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Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry‐driven hit‐to‐lead optimization and in‐depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter‐gene with IC50 values as low as 200 and 11 × 10−9m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI‐tobramycin (Tob) combination against PA biofilms using a tailor‐made squalene‐derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32‐fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker‐mediated therapy against PA infections opening up avenues for preclinical development. [ABSTRACT FROM AUTHOR]

Metabolic rewiring is a mechanism of adaptation to unfavorable environmental conditions and tumor progression. TRAP1 is an HSP90 molecular chaperone upregulated in human colorectal carcinomas (CRCs) and responsible for downregulation of oxidative phosphorylation (OXPHOS) and adaptation to metabolic stress. The mechanism by which TRAP1 regulates glycolytic metabolism and the relevance of this regulation in resistance to EGFR inhibitors were investigated in patient‐derived CRC spheres, human CRC cells, samples, and patients. A linear correlation was observed between TRAP1 levels and 18F‐fluoro‐2‐deoxy‐glucose (18F‐FDG) uptake upon PET scan or GLUT1 expression in human CRCs. Consistently, TRAP1 enhances GLUT1 expression, glucose uptake, and lactate production and downregulates OXPHOS in CRC patient‐derived spheroids and cell lines. Mechanistically, TRAP1 maximizes lactate production to balance low OXPHOS through the regulation of the glycolytic enzyme phosphofructokinase‐1 (PFK1); this depends on the interaction between TRAP1 and PFK1, which favors PFK1 glycolytic activity and prevents its ubiquitination/degradation. By contrast, TRAP1/PFK1 interaction is lost in conditions of enhanced OXPHOS, which results in loss of TRAP1 regulation of PFK1 activity and lactate production. Notably, TRAP1 regulation of glycolysis is involved in resistance of RAS‐wild‐type CRCs to EGFR monoclonals. Indeed, either TRAP1 upregulation or high glycolytic metabolism impairs cetuximab activity in vitro, whereas TRAP1 targeting and/or inhibition of glycolytic pathway enhances cell response to cetuximab. Finally, a linear correlation between 18F‐FDG PET uptake and poor response to cetuximab in first‐line therapy in human metastatic CRCs was observed. These results suggest that TRAP1 is a key determinant of CRC metabolic rewiring and favors resistance to EGFR inhibitors through regulation of glycolytic metabolism. [ABSTRACT FROM AUTHOR]

Cultured mouse embryonic stem cells are a heterogeneous population with diverse differentiation potential. In particular, the subpopulation marked by Zscan4 expression has high stem cell potency and shares with 2 cell stage preimplantation embryos both genetic and epigenetic mechanisms that orchestrate zygotic genome activation. Although embryonic de novo genome activation is known to rely on metabolites, a more extensive metabolic characterization is missing. Here we analyze the Zscan4+ mouse stem cell metabolic phenotype associated with pluripotency maintenance and cell reprogramming. We show that Zscan4+ cells have an oxidative and adaptable metabolism, which, on one hand, fuels a high bioenergetic demand and, on the other hand, provides intermediate metabolites for epigenetic reprogramming. Our findings enhance our understanding of the metastable Zscan4+ stem cell state with potential applications in regenerative medicine. Synopsis: This study reveals that Zscan4+mESCs express high levels of the mitochondrial enzyme Arg2 and show a flexible mitochondria‐mediated oxidative phenotype. Arg2 enzyme expression correlates with markers of the undifferentiated state.Zscan4+ mESCs utilize additional substrates besides glucose, long‐chain FA and glutamine to fuel respiratory activity.Metabolite profiling of Zscan4+ mESCs, compared with Zscan4− mESCs, shows a high content of methionine and aspartate and a reduction in the oxidation of long‐chain FA in favor of short/middle‐chain FA. [ABSTRACT FROM AUTHOR]

Summary: The present study provided a cross‐cultural validation of the Thinking About Life Experiences Scale‐revised (TALE‐R) in an Italian sample of Facebook users (n = 492; female = 378; male = 114; mean age 26.1) to test for replication and universality of the TALE‐R three‐factor model. Furthermore, it explored the interrelations among gender, age, the scores at the TALE‐R and the frequency of posting textual/visual information about individuals' life events on Facebook. Results at exploratory and confirmatory factor analysis gave empirical support to both of a tripartite model for the functions of autobiographical memory (i.e., directive‐behavior, social‐bonding, and self‐continuity) and measurement invariance of this three‐factor model across gender and age. Further results at linear correlation and regression analyses showed that directive‐behavior and self‐continuity functions of autobiographical memory are significantly related to the ways people use Facebook for personal documentation. Age differences more than gender influence this association. Discussion and conclusion reported both theoretical and empirical implications of the findings of the study. [ABSTRACT FROM AUTHOR]

Streptomyces mobaraensis is a key player for the industrial production of the protein cross‐linking enzyme microbial transglutaminase (MTG). Extra‐cellular activation of MTG by the transglutaminase‐activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross‐linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N‐terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N‐terminally shortened variants clearly indicated the highly conserved Leu40‐Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40‐Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X‐ray crystallography. While no structural data could be obtained for the N‐terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI‐family proteins. Enzymes: Chymotrypsin, EC3.4.21.1; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), EC3.4.24.77; streptogrisin‐A (SGPA), EC3.4.21.80; streptogrisin‐B (SGPB), EC3.4.21.81; subtilisin BPN', EC3.4.21.62; transglutaminase, EC2.3.2.13; transglutaminase‐activating metalloprotease (TAMP), EC3.4.‐.‐; tri‐/tetrapeptidyl aminopeptidase, EC3.4.11.‐; trypsin, EC3.4.21.4. Databases: The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org). [ABSTRACT FROM AUTHOR]

