Β-lactam Compounds Research Articles

Dibenzoxanthene-β-lactam hybrids as potential antioxidant and anticancer agents: Synthesis, biological evaluation, and docking study

In this study, we designed and synthesized a series of β-lactam compounds bearing 14-phenyl-14H-dibenzo[a,j]xanthene moiety attached to the N-1 position of the β-lactam ring through a [2 + 2] cycloaddition process. All the synthesized compounds have been characterized by elemental analysis, Fourier-transform infrared (FT-IR) and nuclear magnetic resonance (1H NMR and 13C NMR) spectral analysis. These derivatives were evaluated for their anticancer and antioxidant activities. The in vitro anticancer activity of new compounds was examined against MCF-7 (breast cancer), HepG2 (liver hepatocellular carcinoma), and TC-1 (mouse lung epithelial) cell lines by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Notably, compounds 4c, 4e and 4 g demonstrated significant anticancer activity against TC-1 cell line. Additionally, compounds 4c, 4f and 4 g exhibited promising levels of efficacy against the MCF-7 cell line. The diphenylpicrylhydrazyl (DPPH) radical scavenging assay indicated that 4h and 4i are more potent antioxidant agents than the Vitamin C, while 4a, 4b, 4c, and 4j exhibited moderate antioxidant capabilities. Furthermore, the docking simulations are consistent with the biological assay results, indicating that the hybrids may bind to protein targets through hydrogen bonding and other nonvalent interactions.

Synthesis of novel isoxazole/dihydroisoxazole tethered β-lactam hybrids via regiospecific 1,3-dipolar cycloaddition methodology on 3-phenylthio-β-lactams

Tethering of two biologically active molecules in a single unit is considered as a captivating solution for the extension of existing antibiotics. β-Lactams which display a vast array of biological properties due to various ring substitutions continue to attract the attention of synthetic chemists. The incorporation of the ‘isoxazole’ nucleus in its appendage is known to enhance the properties of β-lactam compounds. Its inclusion in the compounds offers decreased toxicity, increased efficacy, and improved pharmacokinetics spectrum. In the reported work, we highlight the efficient use of 1,3-dipolar cycloaddition reaction to synthesize novel isoxazole/dihydroisoxazole ring-linked β-lactam hybrids using 3-propargyloxy-β-lactams/3-allyl-β-lactams as synthons. The methodology devised has been found to be highly regioselective and generalized for the selected substrates to provide a variety of novel hybrid compounds under elementary reaction conditions. The synthesized compounds have been well characterized by various spectroscopic techniques and optimized reaction conditions for both types of hybrid compounds have been studied.

Resistance to ceftazidime-avibactam and other new β-lactams in Pseudomonas aeruginosa clinical isolates: a multi-center surveillance study.

Multidrug-resistant Pseudomonas aeruginosa infections are a growing threat due to the limited therapeutic options available. Ceftazidime-avibactam (CZA) is among the last-resort antibiotics for the treatment of difficult-to-treat P. aeruginosa infections, although resistance due to the acquisition of transferable β-lactamase genes is increasing. With this work, we report that CZA represents a highly active antipseudomonal β-lactam compound (after cefiderocol), and that metallo-β-lactamases (VIM-type) and extended-spectrum β-lactamases (GES and PER-type) production is the major factor underlying CZA resistance in isolates from Southern Italian hospitals. In addition, we reported that such resistance mechanisms were mainly carried by the international high-risk clones ST111 and ST235.

Modelling lethality and teratogenicity of zebrafish (Danio rerio) due to β-lactam antibiotics employing the QSTR approach

ABSTRACT Nowadays, β-lactam antibiotics are one of the most consumed OTC (over-the-counter) medicines in the world. Its frequent use against several infectious diseases leads to the development of antibiotic resistance. Another unavoidable risk factor of β-lactam antibiotics is environmental toxicity. Numerous terrestrial as well as aquatic species have suffered due to the excessive use of these pharmaceuticals. In this present study, we have performed a toxicity assessment employing a novel in silico technique like quantitative structure–toxicity relationships (QSTRs) to explore toxicity against zebrafish (Danio rerio). We have developed single as well as inter-endpoint QSTR models for the β-lactam compounds to explore important structural attributes responsible for their toxicity, employing median lethal (LC50) and median teratogenic concentration (TC50) as the endpoints. We have shown how an inter-endpoint model can extrapolate unavailable endpoint values with the help of other available endpoint values. To verify the models’ robustness, predictivity, and goodness-of-fit, several universally popular metrics for both internal and external validation were extensively employed in model validation (single endpoint models: r 2 = 0.631 − 0.75, Q 2 F1 = 0.607 − 0.684; inter-endpoint models: r 2 = 0.768 − 0.84, Q 2 F1 = 0.678 − 0.76). Again, these models were engaged in the prediction of these two responses for a true external set of β-lactam molecules without response values to prove the reproducibility of these models.

