Publications

The field of G protein-coupled receptor (GPCR) research has greatly benefited from the spatiotemporal resolution provided by light controllable, i.e., photoswitchable ligands. Most of the developed tools have targeted the Rhodopsin-like family (Class A), the largest family of GPCRs. However, to date, all such Class A photoswitchable ligands were designed to act at the orthosteric binding site of these receptors. Herein, we report the development of the first photoswitchable allosteric modulators of Class A GPCRs, designed to target the M-1 muscarinic acetylcholine receptor. The presented benzyl quinolone carboxylic acid (BQCA) derivatives, Photo-BQCisA and Photo-BQCtrAns, exhibit complementary photopharmacological behavior and allow reversible control of the receptor using light as an external stimulus. This makes them valuable tools to further investigate M-1 receptor signaling and a proof of concept for photoswitchable allosteric modulators at Class A receptors.

JTD Keywords: Agonist, Allosterism, Gpcr, Muscarinic ligands, Photopharmacology, Photoswitc, Selective activation, Serie

The cellular prion protein (PrPC) plays many roles in the developing and adult brain. In addition, PrPC binds to several amyloids in oligomeric and prefibrillar forms and may act as a putative receptor of abnormal misfolded protein species. The role of PrPC in tau seeding and spreading is not known. In the present study, we have inoculated well-characterized sarkosyl-insoluble fractions of sporadic Alzheimer's disease (sAD) into the brain of adult wild-type mice (Prnp(+/+)), Prnp(0/0) (ZH3 strain) mice, and mice over-expressing the secreted form of PrPC lacking their GPI anchor (Tg44 strain). Phospho-tau (ptau) seeding and spreading involving neurons and oligodendrocytes were observed three and six months after inoculation. 3Rtau and 4Rtau deposits from the host tau, as revealed by inoculating Mapt(0/0) mice and by using specific anti-mouse and anti-human tau antibodies suggest modulation of exon 10 splicing of the host mouse Mapt gene elicited by exogenous sAD-tau. However, no tau seeding and spreading differences were observed among Prnp genotypes. Our results show that PrPC does not affect tau seeding and spreading in vivo.

JTD Keywords: Alpha-synuclein, Alzheimer's disease, Amyloid-beta oligomers, Expression, Impairmen, Mapt, Mice, Paired helical filaments, Pathological tau, Prnp, Propagation, Prpc, Seeding, Spreadin, Synaptic plasticity, Tau, Tauopathies

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

JTD Keywords: Antibodies, Beta-lactamase, Cell, Enterica, Escherichia-coli, Expression, Genes, Infection, Intimin, Mous, Mutational analysis, Pcr, Salmonella, Var typhimurium

Instrumental Odour Monitoring Systems are often based on gas sensor arrays, or eventually on single sensor solutions for major odorants. In this work, we investigate the use of a portable Ion Mobility Spectrometer (IMS) as an Instrumental Odour Monitoring System (IOMS) for monitoring odorous emissions from wastewater treatment plants (WWTPs). This preliminary study was carried out on two plants in Pinedo (Valencia), i.e., P1 and P2. Three field campaigns (JanuaryJune-July) captured seasonal and wastewater variations, employing chemometric analysis and Principal Component Analysis (PCA) to distinguish Water line and Sludge line emissions. Random Forest (RF) and Partial Least Squares Discriminant Analysis (PLS-DA) were used to develop an odour classification model. External validation achieved an 87% accuracy for P1 July and results for P2 January-June. These results prove the IMS potential to be used for enhanced odour emission classification, possibly in combination with other monitoring techniques.

JTD Keywords: Discrimination, Ioms, Odour classification, Pc, Pls-da, Source

Clinical relevance of miRNAs as biomarkers is growing due to their stability and detection in biofluids. In this, diagnosis at asymptomatic stages of Alzheimer ' s disease (AD) remains a challenge since it can only be made at autopsy according to Braak NFT staging. Achieving the objective of detecting AD at early stages would allow possible therapies to be addressed before the onset of cognitive impairment. Many studies have determined that the expression pattern of some miRNAs is dysregulated in AD patients, but to date, none has been correlated with downregulated expression of cellular prion protein (PrP C ) during disease progression. That is why, by means of cross studies of miRNAs up -regulated in AD with in silico identification of potential miRNAs-binding to 3 ' UTR of human PRNP gene, we selected miR-519a-3p for our study. Then, in vitro experiments were carried out in two ways. First, we validated miR-519a-3p target on 3 ' UTR- PRNP , and second, we analyzed the levels of PrP C expression after using of mimic technology on cell culture. In addition, RT-qPCR was performed to analyzed miR519a-3p expression in human cerebral samples of AD at different stages of disease evolution. Additionally, samples of other neurodegenerative diseases such as other non -AD tauopathies and several synucleinopathies were included in the study. Our results showed that miR-519a-3p overlaps with PRNP 3 ' UTR in vitro and promotes downregulation of PrP C . Moreover, miR-519a-3p was found to be up -regulated exclusively in AD samples from stage I to VI, suggesting its potential use as a novel label of preclinical stages of the disease.

