Advances in Microtechnology for Improved Cytotoxicity Assessment

In vitro cytotoxicity testing is essential in the pharmaceutical and environmental industry to study the effects of potential harmful compounds for human health. Classical assays present several disadvantages: they are commonly based on live-death labelling, are highly time consuming and/or require skilled personnel to be performed. The current trend is to reduce the number of required cells and the time during the analysis, while increasing the screening capability and the accuracy and sensitivity of the assays, aiming single cell resolution. Microfabrication and surface engineering are enabling novel approaches for cytotoxicity assessment, offering high sensitivity and the possibility of automation in order to minimize user intervention. This review aims to overview the different microtechnology approaches available in this field, focusing on the novel developments for high-throughput, dynamic and real time screening of cytotoxic compounds.Funding support from: University of the Basque Country (PIF16/204), the funding support from Gobierno de España, Ministerio de Economía y Competitividad, with Grant No. BIO2016-80417-P (AEI/FEDER, UE) and Gobierno Vasco under grand IT1271-19

A study of the artistic corpus of red cave paintings in El Buxu cave (Cangas de Onís, Asturias, Spain)

El Buxu cave, which is located in the village of Cardes (Cangas de Onís, Asturias), has been studied since the 1980s, with multiple excavations taking place inside the cave. This work has uncovered a complete artistic corpus, marking out several phases of occupation, with paintings dating to the first phases of the Upper Palaeolithic, Solutrean and Lower and Middle Magdalenian periods. This paper presents a new review of its rock art, documenting all of the red paintings inside the cave, most of which have never been published up until this point. The most notable inclusion is the new description of a zoomorphic figure painted in red, which has previously been interpreted as an aurochs, but whose features are in fact closer to those of a deer or reindeer. In addition, stratigraphic analysis of some of the paintings has revealed that they are overlapped by Solutrean and Magdalenian engravings and black paintings inside the cave. Elemental analysis was performed on series of red pigments and ochre samples, recovered from various strata using X-ray fluorescence spectroscopy. The resulting dataset was treated using Principal Component Analysis, providing a deeper understanding of the composition of the rock art in El Buxu cave, while uncovering potential correlations between the samples ac- cording to their elemental composition. After comparing additional evidences from other red cave paintings in the region with the red pictographs in the cave, along with the stratification of paint pigments and their relationship with the ochre samples in each stratum, it appears that the red paintings comprise the oldest group of pictures inside the cave and can be broadly dated to the pre-Magdalenian cultural period.Our special aknowledgment to the National University of Distance Education (UNED), for its funding for open-access publishin

Precise Integration of Polymeric Sensing Functional Materials within 3D Printed Microfluidic Devices

This work presents a new architecture concept for microfluidic devices, which combines the conventional 3D printing fabrication process with the stable and precise integration of polymeric functional materials in small footprints within the microchannels in well-defined locations. The approach solves the assembly errors that normally occur during the integration of functional and/or sensing materials in hybrid microfluidic devices. The method was demonstrated by embedding four pH-sensitive ionogel microstructures along the main microfluidic channel of a complex 3D printed microfluidic device. The results showed that this microfluidic architecture, comprising the internal integration of sensing microstructures of diverse chemical compositions, highly enhanced the adhesion force between the microstructures and the 3D printed microfluidic device that contains them. In addition, the performance of this novel 3D printed pH sensor device was investigated using image analysis of the pH colour variations obtained from photos taken with a conventional camera. The device presented accurate and repetitive pH responses in the 2 to 12 pH range without showing any type of device deterioration or lack of performance over time.This research was founded by the University of the Basque Country (ESPPOC 16/65 and PIF16/204), “Ministerio de Ciencia y Educación de España” grant PID2020-120313GB-I00/AIE/10.13039/501100011033, and “Gobierno Vasco” grant IT1633-22

Portable and Raman imaging usefulness to detect decaying on mortars from Punta Begoña Galleries (Getxo, North of Spain)

