Extensive antigenic and genetic variation among foot-and-mouth disease type A viruses isolated from the 1994 and 1995 foci in São Paulo, Brazil

Nine foot-and-mouth disease virus (FMDV) type A isolates recovered from the field FMD foci in São Paulo State, Brazil, during 1994 and 1995 (a period preceding the last reported focus of FMD in 1996 in this state) were compared among themselves and with the reference vaccine strain A(24)Cruzeiro. The techniques used were sandwich ELISA, virus neutralization (VN), polyacrylamide gel electrophoresis (PAGE) of the structural polypeptides and direct sequencing of the VP1-coding region (1D gene). Results of VN were recorded as serological relationships R and those from ELISA were expressed as percentage of the homologous reaction r. ELISA and VN gave comparable results (correlation coefficient, 0.936) allowing assignment of these field viruses to four groups which were distinct from the A(24)Cruzeiro strain. PAGE and ID nucleotide sequencing were also able to distinguish between these viruses. The high level:of genetic and antigenic variation found when comparing the A(24)Cruzeiro vaccine strain and type A strains recovered, from the last identified foci of FMD came from a formerly endemic area where vaccination with polyvalent vaccines (O(1)Campos, A(24)Cruzciro and C(3)Indaial) had been extensively applied. The similarity between the results of the serological and genetic analyses suggest that the antigenic differences found are mainly located in the 1D protein. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage.

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.

Estudo epidemiológico, genotípico e fenotípico de estirpes de Staphylococcus aureus produtoras de biofilmes isoladas do ambiente de ordenha e de casos de mastite bovina

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Selênio em tilápia do Nilo utilizando eletroforese em gel e espectrometria atômica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

PURIFICATION AND CHARACTERIZATION OF SOY COTYLEDON -GLUCOSIDASE

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Physical-chemical and microbiological changes in Cerrado Soil under differing sugarcane harvest management systems

Background: Sugarcane cultivation plays an important role in Brazilian economy, and it is expanding fast, mainly due to the increasing demand for ethanol production. In order to understand the impact of sugarcane cultivation and management, we studied sugarcane under different management regimes (pre-harvest burn and mechanical, unburnt harvest, or green cane), next to a control treatment with native vegetation. The soil bacterial community structure (including an evaluation of the diversity of the ammonia oxidizing (amoA) and denitrifying (nirK) genes), greenhouse gas flow and several soil physicochemical properties were evaluated. Results: Our results indicate that sugarcane cultivation in this region resulted in changes in several soil properties. Moreover, such changes are reflected in the soil microbiota. No significant influence of soil management on greenhouse gas fluxes was found. However, we did find a relationship between the biological changes and the dynamics of soil nutrients. In particular, the burnt cane and green cane treatments had distinct modifications. There were significant differences in the structure of the total bacterial, the ammonia oxidizing and the denitrifying bacterial communities, being that these groups responded differently to the changes in the soil. A combination of physical and chemical factors was correlated to the changes in the structures of the total bacterial communities of the soil. The changes in the structures of the functional groups follow a different pattern than the physicochemical variables. The latter might indicate a strong influence of interactions among different bacterial groups in the N cycle, emphasizing the importance of biological factors in the structuring of these communities. Conclusion: Sugarcane land use significantly impacted the structure of total selected soil bacterial communities and ammonia oxidizing and denitrifier gene diversities in a Cerrado field site in Central Brazil. A high impact of land use was observed in soil under the common burnt cane management. The green cane soil also presented different profiles compared to the control soil, but to at a lesser degree.

