Populus tremula seed fibers. Samples were characterized using FT-IR, SEM, XRD, and TGA-DTA analyses. FT-IR spectrum of the extracted cellulose confirmed that hemicellulose and lignin were removed during alkali and bleaching treatments. SEM images showed the partially roughened surface of the fiber due to the removal of non-cellulosic elements and surface impurities during chemical modification. The crystallinity index values for untreated Populus tremula fibers and extracted cellulose were calculated to be 32.8% and 58.9%, respectively. The obvious increase in the crystallinity index for the extracted cellulose confirmed the removal of amorphous compounds present in raw populus. Alkali-treated populus fibers were more thermally stable than raw fibers. All changes observed after alkali and bleaching treatments evidenced the removal of amorphous contents and non-cellulosic components in raw populus fibers. Extracted cellulose exhibited excellent adsorption capacities of methylene blue (140.4 mg g−1) and crystal violet (154 mg g−1). The pseudo second order equation fitted well the kinetic data indicating a chemi-sorption process. The Freundlich model complied well with the experimental data suggesting that the adsorption of the studied dyes was multilayer.Cellulose is the most widely available biopolymer which is extensively used for several applications including textiles, composites, pharmaceutical, water treatment, etc. In this investigation, cellulose was chemically extracted from  Agricultural coproducts and by-products could be treated for the production of various materials . Most of biomass residues are lignocellulosic matters and cellulose is the main component ,2,3. CelPopulus tremula [Efforts have been made to produce natural cellulose fibers from biomass residues  covering tremula , and per tremula  and we hPopulus tremula has the biggest native range of any species from populus genus, being one of the most widely dispersed trees [Populus tremula chip mixtures and the resulting pulp and paper properties were investigated. Marzena et al. [Populus tremula hybridization with populus tremuloides michaux and populus alba L. on the growth and cellulosic pulp properties for papermaking applications. Rooni et al. [Populus tremula with steam explosion and nitrogen explosive decompression to separate enzymatic hydrolysis and to increase bioethanol and biogas yields. To our knowledge, populus seed fibers is not explored for cellulose production. Herein, the aim of the current study was to extract a natural cellulosic fiber from Populus tremula seed plant. Afterward, the cellulosic extract was fully characterized using different chemical, structural, thermal, and morphological characterizations . The extracted cellulose was then applied as adsorbent of cationic dyes, methylene blue and crystal violet. The adsorption efficiency was evaluated by varying different experimental parameters such as pH, time, temperature, and the initial dye concentration. The theoretical kinetic and isothermal equations were investigated for the analysis of the evaluated experimental data.ed trees . The lited trees  reporteda et al.  investigi et al.  have prePopulus tremula seed fibers were collected from the region of Bizerte (Tunisia) during the summer (July). Glacial acetic acid and sodium hydroxide were used as laboratory grade reagents with pure quality. H2O2 (30%) was used to bleach the raw fibers as an oxidizing agent. Methylene blue  and crystal violet  were purchased from Sigma Aldrich company. Methylene blue and crystal violet solutions were prepared using distilled water.Populus tremula seed fibers was performed with reference to a method reported by Aiqin et al. [w/v) at 80 °C for 2 h, in order to remove the wax and maximum of lignin. To remove the residual lignin and bleach the obtained organic matter, the alkali treated fibers were treated in a mixed solution of CH3COOH and H2O2 (v/v = 1:1) at 90 °C for 2 h with a liquor ratio of 1:50 (w/v). Finally, the resulting product was filtered using a Whatman filter paper and oven-dried at 60 °C for 6 h.The extraction of cellulose from n et al.  with a s−1 with a resolution of 4 cm−1. A JEOL JSM-5400 scanning electron microscope was used to examine the morphological characteristics of the studied samples. Gold was used to cover samples utilizing a vacuum sputter-coater to improve both conductivity and image quality with an accelerating voltage of 20 kV. A PANalytical X’Pert PRO MPD apparatus was employed to depict the XRD patterns in 2 theta range of 10°–90°. The samples were thermally analyzed in air flow, at a heating rate of 10°/min, in a Pt crucible with NETZSCH STA 449F3 instrument.The chemical structures of the studied samples were studied by means of an InfraLum FT-08 apparatus equipped with ATR . The spectra were considered in 32 scans from 400 to 4000 cm−1), and temperature (22–55 °C). The adsorbed quantity of methylene blue or crystal violet was calculated using Equation (1):0 and Ce represent the dye concentration at time = 0 min and at equilibrium, respectively. V is the volume of the dye used for experiment and m is the mass of the adsorbent.Adsorption experiments were conducted in batch mode using Erlenmeyer flasks holding 0.0125 g of extracted cellulose and a volume of 10 mL of methylene blue or crystal violet. The mixture was stirred at 125 rpm. After adsorption, the liquid was filtered and its absorbance was measured at the maximum wavelength . The absorbance was checked at different period of times (0–120 min), methylene blue concentrations , (200), and (040) lattice planes of crystalline cellulose I [ine peak ,40. The lulose I .I200 is the intensity of the highest crystalline peak and amI is the intensity of the predominantly amorphous peak.The crystallinity index (CrI) was calculated using Equation (2):Populus tremula fibers and extracted cellulose are calculated to be 32.8% and 58.9%, respectively. These calculated values, which are relatively low, indicated the presence of amorphous compounds including hemicelluloses and lignin in the composition of populus fiber [The crystallinity index values for untreated us fiber ,43. The us fiber ,45. The Populus tremula fibers and extracted cellulose are shown in Populus tremula fibers and extracted cellulose, respectively. The DTA curve of the untreated populus fibers  [−1) [−1) [−1) [−1) [−1) [−1) [−1 to 115 mg·g−1. This behavior could be explained by the decrease of the interaction between dye molecules and active sites present in cellulose surface revealing a phenomenon of desorption. mg·g−1) , jute pr−1) [−1) , plam tr−1) [−1) , and ric−1) [−1) . It is c−1) [−1) , mango s−1) [−1) , and rej−1) [−1) . The ads2 values (R2 ≥ 0.99) and the excellent coincidence of the calculated adsorption capacities (qth) with those determined experimentally (qexp) strongly suggested convenience of this equation. This suggests that the second order equation fitted well our kinetic data indicating a chemi-sorption process [t versus t1/2  suggesting that the sorption of methylene blue and crystal violet was multilayer. The favorability of the phenomenon could be, also, judged using the parameter “n” computed from Freundlich model. From the data, 1 < n suggests that the sorption of these dye molecules was favorable [The adsorption of methylene blue and crystal violet was analyzed using Langmuir, Freundlich, and Temkin . The fitavorable ,57.L) against 1/T  and crystal violet (154 mg·g−1). The high adsorbed amounts of dyes confirmed the efficiency of using the extracted cellulose to treat contaminated waters. The pseudo second order equation described well the kinetic data suggesting a chemi-sorption process. The Freundlich model fitted well the experimental data indicating that the adsorption of the cationic dyes was a multilayer one. The interaction between the extracted cellulose and cationic dyes is exothermic. The adsorption of methylene blue and crystal violet is non-spontaneous. Further investigations will be extended to assess the extracted cellulose for other purposes including composite material design and to chemically functionalize the resulting cellulose polymeric material with aminated reagents.To sum up the findings, cellulose was chemically extracted from


Acidobacteria, Bacteroidetes, and Actinobacteria. In contrast to these groups, Alphaproteobacteria and Gammaproteobacteria contained fewer carbohydrate-active enzymes and depended either on low-molecular-mass C sources or on mycophagy. This group, however, showed rich gene complements for N2 fixation and nitrate/nitrite reduction—key assimilatory and dissimilatory steps in the deadwood N cycle. We show that N2 fixers can obtain C independently from either plant biopolymers or fungal biomass. The succession of bacteria on decomposing deadwood reflects their ability to cope with the changing quality of C-containing compounds and increasing N content.Deadwood decomposition is responsible for a significant amount of carbon (C) turnover in natural forests. While fresh deadwood contains mainly plant compounds and is extremely low in nitrogen (N), fungal biomass and N content increase during decomposition. Here, we examined 18 genome-sequenced bacterial strains representing the dominant deadwood taxa to assess their adaptations to C and N utilization in deadwood. Diverse gene sets for the efficient decomposition of plant and fungal cell wall biopolymers were found in  The decomposition of deadwood in forests is a complex process during which plant polymers such as cellulose, hemicellulose, and lignin are exploited by members of the microbial community. Since the deadwood microbiome is dominated by fungi, their cell wall constituents represent another important resource, especially in the late phases of decomposition. Deadwood is estimated to contain 8% of the total global forest carbon (C) stock , followed by Picea abies (42.8%) and Abies alba (4.8%). The mean living tree volume is 690 m3 h−1, and the volume of coarse woody debris  ranges from 102 to 310 m3 h−1 with an average of 208 m3 h−1 . The core zone of the forest reserve (42 ha) has never been managed, and any human intervention ceased in 1838 when it was declared a reserve. This reserve thus represents a rare fragment of European temperate virgin forest with deadwood left to spontaneously decompose. The reserve is situated at 730–830 m above sea level and the bedrock is almost homogeneous and consists of finely to medium-grainy porphyritic and biotite granite. The annual average rainfall is 866 mm, and the annual average temperature is 6.2°C  of deadwood and the span of the diameter range. Trees decomposing as standing snags were omitted to exclude CWD with unclear decay lengths. The CWD decay length ranged from <5 to >38 years. In October 2013, four samples were obtained by drilling with an electrical drill vertically covering sapwood and heartwood along the whole decomposing stem. Part of the material was used for the description of fungal and bacterial communities and wood chemistry  and WYA4 medium . Occurrences of bacterial colonies were recorded on Petri dishes incubated at 24°C for 8 weeks and marked with a marker indicating the week of the first appearance. Colony PCR was used to infer the taxonomy of the selected strains that appeared during the course of incubation. PCR premix and cycling conditions were as follows: 2.5 μL of 10× buffer for DyNAzyme DNA Polymerase; 0.75 μL of DyNAzyme II DNA polymerase (2 