Dependence of Induced Biological Damage on the Energy Distribution and Intensity of Clinical Intra-Operative Radiotherapy Electron Beams.

The survival fraction of epithelial HaCaT cells was analysed to assess the biological damage caused by intraoperative radiotherapy electron beams with varying energy spectra and intensities. These conditions were achieved by irradiating the cells at different depths in water using nominal 6 MeV electron beams while consistently delivering a dose of 5 Gy to the cell layer. Furthermore, a Monte Carlo simulation of the entire irradiation procedure was performed to evaluate the molecular damage in terms of molecular dissociations induced by the radiation. A significant agreement was found between the molecular damage predicted by the simulation and the damage derived from the analysis of the survival fraction. In both cases, a linear relationship was evident, indicating a clear tendency for increased damage as the averaged incident electron energy and intensity decreased for a constant absorbed dose, lowering the dose rate. This trend suggests that the radiation may have a more pronounced impact on surrounding healthy tissues than initially anticipated. However, it is crucial to conduct additional experiments with different target geometries to confirm this tendency and quantify the extent of this effect.

Feasibility Study on Measuring the Particulate Matter Level in the Atmosphere by Means of Yagi-Uda-Like Antennas.

In this work, the application of a technique for monitoring changes of the dielectric constant of the atmosphere caused by the presence of pollution is discussed. The method is based on changes in the reflection coefficient of the device induced by these dielectric constant variations of the surrounding medium. To that end, several Yagi-Uda-like antenna designs with different size limitations were simulated by using a Method-of-Moments software and optimized by means of a simulated annealing strategy. It has been found that the larger the optimal elements of the array are allowed to be, the higher the sensitivity reached. Thus, in a trade-off between sensitivity and moderate length (regarding flexibility purposes), the most promising solution has been built. This prototype has been experimentally tested in presence of an artificial aerosol made of PAO (polyalphaolefin) oil and black carbon inclusions of a size of 0.2 µm. As a result, potentials for developing a measurement procedure by means of changes in the characteristic parameters of the antenna led by different concentration levels of suspended particles in the surrounding medium are shown. In this manner, a local mapping of polluted levels could be developed in an easy, real-time, and flexible procedure.

Synthesis, spectroscopy and QM/MM simulations of a biomimetic ultrafast light-driven molecular motor.

A molecular motor potentially performing a continuous unidirectional rotation is studied by a multidisciplinary approach including organic synthesis, transient spectroscopy and excited state trajectory calculations. A stereogenic center was introduced in the N-alkylated indanylidene-pyrroline Schiff base framework of a previously investigated light-driven molecular switch in order to achieve the unidirectional C[double bond, length as m-dash]C rotary motion typical of Feringa's motor. Here we report that the specific substitution pattern of the designed chiral molecule must critically determine the unidirectional efficiency of the light-induced rotary motion. More specifically, we find that a stereogenic center containing a methyl group and a hydrogen atom as substituents does not create a differential steric effect large enough to fully direct the motion in either the clockwise or counterclockwise direction especially along the E→Z coordinate. However, due to the documented ultrafast character and electronic circular dichroism activity of the investigated system, we find that it provides the basis for development of a novel generation of rotary motors with a biomimetic framework and operating on a picosecond time scale.

TLR4-Binding DNA Aptamers Show a Protective Effect against Acute Stroke in Animal Models.

Since Toll-like receptor 4 (TLR4) mediates brain damage after stroke, development of TLR4 antagonists is a promising therapeutic strategy for this disease. Our aim was to generate TLR4-blocking DNA aptamers to be used for stroke treatment. From a random oligonucleotide pool, we identified two aptamers (ApTLR#1R, ApTLR#4F) with high affinity for human TLR4 by systematic evolution of ligands by exponential enrichment (SELEX). Optimized truncated forms (ApTLR#1RT, ApTLR#4FT) were obtained. Our data demonstrate specific binding of both aptamers to human TLR4 as well as a TLR4 antagonistic effect. ApTLR#4F and ApTLR#4FT showed a long-lasting protective effect against brain injury induced by middle cerebral artery occlusion (MCAO), an effect that was absent in TLR4-deficient mice. Similar effects were obtained in other MCAO models, including in rat. Additionally, efficacy of ApTLR#4FT in a model of brain ischemia-reperfusion in rat supports the use of this aptamer in patients undergoing artery recanalization induced by pharmacological or mechanical interventions. The absence of major toxicology aspects and the good safety profile of the aptamers further encourage their future clinical positioning for stroke therapy and possibly other diseases in which TLR4 plays a deleterious role.