JTD Keywords: Activation, Alzheimer's disease, Biomarke, Brain, Cellular prion protein, Developmental expression, Gen, Micrornas, Plasma, Prpc, Tau-aggregation

The mechanical properties of the extracellular matrix dictate tissue behaviour. In epithelial tissues, laminin is a very abundant extracellular matrix component and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111-unlike fibronectin or collagen I-impairs cell response to substrate rigidity and YAP nuclear localization. Blocking the laminin-specific integrin β4 increases nuclear YAP ratios in a rigidity-dependent manner without affecting the cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that β4 integrins establish a mechanical linkage between the substrate and keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects the nuclear YAP mechanoresponses, chromatin methylation and cell invasion in three dimensions. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals.© 2023. The Author(s).

JTD Keywords: actin, cell migration, filaments, force transmission, localization, membrane, motility, proteins, yap, Cell adhesion, Cytoskeleton, Extracellular matrix, Fibronectins, Integrin alpha-6-beta-4, Integrins, Keratins, Laminin

During tumor progression, cancer-associated fibroblasts (CAFs) accumulate in tumors and produce an excessive extracellular matrix (ECM), forming a capsule that enwraps cancer cells. This capsule acts as a barrier that restricts tumor growth leading to the buildup of intratumoral pressure. Combining genetic and physical manipulations in vivo with microfabrication and force measurements in vitro, we found that the CAFs capsule is not a passive barrier but instead actively compresses cancer cells using actomyosin contractility. Abrogation of CAFs contractility in vivo leads to the dissipation of compressive forces and impairment of capsule formation. By mapping CAF force patterns in 3D, we show that compression is a CAF-intrinsic property independent of cancer cell growth. Supracellular coordination of CAFs is achieved through fibronectin cables that serve as scaffolds allowing force transmission. Cancer cells mechanosense CAF compression, resulting in an altered localization of the transcriptional regulator YAP and a decrease in proliferation. Our study unveils that the contractile capsule actively compresses cancer cells, modulates their mechanical signaling, and reorganizes tumor morphology.© 2023. The Author(s).

JTD Keywords: force, migration, yap, Cancer-associated fibroblasts, Cell line, tumor, Fibroblasts, Mechanotransduction, cellular, Neoplasms, Tumor, Tumor microenvironment

Alzheimer's disease is characterized by a combination of several neuropathological hallmarks, such as extracellular aggregates of beta amyloid (Aβ). Numerous alternatives have been studied for inhibiting Aβ aggregation but, at this time, there are no effective treatments available. Here, we developed the tri-component nanohybrid system AuNPs@POM@PEG based on gold nanoparticles (AuNPs) covered with polyoxometalates (POMs) and polyethylene glycol (PEG). In this work, AuNPs@POM@PEG demonstrated the inhibition of the formation of amyloid fibrils, showing a 75% decrease in Aβ aggregation in vitro. As it is a potential candidate for the treatment of Alzheimer's disease, we evaluated the cytotoxicity of AuNPs@POM@PEG and its ability to cross the blood-brain barrier (BBB). We achieved a stable nanosystem that is non-cytotoxic below 2.5 nM to human neurovascular cells. The brain permeability of AuNPs@POM@PEG was analyzed in an in vitro microphysiological model of the BBB (BBB-on-a-chip), containing 3D human neurovascular cell co-cultures and microfluidics. The results show that AuNPs@POM@PEG was able to cross the brain endothelial barrier in the chip and demonstrated that POM does not affect the barrier integrity, giving the green light to further studies into this system as a nanotherapeutic.

