The growth of Escherichia coli cultures under the influence of pheomelanin nanoparticles and a chelant agent in the presence of light.

Growing concern of antibiotic resistance has increased research efforts to find nonspecific treatments to inhibit pathogenic microorganisms. In this regard, photodynamic inactivation is a promising method. It is based on the excitation of a photosensitizer molecule (PS) with UV-Vis radiation to produce reactive oxygen species. The high reactivity of such species nearby the PS leads to oxidation of bacterial cell walls, lipid membranes (lipid peroxidation), enzymes, and nucleic acids, eventually producing cell death. In the last decade, many studies have been carried out with different photosensitizers to suppress the growth of bacteria, fungi, viruses, and malignant tumors. Here, our main motivation is to employ pheomelanin nanoparticles as sensitizers for inhibiting the growth of the Gram-negative bacteria E. coli, exposed to blue and UVA radiation. In order to perform our experiments, we synthesized pheomelanin nanoparticles from L-DOPA and L-cysteine through an oxidation process. We carried out experiments at different particle concentrations and different energy fluences. We found that cultures exposed to UVA at 166 µg/mL and 270 J/cm2, in conjunction with ethylenediaminetetraacetic acid (EDTA) as an enhancer, decreased in the viable count 5 log10. Different reactive oxygen species (singlet oxygen, hydroxyl radicals, and peroxynitrates) were detected using different procedures. Our results suggest that the method reported here is effective against E. coli, which could encourage further investigations in other type of bacteria.

Analyzing structural alterations of mitochondrial intermembrane space superoxide scavengers cytochrome-c and SOD1 after methylglyoxal treatment.

Mitochondria are quantitatively the most important sources of reactive oxygen species (ROS) which are formed as by-products during cellular respiration. ROS generation occurs when single electrons are transferred to molecular oxygen. This leads to a number of different ROS types, among them superoxide. Although most studies focus on ROS generation in the mitochondrial matrix, the intermembrane space (IMS) is also important in this regard. The main scavengers for the detoxification of superoxide in the IMS are Cu, Zn superoxide dismutase (SOD1) and cytochrome-c. Similar to ROS, certain reactive carbonyl species are known for their high reactivity. The consequences are deleterious modifications to essential components compromising cellular functions and contributing to the etiology of severe pathological conditions like cancer, diabetes and neurodegeneration. In this study, we investigated the susceptibility of SOD1 and cytochrome-c to in vitro glycation by the dicarbonyl methylglyoxal (MGO) and the resulting effects on their structure. We utilized experimental techniques like immunodetection of the MGO-mediated modification 5-hydro-5-methylimidazolone, differential scanning calorimetry, fluorescence emission and circular dichroism measurements. We found that glycation of cytochrome-c leads to monomer aggregation, an altered secondary structure (increase in alpha helical content) and slightly more compact folding. In addition to structural changes, glycated cytochrome-c displays an altered thermal unfolding behavior. Subjecting SOD1 to MGO does not influence its secondary structure. However, similar to cytochrome-c, subunit aggregation is observed under denaturating conditions. Furthermore, the appearance of a second peak in the calorimetry diagram indirectly suggests de-metallation of SOD1 when high MGO levels are used. In conclusion, our data demonstrate that MGO has the potential to alter several structural parameters in important proteins of energy metabolism (cytochrome-c) and antioxidant defense (cytochrome-c, SOD1).

Patterns produced by dried droplets of protein binary mixtures suspended in water.

Patterns formed by the evaporation of a drop containing biological molecules have provided meaningful information about certain pathologies. In this context, several works propose the study of protein solutions as a model to understand the formation of deposits of biological fluids. Generally, dry droplets of proteins in a saline solution create complex aggregates. Here, we present an experimental study on the formation of patterns produced by the evaporation of droplet suspensions containing a protein binary mixture. We explore the structural aspect of such deposits by using optical and atomic force microscopy. We found that salt is unnecessary for the formation of complex structures such as crystal clusters, dendritic and undulated branches, and interlocked chains. Such structural features allow us to differentiate among protein binary mixtures. Finally, we discuss the potential use of this finding to reveal the presence of a protein suspensions, the folded and unfolded state of a protein, as well as their structural changes.

Visible light neutralizes the effect produced by ultraviolet radiation in proteins.