Iodine speciation in atmospheric aerosols in the marine boundary layer

Iodine chemistry plays an important role in the tropospheric ozone depletion and the new particle formation in the Marine Boundary Layer (MBL). The sources, reaction pathways, and the sinks of iodine are investigated using lab experiments and field observations. The aims of this work are, firstly, to develop analytical methods for iodine measurements of marine aerosol samples especially for iodine speciation in the soluble iodine; secondly, to apply the analytical methods in field collected aerosol samples, and to estimate the characteristics of aerosol iodine in the MBL. Inductively Coupled Plasma – Mass Spectrometry (ICP-MS) was the technique used for iodine measurements. Offline methods using water extraction and Tetra-methyl-ammonium-hydroxide (TMAH) extraction were applied to measure total soluble iodine (TSI) and total insoluble iodine (TII) in the marine aerosol samples. External standard calibration and isotope dilution analysis (IDA) were both conducted for iodine quantification and the limits of detection (LODs) were both 0.1 μg L-1 for TSI and TII measurements. Online couplings of Ion Chromatography (IC)-ICP-MS and Gel electrophoresis (GE)-ICP-MS were both developed for soluble iodine speciation. Anion exchange columns were adopted for IC-ICP-MS systems. Iodide, iodate, and unknown signal(s) were observed in these methods. Iodide and iodate were separated successfully and the LODs were 0.1 and 0.5 μg L-1, respectively. Unknown signals were soluble organic iodine species (SOI) and quantified by the calibration curve of iodide, but not clearly identified and quantified yet. These analytical methods were all applied to the iodine measurements of marine aerosol samples from the worldwide filed campaigns. The TSI and TII concentrations (medians) in PM2.5 were found to be 240.87 pmol m-3 and 105.37 pmol m-3 at Mace Head, west coast of Ireland, as well as 119.10 pmol m-3 and 97.88 pmol m-3 in the cruise campaign over the North Atlantic Ocean, during June – July 2006. Inorganic iodine, namely iodide and iodate, was the minor iodine fraction in both campaigns, accounting for 7.3% (median) and 5.8% (median) in PM2.5 iodine at Mace Head and over the North Atlantic Ocean, respectively. Iodide concentrations were higher than iodate in most of the samples. In the contrast, more than 90% of TSI was SOI and the SOI concentration was correlated significantly with the iodide concentration. The correlation coefficients (R2) were both higher than 0.5 at Mace Head and in the first leg of the cruise. Size fractionated aerosol samples collected by 5 stage Berner impactor cascade sampler showed similar proportions of inorganic and organic iodine. Significant correlations were obtained in the particle size ranges of 0.25 – 0.71 μm and 0.71 – 2.0 μm between SOI and iodide, and better correlations were found in sunny days. TSI and iodide existed mainly in fine particle size range (< 2.0 μm) and iodate resided in coarse range (2.0 – 10 μm). Aerosol iodine was suggested to be related to the primary iodine release in the tidal zone. Natural meteorological conditions such as solar radiation, raining etc were observed to have influence on the aerosol iodine. During the ship campaign over the North Atlantic Ocean (January – February 2007), the TSI concentrations (medians) ranged 35.14 – 60.63 pmol m-3 among the 5 stages. Likewise, SOI was found to be the most abundant iodine fraction in TSI with a median of 98.6%. Significant correlation also presented between SOI and iodide in the size range of 2.0 – 5.9 μm. Higher iodate concentration was again found in the higher particle size range, similar to that at Mace Head. Airmass transport from the biogenic bloom region and the Antarctic ice front sector was observed to play an important role in aerosol iodine enhancement. The TSI concentrations observed along the 30,000 km long cruise round trip from East Asia to Antarctica during November 2005 – March 2006 were much lower than in the other campaigns, with a median of 6.51 pmol m-3. Approximately 70% of the TSI was SOI on average. The abundances of inorganic iodine including iodine and iodide were less than 30% of TSI. The median value of iodide was 1.49 pmol m-3, which was more than four fold higher than that of iodate (median, 0.28 pmol m-3). Spatial variation indicated highest aerosol iodine appearing in the tropical area. Iodine level was considerably lower in coastal Antarctica with the TSI median of 3.22 pmol m-3. However, airmass transport from the ice front sector was correlated with the enhance TSI level, suggesting the unrevealed source of iodine in the polar region. In addition, significant correlation between SOI and iodide was also shown in this campaign. A global distribution in aerosol was shown in the field campaigns in this work. SOI was verified globally ubiquitous due to the presence in the different sampling locations and its high proportion in TSI in the marine aerosols. The correlations between SOI and iodide were obtained not only in different locations but also in different seasons, implying the possible mechanism of iodide production through SOI decomposition. Nevertheless, future studies are needed for improving the current understanding of iodine chemistry in the MBL (e.g. SOI identification and quantification as well as the update modeling involving organic matters).