A Decade-Long Evaluation of Neonatal Septicaemic Escherichia coli: Clonal Lineages, Genomes, and New Delhi Metallo-Beta-Lactamase Variants.

Longitudinal studies of extraintestinal pathogenic Escherichia coli (ExPEC) and epidemic clones of E. coli in association with New Delhi metallo-β-lactamase (blaNDM) in septicaemic neonates are rare. This study captured the diversity of 80 E. coli isolates collected from septicaemic neonates in terms of antibiotic susceptibility, resistome, phylogroups, sequence types (ST), virulome, plasmids, and integron types over a decade (2009 to 2019). Most of the isolates were multidrug-resistant and, 44% of them were carbapenem-resistant, primarily due to blaNDM. NDM-1 was the sole NDM-variant present in conjugative IncFIA/FIB/FII replicons until 2013, and it was subsequently replaced by other variants, such as NDM-5/-7 found in IncX3/FII. A core genome analysis for blaNDM+ve isolates showed the heterogeneity of the isolates. Fifty percent of the infections were caused by isolates of phylogroups B2 (34%), D (11.25%), and F (4%), whereas the other half were caused by phylogroups A (25%), B1 (11.25%), and C (14%). The isolates were further distributed in approximately 20 clonal complexes (STC), including five epidemic clones (ST131, ST167, ST410, ST648, and ST405). ST167 and ST131 (subclade H30Rx) were dominant, with most of the ST167 being blaNDM+ve and blaCTX-M-15+ve. In contrast, the majority of ST131 isolates were blaNDM-ve but blaCTX-M-15+ve, and they possessed more virulence determinants than did ST167. A single nucleotide polymorphism (SNP)-based comparative genome analysis of epidemic clones ST167 and ST131 in a global context revealed that the study isolates were present in close proximity but were distant from global isolates. The presence of antibiotic-resistant epidemic clones causing sepsis calls for a modification of the recommended antibiotics with which to treat neonatal sepsis. IMPORTANCE Multidrug-resistant and virulent ExPEC causing sepsis in neonates is a challenge to neonatal health. The presence of enzymes, such as carbapenemases (blaNDM) that hydrolyze most β-lactam antibiotic compounds, result in difficulties when treating neonates. The characterization of ExPECs collected over 10 years showed that 44% of ExPECs were carbapenem-resistant, possessing transmissible blaNDM genes. The isolates belonged to different phylogroups that are considered to be either commensals or virulent. The isolates were distributed in around 20 clonal complexes (STC), including two predominant epidemic clones (ST131 and ST167). ST167 possessed few virulence determinants but was blaNDM+ve. In contrast, ST131 harbored several virulence determinants but was blaNDM-ve. A comparison of the genomes of these epidemic clones in a global context revealed that the study isolates were present in close proximity but were distant from global isolates. The presence of epidemic clones in a vulnerable population with contrasting characteristics and the presence of resistance genes call for strict vigilance.

Preparation, Identification and Evaluation of Biological Activity of some new β-Lactam compounds derived from Schiff bases

A series of four- membered rings has been synthesized from Dapsone drug. In the first step examination Dapsone has been taken as initial material and treated with 4-aminoacetophenone to prepare new Azo compound. then Azo compound reacts with various aromatic amines in absolute ethanol to prepare Schiff bases derivatives. In the second step these derivatives react with Chloroacetylchloride in the presence of tri ethyl amine(Et3N) to prepare new four- membered heterocyclic compounds . All these derivatives were tested against different bacteria(Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus anthracis). The high efficacy results of these derivatives proved significantly greater than the efficacy of the same Dapsone drug.These compounds were identified and confirmed by FT- IR, 1HNMRand 13C-NMR.

