Novel tetrapeptide inhibitors of bacterial protein synthesis produced by a Streptomyces sp

In the course of a microbial product screening aimed at the discovery of novel antibiotics acting on bacterial protein synthesis, a complex of three structurally related tetrapeptides, namely, GE81112 factors A, B, and B1, was isolated from a Streptomyces sp. The screening was based on a cell-free assay of bacterial protein synthesis driven by a model mRNA containing natural initiation signals. In this study we report the production, isolation, and structure determination of these novel, potent and selective inhibitors of cell-free bacterial protein synthesis, which stably bind the 30S ribosomal subunit and inhibit the formation of fMet-puromycin. They did not inhibit translation by yeast ribosomes in vitro. Spectroscopic analyses revealed that they are tetrapeptides constituted by uncommon amino acids. While GE81112 factors A, B, and B1 were effective in inhibiting bacterial protein synthesis in vitro, they were less active against Gram-positive and Gram-negative bacterial cells. Cells grown in minimal medium were more susceptible to the compounds than those grown in rich medium, and this is most likely due to competition or regulation by medium components during peptide uptake. The novelty of the chemical structure and of the specific mode of action on the initiation phase of bacterial protein synthesis makes GE81112 a unique scaffold for designing new drugs.     

Probing the conformation of the resting state of a bacterial multidrug ABC transporter,BmrA, by a site‐directed spin labeling approach

Previously published 3‐D structures of a prototypic ATP‐binding cassette (ABC) transporter, MsbA, have been recently corrected revealing large rigid‐body motions possibly linked to its catalytic cycle. Here, a closely related multidrug bacterial ABC transporter, BmrA, was studied using site‐directed spin labeling by focusing on a region connecting the transmembrane domain and the nucleotide‐binding domain (NBD). Electron paramagnetic resonance (EPR) spectra of single spin‐labeled cysteine mutants suggests that, in the resting state, this sub‐domain essentially adopts a partially extended conformation, which is consistent with the crystal structures of MsbA and Sav1866. Interestingly, one of the single point mutants (Q333C) yielded an immobilized EPR spectrum that could arise from a direct interaction with a vicinal tyrosine residue. Inspection of different BmrA models pointed to Y408, within the NBD, as the putative interacting partner, and its mutation to a Phe residue indeed dramatically modified the EPR spectra of the spin labeled Q333C. Moreover, unlike the Y408F mutation, the Y408A mutation abolished both ATPase activity and drug transport of BmrA, suggesting that a nonpolar bulky residue is required at this position. The spatial proximity of Q333 and Y408 was also confirmed by formation of a disulfide bond when both Q333 and T407 (or S409) were replaced jointly by a cysteine residue. Overall, these results indicate that the two regions surrounding Q333 and Y408 are close together in the 3‐D structure of BmrA and that residues within these two sub‐domains are essential for proper functioning of this transporter.     

Copper(I)-imine complexes: Synthesis and catalytic activity in olefin cyclopropanation

Different imine-type ligands, prepared by the condensation of anilines or of α-methylbenzylamine with 2-pyridinecarboxaldehyde (pyim^1,2) or 2-quinolinecarboxaldehyde (quim^1,2) were prepared. These species act as N,N′-bidentate, chelating ligands upon coordination to Cu(I): treatment of [Cu(PPh₃)₃Cl] with an equimolar amount of the ligands resulted in the displacement of two molecules of PPh₃, giving rise to the formation of [Cu(pyim^1,2)(PPh₃)Cl] (1-2) and [Cu(quim^1,2)(PPh₃)Cl] (3-4), respectively. The copper derivatives 1-4 proved to be highly active catalysts in olefin cyclopropanation in the presence of ethyl diazoacetate, even using deactivated olefins (namely, 2-cyclohexen-1-one) as substrate. The X-ray structure of complex 2, [Cu(pyim²)(PPh₃)Cl], is also reported.     

Modelling pollutant removal in a pilot-scale two-stage subsurface flow constructed wetlands

The hydraulic behaviour and effluent pollutant concentrations in a pilot-scale two-stage subsurface flow constructed wetland for treatment of municipal wastewater have been simulated. The experimental pilot plant is located in San Michele di Ganzaria (Eastern Sicily) and consists of four lines of two-stage subsurface flow constructed wetlands for secondary or tertiary treatment of municipal wastewater. The first stage, for each line, consists of a horizontal flow bed, while in the second stage a vertical flow bed operates for two lines and a horizontal flow bed for the other two. Phragmites sp. was used as vegetation in two lines while the other two lines are without plants. The HYDRUS-2D software was applied to describe flow and single-solute transport, while the multi-component reactive transport module CW2D was used to model the transformation and elimination processes of organic matter, nitrogen and phosphorus. Tracer studies and chemical wastewater analyses were carried out to calibrate and validate the transport model. In general, the simulation results obtained show a good match with the measured data for water flow, tracer experiments and pollutant removal processes.     