u μL−1); 0.75 μL of BSA (20 mg mL−1); 0.5 μL of PCR Nucleotide Mix (10 mM); 1 μL of primer eub530f ; 1 μL of primer eub1100br . Plates were incubated at 24°C for 2 weeks. After incubation, 12 mL of 1% Congo Red was poured onto each plate, staining was performed for 30 min, the Congo Red was replaced with 12 mL of 1 M NaCl, the NaCl was left for 30 min, the NaCl was replaced with 12 mL of H2O, and the H2O was left overnight. The presence of a halo around the colonies indicated CMC utilization.The activity of the cell wall-associated fraction of enzymes was measured in cells after 2 weeks of incubation in triplicate in 50 mL of liquid GY-VL55 medium as described previously . Sequences were aligned by the MAFFT online tool .Dereplicated 16S rRNA genes of strains with sequenced genomes were merged with representative OTU sequences , followed by the genera Mucilaginibacter (7%) and Pseudomonas , class Polyangia (n = 1), Planctomycetes (n = 2), Verrucomicrobia (n = 5), and WPS-2 (n = 1)–either were not isolated or failed to grow in subsequent cultures. Except for the strains of Bradyrhizobium (1.5%), each genome represented on average <1% of bacteria across all CWD samples. Up to 26.7% of bacteria were represented in the best-covered CWD samples , Mucilaginibacter strain 454, and Sphingomonas strain 22. The Granulicella strain showed the most CAZymes (n = 187 in 72 CAZy families), representing 3.7% of its total genes. In contrast, six members of the genera Variovorax, Pseudomonas, and Bradyrhizobium contained only 62–75 CAZymes in 28–35 CAZy families, representing 1% of their total genes . Mucilaginibacter strain 454 contained three genes for enzymes involved in chitin degradation, and Luteibacter strain 328 contained two chitinases. Both Pseudomonas strains encoded a single chitinase gene, and genes encoding enzymes targeting chitin were present in both Variovorax strains, Caballeronia strain 276 and Sphingomonas strain 22. An increased number of hemicellulases were present in two Acidobacteria strains, Sphingomonas strain 22, Mucilaginibacter strain 454, and Luteibacter strain 328. CAZymes for the labile substrates cellobiose and xylobiose and α-glucanases were present throughout the isolated strains without evident differences. CAZymes for other labile substrates, pectinases, β-glucanases, and glycoconjugate-degrading enzymes, were preferentially found in high-CAZy-containing Acidobacteria strains, Mucilaginibacter strain 454, Glaciihabitans strain 435, and Sphingomonas strain 22. Two Pseudomonas strains contained lytic polysaccharide monooxygenase genes from CAZy family AA10 for the oxidation of a broad range of polysaccharides. Some of the low-CAZy genomes contained genes for C uptake from alternative sources: the mxaF gene for methanol dehydrogenase, which oxidizes methanol, was present in Variovorax and Caballeronia , and four copies of this gene were present in Bradyrhizobium strain 411. Mesorhizobium together with Caballeronia contained genes for soluble diiron monooxygenases.Functional analysis of the identified CAZymes showed the specific C sources targeted by the strains . Genes fLuteibacter strain 328 showed by far the most active cell wall-associated enzymatic fraction. Except for Luteibacter, chitinase activity was highest in Bradyrhizobium strains, Variovorax strains, and Pseudomonas strains. Strains of Pseudomonas, Variovorax, and Luteibacter were not able to grow on CMC medium but displayed cellobiohydrolase and β-glucosidase activities suitable for the degradation of cellulose-derived oligosaccharides  contained genes for dissimilatory nitrite reduction (nrfC or nrfD), directing N flow into dissimilatory nitrate reduction to the ammonia pathway (DNRA). Similar to denitrification genes, nitrification genes, including those that allow the oxidation of ammonia, were missing in the bacterial genomes examined.Nitrogen cycling-related genes showed a higher occurrence in the genomes of bacteria . BradyrhF. sylvatica. Mapping showed that several strains were restricted to very fresh deadwood (<4 years of decomposition), while others tended to increase in abundance at later decomposition stages  but only inhabited certain CWD samples from the youngest age class. The abundance of other strains, including two Bradyrhizobium strains and Rhizobiales strain 265, peaked later during decomposition. Notably, the aforementioned Alphaproteobacteria represent bacteria with the highest abundance in the examined metagenomes . Therefore, we show the most probable functions of the CAZymes. Among cultivated bacteria, we identified strains with an increased potential for the utilization of complex carbohydrates, including cellulose, chitin, hemicelluloses, and pectin. These strains belong to the phyla Acidobacteria, Bacteroidetes, Actinobacteria, and the genus Sphingomonas from the Alphaproteobacteria. The strong hydrolytic capabilities of these taxa have been reported previously for some of the soil-inhabiting groups  as one of the abundant members in the deadwood community  also contained a single cellulase gene, which suggests that these strains might be independent in recalcitrant substrates utilization. Examination of other N cycle pathways shows that genes encoding dissimilatory steps are less common than those encoding assimilatory steps. Specifically, Sodalis strains  contain genes for DNRA, enabling the transformation of oxidized forms of N into ammonium. The balance between DNRA and denitrification is crucial for the soil N budget (Nelson et al., The ability to fix N2 fixation occur in fresh deadwood as well as in the following decomposition phases, showing the importance of this activity throughout decomposition. As the most readily available plant biopolymers are gradually depleted during decomposition, bacteria with genes for chitin degradation might switch to utilizing C from fungal biomass. Mycophagous bacteria such as Granulicella, Luteibacter, and Burkholderiales indeed tended to be more abundant in the later decomposition stages when fungal biomass in deadwood increases and starts to represent a relatively easily accessible source of both C and N.Bacterial community composition was shown to undergo a significant change between the early and late decomposition stages at a deadwood age of ~15 years (Tláskal et al., Acidobacteria and Bacteroidetes served as decomposers with a high number of expressed CAZymes, while Proteobacteria were recognized as opportunists relying on C uptake from other labile sources (Lladó et al., 2 fixation. Metabolically diverse bacteria thus show specific adaptations to challenging deadwood habitats and complement deadwood decomposition driven by fungi through N enrichment.Different C utilization strategies were described for soil habitats in which https://github.com/TlaskalV/Deadwood-bacterial-isolatesThe above methods indicate the programs used for analysis within the relevant sections. The code for reproducing the sequence processing is provided at Data described in this manuscript, including raw sequences from short read sequencing and genome assembly files have been deposited in NCBI under BioProject accession numbers summarized in the VT and PB conceived the study and wrote the manuscript. VT performed strain isolation, genome processing, and strain growth characterization. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bothrops atrox is a terrestrial pitviper that is distributed across the Amazon region, where it occupies different habitats. Using statistical analyses and functional assays that incorporate individual variation, we analyzed the individual venom variability in B. atrox snakes from four different habitats  in and around the Amazon River in Brazil. We observed venom differentiation between spatially distinct B. atrox individuals from the different habitats, with venom variation due to both common (high abundance) and rare (low abundance) proteins. Moreover, differences in the composition of the venoms resulted in individual variability in functionality and heterogeneity in the lethality to mammals and birds, particularly among the floodplain snakes. Taken together, the data obtained from individual venoms of B. atrox snakes, captured in different habitats from the Brazilian Amazon, support the hypothesis that the differential distribution of protein isoforms results in functional distinctiveness and the ability of snakes with different venoms to have variable toxic effects on different prey.Differences in snake venom composition occur across all taxonomic levels and it has been argued that this variation represents an adaptation that has evolved to facilitate the capture and digestion of prey and evasion of predators.  Snake venoms are complex mixtures of toxic components belonging to multipleprotein families , each ofBothrops atrox (common lancehead) is a terrestrial pitviper widely distributed across the Amazon, from tropical lowlands to the rainforest of northern South America east of the Andes  benzoic acid  and activity were determined according to the absorbance at 425 nm and expressed as Absorbance/min/μg of venom. For SVSP catalytic activity, the chromogenic synthetic substrate benzoyl-arginyl-p-nitroanilide (L-BAPNA)  was used and the hydrolysis of the substrate was expressed as the increase in Absorbance/min/μg of venom. The results represent the mean ± SD of three independent experiments, undertaken in triplicate.SVMP, SVSP, and PLA our lab . Briefly® system , as previously described [2 (0.02 M) and 260 μL of citrated plasma , pipette-mixed, and the clotting time was immediately recorded. PBS and ellagic acid were used as negative and positive controls, respectively. Under such conditions, recalcified chicken plasma treated with PBS only is unclottable for at least 30 min . The results are shown as Coagulant Dose 50% (DC50), defined as the amount of venom (μg) that reduces the CT parameter to 900 s.The procoagulant activity was evaluated in recalcified chicken (White leghorn) plasma, obtained under license CEUAIB n° 13,710-14, Instituto Butantan, by thromboelastometry using a four-channel ROTEMescribed . The pla2 chamber, the dorsal skin was removed, and the hemorrhagic halos were measured. Groups of 5 animals were tested, and control group animals were injected with PBS only or B. jararaca venom (10 µg). Results represent the mean ± SE of the area of hemorrhagic spots (cm2) from mice tested in at least 2 independent experiments. The myotoxic activity was assayed using 50 µg of venom pools in 30 µL of PBS, injected intramuscularly into the gastrocnemius muscle in Swiss mice. After 3 h, the animals were bled via ophthalmic plexus and the sera were assayed for creatine-kinase activity with a commercial kit CK-UV (Bioclin), according to the manufacturer’s instructions. Groups of 5 animals were tested, and animals from the control groups were injected with PBS only or B. jararacussu venom (50 µg). Results represent the mean ± SE of the CK activity in mice sera (U/mL) from mice tested in at least 2 independent experiments.Hemorrhagic and myotoxic activities were carried out as previously described  using ma2 chamber for the mice and injection of sodium pentobarbital overdose for the chicks. The data obtained were graphed on a survival curve plot. The tests were undertaken in two independent experiments.The lethality induced by the venoms was tested in murine (Swiss mice 18–20 g body weight) and avian (three days old Bovan chicks 20–30 g body