Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination.

The sequence-specific transcription factor NF-Y binds the CCAAT box, one of the sequence elements most frequently found in eukaryotic promoters. NF-Y is composed of the NF-YA and NF-YB/NF-YC subunits, the latter two hosting histone-fold domains (HFDs). The crystal structure of NF-Y bound to a 25 bp CCAAT oligonucleotide shows that the HFD dimer binds to the DNA sugar-phosphate backbone, mimicking the nucleosome H2A/H2B-DNA assembly. NF-YA both binds to NF-YB/NF-YC and inserts an α helix deeply into the DNA minor groove, providing sequence-specific contacts to the CCAAT box. Structural considerations and mutational data indicate that NF-YB ubiquitination at Lys138 precedes and is equivalent to H2B Lys120 monoubiquitination, important in transcriptional activation. Thus, NF-Y is a sequence-specific transcription factor with nucleosome-like properties of nonspecific DNA binding and helps establish permissive chromatin modifications at CCAAT promoters. Our findings suggest that other HFD-containing proteins may function in similar ways.

The structural bases for agonist diversity in an Arabidopsis thaliana glutamate receptor-like channel.

Arabidopsis thaliana glutamate receptor-like (GLR) channels are amino acid-gated ion channels involved in physiological processes including wound signaling, stomatal regulation, and pollen tube growth. Here, fluorescence microscopy and genetics were used to confirm the central role of GLR3.3 in the amino acid-elicited cytosolic Ca2+ increase in Arabidopsis seedling roots. To elucidate the binding properties of the receptor, we biochemically reconstituted the GLR3.3 ligand-binding domain (LBD) and analyzed its selectivity profile; our binding experiments revealed the LBD preference for l-Glu but also for sulfur-containing amino acids. Furthermore, we solved the crystal structures of the GLR3.3 LBD in complex with 4 different amino acid ligands, providing a rationale for how the LBD binding site evolved to accommodate diverse amino acids, thus laying the grounds for rational mutagenesis. Last, we inspected the structures of LBDs from nonplant species and generated homology models for other GLR isoforms. Our results establish that GLR3.3 is a receptor endowed with a unique amino acid ligand profile and provide a structural framework for engineering this and other GLR isoforms to investigate their physiology.

Structural determinants for NF-Y subunit organization and NF-Y/DNA association in plants.

NF-Y transcription factor comprises three subunits: NF-YA, NF-YB and NF-YC. NF-YB and NF-YC dimerize through their histone fold domain (HFD), which can bind DNA in a non-sequence-specific fashion while serving as a scaffold for NF-YA trimerization. Upon trimerization, NF-YA specifically recognizes the CCAAT box sequence on promoters and enhancers. In plants, each NF-Y subunit is encoded by several genes giving rise to hundreds of potential heterotrimeric combinations. In addition, plant NF-YBs and NF-YCs interact with other protein partners to recognize a plethora of genomic motifs, as the CCT protein family that binds CORE sites. The NF-Y subunit organization and its DNA-binding properties, together with the NF-Y HFD capacity to adapt different protein modules, represent plant-specific features that play a key role in development, growth and reproduction. Despite their relevance, these features are still poorly understood at the molecular level. Here, we present the structures of Arabidopsis and rice NF-YB/NF-YC dimers, and of an Arabidopsis NF-Y trimer in complex with the FT CCAAT box, together with biochemical data on NF-Y mutants. The dimeric structures identify the key residues for NF-Y HFD stabilization. The NF-Y/DNA structure and the mutation experiments shed light on HFD trimerization interface properties and the NF-YA sequence appetite for the bases flanking the CCAAT motif. These data explain the logic of plant NF-Y gene expansion: the trimerization adaptability and the flexible DNA-binding rules serve the scopes of accommodating the large number of NF-YAs, CCTs and possibly other NF-Y HFD binding partners and a diverse audience of genomic motifs.

Structure and kinetic properties of human d-aspartate oxidase, the enzyme-controlling d-aspartate levels in brain.

d-Amino acids are the "wrong" enantiomers of amino acids as they are not used in proteins synthesis but evolved in selected functions. On this side, d-aspartate (d-Asp) plays several significant roles in mammals, especially as an agonist of N-methyl-d-aspartate receptors (NMDAR), and is involved in relevant diseases, such as schizophrenia and Alzheimer's disease. In vivo modulation of d-Asp levels represents an intriguing task to cope with such pathological states. As little is known about d-Asp synthesis, the only option for modulating the levels is via degradation, which is due to the flavoenzyme d-aspartate oxidase (DASPO). Here we present the first three-dimensional structure of a DASPO enzyme (from human) which belongs to the d-amino acid oxidase family. Notably, human DASPO differs from human d-amino acid oxidase (attributed to d-serine degradation, the main coagonist of NMDAR) showing peculiar structural features (a specific active site charge distribution), oligomeric state and kinetic mechanism, and a higher FAD affinity and activity. These results provide useful insights into the structure-function relationships of human DASPO: modulating its activity represents now a feasible novel therapeutic target.