JTD Keywords: beta-amyloid, blood-brain barrier organ-on-a-chip, cellular uptake, citrate, cytotoxicity, electrocatalytic reduction, gold nanoparticles, hypothesis, nanorods, polyoxometalates, size, stability, surface, Alzheimers-disease, Blood–brain barrier organ-on-a-chip, Gold nanoparticles, Nanovehicle, Polyoxometalates, Β-amyloid

Conventionally, odours emitted by different sources present in wastewater treatment plants (WWTPs) are measured by dynamic olfactometry, where a human panel sniffs and analyzes air bags collected from the plant. Although the method is considered the gold standard, the process is costly, slow, and infrequent, which does not allow operators to quickly identify and respond to problems. To better monitor and map WWTP odour emissions, here we propose a small rotary-wing drone equipped with a lightweight (1.3-kg) electronic nose. The "sniffing drone" sucks in air via a ten-meter (33-foot) tube and delivers it to a sensor chamber where it is analyzed in real-time by an array of 21 gas sensors. From the sensor signals, machine learning (ML) algorithms predict the odour concentration that a human panel using the EN13725 methodology would report. To calibrate and validate the predictive models, the drone also carries a remotely controlled sampling device (compliant with EN13725:2022) to collect sample air in bags for post-flight dynamic olfactometry. The feasibility of the proposed system is assessed in a WWTP in Spain through several measurement campaigns covering diverse operating regimes of the plant and meteorological conditions. We demonstrate that training the ML algorithms with dynamic (transient) sensor signals measured in flight conditions leads to better performance than the traditional approach of using steady-state signals measured in the lab via controlled exposures to odour bags. The comparison of the electronic nose predictions with dynamic olfactometry measurements indicates a negligible bias between the two measurement techniques and 95 % limits of agreement within a factor of four. This apparently large disagreement, partly caused by the high uncertainty of olfactometric measurements (typically a factor of two), is more than offset by the immediacy of the predictions and the practical advantages of using a drone-based system.Copyright © 2022. Published by Elsevier B.V.

JTD Keywords: calibration, chemical sensors, drone, dynamic olfactometry, electronic nose, odourquantification, olfaction, volatile organic-compounds, wwtp, Calibration, Chemical sensors, Drone, Dynamic olfactometry, Electronic nose, Environmental monitoring, Odour quantification, Olfaction, Variable selection methods, Wwtp

Understanding the dopaminergic system is a priority in neurobiology and neuropharmacology. Dopamine receptors are involved in the modulation of fundamental physiological functions, and dysregulation of dopaminergic transmission is associated with major neurological disorders. However, the available tools to dissect the endogenous dopaminergic circuits have limited specificity, reversibility, resolution, or require genetic manipulation. Here, we introduce azodopa, a novel photoswitchable ligand that enables reversible spatiotemporal control of dopaminergic transmission. We demonstrate that azodopa activates D1-like receptors in vitro in a light-dependent manner. Moreover, it enables reversibly photocontrolling zebrafish motility on a timescale of seconds and allows separating the retinal component of dopaminergic neurotransmission. Azodopa increases the overall neural activity in the cortex of anesthetized mice and displays illumination-dependent activity in individual cells. Azodopa is the first photoswitchable dopamine agonist with demonstrated efficacy in wild-type animals and opens the way to remotely controlling dopaminergic neurotransmission for fundamental and therapeutic purposes.

JTD Keywords: azobenzene, behavior, brainwave, d-1, dopamine, gpcr, in vivo electrophysiology, inhibitors, optogenetics, optopharmacology, photochromism, photopharmacology, photoswitch, stimulation, zebrafish, Animals, Animals, wild, Azobenzene, Behavior, Brainwave, Dopamine, Gpcr, In vivo electrophysiology, Ligands, Mice, Optogenetics, Optopharmacology, Photochromism, Photopharmacology, Photoswitch, Receptors, Synaptic transmission, Zebrafish

Nowadays, liquid biopsy represents one of the most promising techniques for early diagnosis, monitoring, and therapy screening of cancer. This novel methodology includes, among other techniques, the isolation, capture, and analysis of circulating tumor cells (CTCs). Nonetheless, the identification of CTC from whole blood is challenging due to their extremely low concentration (1-100 per ml of whole blood), and traditional methods result insufficient in terms of purity, recovery, throughput and/or viability of the processed sample. In this context, the development of microfluidic devices for detecting and isolating CTCs offers a wide range of new opportunities due to their excellent properties for cell manipulation and the advantages to integrate and bring different laboratory processes into the microscale improving the sensitivity, portability, reducing cost and time. This chapter explores current and recent microfluidic approaches that have been developed for the analysis and detection of CTCs, which involve cell capture methods based on affinity binding and label-free methods and detection based on electrical, chemical, and optical sensors. All the exposed technologies seek to overcome the limitations of commercial systems for the analysis and isolation of CTCs, as well as to provide extended analysis that will allow the development of novel and more efficient diagnostic tools.© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.

