Surface Bragg gratings of proteins patterned on integrated waveguides for (bio)chemical analysis.

The incorporation of biomacromolecules onto silicon waveguiding microstructures constitutes a growing trend that pushes towards compact and miniaturized biosensing systems. This paper presents the integration of one-dimensional periodic nanostructures of proteins on the surface of micrometric silicon waveguides for transducing binding events between biomacromolecules. The study demonstrates this new bioanalytical principle by experimental results and theoretical calculations, and proves that rib waveguides (1--1.6-µm width) together with protein gratings (495--515-nm period) display suitable spectral responses for this optical biosensing system. Protein assemblies of bovine serum albumin are fabricated on the surface of silicon nitride waveguides, characterized by electron microscopy, and their response is measured by optical frequency domain reflectometry along the fabrication process and the subsequent stages of the biorecognition assays. Detection and quantification limits of 0.3 and 3.7 µg·mL-1, respectively, of specific antibodies are inferred from experimental dose-response curves. Among other interesting features, the results of this study point towards new miniaturized and integrated sensors for label-free bioanalysis.

Analytical probing of membranotropic effects of antimicrobial copper nanoparticles on lipid vesicles as membrane models.

Copper nanoparticles (CuNPs) are antimicrobial agents that are increasingly being used in several real-life goods. However, concerns are arising about their potential toxicity and thus, appropriate legislation is being issued in various countries. In vitro exploration of the permeability and the distribution of nanoparticles in cell membranes should be explored as the first step towards the investigation of the toxicity mechanisms of metal nanoantimicrobials. In this work, phosphatidylcholine-based large unilamellar vesicles have been explored as mimics of cellular membranes to investigate the effect of ultra-small CuNPs on the physicochemical features of phospholipid membranes. 4 nm-sized CuNPs were synthesized by a wet-chemical route that involves glutathione as a stabilizer, with further characterization by UV-vis absorption spectroscopy, fluorescence spectroscopy, transmission electron microscopy, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy. Two fluorescent membrane probes bearing naphthalene moieties (laurdan and prodan) were used to monitor the bilayer structure and dynamics, as well as to demonstrate the strong membranotropic effects of CuNPs. The fluorescence spectroscopic studies were supported by dynamic light scattering (DLS) measurements and the calcein leakage assay. Additionally, the degree of perturbation of the phospholipid bilayer by CuNPs was compared against that of Cu2+ ions, the latter resulting in negligible effects. The findings suggested that CuNPs are able to damage the phospholipid membranes, leading to their agglomeration or disruption.

UIBVFEDPlus-Light: Virtual facial expression dataset with lighting.

It is well-known that lighting conditions have an important influence on the automatic recognition of human expressions. Although the impact of lighting on the perception of emotions has been studied in different works, databases of facial expressions do not consider intentional lighting. In this work, a new database of facial expressions performed by virtual characters with four different lighting configurations is presented. This database, named UIBVFEDPlus-Light, is an extension of the previously published UIBVFED virtual facial expression dataset. It includes 100 characters, four lighting configurations and a software application that allows one to interactively visualize the expressions, and manage their intensity and lighting condition. Also, an experience of use is described to show how this work can raise new challenges to facial expression and emotion recognition techniques under usual lighting environments. Thus, opening new study perspectives in this area.

Semenogelins Armed in Zn(II) and Cu(II): May Bioinorganic Chemistry Help Nature to Cope with Enterococcus faecalis?

Proteolytic degradation of semenogelins, the most abundant proteins from human semen, results in the formation of 26- and 29-amino acid peptides (SgIIA and SgI-29, respectively), which share a common 15 amino acid fragment (Sg-15). All three ligands are effective Zn(II) and Cu(II) binders; in solution, a variety of differently metalated species exist in equilibrium, with the [NH2, 3Nim] donor set prevailing at physiological pH in the case of both metals. For the first time, the Cu(II)-induced antimicrobial activity of Sg-15 against Enterococcus faecalis is shown. In the case of the two native semenogelin fragment metal complexes, the strong local positive charge in the metal-bound HH motif correlates well with their antimicrobial activity. A careful analysis of semenogelins' metal coordination behavior reveals two facts: (i) The histamine-like Cu(II) binding mode of SgI-29 strongly increases the stability of such a complex below pH 6 (with respect to the non-histamine-like binding of SgIIA), while in the case of the SgI-29 Zn(II)-histamine-like species, the stability enhancement is less pronounced. (ii) The HH sequence is a more tempting site for Cu(II) ions than the HXH one.