weight) models with protocols approved by the Animal Ethical Use Committees of the Instituto Butantan (CEUAIB n° 13710-14). Groups of 6 mice or chicks were injected intraperitoneally with a single dose (200 μg) of venom samples, diluted in 500 μL of PBS. Following injection, the times of death for each group were recorded hourly until the sixth hour, and then at 12, 24, and 48 h after inoculation of the samples. After 48 h, the surviving animals were euthanized, using a CO2 were estimated using 10,000 permutations of the raw data. We then visualized the venom phenotype of each snake in multivariate space using robust principal components analysis (PCA) as implemented in the pcaCoDA function from the robCompositions R package [We tested for significant compositional differentiation using a hierarchical multivariate approach that is robust to the compositional nature of the data . First,  package .p-values for multiple tests.After detection of a significant global association between environment and venom composition, we conducted pairwise posthoc PERMANOVA, as described above, between the venom compositions of all pairs of environments to determine which environments differed significantly. Lastly, we preserved the full dimensionality of the data but removed the data from the simplex using the clr-transformation, to determine which peaks specifically varied across environments. To do this, we use the lm function from R stats, with the clr-transformed abundance of an individual peak as the response and environment of origin as the predictor. We repeated this test for all 26 peaks and used the false-discovery rate (fdr) approach to correct the p ≤ 0.05. Data were analyzed using the GraphPad Prism statistical program .For the functional analyses (in vitro and in vivo assays), firstly, the data were evaluated for a normal distribution (Shapiro–Wilk), and then differences among the means were evaluated by one-way ANOVA, followed by a Tukey post-test (for multiple comparisons). Data that did not meet the normality criteria were analyzed using a non-parametric test . Results represent the mean and standard deviation or standard error, as appropriate, and the level of significance was set at


An integrated theoretical/experimentalstrategy has been appliedto the study of environmental effects on the spectroscopic parametersof 4-(diphenylamino)phtalonitrile (DPAP), a fluorescent molecularrotor. The computational part starts from the development of an effectiveforce field for the first excited electronic state of DPAP and proceedsthrough molecular dynamics simulations in solvents of different polaritiestoward the evaluation of Stokes shifts by quantum mechanics/molecularmechanics (QM/MM) approaches. The trends of the computed results closelyparallel the available experimental results thus giving confidenceto the interpretation of new experimental studies of the photophysicsof DPAP in lipid bilayers. In this context, results show unambiguouslythat both flexible dihedral angles and global rotations are significantlyretarded in a cholesterol/DPPC lipid matrix with respect to the DOPCmatrix, thus confirming the sensitivity of DPAP to probe differentenvironments and, therefore, its applicability as a probe for detectingdifferent structures and levels of plasma membrane organization. Indeed, thecomposition and organization of biological membranes is one of themost relevant topics in molecular biophysics. The modern view identifiesa spatially interlaced combination of liquid ordered (Lo) and liquid disordered (Ld) phases, enriched, respectively,in saturated and unsaturated lipids, together with different amountsof cholesterol.12 This nanostructured dynamic assembly of Ld and Lo phases does not entail definite boundaries butis organized around the cytoskeletal network. Moreover, such a dynamicalmembrane organization was proposed to be relevant for most membraneprocesses, such as formation of protein clusters, signal transduction,endocytosis, and cell polarization and motility.15 In this context, it is not surprising that FMRs havebeen employed to detect the different phases of cell membranes19 and to probe the transition from the gel-like to the liquid-crystalphase or, in general, to gain information on the microviscosity ofthe phospholipid bilayers.20 In these studies,it was assumed that more viscous environments may slow down the FMRintramolecular motions,21 thus leadingto stronger intensities in the corresponding emission spectra andincreased fluorescence lifetimes. An effective relation between solventviscosity (η) and fluorescence quantum yield (ϕ) (or lifetime)is represented by the Forster–Hoffmann model22 , which has beenexperimentally proved to hold over a wide range of viscosity and polarityscales.24 It is worth noting that, in turn, lipidmembrane viscosity can influence crucial membrane-associated functions,including, for example, passive permeability of hydrophobic molecules,active solute transport, and protein–protein interactions.25Fluorescent molecular rotors (FMRs) are a class of chemical speciescapable of modulating their structural and optical properties in responseto changes in the viscosity and polarity of the local environment,a feature that makes them particularly suitable for sensing and imagingapplications.27 as well asfor investigating basic properties of lipid bilayer models, thus potentiallyuncovering the subtle interplay between membrane structural rearrangementsand lipid dynamics.29 Several types of FMRs have beenreported to date: some of them show a more pronounced dependency onthe dielectric properties of the surrounding medium, while othersare mainly affected by the molecular free volume of the solvent residues.32 An optimal combination of strong solvatochromism and viscosity sensitivitywas recently shown by the 4-(diphenylamino)phthalonitrile , in particular, is commonly adoptedfor an effective understanding at the atomic level of the dynamicsthat governs macromolecular functionso and Ld phases. To this end,two different phospholipidic systems have been considered, one consistingof pure 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) and another one of dipalmitoylphosphatidylcholine (DPPC) enrichedwith cholesterol  for differentelectronic states of the molecular probe and its validation throughmolecular dynamics simulations and spectroscopic calculations in differentenvironments. Noted is that the development of a proper molecularmodel for this investigation was strictly required since anomalousdye geometries can lead to artifacts in the spectroscopic calculations,and DPAP excitation energies were found to be rather sensitive toits intramolecular configuration.35 In this work,FF development was tailored toward the DPAP potential energy surfaceand corresponding gradient in the first excited state as evaluatedfrom quantum mechanical calculations at the time-dependent (TD) DFTlevel. The sampling of the first electronically excited state (EES)potential energy surface allowed us to effectively simulate excitedstate properties of DPAP embedded in several solvents  and lipid bilayers andto finally model fluorescence signals as issuing from hundreds ofMD trajectory snapshots at a reasonable computational cost. UsingMD simulations, it was shown that DPAP rotational dynamics is significantlyretarded in more structured lipid bilayers. The obtained results, coming from both computationaland experimental investigations, consistently support the use of DPAPas a probe for detecting different structures and levels of plasmamembrane organization.On these grounds, wecombined MD simulations and fluorescence lifetimeimaging microscopy (FLIM) to study the application of DPAP as a probein detecting local order within lipid bilayers representing simplemodels for both L22.1i, j run over atoms and μruns over the internal coordinates. Rtors and Ftors indicateimproper and flexible torsions, respectively. Deviations from bonds,angles, and rigid dihedral angles equilibrium values  are associated with energy penalties whichdepend on the corresponding force constants . Flexible dihedrals are describedby a sum of cosine functions, with kjμδ, njμ, and γjμ being the force constant, the multiplicity, and the phase factorof the jth cosine. Nonbonded interactions are modeledby using the standard Lennard–Jones and Coulomb potentials.Theclassical force field (intra- and intermolecular terms) usedin this work adopts the following energy expression:36K, L run over the normal coordinates, Ngeom is the number of sampled conformations; Ug is the energy differencebetween the energy of the gth conformation and theone computed on the global minimum(g = 0). GK is the energy gradient with respect to the normal coordinate K, while HKL is the Hessian matrix with respect to K and L. Both GK and HKL are evaluated at g = 0. The constants W, W′, and W″ weight the several termsat each geometry and can be chosen in order to drive the results dependingon the circumstances. The energy, gradient, and Hessian terms, calculatedon the obtained equilibrium geometry, are normalized in order to accountfor the different number of terms and to make the weights independentfrom the number of atoms in the molecule. The first term of Ngeom corresponds to the number of scanned geometriessubmitted to partial QM optimization. Such process is evaluated underthe Frozen Internal Rotation Approximation (FIRA), which assumes thatno relevant geometry rearrangements are experienced by the moleculeduring the scan, except for the scanned dihedral itself. Equilibriumvalues of 37 while Lennard–Jones parameters have beentaken from the OPLS-AA38 force field. Minimumgeometry has been located with the time dependent extension of thedensity functional theory (TD-DFT), using the CAM-B3LYP functionaland the SNSD basis set.40 Environment effectshave been included in the geometry optimization procedure by meansof the conductor-like polarizable continuum model (C-PCM),41 using the butanoic acid (dielectric constantϵ of 2.9931) as solvent in order to reproduce the specific lowdielectric medium of the phospholipidic membranes. Acetonitrile alsohas been considered in some cases (vide infra) toverify the influence of a more polar environment in the computationof chemical properties of interest. Relaxed potential energy surfacescan has been performed to accurately parametrize soft dihedral angles,modifying each torsional angle with not uniformly spaced steps . In particular, more points have been consideredclose to the minima. Vertical electronic transitions have been computedat the CAM-B3LYP/SNSD level using state-specific PCM approaches42 to model fluorescence properties. Computationshave been performed on 200 molecular configurations extracted fromthe classical MD trajectories. Single values have been then averagedto obtain the final fluorescence wavelength. All QM calculations wereperformed with the Gaussian suite of the program (G16).43Force field force constants are analytically computed through theminimization of the Joyce objective function2.2o-xylene, and lipid bilayers were performed using GROMACS 4.6.5.44 The OPLS-AA force field38 was chosen for modeling the o-xylene and acetonitrilesolvents. In the case of cyclohexane instead, the general amber forcefield (GAFF)45 was used because of itsbetter reproduction of the cyclohexane density experimental valuewith respect to OPLS-AA. The DOPC bilayer was hydrated with TIP3P47 water molecules and modeled by means of the CHARMM force