The Antimicrobial Peptide Temporin G: Anti-Biofilm,Anti-Persister Activities,and Potentiator Effect of Tobramycin Efficacy Against Staphylococcus aureus

Bacterial biofilms are a serious threat for human health, and the Gram-positive bacterium Staphylococcus aureus is one of the microorganisms that can easily switch from a planktonic to a sessile lifestyle, providing protection from a large variety of adverse environmental conditions. Dormant non-dividing cells with low metabolic activity, named persisters, are tolerant to antibiotic treatment and are the principal cause of recalcitrant and resistant infections, including skin infections. Antimicrobial peptides (AMPs) hold promise as new anti-infective agents to treat such infections. Here for the first time, we investigated the activity of the frog-skin AMP temporin G (TG) against preformed S. aureus biofilm including persisters, as well as its efficacy in combination with tobramycin, in inhibiting S. aureus growth. TG was found to provoke ~50 to 100% reduction of biofilm viability in the concentration range from 12.5 to 100 µM vs ATCC and clinical isolates and to be active against persister cells (about 70–80% killing at 50–100 µM). Notably, sub-inhibitory concentrations of TG in combination with tobramycin were able to significantly reduce S. aureus growth, potentiating the antibiotic power. No critical cytotoxicity was detected when TG was tested in vitro up to 100 µM against human keratinocytes, confirming its safety profile for the development of a new potential anti-infective drug, especially for treatment of bacterial skin infections.     

Predicting and tuning physicochemical properties in lead optimization: amine basicities

This review describes simple and useful concepts for predicting and tuning the pK(a) values of basic amine centers, a crucial step in the optimization of physical and ADME properties of many lead structures in drug-discovery research. The article starts with a case study of tricyclic thrombin inhibitors featuring a tertiary amine center with pK(a) values that can be tuned over a wide range, from the usual value of around 10 to below 2 by (remote) neighboring functionalities commonly encountered in medicinal chemistry. Next, the changes in pK(a) of acyclic and cyclic amines upon substitution by fluorine, oxygen, nitrogen, and sulfur functionalities, as well as carbonyl and carboxyl derivatives are systematically analyzed, leading to the derivation of simple rules for pK(a) prediction. Electronic and stereoelectronic effects in cyclic amines are discussed, and the emerging computational methods for pK(a) predictions are briefly surveyed. The rules for tuning amine basicities should not only be of interest in drug-discovery research, but also to the development of new crop-protection agents, new amine ligands for organometallic complexes, and in particular, to the growing field of amine-based organocatalysis.     

The Outcomes of Decorated Prolines in the Discovery of Antimicrobial Peptides from Temporin-L

Previously, we identified a potent antimicrobial analogue of temporin L (TL), [Pro³]TL, in which glutamine at position 3 was substituted with proline. In this study, a series of analogues in which position 3 is substituted with non-natural proline derivatives, was investigated for correlations between the conformational properties of the compounds and their antibacterial, cytotoxic, and hemolytic activities. Non-natural proline analogues with substituents at position 4 of the pyrrolidine ring were considered. Structure–activity relationship (SAR) studies of these analogues were performed by means of antimicrobial and cytotoxicity assays along with circular dichroism (CD) and NMR spectroscopic analyses for selected compounds. The most promising peptides were additionally evaluated for their activity against some representative veterinary microbial strains to compare with those from human strains. We identified novel analogues with interesting properties that make them attractive lead compounds.     

Enzymatic saccharification of lignocellulosic materials after treatment with supercritical carbon dioxide

Lignocellulosic materials, such as agricultural residues, are abundant renewable resources for bioconversion to sugars. The sugar cane bagasse was studied here to obtain simple sugars for the production of alcohols and other chemicals. The crystalline structure of cellulose and the lignin that physically seals the surrounding cellulose fibers makes enzymatic hydrolysis difficult by preventing the contact between the cellulose and the enzyme. Two different samples of sugar cane (bagasse pulp and skin) were used and compared with microcrystalline cellulose (Avicel). The investigated samples were pretreated with SC-CO₂ explosion before hydrolysis. The experiments were conducted at 12, 14 and 16 MPa at a temperature of 60 °C. In this process, particles of celluloses within the size range from 0.25 to 0.42 mm were placed in defined amounts inside the experimental vessel, CO₂ was injected and let stand for 5 and 60 min. The explosion pretreatment of cellulosic materials by SC-CO₂ was performed in an apparatus of a static type with 300 ml of volume. The hydrolysis reaction using cellulose enzyme was carried at 55 °C for 8 h. After the pretreatment, the glucose yield increased in 72% to the bagasse sample. The SC-CO₂ pretreatment together with alkali increased the glucose yield in 20% as compared with alkali only. X-ray, microscopy and thermal analysis were used to investigate the effect of the pretreatment.     

