This paper deals with the determination of upper and lower bounds for the three-dimensional undrained stability of shallow tunnels. The tunnel is circular and a distance between its face and its lining is considered. The soil shear strength is modeled using the Tresca criterion. Results of the upper and lower bounds of the stability number are presented, for several geometrical and resistance configurations and their comparison with previous results is made, showing the clear improvement obtained. Finally, equations approaching the stability number are proposed.

Journal of Trace Elements in Medicine and Biology, 68 (2021) 126837. doi:10.1016/j.jtemb.2021.126837

Ion beam analysis of fluorine has applications in research on teeth and bones, materials science, geochemistry and archaeometry. A novel PIGE (particle induced gamma-ray emission) standard free methodology for fluorine content determination for in-depth heterogeneous samples based on the excitation function of the 19F(p,p’$\gamma$)19F nuclear reaction is presented. New precise cross section measurements of this reaction in the proton energy range 2.1 to 4.1 MeV have been performed. In addition, the ERYA-Profiling code, a computer program specially developed for PIGE analysis of in-depth heterogeneous samples, employed this new excitation function in a case study where different fluorine simulated depth profiles probed the capability of insight into fluorine distributions in a given sample, showing the potential of PIGE analysis.

We present relativistic ab initio calculations of fundamental parameters for atomic selenium, based on the Multiconfiguration Dirac-Fock method. In detail, fluorescence yields and subshell linewidths, both of K shell, as well as K$\beta$ to K$\alpha$ intensity ratio are provided, showing overall agreement with previous theoretical calculations and experimental values. Relative intensities were evaluated assuming the same ionization cross-section for the K-shell hole states, leading to a statistical distribution of these initial states. A method for estimating theoretical linewidths of X-ray lines, where the lines are composed by a multiplet of fine-structure levels that are spread in energy, is proposed. This method provides results that are closer to K$\alpha$1,2 experimental width values than the usual method, although slightly higher discrepancies occur for the K$\beta$1,3 lines. This indicates some inaccuracies in the calculation of Auger rates that have a higher contribution for partial linewidths of the subshells involved in the K$\beta$1,3 profile. Apart from this, the calculated value of K$\beta$ to K$\alpha$ intensity ratio, which is less sensitive to Auger rates issues, is in excellent agreement with recommended values.

Nuclear Inst. and Methods in Physics Research, A, 1014 (2021) 165701. doi:10.1016/j.nima.2021.165701

The SOUL, or heme-binding protein HBP/SOUL, family represents a group of evolutionary conserved putative heme-binding proteins that contains a number of members in animal, plant andbacterial species. The structures of the murine form of HEBP1, or p22HBP, and the human form of HEBP2, or SOUL, have been determined in 2006 and 2011 respectively. In this work we discuss the structures of HEBP1 and HEBP2 in light of new X-ray data for heme bound murine HEBP1. The interaction between tetrapyrroles and HEBP1, initially proven to be hydrophobic in nature, was thought to also involve electrostatic interactions between heme propionate groups and positively charged amino acid side chains. However, the new X-ray structure, and results from murine HEBP1 variants and human HEBP1, confirm the hydrophobic nature of the heme-HEBP1 interaction, resulting in Kd values in the low nanomolar range, and rules out any electrostatic stabilization. Results from NMR relaxation time measurements for human HEBP1 describe a rigid globular protein with no change in motional regime upon heme binding. X-ray structures deposited in the PDB for human HEBP2 are very similar to each other and to the new heme-bound murine HEBP1 X-ray structure (backbone rmsd ca. 1 {\AA}). Results from a HSQC spectrum centred on the histidine side chain N$δ$-proton region for HEBP2 confirm that HEBP2 does not bind heme via H42 as no chemical shift differences were observed upon heme addition for backbone NH and N$δ$ protons. A survey of the functions attributed to HEBP1 and HEBP2 over the last 20 years span a wide range of cellular pathways. Interestingly, many of them are specific to higher eukaryotes, particularly mammals and a potential link between heme release under oxidative stress and human HEBP1 is also examined using recent data. However, at the present moment, trying to relate function to the involvement of heme or tetrapyrrole binding, specifically, makes little sense with our current biological knowledge and can only be applied to HEBP1, as HEBP2 does not interact with heme. We suggest that it may not be justified to call this very small family of proteins, heme-binding proteins. The family may be more correctly called “the SOUL family of proteins related to cellular fate” as, even though only HEBP1 binds heme tightly, both proteins may be involved in cell survival and/or proliferation.