field.48The classicalMD simulations for DPAP in acetonitrile, cyclohexane, 49 into a previously equilibrated lipid systemcontaining 200 DOPC lipids and solvated with 5791 water molecules.35 The lipid system was originally built up usingthe CHARMM-GUI membrane builder tool. We carried out an energy minimizationof the initial system configuration using the steepest descent algorithm,and then we performed an equilibration for a few nanoseconds in aNpT ensemble, before carrying out the NVT production run (see detailsbelow). The final rectangular box size was 8.27 nm, 8.27 nm, and 6.27nm.To simulate DPAP in a DOPC bilayer, aDPAP molecule was manually inserted, using VMD software,50 whichcontained 1200 lipids with CHOL in a molar concentration of 30%. Thesystem was coarse grained (CG) for 40 μs at 298 K and 1 atmusing the CG MARTINI force field for lipids.51 The 1200 lipids CG system was initially reduced to 600 lipids with23.7% molar concentration of cholesterol to perform all-atom MD simulation.This well equilibrated system was then further reduced in the currentstudy to perform all-atoms MD simulation with the CHARMM36 force field.52 The final system was comprised of 240 DPPC and75 cholesterol molecules, evenly distributed between the upper andlower layers, and 1 DPAP molecule was manually inserted into one layerusing VMD software.49 A 0.15 M NaCl saltconcentration was added, and the whole system was solvated with TIP3P46 water molecules, with approximately 38 watermolecules per lipid. For the DPAP/DPPC/CHOL system, the final boxedges were 9.28 nm, 9.15 nm, and 7.22 nm.An all-atom DPPC/CHOL bilayer was taken from a previouslywellequilibrated DPPC/CHOL system,–1. Equilibration runs were performedin the isothermal–isobaric ensemble: the systems were heatedto 300 K using the velocity rescale method53 and the Berendsen barostat54  for 500 ps with a time stepof 0.2 fs. Production runs were performed in the NVT ensemble: startingfrom the last configurations of the previous equilibration run, theintegration step was increased to 2 fs and the total simulation timewas set to about 130 ns for solvent systems and about 230 ns for thelipid bilayers. LINCS55 was introducedto fix the fastest degrees of freedom at their equilibrium values.In the case of cyclohexane and DPPC/CHOL lipid, only bonds with hydrogenatoms were kept rigid. Nonbonded interactions were truncated at 1.4nm. Long-range electrostatic interactions were modeled by means ofthe particle mesh Ewald (PME) technique56 with a spline interpolation of the order 4. System coordinates werestored every 500 steps.Note that initialconfigurations were minimized by using the steepestdescent algorithm implemented in GROMACS using an energy thresholdof 10 kJ molCp(t)) were calculated asP2 is thesecond order Legendre polynomial, and p is the vectordefined as the cross product of the ij and jk vectors . Finally, membrane thickness, area per lipid,and deuterium order parameters were calculated using the membraneanalysis tool MEMBPLUGIN.57Autocorrelation functions (2.3sn-glycero-3-phosphocholine) and DPPC (10mg/mL in chloroform) were purchased from Avanti Polar Lipids . Cholesterol and low gelling temperature agarose, BioReagent,for molecular biology, were purchased from Sigma-Aldrich . Liposomes of DOPC and DPPC/cholesterol 70:30 were prepared usingthe standard method.58 As an intermediatestep for liposome preparation, a thin film of lipid was obtained byevaporating 100 μL of chloroform solution containing 1 mg ofDOPC or DPPC/CHOL by placing the sample in a centrifugal evaporatorunder vacuum for 2 h. The lipid film was hydrated by adding 250 μLof PBS at pH 7.45 at room temperature (DOPC) and at 50 °C (DPPC/CHOL).The vesicles were frozen in liquid nitrogen and then thawed at 50°C in a water bath. The freeze–thaw cycle was repeatedfive times. To control liposome size, we performed extrusion usinga filter with 0.8 μm pore size. DPAP was solubilized in DMSOand added in liposome solutions. Agarose gel was used to immobilizeliposomes as described in ref  equipped with an external pulsed diode laser for excitation at405 and 470 nm and a TCSPC acquisition card connected to internal spectral detectors. Laser repetition rate wasset to 40 Hz. The image size was 256 × 256 pixels, and the scanspeed was usually set to 400 Hz (lines per second). The pinhole aperturewas set to 1.0 Airy. Samples were imaged using a 100× 1.5 NAoil immersion objective (Leica Microsystems). Emission was monitoredin the 480–525 nm and 540–580 nm ranges using the built-inacousto-optical beam splitter detection system of the confocal microscope.Acquisitions lasting until about 100–200 photons per pixelwere collected at a photon counting rate of 100–500 kHz.33.1ipso carbon atoms and the aminic nitrogen adoptinga nearly planar conformation.The optimized excited statestructure of the DPAP molecular rotorshown in 2adopts 60 Significant structural rearrangements are observed for the chemicalbonds involving the cyano groups. From the analysis of the DPAP HOMOand LUMO  index63 for both ground and first-excited states. Theextent of the electronic rearrangement is defined as the distancebetween the two centers of the density increment and depletion regions,respectively, upon electronic excitation  isdepicted in Figure S2 in the SupportingInformation). In cyclohexane, the computed CT length is 2.173 Åand the charge is 0.62 e.As a preliminary investigation,we performed a comparison betweenthe internal coordinates which significantly change when going fromthe GS to the EES structure see 1. This ke. Such small differences in the atomic charges slightlyaffect the dipole moment value, going from 21.19 D in butanoic acidto 23.14 D in acetonitrile. Overall, the observed insensitivity ofthe estimated atomic charges values to the surrounding medium allowsfor the employment of the same set of charges  for all the investigated environmentsin the following MD simulations.As mentioned in Table S1 of theSupporting Information. The choice of acetonitrile for this analysisallows for an effective comparison with the GS force field, whichwas developed by considering this environment during the previousparametrization.35 The main differenceshave been observed for the cyano nitrogen atoms . In thiscase, the lower atomic charge values confirm a more pronounced electronicdensity in the EES, as already indicated by the interpretation ofthe geometric parameter alteration. The high degree of intramolecularcharge transfer is reflected also by the important difference betweenthe GS and the EES dipole moments: in acetonitrile, the calculateddipole moment is 12.79 D for the GS and 23.14 D in the EES.A comparison between the GSand EES atomic charges computed inacetonitrile is shown in 1 and dihedral 3 (C2″C1″NC1′). Thelast two dihedral angles (2 and 3) are equivalent.The related DFT potential energy profiles have been used to derivethe torsional potential terms of the DPAP excited-state FF. The resultof the fitting procedure is shown in 1  rule the interconversionamong these conformers, corresponding to the planar and orthogonalgeometry of the considered ring with respect to the central aminemoiety. The situation is reversed for dihedral angles 2/3, with the orthogonal conformer being energeticallydisfavored. In such a case, the four energy minima are located atabout ±45° and ±135°. Moreover, in both the energypanels of Tables S2–S7).Dihedral angle 35 All the energy barriers are higher for the EES for both the investigatedtorsions and their relative height is exchanged: in the GS, the highestenergy barrier for dihedral 1 corresponds to the orthogonalconfiguration, whereas it is the planar conformation in the EES. Areversed situation is observed for dihedral 2/3. This remarkable trend is due to the electron delocalization whichinvolves the two unsubstituted rings in the EES, which leads, in turn,to an extra stabilization energy when dihedral 2/3 is nearly planar.As a further analysis, the potential energy profiles ofthe scannedflexible torsions can be compared with the corresponding ones obtainedfor the ground electronic state case.3.2o-xylene, and cyclohexane. The different solvation shells experiencedby the analyzed FMR in its first excited state have been describedby means of the radial distribution function (RDF) computed betweenthe dye and the solvent molecules center of mass (COM). The obtainedprofiles, shown in Figure S3, point outwell-defined first solvation shells for cyclohexane and o-xylene. In the former solvent, a first peak of height 1.75 is foundat 6 Å of COM distance, and a second peak with comparable height(1.25) at 9 Å. In the case of o-xylene, thefirst peak has a height of ≈1.5 at 5 Å; the second peakis not well resolved and it spans a large area from 8 to 12 Å.Only a few molecules of acetonitrile instead are able to closely approachthe solute, since the height of the RDF first peak is significantlylower if compared to the previous ones. On the contrary, the secondsolvation shell is easy to detect from the distribution profile beinglocated at approximately 8 Å with a height of 1.1.The FF developed in the previous section has beentested and validatedby means of MD simulations of DPAP in acetonitrile, Figure S4 for the case of acetonitrile, eachdihedral angle selectively populates the four corresponding energyminima. High energy conformations are avoided, according to the DFTenergy profile depicted in Figure S5a shows that in acetonitrile (the less viscous solvent considered)1 ns is enough to observe oscillations of the considered dihedral from 120°to 60° and vice versa. On the other hand, even after 5 ns unequivocallarge amplitude oscillations were not yet detected in the more viscoussolvents cyclohexane and o-xylene , for which similar viscosities are reportedin the literature.65Thedistributions of the three flexible dihedral angles, evaluated inall the MD simulations, confirm the reliability of the first-excitedstate FF for the DPAP molecule. Indeed, as it is shown in Figure S6: the first rotation takesplace after almost 1 ns of simulation and the following at roughly1.4 and 2 ns. It is noteworthy that in the time interval (5 ns) consideredin this figure only small oscillations are allowed for dihedral 1, which is ruled by a low energy barrier of ≈3 kcal/mol.The first 5 ns of simulated time are, instead, not sufficient to overcomethe energy barrier of 7 kcal/mol ruling the dihedral angle 1. Finally, all the energy minima of dihedral 2/3  are populated.From a quantitative point of view, we further confirmed the observedtrend among the three considered solvents by computing along the entireMD trajectories the time autocorrelation function (ACF) of the rotationof both ring 1 torsional angle (τrotdih) and of the whole molecule (τrot). In the latter case, the axis perpendicular to the planedefined by the three ipso carbon atoms C1, C1′, and C1″ was consideredas the reference vector. The calculated quantities, collected in o-xylene.Focusing on the acetonitrile(ACN) case, where a higher flexibilityof the DPAP internal dynamics is allowed, we noted that the torsionalangles rotate simultaneously in order to decrease the steric hindrancebetween the aromatic rings, this meaning that the three torsions arehighly coupled. This phenomenon was already pointed out in the previouswork on DPAP ground state and can be better appreciated by lookingat Figure S3: in the first solvation shell o-xylenemolecules are closer to the solute center of mass than