This article reports the development and initial validation of a multidimensional measure of employability based on the theoretical model of Lo Presti and Pluviano (2016). Four different studies were designed and implemented. Study 1 was a qualitative study that involved a group of 15 labour market experts and aimed at developing the items pool. In Study 2, an exploratory factor analysis of 526 employees was carried out to examine the structure of the employability measure as previously obtained. Study 3 aimed at verifying the employability measure that had emerged from Study 2 through confirmatory factor analysis of 699 employees, resulting in a 28‐item shortened version encompassing the original four employability dimensions. Finally, in Study 4, concurrent and predictive validity of the definitive version of the employability measure were tested on a sample of 712 employees. Implications for vocational guidance and human resource management, as well as future employability research, are discussed. [ABSTRACT FROM AUTHOR]

The protein cross‐linking enzyme transglutaminase from Streptomyces mobaraensis (MTG) is frequently used to modify therapeutic proteins. In order to reveal the binding mode of glutamine donor substrates, we have now crystallized MTG covalently linked to large inhibitory peptides. A series of peptide structures were examined but DIPIGSKMTG, which was chloroacetylated at serine, was the only inhibitory molecule that resulted in an interpretable density map. We found that, besides the warhead (modified Ser6), Ile4 and Gly5 of the inhibitory peptide occupy the tight but extended hydrophobic bottom of the MTG‐binding cleft. Both termini of the peptide protrude along the cleft walls almost perpendicular to the bottom of the extended cleft. This peptide model suggests a zipper‐like cross‐linking mechanism of self‐assembled substrate proteins by MTG. Microbial transglutaminase was irreversibly inhibited by chloroacetylated serine peptides derived from glutamine donor sites of natural substrate proteins. A crystal structure revealed close interaction between the warhead (glutamine)‐containing tripeptide IGS* (IGQ) and the catalytic cleft bottom, while the peptide termini point to the cleft entry. Such orientation gives rise to the assumption that transglutaminase preferably cross‐links self‐assembled substrate proteins. [ABSTRACT FROM AUTHOR]

The Dispase autolysis‐inducing protein (DAIP) is produced by Streptomyces mobaraensis to disarm neutral metalloproteases by decomposition. The absence of a catalytic protease domain led to the assumption that the seven‐bladed β‐propeller protein DAIP causes structural modifications, thereby triggering autolysis. Determination of protein complexes consisting of DAIP and thermolysin or DAIP and a nonfunctional E138A bacillolysin variant supported this postulation. Protein twisting was indicated by DAIP‐mediated inhibition of thermolysin while bacillolysin underwent immediate autolysis under the same conditions. Interestingly, an increase in SYPRO orange fluorescence allowed tracking of the fast degradation process. Similarly rapid autolysis of thermolysin mediated by DAIP was only observed upon the addition of amphiphilic compounds, which probably amplify the induced structural changes. DAIP further caused degradation of FITC‐labeled E138A bacillolysin by trypsin, as monitored by a linear decrease in fluorescence polarization. The kinetic model, calculated from the obtained data, suggested a three‐step mechanism defined by (a) fast DAIP–metalloprotease complex formation, (b) slower DAIP‐mediated protein twisting, and (c) fragmentation. These results were substantiated by crystallized DAIP attached to a C‐terminal helix fragment of thermolysin. Structural superposition of the complex with thermolysin is indicative of a conformational change upon binding to DAIP. Importantly, the majority of metalloproteases, also including homologs from various pathogens, are highly conserved at the autolysis‐prone peptide bonds, suggesting their susceptibility to DAIP‐mediated decomposition, which may offer opportunities for pharmaceutical applications. Databases: The atomic coordinates and structure factors (PDB ID: 6FHP) have been deposited in the Protein Data Bank (http://www.pdb.org/). Enzymes: Aureolysin, EC 3.4.24.29; bacillolysin (Dispase, Gentlyase), EC 3.4.24.28; lasB (elastase), EC 3.4.24.4; subtilisin, EC 3.4.21.62; thermolysin, EC 3.4.24.27; transglutaminase, EC 2.3.2.13; trypsin, EC 3.4.21.4; vibriolysin (hemagglutinin(HA)/protease), EC 3.4.24.25. The Dispase autolysis‐inducing protein (DAIP) from Streptomyces mobaraensis adopts a seven‐bladed β‐propeller fold mediating metalloproteases self‐degradation. The present study now reveals strong attraction of bacillolysin and thermolysin by DAIP, complex formation, and protein twisting followed by fragmentation as indicated by SYPRO orange and FITC‐labeled bacillolysin. The biochemical data are completed by crystallized DAIP in complex with a C‐terminal thermolysin fragment. [ABSTRACT FROM AUTHOR]

