Small-strain shear stiffness of compacted bentonites for engineered barrier system

The shear modulus (G) of two different bentonites was measured by means of a resonant column apparatus. The samples were compacted at different dry densities and degrees of saturation and tested with different confinement pressures and strains levels for studying the influence of these parameters on the shear modulus. The results show similar tendencies in both bentonites: the shear modulus increases as the dry density increases and exhibits maximum shear modulus when degree of saturation is around 80%. An empirical equation, taking into account the microstructure of the clays, is used to evaluate the shear modulus at small strains as a function of dry density and degree of saturation. Although the values of the shear modulus measured are similar in both bentonites for a given stress and degree of saturation, there is difference in the elastic strain limit of the soil. Bentonite clay is going to be part of the Engineered Barrier System (EBS) in deep geological disposal facilities for the long-term confinement of spent nuclear fuel. In order to fully understand their long-term performance, their behaviour in shearing conditions should be assessed.Peer ReviewedPostprint (author's final draft

Evaluation of the damping ratio of compacted sodium and calcium bentonites in unsaturated conditions

Bentonites are going to be part of the Engineered Barrier System (EBS) in deep geological disposal facilities for the safe disposal of spent nuclear fuel. Some of these repositories might be constructed in tectonically active locations, and some other repository locations might have seismic risks in future related to climate changes (e.g. glaciations). The damping ratio is one of the parameters considered in dynamic analysis, and it can be measured by different methods. In this work, the damping ratio was measured in two different bentonites with the resonant column device and in one of these bentonites, it was also measured with the hollow cylinder, simple shear and triaxial tests in unloading–reloading paths. The results are presented in Pintado et al. (2019; 2023). The tests were carried out at different laboratories. The samples were compacted at different dry densities and degrees of saturation and tested with different confinement pressures and strain levels to study the influence of the shear strain, degree of saturation, dry density and confinement pressure and also the influence of the test method. The two studied bentonites had different plasticity indices which was also considered in the analysis. The results showed a clear dependence of the damping ratio on the confinement pressure and the shear strain but not as clear on the degree of saturation, the dry density and the plasticity index. The damping ratio measured by the hollow cylinder test followed the tendency of the resonant column results. The triaxial test presented larger values of damping ratios than following the tendency of the hollow cylinder and resonant column tests. The simple shear test did not follow the tendency of the other tests, presenting lower damping ratio values. All tests presented large scatter.Peer ReviewedObjectius de Desenvolupament Sostenible::7 - Energia Assequible i No ContaminantPostprint (published version

The Second Generation Antibody-Drug Conjugate SYD985 Overcomes Resistances to T-DM1

[eng] Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer. T-DM1 consists of trastuzumab covalently linked to the cytotoxic maytansinoid DM1 via a non-cleavable linker. Despite its efficacy, primary or acquired resistance frequently develops, particularly in advanced stages of the disease. Second generation ADCs targeting HER2 are meant to supersede T-DM1 by using a cleavable linker and a more potent payload with a different mechanism of action. To determine the effect of one of these novel ADCs, SYD985, on tumors resistant to T-DM1, we developed several patient-derived models of resistance to T-DM1. Characterization of these models showed that previously described mechanisms-HER2 downmodulation, impairment of lysosomal function and upregulation of drug efflux pumps-account for the resistances observed, arguing that mechanisms of resistance to T-DM1 are limited, and most of them have already been described. Importantly, SYD985 was effective in these models, showing that the resistance to first generation ADCs can be overcome with an improved design

Therapy-induced senescence enhances the efficacy of HER2-targeted antibody-drug conjugates in breast cancer

[eng] Antibody-drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a tras-tuzumab-based ADC that relies on lysosomal processing to release the payload, is approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as [vic-]trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/ 6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression.Significance: Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells

Exclusion of NFAT5 from Mitotic Chromatin Resets Its Nucleo-Cytoplasmic Distribution in Interphase

The transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5.Using confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD). NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin.Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export signals acting in interphase, resets the cytoplasmic localization of NFAT5 and prevents its nuclear accumulation and association with DNA in the absence of hypertonic stress

Neutrophil depletion reduces edema formation and tissue loss following traumatic brain injury in mice

Background: Brain edema as a result of secondary injury following traumatic brain injury (TBI) is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear. Methods: In this study we used controlled cortical impact (CCI) as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using an anti-Gr-1 antibody was compared to that in mice with intact neutrophil count. The effect of neutrophil depletion on blood-brain barrier function was assessed by Evan's blue dye extravasation, and analysis of brain water content was used as a measurement of brain edema formation (24 and 48 hours after CCI). Lesion volume was measured 7 and 14 days after CCI. Immunohistochemistry was used to assess cell death, using a marker for cleaved caspase-3 at 24 hours after injury, and microglial/macrophage activation 7 days after CCI. Data were analyzed using Mann-Whitney test for non-parametric data. Results: Neutrophil depletion did not significantly affect Evan's blue extravasation at any time-point after CCI. However, neutrophil-depleted mice exhibited a decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Furthermore, brain tissue loss was attenuated in neutropenic mice at 7 and 14 days after injury. Additionally, these mice had a significantly reduced number of activated microglia/macrophages 7 days after CCI, and of cleaved caspase-3 positive cells 24 h after injury. Conclusion: Our results suggest that neutrophils are involved in the edema formation, but not the extravasation of large proteins, as well as contributing to cell death and tissue loss following TBI in mice

