Interpreting rheology and electrical conductivity: it all boils down to which particle size

Hypothesis: The electrical charges that develop on the surface of the ceramic particles upon contact with water, due to the interaction with ions in solution, result in a liquid-solid interface, which utterly modifies the properties of individual particles and the way they interact with each other to form a structure. This work explores a new approach to the relationships between structure and stability of suspensions. Experiments: For this purpose, suspensions with a constant 0.35 volume fraction of α-alumina particles, neither spherical nor smooth, and controlled ionic strength (0–90 mM KCl) were prepared and characterized in terms of flow behaviour, electrical conductivity and particle’s electrokinetic mobility. Findings: Electrical conductivity (132 µS/cm < conductivity < 5730 µS/cm) and rheology measurements (10−2 Pa s < viscosity < 104 Pa s) were found to complement each other to produce a more accurate picture of the suspension’s structure. Deviations of experimental data from well-accepted behavioural models were elucidated when the surface area equivalent particle size was used. With the electrical double layer thickness obtained from electrical conductivity measurements, this enabled the interpretation of the relationship between the suspension’s viscosity and the particles electrical conductivity, which provides a criterion for the stability of concentrated colloidal suspensions.publishe

A Novel Function

Funding Information: This work was supported by Fundação para a Ciência e Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES), through the grant number PTDC/QUI/64248/2006 (to A.S.P.), the Radiation Biology and Biophysics Doctoral Training Programme—RaBBiT (PD/00193/2012), Applied Molecular Biosciences Unit—UCIBIO (UIDP/04378/2020, i4HB—Institute for Health and Bioeconomy (LA/P/0140/2020) and CEFITEC (UIDB/00068/2020). A.V.A. (PD/BD/135477/2017 and COVID/BD/152498/2022) is supported by the RaBBiT programme. This work benefited from STSM funding by COST Action (CA15126 MOBIEU) and by the project CALIPSOplus under the Grant Agreement 730872 from the EU Framework Programme for Research and Innovation HORIZON 2020. Publisher Copyright: © 2022 by the authors.Encapsulins are protein nanocages capable of harboring smaller proteins (cargo proteins) within their cavity. The function of the encapsulin systems is related to the encapsulated cargo proteins. The Myxococcus xanthus encapsulin (EncA) naturally encapsulates ferritin-like proteins EncB and EncC as cargo, resulting in a large iron storage nanocompartment, able to accommodate up to 30,000 iron atoms per shell. In the present manuscript we describe the binding and protection of circular double stranded DNA (pUC19) by EncA using electrophoretic mobility shift assays (EMSA), atomic force microscopy (AFM), and DNase protection assays. EncA binds pUC19 with an apparent dissociation constant of 0.3 ± 0.1 µM and a Hill coefficient of 1.4 ± 0.1, while EncC alone showed no interaction with DNA. Accordingly, the EncAC complex displayed a similar DNA binding capacity as the EncA protein. The data suggest that initially, EncA converts the plasmid DNA from a supercoiled to a more relaxed form with a beads-on-a-string morphology. At higher concentrations, EncA self-aggregates, condensing the DNA. This process physically protects DNA from enzymatic digestion by DNase I. The secondary structure and thermal stability of EncA and the EncA−pUC19 complex were evaluated using synchrotron radiation circular dichroism (SRCD) spectroscopy. The overall secondary structure of EncA is maintained upon interaction with pUC19 while the melting temperature of the protein (Tm) slightly increased from 76 ± 1 °C to 79 ± 1 °C. Our work reports, for the first time, the in vitro capacity of an encapsulin shell to interact and protect plasmid DNA similarly to other protein nanocages that may be relevant in vivo.publishersversionpublishe

The Conformation of the N-Terminal Tails of Deinococcus grandis Dps Is Modulated by the Ionic Strength

