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.

{Crocodylomorphs were a diverse clade in the Late Jurassic of Portugal, with six taxa reported to date. Here we describe 126 isolated teeth recovered by screen-washing of sediments from Valmitão (Lourinhã, Portugal, late Kimmeridgian–Tithonian), a vertebrate microfossil assemblage in which at least five distinct crocodylomorph taxa are represented. Ten morphotypes are described and attributed to five clades (Lusitanisuchus, Atoposauridae, Goniopholididae, Bernissartiidae and an undetermined mesoeucrocodylian). Four different ecomorphotypes are here proposed according to ecological niches and feeding behaviours: these correspond to a diet based on arthropods and small vertebrates (Lusitanisuchus and Atoposauridae), a generalist diet (Goniopholididae), a durophagous diet (Bernissartiidae) and a carnivorous diet. Lusitanisuchus mitracostatus material from Guimarota is here redescribed to achieve a better illustration and comparison with the new material.This assemblage shares similar ecomorphotypes with other Mesozoic west-central European localities, where a diversity of crocodylomorphs lived together, avoiding direct ecological competition through niche partitioning. The absence of large marine crocodylomorphs, present in other contemporaneous assemblages, is here interpreted as evidence that the Valmitão assemblage was deposited in a freshwater environment, although sample bias cannot be completely ruled out. These affinities are further supported by the presence of lanceolate and leaf-shaped teeth associated with continental clades.}

Three new additional skull specimens of Talarurus plicatospineus have been recovered from the Upper Cretaceous (Cenomanian–Santonian) Bayanshiree Formation, of Bayan Shiree cliffs, eastern Gobi Desert, Mongolia. The skulls feature unique characters such as an anteriorly protruded single internarial caputegulum, around 20 flat or concave nasal-area caputegulae surrounded by a wide sulcus, a vertically oriented elongate loreal caputegulum with a pitted surface, an elongate lacrimal caputegulum positioned above the posterodorsal border of the maxilla, two longitudinally arranged large frontoparietal caputegulae surrounded by smaller rhomboid caputegulae, small but elongate medial supraorbital caputegulae, a posterior supraorbital caputegulum that is four times larger than the anterior one, up to three transverse parallel grooves on the dorsal surface of the posterior supraorbital caputegulum, postocular caputegulae along the ventral to posterior rim of the orbit that extend almost to the anteroventral margin of the squamosal horn, a longitudinal furrow tapering towards the apex of the squamosal horn, a lateral nuchal caputegulum four to five times larger than other nuchal caputegulae, and a pterygovomerine keel with a ventral margin that is dorsally positioned to the alveolar ridge. The phylogenetic analysis result showed that Talarurus is sister to the clade that includes the derived Asian ankylosaurines (Saichania chulsanensis, Tarchia kielanae, and Zaraapelta nomadis). It also shows that there was dispersal of ankylosaurines from Asia into western North America before the Cenomanian. Moreover, the rostral differences between Talarurus and Tsagantegia, another ankylosaur from the same formation, suggest possible niche partitioning between these taxa.

In the current literature we can find mainly two approaches to the
SDE regime switching modeling. The traditional one, the externally induced
regime switching diffusions is described by the switching being derived from
a separate continuous time Markov process, with a finite, or denumerable,
state space { indexing the regimes { the random times of the regime switches
being exactly the jump times of the finite valued Markov process. There is a
first alternative approach in which the regime switching occurs whenever the
trajectory enters in some prescribed region on the state space; the regions we
consider will be mainly open intervals defined by unknown thresholds for the
trajectories; thresholds that, in principle, should also be estimated. In this
approach the partitioning of the the state space is already defined in the drift
and volatility of the SDE. In a second alternative approach the switching occurs
in a random way but at some random times defined when the trajectories hit
some prescribed thresholds, that again, must be estimated. We may designate
these two alternative approaches as auto-induced regime switching diffusions
as there is no external noise source to force the switching occurrence. We prove
a generalization of an existence result of the existence of auto-induced regime
switching SDE solutions for irregular coefficients and a result that encompasses
some of the cases of both externally and auto-induced regime switching SDE
solutions.