How Methylation Modifies the Photophysics of the Native All- trans-Retinal Protonated Schiff Base: A CASPT2/MD Study in Gas Phase and in Methanol.

A comparison between the free-energy surfaces of the all- trans-retinal protonated Schiff base (RPSB) and its 10-methylated derivative in gas phase and methanol solution is performed at CASSCF//CASSCF and CASPT2//CASSCF levels. Solvent effects were included using the average solvent electrostatic potential from molecular dynamics method. This is a QM/MM (quantum mechanics/molecular mechanics) method that makes use of the mean field approximation. It is found that the methyl group bonded to C10 produces noticeable changes in the solution free-energy profile of the S1 excited state, mainly in the relative stability of the minimum energy conical intersections (MECIs) with respect to the Franck-Condon (FC) point. The conical intersections yielding the 9- cis and 11- cis isomers are stabilized while that yielding the 13- cis isomer is destabilized; in fact, it becomes inaccessible by excitation to S1. Furthermore, the planar S1 minimum is not present in the methylated compound. The solvent notably stabilizes the S2 excited state at the FC geometry. Therefore, if the S2 state has an effect on the photoisomerization dynamics, it must be because it permits the RPSB population to branch around the FC point. All these changes combine to speed up the photoisomerization in the 10-methylated compound with respect to the native compound.

A new QM/MM method oriented to the study of ionic liquids.

The interest on room temperature ionic liquids has grown in the last decades because of their use as all-purpose solvent and their low environmental impact. In the present work, a new theoretical procedure is developed to study pure ionic liquids within the framework of the quantum mechanics/molecular mechanics method. Each type of ion (cation or anion) is considered as an independent entity quantum mechanically described that follows a differentiated path in the liquid. The method permits, through an iterative procedure, the full coupling between the polarized charge distribution of the ions and the liquid structure around them. The procedure has been tested with 1-ethyl-3-methylimidazolium tetrafluoroborate. It was found that, similar to non-polar liquids and as a consequence of the low value of the reaction field, the cation and anion charge distributions are hardly polarized by the rest of molecules in the liquid. Their structure is characterized by an alternance between anion and cation shells as evidenced by the coincidence of the first maximum of the anion-anion and cation-cation radial distribution functions with the first minimum of the anion-cation. Some degree of stacking between the cations is also found.

eIF4F complex disruption causes protein synthesis inhibition during hypoxia in nerve growth factor (NGF)-differentiated PC12 cells.

Poor oxygenation (hypoxia) influences important physiological and pathological situations, including development, ischemia, stroke and cancer. Hypoxia induces protein synthesis inhibition that is primarily regulated at the level of initiation step. This regulation generally takes place at two stages, the phosphorylation of the subunit α of the eukaryotic initiation factor (eIF) 2 and the inhibition of the eIF4F complex availability by dephosphorylation of the inhibitory protein 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1). The contribution of each of them is mainly dependent of the extent of the oxygen deprivation. We have evaluated the regulation of hypoxia-induced translation inhibition in nerve growth factor (NGF)-differentiated PC12 cells subjected to a low oxygen concentration (0.1%) at several times. Our findings indicate that protein synthesis inhibition occurs primarily by the disruption of eIF4F complex through 4E-BP1 dephosphorylation, which is produced by the inhibition of the mammalian target of rapamycin (mTOR) activity via the activation of REDD1 (regulated in development and DNA damage 1) protein in a hypoxia-inducible factor 1 (HIF1)-dependent manner, as well as the translocation of eIF4E to the nucleus. In addition, this mechanism is reinforced by the increase in 4E-BP1 levels, mainly at prolonged times of hypoxia.

A Theoretical Study of Solvent Effects on the Structure and UV-vis Spectroscopy of 3-Hydroxyflavone (3-HF) and Some Simplified Molecular Models.

Solvent effects on the UV-vis spectra of 3-hydroxyflavone and other structurally related molecules (3-hydroxychromen-4-one, 3-hydroxy-4-pyrone, and 4-pyrone) have been studied by combining time-dependent density functional theory (TDDFT) and the polarizable continuum method (PCM). Among the first five excited states of the four considered molecules, electronic states of n → π* and π → π* nature appear. In general, the stability of the n → π* states decreases as the π space becomes larger in such a way that only for 4-pyrone and 3-hydroxy-4-pyrone are they the first excited states. In addition, they become less stabilized in ethanol solution than the ground state, and this causes blueshift transitions in solution. The opposite trend is found for the π → π* excited states. They are less energetic with the π-system size and when passing from gas phase to solution. The solvent shift also depends strongly on the size of the π systems and on the formation of an intramolecular hydrogen bond; thus, it decreases when going from 4-pyrone to 3-hydroxyflavone. The performance of the three versions (cLR, cLR2, and IBSF) of the specific-state PCM method in predicting transition energies are compared.

