Esterase Activity of Serum Albumin Studied by 1H NMR Spectroscopy and Molecular Modelling

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (¹H NMR) has been applied to the study of true esterase activity of albumin, using the example of bovine serum albumin (BSA) and p-nitrophenyl acetate (NPA). The site of BSA esterase activity was then determined using molecular modelling methods. According to the data obtained, the accumulation of acetate in the presence of BSA in the reaction mixture is much more intense as compared with the spontaneous hydrolysis of NPA, which indicates true esterase activity of albumin towards NPA. Similar results were obtained for p-nitophenyl propionate (NPP) as substrate. The rate of acetate and propionate release confirms the assumption that there is a site of true esterase activity in the albumin molecule, which is different from the site of the pseudo-esterase activity Sudlow II. The results of molecular modelling of BSA and NPA interaction make it possible to postulate that Sudlow site I is the site of true esterase activity of albumin.     

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (K_D) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.     

Hyaluronan/Diethylaminoethyl Chitosan Polyelectrolyte Complexes as Carriers for Improved Colistin Delivery

Improving the therapeutic characteristics of antibiotics is an effective strategy for controlling the growth of multidrug-resistant Gram-negative microorganisms. The purpose of this study was to develop a colistin (CT) delivery system based on hyaluronic acid (HA) and the water-soluble cationic chitosan derivative, diethylaminoethyl chitosan (DEAECS). The CT delivery system was a polyelectrolyte complex (PEC) obtained by interpolymeric interactions between the HA polyanion and the DEAECS polycation, with simultaneous inclusion of positively charged CT molecules into the resulting complex. The developed PEC had a hydrodynamic diameter of 210–250 nm and a negative surface charge (ζ-potential = −19 mV); the encapsulation and loading efficiencies were 100 and 16.7%, respectively. The developed CT delivery systems were characterized by modified release (30–40% and 85–90% of CT released in 15 and 60 min, respectively) compared to pure CT (100% CT released in 15 min). In vitro experiments showed that the encapsulation of CT in polysaccharide carriers did not reduce its antimicrobial activity, as the minimum inhibitory concentrations against Pseudomonas aeruginosa of both encapsulated CT and pure CT were 1 μg/mL.     

Balancing–sequencing procedure for a mixed model assembly system in case of finite buffer capacity

In the last decades, the necessity to make production more versatile and flexible has forced assembly line production systems to change from fixed assembly lines to mixed model assembly lines, where the output products are variations of the same base product and only differ in specific customizable attributes. Such assembly lines allow reduced setup time, since products can be jointly manufactured in intermixed sequences (Boysen, Flieder, Scholl. Jena Research Papers in Business and Economics, Friedrich-Schiller-Universitat Jena, 1;1–11, 2007a; Boysen, Flieder, Scholl. Jena Research Papers in Business and Economics, Friedrich-Schiller-Universitat Jena, 2;1–33, 2007b). Unfortunately, the installation of customization options typically leads to variations in process times, and when the cycle is exceeded within a certain station, an overload is created, forcing other stations to wait and idle. Normally, process time variation in an un-paced line are absorbed by buffers, but in some industrial application the buffer dimensions are critical not only for the reduction of work in progress but also in reducing other constrains (space, technology, model dimensions, etc.). The problem of balancing mixed model assembly lines (MALBP), in the long term, and that of sequencing mixed model assembly lines (MMS), in the short term (Merengo, Nava, Pozetti. Int J Prod Res 37:2835–2860, 1999), are the two major problems to solve. The object of this paper is to illustrate an innovative balancing–sequencing step-by-step procedure that aims to optimize the assembly line performance and at the same time contain the buffer dimensions in function of different market demand and production mix. The model is validated using a simulation software and an industrial application is presented.     

Description of the microflora of sourdoughs by culture-dependent and culture-independent methods

Four types of sourdoughs (L, C, B, Q) from artisanal bakeries in Northern Italy were studied using culture-dependent and culture-independent methods. In all samples, the yeast numbers ranged from 160 to 10⁷ cfu/g, and the numbers of lactic acid bacteria (LAB) ranged from 10³ to 10⁹ cfu/g. The isolated LAB were sequenced, and a similarity was noted between two samples (C, Q), both in terms of the species that were present and in terms of the percentage of isolates. In these two samples, Lactobacillus plantarum accounted for 73% and 89% of the bacteria, and Lactobacillus brevis represented 27% and 11%. In the third sample (B), however, the dominant LAB isolate was Lb. brevis (73%), while Lb. plantarum accounted for only 27%. The fourth sourdough (L) was completely different from the others. In this sample, the most prominent isolate was Weisella cibaria (56%), followed by Lb. plantarum (36%) and Pediococcus pentosaceus (8%). In three out of four samples (L, C and Q), all of the yeasts isolated were identified as Saccharomyces cerevisiae, yet only Candida humilis (90%) and Candida milleri (10%) were isolated in the fourth sample (B). The microbial ecology of the sourdoughs was also examined with direct methods. The results obtained by culture-independent methods and DGGE analysis underline a partial correspondence between the DNA and RNA analysis. These results demonstrate the importance of using a combined analytical approach to explore the microbial communities of sourdoughs.     