A MICROFLUIDIC METHOD TO MEASURE DIFFUSION IN HYDROGELS

A novel microfluidic method is proposed for studying diffusion of small molecules in a hydrogel. Microfluidic devices were prepared with semi-permeable microchannels defined by crosslinked poly(ethylene glycol) (PEG). Uptake of dye molecules from aqueous solutions flowing through the microchannels was observedoptically and diffusion of the dye into the hydrogel was quantified. To complement the diffusion measurements from the microfluidic studies, nuclear magnetic resonance(NMR) characterization of the diffusion of dye in the PEG hydrogels was performed. The diffusion of small molecules in a hydrogel is relevant to applications such asdrug delivery and modeling transport for tissue-engineering applications. The diffusion of small molecules in a hydrogel is dependent on the extent of crosslinking within the gel, gel structure, and interactions between the diffusive species and the hydrogel network. These effects were studied in a model environment (semi-infinite slab) at the hydrogelfluid boundary in a microfluidic device. The microfluidic devices containing PEG microchannels were fabricated using photolithography. The unsteady diffusion of small molecules (dyes) within the microfluidic device was monitored and recorded using a digital microscope. The information was analyzed with techniques drawn from digital microscopy and image analysis to obtain concentration profiles with time. Using a diffusion model to fit this concentration vs. position data, a diffusion coefficient was obtained. This diffusion coefficient was compared to those from complementary NMR analysis. A pulsed field gradient (PFG) method was used to investigate and quantify small molecule diffusion in gradient (PFG) method was used to investigate and quantify small molecule diffusion in hydrogels. There is good agreement between the diffusion coefficients obtained from the microfluidic methods and those found from the NMR studies. The microfluidic approachused in this research enables the study of diffusion at length scales that approach those of vasculature, facilitating models for studying drug elution from hydrogels in blood-contacting applications.

Proteome analysis of human plasma and amniotic fluid by Off-Gel isoelectric focusing followed by nano-LC-MS/MS

This paper presents a comparative proteomic analysis of human maternal plasma and amniotic fluid (AF) samples from the same patient at term of pregnancy in order to find specific AF proteins as markers of premature rupture of membranes, a complication frequently observed during pregnancy. Maternal plasma and the corresponding AF were immunodepleted in order to remove the six most abundant proteins before the systematic analysis of their protein composition. The protein samples were then fractionated by IEF Off-Gel electrophoresis (OGE), digested and analyzed with nano-LC-MS/MS separation, revealing a total of 73 and 69 proteins identified in maternal plasma and AF samples, respectively. The proteins identified in AF have been compared to those identified in the mother plasma as well as to the reference human plasma protein list reported by Anderson et al. (Mol. Cell. Proteomics 2004, 3, 311-326). This comparison showed that 26 proteins were exclusively present in AF and not in plasma among which 10 have already been described to be placenta or pregnancy specific. As a further validation of the method, plasma proteins fractionated by OGE and analysed by nano-LC-MS/MS have been compared to the Swiss 2-D PAGE reference map by reconstructing a map that matches 2-D gel and OGE experimental data. This representation shows that 36 of 49 reference proteins could be identified in both data sets, and that isoform shifts in pI are well conserved in the OGE data sets.

