Systemic lupus erythematosus (SLE) is a multifactorial chronic autoimmune disease, marked by the presence of autoantibodies to nuclear antigens belonging to different isotype classes. For several years, IgE antibodies have been incriminated in the development of allergic diseases and parasitic infections and different anti-IgE therapies have been developed to encounter the pathogenic role of IgE in these pathologies. Recently, multiple studies showed the presence of elevated total IgE levels and demonstrated a pathogenic role of autoreactive IgE in SLE. This review aims to summarize the findings incriminating IgE and autoreactive IgE in the pathophysiology of SLE, to describe their functional outcomes on their targeted cells as well as to discuss different IgE-related therapeutic modalities that emerged and that may be beneficial for SLE patient care. Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease that predominantly affects women of childbearing age. The etiology of SLE is multifactorial and includes environmental, genetic and hormonal factors. SLE evolves by flares that can be triggered by environmental stimuli such as sun exposure or viral infections . The disThese autoantibody and CIC-dependent pathways lead to the activation of several innate immune cell types. For instance, they trigger neutrophil and platelet activation to extrude DNA by NETosis (Neutrophil Extracellular Traps) and microparticles, respectively, increasing autoantigen bioavailability and CIC formation. Autoantibodies and CIC trigger the production by the monocyte/macrophage family of B cell activating factor of the TNF family (BAFF) that stimulates B cell maturation and thus, autoantibody production. They induce type I interferon (IFN) production by dendritic cells ) also leading to an amplification of their own productions, and they allow basophil recruitment to secondary lymphoid organs where these cells amplify plasmablast number and function ,6,7,8.This simplified pathophysiology scheme of SLE underlines the key role of autoantibodies and CIC in flares and tissue damages occurring during disease course. The isotypes of the CIC-embedded autoantibodies will determine the cellular targets based on the expression by the latter of Fc receptors and the functional outcomes of such pathogenic compound binding. As reviewed before , autoreaThe present review will focus on the last discovered isotype of immunoglobulin, the least represented, namely IgE. As it will be discussed, the ability of IgE to regulate (directly or indirectly) the function of a number of immune cells and its strong activating features on IgE receptor-bearing cells give an immunoregulatory role to this isotype in the SLE environment that identifies it as a very promising therapeutic target.2] tetrameric form of the high affinity receptor for IgE (FcεRI) highly expressed by mast cells and basophils  on the surface of plasmacytoid dendritic cells (pDC), type 2 conventional dendritic cells (cDC2), Langerhans cells, monocytes/macrophages and eosinophils [Autoreactive IgE in SLE patient serum have been described over four decades ago  as was tasophils . The Fcε motives ,17. The inophils ,18.IgE can bind to its lower affinity receptor FcεRII/CD23 as well, a C-type lectin, known to regulate IgE synthesis either positively  or negatively (membrane-bound on B cells) ,19. FcεRThe primary physiological role attributed to IgE is to confer protection against some parasite infections (either endo- or ecto-parasites) ,21 and s−/−Lyn [−/−FcγRIIb and −/− × YaaFcγRIIb [−/−Igh7). In all three cases, IgE deficiency led to prevent, dampen or delay, respectively, the development of lupus-like nephritis and its associated immune activity [−/− × Lyn−/−IL-4 mice, where the reduction in the global TH2 environment leads to a decrease in total IgE titers [In atopic individuals, total IgE levels tend to be increased . This feLyn−/− , FcγRIIb/− × Yaa ) were bractivity ,32. Thesactivity  and autoactivity . MoreoveE titers ,36, also−/−Lyn, −/−FcγRIIb and −/− × YaaFcγRIIb lupus-like mouse models as well as in a cohort of 42 SLE patients where their levels were associated with disease activity and especially with active lupus nephritis .Studies of mast cell contribution to SLE pathogenesis in human patients are limited and, to our knowledge, none have addressed their autoreactive IgE-mediated activation during the course of the disease. Mast cells accumulate in the tubulo-interstitial area of the renal cortex in SLE patients with lupus nephritis (LN) ,43. Rece2 or CXCL12 for instance). As previously mentioned, IgE is mandatory to get an optimal lupus-like disease development in various murine models and both autoreactive IgE and basophil activation are associated with disease activity in all tested mouse models and in SLE patients [−/−Lyn mouse model, IgE deficiency prevents this accumulation, further suggesting the direct link between autoreactive IgE and basophil activation and accumulation in lymph nodes in the SLE context , bullous pemphigoid, allergic asthma or atopic dermatitis ,63,64,65Preventing IgE binding to FcεRI would disable anti-dsDNA IgE-mediated activation of basophils and pDC as described above. Omalizumab is a humanized anti-human IgE monoclonal antibody that holds this feature. Approved by several drug administrations for use in asthma and chronic spontaneous urticaria, Omalizumab efficiently neutralizes circulating IgE, and then prevents their binding to FcεRI and lowers their circulating concentrations decreasing FcεRI expression on basophils, mast cells and dendritic cells ,67,68,69Ligelizumab is another anti-IgE humanized IgG1 monoclonal antibody developed more recently. Like Omalizumab, Ligelizumab binds to free IgE but with a higher affinity than Omalizumab (88-fold stronger affinity) and decreases more effectively IgE levels than Omalizumab ,74. LigeTargeting directly IgE synthesis to avoid long term repetitive injections of biotherapies (that may lead ultimately to resistance) could represent an interesting option in SLE (see above) .New strategies limiting IgE synthesis have also recently emerged targeting the membrane-bound form of IgE on B cells. Indeed, another monoclonal IgG1 antibody has been developed, Quilizumab, that targets the M1 prime (M1′) transmembrane domain of IgE which is not present in secreted IgE . The binhi cells [Another approach to disrupt the IgE-mediated amplification loop of SLE would be to directly target the effector cells for which activation depends on IgE, namely basophils and pDC. CSL362 is a humanized anti-CD123 monoclonal antibody that prevents the binding of IL-3 to its receptor, thus inhibiting IL-3-dependent signaling, and that induces antibody-dependent cell-mediated cytotoxicity (ADCC) of CD123hi cells . Despitehi cells . Subcutahi cells . Of notehi cells ,79.Since their discovery, autoreactive IgE in SLE showed their pathogenic effects that are explained by their ability to activate both basophils and pDC. Their prevalence in SLE patients, their ability to overstimulate already dysregulated pathways in SLE and their association with disease activity clearly identify them as promising therapeutic targets. Development of clinical trials targeting this pathogenic entity and/or FcεRI-bearing cells should lead to real advancement in management of disease flares and SLE patient care.


Protein misfolding induced by missense mutations is the source of hundreds of conformational diseases. The cell quality control may eliminate nascent misfolded proteins, such as enzymes, and a pathological loss-of-function may result from their early degradation. Since the proof of concept in the 2000s, the bioinspired pharmacological chaperone therapy became a relevant low-molecular-weight compound strategy against conformational diseases. The first-generation pharmacological chaperones were competitive inhibitors of mutant enzymes. Counterintuitively, in binding to the active site, these inhibitors stabilize the proper folding of the mutated protein and partially rescue its cellular function. The main limitation of the first-generation pharmacological chaperones lies in the balance between enzyme activity enhancement and inhibition. Recent research efforts were directed towards the development of promising second-generation pharmacological chaperones. These non-inhibitory ligands, targeting previously unknown binding pockets, limit the risk of adverse enzymatic inhibition. Their pharmacophore identification is however challenging and likely requires a massive screening-based approach. This review focuses on second-generation chaperones designed to restore the cellular activity of misfolded enzymes. It intends to highlight, for a selected set of rare inherited metabolic disorders, the strategies implemented to identify and develop these pharmacologically relevant small organic molecules as potential drug candidates. The tridimensional architecture of proteins, determined by their linear amino acid sequence, is essential to their native biological properties, trafficking and functions. Proper protein folding is thus a highly regulated process. For secretory or membrane-associated proteins, the folding process takes place in the endoplasmic reticulum (ER), while folding of cytosolic proteins takes place in cytosol. Yet, upon environmental and metabolic stress, or due to missense mutations, misfolded proteins may accumulate within cells leading to dysregulated cell phenotypes at the origin of hundreds of different protein conformational diseases .Protein folding, along with protein synthesis, disaggregation, trafficking and degradation, is monitored by the proteostasis (protein homeostasis) network . ProteosThe biological concept of molecular chaperoning may be applied to help misfolded mutant proteins that retain some level of functionality escaping the cell quality control mechanism. Despite of its intrinsic pharmacological limitations, in particular due to its lack of protein specificity, this concept has inspired several chemical approaches for protein chaperoning. Besides the use of proteostasis regulators, they represent promising low-molecular-weight compound strategies to treat conformational diseases.N-oxide, used to modify the solvent conditions. Minimizing the protein-water interface, chemical chaperones frequently do not directly bind to the misfolded protein but rather promote their proper folding in destabilizing their unfolded form . Ye. Yein via priori known and pharmacophore identification likely requires a massive, in silico and/or in vitro, screening-based approach. This issue may explain why, so far, only a few second-generation PCs have been developed. Beyond sapropterin, a phenylalanine hydroxylase endogenous ligand used since 2007 in the treatment of phenylketonuria , Web of Science (https://apps.webofknowledge.com) and Pubmed (https://www.ncbi.nlm.nih.gov/pubmed/). The chosen keywords were: “pharmacological chaperones” and “enzyme”. From the obtained references, we included those mentioning non-inhibitory or weak inhibitory pharmacological