Multicriteria analysis of the environmental and economic performance of circularity strategies for concrete waste recycling in Spain.

Construction and demolition waste (CDW) is identified by multiple circular economy (CE) policies as a key sector for implementing circularity strategies due to the high volume of waste produced and the large consumption of raw materials. However, CE is not widely applied in the sector because of the lack of solid estimations on its environmental and economic viability. The main aim of this study was to propose a set of methodological steps to identify the optimal circularity alternatives for CDW products based on a multicriteria analysis of their environmental and economic performance. This methodology is applied to evaluate concrete waste. In specific, high-grade applications of concrete waste were analyzed comprising the processing into recycled coarse aggregates (RCA) for their use in structural and non-structural concrete. Multiple scenarios with different RCA replacements (20%, 30% and 100%) and different types of sorting and recycling (on-site and off-site) were evaluated in accordance with the specific site conditions of the region of Catalonia, Spain. The Life Cycle Analysis methodology (LCA) was used to perform the environmental analysis, while a detailed cost analysis was conducted for the economic aspect. The multicriteria method VIKOR was used for the selection of alternatives considering three different criteria. The results of this study showed environmental and economic advantages of CE scenarios based on the use of RCA over conventional concrete, mainly due to the influence of landfilling and transport distances. RCA produced on-site showed a better performance than RCA from fixed plants.

SureClick® (Darbepoetin alfa) can improve perceived satisfaction and competence for anemia treatment and increase self-administration in non-dialyzed patients with chronic kidney disease.

BACKGROUND AND AIMS: SureClick® is a prefilled pen for administration of darbepoetin alfa (DA) that is ready-to-use. We explored patient satisfaction with SureClick® compared with prefilled syringes (PFS). METHODS: Multicenter, prospective, 6-months, observational study in non-dialyzed patients with chronic kidney disease (CKD) treated with DA in PFS who switched to SureClick® at baseline. Main outcomes were: change in Anemia Treatment Satisfaction Questionnaire (ATSQ-S), Perceived Competence for Anemia Scale (PCAS) and self-administration rate. RESULTS: We enrolled 132 patients with a mean(SD) age of 71.3 (14.6) years, 57.6% women. Mean(SD) ATSQ-S scores at baseline and final records were 25.5 (7.9) and 31.6 (4.9) (on a scale from 0 to 36 maximum satisfaction-, mean change: 6.2, 95%CI: 4.6-7.8, p<0.0001). The PCAS also increased significantly (4.3 (2.0) vs 5.6 (1.6), on a scale from 1 to 7 maximum competence, p<0.0001). At baseline 47.7% of patients self-administered DA with PFS, vs 74.2% with SureClick® (p<0.001). No significant changes in hemoglobin were observed (11.4 (0.5) vs 11.6 (1.3) g/dl, p=0.193). Two patients (1.5%) had adverse reactions to SureClick® (pain on application). CONCLUSIONS: Our results suggest that the change from PFS to SureClick® could increase patient satisfaction and perceived competence in anemia management in non-dialyzed CKD patients, and could increase the self-administration rate, thereby reducing use of health resources.

Novel triazolopyridylbenzamides as potent and selective p38α inhibitors.

A new class of p38α inhibitors based on a biaryl-triazolopyridine scaffold was investigated. X-ray crystallographic data of the initial lead compound cocrystallised with p38α was crucial in order to uncover a unique binding mode of the inhibitor to the hinge region via a pair of water molecules. Synthesis and SAR was directed towards the improvement of binding affinity, as well as ADME properties for this new class of p38α inhibitors and ultimately afforded compounds showing good in vivo efficacy.

Indolin-2-one p38α inhibitors I: design, profiling and crystallographic binding mode.

The use of structure-based design and molecular modeling led to the discovery of indolin-2-one derivatives as potent and selective p38α inhibitors. The predicted binding mode was confirmed by X-ray crystallography.

Design, synthesis, and structure-activity relationships of aminopyridine N-oxides, a novel scaffold for the potent and selective inhibition of p38 mitogen activated protein kinase.

A novel series of aminopyridine N-oxides were designed, synthesized, and tested for their ability to inhibit p38alpha MAP kinase. Some of these compounds showed a significant reduction in the LPS-induced TNFalpha production in human whole blood. Structure-activity relationship studies revealed that N-oxide oxygen was essential for activity and was probably a determinant factor for a marked selectivity against other related kinases. Compound 45 was identified as a potent and selective p38alpha inhibitor with an appropriate balance between potency and pharmacokinetics. In vivo efficacy of 45 was demonstrated in reducing TNFalpha levels in an acute murine model of inflammation (ED(50) = 1 mg/kg in LPS-induced TNFalpha production when dosed orally 1.5 h prior to LPS administration). The oral efficacy of 45 was further demonstrated in a chronic model of adjuvant arthritis in rats with established disease when administered orally (ED(50) = 4.5 mg/kg).

The coat protein of Rabbit hemorrhagic disease virus contains a molecular switch at the N-terminal region facing the inner surface of the capsid.