Enzyme-Linked Oligonucleotide Assay (ELONA).

Aptamers are single-stranded oligonucleotides able to recognize a target with high affinity and specificity. Aptamers are used in different diagnostics applications, highlighting, among all, variations of the traditional enzyme-linked immunosorbent assay (ELISA). In this chapter, we show the procedures for the development of two types of indirect ELONA: a sandwich ELONA and a direct ELONA coupled to either real-time quantitative PCR as a direct and sensitive readout.

An Aptamer against MNK1 for Non-Small Cell Lung Cancer Treatment.

Lung cancer is the leading cause of cancer-related death worldwide. Its late diagnosis and consequently poor survival make necessary the search for new therapeutic targets. The mitogen-activated protein kinase (MAPK)-interacting kinase 1 (MNK1) is overexpressed in lung cancer and correlates with poor overall survival in non-small cell lung cancer (NSCLC) patients. The previously identified and optimized aptamer from our laboratory against MNK1, apMNKQ2, showed promising results as an antitumor drug in breast cancer in vitro and in vivo. Thus, the present study shows the antitumor potential of apMNKQ2 in another type of cancer where MNK1 plays a significant role, such as NSCLC. The effect of apMNKQ2 in lung cancer was studied with viability, toxicity, clonogenic, migration, invasion, and in vivo efficacy assays. Our results show that apMNKQ2 arrests the cell cycle and reduces viability, colony formation, migration, invasion, and epithelial-mesenchymal transition (EMT) processes in NSCLC cells. In addition, apMNKQ2 reduces tumor growth in an A549-cell line NSCLC xenograft model. In summary, targeting MNK1 with a specific aptamer may provide an innovative strategy for lung cancer treatment.

Selection and characterization of DNA aptamers for highly selective recognition of the major allergen of olive pollen Ole e 1.

In this study, single-stranded DNA aptamers with binding affinity to Ole e 1, the major allergen of olive pollen, were selected using systematic evolution of ligands by exponential enrichment (SELEX) method. Binding of the aptamers was firstly established by enzyme-linked oligonucleotide assay (ELONA) and aptaprecipitation assays. Additionally, aptamer-modified monolithic capillary chromatography was used in order to evaluate the recognition of this allergenic protein against other non-target proteins. The results indicated that AptOle1#6 was the aptamer that provided the highest affinity for Ole e 1. The selected aptamer showed good selective recognition of this protein, being not able to retain other non-target proteins (HSA, cyt c, and other pollen protein such as Ole e 9). The feasibility of the affinity monolithic column was demonstrated by selective recognition of Ole e 1 in an allergy skin test. The stability and reproducibility of this monolithic column was suitable, with relative standard deviations (RSDs) in retention times and peak area values of 7.8 and 9.3%, respectively (column-to-column reproducibility). This is the first study that describes the design of an efficient DNA aptamer for this relevant allergen.

Potential Therapeutic Use of Aptamers against HAT1 in Lung Cancer.

Lung cancer is one of the leading causes of death worldwide and the most common of all cancer types. Histone acetyltransferase 1 (HAT1) has attracted increasing interest as a potential therapeutic target due to its involvement in multiple pathologies, including cancer. Aptamers are single-stranded RNA or DNA molecules whose three-dimensional structure allows them to bind to a target molecule with high specificity and affinity, thus making them exceptional candidates for use as diagnostic or therapeutic tools. In this work, aptamers against HAT1 were obtained, subsequently characterized, and optimized, showing high affinity and specificity for HAT1 and the ability to inhibit acetyltransferase activity in vitro. Of those tested, the apHAT610 aptamer reduced cell viability, induced apoptosis and cell cycle arrest, and inhibited colony formation in lung cancer cell lines. All these results indicate that the apHAT610 aptamer is a potential drug for the treatment of lung cancer.

Improved demonstration of immunohistochemical prognostic markers for survival in follicular lymphoma cells.

Follicular lymphoma (FL) is one of the most common forms of the low-grade non-Hodgkin's lymphoma in adults, with a characteristic translocation, t(14;18)(q32;q21) that deregulates the expression of the BCL2 gene. The clinical course of FL patients is variable, whereby a subset of patients survive for long periods even without relapses, whereas the majority have frequent relapses with shorter survival. We have analyzed a series of 186 FLs, studying the correlation between clinical outcome and the tumor cell expression of a set of immunohistochemical markers, using an automated procedure for tissue microarrays to reduce the subjectivity of scoring. The results identified several markers associated with differences in overall survival (OS) in univariate analyses, such as Cyclin E, Mdm2, CD10, p21, IgD, Bcl-xL, CD30, and E2F6. Cases with a higher level of expression of Cyclin E, Mdm2, p21, IgD, Bcl-xL, CD30, and E2F6 were associated with a significantly shorter OS. On the other hand, strong CD10 expression was linked to a significantly better outcome. A Cox model was then constructed, integrating the Follicular Lymphoma International Prognostic Index (FLIPI) score and a restricted selection of three immunohistochemical markers: Cyclin E, Mdm2, and CD10 expression. A potentially useful finding is that the integrated FLIPI plus immunohistochemical model can be used to identify a subset of 26 patients (almost 20% of the total series), with a survival probability of 100% at 5 years. This not only confirms that a group of FL cases may have a very good clinical course, but also indicates that this group can be identified using this integrated clinical and immunohistochemical approach.

Study on the conformational equilibrium of the alanine dipeptide in water solution by using the averaged solvent electrostatic potential from molecular dynamics methodology.

The ASEP/MD method has been employed for studying the solvent effect on the conformational equilibrium of the alanine dipeptide in water solution. MP2 and density functional theory (DFT) levels of theory were used and results were compared. While in gas phase cyclic structures showing intramolecular hydrogen bonds were found to be the most stable, the stability order is reversed in water solution. Intermolecular interaction with the solvent causes the predominance of extended structures as the stabilizing contacts dipeptide-water are favoured. Free-energy differences in solution were calculated and PPII, α(R), and C5 conformers were identified as the most stable at MP2 level. Experimental data from Raman and IR techniques show discrepancies about the relative abundance of α(R) y C5, our results support the Raman data. The DFT level of theory agrees with MP2 in the location and stability of PPII and α(R) forms but fails in the location of C5. MP2 results suggest the possibility of finding traces of C7eq conformer in water solution, in agreement with recent experiments.

DNA Aptamers against Vaccinia-Related Kinase (VRK) 1 Block Proliferation in MCF7 Breast Cancer Cells.

Vaccinia-related kinase (VRK) 1 is a serin/threonine kinase that plays an important role in DNA damage response (DDR), phosphorylating some proteins involved in this process such as 53BP1, NBS1 or H2AX, and in the cell cycle progression. In addition, VRK1 is overexpressed in many cancer types and its correlation with poor prognosis has been determined, showing VRK1 as a new therapeutic target in oncology. Using in vitro selection, high-affinity DNA aptamers to VRK1 were selected from a library of ssDNA. Selection was monitored using the enzyme-linked oligonucleotide assay (ELONA), and the selected aptamer population was cloned and sequenced. Three aptamers were selected and characterized. These aptamers recognized the protein kinase VRK1 with an affinity in the nanomolar range and showed a high sensibility. Moreover, the treatment of the MCF7 breast cell line with these aptamers resulted in a decrease in cyclin D1 levels, and an inhibition of cell cycle progression by G1 phase arrest, which induced apoptosis in cells. These results suggest that these aptamers are specific inhibitors of VRK1 that might be developed as potential drugs for the treatment of cancer.

Radiofrequency at 2.45 GHz increases toxicity, pro-inflammatory and pre-apoptotic activity caused by black carbon in the RAW 264.7 macrophage cell line.

Environmental factors such as air pollution by particles and/or electromagnetic fields (EMFs) are studied as harmful agents for human health. We analyzed whether the combined action of EMF with fine and coarse black carbon (BC) particles induced cell damage and inflammatory response in RAW 264.7 cell line macrophages exposed to 2.45 GHz in a gigahertz transverse electromagnetic (GTEM) chamber at sub-thermal specific absorption rate (SAR) levels. Radiofrequency (RF) dramatically increased BC-induced toxicity at high doses in the first 24 h and toxicity levels remained high 72 h later for all doses. The increase in macrophage phagocytosis induced after 24 h of RF and the high nitrite levels obtained by stimulation with lipopolysaccharide (LPS) endotoxin 24 and 72 h after radiation exposure suggests a prolongation of the innate and inflammatory immune response. The increase of proinflammatory cytokines tumor necrosis factor-α, after 24 h, and of interleukin-1ß and caspase-3, after 72 h, indicated activation of the pro-inflammatory response and the apoptosis pathways through the combined effect of radiation and BC. Our results indicate that the interaction of BC and RF modifies macrophage immune response, activates apoptosis, and accelerates cell toxicity, by which it can activate the induction of hypersensitivity reactions and autoimmune disorders.

DNA aptamers targeting Leishmania infantum H3 protein as potential diagnostic tools.

Leishmaniasis is a disease caused by a parasite of the genus Leishmania that affects millions of people worldwide. These parasites are characterized by the presence of a DNA-containing granule, the kinetoplastid, located in the single mitochondrion at the base of the cell's flagellum. Interestingly, these flagellates do not condense chromatin during mitosis, possibly due to the specific molecular features of their histones. Although histones are extremely conserved proteins, kinetoplastid core histone sequences diverge significantly from those of higher eukaryotes. This divergence makes kinetoplastid core histones potential diagnostic and/or therapeutic targets. Aptamers are short single-stranded nucleic acids that are able to recognize target molecules with high affinity and specificity. Their binding capacity is a consequence of the particular three-dimensional structure acquired depending on their sequence. These molecules are currently used for detection, diagnosis and therapeutic purpose. Starting from a previously obtained ssDNA aptamer population against rLiH3 protein we have isolated two individual aptamers, AptLiH3#4 and AptLiH3#10. Next, we have performed ELONA, Western blot and slot blot assays to establish aptamer specificity and affinity for LiH3 histone. In addition, ELONA assays using peptides corresponding to overlapped sequences of LiH3 were made to map the aptamers:LiH3 interaction. Finally, different assays using aptamers were performed in order to evaluate the possibility of using these aptamers as sensing molecule to recognize the endogenous protein LiH3. Our results indicate that both aptamers have high affinity and specificity for the target and are able to detect the endogenous LiH3 histone protein in promastigotes lysates. In silico analysis reveals that these two aptamers have different potential secondary structure among them, however, both of them are able to recognize the same peptide sequences present in the protein. In conclusion, our findings indicate that these aptamers could be used for LiH3 histone detection and, in consequence, as potential biosensing molecules in a diagnostic tool for leishmaniasis.

Subcutaneous Immunization of Leishmania HSP70-II Null Mutant Line Reduces the Severity of the Experimental Visceral Leishmaniasis in BALB/c Mice.

Leishmania infantum parasites cause a severe form of visceral leishmaniasis in human and viscerocutaneous leishmaniasis in dogs. Recently, we reported that immunization with an attenuated L. infantum cell line, lacking the hsp70-II gene, protects against the development of murine cutaneous leishmaniasis. In this work, we analyzed the vaccine potential of this cell line towards the long-term protection against murine visceral leishmaniasis. This model shows an organ-dependent evolution of the disease. The infection can resolve in the liver but chronically affect spleen and bone marrow. Twelve weeks after subcutaneous administration of attenuated L. infantum, Bagg Albino (BALB/c) mice were challenged with infective L. infantum parasites expressing the luciferase-encoding gene. Combining in vivo bioimaging techniques with limiting dilution experiments, we report that, in the initial phase of the disease, vaccinated animals presented lower parasite loads than unvaccinated animals. A reduction of the severity of liver damage was also detected. Protection was associated with the induction of rapid parasite-specific IFN-γ production by CD4+ and CD8+ T cells. However, the vaccine was unable to control the chronic phase of the disease, since we did not find differences in the parasite burdens nor in the immune response at that time point.

Eukaryotic initiation factors (eIF) 2alpha and 4E expression, localization, and phosphorylation in brain tumors.

Increased protein synthesis is regulated, in part, by two eukaryotic translation initiation factors (eIFs): eIF4E and eIF2alpha. One or both of these factors are often overexpressed in several types of cancer cells; however, no data are available at present regarding eIF4E and eIF2alpha levels in brain tumors. In this study, we analyzed the expression, subcellular localization and phosphorylation states of eIF4E and eIF2alpha in 64 brain tumors (26 meningiomas, 16 oligodendroglial tumors, and 22 astrocytomas) and investigated the correlation with the expression of MIB-1, p53, and cyclin D1 proteins as well. There are significant differences in the phosphorylated eIF4E levels between the tumors studied, being the highest in meningiomas and the lowest in the oligodendroglial tumors. Relative to subcellular localization, eIF4E is frequently found in the nucleus of the oligodendroglial tumors and rarely in the same compartment of the meningiomas, whereas eIF2alpha showed an inverse pattern. Finally, cyclin D1 levels directly correlate with the phosphorylation status of both factors. The different expression, phosphorylation, or/and subcellular distribution of eIF2alpha and eIF4E within the brain types of tumors studied could indicate that different pathways are activated for promoting cell cycle proliferation, for instance, leading to increased cyclin D1 expression.

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis.

Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine-nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E-eIF4G complexes; however, they did not induce eIF2alpha phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E-eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.