chaperones and we excluded those describing inhibitory pharmacological chaperones or pharmacological chaperones without explicit inhibition data.We implemented the following bibliographic methodology to survey the literature. Three bibliographic databases were used: Scifinder  consisting in the periodic infusion of recombinant enzymes. This approach is however notably expensive and inefficient for neurological symptoms. ERT is in addition susceptible to trigger an immune response against the administrated enzyme. Substrate reduction therapy (SRT) is also a validated strategy based on the inhibition of the enzyme catalyzing the biosynthesis of the accumulated metabolite. However, this approach, requiring the selective targeting of a specific transferase, has so far only shown clinical results for Gaucher and Niemann-Pick type C diseases . FinallyGBA1 gene, leading to the deficiency in the enzyme β-glucocerebrosidase , also called glucosylceramidase or acid β-glucosidase. The misfolded Gcase, unable to properly hydrolyze glucosylceramide (GlcCer) in macrophages of spleen, liver and bone marrow, leads to 3 phenotypes depending on GlcCer accumulation rate and age of patients. Type 1 GD (GD1), the most common (90–95%) and also the least severe manifestation, is characterized by variable physical symptoms such as splenomegaly, hepatomegaly and bone pain but also hematologic symptoms. Type 2 GD (GD2), the most severe form, is distinguished by acute neurological involvements at an early age leading to death. Type 3 GD (GD3), is a heterogeneous phenotype characterized by more or less severe neurological impairments, developed at a later age than type 2 and can be associated with some of type 1 symptoms. To date, only GD1 can be cured by ERT (imiglucerase) as a first-line treatment or by SRT with an oral treatment alternative (miglustat or eliglustat) pyrimidine-3-carboxamide (ML198 (or NCGC00188758), In 2007, a quantitative high throughput screening (qHTS) campaign was initiated by the NIH. Starting from the observation that iminosugar derivatives are non-specific and have relatively short half-lives in cells , SidransN-[2- (methyl-phenylamino)-2-oxoethyl]-benzamide amino]-2-oxoethoxy]-NCGC607, , a non-iIn vitro and in vivo ADME  studies were performed and showed good stability, oral absorption but also capacity to cross the blood-brain barrier pyrimidine-3-carboxamide, Another pyrazolopyrimidine, LTI-291 -N-methyl)-N-methyl-2-(pyridin-3-yl)quinazolin-4-amine, Quinazoline derivatives were identified as non-sugar inhibitory PCs of Gcase from several HTS ,61,62. TSeveral promising Gcase non-inhibitory PCs have been uncovered and investigated for GD1, GD2 but also Parkinson disease and further pharmaco-kinetic and -dynamic studies are needed to develop the most effective analogs as well as mechanistic studies in order to identify the allosteric binding sites involved. GLA encoding for the lysosomal α-galactosidase A . More than 800 different GLA mutations are recorded, including almost 590 missense/nonsense mutations (from HGMD). The glycosphingolipids burden caused by the enzyme malfunction induces several cell components dysfunctions, ultimately resulting in oxidative stress, inflammation and apoptosis pyrimidin-4(1H)-one as being able to increase the activity and stability of protein steady-state by promoting the tetrameric form [l-Phe blood concentration. Therefore, given the structural similarity with BH4, it is likely that these compounds act as competitive inhibitors with respect to BH4. Since the identified PCs are recognized or probable inhibitors, at present only the co-factor BH4 can be considered as a non-inhibitory PC.As the BH4 treatment cannot be extended to all mutations, there is a need to develop new PCs with wider applications. A screening using DSF  allowed the identification of 5,6-dimethyl-3-(4-methyl-2-pyridinyl)-2-thioxo-2,3-dihydrothieno[2,3-ric form . Additioric form , performCongenital erythropoietic porphyria , is an ultra-rare autosomal recessive disease affecting 1:1,000,000 persons. CEP is caused by dysfunctions of the uroporphyrinogen III synthase , an enzyme involved in the heme biosynthetic pathway ,111. TheUROS gene mutations which lead to unfolding, instability and rapid degradation of the mutated UROIIIS protein. Among these mutations (less than 50 according HGMD), the missense p.C73R is the most frequent one (about one-third of reported CEP cases) and the corresponding mutated proteins have an enzymatic stability <1% compared to the wild-type [In most cases, CEP is due to ild-type .in silico docking of 25,000 compounds on a human isoform of UROIIIS, an allosteric binding site was identified which was targeted by 18.4% of all tested compounds [in vitro and in vivo. In 2018, ciclopirox was designated by the US Food & Drug Administration, as well as by the European Medicines Agency and the European Commission in Europe as an orphan medicinal product for the treatment of the CEP.Recently Millet and coll. performed a full screening combining stability, functional, structural assays, and drug repurposing to identify new UROIIIS PCs. Starting from ompounds . No mutacblB type  is an inherited rare disease caused by mutations of the gene (MMAB) encoding for the enzyme ATP:cob(I)alamin adenosyltransferase . Patients affected by MMA cblB display, during infancy or early childhood, different features including neonatal ketoacidosis, lethargy, failure to thrive and encephalopathy. The estimated incidence of MMA is 1:50,000 to 1:100,000 people. The portion of MMA attributed to cblB type is ~13% [12).Methylmalonic aciduria  is ~13% . ATR is MMAB gene in cblB type patients, some expressed mutant proteins display reduced substrate and cofactor affinity (p.R191W), negligible activity and presumed instability in vivo , as well as destabilizing mutations that retain some residual activity. More than half of the reported mutations in MMAB cblB are missense mutations leading to destabilization and loss of activity of the protein [The therapeutic options, in the absence of cure, involves a protein-restricted diet and hydroxycobalamin (OHCbl) supplementation even if less than half of the patients show response to it . Among t protein ,117,118. protein .M for ATP and KD for cob(I)alamin similar to wild-type enzyme, but exhibits a 40% reduction in specific activity [N-(((4-chlorophenyl)-carbamothioyl)amino)-2-phenylacetamide, In 2013, Pérez and coll.  carried activity . CompounMolecular docking for compound V identified a most probable binding site located at the C-terminal end of each subunit of the trimeric ATR and adjacent to the binding site of cob(I)alamin but distant from the ATP binding site.Compound V was also evaluated on a cellular disease model using patient-derived transformed fibroblasts presenting the p.I96T mutation. A significant increase in ATR activity (5.4 fold) was observed after incubation with compound V (80 µM) for 72h. Noteworthy, when OHCbl (1 µg/mL) was co-administrated with compound V, the mutant ATR activity was further improved (~17 fold). This synergistic effect of both substances is supported by the proximity of the binding sites of these two molecules in the docked structure of compound V.in vivo.Compound V was also orally administrated to wild-type mice for 12 days and increase of ATR protein level in two disease-relevant organs (liver and brain) supported its stabilizing effect More recently, the effect of compound V on six ATR mutants, including the most common (p.R186W) was evaluated. In a cellular model, compound V increased the stability and the activity of all expressed ATR variants and a synergistic effect was also observed with OHCbl .H-pyrazol-3-yl)-benzene-1,3-diol) as a potential PC on the same mutants [In the same work, Pérez’s group also identified compound VI (4-(4-(4-fluorophenyl)-5-methyl-1 mutants  but the  mutants . NeverthN-glycosylation disorders. This metabolic disorder is caused by the mutation in PMM2 gene encoding for phosphomannomutase 2  [The congenital disorder of glycosylation due to phosphomannomutase deficiency  is the most common of 5.4.2.8) . CurrentPMM2 is a homodimeric enzyme that catalyzes the isomerization of mannose 6-phosphate into mannose 1-phosphate required for lipid-linked oligosaccharide synthesis. This enzyme requires activation by a sugar phosphate, either α-mannose-1,6-bisphoshate or α-glucose-1,6-bisphoshate .PMM2 gene causing PPM2-CGD have been reported so far and more than 85% of these mutations are missense (from HGMD). Certain PMM2 folding mutations, despite of producing unstable proteins, preserve some level of catalytic activity, suggesting that PMM2-CDG is a conformational disease [More than 110 mutations of  disease . Most pa disease .Based on the role of α-glucose-1,6-bisphoshate as a co-activator of PMM2 activity, Cubellis and coll. showed that this compound also stabilizes several pathological mutants ,124,125.To date, the only non-inhibitory PC described for PMM2-CDG was identified from a DSF-based HTS . This coTM) against FD demonstrated the full clinical potential of the so-called first-generation PCs.Twenty years after its formal conceptualization, PC strategy has rapidly evolved toward a promising small organic molecule-based therapeutic option for many conformational diseases. The marketing in 2018 of 1-deoxygalactonojirimycin (GalafoldHowever, such first-generation PCs are active site-specific chaperones, i.e., competitive inhibitors with respect to the substrate, being used at a sub-inhibitory concentration. The therapeutic implementation of such drugs requires a complex discontinuous regimen to rescue the enzyme cellular activity without provoking counterproductive inhibition. The emerging development of second-generation chaperones, defined as non-inhibitory PCs, is thus an extremely promising strategy.Among the 25 conformational diseases due to enzyme misfolding that were tentatively addressed with PC strategy, only eight gave rise to the report of non-inhibitory PCs. Like for the first-generation PCs, the most studied conformational diseases are LSDs and more particularly Gaucher and Fabry diseases. Yet, very encouraging results were also described for less common pathologies.The finding of small PC molecules devoid of any inhibition towards the target misfolded enzyme remains highly demanding but the promising advances of recent years indicate that this challenge can be met. Further achievements likely imply a deeper understanding of PC mechanisms of action, both at a molecular and a cellular level. Characterization of the allosteric binding sites and development of biological assays for the straightforward quantification of PC efficacy are key steps along this process. Their development would contribute to the easier development new second-generation PCs with strong therapeutic potential. Several successes in the discovery of new non-inhibitory PCs already exist. The repositioning of ciclopirox against CEP is an encouraging approach highlighting the therapeutic potential of these second-generation chaperones.