To function adequately, many if not all proteins involved in macromolecular assemblies show conformational polymorphism as an intrinsic feature. This general strategy has been described for many essential cellular processes. Here we describe this structural polymorphism in a viral protein, the coat protein of Rabbit hemorrhagic disease virus (RHDV), which is required during virus capsid assembly. By combining genetic, structure modeling, and cryo-electron microscopy and image processing analysis, we have established the mechanism that allows RHDV coat protein to switch among quasi-equivalent conformational states to achieve the appropriate curvature for the formation of a closed shell. The RHDV capsid structure is based on a T = 3 lattice, containing 180 copies of identical subunits, similar to those of other caliciviruses. The quasi-equivalent interactions between the coat proteins are achieved by the N-terminal region of a subset of subunits, which faces the inner surface of the capsid shell. Mutant coat protein lacking this N-terminal sequence assembles into T = 1 capsids. Our results suggest that the polymorphism of the RHDV T = 3 capsid might bear resemblance to that of plant virus T = 3 capsids.

Engineering photoassimilate partitioning in tobacco plants improves growth and productivity and provides pathogen resistance.

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. To study the in vivo role and function of the maize PRms protein, tobacco plants were transformed with the PRms cDNA under the control of the CaMV35S promoter. Transgenic tobacco plants grow faster and yield more leaf and seed biomass. By using immunoelectron microscopy, we found that PRms is associated with plasmodesmata in leaves of transgenic tobacco plants. Furthermore, we found that activation of sucrose efflux from photosynthetically active leaves and accumulation of higher levels of sucrose in leaf tissues are characteristic features of PRms tobacco plants. This, in turn, results in the constitutive expression of endogenous tobacco PR genes and resistance to phytopathogens. The expression of multiple plant defense genes can then be achieved by using a single transgene. These data provide a new approach for engineering disease-resistant plants while simultaneously improving plant yield and productivity through the modification of photoassimilate partitioning.

The direct activation of MIK, a germinal center kinase (GCK)-like kinase, by MARK, a maize atypical receptor kinase, suggests a new mechanism for signaling through kinase-dead receptors.

Signaling by receptor protein kinases (RPKs) involves their dimerization and transphosphorylation. However, atypical RPKs with kinase-defective domains have been described recently. Some of them are essential for proper signaling in animal systems, although the precise mechanism involved is unknown in most cases. Here we describe the cloning and characterization of an atypical plant receptor kinase from maize, MARK, which does not phosphorylate in vitro. A yeast two-hybrid approach has allowed us to identify a new germinal center kinase (GCK)-related protein, MIK, that interacts with MARK. Interestingly, the interaction of the intracellular domain of MARK with the regulator domain of MIK strongly induces MIK kinase activity. As some GCK-related proteins connect cell-surface receptors to the intracellular MAPK cascades, the activation of MIK by direct interaction with MARK could illustrate a new mechanism for signaling through atypical RPKs.

Involvement of intramolecular interactions in the regulation of G protein-coupled receptor kinase 2.

The G protein-coupled receptor (GPCR) kinase GRK2 phosphorylates G protein-coupled receptors in an agonist-dependent manner. GRK2 activity is modulated through interactions of diverse domains of the kinase with G protein betagamma subunits, several lipids, anchoring proteins, and activated receptors. We report that kinase activity toward either GPCR (rhodopsin) or a synthetic peptide substrate is enhanced in the presence of GST-GRK2 fusion proteins or peptides corresponding to either N- or C-terminal sequences of GRK2. This direct stimulatory action of intrinsic domains on GRK2 activity does not add to the effect of other regulators, such as Gbetagamma subunits, and strongly suggests the existence of some mode of autoregulation. The existence of regulatory intramolecular interactions in GRK2 is supported by the facts that a C-terminal peptide protects the N-terminal region from proteolytic cleavage and that two domains of GRK2 independently coexpressed in cells associate as assessed by immunoprecipitation. Molecular modeling suggests that intramolecular interactions among the N-terminal, C-terminal and kinase domains would keep GRK2 in a constrained conformation characteristic of an inactive, basal state. Our model proposes that disruption of such intramolecular contacts by intermolecular interactions with regulatory proteins (mimicked by exogenously added kinase fragments in vitro) would promote the conformational changes required to bring about GRK2 translocation and activation.

Three-dimensional structure of penicillium chrysogenum virus: a double-stranded RNA virus with a genuine T=1 capsid.