The damage produced by UV-C radiation (100-280nm) in organisms and cells is a well known fact. The main reactions of proteins to UV-C radiation consist in the alteration of their secondary structures, exposure of hydrophobic residues, unfolding and aggregation. Furthermore, it has been found that electromagnetic radiation of lower energy (visible light, where wavelengths are between 400 and 750nm) also induces different disturbances in biomolecules. For instance, it has been observed that blue visible light from emitting diodes (LEDs) produces severe damage in murine cone photoreceptor-derived cells, and it can be even more harmful for some organisms than UV radiation. Recently, it has been found that the exposure of proteins to green and red light produces conformational changes, considerably increasing their cohesion enthalpies. This is presumably due to the strengthening of the hydrogen bonds and the formation of new ones. Therefore, it seems that visible light acts contrary to what it is observed for UV-C: instead of unfolding the proteins it folds them further, halting the damage produced by UV-C. This can be understood if we consider the modification of the folding energy-landscape; visible light induces the descent of the proteins into deeper states impeding the unfolding produced by UV-C.

Effects of green and red light in ßL-crystallin and ovalbumin.

The effects of visible light on biological systems have been widely studied. In particular, the alterations of blue light on the ocular lens have recently attracted much attention. Here, we present a study about the effects produced by green and red light on two different proteins: ßL-crystallin and ovalbumin. Based on differential scanning calorimetry (DSC), circular dichroism (CD), dynamic light scattering (DLS), and fluorescence emission measurements, we found that both wavelengths induce structural changes in these proteins. We also observed that ßL-crystallin aggregates. Our work may advance our understanding about conformational and aggregation processes in proteins subjected to visible radiation and the possible relationship with cataracts. While blue light has been considered the only harmful component in the visible espectrum, our findings show the possibility that lower energy components may be also of some concern.

Sterilization by Cooling in Isochoric Conditions: The Case of Escherichia coli.

High hydrostatic pressure (HHP) affects the structure, metabolism and survival of micro-organisms including bacteria. For this reason HHP is a promising treatment in the food industry. The aim of this work is to evaluate the effect of high pressure, under isochoric cooling conditions, on Escherichia coli, where such high pressure develops due to the fact water cannot expand. We combine survival curves obtained by spectrophotometry and images of atomic force microscopy in this study. Our results show that cooling at -20 and -30°C leads to a partial destruction of a Escherichia coli population. However, cooling at -15°C causes a total extermination of bacteria. This intriguing result is explained by the phase diagram of water. In the first case, the simultaneous formation of ice III and ice Ih crystals provides a safe environment for bacteria. In the second case (-15°C) Escherichia coli remains in a metastable and amorphous free-of-crystals liquid subjected to high pressure. Our work is the first experimental study carried out to inactivate Escherichia coli under isochoric cooling conditions. Unlike HHP, which is based on the application of an external load to augment the pressure, this technique only requires cooling. The method could be used for annihilation of other Escherichia coli strains and perhaps other micro-organisms.

Water under inner pressure: a dielectric spectroscopy study.

Water is the most studied substance on Earth. However, it is not completely understood why its structural and dynamical properties give rise to some anomalous behaviors. Some of them emerge when experiments at low temperatures and/or high pressures are performed. Here we report dielectric measurements on cold water under macroscopically constrained conditions, i.e., water in a large container at constant volume that cannot freeze below the melting point. The inner pressure in these conditions shifts the α relaxation peak to similar frequencies as seen in ice Ih. At 267 K we observe a peculiar response possibly due to the Grotthuss mechanism. At 251 K (the triple point) ice III forms.

Phenomenological study of the ionisation density-dependence of TLD-100 peak 5a.

Horowitz and collaborators have reported evidence on the structure of TLD-100 peak 5. A satellite peak, called 5a, has been singled out as arising from localised electron-hole recombination in a trap/luminescent centre, its emission mechanism would be geminate recombination and, therefore, its population would depend on incident radiation ionisation density. We report a phenomenological study of peak 4, 5a and 5 strengths for glow curves previously measured at UNAM for gammas, electrons and low-energy ions. The deconvolution procedure has followed strict rules to assure that the glow curve, where the presence of peak 5a is not visually noticeable, is decomposed in a consistent fashion, maintaining fixed widths and relative temperature difference between all the peaks. We find no improvement in the quality of the fit after inclusion of peak 5a. The relative contribution of peak 5a with respect to peak 5 does not seem to correlate with the radiation linear energy transfer.