Diurnal variations of airborne pollen concentration and the effect of ambient temperature in three sites of Mexico City.

Pollen is an important cause of allergic respiratory ailments in the Mexico City Metropolitan Area (MCMA). However, very little is known if ambient air temperature correlates with the early blooming of plants observed in other urban areas around the world. A research study was conducted during the dry season of 2012-2013 at three representative sites of the MCMA with different urban characteristics with the aim to understand the relationships between the profusion and diversity of pollen against temperature and other meteorological variables and degree of urbanization. Pollen samples were collected using a Hirst-type trap sampler in the sites: Merced (highly urbanized), Iztapalapa (medium-high urbanized) and Coyoacan (moderately urbanized). Urbanization levels were determined using a composite index based on population density, proportion of surface covered by construction and asphalt, and urban heat island intensity. A set of representative pollen sampling tapes were assayed under a light microscope at magnification of ×1,000 and converted to grains per cubic meter. The most representative pollen types found in the three sites were, regardless of urbanization levels were: Fraxinus, Cupressaceae/Taxodiaceae, Casuarina, Alnus, Myrtaceae, and Pinus. Total pollen concentration was greatest in the moderately urbanized area, although earlier blooming took place at the highly urbanized zone. Total pollen concentration in the medium-high urbanized site has the lowest because the green areas in this zone of MCMA are few. In a diurnal basis, the most abundant pollen types peaked near midday or in the afternoon evening at the three sites. A Spearman test showed a positive correlation among bihourly pollen concentrations, temperature and relative humidity in all sites, but wind speed just correlated in Iztapalapa and Coyoacan. The results obtained suggest that Urban Heat Island Intensity can disturb flowering periods and pollen concentrations, largely in the highly urbanized areas. A principal components analysis established that the concentrations of each pollen type differed across the urbanization gradients. Additionally, it was found that a large number of allergenic pollens are produced by ornamental trees, some only recently introduced by urban planners.

Calculation methods to perform mass balance of endocrine disrupting compounds in a submerged membrane bioreactor: fate and distribution of estrogens during the biological treatment.

The purpose of this paper is to report the study of the fate and distribution of three endocrine disrupting compounds (estrogens); Estrone (E1), 17ß-estradiol (E2), and 17α-ethinylestradiol (EE2) in a laboratory scale submerged membrane bioreactor (SMBR). For this matter, both aqueous and solids phases were analyzed for the presence of E1, E2 and EE2. The outcome of this study was that three SMBRs showed enhanced elimination of estrogens in different operational conditions; the estrogen removal was close to 100% in SMBR. Additionally, E1, E2 and EE2 were detected in SMBR sludge at concentrations of up to 41.2, 37.3 and 36.9 ng g(-1) dry weight, respectively. The estrogen removal in the SMBRs was directly influenced by a combination of simultaneous biodegradation-adsorption processes, indicating that the main removal mechanism of the estrogens in the SMBRs is the biodegradation process. The E1, E2 and EE2 were biologically degraded in the SMBR (87-100%). The sorption of estrogens onto activated sludge was from 2%. Therefore, a high potential for estrogen removal by biodegradation in the SMBR was observed, allowing less estrogen concentration in the dissolved phase available for the adsorption of these compounds onto biological flocs. Two different methods were carried out for mass balance calculations of estrogens in SMBR. For the first method, the measured data was used in both liquid and solid phases, whereas for the second one, it was in aqueous phase and solid-water distribution coefficients (K(d)) value of E1, E2 and EE2. The purpose of these methodologies is to make easier the identification of the main mechanisms involved in the removal of E1, E2 and EE2 in a SMBR. Both methods can be applied in order to determine the mechanism, fate and distribution of estrogens in a SMBR.

A comparison of biodegradation kinetic models applied to estrogen removal with nitrifying activated sludge.

The biological degradation of estrone (E1), estradiol (E2) and ethinylestradiol (EE2) was studied in batch experiments at typical concentration levels using nitrifying activated sludge from a membrane bioreactor (MBR). Since first-order, pseudo first-order and Monod-type kinetics were observed. Pseudo first order kinetic was reformulated using only the soluble concentrations S and assuming adsorption coefficient K(D) of the estrogens. For the adsorption coefficients K(D) determination, activated sludge from MBR was spiked with the respective target compounds and stirred. Finally, the water was analyzed. The K(D) values of estrogens ranged from 0.323 to 0.474 L/g. Greater than 98% of E1, E2 and EE2 were found to be removed in batch reactors. The measured data were linearly regressed giving R(2) values ranging from 0.748 to 0.990. According to these results, the biodegradation kinetics were adjusted to pseudo first-order assuming adsorption coefficient K(D) and Monod-type kinetic. The biodegradation rate constant k of the estrogens were: E1 and E2 > 78.52 L/g(VSS) d and 12.41 L/g(VSS) d for EE2. Monod-type kinetic indicates that these compounds are biodegradated by co-metabolism. E2 was oxidized into E1.