JTD Keywords: cancer detection, cancer diagnosis, cancer-cells, capture, chip, circulating tumor cells, enrichment, liquid biopsy, microchannel, separation, ultra-fast, Cancer detection, Cancer diagnosis, Circulating tumor cells, Label-free isolation, Liquid biopsy, Microfluidics

The mechanical signals sensed by the alveolar cells through the changes in the local matrix stiffness of the extracellular matrix (ECM) are determinant for regulating cellular functions. Therefore, the study of the mechanical response of lung tissue becomes a fundamental aspect in order to further understand the mechanosensing signals perceived by the cells in the alveoli. This study is focused on the development of a finite element (FE) model of a decellularized rat lung tissue strip, which reproduces accurately the mechanical behaviour observed in the experiments by means of a tensile test. For simulating the complex structure of the lung parenchyma, which consists of a heterogeneous and non-uniform network of thin-walled alveoli, a 3D model based on a Voronoi tessellation is developed. This Voronoi-based model is considered very suitable for recreating the geometry of cellular materials with randomly distributed polygons like in the lung tissue. The material model used in the mechanical simulations of the lung tissue was characterized experimentally by means of AFM tests in order to evaluate the lung tissue stiffness on the micro scale. Thus, in this study, the micro (AFM test) and the macro scale (tensile test) mechanical behaviour are linked through the mechanical simulation with the 3D FE model based on Voronoi tessellation. Finally, a micro-mechanical FE-based model is generated from the Voronoi diagram for studying the stiffness sensed by the alveolar cells in function of two independent factors: the stretch level of the lung tissue and the geometrical position of the cells on the extracellular matrix (ECM), distinguishing between pneumocyte type I and type II. We conclude that the position of the cells within the alveolus has a great influence on the local stiffness perceived by the cells. Alveolar cells located at the corners of the alveolus, mainly type II pneumocytes, perceive a much higher stiffness than those located in the flat areas of the alveoli, which correspond to type I pneumocytes. However, the high stiffness, due to the macroscopic lung tissue stretch, affects both cells in a very similar form, thus no significant differences between them have been observed. © 2021 The Authors

JTD Keywords: rat, scaffolds, stiffness, Afm, Animal cell, Animal experiment, Animal model, Animal tissue, Article, Biological organs, Cell function, Cells, Computational geometry, Cytology, Extracellular matrices, Extracellular matrix, Extracellular-matrix, Geometry, High stiffness, Human, Lung alveolus cell type 1, Lung alveolus cell type 2, Lung parenchyma, Lung tissue, Male, Mechanical behavior, Mechanical modeling, Mechanical simulations, Mechanosensing, Model-based opc, Nonhuman, Physical model, Rat, Rigidity, Stiffness, Stiffness matrix, Tensile testing, Thin walled structures, Three dimensional finite element analysis, Tissue, Type ii, Voronoi tessellations

Pseudomonas aeruginosa biofilms and the capacity of the bacterium to coexist and interact with a broad range of microorganisms have a substantial clinical impact. This review focuses on the main traits of P. aeruginosa biofilms, such as the structural composition and regulatory networks involved, placing particular emphasis on the clinical challenges they represent in terms of antimicrobial susceptibility and biofilm infection clearance. Furthermore, the ability of P. aeruginosa to grow together with other microorganisms is a significant pathogenic attribute with clinical relevance; hence, the main microbial interactions of Pseudomonas are especially highlighted and detailed throughout this review. This article also explores the infections caused by single and polymicrobial biofilms of P. aeruginosa and the current models used to recreate them under laboratory conditions. Finally, the antimicrobial and antibiofilm strategies developed against P. aeruginosa mono and multispecies biofilms are detailed at the end of this review.

JTD Keywords: aeruginosa models, antibiotic-resistance, antimicrobials, bacterial biofilms, biofilms, c-di-gmp, chronic infections, enterococcus-faecalis, extracellular dna, in-vitro, lectin pa-iil, p, p. aeruginosa models, polymicrobial, polymicrobial interactions, staphylococcus-aureus, Antimicrobials, Biofilms, Chronic infections, P. aeruginosa models, Polymicrobial, Pseudomonas aeruginosa, Urinary-tract-infection

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway involves a three-step cascade of kinases that transduce signals and promote processes such as cell growth, development, and apoptosis. An aberrant response of this pathway is related to the proliferation of cell diseases and tumors. By using simulation modeling, we document that the protein arginine methyltransferase 5 (PRMT5) modulates the MAPK pathway and thus avoids an aberrant behavior. PRMT5 methylates the Raf kinase, reducing its catalytic activity and thereby, reducing the activation of ERK in time and amplitude. Two minimal computational models of the epidermal growth factor (EGF)-Ras-ERK MAPK pathway influenced by PRMT5 were proposed: a first model in which PRMT5 is activated by EGF and a second one in which PRMT5 is stimulated by the cascade response. The reported results show that PRMT5 reduces the time duration and the expression of the activated ERK in both cases, but only in the first model PRMT5 limits the EGF range that generates an ERK activation. Based on our data, we propose the protein PRMT5 as a regulatory factor to develop strategies to fight against an excessive activity of the MAPK pathway, which could be of use in chronic diseases and cancer.