Juglans cinerea) is a species for which hybridization has only recently begun being explored. This North American hardwood tree is threatened due to Ophiognomonia clavigignenti-juglandacearum (Ocj), the causal fungus of butternut canker disease (BCD), first observed in 1967. Observational evidence in some wild J. cinerea populations indicates that naturalized hybrids of J. cinerea with Japanese walnut (Juglans ailantifolia) may be more tolerant to BCD than non-admixed J. cinerea, but this has not been formally tested in a controlled trial. We aimed to examine potential BCD tolerance within and between J. cinerea and J. cinerea × J. ailantifolia hybrids and to determine if there is a difference in canker growth between BCD fungal isolates. Five-year-old J. cinerea and hybrid trees were inoculated with two Ocj fungal isolates collected from natural infections found in two different sites in Indiana, United States, and a blank control (agar only). Measurements of both artificially induced and naturally occurring cankers were taken at 8-, 12-, 20-, 24-, and 32-month post-inoculation. Differences in canker presence/absence and size were observed by fungal isolate, which could help explain some of the differences in BCD severity seen between J. cinerea populations. Smaller and fewer cankers and greater genetic gains were seen in hybrid families, demonstrating that hybrids warrant further evaluation as a possible breeding tool for developing BCD-resistant J. cinerea trees.Hybridization is a potential tool for incorporating stress tolerance in plants, particularly to pests and diseases, in support of restoration and conservation efforts. Butternut ( Native and non-native diseases and pests are increasingly threatening ecosystems, especially forests, across the globe . This isCastanea dentata (Marsh.) Borkh.], which has been crossed with the Chinese chestnut (Castanea mollissima Blume) in pursuit of resistance to chestnut blight . These results in J. cinerea can be compared to hybrids and other diseases in Juglans. In the aforementioned study, hybrids of J. nigra with four other Juglans species consistently showed greater anthracnose tolerance than their highly susceptible J. nigra parent (Juglans regia L.) and iron walnut (Juglans sigllata Dode) showed similar or even greater susceptibility to walnut bacterial blight (Xanthomonas arboricola pv. juglandis Pierce) than both their progenitors (Agrobacterium tumefaciens Smith & Townsend) has also been documented in hybrids of northern California black walnut [Juglans hindsii (Jeps.) Jeps. ex R.E. Sm.] and J. regia  than the two native North American progenitors  and third (B3) backcross hybrid lines developed at TACF’s Meadowview Research Farm  were found to have average blight areas (B2) or blight ratings (B3), significantly different and intermediate to their American and Chinese chestnut progenitors, but not different from those of the F1 generation , there was no significant difference between any of the progenitors or hybrids in a third site (TN). Given such genotype × environment variation, it is essential that future work test J. cinerea and hybrid families in common garden plots across multiple sites in order to assess the durability of possible BCD resistance. Efforts have also been pursued to develop Dutch elm disease (Ophopstoma spp.) resistant hybrids for restoring the endangered American elm (Ulmus americana L.) and several other Ulmus spp. affected by the disease , both underscore the need for BCD tolerance screenings on multiple different sites to understand possible genotype × environment interactions. Ocj epidemiology. It is likely that the successful restoration of J. cinerea will not be accomplished solely through the integration of genetic BCD resistance, but in combination with appropriate site selection and silvicultural practices  and J. ailantifolia (more flood tolerant) progenitors. Phenotypically, J. ailantifolia progenitor, although some hybrids did occupy the same space as their J. cinerea progenitor. The concerns of dissimilar hybrid and J. cinerea phenotypes, along with the intermediate environmental tolerances of the hybrids, could limit their ability to act as a suitable replacement for J. cinerea, potentially changing the distribution of the species. However, in a phenotypical study of C. dentata hybrids and their progenitors, which are at a much more advanced breeding stage than J. cinerea hybrids, 96% of hybrid trees in the third backcross generation were distinctly different from their C. mollissima progenitor, and closely resembled C. dentata . This is promising for the development of resistance breeding programs using hybrids, but possibly J. cinerea as well. Hybridization in J. cinerea is one of just a few examples in plants where hybrids are being considered not only for preserving a species’ economic value (timber and nut production), but also for ecological (restoration and conservation) and cultural purposes . Thus, this study provides further evidence that hybrids represent a potentially effective tool for incorporating disease resistance to aid in restoration of threatened tree species.Differences in canker occurrence and size by The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AB analyzed the data, interpreted the results, and wrote the original manuscript. JM designed and executed the experiment, contributed to interpretation of the results, and revised the manuscript. SH and DJ contributed to interpretation of the results and revised the manuscript. 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.


Previous studies have confirmed the feasibility of the cortical bone trajectory (CBT) technique. However, there are few reports on spinous process violation and screw penetration during the screw insertion. The purpose of this study was to evaluate the incidence of spinous process violation and screw penetration through the pedicle during CBT screw insertion.Computed tomography (CT) scans with normal lumbar structures were consecutively obtained and three-dimensional (3D) reconstructions of the lumbar spine were created. Bilateral CBT screw placement was simulated on each segment using a screw diameter of 4.5 mm, 5.0 mm, or 5.5 mm. Incidences of these complications were recorded and analyzed.A total of 90 patients were enrolled. Spinous process violation was observed in 68.3, 53.3, 25.5, 1.7, and 0% from L1 to L5, respectively, using 4.5 mm screws. A significant difference was found among the five segments but this was unconnected to gender or screw diameter. The incidence of screw penetration through the inner wall decreased from L1 to L4; in turn, L1 (16.7–35.5%), L2 (12.7–34.4%), L3 (2.8–23.8%) and L4 (1.1–6.7%). This trend was reversed in L5 (6.7–16.7%). Moreover, screw penetration through the outer wall was rare. The incidence of screw penetration varied with screw size as well as lumbar level, but not with gender.There are more difficulties of CBT screw fixation in upper lumbar spine. The low rate of screw penetration, using 4.5 mm screws, suggests the safety for CBT fixation in the lumbar spine. Larger screws (5.0 mm or 5.5 mm) are more recommended for use in the lower lumbar spine. Moreover, CBT fixation in L5 deserves greater attention because of the unique morphology of the pedicle. The pedicle screw fixation technique is widely used as an effective surgical method for spinal segmental fixation. Traditional pedicle screw fixation has been considered to be the optimal method for it has high level of stability. However, this technique is invasive and requires significant soft tissue dissection. Moreover, it should be noted that screw loosening or dislocation is a common problem in traditional trajectory surgery in elderly patients with severe osteoporosis –3. To ovHowever, the CBT technique has some limitations. In real surgeries, the posterior structures (mainly the spinous processes) are often considered to be a significant obstruction to screw placement in this trajectory, especially in the upper lumbar spine. Thus, partial resection of the spinous processes and supraspinous ligament is often required, which may cause damage to the posterior ligamentous complex participating in spinal stability. In addition, due to the special trajectory of the CBT screw, there remains a risk of screw penetration through the pedicle in patients with small pedicles, which can result in nerve injuries and unstable fixation. None of these situations should be overlooked during the operation.Presently, although morphometric and biochemical studies have confirmed the feasibility of the CBT technique, there remains a lack of related reports on spinous process violation and screw penetration during the screw insertion process. Therefore, the purpose of this study was to evaluate the risk of the above complications in CBT screw fixation and provide a reference for the clinical application of this technique using three-dimensional (3D) screw insertion simulation software.All lumbar spinal CT scans (containing L1–5) for either trauma evaluation or for preoperative surgical planning at our institution were retrospectively reviewed between January 2017 and June 2018. Patients with a lumbar fracture, compression of the vertebral body, ankylosing spondylitis, deformity, lumbar tumor or infection, or history of spinal surgery, were excluded. Patients with age of < 40 were excluded.All CT scans were performed on a 16-slice CT scanner . Scan parameters included 120 kV, 200 mA, a 512 × 512 matrix, a layer thickness of 1 mm, and a pitch of 1 mm.CT images of the lumbar spine were manipulated using Mimics software  and underwent 3D reconstruction. After the removal of unnecessary anatomic structures, normal 3D lumbar models were obtained  was used for all statistical analyses. The chi-squared test or Fisher’s exact test was utilized to compare the incidence of the above complications by gender, among the three screw diameters, and among the five levels. A According to the selection criteria, 90 patients  were enrolled in this study. All CT scans underwent 3D reconstruction and showed successful screw placement. Given the lack of any statistical difference between the two sides, one screw insertion was regarded as a separate study object. Thus, 900 screw insertions were contained in this simulation (180 screws in each segment).p < 0.001); however, this appeared to be unconnected to gender (p > 0.05) or screw diameter (p > 0.05).Table p < 0.001). Moreover, different screw sizes also resulted in an apparent difference in the incidence on L1-L5 (p < 0.05). In addition, screw penetration through the outer wall was rare compared to that through the inner wall, and did not tend to occur in the lower lumbar spine. The incidence of screw penetration through the outer wall also varied with screw size and lumbar level. Notably, there was no significant difference in screw penetration by gender.In general, the incidence of screw penetration through the pedicle was not high and differed between the inner and outer walls. The incidence of screw penetration through the inner wall with varying screw sizes  showed a downward trend from L1 to L4; in turn, L1 (16.7–35.5%), L2 (12.7–34.4%), L3 (2.8–23.8%) and L4 (1.1–6.7%). However, the incidence in L5 (6.7–16.7%) reversed this downward trend. As shown in Table The CBT screw fixation technique, as a modified fixation method for traditional pedicle screw fixation, has attracted increasing attention and has already been applied in clinical surgery , 8. To aGiven the lack of sufficient clinical data regarding these two complications, computer simulation software can be of great assistance in evaluating their incidence. Such software enables trajectories to be adjusted without breaking the specimen, and can test a large number of patients in visible models. Through 3D reconstruction of the lumbar spine and simulation of CBT screw placement, evaluation of the incidence of the two complications in each segment can be undertaken. Additionally, simulated placements are conducive to determining the entry point, insertion angle, and appropriate screw dimensions prior to a real operation, and help reduce the incidence of intraoperative complications.In real CBT surgery, the unique trajectory of pedicle screw fixation often causes spinous process violation, where the posterior ligamentous complex can be a significant barrier to screw placement. To solve this problem, partial resection of the spinous process and supraspinous ligament is usually required before screw insertion, especially in upper lumbar surgery. Cheng et al. confirmed this in a cadaveric study . They deScrew penetration through the pedicle is among the common complications of pedicle screw fixation and carries the risk of neurovascular injury and pedicle fracture . ThroughNotably, the incidence of screw penetration through the L5 pedicle inner wall was higher than in L4, which was not consistent with the changes in lumbar pedicle size. The reasons for this may be as follows. First, the distinctive morphology of the L5 pedicle plays an essential role. The greater pedicle axial angle and deeper lateral recess compared to other levels increase the likelihood of screw penetration in L5 fixation . LikewisIt is generally accepted that screw purchase is positively related to screw diameter and length , 19. A cThis study had some limitations. First, it was based on a computer simulation which is not as realistic as a cadaveric study. Second, there was no consideration of the size of the screw head and insertion tools, which inevitably led to an underestimation of the rate of spinous process violation. Third, the subjectivity of the operator may have a bias on the results. We were able to adjust the entry points and insertion angles several times before determining the correct trajectory for each case; this is not possible during an actual operation. Fourth, the relatively small sample size could be problematic in terms of statistical analysis. Finally, race, age, height, and weight were not considered in this study because of the restrictions of sample. In spite of these deficiencies, our study indicated the feasibility and safety of CBT screw fixation, especially in the lower lumbar spine.In conclusion, due to the high incidence of spinous process violation, there are more difficulties of CBT fixation in upper lumbar spine and risk of posterior element damage is higher. In addition, the low rate of screw penetration through the pedicle, with a screw diameter of 4.5 mm, suggests the safety of this method when carried out with small screws. Larger screws (5 mm or 5.5 mm) are more suitable for use in the lower lumbar spine under certain conditions, to provide stronger screw purchase. Moreover, CBT fixation in L5 deserves greater attention due to the unique morphology of the pedicle and its trajectory. In short, preoperative measurement and evaluation are of great importance in the choice of surgical trajectory or screw size before a real operation.