Proteomics in cardiovascular surgery

Proteomics describes, analogous to the term genomics, the study of the complete set of proteins present in a cell, organ, or organism at a given time. The genome tells us what could theoretically happen, whereas the proteome tells us what does happen. Therefore, a genomic-centered view of biologic processes is incomplete and does not describe what happens at the protein level. Proteomics is a relatively new methodology and is rapidly changing because of extensive advances in the underlying techniques. The core technologies of proteomics are 2-dimensional gel electrophoresis, liquid chromatography, and mass spectrometry. Proteomic approaches might help to close the gap between traditional pathophysiologic and more recent genomic studies, assisting our basic understanding of cardiovascular disease. The application of proteomics in cardiovascular medicine holds great promise. The analysis of tissue and plasma/serum specimens has the potential to provide unique information on the patient. Proteomics might therefore influence daily clinical practice, providing tools for diagnosis, defining the disease state, assessing of individual risk profiles, examining and/or screening of healthy relatives of patients, monitoring the course of the disease, determining the outcome, and setting up individual therapeutic strategies. Currently available clinical applications of proteomics are limited and focus mainly on cardiovascular biomarkers of chronic heart failure and myocardial ischemia. Larger clinical studies are required to test whether proteomics may have promising applications for clinical medicine. Cardiovascular surgeons should be aware of this increasingly pertinent and challenging field of science.

Epidemic of Salmonellosis in Passerine Birds in Switzerland With Spillover to Domestic Cats

A die-off of passerine birds, mostly Eurasian siskins (Carduelis spinus), occurred in multiple areas of Switzerland between February and March 2010. Several of the dead birds were submitted for full necropsy. Bacteriological examination was carried out on multiple tissues of each bird. At gross examination, common findings were light-tan nodules, 1 to 4 mm in diameter, scattered through the esophagus/crop. Histologically, a necroulcerative transmural esophagitis/ingluvitis was observed. Bacterial cultures yielded Salmonella enterica subsp. enterica serovar Typhimurium. At the same time, 2 pet clinics reported an unusual increase of domestic cats presented with fever, anorexia, occasionally dolent abdomen, and history of presumed consumption of passerine birds. Analysis of rectal swabs revealed the presence of S. Typhimurium in all tested cats. PFGE (pulsed field electrophoresis) analysis was performed to characterize and compare the bacterial isolates, and it revealed an indistinguishable pattern between all the avian and all but 1 of the feline isolates. Cloacal swabs collected from clinically healthy migrating Eurasian siskins (during autumn 2010) did not yield S. Typhimurium. The histological and bacteriological findings were consistent with a systemic infection caused by S. Typhimurium. Isolation of the same serovar from the dead birds and ill cats, along with the overlapping results of the PFGE analysis for all the animal species, confirmed a spillover from birds to cats through predation. The sudden increase of the number of siskins over the Swiss territory and their persistency during the whole winter of 2009-2010 is considered the most likely predisposing factor for the onset of the epidemic.

Conjugal Transfer of the Tn916-like Transposon TnFO1 from Enterococcus faecalis Isolated from Cheese to Other Gram-positive Bacteria

A Tn916-like transposon (TnFO1) was found in the multiple antibiotic resistant Enterococcus faecalis strain FO1 isolated from a raw milk cheese. In this strain, the tetracycline determinant was localized by DNA-DNA hybridization with a tetM nucleotide probe on the chromosome and on a 30-kb plasmid. The transposon TnFO1 was identified and characterized by DNA-DNA hybridization experiments with the five internal HincII fragments of Tn916. The tetracycline resistance determinant was identified by its complete nucleotide sequence as TetM. Transposon TnFO1 was also detected in its circular form by DNA-DNA hybridization and PCR amplification. Both ends including the joining region of the closed circular transposon TnFO1 were sequenced. TnFO1 could be transferred by conjugation from Enterococcus faecalis into Enterococcus faecalis, Lactococcus lactis subsp. lactis biovar. diacetylactis, Listeria innocua, Leuconostoc mesenteroides and Staphylococcus aureus, and from Lactococcus lactis subsp. lactis biovar. diacetylactis into Listeria innocua. Pulsed-field electrophoresis of genomic DNA from E. faecalis FO1 transconjugants showed that transposon TnFO1 integrated at different sites.