Abstract: Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPIp), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPIp exhibits a rigid, thermo‐resistant double‐psi‐beta‐barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln‐6 as the only amine acceptor site on SPIp accessible for MTG. Substitution of Lys‐7 demonstrated that small and hydrophobic residues in close proximity to Gln‐6 favor MTG‐mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPIp for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG‐substrates used in biomedical applications. [ABSTRACT FROM AUTHOR]

ATG16L1 plays a major role in autophagy. It acts as a molecular scaffold which mediates protein-protein interactions essential for autophagosome formation. The ATG12~ATG5-ATG16L1 complex is one of the key complexes involved in autophagosome formation. Human ATG16L1 comprises 607 amino acids with three functional domains named ATG5BD, CCD and WD40, where the C-terminal WD40 domain represents approximately 50% of the full-length protein. Previously, structures of the C-terminal WD40 domain of human ATG16L1 as well as of human ATG12~ATG5 in complex with the ATG5BD of ATG16L1 have been reported. However, apart from the ATG5BD, no structural information for the N-terminal half, including the CCD, of human ATG16L1 is available. In this study, the authors aimed to structurally characterize the N-terminal half of ATG16L1. ATG16L111-307 in complex with ATG5 has been purified and crystallized in two crystal forms. However, both crystal structures revealed degradation of ATG16L1, resulting in crystals comprising only full-length ATG5 and the ATG5BD of ATG16L1. The structures of ATG5-ATG5BD in two novel crystal forms are presented, further supporting the previously observed dimerization of ATG5-ATG16L1. The reported degradation points towards a high instability at the linker region between the ATG5BD and the CCD in ATG16L1. Based on this observation and further biochemical analysis of ATG16L1, a stable 236-amino-acid subfragment comprising residues 72-307 of the N-terminal half of ATG16L1, covering the residual, so far structurally uncharacterized region of human ATG16L1, was identified. Here, the identification, purification, biochemical characterization and crystallization of the proteolytically stable ATG16L172-307 subfragment are reported. [ABSTRACT FROM AUTHOR]

Autophagy-related protein ATG16L1 is a component of the mammalian ATG12~ATG5/ ATG16L1 complex, which acts as E3-ligase to catalyze lipidation of LC3 during autophagosome biogenesis. The N-terminal part of ATG16L1 comprises the ATG5-binding site and coiled-coil dimerization domain, both also present in yeast ATG16 and essential for bulk and starvation induced autophagy. While absent in yeast ATG16, mammalian ATG16L1 further contains a predicted C-terminal WD40-domain, which has been shown to be involved in mediating interaction with diverse factors in the context of alternative functions of autophagy, such as inflammatory control and xenophagy. In this work, we provide detailed information on the domain boundaries of the WD40-domain of human ATG16L1 and present its crystal structure at a resolution of 1.55 Å. [ABSTRACT FROM AUTHOR]

Background and Purpose: N6 -Isopentenyladenosine (i6A) is a modified nucleoside exerting in vitro and in vivo antiproliferative effects. We previously demonstrated that the actions of i6A correlate with the expression and activity of farnesyl pyrophosphate synthase (FPPS), a key enzyme involved in the mevalonate (MVA) pathway, which is aberrant in brain cancer. To develop new anti-glioma strategies, we tested related compounds exhibiting greater activity than i6A.Experimental Approach: We designed and synthesized i6A derivatives characterized by the introduction of diverse chemical moieties in the N6 position of adenosine and tested for their efficacy in U87 cells and in primary glioma cultures, derived from patients. NMR-based structural analysis, molecular docking calculations and siRNA mediated knockdown were used to clarify the molecular basis of their action, targeting FPPS protein.Key Results: CM223, the i6A derivative including a benzyl moiety in N6 position of adenine, showed marked activity in selectively targeting glioma cells, but not normal human astrocytes. This was due to induction of intrinsic pathways of apoptosis and inhibition of proliferation, along with blockade of FPPS-dependent protein prenylation, which counteracted oncogenic signalling mediated by EGF receptors.Conclusion and Implications: The biological effects together with structural data on interaction of CM223 with FPPS, provided additional evidence for the correlation of the i6A/CM223 antitumor activity with FPPS modulation. Because the MVA pathway is an important promising target, CM223 and its derivatives should be considered interesting active molecules in antiglioma research. [ABSTRACT FROM AUTHOR]

Enteropathogenic Yersinia expresses several invasins that are fundamental virulence factors required for adherence and colonization of tissues in the host. Within the invasin-family of Yersinia adhesins, to date only Invasin has been extensively studied at both structural and functional levels. In this work, we structurally characterize the recently identified inverse autotransporter InvasinE from Yersinia pseudotuberculosis (formerly InvasinD from Yersinia pseudotuberculosis strain IP31758) that belongs to the invasin-family of proteins. The sequence of the C-terminal adhesion domain of InvasinE differs significantly from that of other members of the Yersinia invasin-family and its detailed cellular and molecular function remains elusive. In this work, we present the 1.7 Å crystal structure of the adhesion domain of InvasinE along with two Immunoglobulin-like domains. The structure reveals a rod shaped architecture, confirmed by small angle X-ray scattering in solution. The adhesion domain exhibits strong structural similarities to the C-type lectin-like domain of Yersinia pseudotuberculosis Invasin and enteropathogenic/enterohemorrhagic E. coli Intimin. However, despite the overall structural similarity, the C-type lectin-like domain in InvasinE lacks motifs required for Ca2+/carbohydrate binding as well as sequence or structural features critical for Tir binding in Intimin and β1-integrin binding in Invasin, suggesting that InvasinE targets a distinct, yet unidentified molecule on the host-cell surface. Although the biological role and target molecule of InvasinE remain to be elucidated, our structural data provide novel insights into the architecture of invasin-family proteins and a platform for further studies towards unraveling the function of InvasinE in the context of infection and host colonization. [ABSTRACT FROM AUTHOR]