Biomimetic Dispersive Solid-Phase Microextraction: A Novel Concept for High-Throughput Estimation of Human Oral Absorption of Organic Compounds.

There is a quest for a novel in vitro analytical methodology that is properly validated for the prediction of human oral absorption and bioaccumulation of organic compounds with no need of animal models. The traditional log P parameter might not serve to predict bioparameters accurately inasmuch as it merely accounts for the hydrophobicity of the compound, but the actual interaction with the components of eukaryotic cells is neglected. This contribution proposes for the first time a novel biomimetic microextraction approach capitalized on immobilized phosphatidylcholine as a plasma membrane surrogate onto organic polymeric sorptive phases for the estimation of human intestinal effective permeability of a number of pharmaceuticals that are also deemed contaminants of emerging concern in environmental settings. A comprehensive exploration of the conformation of the lipid structure onto the surfaces is undertaken so as to discriminate the generation of either lipid monolayers or bilayers or the attachment of lipid nanovesicles. The experimentally obtained biomimetic extraction data is proven to be a superb parameter against other molecular descriptors for the development of reliable prediction models of human jejunum permeability with R2 = 0.76, but the incorporation of log D and the number of aromatic rings in multiple linear regression equations enabled improved correlations up to R2 = 0.88. This work is expected to open new avenues for expeditious in vitro screening methods for oral absorption of organic contaminants of emerging concern in human exposomics.

CH vs. HC-Promiscuous Metal Sponges in Antimicrobial Peptides and Metallophores.

Histidine and cysteine residues, with their imidazole and thiol moieties that deprotonate at approximately physiological pH values, are primary binding sites for Zn(II), Ni(II) and Fe(II) ions and are thus ubiquitous both in peptidic metallophores and in antimicrobial peptides that may use nutritional immunity as a way to limit pathogenicity during infection. We focus on metal complex solution equilibria of model sequences encompassing Cys-His and His-Cys motifs, showing that the position of histidine and cysteine residues in the sequence has a crucial impact on its coordination properties. CH and HC motifs occur as many as 411 times in the antimicrobial peptide database, while similar CC and HH regions are found 348 and 94 times, respectively. Complex stabilities increase in the series Fe(II) < Ni(II) < Zn(II), with Zn(II) complexes dominating at physiological pH, and Ni(II) ones-above pH 9. The stabilities of Zn(II) complexes with Ac-ACHA-NH2 and Ac-AHCA-NH2 are comparable, and a similar tendency is observed for Fe(II), while in the case of Ni(II), the order of Cys and His does matter-complexes in which the metal is anchored on the third Cys (Ac-AHCA-NH2) are thermodynamically stronger than those where Cys is in position two (Ac-ACHA-NH2) at basic pH, at which point amides start to take part in the binding. Cysteine residues are much better Zn(II)-anchoring sites than histidines; Zn(II) clearly prefers the Cys-Cys type of ligands to Cys-His and His-Cys ones. In the case of His- and Cys-containing peptides, non-binding residues may have an impact on the stability of Ni(II) complexes, most likely protecting the central Ni(II) atom from interacting with solvent molecules.

Patterned Biolayers of Protein Antigens for Label-Free Biosensing in Cow Milk Allergy.

