New Insights into Glycopeptide Antibiotic Binding to Cell Wall Precursors using SPR and NMR Spectroscopy

Glycopeptide antibiotics, such as vancomycin and teicoplanin, are used to treat life‐threatening infections caused by multidrug‐resistant Gram‐positive pathogens. They inhibit bacterial cell wall biosynthesis by binding to the D ‐Ala‐D ‐Ala C‐terminus of peptidoglycan precursors. Vancomycin‐resistant bacteria replace the dipeptide with the D ‐Ala‐D ‐Lac depsipeptide, thus reducing the binding affinity of the antibiotics with their molecular targets. Herein, studies of the interaction of teicoplanin, teicoplanin‐like A40926, and of their semisynthetic derivatives (mideplanin, MDL63,246, dalbavancin) with peptide analogues of cell‐wall precursors by NMR spectroscopy and surface plasmon resonance (SPR) are reported. NMR spectroscopy revealed the existence of two different complexes in solution, when the different glycopeptides interact with Ac2Kd AlaD AlaOH. Despite the NMR experimental conditions, which are different from those employed for the SPR measurements, the NMR spectroscopy results parallel those deduced in the chip with respect to the drastic binding difference existing between the D ‐Ala and the D ‐Lac terminating analogues, confirming that all these antibiotics share the same primary molecular mechanism of action and resistance. Kinetic analysis of the interaction between the glycopeptide antibiotics and immobilized AcKd AlaD AlaOH by SPR suggest a dimerization process that was not observed by NMR spectroscopy in DMSO solution. Moreover, in SPR, all glycopeptides with a hydrophobic acyl chain present stronger binding with a hydrophobic surface than vancomycin, indicating that additional interactions through the employed surface are involved. In conclusion, SPR provides a tool to differentiate between vancomycin and other glycopeptides, and the calculated binding affinities at the surface seem to be more relevant to in vitro antimicrobial activity than the estimations from NMR spectroscopy analysis.     

Optimization of a lateral flow immunoassay for the ultrasensitive detection of aflatoxin M1 in milk

A high sensitive immunoassay-based lateral flow device for semi-quantitatively determine aflatoxin M₁ (AFM₁) in milk was developed. Investigation and optimization of the competitor design and of the gold-labelling strategy allowed the attainment of the ultra-sensitive assessment of AFM₁ contamination at nanograms per litre level (LOD 20 ng L⁻¹, IC₅₀ 99 ng L⁻¹), as requested by European regulations. A one order of magnitude detectability enhancement in comparison to previously reported gold colloid immunochromatographic assays for this toxin was obtained.     

Solid phase extraction of penicillins from milk by using sacrificial silica beads as a support for a molecular imprint

We have prepared molecularly imprinted beads with molecular recognition capability for target molecules containing the penicillanic acid substructure. They were prepared by (a) grafting mesoporous silica beads with 6-aminopenicillanic acid as the mimic template, (b) filling the pores with a polymerized mixture of methacrylic acid and trimethylolpropane trimethacrylate, and (c) removing the silica support with ammonium fluoride. The resulting imprinted beads showed good molecular recognition capability for various penicillanic species, while antibiotics such as cephalosporins or chloramphenicol were poorly recognized. The imprinted beads were used to extract penicillin V, nafcillin, oxacillin, cloxacillin and dicloxacillin from skimmed and deproteinized milk in the concentration range of 5–100 μg·L⁻¹. The extracts were then analyzed by micellar electrokinetic chromatography by applying reverse polarity staking as an in-capillary preconcentration step, and this resulted in a fast and affordable method within the MRL levels, characterized by minimal pretreatment steps and recoveries of 64–90 %.

Penicillanic acid-imprinted beads prepared in preformed porous silica by an imprinting &; etching approach show selectivity towards β-lactams antibiotics. Molecularly imprinted solid phase extraction/micellar electrokinetic chromatography coupled with in-capillary preconcentration resulted in a fast and affordable method for penicillins in milk at MRL levels.

     

Solid phase extraction of penicillins from milk by using sacrificial silica beads as a support for a molecular imprint

We have prepared molecularly imprinted beads with molecular recognition capability for target molecules containing the penicillanic acid substructure. They were prepared by (a) grafting mesoporous silica beads with 6-aminopenicillanic acid as the mimic template, (b) filling the pores with a polymerized mixture of methacrylic acid and trimethylolpropane trimethacrylate, and (c) removing the silica support with ammonium fluoride. The resulting imprinted beads showed good molecular recognition capability for various penicillanic species, while antibiotics such as cephalosporins or chloramphenicol were poorly recognized. The imprinted beads were used to extract penicillin V, nafcillin, oxacillin, cloxacillin and dicloxacillin from skimmed and deproteinized milk in the concentration range of 5–100 μg·L^?1. The extracts were then analyzed by micellar electrokinetic chromatography by applying reverse polarity staking as an in-capillary preconcentration step, and this resulted in a fast and affordable method within the MRL levels, characterized by minimal pretreatment steps and recoveries of 64–90 %.

