabilizers are cationic and amphiphilic molecules or chelators that, by interacting with polyanionic lipopolysaccharides or by capturing outer layer cations, destabilize the membrane wall. Consequently, the outer membrane becomes more prone to being crossed by xenobiotic. Examples of outer membrane permeabilizers are polymyxins, such as polymyxin B 25, , colistiAlternative strategies to design or to identify new small molecules that could facilitate the diffusion of antibiotics, increasing their intracellular concentration, are of great demand . In thisE. coli with a concentration-dependent mechanism of action. This study demonstrated that the use of GBP leads to a cell with fragmentation at a high concentration. This cationic peptide works by disrupting the membrane barrier and the E. coli ion-channel. The result is a loss of ions Ca2+, K+, and Mg2+. The use of GBP had improved the sensitivity of E. coli to erythromycin and rifampicin. Indeed, the aforementioned drugs resulted as unable to penetrate the intact outer membrane of Gram-negative bacteria. On the contrary, when the membrane is damaged  they are capable of getting inside the bacterial cell, engaging the target(s) for which they were originally designed. These observations support the notion that GBP works as permeabilizers, increasing the membrane permeability, and in turn, facilitating the penetration of antibiotics [Recently, it has been reported that a glycine basic peptide (GBP) exerts its antibacterial activity on the membrane of ibiotics .S. aureus, P. aeruginosa, and E. coli. At the intestinal level, menadione is converted to vitamin K2, being characterized by antibacterial activity only against the P. aeruginosa strain. Surprisingly, menadione, in combination with antibiotics from the aminoglycoside family, provided a reduction of the inhibitory concentration of these antibiotics and suggested a synergistic effect in combination therapy [Another study was conducted regarding the evaluation of menadione 27,  and its  therapy .Another class of interesting molecules endowed with antibiotic adjuvant potential is represented by endogenous antimicrobial peptides (AMPs), which are factors secreted by host cells and organs  representing the so-called defensive line in innate immunity against pathogens. The mechanism of action of AMPs is based on their ability to destabilize the outer cell membrane of prokaryotes by the formation of an amphipathic α-helix or short β-sheet structures , which aK. pneumoniae is 70 µg/mL, while the use of erythromycin in combination with compound CSA-8 leads to the reduction of its MIC to 1 μg/mL. It was also analyzed the anti-bactericidal activity of CSA-13 on 60 carbapenem-resistant strains. In this study, the authors demonstrated that by combining CSA-13 with antibiotics, synergy was achieved with colistin (55%) and tobramycin (35%), while no antagonism was observed [In order to overcome the aforementioned issues linked to the use of AMPs, a new class of adjuvant, namely “Caragenins”, was developed. Caragenins are cationic steroidal antibiotics (CSA), in which the alkoxy groups of the sterol core structure is substituted by an aminoalkyl function. This structural modification makes “Caragenins” resistant to the action of proteases since they do not present peptidic bonds in their structure and they can be produced in large amounts. Moreover, CSA are able to stably incorporate into membranes, having the ability to form complexes with phospholipids ,91. On tobserved ,89. It iOn the route to the identification of novel antibiotics active against drug-resistant strains, the motivation to consider alternative cellular pathways as a source of targets for the development of new antibacterial adjuvant classes represent an interesting alternative to classical approaches.The resistance to antibiotics often causes latent and persistent infections that are very challenging to deal with. During persistence inside the host, pathogens face extremely hostile environments, thus requiring an extensive re-programming of the bacterial metabolism functions, in order to survive such adverse conditions. Therefore, to target key metabolic functions that are relevant for the pathogens to survive in such conditions, could result in better antibiotic treatments and increase susceptibility to traditional antibiotics.In this context, the main factor for the degeneration of a patient’s health during a bacterial infection is bacterial virulence. Over the last decade, a new approach has emerged, designed to hit bacterial virulence, or pathogenicity ,93,94,95The rationale behind the exploitation of amino acid biosynthesis as a target for antimicrobial adjuvant development is the observation that some pathogens spend part of their life cycle in extremely harsh conditions, such as macrophages or the gastric mucosa, where survival and proliferation require powerful adaptation mechanisms involving metabolic pathways ,100. In Among the possible new drug targets are the enzymes involved in the cysteine biosynthesis.It has been observed that the importance of cysteine biosynthetic enzymes varies within the life cycle of pathogens: their activity can be dispensable during growth in vitro or acute infections but becomes indispensable during the persistence phase ,102. MolMany studies on the response of microorganisms to environmental stress  point to an upregulation of many genes of the cysteine regulon.S. typhimurium led to the conclusion that an unpaired oxidative stress response, due to inhibition of cysteine biosynthesis, causes a decrease in antibiotic resistance, in both vegetative and swarm cell populations. Antibiotic-induced oxidative stress, which is widely recognized as a general mechanism of action of many antibacterials [Thus, a role for cysteine biosynthesis in the development of antibiotic resistance has recently been pointed out ,106. Invcterials , could ecterials .Most bacteria and plants carry out cysteine biosynthesis by the reductive sulfate assimilation pathway (RSAP), a multistep reduction of sulfate that culminates in the incorporation of bisulfide into cysteine using an activated form of serine .RSAP begins with the transportation of sulfate inside the cell, followed by its reduction to bisulfide. This process is highly energy-consuming and is tuned to cellular needs. Bacteria find sulfur in the form of sulfate in the environment and actively transport it through plasma membrane.O-acetylserine sulfydrylase (OASS), which catalyzes the last step of cysteine biosynthesis [After the reduction of sulfate to bisulfide, a toxic compound whose concentration inside the cell is kept between 20 and 160 µM, the latter is incorporated into cysteine by a member of a large enzyme family, known as cysteine synthase complex (CSC). The CSC is composed by the enzyme serine acetyl transferase (SAT) and ynthesis .O-acetyl-l-serine (OAS), the activated form of serine, a cysteine precursor where the β-hydroxyl group of the amino acid is acetylated or phosphorylated to generate a better living group in the β-elimination reaction.SAT is catalytically active in the complex and produces N-acetylserine (NAS), the natural inducer of the cysteine regulon signaling. SAT catalyzes the reaction by which an acetyl group from acetyl-CoA is transferred to the hydroxyl one of l-serine to form OAS and CoA. The C-terminus tail of SAT is fundamental for function and regulation, being responsible for an intrasteric inhibition in the presence of cysteine by binding to OASS for the formation of CSC [OAS is unstable and spontaneously converts to n of CSC .Salmonella typhimurium. Later it was observed how many pathogens possess two isoforms of the enzyme: O-acetylserine sulfydrylase (OASS-A encoded by cysK) and O-phosphoserine sulfydrylase , which differ for peculiar functional and structural properties. In S. typhimurium, both OASS-A and B isoforms use O-acetylserine (OAS) as a substrate and S2− as a