Punta Begoña Galleries were built in 1918 in Getxo (Basque Country, North of Spain) but were abandoned in 1960. Nowadays, their conservation state is very poor. In this work, portable Raman spectroscopy was applied to evaluate the original composition and possible deterioration products of the mortars used in the inner walls and those covering the concrete of the ceilings allowing us to select the most appropriate sampling points. In the laboratory, Raman microscopy and Raman imaging, assisted with scanning electron microscopy equipped with an energy dispersive spectrometer (SEM‐EDS), X‐ray diffraction and energy dispersive X‐ray fluorescence (ED‐XRF) imaging, allowed to identify the key compounds to understand the deterioration processes taking place in the mortars of the galleries. The main components of the mortars from the walls were calcite and gypsum. In some cases, alite (Ca3SiO5) and belite (Ca2SiO4) were identified; these components are characteristic of Portland cement clinker. The main components of the mortar covering the concrete were calcite, quartz, aragonite and gypsum. The aragonite identification confirmed the use of beach sand as the aggregate in the mortar. The concrete from the ceiling of the lower gallery is covered with three different mortar layers; the outermost layer is covered with a black crust. In the three mortars, the main components are similar to those used in the mortar covering the concrete from the upper gallery. Thanks to Raman, ED‐XRF and SEM‐EDS imaging, it was possible to map the distribution of the main components through the three mortar layers and also to identify the presence of dolomite {[CaMg(CO3)2]}, which was not possible to detect following single‐point micro‐Raman analyses.This work has been funded by the Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF) through the project DISILICA‐1930 (ref. BIA2014‐59124‐P) and by the cooperation agreement between the University of the Basque Country (UPV/EHU) and the City Council of Getxo (OTRI2014‐0639). C. García‐Florentino is grateful to the University of the Basque Country (UPV/EHU), which funded her predoctoral fellowship. Technical support provided by Raman‐LASPEA Laboratory and General X‐ray Service of the SGIKer (UPV/EHU, Ministry of Economy and Competitiveness of Spain, Basque Government, ERDF and European Social Fund) is also gratefully acknowledged

Microfluidic chip with pillar arrays for controlled production and observation of lipid membrane nanotubes

Lipid membrane nanotubes (NTs) are a widespread template forin vitrostudies of cellular processes happening at high membrane curvature. Traditionally NTs are manufactured one by one, using sophisticated membrane micromanipulations, while simplified methods for controlled batch production of NTs are in growing demand. Here we propose a lab-on-a-chip (LOC) approach to the simultaneous formation of multiple NTs with length and radius controlled by the chip design. The NTs form upon rolling silica microbeads covered by lipid lamellas over the pillars of a polymer micropillar array. The array's design and surface chemistry set the geometry of the resulting free-standing NTs. The integration of the array inside a microfluidic chamber further enables fast and turbulence-free addition of components, such as proteins, to multiple preformed NTs. This LOC approach to NT production is compatible with the use of high power objectives of a fluorescence microscope, making real-time quantification of the different modes of the protein activity in a single experiment possible.This work was partially supported by the Spanish Ministry of Science, Innovation, and Universities grants PGC2018-099971-B-I00, EUR2019-103830, RYC-2014-01419 and BIO2016-80417-P and the Basque Government grants IT1270-19 and IT1271-19. JMMG and MGH acknowledge the predoctoral fellowships from the University of the Basque Country (UPV/EHU). The authors are grateful for the technical support provided by SGIker (UPV/EHU and ERDF, EU) for the SEM experiments

An electroactive and thermo-responsive material for the capture and release of cells

Non-invasive collection of target cells is crucial for research in biology and medicine. In this work, we combine a thermo-responsive material, poly(N-isopropylacrylamide), with an electroactive material, poly(3,4-ethylene-dioxythiopene):poly(styrene sulfonate), to generate a smart and conductive copolymer for the label-free and non-invasive detection of the capture and release of cells on gold electrodes by electrochemical impedance spectroscopy. The copolymer is functionalized with fibronectin to capture tumor cells, and undergoes a conformational change in response to temperature, causing the release of cells. Simultaneously, the copolymer acts as a sensor, monitoring the capture and release of cancer cells by electrochemical impedance spectroscopy. It is expected that this platform has the potential to play a role in top-notch label-free electrical monitoring of human cells obtaining in clinic.This project has received funding from University of the Basque Country (PIF16/204 and MOV19/41), the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 842356, Gobierno de España, Ministerio de Economía y Competitividad, with Grant No. BIO2016-80417-P (AEI/FEDER, UE) and Gobierno Vasco Dpto. Educación for the consolidation of the research groups (IT1271-19)