JTD Keywords: cancer, cell response modulation, computational model, egf-ras-erk signaling route, mapk pathway, methylation, Arginine methyltransferase 5, Cancer, Cell response modulation, Colorectal-cancer, Computational model, Egf-ras-erk signaling route, Epidermal-growth-factor, Factor receptor, Histone h3, Kinase cascade, Mapk pathway, Methylation, Negative-feedback, Pc12 cells, Prmt5, Protein, Signal-transduction

© 2020 Elsevier B.V. The current situation of the Covid-19 pandemic is indicated by a huge number of infections, high lethality, and rapid spread. These circumstances have stopped the activity of almost the entire world, affecting severely the global economy. A rapid diagnosis of the Covid-19 and a generalized testing protocol is essential to fight against the pandemic and to maintain health control in the population. Principal biosensing and diagnostic technologies used to monitor the spread of the SARS-CoV-2 are based on specific genomic analysis and rapid immune tests, both with different technology platforms that include advantages and disadvantages. Most of the in vitro diagnosis companies are competing to be the first on validating under different regulations their technology for placing their platforms for Covid-19 detection as fast as possible in this big international market. A comprehensive analysis of the commercialized technologies for the genomic based sensing and the antibody/antigen detection methods devoted to Covid-19 diagnosis is described in this review, which have been detailed and listed under different countries regulations. The effectiveness of the described technologies throughout the different stages of the disease and a critical comparison of the emerging technologies in the market to counterattack this pandemic have been discussed.

JTD Keywords: covid-19, in vitro diagnosis (ivd), lateral flow immunoassay, point of care (poc), reverse transcriptase polymerase chain reaction (rt-pcr), sars-cov-2, Covid-19, In vitro diagnosis (ivd), Lateral flow immunoassay, Point of care (poc), Reverse transcriptase polymerase chain reaction (rt-pcr), Sars-cov-2

Background & Aims: MicroRNAs (miRNAs) circulate in several body fluids and can be useful biomarkers. The aim of this study was to identify blood-circulating miRNAs associated with cirrhosis progression and acute-on-chronic liver failure (ACLF). Methods: Using high-throughput screening of 754 miRNAs, serum samples from 45 patients with compensated cirrhosis, decompensated cirrhosis, or ACLF were compared with those from healthy individuals (n = 15). miRNA levels were correlated with clinical parameters, organ failure, and disease progression and outcome. Dysregulated miRNAs were evaluated in portal and hepatic vein samples (n = 33), liver tissues (n = 17), and peripheral blood mononuclear cells (PBMCs) (n = 16). Results: miRNA screening analysis revealed that circulating miRNAs are dysregulated in cirrhosis progression, with 51 miRNAs being differentially expressed among all groups of patients. Unsupervised clustering and principal component analysis indicated that the main differences in miRNA expression occurred at decompensation, showing similar levels in patients with decompensated cirrhosis and those with ACLF. Of 43 selected miRNAs examined for differences among groups, 10 were differentially expressed according to disease progression. Moreover, 20 circulating miRNAs were correlated with model for end-stage liver disease and Child-Pugh scores. Notably, 11 dysregulated miRNAs were associated with kidney or liver failure, encephalopathy, bacterial infection, and poor outcomes. The most severely dysregulated miRNAs (i.e. miR-146a5p, miR-26a-5p, and miR-191-5p) were further evaluated in portal and hepatic vein blood and liver tissue, but showed no differences. However, PBMCs from patients with cirrhosis showed significant downregulation of miR-26 and miR-146a, suggesting a extrahepatic origin of some circulating miRNAs. Conclusions: This study is a repository of circulating miRNA data following cirrhosis progression and ACLF. Circulating miRNAs were profoundly dysregulated during the progression of chronic liver disease, were associated with failure of several organs and could have prognostic utility. Lay summary: Circulating miRNAs are small molecules in the blood that can be used to identify or predict a clinical condition. Our study aimed to identify miRNAs for use as biomarkers in patients with cirrhosis or acute-on-chronic liver failure. Several miRNAs were found to be dysregulated during the progression of disease, and some were also related to organ failure and disease-related outcomes. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL).