UIDB/04378/2020 LA/P/0140/2020 UID/Multi/04349/2019 LISBOA-01-0145-FEDER-022096 PTDC/BIA-PRO/111485/2009 PTDC/QUI/64248/2006 PD/00193/2012—UCIBIO (UIDB/04378/2020), UIDB/00068/2020 PD/BD/135476/2017 COVID/BD/152497/2022 PD/BD/135477/2017 COVID/BD/152498/2022 Grant Agreement 730872DNA-binding proteins from starved cells (Dps) are homododecameric nanocages, with N-and C-terminal tail extensions of variable length and amino acid composition. They accumulate iron in the form of a ferrihydrite mineral core and are capable of binding to and compacting DNA, forming low-and high-order condensates. This dual activity is designed to protect DNA from oxidative stress, resulting from Fenton chemistry or radiation exposure. In most Dps proteins, the DNA-binding properties stem from the N-terminal tail extensions. We explored the structural characteristics of a Dps from Deinococcus grandis that exhibits an atypically long N-terminal tail composed of 52 residues and probed the impact of the ionic strength on protein conformation using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism and small-angle X-ray scattering. A novel high-spin ferrous iron-binding site was identified in the N-terminal tails, using Mössbauer spectroscopy. Our data reveals that the N-terminal tails are structurally dynamic and alter between compact and extended conformations, depending on the ionic strength of the buffer. This prompts the search for other physiologically relevant modulators of tail conformation and hints that the DNA-binding properties of Dps proteins may be affected by external factors.publishersversionpublishe

Excited states of bromopyrimidines probed by vuv photoabsorption spectroscopy and theoretical calculations

Funding Information: Funding: J.P.‐d.‐S. acknowledges the Portuguese National Funding Agency FCT‐MCTES through PhD grant PD/BD/142768/2018, together with R.R., J.A., M.M. and F.F.d.S. through the researcher grant PTDC/FIS‐AQM/31215/2017 and AIL through the researcher grant PTDC/FIS‐ AQM/31281/2017. This work was also supported by Radiation Biology and Biophysics Doctoral Training Programme (RaBBiT, PD/00193/2012); UIDB/04378/2020 (UCIBIO); and UIDB/00068/2020 (CEFITEC). The research leading to this result has been supported by the project CALIPSOplus under the Grant Agreement 730872 from the EU Framework Programme for Research and Innovation HORIZON 2020.We report absolute photoabsorption cross sections for gas‐phase 2‐ and 5‐ bromopyrimidine in the 3.7–10.8 eV energy range, in a joint theoretical and experimental study. The measurements were carried out using high‐resolution vacuum ultraviolet synchrotron radiation, with quantum chemical calculations performed through the nuclear ensemble approach in combination with time‐dependent density functional theory, along with additional Franck–Condon Herzberg–Teller calculations for the first absorption band (3.7–4.6 eV). The cross sections of both bromopyrimidines are very similar below 7.3 eV, deviating more substantially from each other at higher energies. In the 7.3–9.0 eV range where the maximum cross‐section is found, a single and broad band is observed for 5‐bromopyrimidine, while more discernible features appear in the case of 2‐bromopyrimidine. Several π* ← π transitions account for the most intense bands, while weaker ones are assigned to transitions involving the nitrogen and bromine lone pairs, the antibonding σ*Br orbital, and the lower‐lying Rydberg states. A detailed comparison with the available photo‐absorption data of bromobenzene is also reported. We have found significant differences regarding the main absorption band, which is more peaked in bromobenzene, becoming broader and shifting to higher energies in both bromopyrimidines. In addition, there is a significant suppression of vibrational structures and of Rydberg states in the pair of isomers, most noticeably for 2‐bromopyrimidine.publishersversionpublishe

Targeted transcript quantification in single disseminated cancer cells after whole transcriptome amplification