Individual mitochondrion characterization: a comparison of classical assays to capillary electrophoresis with laser-induced fluorescence detection.

A method has been developed that uses capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF) for measuring protein abundance in individual mitochondria collected from a discontinuous density gradient and labeled with Mitotracker Green. From these measurements we determined the distribution of protein content per mitochondrion and the relative abundance of mitochondrial proteins in density gradient fractions. In addition, this method is useful for counting mitochondria and, as a consequence, determining the number of mitochondria per unit volume or estimate mitochondria copy number per cell. It was determined that mitochondria accumulate in two interfaces defined by consecutive layers of 35% Metrizamide, 17% Metrizamide, and 6% Percoll. The presence of mitochondria in these interfaces was also confirmed using a modified Lowry assay that prevents interference from Metrizamide and Percoll and determines total protein content, and a succinate dehydrogenase assay that uses dichloroindophenol as an electron acceptor and that specifically indicates abundance of mitochondria. The CE-LIF analysis of mitochondrial properties, based on the individual mitochondrial determinations, has a wider scope than the average values determined by enzymatic or bulk protein assays.

Determination of properties of individual liposomes by capillary electrophoresis with postocolumn laser-induced fluorescence detection.

Individual liposome measurements by capillary electrophoresis with postcolumn laser-induced fluorescence detection facilitated the determination of liposome property distributions, two-dimensional plots, and an improved characterization of a liposomal preparation. This advancement in liposome analysis was feasible by using a high-sensitivity postcolumn laser-induced fluorescence detector wired for millisecond response. For each individual liposome containing fluorescein, peak height and migration time were determined. From these measurements the individual entrapped volumes and electrophoretic mobilities were determined. Distribution analysis of these properties facilitated comparison of various liposome dilutions and indicated that the method is reproducible and unaffected by the density of liposomes (10(7)-10(9) liposomes/mL) in the suspension. Furthermore, liposomes showed entrapped volumes that vary from 0.3 to 13 fL with apparent radius varying from 370 nm to 1.8 microns. Two-dimensional plots of reduced mobility versus kappa R (Debye parameter x liposome radius) revealed that the liposomes resuspended from a dried film of phospholipids are heterogeneous in regard to the surface charge density of individual liposomes. The described method has the potential of becoming a new tool for characterization of commercial liposomal preparations and theoretical studies.

Separation of free amino acids in human plasma by capillary electrophoresis with laser induced fluorescence: potential for emergency diagnosis of inborn errors of metabolism.

Free amino acids (AAs) in human plasma are derivatized with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) and analyzed by capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The labeling procedure is significantly improved over results reported previously. Derivatization can be completed in 40 min, with concentrations as low as 4 x 10(-8) M successfully labeled in favourable cases. Twenty-nine AAs (including 2 internal standards) are identified and can be reproducibly separated in 70 min. Migration time RSD values for 23 of these AAs were calculated and found in the range from 0.5 to 4%. The rapid derivatization procedure and the resolution obtained in the separation are sufficient for a semi-quantitative, emergency diagnosis of several inborn errors of metabolism (IEM). Amino acid profiles for both normal donor plasma samples and plasma samples of patients suffering from phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia, and citrullinemia are studied.

Thinner abuse alters optokinetic nystagmus parameters.

BACKGROUND: Deliberate self-inhalation of solvents such as thinner is a recognized problem in underdeveloped countries, with chronic abuse resulting in neurological impairment. In this article, we use electronystagmography (ENG) to study optokinetic nystagmus abnormalities (OKN) that may be induced by thinner consumption. METHODS: Twenty-five patients exposed to thinner for 5-20 years, in an irregular fashion of consumption, were recruited from a toxicologic center. Twenty-five control subjects were invited to participate as volunteers matched by age (+/-2 years) and gender. At the time of evaluation, all had abstained from intoxicants for at least 4 weeks. ENG recordings were performed by clinicians masked to the patient's group. Clockwise and counterclockwise stimulation were performed at 20 and 40 degrees /sec. RESULTS: None of the patients showed spontaneous nystagmus during the test period. Differences between thinner abusers and controls on clockwise and counterclockwise OKN on number of beats of nystagmus elicited on the 40 degrees /sec velocity were identified. The thinner abusers group showed a lesser number of nystagmus (p level was 0.02 and 0.005, respectively). CONCLUSIONS: The present results confirm the sensibility of OKN as an early marker of solvent abuse. These results were obtained in middle-term chronic exposure to solvent mixtures and are in favor of both cortical and brainstem dysfunction.