Rifaximin-mediated changes to the epithelial cell proteome: 2-D gel analysis

Diarrhea remains a significant cause of worldwide morbidity and mortality. Over 4 million children die of diarrhea annually. Although antibiotics can be used as prophylaxis or for treatment of diarrhea, concern remains over antibiotic resistance. Rifaximin is a semi-synthetic rifamycin derivative that can be used to treat symptoms of infectious diarrhea, inflammatory bowel syndrome, bacterial overgrowth of the small bowel, pouchitis, and fulminant ulcerative colitis. Rifaximin is of particular interest because it is poorly adsorbed in the intestines, shows no indication of inducing bacterial resistance, and has minimal effect on intestinal flora. In order to better understand how rifaximin functions, we sought to compare the protein expression profile of cells pretreated with rifaximin, as compared to cells treated with acetone, rifamycin (control antibiotic), or media (untreated). 2-D gel electrophoresis identified 38 protein spots that were up- or down-regulated by over 2-fold in rifaximin treated cells compared to controls. 16 of these spots were down-regulated, including keratin, annexin A5, intestinal-type alkaline phosphatase, histone h4, and histone-binding protein RbbP4. 22 spots were up-regulated, including heat shock protein HSP 90 alpha, alkaline phosphatase, and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. A better understanding of the functionality of rifaximin will identify additional potential uses for rifaximin and determine for whom the drug is best suited. ^

PEECE II mesocosm experiment: Dynamics of extracellular enzyme activities in seawater under changed atmospheric pCO2, 2011

As part of the PeECE II mesocosm project, we investigated the effects of pCO2 levels on the initial step of heterotrophic carbon cycling in the surface ocean. The activities of microbial extracellular enzymes hydrolyzing 4 polysaccharides were measured during the development of a natural phytoplankton bloom under pCO2 conditions representing glacial (190 µatm) and future (750 µatm) atmospheric pCO2. We observed that (1) chondroitin hydrolysis was variable throughout the pre-, early- and late-bloom phases, (2) fucoidanase activity was measurable only in the glacial mesocosm as the bloom developed, (3) laminarinase activity was low and constant, and (4) xylanase activity declined as the bloom progressed. Concurrent measurements of microbial community composition, using denaturing-gradient gel electrophoresis (DGGE), showed that the 2 mesocosms diverged temporally, and from one another, especially in the late-bloom phase. Enzyme activities correlated with bloom phase and pCO2, suggesting functional as well as compositional changes in microbial communities in the different pCO2 environments. These changes, however, may be a response to temporal changes in the development of phytoplankton communities that differed with the pCO2 environment. We hypothesize that the phytoplankton communities produced dissolved organic carbon (DOC) differing in composition, a hypothesis supported by changing amino acid composition of the DOC, and that enzyme activities responded to changes in substrates. Enzyme activities observed under different pCO2 conditions likely reflect both genetic and population-level responses to changes occurring among multiple components of the microbial loop.

PeECE III - Pelagic Ecosystem CO2 Enrichment Study, Raunefjord, Bergen, Norway, 2005

The effects of CO2-induced seawater acidification on plankton communities were also addressed in a series of 3 mesocosm experiments, called the Pelagic Ecosystem CO2 Enrichment (PeECE I-III) studies, which were conducted in the Large-Scale Mesocosm Facilities of the University of Bergen, Norway in 2001, 2003 and 2005, respectively. Each experiment consisted of 9 mesocosms, in which CO2 was manipulated to initial concentrations of 190, 350 and 750 µatm in 2001 and 2003, and 350, 700 and 1050 µatm in 2005. The present dataset concerns PeECE III.