Mathematics has a strong presence in the school curriculum, often justified by its usefulness in social life, in the world of work and by its connections with other sciences. This interdisciplinary connection, in particular when it requires constructing and refining mathematical models and discussing their applications to solve problems of other sciences, can assist students to understand why mathematics is so important in school. In the development of interdisciplinary activities, the characteristics of the tasks emerge as an important aspect. The emphasis is on the use of technological materials and the way they can support the development of concepts, provide different representations and support deeper understandings, and offer a multifaceted support to collect data and simulate experiences. Based on these assumptions, the aim of this chapter is to present, analyse and discuss tasks that promote interdisciplinary technological approaches from a mathematical point of view. In this chapter we assume interdisciplinarity as a complex construct, and in order to clarify its meaning we will discuss several types of conceptions, from multidisciplinary, to interdisciplinary, and to transdisciplinary. We will then address related concepts, such as modelling and STEM, highlighting similarities and differences between them, to reach an understanding of interdisciplinarity. In the process of the interdiciplinary approach, digital technologies arise as a central element. Based on a set of tasks on mathematics and on different sciences, we discuss what can change on an interdisciplinary approach to the teaching and learning of mathematical content and on the articulation between subjects.

We present a new algorithm for solving large-scale unconstrained optimization problems that uses cubic models, matrix-free subspace minimization, and secant-type parameters for defining the cubic terms. We also propose and analyze a specialized trust-region strategy to minimize the cubic model on a properly chosen low-dimensional subspace, which is built at each iteration using the Lanczos process. For the convergence analysis we present, as a general framework, a model trust-region subspace algorithm with variable metric and we establish asymptotic as well as complexity convergence results. Preliminary numerical results, on some test functions and also on the well-known disk packing problem, are presented to illustrate the performance of the proposed scheme when solving large-scale problems.

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High-strength concrete (HSC) slab-column connections with relatively low concrete strengths compared to today’s capabilities have been tested under seismic-type loading in the past. Herein, the hybrid use of HSC with compressive strength approximately 120 MPa and normal-strength concrete (NSC) is investigated through three reversed horizontal cyclic-loading tests with different geometries of the HSC region and a reference NSC specimen. The results show that HSC applied in the vicinity of the column can significantly enhance the seismic performance of slab-column connections. The best result in terms of drift capacity and economic use of HSC was achieved in the case of full-depth HSC extended from the column’s face up to 2.5 times the effective depth. Drift ratios up to 3.0% were achieved. A comparison with previous tests showed that the hybrid use of HSC and NSC can achieve similar results to the provision of punching shear reinforcement.

Abstract Fiber reinforced polymer (FRP) composites can be efficient for flexural strengthening of flat slabs if debonding of the FRP is postponed. However, with the increase of the flexural capacity, the flat slab becomes more susceptible to punching shear failure. In this context, four flexural or simultaneous flexural and punching shear retrofitting systems are investigated in this study to strengthen a flexure-deficient flat slab. Externally Bonded Reinforcement on Grooves (EBROG) and externally bonded reinforcement (EBR) methods are used for flexural strengthening in two cases: slabs without punching shear reinforcement and with post-installed shear bolts as shear reinforcement. According to the results, flexural strengthening of the slab using the EBR and EBROG techniques increased its load capacity by 12% and 21%, respectively. Simultaneous flexural and shear strengthening of the slab using the EBROG technique was the most effective, leading to a 57% enhancement of the load capacity. For specimens whose failure was governed by punching, comparing the results with code predictions showed that Eurocode and ACI (and the respective guide documents fib bulletin 90 and ACI 440.2R) overestimated the capacity of these specimens. In cases where failure was governed by flexure, a simple application of the yield line theory predicted reasonably well the load capacity of the specimens.

Abstract This paper presents a critical review of the state of the art of experimental research concerning the seismic response of reinforced concrete flat slab frames. After a summary of tests on connections, the paper examines tests carried out on frames with gravity and cyclic lateral loading, and shake table tests; scaled specimens and one real scale study are included. A discussion of the results reached so far is provided focusing on the global response, the different load types and effects; the ultimate rotations at failure in relation to the gravity shear and a classification of different failure modes for different types of connections. Based on this analysis, the research needs are highlighted. An experimental program launched to address these open questions is described. Further open topics are highlighted.