Prevalence and molecular epidemiology of Staphylococcus aureus nasal colonization in four nursing home residents in Crete, Greece.

Nursing homes are considered as reservoirs for methicillin-resistant Staphylococcus aureus (MRSA). The present study investigated the point prevalence and molecular epidemiology of S. aureus colonization among nursing home residents. The study population comprised of 227 residents, living in four nursing homes of the Heraklion, Crete, Greece area, between January and December 2015. From each nursing home, swabs from the anterior nares of all eligible participants were obtained within a 2-week period. The isolated S. aureus strains were identified and screened by standard microbiological and molecular epidemiological methods. S. aureus carriage was found in 62 out of 227 participants (38.4%) with 33 out of 62 (53.2%) being MRSA. The median age was 83 years (range 52-103). Females were more frequently colonized [47 (75.8%)]. All 33 methicillin resistant Staphylococcus aureus (MRSA) isolates were mecA-positive carrying SCCmec type IV, 30 (91%) the fnbA, and 17 (51.5%) the PVL genes. Thirty-two (97%) belonged to a single pulsotype C; among them, the PVL-positives belonged to ST80 clone, whereas, the PVL-negatives to ST225. Among the 33 MRSA isolates, 32 (97%) were clindamycin-resistant, carrying the ermA gene. Methicillin-susceptible Staphylococcus aureus (MSSA) strains showed polyclonality and 76% were PVL-positive. In conclusion the present study has shown that nursing homes in our area can be regarded as important reservoirs for community-associated MRSA (CA-MRSA).

Trioxsalen derivatives with lipoxygenase inhibitory activity.

Trioxsalen (TRX) is a 4,5',8-trimethylated psoralen analog presenting interesting biological activities when irradiated with UVA light. A series of TRX derivatives, which where obtained by its chemical modification and incorporation of a variety of unsaturated functions at position 4' of the psoralen ring-system, were evaluated for their antioxidant activity and their inhibitory activity on soybean lipoxygenase (LOX) and lipid peroxidation. The reducing properties of the compounds were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay and found to be very low, in the range 0-14%, with the exception of the hydroxamic acid 6 which showed almost identical activity to BHT. TRX derivative 3 significantly inhibited LOX, with IC(50) 9.4 muM. With the exception of TRX, all tested analogs inhibited lipid peroxidation in the range of 35-91%. The most potent compound, namely TRX derivative 3, was studied for its anti-inflammatory activity in vivo on rat paw edema induced by carrageenan, and was found to be of almost identical activity to indomethacin. The results of the biological tests are discussed in terms of structural characteristics.

Capillary electrophoresis: a superior miniaturized tool for analysis of the mono-, di-, and oligosaccharide constituents of glycan moieties in proteoglycans.

Proteoglycans are a major family of glycoconjugates which participate in and regulate several cellular events and biological functions. Their glycan chains determine their physicochemical and biological properties. Capillary electrophoresis, because of its high resolving power and sensitivity, has been successfully used for the analysis of carbohydrates. The monosaccharide constituents, the disaccharide sulfation pattern, and the uronic acid distribution within glycan chains of proteoglycans determine their interactions with matrix effectors and are responsible for numerous effects. Determination of the chemical composition and identification of key structural components and domains of glycans are, therefore, essential in understanding the biological functions of proteoglycans. In this report an overview of the capillary electrophoresis methods used to analyze and characterize the structure of the glycan chains of proteoglycans is presented.

Analysis of glycosaminoglycan-derived disaccharides in biologic samples by capillary electrophoresis and protocol for sequencing glycosaminoglycans.

Glycosaminoglycans are biologically significant carbohydrates which either as free chains (hyaluronan) or constituents of proteoglycans (chondroitin/dermatan sulfates, heparin, heparan sulfate and keratan sulfate) participate and regulate several cellular events and (patho)physiological processes. Capillary electrophoresis, due to its high resolving power and sensitivity, has been successfully used for the analysis of glycosaminoglycans. Determination of compositional characteristics, such as disaccharide sulfation pattern, is a useful prerequisite for elucidating the interactions of glycosaminoglycans with matrix effective molecules and, therefore, essential in understanding the biological functions of proteoglycans. The interest in the field of characterization of such biologically important carbohydrates is soaring and advances in this field will signal a new revolution in the area of glycomics equivalent to that of genomics and proteomics. This review focuses on the capillary electrophoresis methods used to determine the disaccharide pattern of glycosaminoglycans in various biologic samples as well as advances in the sequence analysis of glycosaminoglycans using both chromatographic and electrophoretic techniques.

A capillary zone electrophoresis method for determining N-acetylneuraminic acid in glycoproteins and blood sera.