JTD Keywords: aclf, acute-on-chronic liver failure, alt, alanine aminotransferase, ast, aspartate aminotransferase, biomarkers, chronic liver disease, cxcl10, c-x-c motif chemokine ligand 10, ef clif, european foundation for the study of chronic liver failure, foxo, forkhead box o, inr, international normalised ratio, ldh, lactate dehydrogenase, liver decompensation, mapk, mitogen-activated protein kinase, meld, model for end-stage liver disease, nash, non-alcoholic steatohepatitis, non-coding rnas, pbmcs, peripheral blood mononuclear cells, pca, principal component analysis, tgf, transforming growth factor, tips, transjugular intrahepatic portosystemic shunt, Biomarkers, Chronic liver disease, Expression, Liver decompensation, Markers, Mir-146a, Non-coding rnas, Qpcr, quantitative pcr

© 2020 American Chemical Society. The performance of supramolecular nanocarriers as drug delivery systems depends on their stability in the complex and dynamic biological media. After administration, nanocarriers are challenged by physiological barriers such as shear stress and proteins present in blood, endothelial wall, extracellular matrix, and eventually cancer cell membrane. While early disassembly will result in a premature drug release, extreme stability of the nanocarriers can lead to poor drug release and low efficiency. Therefore, comprehensive understanding of the stability and assembly state of supramolecular carriers in each stage of delivery is the key factor for the rational design of these systems. One of the main challenges is that current 2D in vitro models do not provide exhaustive information, as they fail to recapitulate the 3D tumor microenvironment. This deficiency in the 2D model complexity is the main reason for the differences observed in vivo when testing the performance of supramolecular nanocarriers. Herein, we present a real-time monitoring study of self-assembled micelles stability and extravasation, combining spectral confocal microscopy and a microfluidic cancer-on-a-chip. The combination of advanced imaging and a reliable 3D model allows tracking of micelle disassembly by following the spectral properties of the amphiphiles in space and time during the crucial steps of drug delivery. The spectrally active micelles were introduced under flow and their position and conformation continuously followed by spectral imaging during the crossing of barriers, revealing the interplay between carrier structure, micellar stability, and extravasation. Integrating the ability of the micelles to change their fluorescent properties when disassembled, spectral confocal imaging and 3D microfluidic tumor blood vessel-on-a-chip resulted in the establishment of a robust testing platform suitable for real-time imaging and evaluation of supramolecular drug delivery carrier's stability.

JTD Keywords: cancer-on-a-chip, complex, delivery, endothelial-cells, in-vitro, microfluidic, model, nanoparticle, penetration, shear-stress, stability, supramolecular, Cancer-on-a-chip, Cell-culture, Micelle, Microfluidic, Nanoparticle, Stability, Supramolecular

Tackling the first stages of the chondrogenic commitment is essential to drive chondrogenic differentiation to healthy hyaline cartilage and minimize hypertrophy. During chondrogenesis, the extracellular matrix continuously evolves, adapting to the tissue adhesive requirements at each stage. Here, we take advantage of previously developed nanopatterns, in which local surface adhesiveness can be precisely tuned, to investigate its effects on prechondrogenic condensation. Fluorescence live cell imaging, immunostaining, confocal microscopy and PCR analysis are used to follow the condensation process on the nanopatterns. Cell tracking parameters, condensate morphology, cell–cell interactions, mechanotransduction and chondrogenic commitment are evaluated in response to local surface adhesiveness. Results show that only condensates on the nanopatterns of high local surface adhesiveness are stable in culture and able to enter the chondrogenic pathway, thus highlighting the importance of controlling cell–substrate adhesion in the tissue engineering strategies for cartilage repair.

JTD Keywords: Dendrimer, Nanopatterning, RGD, Mesenchymal cell condensation, Cell–cell interactions, YAP, Chondrogenesis

