Olive quick decline syndrome (OQDS) is a disease that has been seriously affecting olive trees in southern Italy since around 2009. During the disease, caused by Xylella fastidiosa subsp. pauca sequence type ST53 (Xf), the flow of water and nutrients within the trees is significantly compromised. Initially, infected trees may not show any symptoms, making early detection challenging. In this study, young artificially infected plants of the susceptible cultivar Cellina di Nardò were grown in a controlled environment and co-inoculated with additional xylem-inhabiting fungi. Asymptomatic leaves of olive plants at an early stage of infection were collected and analyzed using nuclear magnetic resonance (NMR), hyperspectral reflectance (HSR), and chemometrics. The application of a spectranomic approach contributed to shedding light on the relationship between the presence of specific hydrosoluble metabolites and the optical properties of both asymptomatic Xf-infected and non-infected olive leaves. Significant correlations between wavebands located in the range of 530-560 nm and 1380-1470 nm, and the following metabolites were found to be indicative of Xf infection: malic acid, fructose, sucrose, oleuropein derivatives, and formic acid. This information is the key to the development of HSR-based sensors capable of early detection of Xf infections in olive trees.

Intrathecal delivery of Nusinersen-an antisense oligonucleotide that promotes survival motor neuron (SMN) protein induction-is an approved therapy for spinal muscular atrophy (SMA). Here, we employed nuclear magnetic resonance (NMR) spectroscopy to longitudinally characterize the unknown metabolic effects of Nusinersen in the cerebrospinal fluid (CSF) of SMA patients across disease severity. Modulation of amino acid metabolism is a common denominator of biochemical changes induced by Nusinersen, with distinct downstream metabolic effects according to disease severity. In severe SMA1 patients, Nusinersen stimulates energy-related glucose metabolism. In intermediate SMA2 patients, Nusinersen effects are also related to energy homeostasis but involve ketone body and fatty acid biosynthesis. In milder SMA3 patients, Nusinersen mainly modulates amino acid metabolism. Moreover, Nusinersen modifies the CSF metabolome of a more severe clinical group towards the profile of untreated SMA patients with milder disease. These findings reveal disease severity-specific neurometabolic signatures of Nusinersen treatment, suggesting a selective modulation of peripheral organ metabolism by this CNS-directed therapy in severe SMA patients.

Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin alpha, with several human isoforms; among them, importin alpha 3 (Imp alpha 3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered protein (IDP), evolved as a paralogue of nuclear protein 1 (NUPR1), which is involved in chromatin remodeling and DNA repair. It is predicted that NUPR1L has a nuclear localization sequence (NLS) from residues Arg51 to Gln74, in order to allow for nuclear translocation. We studied in this work the ability of intact NUPR1L to bind Imp alpha 3 and its depleted species, increment Imp alpha 3, without the importin binding domain (IBB), using fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular docking techniques. Furthermore, the binding of the peptide matching the isolated NLS region of NUPR1L (NLS-NUPR1L) was also studied using the same methods. Our results show that NUPR1L was bound to Imp alpha 3 with a low micromolar affinity (similar to 5 mu M). Furthermore, a similar affinity value was observed for the binding of NLS-NUPR1L. These findings indicate that the NLS region, which was unfolded in isolation in solution, was essentially responsible for the binding of NUPR1L to both importin species. This result was also confirmed by our in silico modeling. The binding reaction of NLS-NUPR1L to increment Imp alpha 3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Imp alpha 3, confirming that the IBB could act as an auto-inhibition region of Imp alpha 3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in NUPR1L and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation.

Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin alpha, of which there are several human isoforms; among them, importin alpha 3 (Imp alpha 3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54-74) of NUPR1 towards Imp alpha 3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Imp alpha 3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Imp alpha 3 with low micromolar affinity (1.7-27 mu M). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i,i+ 1) nuclear Overhauser effect (NOE) in the 2D-H-1-NMR (two-dimensional-proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Imp alpha 3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure.

The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis. Two of the three GTs were previously annotated as GTs, but the third GT was identified in this study. We determined the GT functions by comparing the WT glycan structures from PBCV-1 with those from a set of PBCV-1 spontaneous GT gene mutants resulting in antigenic variants having truncated glycan structures. According to our working model, the virus gene a064r encodes a GT with three domains: domain 1 has a -l-rhamnosyltransferase activity, domain 2 has an -l-rhamnosyltransferase activity, and domain 3 is a methyltransferase that decorates two positions in the terminal -l-rhamnose (Rha) unit. The a075l gene encodes a -xylosyltransferase that attaches the distal d-xylose (Xyl) unit to the l-fucose (Fuc) that is part of the conserved N-glycan core region. Last, gene a071r encodes a GT that is involved in the attachment of a semiconserved element, -d-Rha, to the same l-Fuc in the core region. Our results uncover GT activities that assemble four of the nine residues of the PBCV-1 MCP N-glycans.

Scientists applying magnetic resonance techniques to cultural heritage are now a quite vast and international community, even if these applications are not yet well known outside this community. Not only laboratory experiments but also measurements in the field are now possible, with the use of portable nuclear magnetic resonance (NMR) instruments that enable non-invasive and non-destructive studies on items of any size, of high artistic and historical value as well as diagnosis of their conservation state. The situation was completely different in the second half of the 1990s when our group started working on applications of NMR to cultural heritage, by combining the knowledge of NMR for fluids in porous media at the University of Bologna, with the skilfulness of the chemists for cultural heritage of CNR and University of Florence, and Safeguarding Cultural Heritage Department of Aosta. Since then, our interest has been mainly devoted to develop methods to study the structure of pore space and their changes as a result of the decay, as well as to evaluate performance of the protective and conservative treatments of porous materials like stone, ceramic, cements and wood. In this paper, we will review the pathway that led us from the first tentative experiments, in the second half of the 1990s to the current work on these topics.

Highly active metallocenes and other single site catalysts as well as Grubbs and Schrock metathesis catalytic systems have opened up the possibility to polymerize cycloolefins or to copolymerize them with ethene or propene. The polymers obtained show exciting structures and properties. Cycloolefins such as cyclopentene, cyclooctene, norbornene, and their substituted derivatives are incorporated into the polymer chain either by double bonds or by ring-opening metathesis polymerization. Materials with elastomeric properties or tactic polymers with high glass transition temperatures and melting points are obtained with a wide range of microstructures. For example, cycloolefin copolymers and other homo- and copolymers of norbornene are of great academic and industrial interest because of their properties and applications in optoelectronics, lenses, and coatings