Fusicoccin Activates KAT1 Channels by Stabilizing Their Interaction with 14-3-3 Proteins.

Plants acquire potassium (K+) ions for cell growth and movement via regulated diffusion through K+ channels. Here, we present crystallographic and functional data showing that the K+ inward rectifier KAT1 (K+Arabidopsis thaliana 1) channel is regulated by 14-3-3 proteins and further modulated by the phytotoxin fusicoccin, in analogy to the H+-ATPase. We identified a 14-3-3 mode III binding site at the very C terminus of KAT1 and cocrystallized it with tobacco (Nicotiana tabacum) 14-3-3 proteins to describe the protein complex at atomic detail. Validation of this interaction by electrophysiology shows that 14-3-3 binding augments KAT1 conductance by increasing the maximal current and by positively shifting the voltage dependency of gating. Fusicoccin potentiates the 14-3-3 effect on KAT1 activity by stabilizing their interaction. Crystal structure of the ternary complex reveals a noncanonical binding site for the toxin that adopts a novel conformation. The structural insights underscore the adaptability of fusicoccin, predicting more potential targets than so far anticipated. The data further advocate a common mechanism of regulation of the proton pump and a potassium channel, two essential elements in K+ uptake in plant cells.

The phosphorylatable Ser320 of NF-YA is involved in DNA binding of the NF-Y trimer.

Nuclear factor Y (NF-Y) is a transcription factor trimer binding to the functionally important CCAAT box, present in promoters of growth-promoting and cell cycle-regulated genes. The regulatory nuclear factor YA (NF-YA) subunit confers sequence-specificity to the histone-like nuclear factor YB/YC dimer. NF-YA harbors 2 serines-Ser320 and Ser326-shown to be phosphorylated by cyclin-dependent kinase 2. High-throughput proteomics data indicate that they are phosphorylated in vivo. Specifically, Ser320 makes structural contacts with the DNA phosphate backbone; Ser320-P is the major NF-YA phosphorylation isoform following overexpression in HeLa cells, increasing upon mitotic arrest. EMSA with recombinant Ala and Glu mutants confirm a role of Ser320, but not Ser326, in stabilization of DNA binding. Transactivation assays of the CCAAT-dependent MDR1 and RHOB promoters show loss in transcription function for Ser320Glu and Ser320Ala NF-YA mutants. Phylogenetic analysis of NF-YA proteins indicates that Ser320 is indeed evolutionarily conserved. We conclude that phosphorylation of this residue belongs to the core mechanisms of DNA-binding control, possibly driven by the necessity to unfasten binding of or to evict NF-Y from CCAAT sites under specific conditions of growth regulation.-Bernardini, A., Lorenzo, M., Nardini, M., Mantovani, R., Gnesutta, N. The phosphorylatable Ser320 of NF-YA is involved in DNA binding of the NF-Y trimer.

Pulmonary nodules precision localization techniques.

The advent of helical high-resolution CT scanners, the application of screening programs and the follow-up of patient with oncological history, led to an increasing number of diagnosis of small pulmonary nodule (less than 10 mm in maximum diameter), partially solid nodule or completely ground glass ones. Their management is controversial. Excisional biopsy by mean of video-assisted thoracic surgery is often a viable choice but to locate these lesions intraoperatively can be impossible without the aid of preoperative or intraoperative localization techniques. In this brief review we will analyze the benefit of adopting localization techniques prior to pulmonary resection for small pulmonary lesions and face the advantages and problems with the main techniques described in the literatures.

Precision surgery in lung metastasectomy.

The value of pulmonary metastasis (PM) resection in the context of controlled primary tumor sites was shown to improve survival of patients if complete resection could be achieved. The surgeon's approach can be modulated by various parameters pertaining to safety margins including local growth properties, size, spread and location of PMs. Lymph node dissection and assessment is recommended although its impact on survival remains unclear. Thoracoscopic surgery combined to thin slice chest CT scans has shown results comparable to thoracotomy in patients with few PMs. The management of PMs should therefore be discussed on an individual, interdisciplinary basis to offer the best possible oncological and surgical results as well as to maximize long term patient survival rates.