ABSTRACT Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal-cell biotechnology. Difficult-to-express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full-length protein constructs. Post-translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time-, labor-, and cost-intensive. It is clearly desirable to find an automated and miniaturized fast multi-sample screening method for protein expression in such systems. With this in mind, in this paper a high-throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide-binding Oligomerization Domain-containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid-based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells. Biotechnol. Bioeng. 2016;113: 1975-1983. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The bacterial flagellum enables directed movement of Salmonella enterica towards favorable conditions in liquid environments. Regulation of flagellar synthesis is tightly controlled by various environmental signals at transcriptional and post-transcriptional levels. The flagellar master regulator FlhD4C2 resides on top of the flagellar transcriptional hierarchy and is under autogenous control by FlhD4C2-dependent activation of the repressor rflM. The inhibitory activity of RflM depends on the presence of RcsB, the response regulator of the RcsCDB phosphorelay system. In this study, we elucidated the molecular mechanism of RflM-dependent repression of flhDC. We show that RcsB and RflM form a heterodimer that coordinately represses flhDC transcription independent of RcsB phosphorylation. RcsB-RflM complex binds to a RcsB box downstream the P1 transcriptional start site of the flhDC promoter with increased affinity compared to RcsB in the absence of RflM. We propose that RflM stabilizes binding of unphosphorylated RcsB to the flhDC promoter in absence of environmental cues. Thus, RflM is a novel auxiliary regulatory protein that mediates target specificity of RcsB for flhDC repression. The cooperative action of the RcsB-RflM repressor complex allows Salmonella to fine-tune initiation of flagellar gene expression and adds another level to the complex regulation of flagellar synthesis. [ABSTRACT FROM AUTHOR]

Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site-specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties. [ABSTRACT FROM AUTHOR]

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The iron chelator deferasirox (DFX) prevents complications related to transfusional iron overload in several haematological disorders characterized by marrow failure. It is also able to induce haematological responses in a percentage of treated patients, particularly in those affected by myelodysplastic syndromes. The underlying mechanisms responsible for this feature, however, are still poorly understood. In this study, we investigated the effect of DFX-treatment in human haematopoietic/progenitor stem cells, focussing on its impact on the redox balance, which proved to control the interplay between stemness maintenance, self-renewal and differentiation priming. Here we show, for the first time, that DFX treatment induces a significant diphenyleneiodonium-sensitive reactive oxygen species (ROS) production that leads to the activation of POU5F1 (OCT4), SOX2 and SOX17 gene expression, relevant in reprogramming processes, and the reduction of the haematopoietic regulatory proteins CTNNB1 (β-Catenin) and BMI1. These DFX-mediated events were accompanied by decreased CD34 expression, increased mitochondrial mass and up-regulation of the erythropoietic marker CD71 (TFRC) and were compound-specific, dissimilar to deferoxamine. Our findings would suggest a novel mechanism by which DFX, probably independently on its iron-chelating property but through ROS signalling activation, may influence key factors involved in self-renewal/differentiation of haematopoietic stem cells. [ABSTRACT FROM AUTHOR]

A collection of 30 ceramic samples, 16 of coarse wares and 14 of fine painted wares, have been investigated by optical microscopy, scanning electron microscopy, inductively coupled plasma optical emission spectrometry, inductively coupled plasma mass spectrometry and neutron activation analysis. Further samples of clayey sediments, both locally outcropping and found within the settling tank, have been submitted to the same analytical techniques for comparison with the ceramic collection. The results demonstrated that local clayey sediments were used as received for the production of coarse wares. The same raw materials were sieved and/or refined by decantation for the production of fine painted wares, which, in fact, provided results that were perfectly comparable with those for the clayey raw materials found within the settling tank. The Faragola productions were distinguished from the neighbouring Apulian productions according to petrographic features (the presence of leucite-bearing volcanic rocks and Mn-rich wads) and their bulk chemical composition. [ABSTRACT FROM AUTHOR]

Uvulopalatopharyngoplasty (UPPP) is an operation that is frequently performed for the treatment of obstructive sleep apnea (OSA). While UPPP usually eliminates or decreases snoring and often reduces excessive daytime sleepiness, the decrease in the number of episodes of apnea and hypopnea, and the improvement in oxygen saturation (SaO2) have been less predictable. We compared preoperative and postoperative polysomnography (PSG) in 27 patients with OSA and found that no single PSG parameter could accurately reflect the changes in respiration seen after UPPP. We suggest that a combination of indices including the apnea index, the apnea and hypopnea index, the frequency and severity of decreases in SaO2, and the lowest SaO2 be used to assess the effect of UPPP. Using this combination we determined that 30% of our patients were markedly improved, 33% were somewhat improved, and 37% were unimproved. To rely solely on the patient's subjective improvement often results in overestimating the therapeutic results of surgery, whereas to rely only on one PSG parameter may underestimate or overestimate the degree of improvement. [ABSTRACT FROM AUTHOR]