This paper focuses on creating one-dimensional diffractive grooved structures of antigen proteins on glass substrates for the label-free detection of antibodies to dairy allergens. In particular, the fabrication of protein structures is carried out by combining microcontact printing with physisorption, imines coupling, and thiol-ene click chemistry. The work first sets up these patterning methods and discusses and compares the main aspects involved in them (structure, biolayer thickness, functionality, stability). Homogeneous periodic submicron structures of proteins are created and characterized by diffractive measurements, AFM, FESEM, and fluorescence scanning. Then, this patterning method is applied to proteins involved in cow milk allergy, and the resulting structures are implemented as optical transducers to sense specific immunoglobulins G. In particular, gratings of bovine serum albumin, casein, and ß-lactoglobulin are created and assessed, reaching limits of detection in the range of 30-45 ng·mL-1 of unlabeled antibodies by diffractive biosensing.

Denaturing for Nanoarchitectonics: Local and Periodic UV-Laser Photodeactivation of Protein Biolayers to Create Functional Patterns for Biosensing.

The nanostructuration of biolayers has become a paradigm for exploiting nanoscopic light-matter phenomena for biosensing, among other biomedical purposes. In this work, we present a photopatterning method to create periodic structures of biomacromolecules based on a local and periodic mild denaturation of protein biolayers mediated by UV-laser irradiation. These nanostructures are constituted by a periodic modulation of the protein activity, so they are free of topographic and compositional changes along the pattern. Herein, we introduce the approach, explore the patterning parameters, characterize the resulting structures, and assess their overall homogeneity. This UV-based patterning principle has proven to be an easy, cost-effective, and fast way to fabricate large areas of homogeneous one-dimensional protein patterns (2 min, 15 × 1.2 mm, relative standard deviation ≃ 16%). This work also investigates the implementation of these protein patterns as transducers for diffractive biosensing. Using a model immunoassay, these patterns have demonstrated negligible signal contributions from non-specific bindings and comparable experimental limits of detection in buffer media and in human serum (53 and 36 ng·mL-1 of unlabeled IgG, respectively).

Large-Scale Nanogrooved Photonic Crystals for Label-Free Biosensing by Guided-Mode Resonance.

We have developed large-scale one-dimensional photonic crystals from standard recordable Blu-ray disks, tailored to sense unlabeled biorecognition events on their surface. These materials rely on coating, with layers of 80 nm of titanium oxide, nanogrooved polycarbonate plates obtained from regular disks. As a result, they present guided-mode resonances that we have demonstrated that can be exploited to quantify biorecognition events by means of the bandgap positions in the transmission spectra. These photonic crystals have displayed well-correlated dose-response curves in immunoassays to quantify IgGs, C-reactive protein, and lactate dehydrogenase. The detection limit reached is 16 ng/mL, 2µg/mL, and 18 ng/mL, respectively. Herein we describe the experimental procedures and methods to fabricate and functionalize these photonic crystals, perform immunoassays on them, set up an optical system to measure their response, and process the resulting data to perform bioanalytical determinations in label-free format.

Understanding How CNNs Recognize Facial Expressions: A Case Study with LIME and CEM.

Recognizing facial expressions has been a persistent goal in the scientific community. Since the rise of artificial intelligence, convolutional neural networks (CNN) have become popular to recognize facial expressions, as images can be directly used as input. Current CNN models can achieve high recognition rates, but they give no clue about their reasoning process. Explainable artificial intelligence (XAI) has been developed as a means to help to interpret the results obtained by machine learning models. When dealing with images, one of the most-used XAI techniques is LIME. LIME highlights the areas of the image that contribute to a classification. As an alternative to LIME, the CEM method appeared, providing explanations in a way that is natural for human classification: besides highlighting what is sufficient to justify a classification, it also identifies what should be absent to maintain it and to distinguish it from another classification. This study presents the results of comparing LIME and CEM applied over complex images such as facial expression images. While CEM could be used to explain the results on images described with a reduced number of features, LIME would be the method of choice when dealing with images described with a huge number of features.

Uracil Derivatives for Halogen-Bonded Cocrystals.

Among non-covalent interactions, halogen bonding is emerging as a new powerful tool for supramolecular self-assembly. Here, along with a green and effective method, we report three new halogen-bonded cocrystals containing uracil derivatives and 1,2,4,5-tetrafluoro-3,6-diiodobenzene as X-bond donor coformer. These multicomponent solids were prepared both by solvent-drop grinding and solution methods and further characterized by powder and single-crystal X-ray diffraction, Fourier-transformed infrared spectroscopy, and thermal methods (TGA-DSC). In order to study the relative importance of hydrogen versus halogen bonds in the crystal packing, computational methods were applied.