Prevalence and risk factors for hepatic steatosis (HS) in the human immunodeficiency virus (HIV)-positive population of western countries are controversially discussed and potentially confounded by coinfection with viral hepatitis. Significant HS (more than 10% of hepatocytes) can be accurately assessed using controlled attenuation parameter (CAP) determination. Aim of this study was to assess prevalence and factors associated with significant HS in HIV monoinfected patients. A total of 364 HIV-infected patients (289 monoinfected) were included in this prospective, cross-sectional study. All patients underwent CAP determination. Steatosis was classified as S1 (significant steatosis) with CAP > 238 dB/m, S2 with CAP > 260 dB/m, and S3 with CAP > 292 dB/m. Multivariable logistic regression analyses were performed to assess the factors associated with HS in this cohort. Significant HS was detected in 118 monoinfected patients (149 in the total cohort). In the total cohort as well as in the monoinfected patients alone, HS grade distribution showed a similar pattern (S1:29%, S2:34%, and S3:37%). Interestingly, patients with HS had a longer history of HIV infection and combined antiretroviral therapy (cART). Interalia, age, gender, ethnicity, and metabolic factors were strongly associated with HS, while body mass index (BMI), triglyceride, and glycated hemoglobin (HbA1c) levels were independently associated with significant HS. HS is highly prevalent among HIV monoinfected patients. Although metabolic risk factors, such as obesity and poorly controlled diabetes, are independently associated with HS in HIV monoinfected patients, cART and control of HIV seem to play an indirect role in the development of HS, probably through the return-to-health effect.

JTD Keywords: CAP, cART, HIV monoinfection, liver injury, NAFLD

The term ‘prion-like’ is used to define some misfolded protein species that propagate intercellularly, triggering protein aggregation in recipient cells. For cell binding, both direct plasma membrane interaction and membrane receptors have been described for particular amyloids. In this respect, emerging evidence demonstrates that several β-sheet enriched proteins can bind to the cellular prion protein (PrPC). Among other interactions, the physiological relevance of the binding between β-amyloid and PrPC has been a relevant focus of numerous studies. At the molecular level, published data point to the second charged cluster domain of the PrPC molecule as the relevant binding domain of the β-amyloid/PrPC interaction. In addition to β-amyloid, participation of PrPC in binding α-synuclein, responsible for neurodegenerative synucleopathies, has been reported. Although results indicate relevant participation of PrPC in the spreading of α-synuclein in living mice, the physiological relevance of the interaction remains elusive. In this comment, we focus our attention on summarizing current knowledge of PrPC as a receptor for amyloid proteins and its physiological significance, with particular focus on α-synuclein.

JTD Keywords: α-synuclein, Charged cluster domain, Interneuronal transport, LAG3, Neurodegeneration, PrPC, Parkinson disease

DEP manipulation of cells present in real samples is challenging. We show in this work that an interdigitated DEP chip can be used to trap and wash a population of the food-spoiling yeast Zygosaccharomyces rouxii that contaminates a sample of apple juice. By previously calibrating the chip, the yeast population loaded is efficiently trapped, washed and recovered in a small-volume fraction which, in turn, can be used for efficient PCR detection of this yeast. DEP washing of yeast cells gets rid of PCR inhibitors present in apple juice and facilitates PCR analysis. This and previous works on the use of DEP chips to improve PCR analysis show that a potential use of DEP is to be used as a treatment of real samples prior to PCR.

JTD Keywords: Dielectrophoresis, PCR, Saccharomyces, Yeast

Calcium phosphate cements (CPCs) were meant to produce hydroxyapatite (HA), which is the calcium phosphate that usually results when the cements are mixed with or immersed in aqueous media. The golden age of CPCs was in the late 1990s and the beginning of the 21st century, when they were presented as promising bone substitutes and drug delivery systems. The different reactions that take part in the cement self-setting process depend on many experimental factors – the composition of the cement, the stability of the different components, pH, liquid-to-powder ratio (LPR), and temperature, among others. CPCs have demonstrated fair efficiency for bone regeneration. Cements have gradually been embraced in the wider field of composites by hybridizing their compositions in order that they may adapt to the new trends.

JTD Keywords: Calcium phosphate cements (CPCs), Cements, Hydroxyapatite (HA), Liquid-to-powder ratio (LPR)

Background: Epidermal Growth Factor (EGF) is a key regulatory growth factor activating many processes relevant to normal development and disease, affecting cell proliferation and survival. Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer. Results: By applying a procedure for cross-platform data meta-analysis based on RankProd and GlobalAncova tests, we establish a well validated gene set with transcript levels altered after EGF treatment. We use this robust gene list to build higher order networks of gene interaction by interconnecting associated networks, supporting and extending the important role of the EGF signaling pathway in cancer. In addition, we find an entirely new set of genes previously unrelated to the currently accepted EGF associated cellular functions. Conclusions: We propose that the use of global genomic cross-validation derived from high content technologies (microarrays or deep sequencing) can be used to generate more reliable datasets. This approach should help to improve the confidence of downstream in silico functional inference analyses based on high content data.

