13 CO2 labelling as a tool for elucidating the mechanism of cuticle development: a case of Clusia rosea.

The plant cuticle is an important plant-atmosphere boundary, the synthesis and maintenance of which represents a significant metabolic cost. Only limited information regarding cuticle dynamics is available. We determined the composition and dynamics of Clusia rosea cuticular waxes and matrix using 13 CO2 labelling, compound-specific and bulk isotope ratio mass spectrometry. Collodion was used for wax collection; gas exchange techniques to test for any collodion effects on living leaves. Cutin matrix (MX) area density did not vary between young and mature leaves and between leaf sides. Only young leaves incorporated new carbon into their MX. Collodion-based sampling discriminated between epicuticular (EW) and intracuticular wax (IW) effectively. Epicuticular differed in composition from IW. The newly synthetised wax was deposited in IW first and later in EW. Both young and mature leaves synthetised IW and EW. The faster dynamics in young leaves were due to lower wax coverage, not a faster synthesis rate. Longer-chain alkanes were deposited preferentially on the abaxial, stomatous leaf side, producing differences between leaf sides in wax composition. We introduce a new, sensitive isotope labelling method and demonstrate that cuticular wax is renewed during leaf ontogeny of C. rosea. We discuss the ecophysiological significance of the new insights.

Rapid and straight forward mass spectrometric determination of nitrocellulose in smokeless powder by DART-Q-ToF-MS/MS.

Smokeless powders (SPs) are a group of low-explosives primarily used as propellant in various munitions and are frequently employed as explosive charges in bombings and terrorist attacks. However, the reliable determination of nitrocellulose (NC), one of the main components of SP remains an analytical challenge, especially in post-blast residues. While highly desirable because of its selectivity, the mass spectrometric (MS) detection of NC is hindered by its polymeric nature and broadly distributed molecular weight, as well as its poor ionizability in many common MS ion-sources. Direct Analysis in Real Time (DART) - MS allows for the rapid and simple analysis of samples and poses a means of circumventing the problems associated with detection of NC. Analytes in DART readily form adducts, which enables the straight-forward detection of nitrate esters such as glucose trinitrate and cellobiose hexanitrate, the respective monomeric and dimeric subunit of NC, and obviates the need for prior derivatization. Therefore, this method is well suited to rapidly and reliably determine the presence of NC in bulk, as well as consumed SP, as could be shown in this proof-of-concept study for a set of three single-base SPs.

DNA immobilization and detection using DNA binding proteins.

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNA binding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNA binding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNA binding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNA binding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNA binding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.

Patterned Photonic Nitrocellulose for Pseudopaper ELISA.

We report an enzyme-link immunosorbent assay (ELISA) based on patterned pseudopaper that is made of photonic nitrocellulose for highly sensitive fluorescence bioanalysis. The pseudopaper is fabricated using self-assembled monodisperse SiO2 nanoparticles that are patterned on a polypropylene substrate as template. The self-assembled nanoparticles have a close-packed hexagonal (opal) structure, so the resulting nitrocellulose has a complementary (inverse opal) photonic structure. Owing to the slow-photon effect of the photonic structure, fluorescence emission for ELISA is enhanced by up to 57-fold without increasing the assay time or complexity. As the detection signal is significantly amplified, a simple smartphone camera suffices to serve as the detector for rapid and on-site analysis. As a demonstration, human IgG is quantitatively analyzed with a detection limit of 3.8 fg/mL, which is lower than that of conventional ELISA and paper-based ELISA. The consumption of sample and reagent is also reduced by 33 times compared with conventional ELISA. Therefore, the pseudopaper ELISA based on patterned photonic nitrocellulose is promising for sensitive, high-throughput bioanalysis.

Bebé colodión autorresolutivo: nueva mutación en el gen ALOX12B / Self-healing Collodion Baby: A New Mutation in the ALOX12BGene

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A rapid method for the identification of nitrocellulose in high explosives and smokeless powders using GC-EI-MS.

