Hospital School Program: The Right to Education for Long-Term Care Children.

Education and health are two inseparable aspects of a single dynamic which aims to support and increase the physical and mental well-being of children and young people. Children must be guaranteed two rights: the right to study and the right to health. Schools capable of reconciling these two fundamental needs are represented by school in hospital and home schooling. Thanks to this flexible teaching method, it is possible to support the child and his or her family during hospitalization, and to prevent consequences such as school failure and dropout. Hospitalization is always a traumatic event for children, in which white coats are unknown figures, perceived all the more threatening the younger the child: a threat to one's integrity, loss of autonomy, distorted perception of time, loss of confidence, and a sense of abandonment. Therefore, it is important to create a communicative basis that facilitates the child's adaptation to the new hospital environment and establishes continuity during this period of time. Teachers play a significant role within the context of such difficulties. They need to understand patients' emotions and act as a bridge between the small inpatient room of the child and the outside world. In this article we examined: (1) the School in Hospital and the reasons why it is a valid resource for the psychophysical rehabilitation of the student in a hospital; (2) the role of the teacher in hospital and the difficult context in which the teacher has to work; and (3) how the school in hospital was challenged by the SARS-CoV2 pandemic.

Characterization of bacterial NMN deamidase as a Ser/Lys hydrolase expands diversity of serine amidohydrolases.

NMN deamidase (PncC) is a bacterial enzyme involved in NAD biosynthesis. We have previously demonstrated that PncC is structurally distinct from other known amidohydrolases. Here, we extended PncC characterization by mutating all potential catalytic residues and assessing their individual roles in catalysis through kinetic analyses. Inspection of these residues' spatial arrangement in the active site, allowed us to conclude that PncC is a serine-amidohydrolase, employing a Ser/Lys dyad for catalysis. Analysis of the PncC structure in complex with a modeled NMN substrate supported our conclusion, and enabled us to propose the catalytic mechanism.

Characterization of human nicotinate phosphoribosyltransferase: Kinetic studies, structure prediction and functional analysis by site-directed mutagenesis.

Nicotinate phosphoribosyltransferase (NaPRT, EC 2.4.2.11) catalyzes the conversion of nicotinate (Na) to nicotinate mononucleotide, the first reaction of the Preiss-Handler pathway for the biosynthesis of NAD(+). Even though NaPRT activity has been described to be responsible for the ability of Na to increase NAD(+) levels in human cells more effectively than nicotinamide (Nam), so far a limited number of studies on the human NaPRT have appeared. Here, extensive characterization of a recombinant human NaPRT is reported. We determined its major kinetic parameters and assayed the influence of different compounds on its enzymatic activity. In particular, ATP showed an apparent dual stimulation/inhibition effect at low/high substrates saturation, respectively, consistent with a negative cooperativity model, whereas inorganic phosphate was found to act as an activator. Among other metabolites assayed, including nucleotides, nucleosides, and intermediates of carbohydrates metabolism, some showed inhibitory properties, i.e. CoA, several acyl-CoAs, glyceraldehyde 3-phosphate, phosphoenolpyruvate, and fructose 1,6-bisphosphate, whereas dihydroxyacetone phosphate and pyruvate exerted a stimulatory effect. Furthermore, in light of the absence of crystallographic data, we performed homology modeling to predict the protein three-dimensional structure, and molecular docking simulations to identify residues involved in the recognition and stabilization of several ligands. Most of these residues resulted universally conserved among NaPRTs, and, in this study, their importance for enzyme activity was validated through site-directed mutagenesis.

Probing pH-dependent dissociation of HdeA dimers.

HdeA protein is a small, ATP-independent, acid stress chaperone that undergoes a dimer-to-monomer transition in acidic environments. The HdeA monomer binds a broad range of proteins to prevent their acid-induced aggregation. To understand better HdeA's function and mechanism, we perform constant-pH molecular dynamics simulations (CPHMD) to elucidate the details of the HdeA dimer dissociation process. First the pK(a) values of all the acidic titratable groups in HdeA are obtained and reveal a large pK(a) shift only for Glu(37). However, the pH-dependent monomer charge exhibits a large shift from -4 at pH > 6 to +6 at pH = 2.5, suggesting that the dramatic change in charge on each monomer may drive dissociation. By combining the CPHMD approach with umbrella sampling, we demonstrate a significant stability decrease of the HdeA dimer when the environmental pH changes from 4.0 to 3.5 and identify the key acidic residue-lysine interactions responsible for the observed pH sensing in HdeA chaperon activity function.

Comparative structural and functional characterization of putative protein effectors belonging to the PcF toxin family from Phytophthora spp.

The PcF Toxin Family (Pfam 09461) includes the characterized phytotoxic protein PcF from Phytophthora cactorum, as well as several predicted protein effectors from other Phytophthora species recently identified by comparative genomics. Here we provide first evidence that such 'putatives', recombinantly expressed in bacteria and purified to homogeneity, similarly to PcF, can trigger defense-related responses on tomato, that is leaf withering and phenylalanine ammonia lyase induction, although with various degrees of effectiveness. In addition, structural prediction by computer-aided homology modeling and subsequent structural/functional comparison after rational engineering of the disulfide-structured protein fold by site-directed mutagenesis, highlighted the surface-exposed conserved amino acid stretch SK(E/C)C as a possible structural determinant responsible for the differential phytotoxicity within this family of cognate protein effectors.

Identification of nicotinamide mononucleotide deamidase of the bacterial pyridine nucleotide cycle reveals a novel broadly conserved amidohydrolase family.

The pyridine nucleotide cycle is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial pyridine nucleotide cycle, was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds of bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in Escherichia coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three-dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and nonfunctional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in the bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.

Homology modeling and deletion mutants of human nicotinamide mononucleotide adenylyltransferase isozyme 2: new insights on structure and function relationship.

Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the formation of NAD by means of nucleophilic attack by 5'-phosphoryl of NMN on the α-phosphoryl group of ATP. Humans possess three NMNAT isozymes (NMNAT1, NMNAT2, and NMNAT3) that differ in size and sequence, gene expression pattern, subcellular localization, oligomeric state and catalytic properties. Of these, NMNAT2, the least abundant isozyme, is the only one whose much-needed crystal structure has not been solved as yet. To fill this gap, we used the crystal structures of human NMNAT1 and NMNAT3 as templates for homology-based structural modeling of NMNAT2, and the resulting raw structure was then refined by molecular dynamics simulations in a water box to obtain a model of the final folded structure. We investigated the importance of NMNAT2's central domain, which we postulated to be dispensable for catalytic activity, instead representing an isozyme-specific control domain within the overall architecture of NMNAT2. Indeed, we experimentally confirmed that removal of different-length fragments from this central domain did not compromise the enzyme's catalytic activity or the overall tridimensional structure of the active site.