Saturn-Shaped Ice Burst Pattern and Fast Basal Binding of an Ice-Binding Protein from an Antarctic Bacterial Consortium.

Ice-binding proteins (IBPs) bind to ice crystals and control their growth, enabling host organisms to adapt to subzero temperatures. By binding to ice, IBPs can affect the shape and recrystallization of ice crystals. The shapes of ice crystals produced by IBPs vary and are partially due to which ice planes the IBPs are bound to. Previously, we have described a bacterial IBP found in the metagenome of the symbionts of Euplotes focardii ( EfcIBP). EfcIBP shows remarkable ice recrystallization inhibition activity. As recrystallization inhibition of IBPs and other materials are important to the cryopreservation of cells and tissues, we speculate that the EfcIBP can play a future role as an ice recrystallization inhibitor in cryopreservation applications. Here we show that EfcIBP results in a Saturn-shaped ice burst pattern, which may be due to the unique ice-plane affinity of the protein that we elucidated using the fluorescent-based ice-plane affinity analysis. EfcIBP binds to ice at a speed similar to that of other moderate IBPs (5 ± 2 mM-1 s-1); however, it is unique in that it binds to the basal and previously unobserved pyramidal near-basal planes, while other moderate IBPs typically bind to the prism and pyramidal planes and not basal or near-basal planes. These insights into EfcIBP allow a better understanding of the recrystallization inhibition for this unique protein.

Target highlights from the first post-PSI CASP experiment (CASP12, May-August 2016).

The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.

Structure-Function Relationships in l-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes.

l-Amino acid deaminase from Proteus myxofaciens (PmaLAAD) is a membrane flavoenzyme that catalyzes the deamination of neutral and aromatic l-amino acids into α-keto acids and ammonia. PmaLAAD does not use dioxygen to re-oxidize reduced FADH2 and thus does not produce hydrogen peroxide; instead, it uses a cytochrome b-like protein as an electron acceptor. Although the overall fold of this enzyme resembles that of known amine or amino acid oxidases, it shows the following specific structural features: an additional novel α+ß subdomain placed close to the putative transmembrane α-helix and to the active-site entrance; an FAD isoalloxazine ring exposed to solvent; and a large and accessible active site suitable to bind large hydrophobic substrates. In addition, PmaLAAD requires substrate-induced conformational changes of part of the active site, particularly in Arg-316 and Phe-318, to achieve the correct geometry for catalysis. These studies are expected to pave the way for rationally improving the versatility of this flavoenzyme, which is critical for biocatalysis of enantiomerically pure amino acids.

[Incidence of biological accidents at work and immune status for vaccine-preventable diseases among resident physicians in specialist training at Ferrara University Hospital]. / Studio di incidenza sugli infortuni biologici nei medici in formazione specialistica dell'Azienda Ospedaliero - Universitaria di Ferrara e stato immunitario nei confronti delle principali infezioni prevenibili.

The hospital as a work environment is particularly characterized by various risks for healthcare workers (HCWs). The main risk is represented by biological accidents, associated with the parenteral transmission of pathogens. Biological injuries can occur during the care service and the manipulation of biological fluids. Hepatitis B (and hepatitis D), hepatitis C and HIV are the most common infections transmitted by biological injuries. Physicians should acquire awareness of the risks associated with their professional activity during their training as medical residents (MRs). Some infectious diseases are preventable by vaccination and the "National Immunization Plan 2017-2019" (PNPV) recommends HCWs vaccination against hepatitis B, influenza, measles -mumps -rubella, chicken pox, and pertussis. Besides, not only HCWs' vaccination can prevent the disease in healthcare professionals, but it also may reduce the transmission to patients. Therefore, active immunization of HCWs by recommended vaccinations plays an important role to prevent disease cases, complications and death in patients. An increased awareness of risk behaviors is the first important point to address in order to reduce biological accidents and infectious diseases transmission, so as to reduce their frequency. Besides, HCWs' vaccination is useful to reinforce protection and to prevent the transmission of some infectious diseases in case of exposure. The aim of this five-year incidence study is to investigate the MRs' biological accidents characteristics and to analyze the MRs' immune status at the University of Ferrara in the period 2011-2015. Data on MRs' biological accidents and immune status at Azienda Ospedaliero-Universitaria of Ferrara in 2011-2015 were analyzed by Microsoft Excel 2007 Software. In this study, the percentage of MRs' biological injuries compared to the total number of MRs showed an annual variability, with a peak in 2011 (11.9%). During the analyzed period, there were 190 biological injuries among the MRs: 81% were percutaneous injuries and blood was the biological fluid involved in 86.3% of cases. The most frequent lesion was puncture (62.6%), occurred in 41.1% of cases by suture needles; 46.3% of accidents occurred in the operating block. These data can be explained by the more invasive and at risk activities carried out in these Operative Units. The high injuries percentage in MRs may be related to less work experience and inadequate training or informations about personal protective equipment use. Among MRs, 93.7%, 93.3% and 96.6% were immune to measles, chicken pox and hepatitis B, respectively; only in the case of rubella, 11.9% of MRs was not immune. This research showed, accordingly to published data, high adhesion to hepatitis B vaccination. However, the healthcare workers' vaccine coverage is still sub-optimal; active immunization by recommended vaccines should be implemented for both parenteral and airborne diseases. As a matter of fact, the recent measles outbreak has involved healthcare workers (4689 cases of measles, 305 in HCWs). Finally, the General Directorates of Health-care settings should improve healthcare personnel adhesion to vaccinations, such as influenza, by promotion activities in the workplace. A proposal in order to achieve coverage objectives could be making vaccinations mandatory, as well as already implemented in other countries.

Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness.

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder ß-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.

Safety of video-assisted thoracic surgery lobectomy for non-small-cell lung cancer in a low-volume unit.

Several studies have demonstrated that for complex surgical procedures, surgeons who treat more patients have better outcomes than their lower-volume counterparts. The aim of this paper is to review the experience with video-assisted thoracic surgery (VATS) lobectomies in our small thoracic unit (group A), to understand whether our short-term results were different to the outcomes obtained by the same surgeon previously working in a high-volume unit (group B). 37 patients underwent VATS lobectomy. Hospital stay was on average 4.5 days (group A) versus 4.1 days (group B). Operative time and the number of 'frozen sections' were higher in group A. Hospital mortality was 0. VATS lobectomies are a safe approach in a low-volume unit formed by a single surgeon with a previous high-volume experience.

Awake uniportal video-assisted thoracic surgery for complications after pneumonectomy.

Since 1998, we started a clinical program for awake video-assisted thoracic surgery in our unit using four-step local anesthesia and sedation. Throughout the years, we experienced several difficult cases, three of them had complications postpneumonectomy. The aim of this paper is to report these three cases.

Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS.

ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD(+)-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38.

Crystal structures of two Bacillus carboxylesterases with different enantioselectivities.

Naproxen esterase (NP) from Bacillus subtilis Thai I-8 is a carboxylesterase that catalyzes the enantioselective hydrolysis of naproxenmethylester to produce S-naproxen (E>200). It is a homolog of CesA (98% sequence identity) and CesB (64% identity), both produced by B. subtilis strain 168. CesB can be used for the enantioselective hydrolysis of 1,2-O-isopropylideneglycerol (solketal) esters (E>200 for IPG-caprylate). Crystal structures of NP and CesB, determined to a resolution of 1.75Å and 2.04Å, respectively, showed that both proteins have a canonical α/ß hydrolase fold with an extra N-terminal helix stabilizing the cap subdomain. The active site in both enzymes is located in a deep hydrophobic groove and includes the catalytic triad residues Ser130, His274, and Glu245. A product analog, presumably 2-(2-hydroxyethoxy)acetic acid, was bound in the NP active site. The enzymes have different enantioselectivities, which previously were shown to result from only a few amino acid substitutions in the cap domain. Modeling of a substrate in the active site of NP allowed explaining the different enantioselectivities. In addition, Ala156 may be a determinant of enantioselectivity as well, since its side chain appears to interfere with the binding of certain R-enantiomers in the active site of NP. However, the exchange route for substrate and product between the active site and the solvent is not obvious from the structures. Flexibility of the cap domain might facilitate such exchange. Interestingly, both carboxylesterases show higher structural similarity to meta-cleavage compound (MCP) hydrolases than to other α/ß hydrolase fold esterases.

Aminoacetone oxidase from Streptococcus oligofermentans belongs to a new three-domain family of bacterial flavoproteins.