primary sulfur source. The OASS isoenzymes are differently expressed depending on the environment conditions  [l-serine external aldimine. In the second half-reaction, the sulfur source attacks the α-aminoacrylate in order to give l-Cys [Proteus mirabilis [E. coli [S. typhimurium [Depending on the organism and growth conditions, the last step of cysteine biosynthesis is catalyzed by different sulfydrylases, that share high homology but also show some functional and structural differences. The first identified OASS isoform (OASS-A) was firstly isolated and characterized from ronment) . They arve l-Cys . Since iirabilis , transcrirabilis , toxin a[E. coli , and anthimurium ,106 , which is a cell–cell communication mechanism controlling phenotype manifestations, such as virulence . This syQS has been identified in a broad range of both Gram-negative and Gram-positive bacteria. It has a great importance in many pathogenic species and has been shown to also play a role in biofilm formation . In GramAzithromycin, which does not have a bactericidal effect on pseudomonas aeruginosa and interferes with its QS pathways, was studied in clinical trials . In theipseudomonas quinolone signal (PQS) system of P. aeruginosa can be considered a potential target. The biosynthesis of the PQS signal 30 requires the action of a set of enzymes PqsABCDEH and its autocatalytic receptor PqsR (MvfR). It has been demonstrated that compound 31 acts as PqsD transition state analogues and reduces bacterial biofilm formation. Later, it was shown that numerous compounds, such as 32, 33, 34, and 35 , which are able to interact with negative charges present in the surface of the membrane, and hydrophobic substituents, which interact with and disrupt the lipid chains of the bacterial membrane. Through the antibiotic adjuvant strategy, a polypharmacy approach on bacteria can be performed, not only by providing the entrance of the antibiotics inside the cell but also by de-energizing the efflux pumps used by the bacteria to escape antibiotic action.The identification of molecules that inhibit sulfur metabolism, as they are expected to strongly decrease bacterial fitness at infection loci, is a promising strategy to increase the efficacy of existing antibiotics acting as potential antibiotic adjuvants, thus providing a new life to old molecules.It is important to note that the list of antibiotic adjuvant approaches described in this review is not complete, indeed, (i) among the known pathways there are many undrugged targets, which still require the development of a probe in order to be validated and in turn, represent new pharmaceutical targets. (ii) In the advent of the “omic” era, there are undiscovered pathways that could be targeted.In conclusion, in the near future, AMR will continue to be one of the main threats for global health, which will require continuous and significant efforts at different social levels. Therefore, the identification of new strategies to limit or to overcome the occurrence of resistance strains will be a long journey, where antibiotic adjuvants counteracting antibiotic resistances will cover a significant area of the AMR fields.


Mechanically stretching the metallocycle-C60 junction with a scanning tunneling microscopy break junction technique causes the release of the C60 guest from the metallocycle, and consequently the conductance switches back to the free-host level. Metallocycle hosts with different shapes and cavity sizes show different degrees of flexibility to accommodate the C60 guest in response to mechanical stretching. DFT calculations provide further insights into the electronic structures and charge transport properties of the molecular junctions based on metallocycles and the metallocycle-C60 complexes.Host−guest interactions are of central importance in many biological and chemical processes. However, the investigation of the formation and decomplexation of host−guest systems at the single-molecule level has been a challenging task. Here we show that the single-molecule conductance of organoplatinum(II) metallocycle hosts can be enhanced by an order of magnitude by the incorporation of a C 60 molecule significantly enhances the conductance of an organoplatinum metallocycle; mechanical stretching of the junction releases the guest, returning the conductance to free-host level.Studying the single-molecule behavior of host-guest complexes can provide fundamental insights into their supramolecular interactions. Here, the authors use the scanning tunneling microscopy break junction technique to show that encapsulation of a C Host–guest interactions have now been exploited in a wide range of disciplines, including chemosensing10, catalysis11, drug delivery12, nanosciences14, and molecular devices16. These previous works focused on host–guest ensemble behaviors of bulk materials. Studying how host–guest complexes behave at the single-molecule level opens new prospects to understand the fundamentals of supramolecular chemistry by providing critical information about the strength and flexibility of host–guest interactions.The discovery of crown ether and cryptand in 1967 as macrocyclic ligands for alkali metal cations laid the foundation of modern host−guest and supramolecular chemistry27. The technique has been recently extended to supramolecular systems including charge–transfer pairs28, π-stacked dimers29, rotaxanes31, or host–guest interactions32. Mechanically controllable break junction (MCBJ) or STMBJ experiments are well-developed to reveal the electrical and mechanical properties of the molecular structure and junction conductances38.The scanning tunneling microscopy break junction (STMBJ) technique allows measurement of charge transport properties of single-molecules involving covalent bonds60 with STMBJ. The coordination-driven self-assembly yields different metallocycles with precisely controlled shape and cavity size, which allows us to modulate the strength of the host–guest interaction. The conductance of the metallocycles with a suitable cavity size is enhanced by an order of magnitude when complexed with the C60 guest. The molecular conductance properties of the metallocycles and their host–guest complexes are further rationalized by theoretical calculations.The direct monitoring of the size-dependent decomplexation was obtained by mechanically stretching the host-guest complexes at the single-molecule level, which switches the conductance back to the free-host level. This demonstrates the capability of host-guest supramolecular systems to modulate molecular conductance.Herein, we measure the conductance and study the charge transport in three supramolecular metallocycles and their host–guest complexes with C5, 6, and 7 with different shape and cavity size were designed and synthesized for the host–guest interaction studies and conductance measurements . The uncoordinated pyridine groups present in these metallocycles are used to contact the gold metal electrodes to form single-molecule junctions.Three organoplatinum(II) metallocycles, nts Fig. , via the1H NMR, 31P{1H} NMR, 1H−1H COSY NMR spectroscopy, and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) -optimized structures of 60 under ambient conditions (see the Methods section). The lone pair electrons of the free pyridine nitrogen atoms of metallocycles can bind to the Au tip and substrate to form molecular junctions by repeatedly moving the STM tip into and out of contact with the gold substrate functionalized with the supramolecular metallocycles44. Conductance–distance traces were recorded during the stretching process for each sample, and plateaus in these traces were related to the formation and breakdown of single-molecule junctions. Thousands of conductance traces were used to construct a conductance histogram, where the peak position measures the average conductance of a single molecule  single-crystal substrate to measure the single-molecule conductance