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BackgroundThe Global Influenza Hospital Surveillance Network is an international platform whose primary objective is to study severe cases of influenza requiring hospitalization.MethodsDuring the 2015-2016 influenza season, 11 sites in the Global Influenza Hospital Surveillance Network in nine countries (Russian Federation, Czech Republic, Turkey, France, China, Spain, Mexico, India, and Brazil) participated in a prospective, active-surveillance, hospital-based epidemiological study. Influenza infection was confirmed by reverse transcription-polymerase chain reaction. Influenza vaccine effectiveness (IVE) against laboratory-confirmed influenza was estimated using a test-negative approach.Results9882 patients with laboratory results were included of which 2415 (24.4%) were positive for influenza, including 1415 (14.3%) for A(H1N1)pdm09, 235 (2.4%) for A(H3N2), 180 (1.8%) for A not subtyped, 45 (0.5%) for B/Yamagata-lineage, 532 (5.4%) for B/Victoria-lineage, and 33 (0.3%) for B not subtyped. Of included admissions, 39% were

Influenza-like illness occurs annually worldwide, with peak timing and severity varying seasonally, resulting in significant annual mortality.

The development of the so-called antibiotic adjuvants as inhibitors of non-essential targets represents an inno- vative and attractive approach to counteract antimicrobial resistance (AMR). Most bacteria rely on the reductive sulfate assimilation pathway (RSAP) to synthesize cysteine, which is a building block for many important bio- molecules. Cysteine biosynthetic enzymes are colonization factors that are dispensable during growth in rich media but might become indispensable during host colonization. Being this pathway absent in mammals, it might represent a promising target for drug intervention. We have focused our attention on compounds targeting serine acetyltransferase (SAT), which is one of the key enzymes involved in the L-cysteine biosynthesis, catalyzing the rate-limiting step of the whole process. In a previous communication, we have reported the discovery through a virtual screening of a new compound (1) with promising SAT inhibitory activity. The capability of this compound to interfere with bacterial growth in the cell assays prompted us to carry out a medicinal chemistry campaign to further investigate its potential. We herein report the synthesis of compound 1 analogues to define the structure–activity relationships (SAR) of this series of potential SAT inhibitors regarding the target binding and general toxicity. Despite the improvement in the inhibitory activity of some molecules, the toxicity profile needs to be fine-tuned, and these findings will be used to drive the synthesis of new analogues.

The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pKa values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein.

We have used fluorescence spectroscopy techniques such as fluorescence correlation spectroscopy and fluorescence anisotropy decay on a wide time range, from nanoseconds to seconds, to investigate the unfolding kinetics induced by guanidinium chloride of GFPMut2 and its point mutation H148G, which has proved to be relevant for GFP photochemistry and photophysics. The mutation affects the unfolding kinetics of GFP leading to a much faster process at alkaline pH values, where protonation dynamics is negligible, that can be ascribed to a twofold role of His148, either as a proton shutter towards the chromophore and as a conformation stabiliser. For both mutants a soft region located near beta-strand 3 is found that starts to gain flexibility in the ns range at denaturant concentrations far lower than those required to turn off the chromophore fluorescence, as derived from the anisotropy decay of an extrinsic probe covalently bound to the proteins. © 2011 Elsevier B.V. All rights reserved.

New probes for kinetic intracellular measurements in the millisecond range are desirable to monitor protein biochemical dynamics essential for catalysis, allosteric regulation and signalling. Good candidates to this aim are the photo-switchable mutants of the Green Fluorescent Protein, whose anionic fluorescence, primed by blue light, is markedly enhanced under an additional excitation at a shorter wavelength and relaxes within few milliseconds. The aim of this report is to study how the brightness enhancement kinetics depends on the physical-chemical and spectroscopic parameters and to provide proof-of-concepts experiments for the use of the fluorescence enhancement in conditions in which the protein diffusion is hindered and thereby photo-bleaching can be a limiting critical issue. Future, direct applications of photo-chromic mutants for modulated excitation imaging would in fact require such a detailed knowledge. We present here an extensive study of the photo-switching mechanism of the E222Q mutant of GFPMut2 (Mut2Q), pumped by visible 488 nm light and probed at 400-420 nm, as a function of pH, viscosity, temperature and light intensity. In solution, two characteristic photo-switching times are found by means of modulated double beam fluorescence correlation spectroscopy in the 1-30 ms range, depending on the solution pH. The photo-switching kinetics is solved in terms of the eigenvalues and the eigenvectors of a specific energy diagram and directly used to fit the data, suggesting that the observed photo-switching amplitudes and kinetics are related to a single three-levels transition loop. Finally we give in-vitro examples of the use of modulated excitation microscopy, based on fluorescence enhancement amplitude and kinetics detection, on Mut2Q protein samples immobilized in acrylamide gels.

GFP mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Because serine 65, threonine 203, glutamate 222, and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, and S72A GFP) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components are found in the fluorescence autocorrelation functions of GFPmut2: a 10-100 micros pH-dependent component and a 100-500 micros laser-power-dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a 3-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the histidine 148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, is due to an excited-state process that is probably related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.