Deciphering the H-Bonding Preference on Nucleoside Molecular Recognition through Model Copper(II) Compounds.

The synthetic nucleoside acyclovir is considered an outstanding model of the natural nucleoside guanosine. With the purpose of deepening on the influence and nature of non-covalent interactions regarding molecular recognition patterns, three novel Cu(II) complexes, involving acyclovir (acv) and the ligand receptor N-(2-hydroxyethyl)ethylenediamine (hen), have been synthesized and thoroughly characterized. The three novel compounds introduce none, one or two acyclovir molecules, respectively. Molecular recognition has been evaluated using single crystal X-ray diffraction. Furthermore, theoretical calculations and other physical methods such as thermogravimetric analysis, infrared and UV-Vis spectroscopy, electron paramagnetic resonance and magnetic measurements have been used. Theoretical calculations are in line with experimental results, supporting the relevance of the [metal-N7(acv) + H-bond] molecular recognition pattern. It was also shown that (hen)O-H group is used as preferred H-donor when it is found within the basal coordination plane, since the higher polarity of the terminal (hen)O-H versus the N-H group favours its implication. Otherwise, when (hen)O-H occupies the distal coordination site, (hen)N-H groups can take over.

Silver(I)-mediated base pairing in DNA involving the artificial nucleobase 7,8-dihydro-8-oxo-1,N6-ethenoadenine.

The artificial nucleobase 7,8-dihydro-8-oxo-1,N6-ethenoadenine (X) was investigated with respect to its ability to engage in Ag(I)-mediated base pairing in DNA. Spectroscopic data indicate the formation of dinuclear X-Ag(I)2-X homo base pairs and mononuclear X-Ag(I)-C base pairs (C, cytosine). Density functional theory calculations and molecular dynamics simulations indicate that the nucleobase changes from its lactam tautomeric form prior to the formation of the Ag(I)-mediated base pair to the lactim form after the incorporation of the Ag(I) ions. Fluorescence spectroscopy indicates that the two Ag(I) ions of the homo base pair are incorporated sequentially. Isothermal titration calorimetry confirms that the affinity of one of the Ag(I) ions is about tenfold higher than that of the other Ag(I) ion. The computational analysis by means of density functional theory confirms a much larger reaction energy for the incorporation of the first Ag(I) ion. The thermal stabilization upon the formation of the dinuclear Ag(I)-mediated homo base pair exceeds the one previously observed for the closely related nucleobase 1,N6-ethenoadenine by far, despite very similar structures. This additional stabilization may stem from the presence of water molecules engaged in hydrogen bonding with the additional oxygen atom of the artificial nucleobase X. The highly stabilizing Ag(I)-mediated base pair is a valuable addition to established dinuclear metal-mediated base pairs.

BIO bragg gratings on microfibers for label-free biosensing.

Discovering nanoscale phenomena to sense biorecognition events introduces new perspectives to exploit nanoscience and nanotechnology for bioanalytical purposes. Here we present Bio Bragg Gratings (BBGs), a novel biosensing approach that consists of diffractive structures of protein bioreceptors patterned on the surface of optical waveguides, and tailored to transduce the magnitude of biorecognition assays into the intensity of single peaks in the reflection spectrum. This work addresses the design, fabrication, and optimization of this system by both theoretical and experimental studies to explore the fundamental physicochemical parameters involved. Functional biomolecular gratings are fabricated by microcontact printing on the surface of tapered optical microfibers, and their structural features were characterized. The transduction principle is experimentally demonstrated, and its quantitative bioanalytical prospects are assessed in a representative immunoassay, based on patterned protein probes and selective IgG targets, in label-free conditions. This biosensing system involves appealing perspectives to avoid unwanted signal contributions from non-specific binding, herein investigated in human serum samples. The work also proves how the optical response of the system can be easily tuned, and it provides insights into the relevance of this feature to conceive multiplexed BBG systems capable to perform multiple label-free biorecognition assays in a single device.

