Reynaldo C. Pless

Comparative studies on Enterococcus, Clostridium perfringens and Staphylococcus aureus as quality indicators in tropical seawater at a Pacific Mexican beach resort.

Three microorganisms were assayed to evaluate the microbiological quality in the seawater at a resort on the Mexican Pacific coast, and to test for possible associations among the titers of the various bacteria, their possible correlations with environmental conditions, and with the location of potential wastewater outflows. Significant microorganism levels were found (at Caletilla beach, Hornos beach, and Papagayo beach, respectively: for Enterococcus 157, 153, and 149, for C. perfringens 35, 89, and 56, for S. aureus 244,137, and 279CFU/100ml), often in excess of the presently set guideline values. In general, bacterial titers were higher during rainy season than in dry season. For S. aureus, in both seasons, highest concentrations were found at 3pm, the time of highest tourist presence at the beaches. Our results argue for the use of these three microorganisms as part of a set of indicators in the routine microbiological evaluation of Mexican beachwaters.

Effects of Tempering Time, Ca(OH)2 Concentration, and Particle Size on the Rheological Properties of Extruded Corn Flour

Extrusion was used for obtaining corn masa. Particle size, Ca(OH)2 concentration, and tempering time had significant effects on the viscosity of extruded flours. Ground corn tempered for different periods of time (0.016–10 h) increased viscosity without application of heat. This behavior can be explained by the release of starches from the protein matrix. Viscoelastic properties of masas showed storage modulus (G′) > loss modulus (G″) for all samples. G′ and G″ increased as a function of tempering time, indicating higher water absorption capacity (WAC). The same behavior was found for Ca(OH)2 concentration, suggesting formation of cross-links between starch and polymers. Viscosity of masas modeled by the power law showed a value of n close to 0.1, suggesting that the dispersed solid phase was greater than the liquid phase. Index n and consistency coefficient K were associated with water absorption and viscosity, respectively. Regarding Ca(OH)2, the higher the Ca(OH)2 concentration, the lower the index n; ...

Protonation of Nucleobases in Single- and Double-Stranded DNA

Single‐stranded model oligodeoxyribonucleotides, each containing a single protonatable base—cytosine, adenine, guanine, or 5‐methylcytosine—centrally located in a background of non‐protonatable thymine residues, were acid‐titrated in aqueous solution, with UV monitoring. The basicity of the central base was shown to depend on the type of the central base and its nearest neighbours and to rise with increasing oligonucleotide length and decreasing ionic strength of the solution. More complex model oligonucleotides, each containing a centrally located 5‐methylcytosine base, were comparatively evaluated in single‐stranded and double‐stranded form, by UV spectroscopy and high‐field NMR. The N3 protonation of the 5‐methylcytosine moiety in the double‐stranded case occurred at much lower pH, at which the duplex was already experiencing general dissociation, than in the single‐stranded case. The central guanine:5‐methylcytosine base pair remained intact up to this point, possibly due to an unusual alternative protonation on O2 of the 5‐methylcytosine moiety, already taking place at neutral or weakly basic pH, as indicated by UV spectroscopy, thus suggesting that 5‐methylcytosine sites in double‐stranded DNA might be protonated to a significant extent under physiological conditions.

124 Evaluation of different DNA polymerases for their effectiveness in using a cytosine analogue during real-time PCR amplification

GC-rich regions of the DNA are of special interest, as they generally occur within transcribed or control regions of the genome. However, the high GC-content makes these sequences prone to the formation of hairpin structures, which may persist even at the relatively high temperature of the extension step in PCR protocols, making their amplification difficult in many instances. Conversion to a different nucleotidic alphabet, resulting in thermodynamically weaker versions of the G:C base pair, may provide a resolution of this problem. The 7-deaza analogue of the guanine base has been tried in this context; however, it appears that the c7G:C base pair, while less stable than the canonical G:C pair, is still too strong, so that problems due to hairpin formation persist. N4-alkylated cytosines form specific, but much weaker base pairs with guanines, and may represent a more effective solution to the problem, provided not only that they are stably inserted into the newly formed DNA during the extension step, but also correctly read in subsequent extensions. We have examined the dCTP analogue, N4methyl-dCTP, for its ability to sustain a PCR, both with HotStart Taq DNA polymerase and with Pfu exo− DNA polymerase, amplifying a 200-bp amplicon within the pUC18 sequence. The Taq enzyme produced the expected product with the nucleotide complement dATP/dGTP/dTTP/N4methyl-dCTP, albeit in reduced yield, compared to the yield obtained with the use of dCTP or of dCTP/N4methyl-dCTP mixtures. A slowdown thermal protocol (Frey et al., 2008, Nature Protocols, 3, 1312–1318), using successively lower hybridization temperatures and slow temperature ramps, proved beneficial in giving a high yield of the desired amplicon, with the expected size and the correct nucleotide sequence. The all-N4-methylC amplicon showed a T m reduced by 11 °C, compared to the amplicon obtained with the canonical set of nucleotides, while amplifications performed with mixtures of dCTP and N4methyl-dCTP gave products of the expected size, showing intermediate melting temperatures (see Figure), all of which attests to the lower thermal stability of the N4-methylC:G pair, compared to the G:C base pair. In contrast, the amplicon synthesized with the dATP/c7dGTP/dTTP/dCTP nucleotide set showed a Tm reduction of only 5°C. The Pfu exo−enzyme was less capable of sustaining PCR with the N4-methyl nucleotide, providing a small yield of 200-bp amplicon only in the presence of higher concentrations of N4-methyl-dCTP.

Toward visually inferring the underlying causal mechanism in a traffic-light-controlled crossroads

The analysis of the events taking place in a crossroads offers the opportunity to avoid harmful situations and the potential to increase traffic efficiency in modern urban areas. This paper presents an automatic visual system that reasons about the moving vehicles being observed and extracts high-level information, useful for traffic monitoring and detection of unusual activity. Initially, moving objects are detected using an adaptive background image model. Then, the vehicles are tracked down by an iterative method where the features being tracked are updated frame by frame. Next, paths are packed into routes using a similarity measure and a sequential clustering algorithm. Finally, the crossroads activity is organized into states representing the underlying mechanism that causes the type of motion being detected. We present the experimental evidence that suggests that the framework may prove to be useful as a tool to monitor traffic-light-controlled crossroads.