Adult haematopoietic stem/progenitor cells ( HSPCs) constitute the lifespan reserve for the generation of all the cellular lineages in the blood. Although massive progress in identifying the cluster of master genes controlling self-renewal and multipotency has been achieved in the past decade, some aspects of the physiology of HSPCs still need to be clarified. In particular, there is growing interest in the metabolic profile of HSPCs in view of their emerging role as determinants of cell fate. Indeed, stem cells and progenitors have distinct metabolic profiles, and the transition from stem to progenitor cell corresponds to a critical metabolic change, from glycolysis to oxidative phosphorylation. In this review, we summarize evidence, reported in the literature and provided by our group, highlighting the peculiar ability of HSPCs to adapt their mitochondrial oxidative/bioenergetic metabolism to survive in the hypoxic microenvironment of the endoblastic niche and to exploit redox signalling in controlling the balance between quiescence versus active cycling and differentiation. Especial prominence is given to the interplay between hypoxia inducible factor-1, globins and NADPH oxidases in managing the mitochondrial dioxygen-related metabolism and biogenesis in HSPCs under different ambient conditions. A mechanistic model is proposed whereby 'mitochondrial differentiation' is a prerequisite in uncommitted stem cells, paving the way for growth/differentiation factor-dependent processes. Advancing the understanding of stem cell metabolism will, hopefully, help to (i) improve efforts to maintain, expand and manipulate HSPCs ex vivo and realize their potential therapeutic benefits in regenerative medicine; (ii) reprogramme somatic cells to generate stem cells; and (iii) eliminate, selectively, malignant stem cells. Linked Articles This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8 [ABSTRACT FROM AUTHOR]

The recently introduced Cu(I)-catalyzed azide-alkyne 1,3-dipolar Huisgen cycloaddition as a prototypic 'click chemistry reaction' presented an opportunity for introducing the 1,4-disubstituted-[1,2,3]triazolyl moiety as a new isostere for peptide bonds in the backbone. Previous study in our lab focused on the synthesis of a model i-to-i+4 side chain-to-side chain 1,4- and 4,1-disubstituted-[1,2,3]triazolyl-bridged cyclo-nonapeptide I (Scheme 1) as analogues of its structurally related helical i-to-i+4 lactam-bridged cyclo-nonapeptide [Lys13(& 1),Asp17(& 2)]parathyroid hormone related peptide (PTHrP)(11-19)NH21 a truncated version of the α-helical and potent parathyroid hormone receptor 1 agonist [Lys13(& 1),Asp17(& 2)]PTHrP(1-34)NH2,2,3 Nα-Ac-Lys-Gly-Lys(& 1)-Ser-Ile-Gln-Asp(& 2)-Leu-Arg-NH2]. Systematic [1,2,3]triazolyl-containing bridge structure-conformation relationship studies in hexafluoroacetone/water mixture included incorporation of bridges varied in size and position and orientation of the triazolyl ring within the bridge. These studies revealed that the size of methylene bridge flanking triazolyl moiety is critical to reproduce in the heterodetic cyclo-nonapeptides. The conformational features of the analogues cyclo-nonapeptide in which Lys13 and Asp17 are bridged by the isosteric lactam. Here, we extend our conformational analysis to dimethyl sulfoxide/water mixture in an effort to characterize inherent conformational properties of the heterodetic cyclopeptides that are solvent independent. Our present study shows that the physicochemical properties of the structure-supporting solvent cannot override the effect of the size of methylene bridge to form helical mimetic structures. Moreover, we prove that [1,2,3]triazolyl ring is not a simple bioisostere of lactam bond, but it affects the secondary structure of the peptide, in relation to its positioning orientation. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 535-545, 2012. [ABSTRACT FROM AUTHOR]

This study examines the interpersonal problems profiles of obese individuals by cluster analysing the interpersonal problems circumplex scores of participants. The Inventory of Interpersonal Problems-Short Circumplex (IIP-32) was completed by 368 treatment-seeking obese individuals. These data were cluster analysed, and groups of obese subjects defined by varying interpersonal problems were compared with regard to psychological distress, self-esteem, body dissatisfaction, quality of life and binge behaviours. Cluster analyses of the IIP-32 resulted in four clusters, which occupied two quadrants of the interpersonal circumplex. Several differences in body mass index, psychological distress, quality of life and body dissatisfaction emerged across the four interpersonal groups. Although obese individuals reported elevated interpersonal distress, these subjects are not homogeneous with regard to interpersonal problems. Psychiatric co-morbidity and psychological distress may explain these interpersonal differences. These findings underscore the importance for clinicians to assess carefully patients' interpersonal functioning, especially with respect to treatment-seeking obese patients. Copyright © 2011 John Wiley & Sons, Ltd. Key Practitioner Message Research has not consistently specified the types of interpersonal problems most frequently experienced by treatment-seeking obese individuals., Most obese individuals share a friendly-dominant interpersonal style., This research supports the importance of measuring and targeting interpersonal variables in the design and evaluation of obesity treatment programmes. [ABSTRACT FROM AUTHOR]