of the metallocycles and their host–guest complexes with Cule Fig. .Fig. 2Co5, 6, 7, and those in the presence of C60, denoted as [5 + C60], [6 + C60], and [7 + C60], respectively. A conductance plateau was typically observed for the free metallocycles and the conductance followed the order as 5>6> 7  show no conductance peak in the histogram within the measured conductance range , and after rinsing with copious amounts of solvent, the coverage of C60 on Au surface was too low to be measured by break junction methods50. However, C60 featured two conductance peaks at ~0.2 G0 and 0.5 G0, with a higher concentration of C60 (7 mM), which is in agreement with reported values , with different shapes and cavity sizes, possess different degrees of host–guest interactions with the C60 guest. In contrast to [7 + C60], [5 + C60] and [6 + C60] show higher conductance values, which are greater than those of the free 5 and 6 molecules by about one order of magnitude. This provides direct evidence of large enhancement of charge transport when a C60 guest is incorporated. This finding led us to explore in situ controlling of the host–guest interaction by mechanically stretching the molecular junction. Mechanical switching has been demonstrated in single molecules38, but mechanical control of host–guest interactions is a new and attractive strategy. The 2D conductance histograms and corresponding average plateau lengths of 5, 6, 7, [5 + C60], [6 + C60], and [7 + C60] are shown in Fig. 5, 6) are comparable, but slightly larger than those of the host–guest complexes ([5 + C60] and [6 + C60]). Figure 60 complexes at different stretching distances. For [5 + C60], as the junction is stretched by a longer distance, the relative peak area of the lower conductance becomes increasingly larger, which is consistent with the transition from the high to low plateaus shown in individual traces in Fig. 5 is still present after C60 is released and the conductance switches back to the free host level. For [6 + C60], the relative peak area of the high conductance also decreases with stretching, but the distance dependence is substantially smaller than that for [5 + C60]. The distinct distance dependence gives a higher conductance band that persists for 0.2–0.3 nm and 0.5–0.6 nm for [5 + C60] and [6 + C60], respectively  comparable with the van der Waals diameter of C60 (10.34 Å), a very subtle structural deformation of the host by 0.1~0.2 nm could be destructive for the host–guest interaction. However, for metallocycle 6 with a moderately larger cavity size (14.10 Å), the host could stand a structural deformation as large as 0.5~0.6 nm, while still maintaining the host–guest complex. Such information can not be obtained on conventional studies of bulk materials.The maximum mechanical stretching force of the STM tip for the pyridine contact is ~0.8 nN52. The resulting 1D and 2D histograms of 5, 6, [5 + C60], and [6 + C60] are shown in Supplementary Fig. To provide a more complete view of the overall break junction process over a wider conductance range, we measured the conductance from the quantum point contact formed between the electrodes to the noise level measured when the electrodes are widely separated using a logarithmic amplifier5, [5 + C60], 6, and [6 + C60] are 1.38 nm, 1.38 nm, 1.41 nm, and 1.42 nm, respectively, suggesting similar junction breakdown configurations for the metallocycles before and after encapsulation of C60. From the DFT optimized molecular length of 5 (1.79 nm) and 6 (1.94 nm), the angle between the center axis of the metallocycle and the substrate plane is estimated to be ~50° for both metallocycles at junction breakdown. These results indicate that during the break junction measurements the metallocyclic molecules were lifted and distorted by the retreating STM tip.The distributions of relative displacement from the point of contact to junction breakdown are shown in Supplementary Fig. 5 and 7, no distinct absorbance changes were observed upon adding the metallocycle into the C60 solution. In contrast, the complexation of 6 with C60 resulted in the appearance of a new shoulder band at 366 nm, which was consistent with a charge transfer (CT) absorption band53. The linear Benesi–Hildebrand plot suggests a 1:1 host–guest binding ratio of 6 with C60 and the binding constant was calculated to be 4.1 × 105 M−1. These results suggest that the driving force of forming the target host–guest complexes is derived from the donor–acceptor interactions between the electron-rich metallocylic skeleton and the electron-deficient of C6054. The failure to observe potential CT absorption band from the complexation of 5 with C60 is possibly caused by the relatively weak and shallow interaction between them. This is supported by the DFT calculation results discussed below. Although the absorption spectral analysis implies that the interaction between 5 and C60 is weaker compared with that between 6 and C60, the metallocycle 5 can still hold C60 with a shallower host–guest configuration according to our DFT calculations. This allows the complexed molecular wire, either in the case of [5 + C60] or [6 + C60], to open up the C60-related current path to enhance the conductance .UV−vis absorption spectral titration experiments were carried out to investigate the host–guest interactions in solution  is consistent with that of the powder sample (1.0/2.1/2.7/2.7) within experimental error.In our previous work, Pt(II)-based self-assembled supramolecular rectangle, square, three-dimensional cage and prism have been investigated by scanning tunneling microscopy on Au(111) surfaces, and demonstrated to be intact after adsorption on Au(111) surfaces5 and Au(111)/6 are shown in Supplementary Fig. 57, which are 1.2 nm and 1.3 nm for 5 and 6, respectively, close to the STMBJ values.AFM images of Au(111)/5 and 6 with C60 were optimized by DFT calculations. The C60 molecule is deeply embedded into the metallocycle framework of 6  and the lowest unoccupied molecular orbital (LUMO) plots of the [5 + C60] and [6 + C60] complexes are displayed in Fig. 60 contributes to the LUMO, suggesting the presence of donor–acceptor interactions in the metallocycle-C60 complexes.The host–guest complexes of 60 guest binds to the metallocycle host, the transmission spectra T(E) were obtained by combining the DFT package SIESTA58 and with the quantum transport code Gollum59 (see details in the Methods section). Based on the relaxed geometries of the gold-molecule-gold junctions estimated from experimental nanogap distance in Supplementary Fig. 60 and the smaller angles  is close to that of the undistorted 5 with larger angles (~65°), while the large conductance discrepancy between red ([5 + C60]) and yellow (5′) curves in Supplementary Fig. 6′ with smaller tilt angles is higher than that of 6 with larger angles, while Supplementary Fig. 6′ is much closer to that of 6. This demonstrates that the smaller distortion of this host  tends to appear near EF. This effect is clearly present in the red curves of Fig. 60 LUMO are pinned close to the DFT-predicted Fermi energy. In contrast, the resonances between −1.0 and −0.8 eV for [5 + C60] and between −1.0 and −0.5 eV for [6 + C60] are due to states located on the host. Therefore, these HOMO resonances are sensitive to the gating effect of the negatively charged C60 guest  metallocycles with precisely controlled shape and cavity size were synthesized by coordination-driven self-assembly. These compounds, together with C60 were immobilized on Au (111) single-crystal electrode by two separate steps. (1) The electrochemically cleaned Au (111) single crystal electrode was first annealed with hydrogen flame briefly, immersed in acetone  containing 10 μM 5/6 or methanol  containing 10 μM 7, incubated for 2 h, then rinsed with acetone (for 5 and 6) or methanol (for 7) and dried with nitrogen gas. (2) After the STMBJ measurements were performed on the