The crystal structure of the H116Q mutant of NDM-1: An enzyme devoid of zinc ions

New Delhi metallo-β-lactamase 1 (NDM-1) is an important causative factor of antimicrobial resistance due to its efficient hydrolysis of a broad range of β-lactam compounds. The two zinc ions at the active site play essential roles in the NDM-1 catalytic activities. In a previous work, H116, one of the three ligands at the Zn1 site, was mutated in order to investigate the nature of zinc ion chelation. We report here the crystal structure of the NDM-1 H116Q mutant, that was designed to convert a B1 di-zinc enzyme into a B3 type, which either still binds two zinc ions or binds only one at the Zn2 site. The effect of mutation on the overall structure is minimal. Unexpectedly, no zinc ion was observed in the crystal structure. The Zn2-site ligating residue C221 forms a covalent bond with the nearby K121, a residue important in maintaining the active-site structure. The largest conformational changes were found at main-chain and side-chain atoms at residues 232–236 (loop 10), the proper configuration of which is known to be essential for substrate binding. The catalytic-site mutation caused little local changes, yet the effects were amplified and propagated to the substrate binding residues. There were big changes in the ψ angles of residues G232 and L234, which resulted in the side chain of N233 being displaced away from the substrate-binding site. In summary, we failed in turning a B1 enzyme into a B3 enzyme, yet we produced a zinc-less NDM-1 with residual activities.

Synthesis, Characterisation and Mechanism of Action of Anticancer 3-Fluoroazetidin-2-ones.

The stilbene combretastatin A-4 (CA-4) is a potent microtubule-disrupting agent interacting at the colchicine-binding site of tubulin. In the present work, the synthesis, characterisation and mechanism of action of a series of 3-fluoro and 3,3-difluoro substituted β-lactams as analogues of the tubulin-targeting agent CA-4 are described. The synthesis was achieved by a convenient microwave-assisted Reformatsky reaction and is the first report of 3-fluoro and 3,3-difluoro β-lactams as CA-4 analogues. The β-lactam compounds 3-fluoro-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxy phenyl)azetidin-2-one 32 and 3-fluoro-4-(3-fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one) 33 exhibited potent activity in MCF-7 human breast cancer cells with IC50 values of 0.075 µM and 0.095 µM, respectively, and demonstrated low toxicity in non-cancerous cells. Compound 32 also demonstrated significant antiproliferative activity at nanomolar concentrations in the triple-negative breast cancer cell line Hs578T (IC50 0.033 μM), together with potency in the invasive isogenic subclone Hs578Ts(i)8 (IC50 = 0.065 μM), while 33 was also effective in MDA-MB-231 cells (IC50 0.620 μM). Mechanistic studies demonstrated that 33 inhibited tubulin polymerisation, induced apoptosis in MCF-7 cells, and induced a downregulation in the expression of anti-apoptotic Bcl2 and survivin with corresponding upregulation in the expression of pro-apoptotic Bax. In silico studies indicated the interaction of the compounds with the colchicine-binding site, demonstrating the potential for further developing novel cancer therapeutics as microtubule-targeting agents.

Covalent docking and molecular dynamics simulations reveal the specificity-shifting mutations Ala237Arg and Ala237Lys in TEM beta-lactamase.

The rate of modern drug discovery using experimental screening methods still lags behind the rate at which pathogens mutate, underscoring the need for fast and accurate predictive simulations of protein evolution. Multidrug-resistant bacteria evade our defenses by expressing a series of proteins, the most famous of which is the 29-kilodalton enzyme, TEM β-lactamase. Considering these challenges, we applied a covalent docking heuristic to measure the effects of all possible alanine 237 substitutions in TEM due to this codon's importance for catalysis and effects on the binding affinities of commercially-available β-lactam compounds. In addition to the usual mutations that reduce substrate binding due to steric hindrance, we identified two distinctive specificity-shifting TEM mutations, Ala237Arg and Ala237Lys, and their respective modes of action. Notably, we discovered and verified through minimum inhibitory concentration assays that, while these mutations and their bulkier side chains lead to steric clashes that curtail ampicillin binding, these same groups foster salt bridges with the negatively-charged side-chain of the cephalosporin cefixime, widely used in the clinic to treat multi-resistant bacterial infections. To measure the stability of these unexpected interactions, we used molecular dynamics simulations and found the binding modes to be stable despite the application of biasing forces. Finally, we found that both TEM mutants also bind strongly to other drugs containing negatively-charged R-groups, such as carumonam and ceftibuten. As with cefixime, this increased binding affinity stems from a salt bridge between the compounds' negative moieties and the positively-charged side chain of the arginine or lysine, suggesting a shared mechanism. In addition to reaffirming the power of using simulations as molecular microscopes, our results can guide the rational design of next-generation β-lactam antibiotics and bring the community closer to retaking the lead against the recurrent threat of multidrug-resistant pathogens.