Abstract: The DDB1–DDB2–CUL4–RBX1 complex serves as the primary detection device for UV-induced lesions in the genome. It simultaneously functions as a CUL4 type E3 ubiquitin ligase. We review the current understanding of this dual function ubiquitin ligase and damage detection complex. The DDB2 damage binding module is merely one of a large family of possible DDB1–CUL4 associated factors (DCAF), most of which are substrate receptors for other DDB1–CUL4 complexes. DDB2 and the Cockayne-syndrome A protein (CSA) function in nucleotide excision repair, whereas the remaining receptors operate in a wide range of other biological pathways. We will examine the modular architecture of DDB1–CUL4 in complex with DDB2, CSA and CDT2 focusing on shared architectural, targeting and regulatory principles. [Copyright &y& Elsevier]

Dyslipidemia is a well-established condition proved to accelerate the progression of chronic kidney disease leading to tubulo-interstitial injury. However, the molecular aspects of the dyslipidemia-induced renal damage have not been fully clarified and in particular the role played by low-density lipoproteins (LDLs). This study aimed to examine the effects of native non-oxidized LDL on cellular oxidative metabolism in cultured human proximal tubular cells. By means of confocal microscopy imaging combined to respirometric and enzymatic assays it is shown that purified native LDL caused a marked increase of cellular reactive oxygen species (ROS) production, which was mediated by activation of NADPH oxidase(s) and by mitochondrial dysfunction by means of a ROS-induced ROS release mechanism. The LDL-dependent mitochondrial alterations comprised inhibition of the respiratory chain activity, enhanced ROS production, uncoupling of the oxidative phosphorylation efficiency, collapse of the mtΔΨ, increased Ca uptake and loss of cytochrome c. All the above LDL-induced effects were completely abrogated by chelating extracellular Ca as well as by inhibition of the Ca-activated cytoplas-mic phospholipase A2, NADPH oxidase and mitochondrial permeability transition. We propose a mechanicistic model whereby the LDL-induced intracellular redox unbalance is triggered by a Ca inward flux-dependent commencement of cPLA2 followed by activation of a lipid- and ROS-based cross-talking signalling pathway. This involves first oxidants production via the plasmamembrane NADPH oxidase and then propagates downstream to mitochondria eliciting redox- and Ca-dependent dysfunctions leading to cell-harming conditions. These findings may help to clarify the mechanism of dyslipidemia-induced renal damage and suggest new potential targets for specific therapeutic strategies to prevent oxidative stress implicated in kidney diseases. [ABSTRACT FROM AUTHOR]

Deposition of senile plaques composed of fibrillar aggregates of Aβ-amyloid peptide is a characteristic hallmark of Alzheimer’s disease. A widely employed approach in the study of anti-Alzheimer agents involves the identification of substances able to prevent amyloid aggregation, or to disaggregate the amyloid fibrils through a direct structural interaction with the soluble or aggregated forms of the peptide. Here, we report the synthesis of a set of 1,3-dihydro-3,6-disubstituted-imidazo[1,5-c]thiazole-5,7-dione derivatives supporting different alkyl, aryl and alkylamine side chains. The ability of these compounds to interact with the Aβ(25-35) peptide was evaluated using circular dichroism, nuclear magnetic resonance and thioflavin fluorescence spectroscopy. A molecular model for Aβ(25-35)–ligand interactions was calculated by molecular docking procedures. Our data show that the ability of the synthesized compounds to modify the structural behaviour of Aβ(25-35) varies as a function of the overall structural features of the ligands rather contributions from specific individual substituents. [ABSTRACT FROM AUTHOR]

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Abstract: The ubiquitous tripeptide Gly-Gly-Gln in class 1 polypeptide release factors triggers polypeptide release on ribosomes. The Gln residue in both bacterial and yeast release factors is N5-methylated, despite their distinct evolutionary origin. Methylation of eRF1 in yeast is performed by the heterodimeric methyltransferase (MTase) Mtq2p/Trm112p, and requires eRF3 and GTP. Homologues of yeast Mtq2p and Trm112p are found in man, annotated as an N6-DNA-methyltransferase and of unknown function. Here we show that the human proteins methylate human and yeast eRF1.eRF3.GTP in vitro, and that the MTase catalytic subunit can complement the growth defect of yeast strains deleted for mtq2. Structured summary: MINT-6571489: HemK2α (uniprotkb:Q9Y5N5) binds (MI:0407) to hTrm112 (uniprotkb:Q9UI30) by pull down (MI:0096) [Copyright &y& Elsevier]

The GTP-binding protein Rap1 regulates integrin-mediated and other cell adhesion processes. Unlike most other Ras-related proteins, it contains a threonine in switch II instead of a glutamine (Gln61 in Ras), a residue crucial for the GTPase reaction of most G proteins. Furthermore, unlike most other GTPase-activating proteins (GAPs) for small G proteins, which supply a catalytically important Arg-finger, no arginine residue of RapGAP makes a significant contribution to the GTPase reaction of Rap1. For a detailed understanding of the reaction mechanism, we have solved the structure of Rap1 in complex with Rap1GAP. It shows that the Thr61 of Rap is away from the active site and that an invariant asparagine of RapGAPs, the Asn-thumb, takes over the role of the cis-glutamine of Ras, Rho or Ran. The structure and biochemical data allow to further explain the mechanism and to define the important role of a conserved tyrosine. The structure and biochemical data furthermore show that the RapGAP homologous region of the tumour suppressor Tuberin is sufficient for catalysis on Rheb. [ABSTRACT FROM AUTHOR]