cyclohexanemolecules, thus obstructing to a larger extent the DPAP internal andglobal movements. Moreover, from inspection of rot (or τrotdih) and the experimental fluorescence lifetime τfl emerges, thus corroborating the fact that more viscous andhindering environments promote radiative processes, as a consequenceof the obstruction to the rotation of the dye subgroup adopting thetwisted intramolecular charge-transfer (TICT) state. This remarkablefinding , which relates an experimental quantity with a computational prediction,could lead to interesting implications, considering that FMRs areoften used within highly viscous media such as silica-based nanoparticles,in order to increase fluorescence lifetimes and to be fruitfully usedfor imaging applications.66This result is in linewith the different solvation shells sampledduring the MD simulations, already seen in 33 proving that the fluorescenceis highly solvatochromic since it shows a red-shift of up to 120 nmgoing from the less polar cyclohexane (having a ϵ of 2.016)to the most polar solvent acetonitrile. According to our procedure differs from the experiment by only 1 nm.In the low-polarity cyclohexane solvent the theoretical predictionof the DPAP emission wavelength (442 nm) is close to the correspondingexperimental quantity (431 nm). Considering the results of our previouswork (where an absorption peak at 325 nm was found), we can also estimatethe Stokes shift provided by the CAM-B3LYP/SNSD/PCM model, leadingto a value of 117 nm, in fair agreement with the experimental one(107 nm). In the case of Finally, the emissionwavelength computed for acetonitrile (thehighest polarity solvent) is 682 nm, which overestimates the experimentalvalue by more than 100 nm. Also the Stokes shift is overestimated,suggesting that the polarity effect in this case is amplified. However,beside that, it is worth noting that the polarity-sensitivity of DPAPis correctly described at the CAM-B3LYP/SNSD/PCM level, thus showingthat this model is able to reproduce the experimental trend. Thisstatement can be easy confirmed even if the related statistical errorsreported in o-xylene, thestructured surrounding solvation shells prevent DPAP from large amplitudemotions, which can lead to larger deviations of the computed fluorescenceenergy. This is graphically displayed by the distribution of the computedemission wavelengths for each liquid, as shown in The larger errors observed for ACNwith respect to the other solventscan be rationalized by noting that the corresponding RDF profile issomewhat less structured, as discussed in 1 is relatedto the corresponding emission wavelength. Higher values in the emissionenergy are associated with the considered torsional angle within theinterval 70–120° , as described in the d andLo membrane phase models, respectively. Once provided tothe liposome solutions, DPAP was readily embedded in the lipid membranes,owing to its highly hydrophobic character that makes it highly insolublein aqueous solution.33 As usual in FMRimaging applications, we focused specifically on the fluorescencelifetime of the dye, since other optical features of the recordedspectra, such as emission intensities and wavelengths, are stronglydependent on local concentration or less sensitive to the microviscosityof the environment. In particular, in order to probe the nature ofthe lipid phase through DPAP emission lifetime, we adopted the phasorapproach69 to confocal fluorescence lifetimeimaging microscopy (ph-FLIM) as a convenient means to spatially mapthe phase order in lipid bilayers. Besides, this is a propedeuticstep toward probing local order in living cells. The phasor analysisis represented in a polar 2D plot (phasor plot70) the cosine  and sine  Fourier transforms of the normalized emission decay collected ineach pixel i, j of an image. Formonoexponential decays, the phasor  lies on a semicircle  of radius 1/2 and center; for multiexponential decays, the phasor lies inside the semicircle.Experimentally, we applied confocal ph-FLIM to DPAP embedded on multilamellarvesicles characterized by homogeneous Ld  orLo phases . Notably, Ld or Lo phases were found to be characterized by well-distinguishablephasors localized in the phasor plot (fl = 6.75 ns), the phasor cloud of the more rigidLo phase mapped closer to the  point as compared tothe Ld phase (τfl = 1.93 ns). On the phasorplot, the combinations of distinguishable photophysical states, suchas those determined by Ld and Lo phases, followa vectorial addition rule, regardless of the number of exponentials.71 Therefore, we may hypothesize that interleaved Ld andLo phases, such as those expected in the plasma membraneof the cell, would fall along the segment that connects the two “reference”phasors. Accordingly, by assuming there is a correlation between lipidcomposition and structural order, ph-FLIM applied to DPAP could, inprinciple, help in determining the composition of the membrane withthe submicrometer resolution typical of confocal microscopy.The sensitivity of DPAP photophysicsto local viscosity, as observed in previous studies,sor plot 7. The di3.42 and a membrane thickness of 38.1 Å, whilefor DPAP/DPPC/CHOL the area per lipid (DPPC) was 60.0 Å2 and the membrane thickness was 40.0 Å. Once embedded withinthe bilayer in one of the leaflet, DPAP remained in the hydrophobicregion under the lipid headgroup surface, as shown by the densitydistributions displayed in Figure S8. InDPPC/CHOL, DPAP partitioned between the lipid phosphate groups andcholesterol . Besides, to also better assess the lateral distribution ofthe FMR within the membrane bilayers, we analyzed the radial distributionfunctions between DPAP and either the phosphate  orcholesterol hydroxyl groups , selecting only the lipidsbelonging to the upper leaflet where DPAP was embedded. As shown in Figure S9, DOPC and DPPC lipids appeared similarlystructured around DPAP with a peak centered at about 0.9 nm, whilethe cholesterol distribution featured a broader distribution peakedat 1.5 nm.The influence of lipid membrane viscosity on DPAPdynamics was further investigated by means of MD simulations. A DPAPmolecule was manually embedded into pre-equilibrated planar DOPC andDPPC/CHOL model bilayers, thus reproducing the experimental setup were evaluatedto provide some information about the lipid structural order withinthe bilayer. Results are reported in 74 and show the more orderedstructure of the latter with respect to the former, as due also tothe condensing effect of cholesterol. When considering lipids in contactwith DPAP, we noticed only slight deviations of the order parameterswith respect to both bulk lipids, within the statistical noise. Thesefindings highlight that DPAP does not significantly perturb the underlyinglipid bilayer structure, a desirable feature for a molecular probe.Furthermore, we analyzed the structural effect dueto the presence of DPAP within both the DOPC and DPPC/CHOL membranemodels by evaluating the deuterium order parameter of the lipids.In particular, in both systems, we considered either the full setof lipids  or only the lipids in proximity toDPAP (within a distance of 5 Å from the FMR). The order parametersfor each carbon atom of the two lipid alkyl chains  and of the whole molecule (τrot) in DOPC and DPPC/CHOLbilayer are reported. First, we observed that DPAP rotational dynamicsrecorded in the membrane systems was greatly retarded if comparedto the one measured in solution, owing to the enhanced viscosity ofthe lipid matrix environment. In DOPC bilayer, both relaxation timeswere about ∼10 ns (rot) were further retarded to about 44 ns. Interestingly, consideringDPAP fluorescence lifetimes  we also noticed a comparabletrend, thus confirming that, within a similar chemical environment, local viscosity effectively hinders rotationalmotions and, in turn, induces longer emission lifetimes. This is particularlyrelevant in view of microscopic techniques aiming at imaging subcellularcompartments through the fluorescence lifetime, a convenient concentration-independentoptical property.In t ∼10 ns 4. In theFurthermore, DPAP emission was evaluatedcomputationally in DOPCand DPPC/CHOL bilayers. The maximum emission peak wavelength of DPAPin DOPC and DPPC/CHOL were located at 496(±31) and 503(±38)nm, respectively. Therefore, the predicted emission wavelength wassimilar for the two phospholipidic bilayers, the small discrepancybeing ascribable to the different DPAP configurations sampled duringthe classical MD simulations. Moreover, the obtained emission valuesfall into the wavelength ranges considered for the evaluation of thefluorescence lifetime see 2.4, thus4o) and disordered (Ld) phasesin plasma membranes. For this purpose, we used a combined experimentaland computational approach, which can be easily extended to studycomplex phenomena occurring upon interaction between lipids and molecularprobes. In the first part, a molecular model of DPAP was developedand validated. A purposely tailored classical force field for modelingthe first electronic excited state of DPAP was obtained using TD-DFTcalculations as reference data. Three different solvents  were considered to assessthe force field through MD simulations of DPAP in solution while inits excited-state. We observed that the rotor internal and globaldynamics is affected by the viscosity of the environment, besidesother specific solute–solvent interactions. The correspondingfluorescence spectra, as computed by means of TD-DFT calculationson hundreds of uncorrelated MD snapshots, reproduced the solvatochromictrend fairly well and the Stokes shift of the emission signals.In this work, we analyzed the FMR DPAPas a molecular probe fordetecting ordered (Ld and Lo phases,respectively. Using confocal fluorescence lifetime imaging microscopy,we obtained a significantly different optical response providing aτfl of about 2 and 7 ns for DOPC and DPPC/CHOL, respectively.MD simulations of DPAP within the same membrane systems revealed thatboth internal and global rotations of the probe were significantlyretarded with respect to the tested solutions, particularly in theLo phase model. Notably, results showed a consistent correlationbetween the fluorescence lifetime and rotational dynamics within thelipid matrix systems under consideration. Therefore, our work highlights,once more, the sensitivity of DPAP toward its microenvironment andsuggests its use as a probe for the detection of lipid structuresas those characterizing plasma membrane organization.In the second part, DPAP was investigated when embedded withintwo different lipid bilayers, namely, pure DOPC and DPPC with cholesterol,as membrane models for the L