Moulds and ochratoxin A on surfaces of artisanal and industrial dry sausages

The use of moulds as a seasoning for sausage can have both desirable and undesirable consequences. The desirable consequences are the creation of a successful product that appeals to consumers. The undesirable consequences are due to the growth of undesirable moulds that produce highly toxic secondary metabolites referred to as mycotoxins. The aim of the paper was to investigate the presence of moulds producing ochratoxin A (OTA) on the surface of sausages from northern Italy. A total of 757 mould strains were isolated from sausage casings. The most frequently identified species were Penicillium nalgiovense, Penicillium oxalicum, Eurotium amstelodami, Penicillium olsonii, Penicillium chrysogenum, Penicillium verrucosum, Penicillium viridicatum, and Eupenicillium crustaceum. Aspergillus ochraceus was detected in only one production lot. Approximately 45% of these samples were positive for the presence of OTA. On the casings of the investigated sausages, the lowest and highest OTA values were 3 and 18 μg/kg, respectively. The OTA concentration was reduced to below the limit of detection (LOD) by brushing and washing the sausages prior to sale. From these data it appears that the presence of OTA on the surface of sausage (on the casings) is not indicative of any health risk for human consumption of sausage, since OTA was not identified inside the dry meat.     

An improved electronic colon cleansing method for detection of colonic polyps by virtual colonoscopy

Electronic colon cleansing (ECC) aims to segment the colon lumen from a patient abdominal image acquired using an oral contrast agent for colonic material tagging, so that a virtual colon model can be constructed. Virtual colonoscopy (VC) provides fly-through navigation within the colon model, looking for polyps on the inner surface in a manner analogous to that of fiber optic colonoscopy. We have built an ECC pipeline for a commercial VC navigation system. In this paper, we present an improved ECC method. It is based on a partial-volume (PV) image-segmentation framework, which is derived using the well-established statistical expectation-maximization algorithm. The presented ECC method was evaluated by both visual inspection and computer-aided detection of polyps (CADpolyp) within the cleansed colon lumens obtained using 20 patient datasets. Compared to our previous ECC pipeline, which does not sufficiently consider the PV effect, the method presented in this paper demonstrates improved polyp detection by both visual judgment and CADpolyp measure.     

Preparation of sintered Nd-Fe-B magnets by pressless process

Pressless process used for the preparation of sintered Nd-Fe-B magnets has been studied. The effect of the average particle size D _av, filling density ρ_f, and powder-texturing conditions for obtaining density ρ and desired magnetic hysteretic properties of sintered magnets has been investigated for both traditional technology (TT) and low-oxygen technology (LOT). The ρ_f magnitude ensuring the optimum relation between the density ρ of sintered magnets and their degree of texture was shown to be 2.5–3.0 and 2.2 g/cm³ for the TT and LOT, respectively. At lower ρ_f magnitudes, no required density of sintered magnets is reached, whereas at higher filling densities, a low level of texture and low remanence are realized. Optimum parameters of pulsed field inducing the high degree of texture in powders have been determined. The following properties were achieved for magnets prepared by pressless LOT: B _r ≥ 14.2 kG, H _c ≥ 8 kOe, and (BH)_max ≥ 47.9 MG Oe.     

Fast-response p-i-n photodiodes for 0.9–2.4 μm wavelength range

Fast-response, uncooled p-i-n photodiodes with a long-wavelength spectral sensitivity boundary at λ = 2.4 μm have been created on the basis of GaSb/GaInAsSb/GaAlAsSb heterostructures. A low doping level (10¹⁴–10¹⁵ cm^?3) in the active layer ensured a low capacitance of the photodiode structure (below 1 pF at a sensitive area diameter of 100 μm) and a record small response time (on a level of 100–150 ps). The photodiode pass band reaches up to 2 GHz. The proposed devices are characterized by a small dark current level (500–1000 nA at a reverse bias voltage of 1–3 V) and a detection ability reaching 9 × 10¹⁰ cm Hz^1/2 W^?1 in a spectral interval of maximum sensitivity within 1.9–2.2 μm.