metallocycle modified Au electrode, the resulting electrode was immersed in 1,1,2,2-tetrachloroethane  containing 10 μM C60, incubated for 1 h, then rinsed with 1,1,2,2-tetrachloroethane and dried with nitrogen gas for further STMBJ measurements.The metallocycles and C21 were carried out by a scanning tunneling microscope  and a STM scanner (Molecular Imaging). The molecules modified on Au (111) single-crystal electrode was measured in the air with a small bias (0.1 V) applied between the STM substrate (Au (111) single-crystal electrode) and the STM tip that was freshly prepared by cutting a gold wire . The STM tip was repeatedly brought into contact and retracted from the substrate, during which thousands of conductance distance traces were collected to construct conductance histograms. For each analyte, the STM conductance measurement was performed three time as repeating experiments.The break junction measurements62. No symmetry constraints were used in the optimization (nosymm keyword was used). Conductance calculations and corresponding geometry optimizations were performed by using the DFT code SIESTA58, with a local density approximation , double-ζ polarized basis set for elements in molecules, double-ζ for gold element, cutoff energy of 200 Ry, 0.02 eV/A force tolerance, and 1 × 1 k points in the transverse directions. To compute the electrical conductance of the molecules, they were each placed between pyramidal gold electrodes. For each structure, the transmission coefficient T(E) describing the propagation of electrons of energy E from the left to the right electrode was calculated by GOLLUM59 code based on the resulting Hamiltonian and overlap matrices from SIESTA and the formulae of transport theory:ΓL,R(E) = i−ΣL,R†(E))/2 is the imaginary part of the self-energies ΓL,R(E). ΓL,R determines the width of transmission resonances and self-energies describe the contact between the molecule and left (L) and right (R) electrodes. G is the retarded Green’s function of the molecule in the presence of the electrodes. The conductance is extracted from the transmission spectrum and evaluated by the following formulae:G0 = 2e2/h is the conductance quantum; h is the Planck’s constant; e is the charge of a proton; EF is the Fermi energy. Finally, the room temperature electrical conductance was computed from the formula (2).DFT calculations were conducted using the B3LYP exchange correlation function and implemented in the Gaussian 09 package. The electronic structures were optimized using a general basis set with the Los Alamos effective core potential LANL2DZ basis set for Pt and 6–31 G* for other atomsSupplementary InformationPeer Review File


The influence of the kind of silsesquioxane and of the copolymerization conditions on the reaction performance and on the properties of the copolymers was studied. In the case of copolymerization of E/POSS-6-3, the positive comonomer effect was observed, which was associated with the influence of POSS-6-3 on transformation of the bimetallic ion pair to the active catalytic species. Functionality of silsesquioxanes and polymerization parameters affected the polyhedral oligomeric silsesquioxanes (POSS) contents in the copolymers which varied in the range of 1.33–7.43 wt %. Tri-alkenyl-silsesquioxanes were incorporated into the polymer chain as pendant groups while the tetra-alkenyl-silsesquioxane derivative could act as a cross-linking agent which was proved by the changes in the contents of unsaturated end groups, by the glass transition temperature values, and by the gel contents (up to 81.3% for E/POSS-10-4). Incorporation of multi-alkenyl-POSS into the polymer chain affected also the melting and crystallization behaviors.The copolymers of ethylene (E) with open-caged  One of many advantages of polyhedral oligomeric silsesquioxanes (POSS) is their structural diversity. The POSS compounds have various structures of their silicon–oxygen cores as well as various kinds and numbers of substituents attached to them ,2. The PIn the case of the multi-functional POSS comonomers  the organic–inorganic polymers can be prepared in different ways, depending on the kinds of their reactive groups ,21,22,23The multi-functional POSS derivatives are useful in preparation of (co)polymers with different structural properties. Matějka et al.  reportediso-butyl-substituted tri-alkenyl-silsesquioxanes (POSS-6-3 and POSS-10-3) and phenyl-substituted tetra-alkenyl-silsesquioxane (POSS-10-4), which contained the incompletely condensed silicon–oxygen T4D3 and M4T8 cages, respectively zirconium dichloride catalyst (rac-Et(Ind)2ZrCl2) activated by modified methylaluminoxane (MMAO) was applied in the (co)polymerization processes.The 1H and 13C NMR), Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), and gel permeation chromatography (GPC). The influence of POSS on the catalytic system activated by MMAO was investigated by 1H NMR. Based on the obtained results, the compositions and structural properties of copolymers were comprehensively analyzed and discussed. We also paid attention to thermal properties of copolymers, including their melting and crystallization behaviors.The aim of this work was to study the influence of the kind of POSS as well as of the polymerization conditions, such as concentration of the POSS comonomer in the feed, ethylene pressure, and reaction time, on the performance of ethylene/POSS copolymerization and on the structures and properties of the obtained copolymers. The copolymers were characterized by the following methods: nuclear magnetic resonance (rac-ethylenebis-(1-η5-indenyl)-zirconium dichloride (rac-Et(Ind)2ZrCl2, Sigma-Aldrich, Saint Louis, MO, USA), 1,2-dichlorobenzene-d4 , hydrochloric acid , 2,2′-metylenebis(4-methyl-6-tert-butylfenol) , and methanol  were used as purchased. Toluene-d8  was dried over molecular sieves (4 Å) and degassed in vacuo, and then it was stored and handled in vacuo.Toluene was purchased from Chempur  and it was refluxed over sodium and distilled under nitrogen prior to use. Modified methylaluminoxane , 2O3. 1,2,4-Trichlorobenzene  was purified by distillation.Ethylene  and nitrogen  were used after passing them through a column with the sodium metal supported on Ali-Bu7(Si7O9)(3OH)) and tetrasilanol silsesquioxane (DDSQ-4OH) with respective chlorosilanes. The obtained products made precursors for hydrosilylation. The procedures were based on the general methodology for alkenyl-substituted silsesquioxanes  × 100%.The gel contents of copolymers were determined by solvent extraction, according to the (Polish) standard PN-EN ISO 10147:2013-06P. The copolymer was extracted with 175 cmrac-Et(Ind)2ZrCl2/MMAO catalytic system, the concentration of the POSS comonomer in the reaction feed was varied in the range from 6.67 × 10−4 to 6.67 × 10−3 mol/dm3 and the ethylene pressure was 0.2 or 0.5 MPa. The results of E/POSS copolymerization are presented in In the case of ethylene copolymerization with POSS, catalyzed by the rac-Et(Ind)2ZrCl2/MMAO catalytic systems were less active in copolymerization of ethylene with POSS-10-3 and POSS-10-4 in comparison to ethylene homopolymerization 2ZrCl2 catalyst. The opposite relation was observed in the case of ethylene copolymerization with monoalkenyl(siloxy)- and monoalkenylsilsesquioxane which contained completely condensed T8 cages + Me–MAO− (b) and Cp2ZrMe+ ← Me–Al≡MAO (c) are the major species at high Al/Zr ratios (200–4000 mol/mol) + Me–MAO− complex only [In the case of MAO-activated metallocene, the ion pairs (here (a) ,36,37,38rac-Et(Ind)2ZrCl2 with MMAO, obtained in a wide range of Al/Zr molar ratios (Al/Zr = 50 to 1000 mol/mol), and of appropriate raw materials for reference. As can be seen from rac-Et(Ind)2ZrCl2 by MMAO caused the down-field shift of the Ind-C51H NMR resonance of the ansa-metallocene catalyst, irrespective of the Al/Zr molar ratios used  and 3 (–0.75 ppm) increased with the increasing Al/Zr molar ratio. In