Charcot–Marie–Tooth disease (CMT) is the most frequent hereditary peripheral neuropathies. It is subdivided in two main groups, demyelinating (CMT1) and axonal (CMT2). CMT1 forms are the most frequent. The goal of this review is to present published data on 1—cellular and animal models having opened new potential therapeutic approaches. 2—exploration of these tracks, including clinical trials. The first conclusion is the great increase of publications on CMT1 subtypes since 2000. We discussed two points that should be considered in the therapeutic development toward a regulatory-approved therapy to be proposed to patients. The first point concerns long term safety if treatments will be a long-term process. The second point relates to the evaluation of treatment efficiency. Degradation of CMT clinical phenotype is not linear and progressive. Among hereditary peripheral neuropathies, the most frequent is Charcot–Marie–Tooth disease (CMT). CMT constitutes a clinically and genetically heterogeneous group of hereditary motor and sensory peripheral neuropathies. On the basis of electrophysiologic properties and histopathology, CMT has been divided into primary peripheral demyelinating (type 1) and primary peripheral axonal (type 2) neuropathies. The demyelinating neuropathies classified as CMT type 1 are characterized by severely reduced motor nerve conduction velocities (NCV) (less than 38 m/s) and segmental demyelination and remyelination with onion bulb formations on nerve. The axonal neuropathies, classified as CMT type 2, are characterized by normal or mildly reduced NCVs and chronic axonal degeneration and regeneration on nerve. Among the CMT1 group, there are X-linked autosomal dominant and autosomal recessive forms of CMT. Distribution of the different mutations has been reported.The typical presenting symptom is a weakness of the feet and ankles. The initial physical findings are depressed or absent tendon reflexes with a weakness of foot dorsiflexion at the ankle. The typical affected adult has a bilateral foot drop, symmetrical atrophy of muscles below the knee (stork leg appearance), pes cavus, atrophy of intrinsic hand muscles, especially the thenar muscles of the thumb, and absent tendon reflexes in both upper and lower extremities. The life span is not decreased. There are only a few epidemiologic studies on the prevalence of CMT disease. The most generally accepted is the study by Skre proposing a prevalence of one affected person in 2500 . Over 80From a recent study of the consortium published in 2015, the frequency of different CMT subtypes ranged from 62% of patients with a genetic diagnosis for the most frequent subtype (CMT1A), up to 0.1% for CMT1D . This reTherapeutic development in CMT1 relies heavily on the use of non-clinical animal models, cell culture models and more recently, patient-derived cell lines. This review will be divided into two chapters. We will describe first the reports on animal and cellular models that have been constructed and that have been recognized as valuable tools by both the research community and regulators. In a second part, we will describe preclinical and clinical trials performed in different CMT1 subtypes.Construction of pertinent animal models is a prerequisite stage in drug development. Several models of CMT type 1, as well as cellular models have been reported. We will describe the characteristics of models that have been used to develop the below mentioned therapies. Other models will be only briefly cited.PMP22 caused by duplication of a 1.6 Mb segment of chromosome 17 short arm encompassing this gene [PMP22 gene in the murine genome [CMT1A is caused by over expression of the gene his gene ,5. TherePMP22 gene, is surrounded by a large amount of DNA. Five lines of transgenic mice carrying increasing copies of the human PMP22 gene (one to seven) and expressing increasing levels of the transgene were obtained [At the same time, using YAC DNA, a French/English collaboration created a mouse model of a CMT1A by the overexpression of PMP22 human gene. The use of YAC DNA for making transgenic mice has the advantage that the gene of interest, in this case the obtained . From hiobtained . Anotherobtained . Thus, lMore recently, Baas et al. described the phenotype of a spontaneous deletion of the C22 line, and named it the C3 line, which is similar to the C61 line .X-linked Charcot–Marie–Tooth disease (CMTX) is an inherited disorder, presenting as peripheral neuropathy that affects males more severely than females . The aveGJB1 gene located on the proximal long arm of the X chromosome [CMTX1 is caused by mutations in the romosome . It encoromosome . Cx32 isromosome ,14,15. PThe first mouse model presenting with a neuropathy similar to that seen in patients is a null allele produced by Gjb1 invalidation. This line presents a peripheral neuropathy and abnormalities in myelin . This moGJB1 gene to create mouse transgenic lines. Five transgenic lines were generated and have been investigated [Therefore, a mouse expressing a human mutated Cx32 was generated. The mutations G12S and S26L, found in non-related CMTX families that affect trafficking of Cx32 (G12S) or connexon activity (S26L), have been introduced into a human BAC containing the stigated .Defects in connexon activity and genomic instability was observed cell lines of the five transgenic lines, suggesting that Cx32 has a role in mitosis. These lines showed locomotor impairment, with the severity of at least the S lines being correlated with the number of copies of the transgene inserted into the murine genome .The CMT1B subtype is caused by mutation in the P0 gene, coding for the major protein of myelin and involved in the folding and stability of myelin sheet . Some muMany different genetically modified P0 mice have been generated. P0 null mutant mice have been considered as a model of patients with DSS that are homozygous for functional null mutations. The heterozygous P0 null mutant mouse represents a late onset, milder neuropathy, and has been considered as a model for CMT1B patients carrying a loss of function mutation in one allele . The majWrabetz 2006 [Mpz gene, has been also generated by Saporta [A P0 R98C “knock-in” mouse model of CMT1B, where a mutation encoding R98C was targeted to the mouse  Saporta . Both heMore recently, Fratta 2019  developeDifferent types of gain of abnormal function produce the diverse neuropathy phenotypes associated with P0, rendering challenging the development of a unique future therapeutic strategy for CMT1B patients. However, it seems that many CMT1B causing mutations activate the UPR . PMP22 gene [CMT1E, which represents a rare subtype of CMT1, is caused by point mutations in the P22 gene ,25, whicP22 gene . AffectePMP22) gene. Thus, two parties described the molecular dissection of the PMP22 promoter to find potential biological upstream sequences to reduce gene transcription.As described above, CMT1A disease is caused by overexpression of peripheral myelin protein-22 , and placebo control. Daily administration of progesterone elevated the steady-state levels of nic rats . Taken tPMP22 expression [PMP22 promoter suggesting that AA may act on PMP22 expression by modulating cyclic AMP pool. It has been previously shown that PMP22 overexpression acts by a nonlinear mechanism, with a threshold level of 70–80%. Below this threshold, overexpression can be tolerated without induction of the pathological phenotype [It has been suggested that ascorbic acid  promotes myelination in axon/Schwann cells . C22 micpression . Furtherhenotype . Furtherhenotype . Becausehenotype . The CMTAt the same time, Burns et al. report a pediatric double blinded trial performed on a cohort of children with CMT1A treated either with a placebo or ascorbic acid at a dosage similar to adults. Placebo and treated children were evaluated using regression curves methods. The two curves presented different slopes, but p-value was at the limit of statistical significancy (0.06) .Two further recent publications suggest the interest of AA in CMT1A. One publication demonstrated that mice invalidated for the transporter regulating blood concentration of AA, SVCT1, presented a demyelinating phenotype similar to CMT . A seconMore recently, the polytherapy product (PXT3003) of Pharnext (France), a mix of three different repurposed molecules , was tested on Schwann cells as well as in the rat model of CMT1A . Their aRecently, curcumin appeared as a potential promising therapy for CMT, but its development is hindered by its unfavorable pharmacokinetics. Recently, cyclodextrin/cellulose nanocrystals of curcumin (Nano-Cur) have been developed to bypass this limitation. Therapeutic potential of Nano-Cur was investigated in vitro in Schwann cells (SCs) and in vivo in the transgenic CMT1A rat model . In vitrSeveral gene silencing therapy approaches are currently developed in CMT1A. As CMT1A is a gene dosage disease, therapies using antisense oligonucleotide (ASO) to lower PMP22 expression mRNA have been proposed. Ionis Pharmaceuticals  has published, in collaboration with US CMT patient association CMTA, promising results  which shPMP22 gene expression and the preservation of both myelin and axons. Interestingly, the same approach was effective in the partial rescue of demyelination even after the onset of disease. Finally, a Korean group  performeGjb1 gene, coding for a connexin (Cx32), is associated with X-linked Charcot–Marie–Tooth (CMTX1). The group of K. Kleopa reports the construction of a lentiviral vector carrying the GJB1 gene under the Schwann cell-specific myelin protein zero (Mpz) promoter. This construction was delivered into the mouse sciatic nerve by a single injection immediately distal to the sciatic notch. Enhanced green fluorescent protein (EGFP) reporter gene expression was quantified, and Cx32 expression was examined on a Cx32 knockout (KO) background [p = 0.00148) and secondary inflammation (p = 0.0178) at 6 months of age compared to mock-treated animals. It has been concluded that gene delivery using a lentiviral vector leads to efficient gene expression specifically in Schwann cells. Restoration of Cx32 expression ameliorates nerve pathology in a disease model and provides a promising approach for future treatments of CMTX1. However, this group reports that this construction can correct phenotypes in a mouse line but poorly or an absence of correction on two other mutations. In addition, the transition of this strategy from animal models to patients could be difficult [As reported, mutation in the ckground . They reifficult .As reported above, transgenic animals expressing a human mutated Gjb1 transgene present polyploidy and abnormal over-duplication of the centrosome and is associated with locomotor deficit. It has been shown that genomic instability and centrosome duplication is linked to CamKII activity . ExploraThe first publication of a potential therapeutic solution for CMT1B was to treat the disorder by oral administration of curcumin . They de+/− mice with Compound 31, a Nav1.8 inhibitor , has been shown to improve animal motor performance, which is associated with an improvement of the amplitude of the plantar compound muscle action potential [In demyelinating CMTs, the alteration of myelin sheath triggers impairment of axonal excitability that is accompanied by alterations in voltage dependent ion currents, leading to a decrease of the peripheral nerve conduction velocity. An ectopic expression of the sensory neuron specific VGSC isoform Nav1.8 has been reported on motor axons of mild and severe CMT1B mice model, respectively, heterozygously  or homozotential . Recentlotential . Thus, NMutations in the main structural proteins of myelin, PMP22, P0, and Connexin32 (Cx32), represent the vast majority of all CMT cases being, respectively, responsible for CMT1A, CMT1B, and CMTX1. Some mutant proteins display abnormal trafficking and do not reach the cell membrane but accumulate in the Endoplasmic Reticulum/Golgi apparatus and undergo endosomal and proteasomal degradation (for review see in ). The baNumerous studies have shown that enhancing ER quality control pathways and the folding/degradation of proteins may provide a feasible and efficient therapeutic strategy in conformational