Single-cell analysis avoids sample processing bias.

Microscale separation tools such as capillary chromatography and capillary electrophoresis (CE) allow the study of metabolism in individual cells. In this work, we demonstrate that single-cell analysis describes metabolism more accurately than analysis of cellular extracts. We incubated HT29 cells (human colon adenocarcinoma) with a fluorescently labeled metabolic probe. This disaccharide, LacNAc, was labeled with a fluorescent dye, tetramethylrhodamine (TMR). The probe was taken up by the cells and metabolized to a number of products that retained the fluorescent label. We then split the cells into two batches. A cellular extract was prepared from one batch and analyzed by CE with laser-induced fluorescence (LIF) detection. The cells from the second batch were used for single-cell analysis by CE-LIF. Separation and detection conditions were identical for extract and single-cell analyses. We found that the electropherogram obtained by averaging the results from a number of single cells differed significantly from the cell extract electropherogram. Differences were due to sample processing during extract preparation. Disruption of the cells liberated enzymes that were compartmentalized within the cell, which allowed non-metabolic reactions to proceed. The accumulation of these non-metabolic products introduced a bias in the cell extract assay. During single-cell analysis, cells were lysed inside the capillary and the separation voltage was applied immediately to separate the enzymes from their substrates and prevent non-metabolic reactions. This paper is the first to report that CE analysis of single cells provides more accurate metabolic information than the CE analysis of a cellular extract.

Instrumentation for chemical cytometry.

Capillary electrophoresis is ideally suited to chemical analysis of individual cells. Small mammalian somatic cells (approximately 15 microns in diameter) can be analyzed by injecting the intact cell into a capillary, lysing the cell, separating and detecting the cellular components, and reconditioning the capillary prior to the next injection. In this paper, we report on technical improvements to single-cell analysis. We designed an inexpensive multipurpose single-cell injector that facilitates the following: (i) monitoring of injection, (ii) reproducible pressure- or electrokinetic-driven injection of the cell, (iii) complete cell lysis by SDS within 30 s of injection, and (iv) pressure-driven capillary reconditioning. Furthermore, we report on the analysis of glycosylation and glycolysis in single human carcinoma cells (HT29 cell line). The reliability and quality of the analysis is confirmed by comparing electropherograms from single cells and those from purified cell extracts.

One-dimensional protein analysis of an HT29 human colon adenocarcinoma cell.

A single HT29 human colon adenocarcinoma cell was introduced into a fused-silica capillary and lysed, and the protein content was fluorescently labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. The labeled proteins were separated by capillary electrophoresis in a submicellar buffer and detected by laser-induced fluorescence in a postcolumn sheath-flow cuvette. Several dozen components were resolved. A number of experiments were done to verify that these components were proteins. Most components of the single-cell electropherogram had the same mobility as components present in the 30-100 kDa fraction of a protein extract prepared from the cell culture. One component was identified as a approximately 100 kDa protein by co-injecting the sample with purified protein obtained from an SDS-PAGE gel. Protein expression varied significantly between cells, but the average expression was consistent with that observed from a protein extract prepared from 10(6) cells.

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.

Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast.

Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-->2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate.

Picomolar analysis of proteins using electrophoretically mediated microanalysis and capillary electrophoresis with laser-induced fluorescence detection.

We report a method for the analysis of picomolar concentration proteins using electrophoretically mediated microanalysis (EMMA) to label proteins on-column with a fluorogenic reagent. Labeling is followed by capillary zone electrophoresis separation and postcolumn detection based on laser-induced fluorescence. The method provides a concentration detection limit (3 sigma) of 3 x 10(-13) M for conalbumin. The method provides separation efficiency of 300,000 theoretical plates. Protein extract from a human colon adenocarcinoma cell line generated a dozen major components and many minor components in a 12-min separation; the protein extract from 2.5 cells was used for this analysis. When compared to UV absorbance detection, the EMMA method provides 7,000,000-fold improvement in detection limit.

Sodium dodecyl sulfate-capillary electrophoresis of proteins in a sieving matrix utilizing two-spectral channel laser-induced fluorescence detection.