turn, the complex 1 gradually disappeared at high Al/Zr molar ratios which was confirmed by the decrease in the intensity of the µ-Me resonance at −1.2 ppm (2 and 3 turned out to be the major species at high Al/Zr ratios (500 and 1000 mol/mol)  [The concentrations of complexes −1.2 ppm b: 3–6. Tmol/mol) b: 6, 7 wctively) ,40,41,42rac-Et(Ind)2ZrMe2/MMAO catalytic system, even at Al/Zr = 50 mol/mol, led to the disappearance of the µ-Me resonance of the complex 1 at –1.2 ppm (1 (inactive form) into 2 or 3 complexes (precursors of the active form). However, the observed metallocene species 2 and 3 were dominant in the catalytic system in this case, even at the low Al/Zr ratio of 50 mol/mol, which is unusual for this group of catalysts [It should be stressed, however, that to the best of our knowledge, the complete ansa-metallocene/MMAO/silsesquioxane system was never studied before by NMR. Meanwhile, it turned out in our research that the addition of the POSS-6-3 comonomer to the –1.2 ppm  b: 7. Thatalysts ,40,41,421. Moreover, POSS-6-3 broke the methyl bridge in complex 1 and caused the active site to be readily accessible to MMAO.Thus, the described results indicated that the POSS-6-3 compound acted not only as a comonomer but it also competed with MMAO for a bimetallic ion pair rac-Et(Ind)2ZrMe2/MMAO catalytic system was further investigated for the adopted Al/Zr molar ratio of 50 mol/mol. The POSS comonomers with longer alkenyl substituents were found not to affect the transformation of complex 1 into complex 2 or 3, which was confirmed by the presence of the µ-Me resonance at –1.2 ppm in 1H NMR  and the structures of copolymers were characterized by the 1H and 13C NMR (The contents of POSS units incorporated into the copolymer chain (C 13C NMR  and FT-I 13C NMR  methods.1H NMR spectrum (iso-butyl non-reactive substituent of the POSS units. A part of the (CH2)x spacer of POSS leads to the signals in the same region of the spectrum as the polyethylene chain at about 1.32 ppm. The peaks assigned to chain unsaturation in copolymers occurred in the olefinic region (5.85–4.55 ppm). In the case of E/POSS copolymers, the 13C NMR spectrum (1 in the spacer), 18.75 ppm (C2 in the spacer), 29.98 ppm (C3 in the spacer), and 34.64 ppm (C4 in the spacer) for a part of the (CH2)x spacer that were unchanged after polymerization and similar to the peaks for the iso-butyl group at 24.16, 26.07, and 27.35 ppm. The new peaks at 34.64 (RCH2–CH) and 36.21 ppm (R3CH) appeared due to the incorporation of POSS into the polymer chain.The signal pattern in the spectrum a is in aspectrum  b showed−1 in the FT-IR spectra of neat POSS comonomers  during polymerization (The copolymers with higher contents of the POSS comonomers were obtained under lower ethylene pressure (pe = 0.2 MPa were characterized by the highest contents of the POSS units (up to 7.43 wt %) (The structures of the POSS comonomers strongly influenced POSS incorporation into the copolymer chain and the copolymers with POSS-10-4 obtained under p43 wt %) , item 16CDDSQ = 11.53 wt %) [Despite the presence of three and four alkenyl substituents in the POSS comonomers, the incorporation degree of the POSS comonomer into the polymer chain was unexpectedly found lower in comparison with the DDSQ comonomer under the same reaction conditions (53 wt %) . In the e values were equal to 162 and 182 for copolymerization of E/POSS-6-3 and E/POSS-10-3, respectively. The resulting lower reactivity of the POSS-10-3 comonomer  could be explained by higher steric hindrance of the longer reactive group. Noteworthy is that the re values in ethylene/tri-alkenyl-POSS copolymerization were comparable to the re values for copolymerization with monoalkenyl(siloxy)- and monoalkenylsilsesquioxanes [e) and monomer dispersity (MD) were also determined - or monoalkenylsilsesquioxanes [e values for E/POSS-10-4 copolymers were comparable to those values for E/DDSQ copolymers [The lowest values of nnditions . It shoupolymers .The monomer dispersity (MD) values were equal to about 100 irrespective of the kind of the POSS comonomer  which suMw) of the E/tri-alkenyl-POSS copolymers synthesized by rac-Et(Ind)2ZlCl2 were much higher in comparison to neat polyethylene, which suggested that chain termination by transfer to the monomer was significantly limited  or by the influence of the POSS comonomer on the formation of the ion pair, which resulted in many “open” active sites of the catalysts for the monomer insertion.Moreover, the ure data ,4,5,6, sMw/Mn = 3.9–4.9) polymers as described above were characterized by the unimodal but relatively broad molecular weight distributions (3.9–4.9) , Table 11H NMR and FT-IR spectra for the E/POSS copolymers revealed the presence of only vinyl and tri-substituted vinylene groups , cyclic units (type II), and copolymers with cross-linked structures (type III) analogously to previously investigated ethylene/DDSQ copolymers .−1 associated with the cyclic structure in the main chain is not observed [In the case of ethylene copolymerization with the POSS comonomers which contain the reactive substituents located close to each other, the formation of cyclic units in the polymer chain could be expected , type IIobserved . NoteworIt is suggested that tetra-alkenyl-POSS acted as a cross-linking agent  increased with the increasing content of POSS in the copolymer. It should be noted that the gel content for tetra-alkenyl-POSS-containing copolymers was higher (from 53.0% to 81.3%) in comparison to copolymers with the DDSQ comonomer (up to 65.0%)  despite Tg values significantly with the increasing POSS content (The incorporation of the POSS-10-4 comonomers into the polymer chain increased the  content  which waTg) of the ethylene copolymers with tri-alkenyl-POSS slightly decreased with the increasing contents of POSS in the copolymer  and peak melting temperature (Tmp), regardless of the kind of the POSS comonomer  and phenyl-substituted tetra-alkenyl-silsesquioxane (POSS-10-4) were for the first time successfully synthesized by coordinative copolymerization with the use of the metallocene catalytic system (rac-Et(Ind)2ZrCl2/MMAO).The hybrid copolymers of ethylene with open-caged copolymer/molZr × 0.5 h) was noted. Moreover, the positive comonomer effect was observed for the first time in the ethylene/POSS copolymerization.The performance of copolymerization was found to be significantly affected by the kind and concentration of the silsesquioxane comonomer in the feed as well as by the ethylene pressure and reaction time. The steric hindrance, inductive effect, and interactions between the open silicon–oxygen cage of the POSS comonomers and the active center of the metallocene catalyst significantly influenced the efficiency of polymerization. In the case of E/POSS-6-3 copolymerization, a very high catalyst activity  at the very low Al/Zr ratio (50) which is atypical for the metallocene catalysts.Based on the The E/POSS copolymers varied in the POSS contents, reaching up to 7.43 wt % in the case of the E/POSS-10-4 copolymer. Incorporation of the POSS comonomers into the polymer chains resulted in the increase in the molecular weights which indicated the limitation of chain termination by transfer to the monomer and suggested the modifying influence of the POSS comonomer on the metallocene active sites. Depending on the functionality of the POSS comonomer, copolymers varied in the structural architecture. Tri-alkenyl-silsesquioxanes were incorporated into the polymer chains as pendant groups. In the case of copolymers with tetra-alkenyl-silsesquioxane, the cross-linked structure was demonstrated and the gel content reached even 81.3%. These structural differences were proved by the changes in the contents of unsaturated end groups and by the glass transition temperature values. The copolymers of ethylene with multi-alkenyl-POSS were also characterized by interesting melting and crystallization behavior.