Abstract: The hypoxia-inducible factor (HIF) transcriptional system enables cell adaptation to limited O2 availability, transducing this signal into patho-physiological responses such as angiogenesis, erythropoiesis, vasomotor control, and altered energy metabolism, as well as cell survival decisions. However, other factors beyond hypoxia are known to activate this pleiotropic transcription factor. The aim of this study was to characterize HIF in human hematopoietic stem cells (HSCs) and evidence is provided that granulocyte colony stimulating factor-mobilized CD34+- and CD133+-HSCs express a stabilized cytoplasmic form of HIF-1α under normoxic conditions. It is shown that HIF-1α stabilization correlates with down-regulation of the tumour suppressor von Hippel-Lindau protein (pVHL) and is positively controlled by NADPH-oxidase-dependent production of reactive oxygen species, indicating a specific O2-independent post-transcriptional control of HIF in mobilized HSCs. This novel finding is discussed in the context of the proposed role of HIF as a mediator of progenitor cell recruitment to injured ischemic tissues and/or in the control of the maintenance of the undifferentiated state. [Copyright &y& Elsevier]

In the attempt to identify a new lead compound able to modify the conformational preferences of the β-amyloid peptides, a set of new compounds characterized by a thiazolidine ring linked to several different aryl moieties were synthesized. The ability of these compounds to prevent the β-amyloid aggregation was evaluated using circular dichroism and nuclear magnetic resonance techniques. Molecular docking procedure allowed an interpretation of spectroscopic in the key of molecular interaction. [ABSTRACT FROM AUTHOR]

The accumulation of β-amyloid peptides into senile plaques is one of the hallmarks of Alzheimer's disease (AD). There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the β-sheet oligomerization process of β-amyloid. Aβ(25-35), the sequence of which is GSNKGAIIGLM, is a highly toxic segment of amyloid β (Aβ)-peptides, which forms fibrillary aggregates. In the present work, two spin-labelled Aβ(25-35) analogues containing the nitroxide group of the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe at the N- or the C-terminus of the peptide sequence, respectively, were synthesized in order to investigate the peptide-membrane interaction. The orientation and associated changes of the peptide conformation in the presence of different artificial membrane models (micelles, liposomes) were evaluated by electron paramagnetic resonance and circular dichroism techniques. The results of this study allowed us to propose a model in which the C-terminal portion of the peptide is highly associated to the membrane, while the N-terminal part extends into the aqueous phase with occasional contacts with the lipid head-group region. Interestingly, the interaction of the C-terminal portion of the peptide is particularly enhanced in the presence of sodium dodecyl sulfate (SDS) molecules. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: The impact of cAMP on ROS-balance in human and mammalian cell cultures was studied. cAMP reduced accumulation of ROS induced by serum-limitation, under conditions in which there was no significant change in the activity of scavenger systems. This effect was associated with cAMP-dependent activation of the NADH-ubiquinone oxidoreductase activity of complex I. In fibroblasts from a patient a genetic defect in the 75kDa FeS-protein subunit of complex I resulted in inhibition of the activity of the complex and enhanced ROS production, which were reversed by cAMP. A missense genetic defect in the NDUFS4 subunit, putative substrate of PKA, suppressed, on the other hand, the activity of the complex and prevented ROS production. [Copyright &y& Elsevier]

MnmE, a Guanine nucleotide-binding protein conserved between bacteria and man, is involved in the modification of tRNAs. Here we provide biochemical and X-ray structural evidence for a new GTP-hydrolysis mechanism, where the G-domains of MnmE dimerise in a potassium-dependent manner and induce GTP hydrolysis. The structure in the presence of GDP-AlFx and potassium shows how juxtaposition of the subunits induces a conformational change around the nucleotide which reorients the catalytic machinery. A critical glutamate is positioned such as to stabilise or activate the attacking water. Potassium provides a positive charge into the catalytic site in a position analogous to the arginine finger in the Ras-RasGAP system. Mutational studies show that potassium-dependent dimerisation and GTP hydrolysis can be uncoupled and that interaction between the G-domains is a prerequisite for subsequent phosphoryl transfer. We propose a model for the juxtaposition of G-domains in the full-length protein and how it induces conformational changes in the putative tRNA-modification centre. [ABSTRACT FROM AUTHOR]

Formins induce the nucleation and polymerisation of unbranched actin filaments via the formin-homology domains 1 and 2. Diaphanous-related formins (Drfs) are regulated by a RhoGTPase-binding domain situated in the amino-terminal (N-terminal) region and a carboxy-terminal Diaphanous-autoregulatory domain (DAD), whose interaction stabilises an autoinhibited inactive conformation. Binding of active Rho releases DAD and activates the catalytic activity of mDia. Here, we report on the interaction of DAD with the regulatory N-terminus of mDia1 (mDiaN) and its release by Rho•GTP. We have defined the elements required for tight binding and solved the three-dimensional structure of a complex between an mDiaN construct and DAD by X-ray crystallography. The core DAD region is an α-helical peptide, which binds in the most highly conserved region of mDiaN using mainly hydrophobic interactions. The structure suggests a two-step mechanism for release of autoinhibition whereby Rho•GTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that Rho•GTP accelerates the dissociation of DAD from the mDiaN•DAD complex. [ABSTRACT FROM AUTHOR]