Gene expression analysis of rare or heterogeneous cell populations such as disseminated cancer cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-amplification of WTA products, quantification of amplified cDNA yields and final qPCR quantification, to identify the most reliable and accurate workflow for quantitation of gene expression of the ERBB2 gene in DCCs. We found that absolute quantification out-performs relative quantification. We then validated the performance of our method on single cells of established breast cancer cell lines displaying distinct levels of HER2 protein. The different protein levels were faithfully reflected by transcript expression across the tested cell lines thereby proving the accuracy of our approach. Finally, we applied our method to breast cancer DCCs of a patient undergoing anti-HER2-directed therapy. Here, we were able to measure ERBB2 expression levels in all HER2-protein-positive DCCs. In summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of ERBB2 in DCCs

p45 NF-E2 regulates syncytiotrophoblast differentiation by post-translational GCM1 modifications in human intrauterine growth restriction

AbstractPlacental insufficiency jeopardizes prenatal development, potentially leading to intrauterine growth restriction (IUGR) and stillbirth. Surviving fetuses are at an increased risk for chronic diseases later in life. IUGR is closely linked with altered trophoblast and placental differentiation. However, due to a paucity of mechanistic insights, suitable biomarkers and specific therapies for IUGR are lacking. The transcription factor p45 NF-E2 (nuclear factor erythroid derived 2) has been recently found to regulate trophoblast differentiation in mice. The absence of p45 NF-E2 in trophoblast cells causes IUGR and placental insufficiency in mice, but mechanistic insights are incomplete and the relevance of p45 NF-E2 for human syncytiotrophoblast differentiation remains unknown. Here we show that p45 NF-E2 negatively regulates human syncytiotrophoblast differentiation and is associated with IUGR in humans. Expression of p45 NF-E2 is reduced in human placentae complicated with IUGR compared with healthy controls. Reduced p45 NF-E2 expression is associated with increased syncytiotrophoblast differentiation, enhanced glial cells missing-1 (GCM1) acetylation and GCM1 desumoylation in IUGR placentae. Induction of syncytiotrophoblast differentiation in BeWo and primary villous trophoblast cells with 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) reduces p45 NF-E2 expression. Of note, p45 NF-E2 knockdown is sufficient to increase syncytiotrophoblast differentiation and GCM1 expression. Loss of p45 NF-E2 using either approach resulted in CBP-mediated GCM1 acetylation and SENP-mediated GCM1 desumoylation, demonstrating that p45 NF-E2 regulates post-translational modifications of GCM1. Functionally, reduced p45 NF-E2 expression is associated with increased cell death and caspase-3 activation in vitro and in placental tissues samples. Overexpression of p45 NF-E2 is sufficient to repress GCM1 expression, acetylation and desumoylation, even in 8-Br-cAMP exposed BeWo cells. These results suggest that p45 NF-E2 negatively regulates differentiation and apoptosis activation of human syncytiotrophoblast by modulating GCM1 acetylation and sumoylation. These studies identify a new pathomechanism related to IUGR in humans and thus provide new impetus for future studies aiming to identify new biomarkers and/or therapies of IUGR.</jats:p

Vapour pressure deficit modulates hydraulic function and structure of tropical rainforests under nonlimiting soil water supply

Atmospheric conditions are expected to become warmer and drier in the future, but little is known about how evaporative demand influences forest structure and function independently from soil moisture availability, and how fast-response variables (such as canopy water potential and stomatal conductance) may mediate longer-term changes in forest structure and function in response to climate change. We used two tropical rainforest sites with different temperatures and vapour pressure deficits (VPD), but nonlimiting soil water supply, to assess the impact of evaporative demand on ecophysiological function and forest structure. Common species between sites allowed us to test the extent to which species composition, relative abundance and intraspecific variability contributed to site-level differences. The highest VPD site had lower midday canopy water potentials, canopy conductance (gc), annual transpiration, forest stature, and biomass, while the transpiration rate was less sensitive to changes in VPD; it also had different height–diameter allometry (accounting for 51% of the difference in biomass between sites) and higher plot-level wood density. Our findings suggest that increases in VPD, even in the absence of soil water limitation, influence fast-response variables, such as canopy water potentials and gc, potentially leading to longer-term changes in forest stature resulting in reductions in biomass