Genotoxicity evaluation of a new phthalazine substituted β-lactam derivative in human lymphocytes.

The aim of present study, to evaluate the genotoxic potential of 1-(4-(3,3-dimethyl-1,6-dioxo-2,3,4,6,11,13-hexahydro-1H-indazolo[1,2b] phthalazine-13yl)phenyl)-2-phenylazetidine-3-yl-acetate which was synthesised assuming that it may be a pharmaceutical raw material and found to inhibit human carbonic anhydrase I, II isozymes. To determine the genotoxic potential of this phthalazine substituted β-lactam compound, chromosomal aberration (CA) and micronucleus (MN) tests were implemented in human peripheral blood lymphocytes. In these tests, lymphocyte cultures were treated with four concentrations (30, 15, 7.5, 3.75 μg/mL) of test compound and simultaneously with negative control (sterile distilled water), solvent control (DMSO) positive control (MMC). According to our results, CA frequencies were significantly increased in two high applied concentrations (30, 15 μg/mL) compared with negative and solvent control. MN frequencies were significantly increased in three applied concentrations (30, 15, 7.5 μg/mL) except lowest concentration (3.75 μg/mL) compared with solvent control. Mitotic indices were also affected by treatment with test compound. The obtained results provide evidence to demonstrate that new phthalazine substituted β-lactam derivative can exert genotoxic and cytotoxic effects in peripheral human lymphocytes especially at high concentrations.

Synthesis, characterization and SAR studies of Novel Series of Spiro β-Lactam of 5-methyl-indole-2,3-dione derivatives as a potential antibacterial and anthelmintic agent

A novel series of spiro cyclo-indolyl β-lactam compounds of the 5-methyl-indole-2,3-dione derivatives has been synthesized from the new Schiff bases Isatin, using the Staudinger synthesis. FT-IR, 1H-NMR, 13C-NMR, mass spectroscopy, and elemental analyses were used to describe the produced chemicals. The anthelmintic potency of the produced compounds was evaluated using standard albendazole. The antibacterial activity also tested for the synthesized compounds by calculating the zone of inhibition using cup plate method and by comparison with the standard Ampicillin against the five different pathogens (Bacillus subtilis (ATCC-1086), Pseudomonas auroginosa (ATCC-1232), Escherichia coli, (ATCC-3273), Proteus mirabilis (ATCC-224), and Staphylococcus aureus (ATCC-449)). The result of anthelmintic potential, when compared to conventional albendazole (PT: 1.324±0.12, DT: 1.421±0.21), synthesized compounds ICP-3B (PT: 1.883±0.24, DT: 1.943±0.02) and ICM-3B (PT: 1.758±0.27, DT: 1.675±0.32) were shown significant activity in terms of paralysis and death time. In the antibacterial study, the compound ICP-3B has shown 20.53mm clear zone of inhibition at 100 µg/mL against the bacteria Bacillus subtilis in comparison to the standard ampicillin (23.04 mm zone). The compound ICM-3B, clearly shows the inhibition zone of 21.27 mm against Bacillus subtilis, 24.64 mm against pseudomonas aeruginosa, 20.62mm against E. coli, 20.41 mm against Proteus mirabilis and 23.65 mm against the bacteria staphylococcus aureus at the level of 100µg/mL. It was confirmed that the compounds were synthesized as expected in the reaction scheme based on the collected spectrum and elemental data. The obtained anthelmintic ant antibacterial results also affirm the potentiality of the synthesized compounds. The further compounds can be synthesised as well as other pharmacological activities can be tested for these compounds with the concept of molecular modelling.

Chemoenzymatic enantioselective route to get (+) and (−) 4-acetoxy-azetidin-2-one by lipase-catalysed kinetic resolution and their applications

4-Acetoxy-azetidin-2-one is an extremely useful intermediate widely applied for the synthesis of several biologically active β-lactam compounds. However, it is available as a racemic mixture that could limit its application in the synthesis of enantiopure products. Herein we evaluated the use of lipases in a kinetic resolution (KR) process to finally obtain 4-acetoxy-zetidin-2-one as separated pure enantiomers. From a preliminary screening on a set of commercial enzymes, Pseudomonas fluorescens emerged as the most suitable lipase that allowed to obtain good conversions and excellent enantiomeric excesses. On the enantiomerically pure 4-acetoxy-azetidin-2-ones some nucleophilic substitutions and N-thio-alkylation reactions were tested in order to evaluate the stereochemical integrity at the C-4 position.