Nitrocellulose (NC) is one of the most common ingredients in explosive mixtures, however because of its non-volatility, its detection using Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has not been achieved until today. A rapid method for the identification of NC in bulk explosives using GC-EI-MS was developed. The sample preparation is simple and takes place in a test tube, employing standard equipment of a forensics laboratory. The protocol was optimized and applied to seven, both high and low, commercial explosives, which contained the substance of interest. Moreover, three explosives in the absence of NC were tested to cross check for false positives. Fourteen different standard explosive substances that are usually found in explosive mixtures were then employed in order to monitor the effect of the method on these compounds and check for interferences. Results showed that NC was detected, by its trimethylsilyl (TMS) derivatives, in all the explosive mixtures analyzed and no false positives were observed. The proposed method showed selectivity for NC, as it had no interference coming from other ingredients of explosive mixtures. The protocol introduced offers considerable improvement in identifying the individual components of an explosive mixture and contributes in successful classification of explosives.

Assessing the evidentiary value of smokeless powder comparisons.

Gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) and physical characteristics data for 726 smokeless reloading powders were analyzed by pairwise comparisons of samples comprising the same product and different products. Pairwise comparisons were restricted to samples having matching kernel shape, color, presence or absence of a perforation and measurements. Discrete results were analyzed for same and different products having matching chemical composition determined from a list of 13 organic components. A continuous score-based likelihood ratio was determined for same and different product comparisons using the Fisher transform of the Pearson correlation between the total ion spectra of the compared samples. Probability distributions for same product and different product comparisons appeared bimodal and were modeled with kernel density distributions. In the discrete and continuous data comparisons, the likelihood ratios for probabilities conditioned on same shape, color, presence/absence of perforation and size were found to provide relatively limited support for either the proposition of same product or different product. Further restricting the pairwise comparisons to samples belonging to the same cluster, as determined by agglomerative hierarchical cluster analysis, provided probability distributions for same product and different product comparisons that were more normal, but did not improve the resulting likelihood ratios. These results inform the forensic analyst regarding the evidentiary value of database search results and direct comparisons of recovered and control samples of smokeless powders.

Capillary electrophoresis fingerprinting of 8-aminopyrene-1,3,6-trisulfonate derivatized nitrocellulose after partial acid depolymerization.

Fine characterization of nitrocellulose (NC) remains a challenge, especially in forensic analysis, and a strategy consisting in obtaining representative fingerprints by a separation technique, as for proteins, is of prime interest. In this work, we first established that NCs (especially of high molar mass) cannot be representatively derivatized by 8-aminopyrene-1,3,6-trisulfonic acid (APTS), because of their poor solubility in the medium required for APTS derivatization. Therefore, a partial acid depolymerization step was considered, prior to derivatization by APTS, in an attempt to generate a mixture of oligosaccharides retaining information on the initial NC sample and/or on the cellulose used to prepare it. Acid depolymerization conditions (time and acid concentration) as well as APTS derivatization conditions (time, temperature, APTS/NC and reducing agent/APTS molar ratios) were investigated for lowly-nitrated NCs. The best compromise between depolymerization yields, speed, and pertinency of the resulting oligosaccharidic mixture was obtained using fuming hydrochloric acid (37%, w/w) at 50 °C for 30 min. The most effective procedure for APTS derivatization of oligosaccharides obtained after partial acid depolymerization of NC was achieved at 70 °C for 2h. The resulting APTS-derivatized oligosaccharides were then separated by capillary electrophoresis (CE) using a background electrolyte composed of 60mM 6-aminocaproic acid, pH 4.5 (adjusted with acetic acid)+0.02% hydroxypropyl methyl cellulose. Finally, for the first time, they were identified using APTS-derivatized cellodextrin standards and by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Discrimination of non-explosive and explosive samples through nitrocellulose fingerprints obtained by capillary electrophoresis.