CoNTe: A Core Network Temporal Blockchain for 5G.

Virtual Network Functions allow the effective separation between hardware and network functionality, a strong paradigm shift from previously tightly integrated monolithic, vendor, and technology dependent deployments. In this virtualized paradigm, all aspects of network operations can be made to deploy on demand, dynamically scale, as well as be shared and interworked in ways that mirror behaviors of general cloud computing. To date, although seeing rising demand, distributed ledger technology remains largely incompatible in such elastic deployments, by its nature as functioning as an immutable record store. This work focuses on the structural incompatibility of current blockchain designs and proposes a novel, temporal blockchain design built atop federated byzantine agreement, which has the ability to dynamically scale and be packaged as a Virtual Network Function (VNF) for the 5G Core.

In-vitro prediction of the membranotropic action of emerging organic contaminants using a liposome-based multidisciplinary approach.

According to ISO 17402:2008 more knowledge is needed on processes controlling bioavailability of organic species so as to close the still existing gap between chemical measurements and biological effects. The bioavailability concept encompasses the investigation of the degree of penetration of target species across biological membranes. In addition, REACH (Registration, Evaluation, Authorisation and restriction of Chemicals) guidelines promote the use of in-vitro methods against conventional ecotoxicological tests because of the ethical controversy of in-vivo tests. This work is aimed at filling the gap by proposing a multidisciplinary approach based on high-resolution and low-resolution empirical techniques, and theoretical quantum mechanics for the in-vitro investigation of the bioavailability and membranotropic effects of organic emerging contaminants, including bioaccumulation, via passive diffusion across lipid bilayers. Phosphatidylcholine (PC) liposomes are selected as biomembrane surrogates, and contaminant effects are explored by (i) fluorescence anisotropy and generalized polarization assays using membrane fluorescence probes (laurdan and prodan) and UV-Vis spectroscopy, (ii) 1H NMR measurements to ascertain supramolecular interactions with PC and (iii) molecular dynamics simulations. In particular, un-regulated model compounds with distinct physico-chemical properties that are representative of three different classes of emerging contaminants in environmental compartments are chosen for validation of the holistic approach: (i) diclofenac as a model of anti-inflammatory drug; (ii) triclosan as an anti-microbial agent; and (iii) bisphenol A as a plastic-borne compound, and compared with chlorpyrifos as a legacy insecticide. Laurdan anisotropic measurements are in good agreement with 1H NMR data and both approaches pinpoint that triclosan and chlorpyrifos are highly bioaccumulative in membranes. Molecular dynamic studies indicate that the lateral diffusion of the lipid bilayer is much lower with the incorporation of either triclosan or chlorpyrifos into the bilayer. The theoretical simulations also allowed estimating absolute bioavailability data under passive diffusion (<0.1%, 63%, 73% and 89% for diclofenac, bisphenol A, triclosan and chlorpyrifos, respectively) given as the percentage of time that a given species is located in the region of the fatty acyl chains. Our findings indicate that PC-based liposome assays serve as a fast and cost-effective in-vitro approach, notwithstanding its low resolution features, for environmental bioavailability studies of emerging contaminants for which insufficient or inconsistent ecotoxicological data are identified in the literature.

Adipato bridged novel hexanuclear Cu(ii) and polymeric Co(ii) coordination compounds involving cooperative supramolecular assemblies and encapsulated guest water clusters in a square grid host: antiproliferative evaluation and theoretical studies.