Microtechnologies for Cell Microenvironment Control and Monitoring

A great breadth of questions remains in cellular biology. Some questions cannot be answered using traditional analytical techniques and so demand the development of new tools for research. In the near future, the development of highly integrated microfluidic analytical platforms will enable the acquisition of unknown biological data. These microfluidic systems must allow cell culture under controlled microenvironment and high throughput analysis. For this purpose, the integration of a variable number of newly developed micro- and nano-technologies, which enable control of topography and surface chemistry, soluble factors, mechanical forces and cell-cell contacts, as well as technology for monitoring cell phenotype and genotype with high spatial and temporal resolution will be necessary. These multifunctional devices must be accompanied by appropriate data analysis and management of the expected large datasets generated. The knowledge gained with these platforms has the potential to improve predictive models of the behavior of cells, impacting directly in better therapies for disease treatment. In this review, we give an overview of the microtechnology toolbox available for the design of high throughput microfluidic platforms for cell analysis. We discuss current microtechnologies for cell microenvironment control, different methodologies to create large arrays of cellular systems and finally techniques for monitoring cells in microfluidic devices.E.A.-H. acknowledges funding from the Basque Government, Department of Education, for predoctoral fellowship 2016. M.G.-H. acknowledges funding from the University of the Basque Country UPV/EHU, PIF16/204 predoctoral fellowship "call for recruitment of research personnel in training". J.E.-E. acknowledges funding from the University of the Basque Country UPV/EHU, postdoctoral fellowship ESPPOC 16/65 "Call for recruitment and specialization of Doctor Researchers 2016". M.M.D.P. and L.B.-D., acknowledge funding support from University of the Basque Country UPV/EHU, UFI11/32, and from Gobierno Vasco under Grupos Consolidados with Grant No. IT998-16. F.B.-L. acknowledges funding support from the Ramon y Cajal Programme (Ministerio de Economia y Competitividad), Spain. F.B.-L. and L.B.-D. acknowledge funding support from the European Union's Seventh Framework Programme (FP7) for Research, Technological Development and Demonstration under Grant agreement No. 604241 as well as Gobierno Vasco, Dpto. Industria, Innovacion, Comercio y Turismo under ELKARTEK 2015 with Grant No. KK-2015/0000088

The NER-related gene GTF2H5 predicts survival in high-grade serous ovarian cancer patients

We aimed to evaluate the prognostic and predictive value of the nucleotide excision repair-related gene GTF2H5, which is localized at the 6q24.2-26 deletion previously reported by our group to predict longer survival of high-grade serous ovarian cancer patients. In order to test if protein levels of GTF2H5 are associated with patients' outcome, we performed GTF2H5 immunohistochemical staining in 139 high-grade serous ovarian carcinomas included in tissue microarrays. Upon stratification of cases into high- and low-GTF2H5 staining categories (> and ≤ median staining, respectively) Kaplan-Meier and log-rank test were used to estimate patients' survival and assess statistical differences. We also evaluated the association of GTF2H5 with survival at the transcriptional level by using the on-line Kaplan-Meier plotter tool, which includes gene expression and survival data of 855 high-grade serous ovarian cancer patients from 13 different datasets. Finally, we determined whether stable short hairpin RNA-mediated GTF2H5 downregulation modulates cisplatin sensitivity in the SKOV3 and COV504 cell lines by using cytotoxicity assays. Low expression of GTF2H5 was associated with longer 5-year survival of patients at the protein (hazard ratio [HR], 0.52; 95% CI, 0.29 to 0.93; p=0.024) and transcriptional level (HR, 0.80; 95% CI, 0.65 to 0.97; p=0.023) in high-grade serous ovarian cancer patients. We confirmed the association with 5-year overall survival (HR, 0.55; 95% CI, 0.38 to 0.78; p=0.0007) and also found an association with progression-free survival (HR, 0.72; 95% CI, 0.54 to 0.96; p=0.026) in a homogenous group of 388 high-stage (stages III-IV using the International Federation of Gynecology and Obstetrics staging system), optimally debulked high-grade serous ovarian cancer patients. GTF2H5- silencing induced a decrease of the half maximal inhibitory concentration upon cisplatin treatment in GTF2H5 -silenced ovarian cancer cells. Low levels of GTF2H5 are associated with enhanced prognosis in high-grade serous ovarian cancer patients and may contribute to cisplatin sensitization

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Materials science and microfabrication: key tools to develop microsystems for chemical and cellular monitoring

372 p.En las últimas décadas, se han realizado avances considerables en el desarrollo de dispositivos miniaturizados "lab-on-a-chip" integrados. Los sistemas miniaturizados pueden aportar información difícil de conseguir con otros medios, por lo que la utilización de protocolos de microfabricación de fácil implementación impulsaría el uso de estas plataformas como tecnologías de monitorización. En esta tesis, se han combinado la microfabricación y la ciencia de los materiales para desarrollar microsistemas para monitorización química y celular, dando lugar a estrategias novedosas como sensores, basadas en la integración de materiales funcionales en sistemas miniaturizados