The effect of temperature on the observed flow-induced structure in semidilute solutions of gently solubilized starches

The effect of temperature on the unexpected shear-thickening behavior exhibited by semidilute solutions of waxy maize starch during a repeated shear-loop experiment was evaluated. Shear-thickening behavior, characterized using power law models, was observed for waxy maize starch, but not for normal maize starch at the same concentration. The shear-thickening region was observed for waxy maize starch at temperatures ranging from 25°C to 80°C and shear rates from 2–100 s^?1 during the initial part of the shear-loop experiment. For waxy maize starch, the power law exponent, n, was found to be 0.539 ± 0.017 in the shear-thinning region. For normal maize starch, the value of n was found to be equal to 0.751 ± 0.015 and only shear-thinning behavior was observed. The values of n for both waxy maize and normal maize starch were found to be temperature independent within the precision of the experimental measurements. The differences in n indicate the differences in the conformational dynamics of waxy maize and normal maize starches in solution.     

Low-level (PPB) determination of cisplatin in cleaning validation (rinse water) samples. II. A high-performance liquid chromatographic method

A high-performance liquid chromatographic (HPLC) method is described for the determination of residual levels of cisplatin from extracts of surfaces with very low surface area; from extracts of surfaces of coupons made of Teflon® (polytetrafluoroethylene, PTFE), stainless steel, and glass; and in aqueous solution collected after rinsing equipment and parts. Initially, the method was developed to determine cisplatin at concentrations ranging from 20 to 200 ng/ml by direct injection. Retaining the same method conditions, the scope of the method was expanded by the addition of a sample preconcentration step, allowing analyses at levels ranging from 0.5 ng to 20 ng/ml. Preconcentration is necessary for the determination of cisplatin in rinse waters at a quantifiable concentration of about 2 PPB. Under these conditions, the detection limit is about 0.2 to 0.3 ng/ml. Residual cisplatin on different types of surfaces, including surfaces with very low surface area, can be determined by swabbing each test surface with a derivatizing solution. The cisplatin recovered in the swabbing solution can be analyzed by HPLC using direct injection or preconcentration, depending on the expected level of cisplatin in the sample. Initial methods were developed to quantitate at a cisplatin concentration of about 100 PPB or higher in solution extracted from surfaces. However, when surface areas are limited because of the size of the parts, solution concentration becomes very low as a result of the minimum volume required for extraction. To support the application of swabbing techniques to surface analysis, stainless steel, Teflon, and glass surfaces were spiked with cisplatin at 2.5 to 20 ng/cm². Satisfactory overall recoveries of 90% ± 10% were obtained from all surfaces. Cisplatin has no ultraviolet/visible (UV/Vis) spectral-active functional group that can be used to detect low levels of cisplatin. Hence, diethyldithiocarbamate (DDTC) was used as a derivatizing agent to increase sensitivity to UV absorption at 340 nm. Diethyldithiocarbamate forms complexes with the platinum in cisplatin to yield a platinum-DDTC (Pt-DDTC) complex with a high molar-extinction coefficient. The Pt(DDTC)² complex thus formed was chromatographically separated and then quantitated by comparison of its detector response to that of a similarly derivatized standard preparation. DDTC also has application as a cleaning agent for cisplatin (e.g., for production equipment cleaning, spill cleanup). Destruction of cisplatin can be effected by the reaction of cisplatin with this cleaning agent. Derivatization of cisplatin will convert active cisplatin to platinum-DDTC on surfaces or in solution. Final cleaning can be accomplished using a water-for-injection rinse. After such a cleaning process, the rinse water, when collected and analyzed, showed levels of free cisplatin less than the detection concentration of 0.2 PPB and a total platinum concentration less than 10 PPB as Pt-DDTC complex.     