The aaoSo gene from Streptococcus oligofermentans encodes a 43 kDa flavoprotein, aminoacetone oxidase (SoAAO), which was reported to possess a low catalytic activity against several different L-amino acids; accordingly, it was classified as an L-amino acid oxidase. Subsequently, SoAAO was demonstrated to oxidize aminoacetone (a pro-oxidant metabolite), with an activity ~25-fold higher than the activity displayed on L-lysine, thus lending support to the assumption of aminoacetone as the preferred substrate. In the present study, we have characterized the SoAAO structure-function relationship. SoAAO is an FAD-containing enzyme that does not possess the classical properties of the oxidase/dehydrogenase class of flavoproteins (i.e. no flavin semiquinone formation is observed during anaerobic photoreduction as well as no reaction with sulfite) and does not show a true L-amino acid oxidase activity. From a structural point of view, SoAAO belongs to a novel protein family composed of three domains: an α/ß domain corresponding to the FAD-binding domain, a ß-domain partially modulating accessibility to the coenzyme, and an additional α-domain. Analysis of the reaction products of SoAAO on aminoacetone showed 2,5-dimethylpyrazine as the main product; we propose that condensation of two aminoacetone molecules yields 3,6-dimethyl-2,5-dihydropyrazine that is subsequently oxidized to 2,5-dimethylpyrazine. The ability of SoAAO to bind two molecules of the substrate analogue O-methylglycine ligand is thought to facilitate the condensation reaction. A specialized role for SoAAO in the microbial defence mechanism related to aminoacetone catabolism through a pathway yielding dimethylpyrazine derivatives instead of methylglyoxal can be proposed.

[A survey on injuries among nurses and nursing students: a descriptive epidemiologic analysis between 2002 and 2012 at a University Hospital]. / Indagine sugli infortune tra il personale infermieristico e gli studenti del corso di laurea in infermieristica: analisi epidemiologica descrittiva nel period 2002-2012 in un'Azienda Ospedaliero-Universitaria.

BACKGROUND: Biological risk is the main occupational hazard in hospitals (40-50% of the total). More than 130,000 injuries occur every year in Italy and nurses are the most affected occupational category. OBJECTIVES: This study evaluated the incidence of injuries related to biological risk in nurses and nursing students in the University Hospital of Ferrara, how they occur, the knowledge on the topic and on behaviour during the department's activity. METHODS: A retrospective study involving a sample of 8 departments (selected for the occurrence of more than 30 biological injuries between 1st January 2002 and 31 December 2012) recorded injuries related to biological risk; subsequently a cross-sectional survey was carried out through a questionnaire administered to nurses and nursing students. RESULTS: 909 biological accidents were reported (81.18% in nurses and 18.82% in students). Blood was the main biological material involved (83.72% of cases), mostly by percutaneous exposure (84.16%). According to the questionnaire, 53% of subjects reported having had at least one injury during their career, and 5.72% did not report it; 46% reported doing risky procedures (re-capping needles) and 95.45% that they had been informed about the correct use of PPE. CONCLUSIONS: The lower percentage of injuries in students could be linked to good university training and to less risky procedures being performed. Re-capping needles remains one of the most dangerous manoeuvers practised. Ongoing training on the correct use of PPE is essential to train prepared and aware health professionals.

Characterization of human DAAO variants potentially related to an increased risk of schizophrenia.

Considering the key role of d-serine in N-methyl-d-aspartate receptor-mediated neurotransmission, it is highly relevant to define the role that enzymes play in d-serine synthesis and degradation. In particular, the details of regulation of the d-serine catabolic human enzyme d-amino acid oxidase (hDAAO) are unknown although different lines of evidence have shown it to be involved in schizophrenia susceptibility. Here we investigated the effect of three single nucleotide polymorphisms and known mutations in hDAAO, i.e., D31H, R279A, and G331V. A very low amount of soluble G331V hDAAO is produced in E. coli cells: the recombinant variant enzyme is fully active. Human U87 glioblastoma cells transiently transfected for G331V hDAAO show a low viability, a significant amount of protein aggregates, and augmented apoptosis. The recombinant D31H and R279A hDAAO variants do not show alterations in tertiary and quaternary structures, thermal stability, binding affinity for inhibitors, and the modulator pLG72, whereas the kinetic efficiency and the affinity for d-serine and for FAD were higher than for the wild-type enzyme. While these effects for the substitution at position 31 cannot be structurally explained, the R279A mutation might affect the hDAAO FAD-binding affinity by altering the "structurally ambivalent" peptide V47-L51. In agreement with the observed increased activity, expression of D31H and R279A hDAAO variants in U87 cells produces a higher decrease in cellular d/(d+l) serine ratio than the wild-type counterpart. In vivo, these substitutions could affect cellular d-serine concentration and its release at synapsis and thus might be relevant for schizophrenia susceptibility.