Hazard assessment of beta-lactams: Integrating in silico and QSTR approaches with in vivo zebrafish embryo toxicity testing

Antibiotics have emerged as a well-known representative of pharmaceuticals and personal care products (PPCPs) by causing public health and environmental problems due to their potential toxicity. β-lactams are the most commonly used antibiotics in the world. This study used zebrafish embryos to evaluate the toxicity of β-lactams. The results showed that 23 β-lactam compounds induced malformation and death in a concentration–response manner. Moreover, this study established and validated quantitative structure–toxicity relationship (QSTR) models for the toxicity of β-lactams in zebrafish. These models performed well and fast in the prediction of the acute toxicity of β-lactams. Structural interpretation indicated that the β-lactam ring, the thiazolidine/dihydrothiazine rings, the side chains, and spatial configuration are the main factors responsible for the toxicity of β-lactams. The results from our previous studies and this study also revealed that the potential biological risks caused by β-lactams and their degradation products could not be ignored. This study provided important data for further environmental risk assessment of β-lactams and regulatory purposes.

N-Thio-β-lactams targeting L,D-transpeptidase-2, with activity against drug-resistant strains of Mycobacterium tuberculosis

Effective treatment of tuberculosis is frequently hindered by the emerging antimicrobial resistance of Mycobacterium tuberculosis. The present study evaluates monocyclic β-lactam compounds targeting the mycobacterial cell wall remodeling. Novel N-thio-β-lactams were designed, synthesized, and characterized on the L,D-transpeptidase-2, a validated target in M. tuberculosis. The candidates were evaluated in biochemical assays identifying five compounds presenting target-specific kinetic constants equal or superior to meropenem, a carbapenem currently considered for tuberculosis therapy. Mass spectrometry in line with the crystal structures of five target-ligand complexes revealed that the N-thio-β-lactams act via an unconventional mode of adduct formation, transferring the thio-residues from the lactam ring to the active-site cysteine of LdtMt2. The resulting stable adducts lead to a long-term inactivation of the target protein. Finally, the candidates were evaluated invitro against a drug-susceptible and multidrug-resistant clinical isolates of M. tuberculosis, confirming the antimycobacterial effect of these novel compounds.

A general strategy for the synthesis of α-trifluoromethyl- and α-perfluoroalkyl-β-lactams via palladium-catalyzed carbonylation.

β-Lactam compounds play a key role in medicinal chemistry, specifically as the most important class of antibiotics. Here, we report a novel one-step approach for the synthesis of α-(trifluoromethyl)-β-lactams and related products from fluorinated olefins, anilines and CO. Utilization of an advanced palladium catalyst system with the Ruphos ligand allows for selective cycloaminocarbonylations to give diverse fluorinated β-lactams in high yields.

Efflux Pump AcrAB Confers Decreased Susceptibility to Piperacillin-Tazobactam and Ceftolozane-Tazobactam in Tigecycline-Non-Susceptible Klebsiella pneumoniae.

IntroductionDrug efflux pumps are critical for resistance in Gram-negative organisms, but there are limited data on the role they play in decreased susceptibility to β-lactam/β-lactamase inhibitor combinations. In this study, we aimed to investigate the impact of efflux pump AcrAB on piperacillin–tazobactam (TZP) and ceftolozane–tazobactam (C/T) susceptibility in tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains.MethodsA tigecycline gradient was used to obtain various TNSKP strains, and in conjunction with the gradient derived strains, a TNSKP clinical strain (TNSKP24) was also included. Minimum inhibitory concentrations (MICs) of antibiotics were determined by the broth microdilution method, and whole-genome sequencing (WGS) was carried out to analyze genomic changes. PCR and sequencing were performed to confirm mutations in ramR, acrR, and the intergenic region of ramR-romA, and qRT-PCR was applied to evaluate levels of gene expression. In-frame acrB knockout and complementation were performed in 3 TNSKP strains.ResultsTwo derivatives of K. pneumoniae K2606 (K2606-4 and K2606-16) and TNSKP24 overexpressed efflux pump AcrAB were obtained for further study. The MICs of TZP and C/T exhibited a 4- to 8-fold increase in K2606-4 and K2606-16, respectively, when compared with K2606 (TZP, 2/4 μg/mL; C/T, 0.25/4 μg/mL). Deletion of acrB decreased the MICs of TZP and C/T by 4- to 16-fold in TNSKP24, K2606-4, and K2606-16, respectively, and complementation of acrB increased the MICs of these agents. MICs of clavulanate, sulbactam, and avibactam in the presence of β-lactam compounds did not change after acrB deletion and subsequent introduction of complementation mutants.ConclusionThis study highlights that decreased susceptibility to TZP and C/T could be caused by the multidrug efflux pump AcrAB in TNSKP strains.