Two new coordination compounds involving hexanuclear Cu(ii), viz., [Cu6(phen)6(µ4-adpt)4(H2O)2](NO3)4·10H2O (1) and polymeric Co(ii), viz., {[(µ2-adpt)4Co(µ2-H2O)2Co(H2O)4]·4H2O}n (2) (phen = 1,10-phenanthroline; adpt = adipate) have been synthesized and characterized using elemental analysis, TGA, spectroscopic (IR, electronic and ESR), PXRD and single crystal X-ray diffraction techniques. Discrete nitrate-water clusters involving the [(H2O)3NO3]- core in 1 and linear (H2O)4 core in 2 provide stability to the layered network of the structures of the compounds. Interestingly, the water clusters in polymer 2 are encapsulated as guests in the voids of the host square grid that extends in 2D architecture. Theoretical studies have revealed the presence of interesting energetically significant cooperativity effects of π-stacking contacts that are responsible for the hexanuclear structure of compound 1. Both complexes significantly inhibit cell viability by inducing apoptotic cell death in the DL cancer cell line with negligible cytotoxicity in normal cells (PBMC). An assessment of ROS (reactive oxygen species) level study revealed a rapid increase of ROS in DL cells indicating cytotoxicity of the compounds against the DL cells. A decrease in MMP (mitochondrial membrane potential) is associated with an opening of the mitochondrial permeability transition pores which corroborates the apoptotic features of 1 and 2. The mode of action of the cytotoxic activities of the compounds has been explored with respect to their in silico docking ability and further inhibition of antiapoptotic proteins as evidenced by western blot analysis. SAR analyses based on pharmacophore modelling reveal that the molecular features of the structures of the compounds play important roles in biological activities.

UIBVFED: Virtual facial expression dataset.

Facial expression classification requires large amounts of data to reflect the diversity of conditions in the real world. Public databases support research tasks providing researchers an appropriate work framework. However, often these databases do not focus on artistic creation. We developed an innovative facial expression dataset that can help both artists and researchers in the field of affective computing. This dataset can be managed interactively by an intuitive and easy to use software application. The dataset is composed of 640 facial images from 20 virtual characters each creating 32 facial expressions. The avatars represent 10 men and 10 women, aged between 20 and 80, from different ethnicities. Expressions are classified by the six universal expressions according to Gary Faigin classification.

Ecotoxicological equilibria of triclosan in Microtox, XenoScreen YES/YAS, Caco2, HEPG2 and liposomal systems are affected by the occurrence of other pharmaceutical and personal care emerging contaminants.

Contaminants of emerging concern may be considered as any chemicals or factors whose unintended continuous release and persistence in the environment may lead to any observable undesirable response of living beings. Still not much is known on reciprocal toxicological impact of given chemicals when present in binary or more complex mixtures. In this work, an attempt was thus undertaken to study the impact of butylparaben, methylparaben and diclofenac on toxicological behavior and properties of triclosan (at varying concentration levels) with respect to Microtox, XenoScreen YES/YAS, Caco-2, HEPG2, and liposomal systems. Having performed analytical and biological studies modeling was done using two modeling approaches, viz., concentration addition (CA) and independent action (IA) at three concentration levels of each chemical studied. The effect of the highest concentration of triclosan studied was impacted by even small amounts of methylparaben and butylparaben in Microtox while diclofenac preferably affected triclosan activity at its lowest concentration level (with CA model). Estrogenic agonistic properties of triclosan were severely impacted by both parabens in an antagonistic way; diclofenac showed in all cases underestimation or synergy at the lowest triclosan concentration studied. Estrogenic antagonistic activity of triclosan was also slightly affected by parabens and by diclofenac in binary mixtures, showing overestimation and antagonist effects. HepG2 cells appeared to be the most resistant to the toxic effect of the mixtures at the concentrations tested and no significant proof of synergy or antagonism could be detected with the MTT assay. The liposome assays on the mixtures followed the same trends obtained with the MTT assay with Caco-2 cells, confirming the validity of the in vitro model used in this research. As studies on emerging contaminants mixtures toxicity are still scarce, research presented here constitute an important part in confirming utility and versatility of emerging contaminants modeling in environmental toxicology.

Synthesis, X-ray characterization and regium bonding interactions of a trichlorido(1-hexylcytosine)gold(iii) complex.

Herein we report the synthesis and X-ray characterization of a gold(iii) complex of 1-hexylcytosine via N(3). The AuCl3N complexes stack on top of each other by reciprocal [AuCl] regium bonding interactions. After the first example 35 years ago, this is the second available structure of a cytosine nucleobase model complexed to gold(iii).