Physics Letters B, 809 (2020) 135748. 10.1016/j.physletb.2020.135748

Siglec-15 is a conserved sialic acid-binding Ig-like lectin expressed on osteoclast progenitors that plays an important role in osteoclast development and function. It is also expressed by tumor-associated macrophages and by some tumors, where it is thought to contribute to the immunosuppressive microenvironment. It was shown previously that engagement of macrophage-expressed Siglec-15 with tumor cells expressing its ligand, sialyl Tn (sTn), triggered production of TGF-$\beta$. In the present study, we have further investigated the interaction between Siglec-15 and sTn on tumor cells and its functional consequences. Based on binding assays with lung and breast cancer cell lines and glycan-modified cells, we failed to see evidence for recognition of sTn by Siglec-15. However, using a microarray of diverse, structurally-defined glycans, we show that Siglec-15 binds with higher avidity to sialylated glycans other than sTn or related antigen sequences. In addition, we were unable to demonstrate enhanced TGF-$\beta$ secretion following co-culture of Siglec-15-expressing monocytic cells lines with tumor cells expressing sTn, or following Siglec-15 cross-linking with monoclonal antibodies. However, we did observe activation of the SYK/MAPK signaling pathway following antibody cross-linking of Siglec-15 that may modulate the functional activity of macrophages.

Understanding the specific molecular interactions between proteins and $\beta$1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for $\beta$1,3-1,4-mixed-linked over $\beta$1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to $\beta$1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the $\beta$1,3-linkage are important for recognition, and identified the tetrasaccharide Glc$\beta$1,4Glc$\beta$1,4Glc$\beta$1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of $\beta$1,3-1,4-mixed-linked glucans. This is mediated by a conformation-selection mechanism of the ligand in the binding cleft through CH-$π$ stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a $\beta$1,3-linkage at the reducing end and at specific positions along the $\beta$1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked $\beta$-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. DATABASE: Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.

The simulation of atomic relaxation relies on data libraries with tabulated partial fluorescence yield values of radiative transitions, commonly derived from the Evaluated Atomic Data Library (EADL)....

In this work, we aim at achieving the most accurate quantitative determination of elements in human tissues by means of X-ray Fluorescence spectrometry using the external calibration approach. A calibration curve built using a set of certified reference materials (CRM) of animal tissue was compared with the one obtained with a set of CRMs of plants and leaves with lower atomic number Z but with correction of the matrix using the scattering peaks of the X-ray tube anode. Finally, a calibration curve combining the two sets of CRMs was built and the accuracy of the quantification using the three methods was compared and a more precise method of quantification was obtained. This improved approach was tested on five paired samples of normal and tumour human tissue. Despite the high heterogeneity of the samples, and given the improvement in accuracy of the measurements, significant differences were found in the elemental concentration of low-Z elements. This journal is

Co-sputtering of SiO2 and high-$ąppa$ Ta2O5 was used to make multicomponent gate dielectric stacks for In-Ga-Zn-O thin-film transistors (IGZO TFTs) under an overall low thermal budget (T = 150 °C). Characterization of the multicomponent layers and of the TFTs working characteristics (employing them) was performed in terms of static performance, reliability, and stability to understand the role of the incorporation of the high-$ąppa$ material in the gate dielectric stack. It is shown that inherent disadvantages of the high-$ąppa$ material, such as poorer interface properties and poor gate insulation, can be counterbalanced by inclusion of SiO2 both mixed with Ta2O5 and as thin interfacial layers. A stack comprising a (Ta2O5)x(SiO2)100 − x film with x = 69 and a thin SiO2 film at the interface with IGZO resulted in the best performing TFTs, with field-effect mobility (µFE) ≈ 16 cm2·V−1·s−1, subthreshold slope (SS) ≈ 0.15 V/dec and on/off ratio exceeding 107. Anomalous Vth shifts were observed during positive gate bias stress (PGBS), followed by very slow recoveries (time constant exceeding 8 × 105 s), and analysis of the stress and recovery processes for the different gate dielectric stacks showed that the relevant mechanism is not dominated by the interfaces but seems to be related to the migration of charged species in the dielectric. The incorporation of additional SiO2 layers into the gate dielectric stack is shown to effectively counterbalance this anomalous shift. This multilayered gate dielectric stack approach is in line with both the large area and the flexible electronics needs, yielding reliable devices with performance suitable for successful integration on new electronic applications.

The current trend for smart, self-sustainable, and multifunctional technology demands for the development of energy harvesters based on widely available and environmentally friendly materials. In this context, ZnSnO3 nanostructures show promising potential because of their high polarization, which can be explored in piezoelectric devices. Nevertheless, a pure phase of ZnSnO3 is hard to achieve because of its metastability, and obtaining it in the form of nanowires is even more challenging. Although some groups have already reported the mixing of ZnSnO3 nanostructures with polydimethylsiloxane (PDMS) to produce a nanogenerator, the resultant polymeric film is usually flat and does not take advantage of an enhanced piezoelectric contribution achieved through its microstructuration. Herein, a microstructured composite of nanowires synthesized by a seed-layer free hydrothermal route mixed with PDMS (ZnSnO3@PDMS) is proposed to produce nanogenerators. PFM measurements show a clear enhancement of d33 for single ZnSnO3 versus ZnO nanowires (23 ± 4 pm/V vs 9 ± 2 pm/V). The microstructuration introduced herein results in an enhancement of the piezoelectric effect of the ZnSnO3 nanowires, enabling nanogenerators with an output voltage, current, and instantaneous power density of 120 V, 13 $μ$A, and 230 $μ$W·cm-2, respectively. Even using an active area smaller than 1 cm2, the performance of this nanogenerator enables lighting up multiple LEDs and other small electronic devices, thus proving great potential for wearables and portable electronics.