Catalyst-Dependent Selective Synthesis of O/S- and S/S-Acetals from Enol Ethers

Enol ethers are reacted with mercaptanes to give the corresponding O/S- or S/S-acetals in medium to high yield. Either product can be formed selectively depending on the acid catalyst and the reaction time applied.     

An epidemiological study of low back pain in professional drivers

The prevalence of low back pain (LBP) was investigated in 598 Italian professional drivers exposed to whole-body vibration (WBV) and ergonomic risk factors (drivers of earth moving machines, fork-lift truck drivers, truck drivers, bus drivers). The control group consisted of a small sample of 30 fire inspectors not exposed to WBV. Personal, occupational and health histories were collected by means of a structured questionnaire. Vibration measurements were performed on representative samples of the machines and vehicles used by the driver groups. From the vibration magnitudes and exposure durations, alternative measures of vibration dose were estimated for each subject. Daily vibration exposure, expressed in terms of 8-h energy-equivalent frequency-weighted acceleration, A(8), averaged 0.28-0.61 (range 0.10-1.18) m s⁻² rms in the driver groups. Duration of exposure to WBV ranged between 1 and 41 years. The 7-day and 12-month prevalence of LBP was greater in the driver groups than in the controls. In the professional drivers, the occurrence of 12-month LBP, high intensity of LBP (Von Korff pain scale score ?5), and LBP disability (Roland & Morris disability scale score ?12) significantly increased with increasing cumulative vibration exposure. Even though several alternative measures of vibration exposure were associated with LBP outcomes, nevertheless a more regular trend of association with LBP was found for vibration dose expressed as ∑a_vit_i (m s⁻² h), in which the frequency-weighted acceleration, a_v, and lifetime exposure duration, t, were given equal weight. In multivariate data analysis, individual characteristics (e.g. age, body mass index) and a physical load index (derived from combining manual materials handling and awkward postures) were significantly associated with LBP outcomes, while psychosocial work factors (e.g. job decision, job support) showed a marginal relation to LBP. This study tends to confirm that professional driving in industry is associated with an increased risk of work-related LBP. Exposure to WBV and physical loading factors at work are important components of the multifactorial origin of LBP in professional drivers.     

Desorption and fragmentation properties of dipeptide samples through252Cf-PDMS and FAB

Summary The desorption and fragmentation of dipeptide smaples was investigated through²⁵²Cf-PDMS and FAB techniques. The results for the dipeptides Ala-Ala (molecular weight 160) and Ala-Leu (molecular weight 202) were discussed and compared to previously reported investigations. The authors of this paper have agreed to not receive the proofs for correction.     

Determination of the content of fatty acid methyl esters (FAME) in biodiesel samples obtained by esterification using 1H-NMR spectroscopy

Three different calibration curves based on (1)H-NMR spectroscopy (300 MHz) were used for quantifying the reaction yield during biodiesel synthesis by esterification of fatty acids mixtures and methanol. For this purpose, the integrated intensities of the hydrogens of the ester methoxy group (3.67 ppm) were correlated with the areas related to the various protons of the alkyl chain (olefinic hydrogens: 5.30-5.46 ppm; aliphatic: 2.67-2.78 ppm, 2.30 ppm, 1.96-2.12 ppm, 1.56-1.68 ppm, 1.22-1.42 ppm, 0.98 ppm, and 0.84-0.92 ppm). The first curve was obtained using the peaks relating the olefinic hydrogens, a second with the parafinic protons and the third curve using the integrated intensities of all the hydrogens. A total of 35 samples were examined: 25 samples to build the three different calibration curves and ten samples to serve as external validation samples. The results showed no statistical differences among the three methods, and all presented prediction errors less than 2.45% with a co-efficient of variation (CV) of 4.66%.     

Bergman Spaces and Carleson Measures on Homogeneous Isotropic Trees

Hastings studied Carleson measures for non-negative subharmonic functions on the polydisk and characterized them by a certain geometric condition relative to Lebesgue measure σ. Cima & Wogen and Luecking proved analogous results for weighted Bergman spaces on the unit ball and other open subsets of \(\mathbb {C}^{n}\). We consider a similar problem on a homogeneous tree, and study how the characterization and properties of Carleson measures for various function spaces depend on the choice of reference measure σ.