This work is focused on a novel procedure to discriminate nitrocellulose-based samples with non-explosive and explosive properties. The nitrocellulose study has been scarcely approached in the literature due to its special polymeric properties such as its high molar mass and complex chemical and structural characteristics. These properties require the nitrocellulose analysis to be performed by using a few organic solvents and in consequence, they limit the number of adequate analytical techniques for its study. In terms of identification of pre-blast explosives, mass spectrometry is one of the most preferred technique because it allows to obtain structural information. However, it has never been used to analyze polymeric nitrocellulose. In this study, the differentiation of non-explosive and explosive samples through nitrocellulose fingerprints obtained by capillary electrophoresis was investigated. A batch of 30 different smokeless gunpowders and 23 different everyday products were pulverized, derivatized with a fluorescent agent and analyzed by capillary electrophoresis with laser-induced fluorescence detection. Since this methodology is specific to d-glucopyranose derivatives (cellulosic and related compounds), and paper samples could be easily found in explosion scenes, 11 different paper samples were also included in the study as potential interference samples. In order to discriminate among samples, multivariate analysis (principal component analysis and soft independent modeling of class analogy) was applied to the obtained electrophoretic profiles. To the best of our knowledge, this represents the first study that achieve a successful discrimination between non-explosive and explosive nitrocellulose-based samples, as well as potential cellulose interference samples, and posterior classification of unknown samples into their corresponding groups using CE-LIF and chemometric tools.

New protocol for the isolation of nitrocellulose from gunpowders: utility in their identification.

In this work, a new approach for the isolation of nitrocellulose from smokeless gunpowders has been developed. A multistep solvent extraction method was needed to purify nitrocellulose contained in gunpowders. For single-base or double-base gunpowders six consecutive solvent extractions were selected: three extractions with methanol (to remove nitroglycerin, 2,4-dinitrotoluene, ethyl-centralite, diphenylamine, and diphenylamine derivatives); one extraction with dichloromethane (to remove colorants and plasticizers of organic nature); one extraction with methanol (to facilitate a final polar extraction); and one extraction with water (to remove ionic components) were necessary at 35 degrees C. For the triple-base gunpowder studied, eight solvent extractions were needed due to a high concentration of the water-soluble nitroguanidine was present. In addition to the same five initial phases used for the single-base and double-base gunpowders, three water extraction phases at a higher temperature (75 degrees C instead of 35 degrees C) were also needed. A final step to solubilize nitrocellulose in methyl ethyl ketone was used to remove inert components (mainly graphite). Nitrocellulose isolated from these propellants was characterized by Fourier-Transformed Infrared Spectroscopy (FTIR spectroscopy). The same FTIR spectra were observed for nitrocelluloses isolated from different types of gunpowders. A comparison of FTIR spectra of nitrocellulose samples of different nitration degree evidenced that the bands regions most affected by this factor were: 3600-3400cm(-1), corresponding to the stretching vibrations of residual hydroxyl groups; 1200-1000cm(-1), attributed to the valence vibrations nuCO of the glucopyranose cycle; and 750-690cm(-1), assigned to vibrations of the nitrate group. In both cases, the bands appearing in these regions were more pronounced in the spectra of nitrocellulose samples of low nitration degree.

Microscopic and spectroscopic features of gunpowder and its documentation in gunshot wounds in charred bodies.

Determining the direction and range of fire of gunshot wounds in charred bodies can be difficult because soot resulting from thermal injury can grossly be identical to soot arising from a contact or close-range firearm discharge. Two charred bodies had gunshot wounds of the head and neck region that were distorted by thermal effect, precluding determination of the direction and range of fire by gross findings alone. By microscopy, deep wound tissue from each charred body had foreign material suggestive of gunpowder. Samples of the foreign material were examined by Fourier transform infrared (FT-IR) microscopy and determined to be cellulose nitrate (nitrocellulose), a main component of gunpowder. In addition, 12 cases of suicide in well-preserved bodies with contact gunshot wounds were examined with FT-IR microscopy, confirming the presence of cellulose nitrate in 6 (50%) of the cases. Identification of cellulose nitrate in the tracks of gunshot wounds can assist in the determination of direction and range of fire when the surface features are charred.

Routine hydrogen isotope measurement of cellulose nitrate by high-temperature pyrolysis--reference materials and precision.