Biogas production and valorization by means of a two-step biological process

The scope of this research work was to investigate biogas production and purification by a two-step bench-scale biological system, consisting of fed-batch pulse-feeding anaerobic digestion of mixed sludge, followed by methane enrichment of biogas by the use of the cyanobacterium Arthrospira platensis. The composition of biogas was nearly constant, and methane and carbon dioxide percentages ranged between 70.5–76.0% and 13.2–19.5%, respectively. Biogas yield reached a maximum value (about 0.4 m³_biogas/kgCOD_i) at 50 days-retention time and then gradually decreased with a decrease in the retention time. Biogas CO₂ was then used as a carbon source for A. platensis cultivation either under batch or fed-batch conditions. The mean cell productivity of fed-batch cultivation was about 15% higher than that observed during the last batch phase (0.035 ± 0.006 g_DM/L/d), likely due to the occurrence of some shading effect under batch growth conditions. The data of carbon dioxide removal from biogas revealed the existence of a linear relationship between the rates of A. platensis growth and carbon dioxide removal from biogas and allowed calculating carbon utilization efficiency for biomass production of almost 95%.     

Vanillin bioproduction from alkaline hydrolyzate of corn cob by Escherichia coli JM109/pBB1

Hydrolysis of corn cob performed for 6 h with 0.5 N NaOH at solid/liquid ratio of 0.084 g/g allowed obtaining a hydrolyzate containing 1171 ± 34 mg/l ferulic acid and 2156 ± 63 mg/l p-coumaric acid that was used as a medium for vanillin bioproduction by the engineered strain Escherichia coli JM109/pBB1. Aiming at maximizing vanillin bioproduction, the effects of medium heat sterilization, one-stage or two-stage pre-cultivation, adaptation of the microorganism to the hydrolyzate and inoculum biomass level were investigated. Biomass pre-cultivated once in unsterilized hydrolyzate was able to effectively convert ferulic and p-coumaric acids to a mixture of vanillin, vanillic acid and vanillyl alcohol provided with the typical vanilla flavor. At initial biomass concentration of 0.5 g_DM/l, maximum values of vanillin concentration (239 ± 15 mg/l), vanillin yield on consumed ferulic acid (0.66 ± 0.03 mol/mol) and vanillin volumetric productivity (10.9 ± 0.7 mg/lh) were obtained after 22 h.     

A dry lab for medical engineers?

Post-operatory wound infections are a very uncommon finding after thyroidectomy. For these reasons international guidelines do not routinely recommend systemic antibiotic prophylaxis. The benefits of this antibiotic prophylaxis is not supported by clinical evidence in the literature. We have conducted a multicentric randomized double-blind trial on 500 patients who had undergone thyroidectomy for goitre or thyroid carcinoma. The 500 patients enrolled in the study (mean age 47 years) were randomized in two subgroups of 250 patients. 250 patients were treated with standard antibiotic prophylaxis with sulbactam/ampicillin 1 fl (3 gr.) 30 min before surgery. No antibiotic prophylaxis was instituted in the remainder 250 patients. Our RCT showed that prophylactic antibiotic treatment is not beneficial in patients younger than eighty years old, with no concomitant metabolic, infective and hematologic disease, with no cardiac valvulopathies, not under steroidal or immunosuppressive treatment, and not severely obese. Our study should be regarded only as a preliminary RCT, and should be followed by a study in which a larger number of patients should be enrolled so that statistically significant data can be obtained.     

Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems

Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses; therefore, the knowledge of the volumetric mass transfer coefficient (k_La) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the k_La values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium, in the absence of biomass. Aeration and agitation were selected as the independent variables using a 2² full factorial design. Both variables showed statistically significant effects on k_La, and the highest values of this parameter in both media for simple fermentation (241 s⁻¹) and extractive fermentation with ATPS (70.3 s⁻¹) were observed at the highest levels of aeration (5 vvm) and agitation (1200 rpm). The k_La values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number (N³D²) and superficial gas velocity (V_s) determined in distilled water (α = 0.39 and β = 0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium (α = 0.38 and β = 0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation (α = 0.50 and β = 1.0). A reasonable agreement was found between the experimental data of k_La for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions.