Germán Villa - Academia.edu (original) (raw)

Papers by Germán Villa

Methods in Molecular Biology, 2020

Many reliable and reproducible methods exist for manufacturing gold nanoparticles with the desire... more Many reliable and reproducible methods exist for manufacturing gold nanoparticles with the desired and specific compositions, structures, arrangements, and physicochemical properties. In this report, we review the key principles guiding the formation and growth of nanoclusters, their evolution into nanoparticles, and the role and contribution of coatings. We describe a range of imaging methods for characterization of nanoparticles at atomic resolution and a range of spectroscopy methods for structural and physicochemical characterization of such nanoparticles. This chapter concludes with a short review of the emergent applications of nanoparticles in biosciences.

Metallomics, 2021

In this report we investigate the toxicity of the ionophore thiomaltol (Htma) and Cu salts to mel... more In this report we investigate the toxicity of the ionophore thiomaltol (Htma) and Cu salts to melanoma. Divalent metal complexes of thiomaltol display toxicity against A375 melanoma cell culture resulting in a distinct apoptotic response at sub-micromolar concentrations, with toxicity of Cu(tma)2 > Zn(tma)2 >> Ni(tma)2. In metal-chelated media, Htma treatment shows little toxicity, but the combination with supplemental CuCl2, termed Cu/Htma treatment, results in toxicity that increases with suprastoichiometric concentrations of CuCl2 and correlates with the accumulation of intracellular copper. Electron microscopy and confocal laser scanning microscopy of Cu/Htma treated cells shows a rapid accumulation of copper within lysosomes over the course of hours, concurrent with the onset of apoptosis. A buildup of ubiquitinated proteins due to proteasome inhibition is seen on the same timescale and correlates with increases of copper without additional Htma.

International Review of Neurobiology, 2020

Amyloid-β (Aβ) senile plaques and neurofibrillary tangles of tau are generally recognized as the ... more Amyloid-β (Aβ) senile plaques and neurofibrillary tangles of tau are generally recognized as the culprits of Alzheimer's disease (AD) and related dementia. About 25 years ago, the amyloid cascade hypotheses postulated a direct correlation of plaques with the development of AD, and it has been the dominant theory since then. In this period, more than 200 clinical trials focused mainly on targeting components of the Aβ cascade have dramatically failed, some of them in Phase III. With a greater than 99.6% failure rate at a cost of several billion from governments, industry, and private funders, therapeutic strategies targeting amyloid and tau are now under scrutiny. Therefore, it is time to reevaluate alternatives to targeting Aβ and tau as effective therapeutic strategies for AD. The diagnosis of AD is currently based on medical examination of symptoms including tests to assess memory impairment, attention, language, and other thinking skills. This is complemented with brain scans, such as computed tomography, magnetic resonance imaging, or positron emission tomography with the help of imaging probes targeting Aβ or tau deposits. This approach has contributed to the tunnel vision focus on Aβ and tau as the main culprits of AD. However, events upstream of these proteopathies (age-related impaired neuronal bioenergetics, lysosome function, neurotrophic signaling, and neuroinflammation, among others) are almost surely where the development of alternative therapeutic interventions should be targeted. Here, we present the current status of therapeutic candidates targeting diverse mechanisms and strategies including Aβ and tau, proteins involved in Aβ production and trafficking (ApoE, α/β/γ-secretases), neuroinflammation, neurotransmitters, neuroprotective agents antimicrobials, and gene and stem cell therapy. There are currently around 33 compounds in Phase III, 78 in Phase II, and 32 more in Phase I trials. With the current world health crisis of increased dementia in a rapidly aging population, effective AD therapies are desperately needed.

Journal of Materials Science, 2020

Gold clusters protected by 3-MBA ligands (MBA = mercaptobenzoic acid, -SPhCO2H) have attracted re... more Gold clusters protected by 3-MBA ligands (MBA = mercaptobenzoic acid, -SPhCO2H) have attracted recent interest for their unusual structures and advantageous ligand-exchange and bioconjugation properties. Azubel et al. first determined the core structure of an Au68-complex, which was estimated to have 32 ligands (3-MBA groups). To explain the exceptional structure-composition and reaction properties of this complex, and its larger homologs, Tero et al. proposed a “dynamic stabilization” via carboxyl O-H--Au interactions. Herein, we report the first results of an integrated LC/MS analysis of unfractionated samples of gold / 3-MBA clusters, spanning the narrow size range 13.4 to 18.1 kDa. Using high-throughput procedures adapted from bio-macromolecule analyses, we show that integrated capillary HPLC-ESI-MS, based upon aqueous-methanol mobile phases and ion-pairing reverse-phase chromatography, can separate several major components from the nanoclusters mixture that may be difficult to ...

Langmuir, 2018

We report an efficient wet-chemical production of self-assembled gold-copper bimetallic nanoparti... more We report an efficient wet-chemical production of self-assembled gold-copper bimetallic nanoparticles (diameter of ~2 nm) into two-dimensional flexible ribbon-like nanostructures. A direct observation of a layered arrangement of particles into the nanoribbons was provided through highresolution transmission electron microscopy and electron tomography. These nanoribbons showed photoluminesce and an efficient photocatalytic activity for the conversion of 4-nitrophenol. Their thermal stability of the nanoribbons was also measured by in situ heating treatment in the electron microscope, confirming that the self-assembled gold-copper nanoribbons efficiently supported up to 350 °C. The final morphology of the nanoparticles and their ability for self-assembly into flexible nanoribbons was dependent on concentration and ratio of precursors. Therefore, these experimental factors were discussed. Remarkably, the presence of copper was found to be critical to trigger the self-assembly of nanoparticles into ordered layered structures. These results in the synthesis and stability of self-assemblies of metallic nanoparticles present a potential extension of the method to produce materials with catalytic applications.

Laboratory Investigation, 2019

The identification of amyloid-β precursor protein (APP) pathogenic mutations in familial early on... more The identification of amyloid-β precursor protein (APP) pathogenic mutations in familial early onset Alzheimer's disease (AD), along with knowledge that amyloid-β (Aβ) was the principle protein component of senile plaques, led to the establishment of the amyloid cascade hypothesis. Down syndrome substantiated the hypothesis, given an extra copy of the APP gene and invariable AD pathology hallmarks that occur by middle age. An abundance of support for the amyloid cascade hypothesis followed. Prion-like protein misfolding and non-Mendelian transmission of neurotoxicity are among recent areas of investigation. Aβ-targeted clinical trials have been disappointing, with negative results attributed to inadequacies in patient selection, challenges in pharmacology, and incomplete knowledge of the most appropriate target. There is evidence, however, that proof of concept has been achieved, i.e., clearance of Aβ during life, but with no significant changes in cognitive trajectory in AD. Whether the time, effort, and expense of Aβ-targeted therapy will prove valuable will be determined over time, as Aβ-centered clinical trials continue to dominate therapeutic strategies. It seems reasonable to hypothesize that the amyloid cascade is intimately involved in AD, in parallel with disease pathogenesis, but that removal of toxic Aβ is insufficient for an effective disease modification.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2019

For its fundamental relevance, transport of water and glycerol across the erythrocyte membrane ha... more For its fundamental relevance, transport of water and glycerol across the erythrocyte membrane has long been investigated before and after the discovery of aquaporins (AQPs), the membrane proteins responsible for water and glycerol transport. AQP1 is abundantly expressed in the human erythrocyte for maintaining its hydrohomeostasis where AQP3 is also expressed (at a level ~30folds lower than AQP1) facilitating glycerol transport. This research is focused on two of the remaining questions: How permeable is AQP3 to water? What is the glycerol-AQP3 affinity under near-physiological conditions? Through atomistic modelling and large-scale simulations, we found that AQP3 is two to three times more permeable to water than AQP1 and that the glycerol-AQP3 affinity is approximately 500/M. Using these computed values along with the data from the latest literature on AQP1 and on erythrocyte proteomics, we estimated the water and glycerol transport rates across the membrane of an entire erythrocyte. We used these rates to predict the time courses of erythrocyte swelling-shrinking in response to inward and outward osmotic gradients. Experimentally, we monitored the time course of human erythrocytes when subject to an osmotic or glycerol gradient with light scattering in a stopped-flow spectrometer. We observed close agreement between the experimentally measured and the computationally predicted time courses of erythrocytes, which corroborated our computational conclusions on the AQP3 waterpermeability and the glycerol-AQP3 affinity.

Nanoscale, Jan 24, 2018

Altered metabolism of biometals in the brain is a key feature of Alzheimer's disease, and bio... more Altered metabolism of biometals in the brain is a key feature of Alzheimer's disease, and biometal interactions with amyloid-β are linked to amyloid plaque formation. Iron-rich aggregates, including evidence for the mixed-valence iron oxide magnetite, are associated with amyloid plaques. To test the hypothesis that increased chemical reduction of iron, as observed in vitro in the presence of aggregating amyloid-β, may occur at sites of amyloid plaque formation in the human brain, the nanoscale distribution and physicochemical states of biometals, particularly iron, were characterised in isolated amyloid plaque cores from human Alzheimer's disease cases using synchrotron X-ray spectromicroscopy. In situ X-ray magnetic circular dichroism revealed the presence of magnetite: a finding supported by ptychographic observation of an iron oxide crystal with the morphology of biogenic magnetite. The exceptional sensitivity and specificity of X-ray spectromicroscopy, combining chemical...

Journal of neurology, neurosurgery, and psychiatry, Jan 20, 2018

editorial commentary support alterations in brain iron homeostasis with potential consequences of... more editorial commentary support alterations in brain iron homeostasis with potential consequences of high oxidative stress condition in affected neurons as fundamental to AD. competing interests None declared. Provenance and peer review Commissioned; internally peer reviewed.

Microscopy research and technique, Jan 11, 2017

In the current work, irregular morphology of Staphylococcus aureus bacteria has been visualized b... more In the current work, irregular morphology of Staphylococcus aureus bacteria has been visualized by phase retrieval employing off-axis electron holography (EH) and 3D reconstruction electron tomography using high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM). Bacteria interacting with gold nanoparticles (AuNP) acquired a shrunken or irregular shape due to air dehydration processing. STEM imaging shows the attachment of AuNP on the surface of cells and suggests an irregular 3D morphology of the specimen. The phase reconstruction demonstrates that off-axis electron holography can reveal with a single hologram the morphology of the specimen and the distribution of the functionalized AuNPs. In addition, EH reduces significantly the acquisition time and the cumulative radiation damage (in three orders of magnitude) over biological samples in comparison with multiple tilted electron expositions intrinsic to electron tomography, as well as the processing ti...

Biometals in Neurodegenerative Diseases, 2017

Abstract Metal ions are key biologically active components critical to neuronal metabolism. Biome... more Abstract Metal ions are key biologically active components critical to neuronal metabolism. Biometals function as cofactors in cell respiration, enzyme function, antioxidants, oxidative stress, and neuronal signaling in synapses. Neurodegenerative diseases like Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis, dementia with Lewy bodies, Creutzfeldt–Jakob, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron diseases are directly related to aging processes, but recent scientific evidence has further linked brain degeneration and neuronal death to altered levels of some biometals and mitochondrial abnormality. Development of neurodegenerative diseases rely on different factors with many layers of complexity, but with some common features including presence of protein aggregates in brain, biometals misbalance, and mitochondrial dysfunction in neurons. Here, we discuss data that support the interplay and complex implications of different biometals and role of mitochondrial dysfunctions in neurodegenerative processes.

Biochemical and biophysical research communications, May 17, 2017

Amyloid-beta (Aβ) peptides, Aβ40 and the more neurotoxic Aβ42, have been the subject of many rese... more Amyloid-beta (Aβ) peptides, Aβ40 and the more neurotoxic Aβ42, have been the subject of many research efforts for Alzheimer's disease. In two recent independent investigations, the atomistic structure of Aβ42 fibril has been clearly established in the S-shaped conformation consisting of three β-sheets stabilized by salt bridges formed between the Lys28 sidechain and the C-terminus of Ala42. This structure distinctively differs from the long-known structure of Aβ40 in the β-hairpin shaped conformation consisting of two β-sheets. Recent in silico investigations based on all-atom models have reached closer agreement with the in vitro measurements of Aβ40 thermodynamics. In this study, we present an in silico investigation of Aβ42 thermodynamics. Using the established force field parameters in seven sets of all-atom simulations, we examined the stability of small Aβ42 oligomers in physiological saline. We computed the elongation affinity of the S-shaped Aβ42 fibril, reaching agreeme...

CrystEngComm, 2017

Figure S1. Energy Disperse X-Ray Spectroscopy. EDX analysis of the MnO2 nanotubes, left, and MnO2... more Figure S1. Energy Disperse X-Ray Spectroscopy. EDX analysis of the MnO2 nanotubes, left, and MnO2 nanotubes decorated with the Co3O4 nanoparticles, right. EDX spectrum of the MnONT and MnONT+Co3O4, left and right respectively, samples analyzed with UHR-FE-SEM HITACHI S5500. In the left spectrum it was clearly appreciated the presence of the characteristic peaks of Mn (0.64, 5.90 and 6.49 keV), O (0.526 keV) and K (3.31 and 3.59 Electronic Supplementary Material (ESI) for CrystEngComm.