Updating known distribution models for forecasting climate change impact on endangered species

To plan endangered species conservation and to design adequate management programmes, it is necessary to predict their distributional response to climate change, especially under the current situation of rapid change. However, these predictions are customarily done by relating de novo the distribution of the species with climatic conditions with no regard of previously available knowledge about the factors affecting the species distribution. We propose to take advantage of known species distribution models, but proceeding to update them with the variables yielded by climatic models before projecting them to the future. To exemplify our proposal, the availability of suitable habitat across Spain for the endangered Bonelli’s Eagle (Aquila fasciata) was modelled by updating a pre-existing model based on current climate and topography to a combination of different general circulation models and Special Report on Emissions Scenarios. Our results suggested that the main threat for this endangered species would not be climate change, since all forecasting models show that its distribution will be maintained and increased in mainland Spain for all the XXI century. We remark on the importance of linking conservation biology with distribution modelling by updating existing models, frequently available for endangered species, considering all the known factors conditioning the species’ distribution, instead of building new models that are based on climate change variables only.Ministerio de Ciencia e Innovación and FEDER (project CGL2009-11316/BOS

Analysing biodiversity and conservation knowledge products to support regional environmental assessments

Two processes for regional environmental assessment are currently underway: the Global Environment Outlook (GEO) and Intergovernmental Platform on Biodiversity and Ecosystem Services (IPBES). Both face constraints of data, time, capacity, and resources. To support these assessments, we disaggregate three global knowledge products according to their regions and subregions. These products are: The IUCN Red List of Threatened Species, Key Biodiversity Areas (specifically Important Bird & Biodiversity Areas [IBAs], and Alliance for Zero Extinction [AZE] sites), and Protected Planet. We present fourteen Data citations: numbers of species occurring and percentages threatened; numbers of endemics and percentages threatened; downscaled Red List Indices for mammals, birds, and amphibians; numbers, mean sizes, and percentage coverages of IBAs and AZE sites; percentage coverage of land and sea by protected areas; and trends in percentages of IBAs and AZE sites wholly covered by protected areas. These data will inform the regional/subregional assessment chapters on the status of biodiversity, drivers of its decline, and institutional responses, and greatly facilitate comparability and consistency between the different regional/subregional assessments

High-Throughput NMR Assessment of the Tertiary Structure of Food Allergens

In vitro component-resolved diagnosis of food allergy requires purified allergens that have to meet high standards of quality. These include the authentication of their conformation, which is relevant for the recognition by specific IgE antibodies from allergic patients. Therefore, highly sensitive and reliable screening methods for the analysis of proteins/allergens are required to assess their structural integrity. In the present study one-dimensional 1H Nuclear Magnetic Resonance (1D 1H-NMR) analysis was adopted for the assessment of overall structural and dynamic properties and authentication of a set of relevant food allergens, including non-specific lipid transfer proteins from apple, peach and hazelnut, 7/8S seed storage globulins from hazelnut and peanut, 11S seed storage globulins from hazelnut and peanut, caseins from cows' and goats' milk and tropomyosin from shrimp.Two sets of 1D 1H-NMR experiments, using 700 MHz and 600 MHz instruments at 298 K were carried out to determine the presence and the extent of tertiary structure. Structural similarity among members of the individual allergen families was also assessed and changes under thermal stress investigated. The nuclear magnetic resonance (NMR) results were compared with structural information available either from the literature, Protein Data Bank entries, or derived from molecular models.1D (1)H-NMR analysis of food allergens allowed their classification into molecules with rigid, extended and ordered tertiary structures, molecules without a rigid tertiary structure and molecules which displayed both features. Differences in thermal stability were also detected. In summary, 1D (1)H-NMR gives insights into molecular fold of proteins and offers an independent method for assessing structural properties of proteins