diseases. Genetic and pharmacological treatments boosting the UPR by inhibiting PP1c/PPP1R15A phosphatase complex have been used successfully in preclinical models of CMT1B. The small molecule IFB-088 , a PP1c/PPP1R15A phosphatase complex inhibitor developed by InFlectis BioScience (France), has been shown to ameliorate myelination and to largely prevent the motor, morphological, and molecular defects of S63del mice . A phase−/−), they showed a significant amelioration of demyelination and a reduction of macrophages in peripheral nerves of young P0het JHD−/− mice. Passive systemic transfer of antibodies (IgGs) into P0het JHD−/− mice restored antibody decoration and reverted/reduced macrophage elevation and impaired nerve histology, suggesting a role of endogenous antibodies in macrophage-mediated demyelination. The group of R. Martini identified low-grade inflammation as a substantial disease modifier in the pathogenesis of distinct CMT1 mouse models (for review see in ). While Previous studies in myelin-mutant mouse models of CMT have demonstrated that low-grade secondary inflammation implicating phagocytosing macrophages amplifies demyelination, Schwann cell dedifferentiation, and perturbation of axons. The cytokine colony stimulating factor-1 (CSF-1) acts as an important regulator of these macrophage-related disease mechanisms, as genetic and pharmacologic approaches to block the CSF-1/CSF-1R signaling result in a significant alleviation of pathological alterations in mutant peripheral nerves. In mouse models of CMT1A and CMTX1, as well as in human biopsies, CSF-1 is predominantly expressed by endoneurial fibroblasts, which are closely associated with macrophages, suggesting local stimulatory mechanisms. These results further corroborate the important role of secondary inflammation in mouse models of CMT1 and might identify specific targets for therapeutic approaches to modulate innate immune reactions.However, this approach raised the question of safety of long-term administration of anti-inflammatory molecules. We will comment this point in conclusion chapter.Muscle atrophy and thus weakness are the main consequences of anomalies in peripheral nerves. Follistatin is a secreted protein that promotes muscle growth and function by sequestering these ligands extracellularly. Acceleron constructed a locally acting, follistatin-based fusion protein, ACE-083, and evaluated the potential of this construction as a novel therapeutic agent for focal or asymmetric myopathies. Intramuscular administration of ACE-083 caused localized, dose-dependent hypertrophy of the injected muscle in wild-type mice and mouse models of Charcot–Marie–Tooth disease (CMT) with no evidence of systemic muscle effects or endocrine perturbation. Importantly, ACE-083 also increased the force of isometric contraction in situ by the injected tibialis anterior muscle in wild type mice and disease models and increased ankle dorsiflexion torque in CMT mice . These rThe company Sarepta Therapeutics , in collaboration with the Nationwide Children’s Hospital , is developing neurotrophin 3 (NT-3), a gene therapy candidate designed to treat CMT, with the lead indication being type 1A . They shIn patients with CMT1A, peripheral nerves display aberrant myelination during postnatal development, followed by slowly progressive demyelination and axonal loss during adult life. It has been shown that myelinating Schwann cells in a rat model of CMT1A exhibit a developmental defect that includes reduced transcription of genes required for myelin lipid biosynthesis. Consequently, lipid incorporation into myelin is reduced, leading to an overall distorted stoichiometry of myelin proteins and lipids with ultrastructural changes of the myelin sheath. Substitution of phosphatidylcholine and phosphatidylethanolamine in the diet is sufficient to overcome the myelination deficit of affected Schwann cells in vivo . This trThe first comment is that the number of publications greatly increased since 2000, revealing the growing interest of CMT to the researchers and clinicians. This is good news for patients that are waiting for a curative treatment.The second comment concerns safety. It is very likely that treatments will be a long-term process. Several preclinical studies, using animal models, demonstrate that treatment should not be stopped, as stopping the treatment results in phenotype degradation. Therefore, it would be interesting to test molecules for which we already have long-term safety data available. As CMT disease is a progressive neurodegenerative disorder that does not affect live span, long-term safety is a crucial problem.The third point relates to the evaluation of treatment efficiency. Degradation of CMT clinical phenotype is not linear but occurs by steps. This implies that natural history of the disorder was reported. This is actually the case for CMT1A subtype but natural history of other CMT1 subtypes need to be more documented. Studies on this point shall allow to propose a consensus process of evaluation, which is not currently the case, as different studies are using different scoring systems. CMTNS is the score that has been generally used. It includes a clinical score, CMTES, and NCV. However, it has been reported that NCV is not correlated to disease severity and is not an accurate measure to evaluate disease evolution. CMTES alone is probably better. In addition, other endpoints, such as measures of fat and fibers in muscle evaluated by MRI, have been proposed. Using a consensus process of evaluation will make comparison of different trials easier. Finally, consensus scores should be proposed for all CMT1 diseases or by subtypes. Answers to these questions will be useful for evaluation of future clinical trials.


Sustainable provision of chemicals and materials is undoubtedly a defining factor in guaranteeing economic, environmental, and social stability of future societies. Among the most sought-after chemical building blocks are volatile fatty acids (VFAs). VFAs such as acetic, propionic, and butyric acids have numerous industrial applications supporting from food and pharmaceuticals industries to wastewater treatment. The fact that VFAs can be produced synthetically from petrochemical derivatives and also through biological routes, for example, anaerobic digestion of organic mixed waste highlights their provision flexibility and sustainability. In this regard, this review presents a detailed overview of the applications associated with petrochemically and biologically generated VFAs, individually or in mixture, in industrial and laboratory scale, conventional and novel applications. Considering that the volume of waste generated all around the world is expected to increase continuously in the upcoming years due to the increasing population, it is crucial to find out feasible waste management routes not only to handle but also to valorize waste. Thanks to the adaptation capabilities of bioengineering, environmentally benign and sustainable production approaches are now in hand that provide us with the ability to convert large diversity of organic residuals and waste streams to value-added resources, returning nutrient to production and application cycle in a circular manner , , 4 produ3.3Nutrients, especially nitrogen and phosphorus in the municipal wastewater treatment plant discharge causes eutrophication in surface waters. VFAs are used as easily degradable and cost effective carbon source for biological nutrient removal processes (BNR) at wastewater treatment plants (WWTPs) and can be utilized efficiently for denitrification and bio-P Process  . ThroughThe most favored VFA carbon sources for EBPR process were acetic and propionic acid , while aThe production of enough VFAs on site to sustain the EBPR process is more cost effective than chemical flocculation process for the phosphate removal as concluded by 4Anaerobic digestion (AD) has rapidly developed in recent years ,224. Bes4.14+-N [Plants require 13 mineral nutrient elements for growth, including macronutrients  and micronutrients , which are all critical nutrients for plants to complete their life cycle. Conventionally, the anaerobic digestion effluents was subjected to mechanical solid–liquid separated and the solid part could be easily transported to markets or fields for reutilization as fertilizer, either directly or after composting . Many ba4+-N . The N c4+-N . 226 sug4+-N . At the 4+-N . Therefo4+-N .4.2Scirpus, Typha, Phragmites, Polygonum, Sagittariaare, Cyperus, and Thalia are competitive and tolerant to eutrophic habitats [Cyperus involucrateus Rottb. and Thalia geniculate L. are fast-growing ornamental plants with such capacity. High growth rate and biomass production of emergent plants reflects their potentially high ability to absorb and accumulate nutrients. Therefore, they have been used worldwide in various types of constructed wetlands for treating several types of wastewater [4-P (48%), NH4+-N (99%), and inorganic N (64%). The pollutant level in the anaerobic digester effluent was significantly reduced. A similar result was reported as constructed wetlands lowered the concentrations of COD (89%) and turbidity (99%), but inhibition of algal biomass growth was observed due to physico-chemical characteristics of the wastewater [Studies suggested that wetland plants can well grow up in certain kinds of anaerobic digestion effluent and reduced the pollutant levels. Several genera of wetland plant species such as habitats ,241. Cypstewater ,242. Repstewater ,244. 228stewater . High costewater ,247. Man4.32) to a solution containing soluble PO4-P (ortho-P) and ammonium, thereby adjusting the pH to 8.3–10 and inducing the precipitation of struvite, MgNH4PO4 · 6H2O [4+-N can be captured [3) reacts with H2SO4/H3PO4 to form ammonia salt that can be used as a fertilizer [Nutrient recovery from digested biodegradable waste as marketable products has become an important task for anaerobic digestion plants to meet both regulatory drivers and market demands . Several4 · 6H2O . Even thcaptured  and suitcaptured ,256. Rec5As presented in this review, regardless of the source of provision, the list of applications associated with VFAs as precursor chemicals and materials is extended. It is not beyond expectation that as the societies develop, industries grow and new markets pop up, new applications for VFAs will be defined. The boost in demands of VFAs should be met by petrochemical or biotechnological routes for VFAs (bio)synthesis. Although rather cheap, available at this time in history and main source of VFA production, fossil-resources with their finite amounts and geographical-concentration, fluctuating prices and environmental related issue are unlikely to have a role in future sustainable development of developed and developing countries. However, there is promising prospective in sight as there is a backup plan for production of VFAs that concerns environmentally benign biotechnological approaches using renewable resources for production of VFAs. Production of these bio-based VFAs from anaerobic digestion of organic-rich waste, residual and by-product streams have recently attracted great attention. Although there is a long way for bio-based VFAs to take over the main share of the VFAs market, these green VFAs not only create value from waste but also guarantees sustainable generation and provision of these precious chemical building blocks for generations to come .