Metallo-β-Lactamase Inhibitors Inspired on Snapshots from the Catalytic Mechanism.

β-Lactam antibiotics are the most widely prescribed antibacterial drugs due to their low toxicity and broad spectrum. Their action is counteracted by different resistance mechanisms developed by bacteria. Among them, the most common strategy is the expression of β-lactamases, enzymes that hydrolyze the amide bond present in all β-lactam compounds. There are several inhibitors against serine-β-lactamases (SBLs). Metallo-β-lactamases (MBLs) are Zn(II)-dependent enzymes able to hydrolyze most β-lactam antibiotics, and no clinically useful inhibitors against them have yet been approved. Despite their large structural diversity, MBLs have a common catalytic mechanism with similar reaction species. Here, we describe a number of MBL inhibitors that mimic different species formed during the hydrolysis process: substrate, transition state, intermediate, or product. Recent advances in the development of boron-based and thiol-based inhibitors are discussed in the light of the mechanism of MBLs. We also discuss the use of chelators as a possible strategy, since Zn(II) ions are essential for substrate binding and catalysis.

Improving the chromatographic selectivity of β-lactam residue analysis in milk using phenyl-column chemistry prior to detection by tandem mass spectrometry.

Analyte isobaric interferences can limit the development of a comprehensive analytical method for the quantitative liquid chromatography-tandem mass spectrometry profiling of an important cohort of veterinary drugs. In this work, a selective chromatographic separation was developed for the analysis of 32 β-lactam antibiotic residues (12 penicillins, 14 cephalosporins, five carbapenems and faropenem) in milk samples. A range of analytical columns with different stationary phases and mobile phases were evaluated for retention and separation of the β-lactam compounds. Results showed that, among the columns tested, only phenyl-hexyl could adequately separate ampicillin from cephalexin and amoxicillin from cefadroxil, which had shown isobaric interferences on a number of stationary phases. Chromatography was performed using a water/acetonitrile binary gradient with formic acid and ammonium acetate. The β-lactam residues were extracted from the milk samples using a water:acetonitrile solution and purified by C18 dispersive solid-phase extraction (d-SPE) clean-up, followed by concentration under nitrogen and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) determination. Analytes were monitored in positive electrospray ionisation mode (ESI(+)). Possible interfering matrix effects were overcome by using 13 internal standards. The method was fully validated according to 2002/657/EC guidelines, showing satisfactory performance characteristics. Under within-laboratory reproducibility conditions, trueness and precision ranged from 91 to 130% and from 1.4 to 38.6%, respectively. Decision limits (CCα) were in the range 2.1-133μgkg-1. Limits of detection (LODs) and quantitation (LOQs) ranged between 0.0090 and 1.5μgkg-1 and from 0.030 to 5.0μgkg-1, respectively.

Combining Biologically Active β-Lactams Integrin Agonists with Poly(l-lactic acid) Nanofibers: Enhancement of Human Mesenchymal Stem Cell Adhesion.

Regulating stem cell adhesion and growth onto functionalized biomaterial scaffolds is an important issue in the field of tissue engineering and regenerative medicine. In this study, new electrospun scaffolds of poly(l-lactic acid) (PLLA), as bioresorbable polymer, and β-lactam compounds agonists of selected integrins, as functional components with cell adhesive properties, are designed. The new β-lactam-PLLA scaffolds contribute significantly in guiding protein translation involved in human bone marrow mesenchymal stem cells (hBM-MSC) adhesion and integrin gene expression. Scanning electron microscopy, confocal laser scanning microscopy, and Western Blot analyses reveal that GM18-PLLA shows the best results, promoting cell adhesion by significantly driving changes in focal adhesion proteins distribution (β1 integrin and vinculin) and activation (pFAK), with a notable increase of GM18-targets subunits integrin gene expression, α4 and β1. These novel functionalized submicrometric fibrous scaffolds demonstrate, for the first time, the powerful combination of selective β-lactams agonists of integrins with biomimetic scaffolds, suggesting a designed rule that could be suitably applied to tissue repair and regeneration.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance.

MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)-uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882-ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR-uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.