Antibiotic overuse is the most important modifiable factor contributing to antibiotic resistance. We conducted an educational campaign in Minya, Egypt targeting prescribers and the public through communications focused on appropriate antibiotic use for acute respiratory infections (ARIs).The entire population of Minya was targeted by the campaign. Physicians and pharmacists were invited to participate in the pre-intervention assessments. Acute care hospitals and a sample of primary healthcare centers in Minya were randomly selected for a pre-intervention survey and all patients exiting outpatient clinics on the day of the survey were invited to participate. The same survey methodology was conducted for the post-intervention assessments. Descriptive comparisons were made through three assessments conducted pre- and post-intervention. We quantitated antibiotic prescribing through a survey administered to patients with an ARI exiting outpatient clinics. Additionally, physicians, pharmacists, and patients were interviewed regarding their attitudes and beliefs towards antibiotic prescribing. Finally, physicians were tested on three clinical scenarios  to measure their knowledge on antibiotic use.Post-intervention patient exit surveys revealed a 23.1% decrease in antibiotic prescribing for ARIs in this population (83.7 to 64.4%) and physicians and pharmacists self-reported less frequently prescribing antibiotics for ARIs on their follow-up surveys. We also found an increase in correct responses to the clinical scenarios and in attitude and belief scores for physicians, pharmacists, and patients regarding antibiotic use in the post-intervention sample.Overall, the samples surveyed after the community-based educational campaign reported a lower frequency of antibiotic prescribing and improved knowledge and attitudes regarding antibiotic misuse compared to the samples surveyed before the campaign. Ongoing interventions educating providers and patients are needed to decrease antibiotic misuse and reduce the spread of antibiotic resistance in Egypt. Antibiotic resistance is a growing public health threat worldwide and a major threat to global health security. Increasing antibiotic resistance is a consequence of selective pressures created by use of antibiotics. Respiratory infections are the leading reason for antibiotic prescriptions in both the adult and pediatric populations. Overuse of antibiotics, such as taking antibiotics when they aren’t needed for viral infections, is a driver of antibiotic resistance, which is a serious problem for public health, individual patients, and healthcare systems. Antibiotics are most frequently prescribed for management of acute respiratory tract infections (ARIs), including rhinosinusitis, pharyngitis, bronchitis, otitis media, and nonspecific ARIs . PublishNumerous interventions to improve antibiotic prescribing practices have been reported from various countries with varying results –5. No siRecent studies in Egypt have shown that antibiotics are used extensively for treatment of ARIs , 12. TheBaseline surveys were conducted in Minya District in Upper Egypt from May–July 2011 at four acute care hospitals and randomly selected 41 primary healthcare units (PHU) (representing 50% of PHUs in Minya). All patients that presented to these facilities during the day of the visit were invited to participate in the survey. Patients with an ARI were interviewed as they were exiting outpatient clinics , or general outpatient clinics) to estimate the frequency of antibiotic prescribing for ARIs. A post-intervention survey was repeated using the same sampling strategy and number of health facilities, from October–December, 2012. Interviews were conducted with adult patients (age 18 years or older) or with a parent or caretaker of pediatric patients (less than 18 years of age). The detailed methods and results of the baseline patient exit survey are published elsewhere [This survey targeted physicians and pharmacists to measure their attitudes and practices related to treating patients with ARIs . All phyIn addition, physicians were presented three clinical scenarios that did not warrant antibiotic therapy , and asked how they would treat these patients. The knowledge of the physicians regarding treatment choices in response to these ARIs was measured.The same data collection tool used to measure physician and pharmacist beliefs and attitudes about antibiotic use and resistance was used for patients who participated in the patient exit interviews in the pre- and post-intervention periods. The same scoring procedures were also used to measure the beliefs and attitudes before and after the intervention.Sample size calculations were estimated in preparation for the pre-intervention surveys assuming that physicians prescribed antibiotics for 60% of ARI visits and that antibiotic prescribing for ARIs would be reduced by 20% post-intervention, with a significance level of 95% and a power of 90%. The details of the sample size calculations are described elsewhere .An intensive campaign to promote appropriate antibiotic prescription was launched in Minya District from August to December 2011. The aim of the campaign was to raise the awareness of physicians, pharmacists, and the general public in the district regarding the importance of rational antibiotic prescribing for ARIs. The primary audience included all physician specialties that might be involved with treatment of patients with ARIs , either in the government or private sector. In addition, pharmacists and their assistants in the public and private sector were also targeted. The campaign targeted all physicians, pharmacists, and the general public in Minya. Random samples of patients from the Minya District were interviewed pre- and post-intervention and physicians and pharmacists were invited to participate in the pre- and post-intervention surveys as described above in order to describe the reach of the campaign throughout the entire district.A social media campaign targeting educated youth was implemented using Facebook and the YouTube channel. A page named after the campaign slogan “Be wise in using antibiotics” was added on Facebook and YouTube. Three moderators were leading a discussion on appropriate antibiotic use in Arabic. Electronic copies of the informational, educational, and communication materials were uploaded on the Facebook page. Videos produced by CDC’s Get Smart campaign were translated and subtitles were added in Arabic after obtaining CDC approval. These videos were uploaded to the YouTube channel. Animated videos were also produced to act as viral videos to bring users to the campaign pages.Multiple focus group discussions were conducted with physicians, pharmacists, non-governmental organizations, and the general public in order to define the key messages and potential communication channels. The message themes for physicians and pharmacists were mainly to improve knowledge regarding appropriate antibiotic prescribing. Specific messages were focused on avoiding antibiotic prescribing for viral ARIs.Informational, educational, and communication materials were developed to target three groups . For physicians and pharmacists, Centers for Disease Control and Prevention fact sheets on ARI management guidelines and algorithms were revised and updated by an international consultant and printed with different color codes for each type of respiratory infection . A notebook