The determination of isotope ratios of non-exchangeable hydrogen in tree-ring cellulose is commonly based on the nitration of wood cellulose followed by online high-temperature pyrolysis and isotope ratio mass spectrometry measurement of cellulose nitrate samples. The application of this method requires a proper calibration using appropriate reference materials whose delta(2)H values have been reliably normalized to the V-SMOW/SLAP scale. In our study, we achieve this normalization by a direct alternating measurement of reference waters (V-SMOW and SLAP) and three cellulose nitrates chosen as reference materials. For that purpose, both water and solid organic samples are introduced into the pyrolysis reactor by silver capsule injection. The analytical precision of the water measurement using the capsule method is +/-1.5 per thousand. The hydrogen isotopic composition of three cellulose nitrate standards measured ranges from -106.7 to -53.9 per thousand. The standard deviation of the calculated means from five measurement periods of those standards is better than 1 per thousand. Twenty-four different measurements of the hydrogen isotope composition of cellulose nitrate were evaluated in order to assess the precision of the described method. We obtained an analytical precision of +/-3.0 per thousand as representative for the 95% confidence interval applicable for routine measurements of cellulose nitrate samples. Evidence was found for significant differences in the behavior of cellulose nitrate and PE foil during the pyrolitic conversion that emphasizes the need for a proper calibration of the routine measurements. This calibration can only be successful if the reference materials used have a very similar chemical composition and undergo the same preparation procedure as the samples.

Degradation of nitrocellulose by fungi.

Three lignocellulolytic fungi, Trametes versicolor, Pleurotus ostreatus, and Coprinus cinereus, and two cellulolytic fungi Trichoderma reesei and Chaetomium elatum were tested for their ability to degrade nitrocellulose. They were provided with different carbon and nitrogen sources in liquid cultures. Nitrocellulose (N content above 12%) was added as nitrogen source (in solution in acetone) alongside amino acids or as sole N source. Either starch or carboxy-methyl cellulose were provided as carbon sources. After 28 days of growth the highest decrease of nitrocellulose was observed with Chaetomium elatum when up to 43% was degraded in a medium containing nitrocellulose as the only nitrogen source. Coprinus cinereus caused a 37% decrease of nitrocellulose when provided with amino acids and starch as co-substrate. In cultures of Trametes versicolor, Pleurotus ostreatus and Trichoderma reesei, only 10%-22% decrease of nitrocellulose was measured in all media. In the presence of nitrocellulose with N content below 12% supplied as 3 mm pellets as the only carbon source, or with nitrocellulose with carboxy-methyl cellulose, the release of nitrite and nitrate from liquid cultures of Chaetomium elatum was measured. Between 6 and 9 days of growth in these media, an increase in both nitrite and nitrate was observed with a loss in weight of nitrocellulose up to 6% achieved after 34 days. The physical nature of the NC pellets may have reduced the rate of degradation in comparison with supplying NC in solution in the cultures.

Solid phase microextraction ion mobility spectrometer interface for explosive and taggant detection.

Ion mobility spectrometry (IMS) is a rugged, inexpensive, sensitive, field portable technique for the detection of organic compounds. It is widely employed in ports of entry and by the military as a particle detector for explosives and drugs of abuse. Solid phase microextraction (SPME) is an effective extraction technique that has been successfully employed in the field for the pre-concentration of a variety of compounds. Many organic high explosives do not have a high enough vapor pressure for effective vapor sampling. However, these explosives and their commercial explosive mixtures have characteristic volatile components detectable in their headspace. In addition, taggants are added to explosives to aid in detection through headspace sampling. SPME can easily extract these compounds from the headspace for IMS vapor detection. An interface that couples SPME to IMS was constructed and evaluated for the detection of the following detection taggants: 2-nitrotoluene (2-NT), 4-nitrotoluene (4-NT), and 2,3-dimethyl-2,3-dinitrobutane (DMNB). The interface was also evaluated for the following common explosives: smokeless powder (nitrocellulose, NC), 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), 2,4,6-trinitrotoluene (2,4,6-TNT), hexahydro-1,3,5-trinitro-s-triazine (RDX), and pentaerythritol tetranitrate (PETN). This is the first peer reviewed report of a SPME-IMS system that is shown to extract volatile constituent chemicals and detection taggants in explosives from a headspace for subsequent detection in a simple, rapid, sensitive, and inexpensive manner.

Trace explosives detection by photoluminescence.