MyBP-C is phosphorylated at up to four sites. In donor heart the 4P and 3P species predominate but in the pressure overload samples the 4P species was much reduced and 3P and 1P species predominated. Total phosphorylation was 2.0 ± 0.2 molsPi/mol MyBP-C (n = 8) compared with 3.4 ± 0.07 (n = 21) in donor heart and 1.1 ± 0.1 (n = 10) in HOCM heart. We conclude that pressure overload may be associated with substantial dephosphorylation of troponin I and MyBP-C.In previous studies of septal heart muscle from HCM patients with hypertrophic obstructive cardiomyopathy  we found that the level of phosphorylation of troponin I (TnI) and myosin binding protein C (MyBP-C) was extremely low yet samples from hearts with HCM or DCM mutations that did not have pressure overload were similar to donor heart controls. We therefore investigated heart muscle samples taken from patients undergoing valve replacement for aortic stenosis, since they have pressure overload that is unrelated to inherited cardiomyopathy. Thirteen muscle samples from septum and from free wall were analyzed (LVOT gradients 30–100 mmHg) The levels of TnI and MyBP-C phosphorylation were determined in muscle myofibrils by separating phosphospecies using phosphate affinity SDS-PAGE and detecting with TnI and MyBP-C specific antibodies. TnI was predominantly monophosphorylated and total phosphorylation was 0.85 ± 0.03 molsPi/mol TnI. This phosphorylation level was significantly different ( Cardiac muscle contractility is modulated by the β-adrenergic system that primarily acts via activation of protein kinase A (PKA). In the cardiac muscle sarcomere the main targets of PKA are myosin binding protein C (MyBP-C) and Troponin I (TnI) and phosphorylation of these proteins plays a vital role in the enhanced contraction and relaxation kinetics induced by adrenergic stimulation . In non-In heart disease the phosphorylation level is often low. For instance in idiopathic (non-ischaemic) end stage heart failure explants, phosphorylation levels are 0.26 molsPi/mol TnI and 0.62 molsPi/mol MyBP-C . In geneThe HCM-associated mutations often cause a hypertrophied interventricular septum that can lead to left ventricular outflow tract obstruction (LVOTO). The septal myectomy operation for patients with HCM is usually indicated to reduce the obstruction when there is a high Aorta/LV pressure difference, typically 100 mmHg . We hypoDonor hearts and the K280N HCM sample were supplied by the Sydney Heart Bank . Donor sBiopsies were taken from septum and free wall of patients undergoing valve replacement surgery to relieve aortic stenosis at the Royal Brompton Hospital, London and Careggi University Hospital, Florence. The ACTC E99K patient sample was kindly supplied by Dr. Lorenzo Monserrat, La Coruña, Spain . AvailabPatients gave written consent with PIS approved by the relevant ethical committee. All samples are anonymised. The investigations conform to the principles of the Declaration of Helsinki.Ethical approval for collection and distribution of the human heart samples was granted by the Research Integrity, Human Research Ethics Committee, University of Sydney (Protocol No. 15401); the Joint UCL/UCLH Ethics Committee Rec No: 04/0035; Outer North East London Research Ethics Committee REC ref: 10/H0701/8; Careggi University Hospital Ref: 2006/0024713 and Comite Ético de Investigatio ń Clìnica de Galicia. Permission for study of the samples was granted by the NHS National Research Ethics Service, South West London REC3 (10/H0803/147).2PO4, 5 mmol/l Na2HPO4 pH 7.0, 0.1 mol/l NaCl, 5 mmol/l MgCl2, 0.5 mmol/l EGTA, 0.1% Triton X-100, 20 mmol/l NaF and 5 mmol/l DTT with 2 μg/ml each of the protease inhibitors E64, chymostatin, leupeptin and pepstatin A). The homogenate was then centrifuged at 16,500 × g for 5 min and the supernatant discarded. The wash process was repeated and then the pellet was dissolved in sample buffer containing 8 M urea, 2 M thiourea, 0.05 M Tris-HCl, pH 6.8, 75 mM DTT, 3% SDS and 0.05% bromophenol blue as decribed  and 50 μM Phos-TagTM acrylamide [from 5 mM stock solution of Phos-TagTM acylamide AAL-107 ] prepared according to suppliers instructions (TnI phosphorylation levels in heart muscle myofirils was measured by Phosphate affinity SDS-PAGE as described by ructions .Gels were probed with the phosphorylation-independent anti-human-cardiac troponin I (hcTnI) clone 14G5 mouse mAb (Abcam plc antibodies), 1/2,000 dilution on Western blots. Secondary antibody was HRP- conjugated anti-rabbit IgG  and the blots were visualized using ECL (GE Biosciences). Chemiluminescence was detected by a cooled CCD camera-based gel imager .To resolve MyBP-C phosphospecies the myofibril samples were run on the gels for 165 min. The current was initially 25 mA, raised to 35 mA once the samples had entered the resolving gel. The gels were Western blotted and probed with a rabbit polyclonal antibody against cMyBP-C residues 2–14 which recognizes total cMyBP-C or with phosphorylation site-specific antibodies .We studied 13 heart muscle biopsies from intraventricular septum and free wall taken from patients undergoing valve surgery to relieve pressure overload and compared them with previously studied donor heart samples and myectomy samples from patients with HOCM . LVOT grThe levels of TnI and MyBP-C phosphorylation were determined in muscle myofibrils by separating phosphospecies using phosphate affinity SDS-PAGE and detecting with TnI and MyBP-C specific, but phosphorylation-independent antibodies previously characterized. This technique measures the proportions of bis-phosphorylated, monophosphorylated and unphosphorylated species of TnI. We previously showed that in donor hearts, 70% of the troponin I is bis-phosphorylated and 21% is monophosphorylated with a calculated total phosphorylation of 1.6 ± 0.06 molsPi/mol TnI. The HOCM samples were just 5% bis-phosphorylated and 30% monophosphorylated with a calculated total phosphorylation level of 0.18 ± 0.02 molsPi/mol TnI .n = 21) and total phosphorylation was 0.85 ± 0.03 molsPi/mol TnI and was not significantly different in septum and free wall samples (see p ≤ 0.0001). Western blotting using antibodies to bis-phosphorylated troponin I confirmed that phosphorylation at serines 22 and 23 was low relative to donor heart samples. Mass spectrometry measurements showed that only Ser22 or Ser23 were phosphorylated with no evidence for phosphorylation at other sites in Troponin I.In the pressure overload samples, we found that TnI was predominantly monophosphorylated  (n = 15) compared with 3.4 ± 0.07 (n = 21) in donor heart and 1.1 ± 0.1 (n = 10) in HOCM heart  . In the n = 15) . Total pCM heart . CompariCM heart . We usedThe ACTC E99K mouse model of HCM develops apical hypertrophy without LVOTO or symptoms of heart failure at 21 weeks ; the Joint UCL/UCLH Ethics Committee Rec No: 04/0035; Outer North East London Research Ethics Committee REC ref: 10/H0701/8; Careggi University Hospital Ref: 2006/0024713 and Comite É t́ico de Investigatio ń Clínica de Galicia. Permission for study of the samples was granted by the NHS National Research Ethics Service, South West London REC3 (10/H0803/147). The patients/participants provided their written informed consent to participate in this study.OC, AM, and SM performed the experimental work. AJ, CP, and SP supplied the tissue samples. All the authors contributed to and approved the final manuscript that was edited by SM.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.