was designed and printed including the fact sheets as page separators. Reminder messages were printed on 12-page desktop calendars including the principles of antibiotic prescriptions for ARIs. To improve awareness on antibiotic resistance, a promotion folder was prepared which delivered information on the burden of antibiotic resistance and its consequences. For the general public, posters communicating specific simple messages to decrease their demand for antibiotics were displayed in doctor clinic waiting areas, pharmacies, community gathering areas, universities, and schools.A 5-day training course by an international infectious disease consultant was held to educate participants on appropriate management of ARIs. The workshop targeted all clinical specialties and primary health care doctors of Minya District that treat ARIs . Educational sessions were also provided to pharmacists and focused mainly on the difference between bacterial and viral infections, and the contribution of antibiotic overuse to the development of antibiotic resistance. A total of 315 physicians and pharmacists attended the training workshops, which represented 70% of physicians and pharmacists employed in the government sector. To ensure district-wide coverage of scientific information, three senior medical doctors and 13 pharmacists that attended the training by the international consultant and showed special interest and skills were recruited to conduct academic detailing and visit and educate private physicians and pharmacists that did not attend the training. They used the educational materials to disseminate specific campaign messages to doctors and pharmacists.Percent change was calculated to descriptively compare antibiotic prescribing for pediatric and adult patients before and after the intervention. To describe the attitudes and beliefs of the patients towards antibiotic use, a scoring system described previously by Taylor et al., 2003  was usedTable The mean knowledge and attitude scores supporting the judicious use of antibiotics improved after comparing responses pre- and post-intervention for both physicians and pharmacists (from 3.8 ± 0.5 to 4.0 ± 0.7 for physicians and from 3.3 ± 0.9 to 4.0 ± 1.2 for pharmacists). .  in the mean attitude scores for both groups of patients (parents of pediatrics and adults) between the pre-and post-intervention periods Table . Both paFollowing an educational intervention, including a public education campaign, provider education, and academic detailing, to improve antibiotic use for ARIs in Minya District, Egypt there were reported improvements in antibiotic prescribing and knowledge. The results of the present study are consistent with evidence from similar settings including two previous studies in hospitals in Thailand that found overall improved antibiotic use and reduced incidence of inappropriate use following educational campaigns , 16. BasThe follow-up surveys of patients leaving outpatient visits for ARIs in Minya demonstrated a decrease in antibiotic prescribing after the educational intervention. Comparing survey results before and after the intervention, prescribing decreased in both pediatric and adult visits for ARI symptoms. Although this is the first study of its kind in Egypt, similar studies assessing antibiotic use interventions have been conducted in other countries. Two studies in the United States implementing educational interventions to address pediatric prescribing led to intervention-attributable declines in antibiotic prescription rates between 11 and 16% , 10. AnoThe current intervention also demonstrated improved knowledge and attitudes regarding appropriate antibiotic use among physicians and pharmacists surveyed following the intervention compared to pre-intervention. A similar study in the United States found that after a statewide educational campaign, desired responses about antibiotic use in case scenarios for upper respiratory infection and bronchitis increased among clinicians . AdditioBased on data from this pilot, the health authorities in Minya District developed policies for appropriate antibiotic use for ARIs that were endorsed by local physicians, and included distribution of the policies and regular auditing and review of physician prescriptions. Additionally, the Ministry of Health and Population in Egypt is exploring feasible methods and approaches to expand the communication campaign in phases to more regions in Egypt using the model applied in Minya and the available information, education, and communication materials.This study is subject to several limitations. First, the intervention was restricted to a single district in Egypt; therefore, the results may not be generalizable outside Minya. Additionally, the baseline surveys were conducted between May and July while the post-intervention surveys were conducted between October and December. Therefore, we cannot control for the temporal and seasonal effects on prescribing frequency and the influence of the survey timing on the pre- and post-intervention assessments. Similarly, although the same survey tool was used in the baseline and post-intervention surveys, the individual patients, pharmacists, and physicians surveyed in these two time periods differed due to the sampling approach, which introduces individual variability between the two populations. Therefore, we could conduct only descriptive comparisons and did not apply statistical methods to analyze the pre- and post-intervention results. Thus, we could not determine if the differences were due to the campaign alone or the influence of other confounding factors. However, since the campaign targeted all physicians and pharmacists in Minya, physicians and pharmacists were invited to participate in the pre- and post-intervention surveys and random samples of all healthcare clinics in Minya were taken for the patient surveys before and after the intervention. This approach allowed us to describe the pre- and post-intervention prescribing frequency and knowledge of antibiotic use throughout Minya. While the campaign targeted the Minya population broadly, we could not confirm that every individual was exposed to the intervention materials, a common limitation of public education campaigns. Finally, there is potential for social desirability bias in the self-reporting of prescribing practices by physicians and pharmacists as well as in responses to the beliefs and attitudes surveys.An educational intervention was implemented in Minya District, including a communication campaign, training of physicians and pharmacists, and provider outreach. Following the campaign, antibiotic prescribing for ARIs decreased and knowledge and attitude scores regarding appropriate use among physicians, pharmacists, and patients improved compared to the surveys conducted pre-intervention. Similar interventions may be effective in comparable settings where baseline antibiotic prescribing frequency is high and where patients are able to obtain antibiotics without prescriptions. Such interventions, resulting in reduced antibiotic prescribing, are needed to reduce selective pressures that are contributing to increasing antibiotic resistance worldwide and threatening global health security. The Ministry of Health and Population in Egypt is considering expansion of the campaign to more regions in Egypt to promote appropriate antibiotic use for ARIs using the Minya model as the basis for implementation.


