Understanding the reaction balances behind the viability PCR protocols based on photoreactive dyes.

Viability-PCR (vPCR) protocols are mainly based on photo-reactive dyes impermeant to intact cell membranes. The absence of cell barriers allows the reagent's interaction with the genetic material after a short incubation period. By light-induced reaction, DNA becomes the unsuitable mould for the polymerases and thus cannot be amplified and detected by PCR. General rules and consensus exist on critical aspects of successful vPCR protocol development. However, the understanding of the vPCR reaction concerning how much reagent is really effective or the proper amount of light has been poorly studied. The convenience of using 600 times more dye than bases pairs exist suggests that although these dyes are DNA intercalating reagents, many organic molecules can adsorb it. Concerning light, no exact references exist about how much energy is needed to activate the azide group of reagents such as propidium monoazide. Therefore, it cannot be calculated in terms of energy how much light needs a vPCR protocol. The general rule is to provide reagents and energy in excess. This work provides different responses (based on experimental results) to both questions, which can contribute to a better understanding of the theoretical basis of vPCR protocols.

Key Factors for Removing Bias in Viability PCR-Based Methods: A Review.

During the last decade, the viability PCR approach has been investigated intensively with the goal of detecting solely the DNA derived from intact cells by removing the bias resulting from dead or damaged cell artifacts. However, since the first published paper in 2003 and despite the various continuous improvements, the stubborn reality is that in most cases the goal has not been achieved. Complete neutralization of nucleic acids derived from dead microorganisms was not possible, at least without affecting live cells. However, recently published works in this field show how neutralization rates can be improved. In this communication, we review recent improvements that enable effective vPCR procedures.

Viability qPCR, a new tool for Legionella risk management.

BACKGROUND: Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. METHODS: The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. RESULTS: In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p<0.001). Otherwise, no significant differences between methods were found in the rest of the groups. CONCLUSIONS: The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy.

Diclofenac Loaded Lipid Nanovesicles Prepared by Double Solvent Displacement for Skin Drug Delivery.

PURPOSE: Herein, we detail a promising strategy of nanovesicle preparation based on control of phospholipid self-assembly: the Double Solvent Displacement. A systematic study was conducted and diclofenac as drug model encapsulated. In vitro skin studies were carried out to identify better formulation for dermal/transdermal delivery. METHODS: This method consists in two solvent displacements. The first one, made in a free water environment, has allowed triggering a phospholipid pre-organization. The second one, based on the diffusion into an aqueous phase has led to liposome formation. RESULTS: Homogeneous liposomes were obtained with a size close to 100 nm and a negative zeta potential around -40 mV. After incorporation of acid diclofenac, we obtained nanoliposomes with a size between 101 ± 45 and 133 ± 66 nm, a zeta potential between 34 ± 2 and 49 ± 3 mV, and the encapsulation efficiency (EE%) was between 58 ± 3 and 87 ± 5%. In vitro permeation studies showed that formulation with higher EE% dispayed the higher transdermal passage (18,4% of the applied dose) especially targeting dermis and beyond. CONCLUSIONS: Our results suggest that our diclofenac loaded lipid vesicles have significant potential as transdermal skin drug delivery system. Here, we produced cost effective lipid nanovesicles in a merely manner according to a process easily transposable to industrial scale. Graphical Abstract ᅟ.

[Role of the forensic doctor in the assessment of brain injury]. / Papel del médico forense en la valoración de los traumatismos craneoencefálicos.

Although the rate of brain injury remains stable, due to medical advances, many patients who have suffered serious brain injury survive the accident, but remain with very important consequences. The role of the forensic doctor is increasingly more relevant in establishing curing periods, injury stabilization, treatments received and accident consequences so that the courts can dictate the corresponding compensations, incapacity levels and penal grades.

[Legal medical evaluation of the sequels derived from moderate-serious cranioencephalic traumas]. / Valoración médico-legal de la secuelas derivadas de traumatismos craneoencefálicos moderados-graves.

Currently, the increasing number of subjects who survive a moderate to severe cranioencephalic traumatism (CET) has led to a greater number of sequels. This has made it necessary to study these sequels more specifically. Thus, we have wanted to manifest which are the most common sequels in the cases of patients who have suffered moderate to severe CET through our professional experience and current research done on this. Furthermore, we indicate the need to become aware of these deficits since these (specifically neuropsychological) are often too specific if we compare them with the generality of the standard used for their evaluation.

Neutral-red reaction is related to virulence and cell wall methyl-branched lipids in Mycobacterium tuberculosis.

Searching for virulence marking tests for Mycobacterium tuberculosis, Dubos and Middlebrook reported in 1948 that in an alkaline aqueous solution of neutral-red, the cells of the virulent H37Rv M. tuberculosis strain fixed the dye and became red in color, whereas the cells of the avirulent H37Ra M. tuberculosis strain remained unstained. In the 1950 and 1960s, fresh isolates of M. tuberculosis were tested for this neutral-red cytochemical reaction and it was reported that they were neutral-red positive, whereas other mycobacteria of diverse environmental origins that were non-pathogenic for guinea pigs were neutral-red negative. However, neutral-red has not really been proven to be a virulence marker. To test if virulence is in fact correlated to neutral-red, we studied a clinical isolate of M. tuberculosis that was originally neutral-red positive but, after more than 1 year passing through culture mediums, turned neutral-red negative. We found that, in comparison to the original neutral-red positive strain, this neutral-red negative variant was attenuated in two murine models of experimental tuberculosis. Lipid analysis showed that this neutral-red negative natural mutant lost the capacity to synthesize pthiocerol dimycocerosates, a cell wall methyl-branched lipid that has been related to virulence in M. tuberculosis. We also studied the neutral-red of different gene-targeted M. tuberculosis mutants unable to produce pthiocerol dimycocerosates or other cell wall methyl-branched lipids such as sulfolipids, and polyacyltrehaloses. We found a negative neutral-red reaction in mutants that were deficient in more than one type of methyl-branched lipids. We conclude that neutral-red is indeed a marker of virulence and it indicates important perturbations in the external surface of M. tuberculosis cells.