$Research paper thumbnail of Structural damage reduction in protected gold clusters by electron diffraction methods$

Advanced structural and chemical imaging, 2017

The present work explores electron diffraction methods for studying the structure of metallic clu... more The present work explores electron diffraction methods for studying the structure of metallic clusters stabilized with thiol groups, which are susceptible to structural damage caused by electron beam irradiation. There is a compromise between the electron dose used and the size of the clusters since they have small interaction volume with electrons and as a consequence weak reflections in the diffraction patterns. The common approach of recording individual clusters using nanobeam diffraction has the problem of an increased current density. Dosage can be reduced with the use of a smaller condenser aperture and a higher condenser lens excitation, but even with those set ups collection times tend to be high. For that reason, the methods reported herein collects in a faster way diffraction patterns through the scanning across the clusters under nanobeam diffraction mode. In this way, we are able to collect a map of diffraction patterns, in areas with dispersed clusters, with short expo...

The Journal of Physical Chemistry B, 2016

Biomedical Optics Express, 2016

In the present work, we report a dry-based application technique of Au/SiO 2 clouds in powder for... more In the present work, we report a dry-based application technique of Au/SiO 2 clouds in powder for rapid ex vivo adenocarcinoma diagnosis through surface-enhanced Raman scattering (SERS); using low laser power and an integration time of one second. Several characteristic Raman peaks frequently used for the diagnosis of breast adenocarcinoma in the range of the amide III are successfully enhanced by breading the tissue with Au/SiO 2 powder. The SERS activity of these Au/SiO 2 powders is attributed to their rapid rehydration upon contact with the wet tissues, which promotes the formation of gold nanoparticle aggregates. The propensity of the Au/SiO 2 cloud structures to adsorb biomolecules in the vicinity of the gold nanoparticle clusters promotes the necessary conditions for SERS detection. In addition, electron microscopy, together with elemental analysis, have been used to confirm the structure of the new Au/SiO 2 cloud material and to investigate its distribution in breast tissues.

Journal of biophotonics, Jan 27, 2016

CdTe quantum dots (QDs) are widely used in bio-applications due to their size and highly efficien... more CdTe quantum dots (QDs) are widely used in bio-applications due to their size and highly efficient optical properties. However internalization mechanisms thereof for the variety of freshly extracted, not cultivated human cells and their specific molecular interactions remains an open topic for discussion. In this study, we assess the internalization mechanism of CdTe quantum dots (3.3 nm) capped with thioglycolic acid using non cultivated oral epithelial cells obtained from healthy donors. Naked gold nanoparticles (40 nm) were successfully used as nanosensors for surface-enhanced Raman spectroscopy to efficiently identify characteristic Raman peaks, providing new evidence indicating that the first interactions of these QDs with epithelial cells occurred preferentially with aromatic rings and amine groups of amino acid residues and glycans from trans-membrane proteins and cytoskeleton. Using an integrative combination of advanced imaging techniques, including ultra-high resolution SE...

Journal of Applied Toxicology, 2015

The antimicrobial properties of silver nanoparticles (AgNPs) have made these particles one of the... more The antimicrobial properties of silver nanoparticles (AgNPs) have made these particles one of the most used nanomaterials in consumer products. Therefore, an understanding of the interactions (unwanted toxicity) between nanoparticles and human cells is of significant interest. The aim of this study was to assess the in vitro cytotoxicity effects of silver nanoclusters (AgNC, &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 2 nm diameter) on peripheral blood mononuclear cells (PBMC). Using flow cytometry and comet assay methods, we demonstrate that exposure of PBMC to AgNC induced intracellular reactive oxygen species (ROS) generation, DNA damage and apoptosis at 3, 6 and 12 h, with a dose-dependent response (0.1, 1, 3, 5 and 30 µg ml(-1)). Advanced electron microscopy imaging of complete and ultrathin-sections of PBMC confirmed the cytotoxic effects and cell damage caused by AgNC. The present study showed that AgNC produced without coating agents induced significant cytotoxic effects on PBMC owing to their high aspect ratio and active surface area, even at much lower concentrations (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;1 µg ml(-1)) than those applied in previous studies, resembling what would occur under real exposure conditions to nanosilver-functionalized consumer products. Copyright © 2015 John Wiley &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp; Sons, Ltd.