Background: Increased death due to the opioid epidemic in the United States has necessitated the development of new strategies to treat addiction. Monoclonal antibodies and antidrug vaccines provide a tool that both aids addiction management and reduces the potential for overdose. Dual drug vaccines formulated by successive conjugation or by mixture have certain drawbacks. The current study examines an approach for combatting the dangers of fentanyl-laced heroin, by using a hapten with one epitope that has domains for both fentanyl and heroin.Results: We evaluated a series of nine vaccines developed from chemically contiguous haptens composed of both heroin- and fentanyl-like domains. Analysis of the results obtained by SPR and ELISA revealed trends in antibody affinity and titers for heroin and fentanyl based on epitope size and linker location. In antinociception studies, the best performing vaccines offered comparable protection against heroin as our benchmark heroin vaccine, but exhibited attenuated protection against fentanyl compared to our fentanyl vaccine.Conclusion: After thorough investigation of this strategy, we have identified key considerations for the development of a chemically contiguous heroin–fentanyl vaccine. Importantly, this is the first report of such a strategy in the opioid–drug–vaccine field. In 2016, the Centers for Disease Control (CDC) estimated that approximately 20.4% of the U.S. population  was suffering from chronic pain [®), a competitive antagonist of µ-opioid receptors. Other treatments for opioid substance abuse focus on mitigating cravings and managing long-term addiction. However, these treatments have an inherent risk for continued abuse. Combatting the complex nature of addiction requires a multifaceted strategy – several elements of the opioid crisis that need to be addressed include, but are not limited to, new non-addictive opioid drugs, innovative treatments to prevent relapse, and preventing and/or reversing overdose.Respiratory depression is a common side effect of opioid overdose and can lead to serious complications, including death. The current treatment for overdose is naloxone  mixing two individual vaccines together (admixture vaccine) –10; or 2As a starting point for our exploration of chemically contiguous drug–hapten conjugates, we envisioned generating a series of structurally diverse heroin–fentanyl haptens with a focus on linker placement and bond distance between each drug’s chemical connections in order to probe the effect of the drug-epitope structure on eliciting an immune response. The second consideration in our chemically contiguous hapten design was the location of the linker to the carrier protein. We chose three arrangements: 1) extending the linker off the phenylamide ring of fentanyl; 2) extending the linker from the phenethyl ring of fentanyl; or 3) anchoring the linker in between the two drug-like portions of our singular epitope. Again, HF-3, HF-5, and HF-8 could share an intermediate, while the fentanyl-like domains of HF-1, HF-2, HF-3, and HF-7  was synthesized according to literature procedure and purified by recrystallization [1 with tert-butyl bromoacetate and subsequent deprotection with trifluoroacetic acid yielded the N-glycine derivative of norheroin, 16. This critical intermediate was used to construct HF-3, HF-5, and HF-8 butanoic acid was accessed via protection and deprotection of the amine with a phthaloyl group . Reductive amination of 4 with commercially available phenethylpiperidin-4-one furnished intermediate 5 in good yield  hydrate followed by sodium periodate to obtain aldehyde 7 butanoic acid, an alkene was installed with a Suzuki reaction, which in turn underwent ozonolysis to give 21, shown in 21 to give 24, completing the fentanyl-like domain . However, increasing the conjugation to a 1 mg scale caused the protein to precipitate immediately upon addition of HF-1 in 9:1 DMF/H2O solution, eliminating the possibility of conjugation. To mitigate this precipitation problem, glycerol was added to the soluble KLH or BSA solution  were performed with activated HF-4, had solubility issues upon addition to carrier protein and therefore needed higher glycerol content to ameliorate protein precipitation and conjugation issues. The linker placement at the phenethyl position greatly improved conjugation compared to the other phenylamide group; HF-4 had a hapten density of 21.1, drastically higher than HF-2 (hapten density: 12.6) and HF-3 (hapten density: 8.3). Gratifyingly, we observed copy numbers on BSA for every synthesized hapten. Immunoconjugates were then prepared with keyhole limpet hemocyanin (KLH) for murine immunization studies.Another trend observed for the chemically contiguous haptens was the increased amount of time needed to activate the carboxylic acids. Typically, fentanyl and heroin haptens reach optimal threshold activation (above 80%) within thirty minutes to several hours. However, these haptens required a minimum of four to twelve hours for at least 50% activation. In addition, bulkier haptens, including The nine haptens were formulated with Alhydrogel  and murine CpG ODN as previously described ,17. BrieFemale BALB/cByJ mice were immunized and bled using the protocol detailed in our previous study, except this study incorporated an extra boost at week 7, bleed at week 8, and antinociception at week 9 . The vac50 affinity continued to improve up to week 8. Individual conjugate vaccines had the highest level of midpoint titers of all groups as well as the best affinity values  had affinity only toward one of the two drugs, not both as initially anticipated. Also of note was that, while HF-4 and HF-6 had excellent affinity toward 6-AM, this did not translate to antibody-fentanyl affinity, which was 40 and 70-fold poorer. As expected this poor affinity to fentanyl was recapitulated as a nonstarter in behavior as seen in the tail flick antinociception assay . Week 8 titers show a slight correlation, although it is was not found to be significant. Moreover, nearly all of the vaccines produced very low titers and affinity to fentanyl compared to fentanyl-KLH vaccine (Figures S8 and S9, We also sought to determine if there was any predictive correlation between the in vitro SPR and ELISA results, and the behavioral results. To this end, nociception EDt titers , we saw HF-7 conjugate vaccine did show promising results as its heroin response was on par with Her-hapten immune response and it provided some efficacy against the fentanyl in heroin mixture. As linker-carrier protein regiochemical placement seemed more controlling for a balanced immune response against both drugs, it will be of interest to explore linker-carrier-protein display off the 3 or 6-position of heroin, thereby more prominently displaying the fentanyl moiety. These studies will be reported in due course.Our chemically contiguous hapten campaign was initiated to investigate the possibility of taking two structurally different opioids, heroin and fentanyl, and combining them into a singular, unique opioid hapten that, when formulated with an adjuvant, would present us with a vaccine capable of generating antibodies to both drugs. Based on this idea, a series of nine vaccines were prepared that enabled us to detect subtle influences on antibody response to each drug. In sum, we found little traction with each hapten’s vaccine efficacy from the standpoint of the chemical connection between the two drugs. Interestingly, immune response seemed to be more influenced by linker-carrier protein regiochemical placement. Although none of the nine haptens outperformed the individual conjugate vaccines against their assigned drug, the 1H NMR, 13C NMR, and high-resolution mass-spectrometry signals.Supporting information features SPR and ELISA experimental results, and additional experimental data. It also includes detailed procedures for all reported intermediates and final compounds, as well as their File 1Supporting experimental results.File 21–53.Experimental procedures for compounds File 31–53.NMR spectra of compounds