Acrylamide is a neurotoxic and carcinogenic organic compound that is able to bind to several biomolecules and form adducts, through nucleophilic addition and in vivo by the Maillard Reaction, interfering with the biological functions of these molecules. Hemoglobin is one of the most abundant intracellular blood proteins, and thus it is of high interest to understand whether the binding of acrylamide can alter its properties. The interaction of acrylamide with hemoglobin was assessed in a 20:1 ratio, and after a 72 h-incubation period, a decrease of ca. 50% in the absorbance of the hemoglobin's Soret band was observed at 37 °C. This together with the analysis of circular dichroism spectra indicate that acrylamide binds in close proximity to the heme group. These perturbations were confirmed to not correspond to the loss of the heme group and were mostly reverted after passing the protein through a size-exclusion chromatographic matrix, suggesting a dominant non-covalent interaction for the observed effect. The thermodynamic parameters of unfolding in the absence and presence of acrylamide, suggest an interaction based on H-bonds and van der Waals forces that slightly stabilizes hemoglobin. The oxygen binding capacity of hemoglobin does not seem to be hindered, as no differences in the Q bands were observed in the adduct.

Increasing atmospheric concentration of N2O has been a concern, as it is a potent greenhouse gas and promotes ozone layer destruction. In the N-cycle, release of N2O is boosted upon a drop of pH in the environment. Here, Marinobacter hydrocarbonoclasticus was grown in batch mode in the presence of nitrate, to study the effect of pH in the denitrification pathway by gene expression profiling, quantification of nitrate and nitrite, and evaluating the ability of whole cells to reduce NO and N2O. At pH 6.5, accumulation of nitrite in the medium occurs and the cells were unable to reduce N2O. In addition, the biochemical properties of N2O reductase isolated from cells grown at pH 6.5, 7.5 and 8.5 were compared for the first time. The amount of this enzyme at acidic pH was lower than that at pH 7.5 and 8.5, pinpointing to a post-transcriptional regulation, though pH did not affect gene expression of N2O reductase accessory genes. N2O reductase isolated from cells grown at pH 6.5 has its catalytic center mainly as CuZ(4Cu1S), while that from cells grown at pH 7.5 or 8.5 has it as CuZ(4Cu2S). This study evidences that an in vivo secondary level of regulation is required to maintain N2O reductase in an active state.

One of the trends in downstream processing comprises the use of ?anything-but-chromatography? methods to overcome the current downfalls of standard packed-bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new approach combining precipitation and magnetic separation was developed and named as affinity magnetic precipitation. A precipitation screening, based on the Hofmeister series, and a commercial precipitation kit were tested with affinity magnetic particles to assess the best condition for antibody capture from human serum plasma and clarified cell supernatant. The best conditions were obtained when using PEG3350 as precipitant at 4°C for 1h, reaching 80% purity and 50% recovery of polyclonal antibodies from plasma, and 99% purity with 97% recovery yield of anti-TNFα mAb from cell supernatants. These results show that the synergetic use of precipitation and magnetic separation can represent an alternative for the efficient capture of antibodies. This article is protected by copyright. All rights reserved

Reduction of N2O to N2 is catalysed by nitrous oxide reductase in the last step of the denitrification pathway. This multicopper enzyme has an electron transferring centre, CuA, and a tetranuclear copper-sulfide catalytic centre, “CuZ”, which exists as CuZ*(4Cu1S) or CuZ(4Cu2S). The redox behaviour of these metal centres in Marinobacter hydrocarbonoclasticus nitrous oxide reductase was investigated by potentiometry and for the first time by direct electrochemistry. The reduction potential of CuA and CuZ(4Cu2S) was estimated by potentiometry to be +275 ± 5 mV and +65 ± 5 mV vs SHE, respectively, at pH 7.6. A proton-coupled electron transfer mechanism governs CuZ(4Cu2S) reduction potential, due to the protonation/deprotonation of Lys397 with a pKox of 6.0 ± 0.1 and a pKred of 9.2 ± 0.1. The reduction potential of CuA, in enzyme samples with CuZ*(4Cu1S), is controlled by protonation of the coordinating histidine residues in a two-proton coupled electron transfer process. In the cyclic voltammograms, two redox pairs were identified corresponding to CuA and CuZ(4Cu2S), with no additional signals being detected that could be attributed to CuZ*(4Cu1S). However, an enhanced cathodic signal for the activated enzyme was observed under turnover conditions, which is explained by the binding of nitrous oxide to CuZ0(4Cu1S), an intermediate species in the catalytic cycle.

BackgroundHalf of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1.
Methods and results
By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced.
Conclusions
SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53.
General Significance
This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.