The Transcription Factor NFAT5 Is Required for Cyclin Expression and Cell Cycle Progression in Cells Exposed to Hypertonic Stress

Background: Hypertonicity can perturb cellular functions, induce DNA damage-like responses and inhibit proliferation. The transcription factor NFAT5 induces osmoprotective gene products that allow cells to adapt to sustained hypertonic conditions. Although it is known that NFAT5-deficient lymphocytes and renal medullary cells have reduced proliferative capacity and viability under hypertonic stress, less is understood about the contribution of this factor to DNA damage responses and cell cycle regulation. Methodology/Principal Findings: We have generated conditional knockout mice to obtain NFAT5−/− T lymphocytes, which we used as a model of proliferating cells to study NFAT5-dependent responses. We show that hypertonicity triggered an early, NFAT5-independent, genotoxic stress-like response with induction of p53, p21 and GADD45, downregulation of cyclins, and cell cycle arrest. This was followed by an NFAT5-dependent adaptive phase in wild-type cells, which induced an osmoprotective gene expression program, downregulated stress markers, resumed cyclin expression and proliferation, and displayed enhanced NFAT5 transcriptional activity in S and G2/M. In contrast, NFAT5−/− cells failed to induce osmoprotective genes and exhibited poorer viability. Although surviving NFAT5−/− cells downregulated genotoxic stress markers, they underwent cell cycle arrest in G1/S and G2/M, which was associated with reduced expression of cyclins E1, A2 and B1. We also show that pathologic hypertonicity levels, as occurring in plasma of patients and animal models of osmoregulatory disorders, inhibited the induction of cyclins and aurora B kinase in response to T cell receptor stimulation in fresh NFAT5−/− lymphocytes. Conclusions/Significance: We conclude that NFAT5 facilitates cell proliferation under hypertonic conditions by inducing an osmoadaptive response that enables cells to express fundamental regulators needed for cell cycle progression.Molecular and Cellular Biolog

Left ventricular remodeling in swine after myocardial infarction: a transcriptional genomics approach

Despite the apparent appropriateness of left ventricular (LV) remodeling following myocardial infarction (MI), it poses an independent risk factor for development of heart failure. There is a paucity of studies into the molecular mechanisms of LV remodeling in large animal species. We took an unbiased molecular approach to identify candidate transcription factors (TFs) mediating the genetic reprogramming involved in post-MI LV remodeling in swine. Left ventricular tissue was collected from remote, non-infarcted myocardium, 3 weeks after MI-induction or sham-surgery. Microarray analysis identified 285 upregulated and 278 downregulated genes (FDR < 0.05). Of these differentially expressed genes, the promoter regions of the human homologs were searched for common TF binding sites (TFBS). Eighteen TFBS were overrepresented >two-fold (p < 0.01) in upregulated and 13 in downregulated genes. Left ventricular nuclear protein extracts were assayed for DNA-binding activity by protein/DNA array. Out of 345 DNA probes, 30 showed signal intensity changes >two-fold. Five TFs were identified in both TFBS and protein/DNA array analyses, which showed matching changes for COUP-TFII and glucocorticoid receptor (GR) only. Treatment of swine with the GR antagonist mifepristone after MI reduced the post-MI increase in LV mass, but LV dilation remained unaffected. Thus, using an unbiased approach to study post-MI LV remodeling in a physiologically relevant large animal model, we identified COUP-TFII and GR as potential key mediators of post-MI remodeling

Accuracy versus precision in boosted top tagging with the ATLAS detector

The identification of top quark decays where the top quark has a large momentum transverse to the beam axis, known as top tagging, is a crucial component in many measurements of Standard Model processes and searches for beyond the Standard Model physics at the Large Hadron Collider. Machine learning techniques have improved the performance of top tagging algorithms, but the size of the systematic uncertainties for all proposed algorithms has not been systematically studied. This paper presents the performance of several machine learning based top tagging algorithms on a dataset constructed from simulated proton-proton collision events measured with the ATLAS detector at √s = 13 TeV. The systematic uncertainties associated with these algorithms are estimated through an approximate procedure that is not meant to be used in a physics analysis, but is appropriate for the level of precision required for this study. The most performant algorithms are found to have the largest uncertainties, motivating the development of methods to reduce these uncertainties without compromising performance. To enable such efforts in the wider scientific community, the datasets used in this paper are made publicly available