In the last decade, robot-assisted trans-axillary thyroidectomy has spread rapidly and has been proven to be a safe and effective procedure. However, several case series have reported new complications that have led to criticism regarding this approach. This study analyzed the incidence of complications in a large cohort of European patients. We enrolled all patients who underwent robot-assisted trans-axillary thyroidectomy from 2012 to 2020 at the University Hospital of Pisa Department of Endocrine Surgery. We analyzed complications and divided them into 2 groups. Group A included conventional complications, such as transient or permanent recurrent laryngeal nerve palsy, transient or permanent hypocalcemia, hemorrhage, and tracheal injury. Group B included unconventional complications, such as brachial plexus palsy, track seeding, seroma, great vessels injury, and skin flap perforation. There were 31 postsurgical complications (5.7%). Group A included 25 complications (4.6%): transient and permanent recurrent laryngeal nerve palsy occurred in 7 patients (1.3%) and in 1 (0.2%), respectively; transient and permanent hypocalcemia occurred in 9 patients (1.7%) and in 1 (0.2%), respectively. Postoperative bleeding occurred in 6 patients (1.1%) and tracheal injury in 1 (0.2%). Group B included 6 complications (1.1%): 1 patient with brachial plexus injury (0.2%), 1 with track seeding (0.2%), and 4 with seroma (0.7%). Robotic trans-axillary thyroidectomy is a safe approach with a risk of postoperative complications comparable to the conventional technique. Almost all complications after a novel introduction are anecdotal. With an accurate patient selection, high-volume institutions with experienced surgeons can perform this technique safely. In the last 2 decades, endoscopic procedures for thyroid surgery with alternative access to the conventional Kocher incision have spread, leading to improvements in the fields of immediate postoperative pain and cosmetic results –3. All oThe da Vinci robotic system , which was approved by the United States Food and Drug Administration in 2006 .In 2007, Chung and colleagues introduced robot-assisted trans-axillary thyroidectomy (RATT) using the da Vinci robotic system , 9. This. However, data reported in the literature suggest a low incidence of these complications [. The incidences of classic postoperative complications with RATT, such as hypoparathyroidism, recurrent laryngeal nerve (RLN) palsy, and postoperative bleeding, are reported to be comparable to those of conventional technique [At the beginning, concerns were raised about new complications associated with the RATT procedure. These include traction injury of the brachial nervous plexus , perforaications . The incechnique .Robotic thyroidectomy has therefore proved to be a safe technique in both postoperative complications and oncologic radicality in the treatment of thyroid carcinomas , 17. HowA prospectively designed database was used for data collection and analysis. We enrolled all patients who underwent RATT from February 2012 to December 2020 at the University Hospital of Pisa Department of Endocrine Surgery. All procedures were performed with the da Vinci robotic system, both the SI and the XI versions, using a single axillary incision and 3 robotic arms. Data regarding patient demographics, extent of surgery, preoperative diagnosis, thyroid volume and diameter of nodules, operating time, postoperative hospital length of stay, and postoperative complications were recorded. Selection criteria for RATT were a sonographically estimated thyroid volume up to 30 mL and a maximal sonographically determined nodule diameter up 60 mm for benign nodules and up to 30 mm for malignant nodules. Exclusion criteria were previous neck surgery, locally advanced cancer, lymph node metastasis at diagnosis, distant metastasis at diagnosis, severe thyroiditis, and Graves’ disease. The choice of hemi-thyroidectomy or total thyroidectomy was in concordance with the American Thyroid Association (ATA) guidelines at the time.We analyzed complications and divided them into 2 groups. Group A included conventional complications, which are defined as complications that could occur after standard open thyroidectomy, such as transient or permanent RLN palsy, transient or permanent hypocalcemia, hemorrhage, or tracheal injury. Group B included nonconventional complications, which are defined as complications of novel introduction mainly related to the remote access or to the position of the patient, such as brachial plexus palsy, surgical track seeding, seroma, great vessels injury, or skin flap perforation.All patients underwent postoperative laryngoscopy to assess motility of the vocal cords. Permanent chordal paralysis was defined as such if it persisted more than 6 months after surgery. Hypoparathyroidism was diagnosed in patients who had a reduction of serum calcium below 8 mg/dL. It was defined permanent if it lasted more than 6 months after the operation and required supplemental calcium and vitamin D oral therapy. Bleeding was defined as a hemorrhage that could be treated by surgical revision or by conservative therapy. Tracheal perforation was diagnosed when a subcutaneous emphysema developed and was confirmed by instrumental imaging. Brachial plexus injuries were identified in patients who presented pain and/or movement deficit in the arm or shoulder ipsilateral to surgical access. Clinical deficits were confirmed electrophysiologically by abnormal nerve conduction (neurapraxia) .2 (range 18–25 kg/m2). Mean diameter of the nodules was 25 mm (range 5–60 mm), and mean thyroid volume was 18.4 mL (range 9–25 mL).Between February 2012 and December 2020, 541 patients underwent RATT at the University Hospital of Pisa Department of Endocrine Surgery, including 537 women (99.3%) and 4 men (0.7%). Mean age was 36.6 years (range 16–73 years), and mean body mass index (BMI) was 21.3 kg/mThe surgical extent included 203 total thyroidectomies (37.5%), 336 lobectomies 62.1%), and 2 isthmusectomies (0.4%). Mean operative time was 95 min for total thyroidectomies, 79 min for lobectomies (taking into considerations 33 lobectomies in which a frozen section was performed), and 60 min for isthmusectomies. Conversion to standard cervicotomy was required in only 1 procedure (0.2%) due to locally advanced cancer infiltrating the internal jugular vein that escaped preoperative diagnosis  Table . Group AGroup B included 6 complications (1.1%). A brachial plexus injury with hypofunctionality of a distal segment of a sensitive branch of ulnar nerve was documented in 1 patient (0.2%). Track seeding of a benign goiter was reported in 1 patient (0.2%), and the relapse was removed through an incision above the pectoralis major muscle under local anesthesia. Seroma occurred in 4 patients (0.7%) and was successfully treated by repeated needle aspiration at the outpatient clinic.In the last 2 decades, RATT has become a widespread approach to treat thyroid diseases but is mostly limited to the Asian countries . Indeed,Overall, higher BMI and different anthropometric characteristics, as well as larger size of nodules and goiters combined with the elevated inherent cost of robotic surgery and the need of training, hinder the diffusion of this approach in United States and Europe , 24. AltFor a safe adoption of a novel technique, surgeons should ensure acceptable outcomes and minimize surgical complications . TherefoThe overall incidence of postoperative complications in our case series was acceptable (5.7%). Most of the complications occurred in the conventional group (4.6%). The rates of transient and permanent RLN injury were 1.3% and 0.2%, respectively, which are comparable to those of conventional thyroidectomy . The robDuring the bilateral procedure, the contralateral RLN may be poorly visualized, especially at the beginning of the experience. Once the superior pedicle is dissected and the lobe is freed from the strap muscles, a gentle traction upward of the lobe and an accurate dissection enables surgeons to identify the contralateral RLN. Moreover, intraoperative nerve monitoring is available even for RATT, and this tool may guide surgeons in RLN identification or surgical strategy . Although very rare, bilateral RLN injury is the most serious complication of bilateral thyroid surgery, leading to airway obstruction and potentially requiring tracheostomy. Notably, this complication did not occur in our patients.We documented 9 patients (1.7%) with transient hypocalcemia that progressively resolved with oral calcium and vitamin D supplementation within 6 months, and permanent hypocalcemia in 1 patient (0.2%). Taking into consideration only bilateral procedures, these complications occurred respectively in 4.4% and 0.5% of cases. Again, we attribute these encouraging data to the 3D-magnified view and to the fine movements of the robotic system which allow a gentle dissection that avoids jeopardizing the parathyroid blood supply.Notwithstanding, some debate persists in literature regarding the rate of transient hypocalcemia. A meta-analysis by Jackson et al. reported that RATT and open thyroidectomy (OT) have a similar complication rate, except for transient hypocalcaemia, which shows a higher risk after RATT . HoweverPostoperative bleeding is a potentially life-threatening event in thyroid surgery due to the risk of airway obstruction and may require emergency surgical treatment. The incidence of postoperative hemorrhage ranges between 1.2 and 1.5% , 30 and Our data suggest that dissection and hemostasis performed by the robotic system are safe. We believe that the effectiveness of the harmonic scalpel helps in obtaining this result, thanks to the absolute immobility and right pressure on the branches produced by the robot and not influenced by the surgeon. Moreover, the working space created to access the thyroid lodge plays a protective role, allowing the blood to evacuate in the vast subcutaneous pocket in the pre-pectoral region, breaking down the risk of airway compression , 21.A serious and threatening complication after thyroid surgery is tracheal injury, which occurred in only 1 patient (0.2%) in our case series, an incidence comparable to that reported by Asian authors . The patRegarding nonconventional complications, we reported 1 traction injury of the ulnar nerve (0.2%) at the beginning of the experience. The patient presented with neurapraxia and underwent a neurologic examination and electromyography that documented a hypofunctionality of the sensory component of the ulnar nerve. After a couple of weeks of physiotherapy rehabilitation, the sensorial deficit almost resolved, although a small area of hyposensitivity of the fifth finger persists.The brachial plexus injury usually occurs during the first 20 to 40 min of surgery and is probably caused by extreme extension of the arm position or extreme flap retraction . A propeAnother unconventional complication is the track seeding along the surgical access. Iatrogenic implantation refers to the desquamation and dissemination of cells that develop into nodular recurrences. Some case reports have described this clinical condition for malignant and benign diseases –36. In oAlbeit rarely documented after conventional thyroidectomy, RATT harbors a higher potential to lead to postoperative seroma formation due to the wide subcutaneous dissection from the axilla to the neck region. Although its incidence is reported elsewhere to occur in almost 2% of patients , we docuOther complications related to the access are the lesions of great vessels. An eventual injury of the external jugular vein can be managed by means of ligation without adverse effects. However, an immediate repair is necessary if an injury occurs to the carotid artery or internal jugular vein (IJV) during the access to the thyroid lodge. More complex carotid injuries require conversion to open surgery to have an adequate operative field and therefore greater control over the injured vessel. Bleeding from the IJV is more easily controllable, unless located in the caudal portion of the neck. In the event of a small tear of the IJV or of a branch, bleeding can be managed by means of direct robot-assisted suture or by application of an endoscopic clip by the assistant surgeon at the bedside .Even chyle leakage can be considered an unconventional complication related to access, unless caused by a lymph nodes dissection (not performed with the robotic approach at our institution). However, all of these complications are anecdotal and never occurred in our case series.Lastly, another complication introduced with the advent of RATT is the skin flap perforation when surgeons create the axillary pocket. Because the skin over the clavicle is very thin and has less fat, a meticulous dissection is necessary with special care not to perforate the skin or cause a subdermal burn by electrocautery . Even thIn light of our findings, we consider RATT as a safe procedure with comparable outcomes to conventional thyroidectomy in properly selected patients, and our data are in agreement with other published reports. Although RATT harbors the potential risk of technique-related complications of novel introduction, these are very rare. Of course, surgeons must keep in mind the possibility that these complications may occur and how to prevent them.The main limitations of our study are the retrospective nature and the lack of a control group. Overall though, the outcomes that emerged despite these limitations demonstrate that the incidence of complications after RATT is very low even in a cohort of European patients, who are known to have a higher BMI, a larger thyroid volume, and a higher incidence of thyroiditis compared with Asian patients, all factors that are considered to increase the incidence of complications after robotic thyroid surgery . To our Moreover, recognizing RATT as a valid option to treat thyroid diseases with outcomes comparable to conventional surgery, we are able to satisfy patients who reject a cervical scar, improving the cosmetic outcome that in some cultures represents a very important issue.We want to underline that this is a case series of a high-volume institution in which RATT has been performed for several years and in which surgeons had gained experience in robotic and endocrine surgery. Indeed, surgeon’s experience plays a role of paramount importance in the management of a potential complication to ensure little or no long-term sequelae.In conclusion, robotic trans-axillary thyroidectomy is a safe approach with a risk of postoperative complications comparable to the conventional technique. Procedure-related complications occur with a very low incidence and are almost all anecdotal. Therefore, high-volume institutions with experienced surgeons who have mastered robotic and endocrine surgery can perform this technique safely with optimal results. To obtain these outcomes, a proper patient selection is mandatory.