Signal integrity and timing issues of VME64x double edge cycles

The Double-Edge Source Synchronous Block Transfer (2eSST) is the latest performance update to the VME64 protocol, approved as an ANSI standard in 2003. This extension has taken the VMEs data transfer rate from the original 40 MByte/s to 320 MByte/s. The architectural change at the base of such an impressive step forward is twofold. Different from all the previous cycles, data transfers are driven synchronously by the producer, without handshaking, and data is latched on both the rising and falling edges of the strobe signal. The double edge clocking effectively doubles the bandwidth and it is normally present on PC motherboards equipped with double data rate RAM chips and high-performance graphic adapters. In these applications, a careful approach to signal integrity has shown to be critical in order to avoid timing violations. In parallel, multidrop bus architecture with long lines and up to 21 slots, these concerns become imperative for successful operations. In this paper we present the tests performed on 2eSST. The Motorola MVME6100 and a custom designed board have been used to characterize the timing with different bus loading conditions and to evaluate the impact of different layout choices. Driving capability and crosstalk immunity of enhanced transceivers specifically developed for 2eSST and traditional components are compared.     

Study of the microstructure of inkjet-printed P3HT:PCBM blend for photovoltaic applications

Recently, great interest has been devoted to cost-effective alternative energy sources such as organic solar cells because of the mechanical flexibility and the versatility of chemical structure, the low cost of fabrication, and ease of processing. As regards this last point, the possibility to deposit organic materials from solutions at low temperatures makes them employable for fabricating printed solar cells by direct printing methods. In this study, we used the inkjet-printing technology to deposit P3HT blends with various fullerene acceptors ([60]PCBM, [70]PCBM and bis[60]PCBM) dissolved in single solvents, 1,2-dichlorobenzene (DCB) and chlorobenzene (CB), and their mixtures. After optimizing the printing parameters (printhead speed, drop emission frequency, and substrate temperature), the effect of the solvents on the morphology of the photoactive layers was analyzed through Raman spectroscopy and atomic force microscopy. Polymer solar cells with the structure glass/ITO/PEDOT:PSS/blend/Ca/Al were fabricated and characterized by current–voltage (I–V) measurements under 100 mW/cm² AM 1.5G illumination. A comparative study of the performances of the devices was performed based on three different fullerene derivatives, correlating them to the microstructure of the printed blend films. The optimal devices were obtained when the blend films were deposited from a mixture of DCB:CB 4:1 by volume: this was in agreement with the most favorable morphology of these films.     

Detection of QTL for milk protein percentage in Italian Friesian cattle by AFLP markers and selective genotyping

We targeted quantitative trait loci (QTL) for milk protein percentage (P%) in two Italian Holstein granddaughter design families using selective genotyping in combination with high throughput amplified fragment length polymorphism (AFLP) markers. A total of 64 extreme high and low sires in respect to estimated breeding value (EBV) for P% (EBVP%) were genotyped with 25 AFLP primer combinations that revealed 305 and 291 polymorphisms in the two families. Association between markers and EBVP% was investigated by a linear model only on bands having paternal origin (105 and 96 AFLP bands in family D and S, respectively). Although no marker was significantly associated with the target trait after correction for multiple comparisons, 17 AFLP markers, significant without correction for multiple tests, were considered suggestive of the presence of a QTL. Eleven of these were successfully located on six Bos taurus (BTA) chromosomes by radiation hybrid or in-silico mapping. Ten of these mapped in the immediate neighbourhood (less than 10 cM) of already described QTL for P%. Suggestive association was verified in four regions by microsatellites analysis: one on BTA 10; one on BTA 28; and two on BTA 18. Microsatellites identified significant effects by single marker and interval mapping analyses on BTA 10 and BTA 28, while they were only suggestive of the presence of QTL on BTA 18. In summary, our results firstly indicate that AFLP markers may be used to seek QTL exploiting a selective genotyping approach in GDD, a wide used experimental design in cattle; secondly, propose two approaches for AFLP mapping, namely in-silico mapping exploiting most updated release from the bovine whole genome sequencing project, and physical mapping exploiting a panel of Bovine/Hamster Radiation Hybrids; and thirdly, provide new information on QTLs for an economic important trait in a never investigated Holstein cattle population. AFLP in combination with selective genotyping can be a useful strategy for QTL searching in minor livestock species, sometimes having large economic impact in marginal areas, where more informative markers are still poorly developed.