We report a method for protein labeling, separation by capillary electrophoresis in a polymer sieving matrix, and detection by laser-induced fluorescence. Different dyes are used to label standard and sample proteins. A two-spectral channel detector resolves fluorescence from the sample and standards. Comparison of the migration time of the sample and standards permits the precise determination of molecular weight, irrespective of variations in run-to-run migration times.

Cationic and anionic polymeric additives for wall deactivation and selectivity control in the capillary electrophoretic separation of proteins in food samples.

Both cationic and anionic polymeric additives were used for the capillary electrophoretic separation of proteins in food samples. The cationic polyelectrolyte polydiallyldimethylammonium chloride was more effective in minimizing protein-wall interactions at pH 3 than at pH 7, presumably due to greater repulsion between the adsorbed polymer and proteins. Improved resolution was observed in the presence of the co-additive sodium octanesulphonate, presumably due to ion-pairing interactions with protein sample components. The anionic polymer dextran sulfate produced relatively high efficiencies, 120,000-180,000 theoretical plates, for protein separation, presumably because the polymer adsorbed to the capillary wall, rendering the surface more hydrophilic. In addition to reduced protein-wall interactions, improved resolution was observed, presumably due to analyte-polymer ion-exchange/ion-pairing interactions. When poly(vinyl sulphonic acid) was used instead of dextran sulfate, broader profiles were obtained and fewer components were resolved, presumably due to reduced wall deactivation that is related to the lower hydrophilicity of poly(vinyl sulphonic acid).

Picomolar assay of native proteins by capillary electrophoresis precolumn labeling, submicellar separation, and laser-induced fluorescence detection.

We report a method for the assay of proteins at concentrations lower than 10(-)(10) M with as little as 200 amol of protein. High sensitivity is accomplished by derivatizing the ε-amino group of the protein's lysine residues with the fluorogenic dye 5-furoylquinoline-3-carboxaldehyde and use of a sheath flow cuvette fluorescence detector. Most proteins have a large number of lysine residues; therefore, a large number of fluorescent molecules can be attached to each protein molecule. In general, precolumn labeling improves sensitivity but degrades resolution due to the inhomogeneity of the reaction products from multiple labeling. However, we demonstrate that, through careful manipulation of the separation and reaction conditions, high sensitivity can be obtained without excessive loss in separation efficiency. Over 190 000 theoretical plates are obtained for fluorescently labeled ovalbumin.

Pseudo-coulometric loading in capillary electrophoresis DNA sequencing.

While injection volumes in capillary electrophoresis are typically in the nanoliter range, it is difficult to physically prepare and manipulate samples much smaller than a microliter. As a result, only a small fraction of the analyte contained with the sample volume is transferred to the capillary. This problem is particularly acute in DNA sequencing applications, where on-column stacking is difficult and where the sequencing sample is relatively expensive to prepare. We report a method that transfers 75% of the DNA contained within a 3 microliters sample onto a capillary for DNA sequencing. This method relies on the use of very low ionic strength formamide to resuspend the DNA after an ethanol precipitation. The use of low ionic strength formamide achieves two tasks. First, it produces a very high resistance sample, which increases the voltage drop across the sample and decreases the field across the capillary. This electric field manipulation ensures that DNA fragments do not migrate down the capillary during the loading process, allowing long injection periods without excessive band-broadening. Second, the low ionic strength of the formamide increases the transference number of the DNA; more of the current passing through the injection tip of the capillary is carried by DNA fragments. In the limit of complete elimination of impurity ions from the loading solvent, current passing through the sample is carried only by DNA fragments and loading becomes a coulometric process.

[The use of years of life lost for measuring the level and change of mortality]. / Los anos de vida perdidos: su utilizacion para medir el nivel y cambio de la mortalidad.

"The use of the new index of years of life lost allows us to relate mortality by age and causes of death to the change of the life expectancy, at birth or between any given ages. This index replaces the use of the multiple decrement life tables for analyzing the impact of the change in mortality by age and cause of death on the life expectancies....The article presents the theoretical derivation of the index, some examples of its use, and a detailed calculation." Examples provided include Mexico, Chile, and Argentina. (SUMMARY IN ENG)

[Comments on some indexes to measure the level and change of mortality]. / Comentarios sobre algunos indices para medir el nivel y el cambio de la mortalidad.

"The level and change of mortality can be measured in several ways not only because [of] the selected index, but also because of the way that the mortality change is measured. This document considers the indexes more frequently used for measuring the level of mortality, including the one of years of life lost recently developed. Each index is analyzed in relation to its accuracy for measuring the level and change of mortality, and advantages and disadvantages of each of them are discussed. The index years of life lost is presented with some details, since it was developed rather recently." (SUMMARY IN ENG)