The ExcelsiusGPS®  is a next-generation spine surgery robotic system recently approved for use in the United States. The objective of the current study is to assess pedicle screw accuracy and clinical outcomes among two of the first operative cases utilizing the ExcelsiusGPS® robotic system and describe a novel metric to quantify screw deviation.Two patients who underwent lumbar fusion at a single institution with the ExcelsiusGPS® surgical robot were included. Pre-operative trajectory planning was performed from an intra-operative CT scan using the O-arm . After robotic-assisted screw implantation, a post-operative CT scan was obtained to confirm ideal screw placement and accuracy with the planned trajectory. A novel pedicle screw accuracy algorithm was devised to measure screw tip/tail deviation distance and angular offset on axial and sagittal planes. Screw accuracy was concurrently determined by a blinded neuroradiologist using the traditional Gertzbein-Robbins method. Clinical variables such as symptomatology, operative data, and post-operative follow-up were also collected.Eight pedicle screws were placed in two L4-L5 fusion cases. Mean screw tip deviation was 2.1 mm (range 0.8–5.2 mm), mean tail deviation was 3.2 mm (range 0.9–5.4 mm), and mean angular offset was 2.4 degrees (range 0.7–3.8 degrees). All eight screws were accurately placed based on the Gertzbein-Robbins scale (88% Grade A and 12% Grade B). There were no cases of screw revision or new post-operative deficit. Both patients experienced improvement in Frankel grade and Karnofsky Performance Status (KPS) score by 6 weeks post-op.The ExcelsiusGPS® robot allows for precise execution of an intended pre-planned trajectory and accurate screw placement in the first patients to undergo robotic-assisted fusion with this technology. Robotics in spinal surgery have become increasingly prevalent and sophisticated over the last two decades –3. SpinaFiducial and surveillance markers are placed in the posterior superior iliac spine (PSIS), outside the surgical field, and a temporary intraoperative CT (ICT) fixture is secured to the skin for patient registration. Notably, pre-operative x-ray, pre-operative CT, or intra-operative CT scans may be utilized for image-guidance. Screw trajectories are planned on the registered images and the trajectory is guided by the floor-mounted robotic arm. Screws are deployed through the rigid tubular robotic arm, thus avoiding the unwieldy patient-mounted frames and flimsy Kirschner wires (K-wires) that previous systems rely upon.Pedicle screw insertion accuracy in robotic assisted spinal surgery has been well studied and reported to be superior compared to conventional fluoroscopic-guided or free-hand techniques –18. The The goal of this study was two-fold: to describe two of the first spinal surgeries with the ExcelsiusGPS® robotic system; and to propose a novel method of assessing pedicle screw accuracy, in the setting of robotic-assistance, by quantifying screw deviations relative to the pre-operatively planned trajectory.The study was a retrospective, institution review board (IRB)-approved review of patients who underwent spinal surgery with the ExcelsiusGPS®  at our institution from October 2017 to December 2017. Patient variables such as demographics, presenting symptoms, Karnofsky Performance Status (KPS), Frankel grade, and ambulatory status were collected. Operative data such as surgical indication, estimated blood loss (EBL), and length of case were also collected.Patients were positioned prone on a Jackson table, with intraoperative neuromonitoring performed throughout the case. After prepping and draping the patient in the usual sterile fashion, the ExcelsiusGPS® fiducial marker  was placed in the right posterior superior iliac spine via a small stab incision. A second surveillance marker was placed in the left PSIS with another stab incision. The ICT was attached to the fiducial marker, in a plane parallel to the floor and just above the patient’s skin. The O-arm  was then used to obtain a 3-dimensional (3D) CT scan. This was transferred to the ExcelsiusGPS® surgical system for screw planning, which includes entry point, trajectory, screw length, and screw width.The reference frame ICT was then removed from the field and a typical open spine exposure was performed. For screw placement, the ExcelsiusGPS® robot was draped sterilely, wheeled into the surgical field, and locked securely to the floor. The navigated instruments, including a position tracker, drill, and navigated screw guide were registered with the system. The surgeon utilized a foot pedal to bring the robotic arm to the desired position, corresponding to the screw trajectory planned. With the end effector in position, a power drill was first used to cannulate a trajectory and the screw is then placed through the stable, rigid end effector. Real-time visualization of screw trajectory and indicators of excessive skive force were available to the surgeon.After instrumentation, laminectomies were performed. Fusion was achieved by using demineralized bone matrix and cancellous autologous bone graft. A post-operative CT scan with 0.5 to 2 mm thickness slices was again performed with the O-arm.Screw accuracy was assessed using the traditional Gertzbein-Robbins Scale and a newly devised methodology. For the Gertzbein-Robbins method , screw aIn addition to the Gertzbein-Robbins scale, each implanted screw was evaluated for deviation from a pre-operatively planned trajectory. Deviation was assessed by comparing a 3D offset from the pre-operative plan to the placed screw. Longitudinal length, which does not reflect medial or lateral breach, was not considered for the final analysis. Assessments were performed using dedicated ExcelsiusGPS® software by an engineer (NC) blinded to the patient’s clinical information. Deviation was defined in three data points: distance of placed screw tip from intended trajectory, distance of placed screw tail from intended trajectory, and angular offset.Screw accuracy was first graded using the Gertzbein-Robbins method , performDeviation of actual screw placement with pre-planned trajectory was also assessed. In all cases, longitudinal screw offset was removed from the final analysis, since screw length is not a predictor of medial/lateral breach. In the first case, the deviated tip distance was < 2 mm in all four screws. In the second case, one screw (left L4) had a > 5 mm deviated tip and tail distance, likely secondary to an entry point that was equivalently offset. Overall, mean screw tip deviation was 2.1 mm (range 0.8–5.2 mm), mean tail deviation was 3.2 mm (range 0.9–5.4 mm), and mean angular offset was 2.4 degrees (range 0.7–3.8 degrees).. Both patients presented with radicular leg pain with accompanying mechanical back pain, pre-operative difficulty with ambulation and neurologic deficits, including lower extremity weakness and bladder dysfunction. Both patients were Frankel grade D and had a KPS of 60 at baseline.Table  The meanSpine surgery robots are becoming increasingly accepted for their various advantages over conventional techniques. For instance, in one study of 112 patients and 536 pedicle screws, the use of the SpineAssist™ surgical robot significantly increased screw accuracy (94.5% vs 91.4%) and decreased radiation exposure (34 s vs 77 s) compared to conventional 2-D fluoroscopy . Our curMost literature on pedicle screw accuracy assessment utilize the Gertzbein-Robbins grading scale, which determines acceptability of screw position based on an optimal transpedicular trajectory without cortical violation . Other CVan Dijk et al. evaluated for the reproducibility of the surgeon’s plan in robotic- guided percutaneous posterior lumbar interbody fusion using the Mazor SpineAssist robot . The autIn another study, Devito et al. performed a similar analysis on planned versus actual placements of 646 screws using the Mazor SpineAssist robot. The authors examined 6 locations on each screw, which defined the entry and exit points from the pedicle and then measured pedicle length in sagittal and axial views to create a 3D positional and orientation accuracy assessment . A mean While most studies on this topic draw comparisons between two fused CT scans (pre-op versus post-op), Tsai et al. proposed an intraoperative robotic classification system to assess the accuracy of K-wire placement as a predictor of pedicle screw accuracy . In theiThe ExcelsiusGPS® robotic system has several technological improvements over prior robotic systems in spinal surgery, particularly as it relates to the usage of K-wires. The Mazor SpineAssist and Renaissance systems rely on an interspinous clamp attached to 3D fiducial markers for patient registration. Robotic arms are then screwed onto the interspinous clamp, and a guide tube allows for cannulation and the insertion of K-wires. The interspinous clamp can be disrupted with inadvertent motion and can become malpositioned during drilling. Since the arms are screwed on the interspinous clamp attachment, they are not rigid enough for direct screw insertion and thus a K-wire must first be used –3, 14. TLimitations of our study include the small sample size and single institution study. One of the current drawbacks is the significant time required for the two O-arm spins, which were done early in our series to assess accuracy and is no longer routinely done. In addition, only medial/lateral  and superior/inferior  deviations were utilized for our accuracy assessment, since the longitudinal depth of the screw is operator dependent and not necessarily a predictor of screw accuracy.The new ExcelsiusGPS® robot has several technological advances over prior spine robots and allows for precise execution of an intended pre-planned trajectory and accurate screw placement in the first patients to undergo robotic-assisted fusion with this technology.