Some field tests in counter-terrorism efforts to detect explosive traces employ chemistries that yield colored products. We have examined a test kit of this kind, ETK(Plus), based on widely used chemistries and employed extensively by the Israel Police. Our investigation focuses on the prospect of gaining sensitivity by replacing the normal colorimetric modality with photoluminescence detection, which, to our knowledge, has not been explored to date. We find two or more orders of magnitude sensitivity gains for all explosives studied, using field-worthy photoluminescence techniques. We have also investigated a general lanthanide-based photoluminescence approach which shows promise and the ability to photoluminescence-detect trace explosives in the presence of intense background color and/or background fluorescence by time-resolved imaging.

Relación cuantitativa entre el nivel de Streptococcus mutaras y Candida albicans en niños con síndrome de Down / Quantitative relationship between salivary level of Streptococcus mutans and Gandida albicans in children with Downs syndrome

Objetivo: El presente trabajo intenta establecer una correlación entre el recuento cuantitativo de Streptococcus mutans y Candida albicans en la saliva de pacientes con síndrome de Down (SD) y en pacientes deficientes mentales (DM), respecto a un grupo normal (C). Diseño del estudio: Este estudio se realizó en 166 niños (49 SD, 60 DM y 57 C). Las muestras de saliva fueron cultivadas en agar TYCSB y agar Sabouraud y posteriormente se realizaron análisis microbiológicos, pruebas de microscopía electrónica de barrido y de agregación. La valoración estadística de los resultados se realizó mediante análisis de la varianza (ANOVA) y test "T de Student" para datos no apareados. Resultados: Se encontró una correlación de 0,45 para el grupo C, 0,55 en los pacientes SD y 0,37 en los pacientes MD, en la comparación entre el recuento de S. mutans y C. albicans, que se confirmó con experimentos in vitro de S. mutans y C. albicans realizados mediante filtros de nitrocelulosa. Conclusiones: Este tipo de asociación entre bacterias y hongos contribuye a justificar que exista un sinergismo patogénico entre diversos microorganismos, con las consiguientes repercusiones infecciosas, que pueden manifestarse tanto de forma local como sistémica, dependiendo del estado inmunitario del paciente afecto (AU)

Associating gunpowder and residues from commercial ammunition using compositional analysis.

Qualitatively identifying and quantitatively determining the additives in smokeless gunpowder to calculate a numerical propellant to stabilizer (P/S) ratio is a new approach to associate handgun-fired organic gunshot residues (OGSR) with unfired powder. In past work, the P/S values of handgun OGSR and cartridges loaded with known gunpowders were evaluated. In this study, gunpowder and residue samples were obtained from seven boxes of commercial 38 caliber ammunition with the goals of associating cartridges within a box and matching residues to unfired powders, based on the P/S value and the qualitative identity of the additives. Gunpowder samples from four of the seven boxes of ammunition could be easily differentiated. When visual comparisons of the cartridge powders were considered in addition to composition, powder samples from all seven boxes of ammunition could be reliably differentiated. Handgun OGSR was also collected and evaluated in bulk as well as for individual particles. In some cases, residues could be reliably differentiated based on P/S and additive identity. It was instructive to evaluate the composition of individual unfired gunpowder and OGSR particles. We determined that both the numerical centroid and dispersity of the P/S measurements provide information for associations and exclusions. Associating measurements from residue particles with those of residue samples collected from a test firing of the same weapon and ammunition appears to be a useful approach to account for any changes in composition that occur during the firing process.

Lymphoproliferative responses of leprosy patients and healthy controls to nitrocellulose-bound M. leprae antigens

The lymphoproliferative responses of 51 leprosy patients and 11 healthy contacts were analyzed using the nitrocellulose-bound specific antigen fractions from the cell-free extract of Mycobacterium leprae. The main proliferation-inducing fraction for peripheral blood mononuclear cells of the healthy contacts was found to be the Fraction II, bearing antigens in the range of 66-45 kDa. However, this fraction failed to induce lymphoproliferation in the leprosy patients, unlike healthy contacts (p < 0.032). The number of responders as well as the strength of the responses to 66-45 kDa proteins were found to be low in the leprosy patients compared to the healthy contacts. Further, preliminary analysis with the subfractions of Fraction II produced a similar pattern, suggesting that the immune response to the antigens in the range of 66-45 kDa M. leprae proteins remains suppressed in subjects with clinical signs and symptoms of the disease.