Although double-stranded (ds) RNA viruses are a rather diverse group, they share general architectural principles and numerous functional features. All dsRNA viruses, from the mammalian reoviruses to the bacteriophage phi6, including fungal viruses, share a specialized capsid involved in transcription and replication of the dsRNA genome, and release of the viral plus strand RNA. This ubiquitous capsid consists of 120 protein subunits in a so-called T=2 organization. The stringent requirements of dsRNA metabolism may explain the similarities observed in capsid architecture among a broad spectrum of dsRNA viruses. We have used cryo-electron microscopy combined with three-dimensional reconstruction techniques and complementary biophysical techniques, to determine the structure at 26A resolution of the Penicillium chrysogenum virus (PcV) capsid. In contrast to all previous studies of dsRNA viruses, PcV capsid is an authentic T=1 capsid with 60 equivalent protein subunits. This T=1 capsid is built with the largest structural protein (110 kDa). Structural comparison between viral particles and capsids devoid of RNA show changes along the inner surface of the capsid, mostly located around the icosahedral 5 and 3-fold axis. Considering that there may be numerous interactions between the inner surface of the protein shell and the underlying RNA, the genome could have an important role in the conformation of the structural subunits. The empty capsid structure suggests a mechanism for transcript release from actively transcribing particles. Furthermore, sequence analysis of the PcV coat protein revealed that both halves of the protein share numerous regions of similar amino acid residues. These results open new perspectives when considering the structural organization of dsRNA virus capsids.

Structural (betaalpha)8 TIM barrel model of 3-hydroxy-3-methylglutaryl-coenzyme A lyase.

This study describes three novel homozygous missense mutations (S75R, S201Y, and D204N) in the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase gene, which caused 3-hydroxy-3-methylglutaric aciduria in patients from Germany, England, and Argentina. Expression studies in Escherichia coli show that S75R and S201Y substitutions completely abolished the HMG-CoA lyase activity, whereas D204N reduced catalytic efficiency to 6.6% of the wild type. We also propose a three-dimensional model for human HMG-CoA lyase containing a (betaalpha)8 (TIM) barrel structure. The model is supported by the similarity with analogous TIM barrel structures of functionally related proteins, by the localization of catalytic amino acids at the active site, and by the coincidence between the shape of the substrate (HMG-CoA) and the predicted inner cavity. The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site. We also report the localization of all missense mutations reported to date and show that these mutations are located in the beta-sheets around the substrate cavity.

Loss of p16INK4a results in increased glucocorticoid receptor activity during fibrosarcoma development.

Glucocorticoids inhibit proliferation of many cell types, but the relationship between the glucocorticoid receptor (GR) and the proteins regulating cell cycle progression is not fully understood. We previously found that during fibrosarcoma (FS) progression, GR displays only modest transcriptional activity in the preneoplastic stages, whereas it is highly active in FS cells. Now, we report that glucocorticoids reduce proliferation throughout FS development. The cyclin-dependent kinase inhibitor p16(INK4a) is frequently absent in many cancers, including FSs. We observed that p16(INK4a) protein expression is lost at the tumor stage of FS progression. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restores p16(INK4a) expression and reverts the phenotype of FS cells to low GR transcriptional activity, similar to that of the p16(INK4a)-expressing preneoplastic stages. Importantly, exogenous p16(INK4a) introduced by cotransfection is sufficient to reduce GR activity in FS cells, without affecting GR activity in p16-positive aggressive fibromatosis cells. Furthermore, GR transcriptional activity is elevated in mouse embryo fibroblasts derived from INK4a(-/-) mice compared with those derived from WT mice, implying that the difference in p16(INK4a) expression is sufficient to modulate GR activity. These results suggest a relationship between steroid hormone receptor activity and cell cycle inhibition, whereby absence of p16(INK4a) protein leads to higher GR transactivation activity and reduced cell sensitivity to dexamethasone. This observation might have important implications for current cancer therapies.

Functional interaction between the Ser/Thr kinase PKL12 and N-acetylglucosamine kinase, a prominent enzyme implicated in the salvage pathway for GlcNAc recycling.

PKL12 (STK16) is a ubiquitously expressed Ser/Thr kinase, not structurally related to the well known subfamilies, with a putative role in cell adhesion control. Yeast two-hybrid protein interaction screening was used to search for proteins that associate with PKL12 and to delineate signaling pathways and/or regulatory circuits in which this kinase participates. One positive clone contained an open reading frame highly similar to N-acetylglucosamine kinase (GlcNAcK) of several species. The PKL12/GlcNAcK interaction was further confirmed both in vitro and in vivo. Protein expression analysis of GlcNAcK using a specific rabbit antiserum displayed a ubiquitous pattern in cell lines and animal tissues. Subcellular localization studies showed that GlcNAcK is a cytoplasmic protein with a dual subcellular localization, distributed between the perinuclear and peripheral cell reservoirs. After overexpression, GlcNAcK localizes in vesicular structures associated mainly with the cell membrane and colocalizes with the PKL12 protein. GlcNAcK is not otherwise a substrate for PKL12 activity and PKL12 does not appear to influence GlcNAcK activity either in vitro or in vivo. In vitro kinase assays have nonetheless revealed that functional GlcNAcK, although not able to modulate autophosphorylation of PKL12, greatly influences PKL12 kinase activity on a defined substrate protein. These results are interpreted to indicate a potential in vivo role for GlcNAcK in PKL12 translocation and a tentative regulatory role for PKL12-mediated phosphorylation on substrate proteins.