Recently, the infra-acetabular screw has been proposed for use in treatment of acetabular fractures as a part of a periacetabular fixation frame. Biomechanical studies have shown that an additional infra-acetabular screw placement can enhance the fixation strength of acetabular fracture internal fixation. Currently, the reported exit point of the infra-acetabular screw has been located at the ischial tuberosity (Screw I). However, our significant experience in placement of the infra-acetabular screw has suggested that when the exit point is located between the ischial tuberosity and the ischial spine (Screw II), the placement of a 3.5 mm infra-acetabular screw may be easier for some patients. We conducted this study in order to determine the anatomical differences between the two different IACs.The raw datasets were reconstructed into 3D models using the software MIMICS. Then, the models, in the STL format model, were imported into the software Geomagic Studio to delete the inner triangular patches. Additionally, the STL format image processed by Geomagic Studio was imported again into MIMICS. Finally, we used an axial perspective based on 3D models in order to study the anatomical parameters of the two infra-acetabular screw corridors with different exit points. Hence, we placed the largest diameter virtual screw in the two different screw corridors. The data obtained from this study presents the maximum diameter, length, direction, and distances between the entry point and center of IPE.In 65.31% males and 40.54% females, we found a screw I corridor with a diameter of at least 5 mm, while a screw II corridor was present in 77.55% in males and 62.16% in females. Compared to screw I, the length of screw II is reduced, the angle with the coronal plane is significantly reduced, and the angle with the transverse plane is significantly increased.For East Asians, changing the exit point of the infra-acetabular screw can increase the scope of infra-acetabular screw use, especially for females. The acetabulum has a complex anatomical structure and is surrounded by some important blood vessels, nerves, and organs. The acetabular fracture surgery is the most complex and challenging surgery in the field of traumatic orthopedics . Hence, In 2010, the infra-acetabular screw was reported to be able to help close the incomplete “frame” system around the acetabulum, including both acetabular columns, supra-acetabular screws, and the true pelvic rim plate . MeanwhiDespite the fact that the infra-acetabular screw has several advantages, its disadvantages include that it is difficult to place the screw as the screw corridor is relatively narrow and it is an unconventional screw corridor. Some researchers believe that some patients are not suitable for infra-acetabular screws . TherefoIn this study, the exit points of the infra-acetabular screw have been located in the ischial tuberosity , 6, 8–10All of the procedures were conducted in accordance with the declaration of Helsinki and relevant policies in China. The study protocol was granted approval by the ethics review board at Nanfang Hospital, Southern Medical University. We obtained verbal consent from all subjects prior to collecting data for this. Overall, 86 subjects that were admitted to our institution without pelvic and acetabular injury or lesions were recruited in this study Table . All subThe raw data sets were reconstructed into 3D models using the software MIMICS 17.0 . The left hemi-pelvis was exported in STL format and imported into the image-processing software Geomagic Studio 2013 . Next, the inner triangular patches, which represented contents of the marrow cavity, were deleted in order to make the marrow cavity hollow in 3D models. After processing in Geomagic Studio 2013, the images were exported in STL format and imported again into MIMICS where all the simulations and measurements were carried out.In recent years, application of computer-aided technology has allowed the rapid development of digital orthopedics. A bio-morphological study of screw corridors around the acetabulum can be performed easily and quickly using computer-aided techniques –13. Our In order to distinguish the screw path in 3D models from the same perspective, we downgraded the transparency of 3D models and turned 3D models at the axial perspective of the infra-acetabular corridor, which is a view that is perpendicular to the cross-section of the two infra-acetabular corridor axis. As a result, a translucent area with a darker outline of each infra-acetabular corridor was clearly seen. The translucent area represented the marrow cavity of the infra-acetabular corridor, the outline of which showed the cortical bone Figs.  and 2.FFirst, we adjusted positions of the 3D model in the axial perspective in order to find the most likely largest translucent area by visual observation. Then, a virtual computer-aided design (CAD) Screw I (2 mm in diameter) was placed perpendicular on the screen at the center of the translucent area. We adjusted the CAD Screw I diameter progressively in order to accurately determine the maximum implant diameter that the transparent area could accommodate. The maximum implant diameter was defined as the largest diameter of the screw that did not penetrate the outline of the translucent area Figs.  and 2. Ap < 0.05. The SPSS statistical software package for Windows (version 19.0) was used for all statistical analyses.All experimental data (continuous variables) were presented as mean and standard deviation (SD) or median and range. An independent sample t-test was utilized to compare data between males and females. Paired sample t-test was used to compare the data between Screw I and Screw II. Linear regression was used to analyze the linear relationships between the two different infra-acetabular parameters and age, body height, and body weight. Statistical significance was set at Among 65.31% 32/49) males and 40.54% (15/37) females, we found a Screw I corridor with a diameter of at least 5 mm. The mean maximum diameter and length of Screw I were 5.30 ± 1.25 mm (2.62–7.58 mm) and 94.57 ± 6.07 mm (78.99–105.91 mm), respectively. The caudal and medial distances of the entry point to the center of IPE of Screw I were 7.49 ± 4.68 mm (− 6.72–21.43 mm) and 12.32 ± 3.12 mm (5.66–23.17 mm), respectively. The angles of Screw I to the transverse, coronal and sagittal planes were 36.48 ± 8.18° (15.95–54.38°), 52.77 ± 8.14° (35.50–72.52°), − 0.49 ± 6.43° (− 18.04–11.20°), respectively. All differences, with the exception of the coronal angle of the Screw I between males and females, were statistically significant  males and 62.16% (23/37) females, we found a screw II corridor with a diameter of at least 5 mm. The mean maximum diameter and length of Screw II were 5.68 ± 1.39 mm (2.56–8.26 mm) and 80.16 ± 4.46 mm (71.17–89.78 mm), respectively. The caudal and medial distance of the entry point to the center of IPE of Screw II was 20.24 ± 5.00 mm (9.67–32.35 mm) and 12.41 ± 3.18 mm (6.09–21.01 mm), respectively. The angles of Screw II to the transverse, coronal and sagittal planes were 12.69 ± 7.46°(0.03–34.19°), 75.20 ± 7.20°(51.33–88.98°), − 1.19 ± 6.91°(− 17.26–8.86°), respectively. The differences in diameter, length, medial distance and angles to the sagittal planes of Screw II between males and females were statistically significant   Tables  and 5.TThe value of an infra-acetabular screw is that it is an important part of the periacetabular fixation “frame”. Biomechanical studies demonstrated that an additional placement of this screw significantly increases the strength of a standard plate fixation for treatment of anterior column fractures , 5. A biAt present, in studies of the infra-acetabular screw, the exit point of the screw was located in the ischial tuberosity. However, our study identified that when the exit point of the screw was located between ischial tuberosity and the ischial spine, there is also an IAC. According to our results, a screw II corridor with a diameter of at least 5 mm was found in 77.55% of males and 62.16% of females, which was increased by 12.24 and 21.62% respectively compared to the Screw I corridor. That demonstrated that the Screw II corridor could increase the proportion of IAC exceeding 5 mm in Eastern population, which enables the placement of a 3.5 mm screw in some patients with a small screw I corridor, particularly in female patients. Most of the Screw II corridor diameters were increased compared to Screw I, with an average increase of 7.20%. Meanwhile, all of the Screw II corridor lengths were shortened, with an average reduction of 15.24%. Length shortening can greatly reduce the difficulty of inserting a long screw into a narrow corridor. In males, the two different infra-acetabular corridors were parallel to the sagittal plane, while angles to the sagittal plane of the two corridors of females were larger than that of males, and the directions were from the medial to the lateral side. Compared to Screw I, the entry point of Screw II is farther away caudally from the center of IPE.Our results demonstrate that due to a narrower corridor of the infra-acetabular screw in Asian populations, some patients are not suitable for use of infra-acetabular screws. Therefore, we recommend that each patient that intends to insert an infra-acetabular screw should have a CT scan and use a digital orthopedic software to perform a preoperative virtual placement of an infra-acetabular screw prior to surgery. In this way, a personalized surgical plan can be developed for patients to improve the safety of the operation. Furthermore, intraoperative navigation and 3D printing technology have been utilized to help place periacetabular screws , 15–17, This was a purely anatomical study that utilized computer-aided technology. Therefore, there were certain unavoidable limitations. CT resolution, the adopted smoothing criteria, selection of seed threshold and sensitivity can all cause the 3D model to not conform to the real specimen. Furthermore, observer errors can cause observation and measurement errors. The sample size may not be enough to represent the entire population. The absence of a correlation between the values found and additional morphometric pelvic parameters can cause preoperative planning of the two different infra-acetabular screws to rely more on CT scans. More importantly, this study lacks clinical support. In the future, clinical research needs to be conducted in order to account for this limitation.Our study provided surgeons with the anatomical parameters of two different IACs in an Eastern population. Compared to previous research, we found that the proportion of Screw I with a diameter of at least 5 mm was greatly reduced, while Screw II could reduce this gap. This may provide an alternative acetabular infra-acetabular screw placement corridor for surgeons.