A simple capillary zone electrophoresis (CZE) method for the determination of the content of the major sialic acid form N-acetylneuraminic acid (Neu5Ac) in glycoproteins was established. The present method utilizes a simplified hydrolysis-purification procedure consisting of mild acid hydrolysis (25 mM trifluoroacetic acid for 2h at 80 degrees C) to release Neu5Ac and ultrafiltration on Centricon-3 membrane to remove the obtained asialoglycoproteins and other macromolecules present in biologic samples. Derivatization with benzoic anhydride at 80 degrees C for 20 min resulted in complete conversion of Neu5Ac to per-O-benzoylated Neu5Ac. CZE analysis was performed using the operating buffer 25mM phosphate, pH 3.5, containing 50% (v/v) acetonitrile as organic modifier at 30 kV, and detection of the per-O-benzoylated Neu5Ac at 231 nm. The method showed excellent repeatability (RDS<1.98%) and a linearity range from 5 microg/mL to 5mg/mL with a detection limit of 2 microM. Application of the method to microanalysis of human alpha(1)-acid glycoprotein and blood serum samples showed excellent agreement with previously published values, suggesting a high precision for the developed CZE method.

Microemulsion electrokinetic capillary chromatography of sulfated disaccharides derived from glycosaminoglycans.

Microemulsion electrokinetic capillary chromatography (MEEKC) is a capillary electrophoresis technique in which neutral and ionized species can be resolved according to their partitioning into moving oil droplets present in the operating buffer. In this report, we present for the first time the application of MEEKC in the analysis of glycosaminoglycans. An efficient method for the separation of the variously sulfated delta-disaccharides obtained following digestion of chondroitin and dermatan sulfates with chondro/ dermato lyases and derivatization with 2-aminoacridone is described. Nonsulfated, mono-, di-, and trisulfated delta-disaccharides were completely separated using the microemulsion octane/butan-1-ol/Sodium dodecyl sulfate (SDS) in 10 mM borate buffer, pH 9.3, at 25 kV. Agreement of the obtained disaccharide composition with literature values showed that MEEKC can be used for the analysis of glycosaminoglycans.

Capillary electrophoresis and enzyme solid phase assay for examining the purity of a synthetic heparin proteoglycan-like conjugate and identifying binding to basic fibroblast growth factor.

Interaction of basic fibroblast growth factor (bFGF) with heparin/heparan sulfate proteoglycans protects the growth factor against proteolytic degradation and is essential for its cellular activity. Although the structural requirements of heparin and heparan sulfate for the high-affinity binding to bFGF have been extensively examined, studies on intact heparin proteoglycans are limited. In this report, the purity and the binding ability of a heparin proteoglycan-like molecule-the heparin-bovine serum albumin (heparin-BSA) conjugate-was examined using capillary zone electrophoresis (CZE). Furthermore, the affinity of bFGF binding to the heparin-BSA conjugate was studied using an enzyme solid-phase assay. Chondroitin sulfate, dermatan sulfate, hyaluronan, heparan sulfate and variously sulfated disaccharides derived from heparin and heparan sulfate were also studied for their ability to compete with the binding of bFGF to heparin. Heparin-BSA conjugate was synthesized by reductive amination and, following precipitation with 1.5 vols of ethanol-sodium acetate, it was obtained free of contaminating heparin. Heparin-BSA-bFGF conjugate was obtained following incubation of heparin-BSA with bFGF for 2 h at 37 degrees C. Intact heparin, heparin-BSA and heparin-BSA-bFGF conjugates were completely resolved by CZE using 50 mM phosphate, pH 3.5, as operating buffer, reversed polarity (30 kV) and detection at 232 nm. Competitive solid phase assay showed that, among the glycosaminoglycans tested, heparin exhibits the highest affinity binding to bFGF (IC(50) = 6.4 nM). Heparan sulfate showed a lower affinity as compared with that of heparin, whereas all other glycosaminoglycans and heparin/heparan sulfate-derived disaccharides tested showed minute effects. The developed CZE method is rapid and accurate and can be easily used to identify bFGF-interacting heparin preparations of biopharmaceutical importance.

Identification of kappa and lambda chains of the major immunoglobulin G subclasses by capillary zone electrophoresis.

Immunoglobulins are present in most tissues and plasma and play crucial role in immune system. Alteration of the levels of the immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3 and IgG4) is an indication of a disturbed immunological response. The aim of the present study was the development of a capillary electrophoresis (CE) method for the analysis of IgG subclasses in respect to their variable kappa and lambda chains. Various analytical conditions and CE modes, including capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and micellar electrokinetic capillary chromatography (MECC) have been thoroughly studied. CZE was found to be the most convenient way to separate IgG subclasses. Three of the human IgG subclasses were resolved using uncoated fused-silica and 50 mM phosphate, pH = 9.3, as operating buffer at 20 kV and detection at 214 nm. IgG1kappa was completely separated from IgG2kappa and IgG3kappa, whereas IgG2kappa co-migrated with IgG4kappa, which is the minor IgG subclass. Under the same conditions IgG4lambda was completely separated from IgG1lambda, IgG2lambda and IgG3lambda, enabling the identification of the various lambda chains. The developed CE method is rapid and can be applied to the identification of the major immunoglobulin G subclasses in respect to their variable kappa and lambda chains.