TrmE is a 50 kDa guanine nucleotide-binding protein conserved between bacteria and man. It is involved in the modification of uridine bases (U34) at the first anticodon (wobble) position of tRNAs decoding two-family box triplets. The precise role of TrmE in the modification reaction is hitherto unknown. Here, we report the X-ray structure of TrmE from Thermotoga maritima. The structure reveals a three-domain protein comprising the N-terminala/ßdomain, the central helical domain and the G domain, responsible for GTP binding and hydrolysis. The N-terminal domain induces dimerization and is homologous to the tetrahydrofolate-binding domain of N,N-dimethylglycine oxidase. Biochemical and structural studies show that TrmE indeed binds formyl-tetrahydrofolate. A cysteine residue, necessary for modification of U34, is located close to the C1-group donor 5-formyl-tetrahydrofolate, suggesting a direct role of TrmE in the modification analogous to DNA modification enzymes. We propose a reaction mechanism whereby TrmE actively participates in the formylation reaction of uridine and regulates the ensuing hydrogenation reaction of a Schiff's base intermediate. [ABSTRACT FROM AUTHOR]

When applied as the nanoparticle formulation the QSI potentiates biofilm eradication capabilities of tobramycin by at least 32-fold. In article number 2004369, Martin Empting and colleagues describe a new generation of dual-loaded nanoparticles enabling efficient eradication of I Pseudomonas aeruginosa i biofilms. [Extracted from the article]

To test the effect of alcohol ingestion and snoring on deep-disordered breathing (SDB), the sleep and respiration of 31 nonobese healthy males ages 30-49 (15 snorers, 16 nonsnorers) were studied overnight after alcohol ingestion. Subjects received placebo, 0.32, 0.65, and 0.81 g alcohol/kg body weight prior to their evening bedtime, with each dose given on one of four nonconsecutive nights in a repeated-measures counterbalanced design. On each night, respiration was assessed by recording respiratory effort from inter-costal surface electromyography (EMG), ventilation from oral and MMI thermistors, and arterial oxygen saturation (SaO2) from an ear oximeter (BIOX III). Snorers had significantly: (a) more total SDB, ( b) more obstructive deep apnea (OSA), and (c) lower minimum SaO2 than nonsnorers after the placebo and each alcohol dose. Snorers had more hypoxic events than nonsnorers after each alcohol dose but not after placebo. Increasing alcohol dose caused a statistically significant ( p= 0.0004) decrease in minimum SaO2 in snorers only, but this decrease was small and probably not clinically important. Alcohol did not cause significant increases in SDB and hypoxic events, and did not have different effects on SDB and hypoxic events for snorers versus nonsnorers. Because this experiment included only nonobese 30-49-year-old males, these results do not imply that alcohol has no significant effects on obese subjects or those older than 50. [ABSTRACT FROM AUTHOR]

This study directly tested the beneficial effect of isolated REM and isolated NREM skepon the recall of narcoleptics. In a within subject design. 10 narcoleptics were instructed to sleep for a certain optimal duration and at a certain optimal time before each session, and were given 12-14 Sessions, one a day, on different lists of a complex associative memory task and a minimally associative memory task. Following the 10-min task, the subject either had 20 min of polygraphically recorded napping or card playing, followed by a free recall test. The results for the complex associative task indicated significant differences between the three conditions for free recall. Recall was significantly better after isolated REM than after isolated NREM sleep or wakefulness and was significantly better after NREM than after wakefulness. The results from the minimally associative task were inconclusive. The results are consistent with the proposed neuronat activity correlates (NAC) theory that REM sleep actively consolidates and/or integrates complex associative information and that NREM sleep passively prey cuts retroactive interference of recently acquired complex associative information. [ABSTRACT FROM AUTHOR]

Somatostatin (SS) is a cyclic tetradecapeptide able to inhibit the release of growth hormone (GH) mainly through the binding to two G‐protein coupled receptor (GPCR) subtypes, SSTR2 and SSTR5. These receptors are overexpressed in approximately 90% of carcinoid tumors. However, the clinical use of somatostatin is limited by its short half‐life in vivo. In order to overcome this severe drawback, a huge number of analogs have been prepared, leading to the development of Octreotide, which is currently used in the clinic, among other applications, to treat various neuroendocrine tumors and, radiolabeled by, for example, 111In, 11C, and 68Ga, for imaging SS‐secreting tumors. Despite the success of Octreotide, there is an unmet need for the development of novel, more stable and selective Octreotide‐derived radiotherapeutics. To this end, the Cu(I)‐catalyzed azide‐alkyne 1,3‐dipolar Huisgen's cycloaddition, the prototypic click reaction, presents a promising opportunity to replace the susceptible disulfide bridge with a durable [1,2,3]triazolyl containing bridge and to introduce conformational constraints increasing specific receptor binding. Herein we report the design and synthesis of a series of i‐to‐i + 5 1,4‐ and 4,1‐disubstituted [1,2,3]triazolyl‐bridged cyclopeptides derived from the Octreotide scaffold and their detailed conformational analysis via NMR spectroscopy. [ABSTRACT FROM AUTHOR]