TM6SF2) E167K genetic variant associates with NAFLD and with reduced plasma triglyceride levels in humans. However, the molecular mechanisms underlying these associations remain unclear. We hypothesized that TM6SF2 E167K affects hepatic very low-density lipoprotein (VLDL) secretion and studied the kinetics of apolipoprotein B100 (apoB100) and triglyceride metabolism in VLDL in homozygous subjects. In 10 homozygote TM6SF2 E167K carriers and 10 matched controls, we employed stable-isotope tracer and compartmental modeling techniques to determine apoB100 and triglyceride kinetics in the 2 major VLDL subfractions: large triglyceride-rich VLDL1 and smaller, less triglyceride-rich VLDL2. VLDL1-apoB100 production was markedly reduced in homozygote TM6SF2 E167K carriers compared with controls. Likewise, VLDL1-triglyceride production was 35% lower in the TM6SF2 E167K carriers. In contrast, the direct production rates for VLDL2-apoB100 and triglyceride were not different between carriers and controls. In conclusion, the TM6SF2 E167K genetic variant was linked to a specific reduction in hepatic secretion of large triglyceride-rich VLDL1. The impaired secretion of VLDL1 explains the reduced plasma triglyceride concentration and provides a basis for understanding the lower risk of cardiovascular disease associated with the TM6SF2 E167K genetic variant.Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation. The transmembrane 6 superfamily member 2 (  Nonalcoholic fatty liver disease (NAFLD) is a condition defined by excessive (>5% by weight) fat accumulation in the liver. As a consequence of the obesity and diabetes epidemic, NAFLD has become the most common liver disorder in developed countries, with more than 100 million adults and children affected in the United States alone , 3, the TM6SF2) rs58542926 variant, encoding for a glutamic acid to lysine substitution at position 167 of the amino acid sequence (E167K), has been identified as one of the most important genetic determinants of hepatic fat content   secretion , or by blocking lipid catabolism or outflow  –16. Of tide (TG) , 17, and VLDL-TG , 17, 18. studies . Howeverd levels . Furtherecretion . These cTM6SF2 E167K genetic variant causes relative phosphatidylcholine (PC) deficiency due to impaired synthesis of this lipid from polyunsaturated fatty acids  and control subjects (n = 10). The 2 groups were matched for BMI, waist circumference, and indices of glucose homeostasis; measures of visceral (VAT) and abdominal subcutaneous (SAT) adipose tissue also did not differ significantly between the groups (P = 0.35 for both). As expected, the TM6SF2 E167K carriers had more than 2-fold higher liver fat content than noncarriers  (We examined homozygote = 0.023) .P = 0.003) and plasma TG  were significantly lower in homozygote TM6SF2 carriers compared with controls  . In contrast, the direct production rates for VLDL2-apoB100 and -TG were not different between carriers and noncarriers  between the 2 groups of study subjects (To test the hypothesis that the = 0.003) . Likewiscarriers . Fractioe groups . There wsubjects .1-TG FCR .Because plasma apoC-III levels are strong predictors of VLDL1-TG FCR  and therTM6SF2 E167K, in particular how it affects hepatic VLDL secretion, and by extrapolation risk of atherosclerotic cardiovascular disease. Our results show clearly that in subjects homozygous for this variant, there is a specific impact on the synthesis and secretion of large TG-rich VLDL1 but no effect on the direct secretion of the smaller, less TG-rich VLDL2. This finding is informative since we know from previous studies that production of these 2 VLDL subfractions is regulated independently  may be critical for the formation of lipid droplets that fuse with VLDL2-sized precursor particles to form VLDL1 or be required for the fusion process per se. The mechanism responsible for the generation of lipid droplets within the ER is not completely clear. It is known that enzymes capable of synthesizing neutral lipids are present in this cellular location , had participated in earlier studies where exome sequencing or genotyping and imputation had been performed to explore genes involved in lipid metabolism. The remaining 8 subjects came from previously examined kinetic study cohorts (TM6SF2 E167K (9 men and 1 woman) and 10 control subjects (9 men and 1 woman), matched for degree of obesity and glycemic status, who were not carriers of TM6SF2 E167K and also were not homozygous for PNPLA3 I143M  > 42 mmol/mol, LDL-cholesterol > 4.5 mmol/L, plasma TG > 2.5 mmol/L, uncontrolled hypertension (>160 mmHg systolic and /or >105 mmHg diastolic blood pressure), use of thiazide diuretics (>25 mg/d), use of medications affecting lipid or glucose metabolism, or high alcohol consumption (>30 g/d for men and >20 g for women). The participants were asked to abstain from alcohol and strenuous physical exercise during leisure time before metabolic investigations.A group of 20 subjects, recruited on the basis of their  cohorts , 42, 43.A3 I143M . Inclusi2H2O (2 g/kg) was given between 1800 and 2200 hours to give an assessment of the degree of the contribution of hepatic DNL to VLDL1 TGs (see refs. 2H5]-glycerol (500 mg) and [2H3]-leucine (7 mg/kg). Blood samples were drawn before tracer injection and at frequent intervals thereafter . Concentrations of glucose  and insulin  were measured in fasting blood samples and HOMA-IR was calculated  was added during the extraction and used for quantification.Lipid extraction of 50 μL of each VLDL fraction was performed using the BUME method . TGs werLiver fat content was measured using proton magnetic spectroscopy (1.5 T whole-body device) , and subTM6SF2 rs58542926 (E167K) and PNPLA3 rs738409 (I148M) status by TaqMan assays  using CFX384 Real Time PCR detection system (Bio-Rad Laboratories). Data analysis was performed using Bio-Rad CFX manager software.The genotypes of participants were confirmed upon recruitment to the present study. DNA was extracted from blood using DNeasy Blood & Tissue Kit (Qiagen) and used to determine 2H3] leucine and [2H5] glycerol, (b) apoB100 and TG pool sizes in VLDL1 and VLDL2, (c) enrichment curve of plasma leucine, (d) enrichment of deuterated leucine in in VLDL1- and VLDL2-apoB100, and (e) enrichment of deuterated glycerol in VLDL1- and VLDL2-TG  . Inputs VLDL2-TG . OutputsP values were calculated using the Kruskal-Wallis test. Correlation coefficients refer to the Spearman’s rank correlation coefficient. Trend analyses for lipidomics data were performed using linear mixed models with number of double bonds as fixed effect and subject as random effect. Data were normalized to mean of control group; P values correspond to tests of the linear effect of double bonds differing from 0. P < 0.05 was considered statistically significant.Statistical calculations were performed using R (version 3.6.3). Data are reported as mean ± SD, and The study protocol was approved by the Ethics Committee of Helsinki University Central Hospital, Helsinki, Finland. ClinicalTrials.gov Identifier: NCT04209816. The study was performed in accordance with the Declaration of Helsinki and the European Medicines Agency Note for guidance on good clinical practice. All study participants signed a written informed consent form before any study procedures were initiated.The authors contributed to the present work as follows: JB, NM, and MRT contributed to conception and design; MA, EB, NM, SS, JR, MS, PR, S Ripatti, AP, RMM, AH, and S Romeo to the acquisition of data or analysis; and JB, MA, EB, CJP, and MRT to the interpretation of data. JB, MA, CJP, and MRT drafted the original and revised manuscripts, and all authors approved the final version to be published.


The activity of Furvina and the most promising quorum-sensing inhibitor (QSI) was evaluated in biofilm prevention and in biofilm control (combined with fusidic acid). The biofilms were further characterized in terms of biofilm mass, viability and membrane integrity. Compound 2 caused the most significant QS inhibition with reductions between 60% and 80%. Molecular docking simulations indicate that this compound interacts preferentially with the protein hydrophobic cleft in the LytTR domain of AgrA pocket. Metabolic inactivations of 40% for S. aureus ALC1742 and 20% for S. aureus ALC1743 were reached. A 24 h-old biofilm formed in the presence of the QSI increased the metabolic inactivation by fusidic acid to 80%, for both strains. The overall results highlight the effects of compound 2 as well as the potential of combining QSI with in-use antibiotics for the management of skin and soft tissues infections.Quorum sensing (QS) plays an essential role in the production of virulence factors, in biofilm formation and antimicrobial resistance. Consequently, inhibiting QS is being considered a promising target for antipathogenic/anti-virulence therapies. This study aims to screen 2-nitrovinylfuran derivatives structurally related to Furvina  for their effects on QS and in biofilm prevention/control. Furvina and four 2-nitrovinylfuran derivatives (compounds  Staphylococcus aureus is an opportunistic pathogen widely associated with skin colonization and biofilm-related infections [N-acyl-homoserine lactones (HSLs), while in Gram-positive they are generally peptides (AIP) [S. aureus, the QS system is designated as accessory gene regulator (Agr) as it is encoded by agr locus. The agr locus consists of two divergent transcriptional units, RNAII and RNAIII, which are driven by P2 and P3 promoters, respectively [BDCA, that are involved in the successful run of the Agr QS circuit [fections . The skifections . Howeverfections . Such phfections . Besidesfections . This mefections . In Grames (AIP) . In S. aectively . The RNA circuit . AgrD is circuit ,8. When  circuit . AgrC is circuit . AgrA ac circuit ,10.S. aureus Agr QS system is biofilm formation. In fact, Agr affects not only biofilm formation but also its maturation [S. aureus biofilm maturation is mostly due to the decreased expression of detachment molecules, such as δ-toxin [S. aureus infections by modulating QS transcriptional regulators to prevent biofilm formation and inhibit virulence factors [Associated with turation . Biofilmturation ,13,14. S δ-toxin . However δ-toxin . This of factors ,17. In f factors ,19,20.Z)-2-bromo-5-(2-bromo-2-nitrovinyl)furan), synthesized from furfural a starting material present in sugar cane bagasse, reported to act as antimicrobial for topical application. Furvina is described to be a broad-spectrum antibiotic and is commercialized in Cuba as Vitrofural®, Furvinol® and Dermofural® [Pseudomonas aeruginosa [P. aeruginosa biofilm formation and hindered the production of several virulence factors [Due to the current limitations of antimicrobial drug discovery programs, a particular focus on repurposing and repositioning of drugs or drug candidates has been given for finding new therapeutic applications ,22,23. Fmofural® ,25. The mofural® . Furthermofural® . In a prruginosa . Furvina factors . S. aureus Agr QS system, using S. aureus bioreporters strains. Furvina and the best QSI was evaluated in silico (molecular docking simulations) and further tests were performed to assess the effects of these compounds on the prevention of biofilm setup and in the increase of biofilm susceptibility to fusidic acid (24 h-old biofilms formed in the presence of the identified QSI or Furvina).In this work, the effects of Furvina and selected 2-nitrovinylfuran derivatives  were evaS. aureus ALC1742 and S. aureus ALC1743 . The cut-off concentration was used in cases of MIC lower than 1000 μg/mL, in order to test the possible effects of a concentration higher than MIC in the Agr QS as well as to corroborate the fact that the observed activity was not due to growth inhibition. Furvina and 2-nitrovinylfuran derivatives were evaluated against planktonic  ALC1743  to obtaiS. aureus Agr QS system. Accordingly, and taking into account that the effect in the S. aureus Agr QS system is measured based on the emission of gfp, the highest fluorescence reduction regards the topmost inhibition of each promoter—P2 for S. aureus ALC1742 and P3 for S. aureus ALC1743, respectively [2 was that showing the highest inhibitory effect (more than 50% for both promoters).Furvina and its chemically related compounds were screened to assess their effects on the ectively . As show2 with both P2 (RNAII) and P3 (RNAIII), molecular docking simulations were carried out in the S. aureus Agr system to identify the preferred binding mode adopted by the compounds in the protein hydrophobic cleft pocket previously identified (LytTR domain of AgrA) [2 were docked to the protein. Docking best poses were then energy minimized to simulate the induced fit effect of binding pocket ligand surrounding residues. Finally, the complex AgrA- compound 2 was aligned with AgrA LytTR domain complexed with P2 (RNAII) (pdb code 3bs1) [In silico docking experiments showed that AgrA-compound 2 complex is stabilized by several interactions with the shallow groove formed by the β10−α2 loop, particularly by the interaction with positively charged residues Lys236, Lys237, Arg233 and polar Asn234. Finally, with hydrophobic residues Val232 and Val235 (2 would be able to interfere in the binding with P2 (RNAII) and P3 (RNAIII), as shown in Based on the in vitro data showing the interference of Furvina and compound of AgrA) . Crystalof AgrA) , resolutde 3bs1)  and P3 (de 3bs1) . In silid Val235 a,b. In td Val235 c,d. The 2 were selected to further assess their effect in the prevention of biofilm formation (24 h exposure) and to