JTD Keywords: Gene-expression measurements, Quality-control maqc, Cancer-cell-lines, Real-time pcr, Oligonucleotide microarrays, Phosphorylation dynamics, In-vivo, Networks, Signal, Technologies

There is an increasing level of interest in non-tuberculous mycobacteria (NTM) due to the increasing reported rates of diseases caused by them. Although it is well known that NTM are widely distributed in the environment it is necessary to identify its reservoirs to prevent possible infections. In this study, we aimed to investigate the occurrence and levels of NTM in cooling towers to provide evidences for considering these settings as possible sources of respiratory infections. In the current study, we detected and quantified the presence of NTM by means of a rapid method in water samples taken from 53 cooling towers of an urban area (Barcelona, Spain). A genus-specific quantitative PCR (Q-PCR) assay with a quantification limit (QL) of 500 cells l(-1) was used. 56% (30) of samples were positive with a concentration range from 4.6 x 10(3) to 1.79 x 10(6) cells l(-1). In some cases (9/30), samples were positive but with levels below the QL. The colonization rate confirmed that cooling towers could be considered as a potential reservoir for NTM. This study also evaluated Q-PCR as a useful method to detect and quantify NTM in samples coming from environmental sources.

JTD Keywords: Real-time PCR, Disease, Identification, Tuberculosis, Pathogens, Waters

A hybrid microfluidic device that uses hydrodynamic forces to separate human plasma from blood cells has been designed and fabricated and the advantageous effects of temperature and flow rates are investigated in this paper. The blood separating device includes an inlet which is reduced by approximately 20 times to a small constrictor channel, which then opens out to a larger output channel with a small lateral channel for the collection of plasma. When tested the device separated plasma from whole blood using a wide range of flow rates, between 50 mu l min(-1) and 200 mu l min(-1), at the higher flow rates injected by hand and at temperatures ranging from 23 degrees C to 50 degrees C, the latter resulting in an increase in the cell-free layer of up to 250%. It was also tested continuously using between 5% and 40% erythrocytes in plasma and whole blood without blocking the channels or hemolysis of the cells. The mean percentage of plasma collected after separation was 3.47% from a sample of 1 ml. The percentage of cells removed from the plasma varied depending on the flow rate used, but at 37 degrees C ranged between 95.4 +/- 1% and 97.05 +/- 05% at 100 mu l min(-1) and 200 mu l min(-1), respectively. The change in temperature also had an effect on the number of cells removed from the plasma which was between 93.5 +/- 0.65% and 97.01 +/- 0.3% at 26.9 degrees C and 37 degrees C, respectively, using a flow rate of 100 mu l min(-1). Due to its ability to operate in a wide range of conditions, it is envisaged that this device can be used in in vitro 'lab on a chip' applications, as well as a hand-held point of care (POC) device.

JTD Keywords: On-a-chip, Cells, Viscosity, Membrane

Transcription Factor binding sites are short and degenerate sequences, located mostly at the promoter of the gene, where some proteins bind in order to regulate transcription. Locating these sequences is an important issue, and many experimental and computational methods have been developed. Algorithms to search binding sites are usually based on Position Specific Scoring Matrices (PSSM), where each position is treated independently. Mapping symbolical DNA to numerical sequences, a detector has been built with a Principal Component Analysis of the numerical sequences, taking into account covariances between positions. When a treatment of missing values is incorporated the Q-residuals detector, based on PCA, performs better than a PSSM algorithm. The performance on the detector depends on the estimation of missing values and the percentage of missing values considered in the model.

JTD Keywords: Binding sites, BPCA, Missing values, Numerical DNA, Principal components analysis, Transcription factors

Background: Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrPsc) of the natural cellular prion protein (PrPc) encoded by the Prnp gene. Although several roles have been attributed to PrPc, its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrPc studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Methodology/Principal Findings: Here we explore the role of PrPc expression in neurotransmission and neural excitability using wild-type, Prnp 2/2 and PrPc-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp 2/2 mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using IlluminaTM microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp 2/2 and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABAA and AMPA-kainate receptors are co-regulated in both Prnp 2/2 and Tg20 mice. Conclusions/Significance: Present results demonstrate that PrPc is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABAA and AMPA-Kainate neurotransmission. New PrPc functions have recently been described, which point to PrPc as a target for putative therapies in Alzheimer’s disease. However, our results indicate that a ‘‘gain of function’’ strategy in Alzheimer’s disease, or a ‘‘loss of function’’ in prionopathies, may impair PrPc function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies.

JTD Keywords: Prions, Prionopathies, Natural cellular prion protein (PrPc), Hippocampus, GABA (A) receptor, Glutamate Receptor