evaluate their effect on biofilm (24 h-old biofilms formed in the presence of Furvina and compound 2)susceptibility to fusidic acid. The selected compounds (Furvina and compound 2) as well as fusidic acid were studied for their ability to interfere with the biofilms in terms of mass removal, metabolic activity and membrane integrity (p < 0.05), which is ~50% higher than the values obtained for compound 2, for both strains. In terms of metabolic inactivation, Furvina also presented the best performance with approximately 75% reduction compared to the ~40% and ~20% reduction obtained by compound 2 for ALC1742 and ALC1743, respectively (p < 0.0001). Furvina and compound ntegrity . Regardi2 was that causing the highest potentiation of fusidic acid action. The combination of Furvina and compound 2 with fusidic acid improved biofilm mass reduction in comparison to the antibiotic alone, and no significant difference was obtained from the combination with Furvina or compound 2 (p > 0.05). While, for ALC1742, Furvina conjugated with fusidic acid reduced the biofilm mass in approximately 38%, compound 2 reduced in 34%, which is significantly higher than the 20% achieved by the antibiotic alone (p < 0.05). For ALC1743, compound 2 and fusidic acid were not able to reduce biofilm mass, while Furvina combined with the antibiotic achieved a 29% reduction. However, this reduction was not significantly different from that caused by the antibiotic alone (23%). Considering the metabolic activity reduction, compound 2 conjugated with fusidic acid caused 80% inactivation, which is significantly higher than the ~70% obtained by fusidic acid alone (p < 0.05 for both strains) or in combination with Furvina (p < 0.05 for both strains). In terms of membrane integrity of the 24-h oldbiofilms, most of the bacteria maintained their membrane intact (more than 70%), for both strains and different concentrations of compound 2 and Furvina  are recognized [S. aureus. The study was extended to the evaluation of the effects of Furvina and the best QSI candidate on the prevention of biofilm formation and on the enhancement of biofilm susceptibility to fusidic acid.Even though the antimicrobial action of Furvina and effects on the QS network of cognized , no data2 were selected for further biological analysis after the QS effect screening. Furvina was selected as a baseline compound based on the recognized activity in the QS of P. aeruginosa, while compound 2 showed the highest fluorescence inhibition, being considered the best QSI candidate. Looking to their chemical structures, it is possible to highlight that they differ on the presence or absence of some functional groups and those differences can be associated with the increased capacity for inhibiting QS. When compared with Furvina, compound 2 has no bromine on the furan moiety and the bromine present in the styryl side chain has been replaced by a methyl group  of AgrA pocket, which has been previously identified to be important for the binding of small molecules [Docking simulations demonstrated that compound olecules . The binolecules . Further2. This result was excepted as the mode of action of Furvina involves the reaction with the cysteine residues of MurA, a protein that is crucial for peptidoglycan synthesis, which in turn is the main component of the bacterial cell wall [2 interferes with S. aureus Agr QS and increases S. aureus biofilm susceptibility to fusidic acid. These results corroborate a previous study showing that QS signaling molecules are involved in many aspects of biofilm dynamics  [2 remains to be understood. Fusidic acid inhibits protein synthesis in Gram-positive bacteria, binding to elongation factor G (EF-G) and locking it to the ribosome [Regarding biofilm prevention, this study demonstrates that Furvina had a higher antimicrobial and antibiofilm activity than compound ell wall . Additiooughing) . Once a oughing) . Even thribosome . Despiteribosome . This isribosome .agr dysfunctional bacteria are less prone to be transmitted between patients, suggesting that whereas agr mutants arise with the use of these combinatorial approaches, they are unlikely to have a selective advantage over wild-type bacteria [agr mutants are mostly associated with bacteremia and not with acute skin infection in immunocompetent individuals [Fusidic acid is a drug that has excellent bioavailability and skin penetration . Furtherbacteria ,44. Impoividuals .agr expression appears to contribute to Staphylococcal pathogenesis in several infection models [Overall, this study proposes that the virulence of staphylococci is affected by the inhibition of the Agr QS system, which is in agreement with previous findings ,47,48,49n models ,47,48,49n models .Furvina and 2-nitrovinylfuran derivatives 1–4 were synthesized as previously described ,44,45. Tv/v; Fisher Scientific, Loughborough, UK) under sterile conditions. Serial dilutions of the compounds, from 1000 μg/mL to 6.25 μg/mL, were prepared when needed. The percentage of DMSO never exceeded 10% (v/v) of the final volume. Fusidic acid was purchased from Sigma-Aldrich and used according to the manufacturer’s recommendations.Furvina and 2-nitrovinylfuran derivatives 1–4 solutions were prepared in dimethyl sulfoxide  at 37 °C. Regarding the study of the effect of the selected compounds in QS, bacteria were cultured without shaking to prevent gfp expression, the parameter measured in this experiment [The periment . However8 CFU/mL). Then, a volume of 180 μL of this cell suspension was added to a sterile, 96-well flat, clear bottomed polystyrene (PS) microtiter plates , already containing 20 μL of compound at concentrations from 6.25 to 1000 μg/mL. Regarding fusidic acid, the range of concentrations tested were 0.0625–32 μg/mL. At the end, the volume of each compound never exceeded 10% (v/v) of the well volume. Microtiter plates were then incubated for 24 h at 37 °C at 150 rpm. Absorbance measurements were performed in the beginning (t = 0 h) and in the end (t = 24 h) of the incubation period using a microplate reader . Cell suspensions with and without DMSO were used as controls. MIC was set as the lowest concentration of the compound at which the final OD was equal or lower than the initial OD (cell growth inhibition). This test was performed three times, with six replicates.The MIC of Furvina, 2-nitrovinylfuran derivatives 1–4 and fusidic acid were determined based on the microdilution method described by Borges et al. . This meS. aureus ALC1742 and ALC1743 was evaluated based on Xiong et al. [7 CFU/mL) at 620 nm. A volume of 180 μL of the cell suspensions was added to a sterile, 96-well flat-bottomed, polystyrene (PS) microtiter plates  containing 20 μL each compound at different concentrations (6.25–1000 μg/mL). At selected time points , the emitted fluorescence (ʎexcitation: 485–12 nm and ʎemission: 510–10 nm) was measured using a microtiter plate reader . The effect on the promoter was measured as fluorescence reduction of the cells when in contact with a specific compound compared to cells in contact with 10% (v/v) DMSO. All experiments were performed three times, with six replicates.The interference of Furvina and 2-nitrovinylfuran derivatives in the QS system of g et al. . Bacteri(a) Ligands PreparationTheoretical 3D models of compounds were built by means of Maestro GUI. Ligand’s global minimum energy conformation was determined by molecular mechanics conformational analysis performed with Macromodel software version 9 . The geo(b) Protein PreparationThe protein structure was obtained from the PDB web site with pdb code 4g4k and was prepared by using the Protein Preparation Wizard module . Water m(c) Docking simulations−1. Depictions were obtained with Pymol  and Maestro.Three protocols were compared: Glide-SP , Glide-XS. aureus ALC1742 and S. aureus ALC1743 biofilm formation. For at, 96-well flat, clear bottomed PS microtiter plates were filled with 180 μL of cells suspension (~1 × 107 CFU/mL) and 20 μL of the compounds at MIC and ½ MIC. Bacterial suspensions with DMSO and without compounds were used as controls. The plates were incubated at 37 °C and 150 rpm for 24 h (biofilm formation). After the incubation period, the content of each well was discarded and washed twice with sterile phosphate buffer saline . Microtiter plates were analyzed in terms of biomass quantification by crystal violet staining , metabolic activity by alamar blue  staining and also in terms of membrane integrity using the Live/Dead Baclight kit.Biofilms were developed according to the modified microtiter plate assay proposed by Stepanovic et al. . Furvina(a) Biomass quantification of biofilm cells by crystal violet stainingv/v) commercial ethanol for 15 min. The content of the microtiter plates was again rejected, and the plate was left to dry. Then, the fixed bacteria were stained for 5 min with 200 μL of 1% CV solution. After that period, the excess of stain was gently withdrawn. Afterwards, the dye bound to the adherent cells was resolubilized with 200 μL of 33% (v/v) glacial acetic acid (Fisher Scientific) and the absorbance was measured at 570 nm using a microtiter plate reader. All tests were performed in triplicate with six replicates. The results were presented in terms of percentage of biofilm mass removal when exposed to the different compounds at the respective concentrations according to Equation (1):control is the OD (λ = 570 nm) for biofilms not exposed and ODcompound is the OD (λ = 570 nm) for biofilms exposed to the selected compounds.Biofilm mass quantification by CV staining was assessed according to Borges et al. . After t(b) Metabolic activity quantification of biofilm cells by alamar blue stainingexcitation = 570 nm and λemission nm = 590 nm in a microtiter plate reader . All tests were performed at least three time with six replicates. Results were obtained in terms of percentage of biofilm metabolic activity reduction when exposed to the different compounds. The percentage of biofilm metabolic activity reduction was determined according to the following Equation (2):control is the fluorescence intensity of not exposed biofilms and FIcompound is the fluorescence intensity for biofilms exposed to the selected compounds.The quantification of the metabolic activity of biofilm cells was performed according to Borges et al. ,63. Brie(c) Membrane integrity of biofilm cellsS. aureus biofilm cells was evaluated by the Live/Dead Baclight kit  after exposure to the selected compounds. This kit is composed by two nucleic acid binding stains: SYTO 9™ and propidium iodide (PI). Briefly, biofilm cells, previously obtained by scraping of the wells of microtiter plate were diluted (1:10) in PBS (pH = 7.4). Then, a volume of 300 μL of each diluted bacterial suspension was filtered through a Nucleopore black polycarbonate membrane  and then stained with 250 μL of SYTO 9™ and 50 μL of PI [2), B is the microscopic field area (cm2), V is the volume of the filtered sample (mL) and D is the dilution factor.The membrane integrity of μL of PI . This miThe percentage of intact cells were presented according to the following equation:TM as a function of concentration.The results were presented as percentage of cells stained with SYTO 9To evaluate the effect of the best compound on biofilm susceptibility to fusidic acid, biofilms were developed in the presence of 1000 μg/mL of the compound (since no MIC for this compound was detected within the concentrations tested). Bacterial suspensions with DMSO and without compound were used as controls. Besides that, Furvina was also tested as baseline compound for comparative purposes. After 24 h incubation at 37 °C and 150 rpm, 24 h-old biofilms were washed twice with PBS (pH = 7.4) and exposed to fusidic acid at 10× MIC. The plates were incubated for 24 h at 37 °C and 150 rpm. The biofilms were analyzed in terms of biomass, metabolic activity and membrane integrity as described above.p < 0.05, statistically significant). All experiments were performed in duplicate with at least three replicates for each condition tested.Statistical analysis was performed using GraphPad Prism software version 5.01 . One-way ANOVA and multiple comparisons were used to test the significance based on a confidence level of ≥95% (